EP1879448A2

EP1879448A2 - Compositions and methods related to serpin spi6

Info

Publication number: EP1879448A2
Application number: EP06748251A
Authority: EP
Inventors: Philip G. Ashton-Rickardt; Manling Zhang
Original assignee: University of Chicago
Current assignee: University of Chicago
Priority date: 2005-02-25
Filing date: 2006-02-24
Publication date: 2008-01-23
Also published as: WO2006091773A3; WO2006091773A2

Abstract

Disclosed are transgenic animals overexpressing serpin Spi6 and transgenic animals comprising a disrupted Spi6 gene. Said animals have been used to study Spi6 function and Spi6 dependent mechanisms. It is suggested to use said animals to identify compounds modulating Spi6, and to use Spi6 or inhibition of Spi6 for therapeutic application.

Description

COMPOSITIONS AND METHODS RELATED TO SERPINS

I. BACKGROUND

1. Proteolysis is crucial to a wide variety of cellular processes including programmed cell death (PCD), necrosis and cell differentiation. Homeostatic regulation of serine proteases is mainly achieved through interaction with inhibitors belonging to the Serine Protease Inhibitor (serpin) superfamily (Silverman et al., 2001). Serpins are involved in many cell metabolism states.

II. SUMMARY

2. Disclosed are compositions and methods related to serpins and cellular and organismal conditions related to serpins.

III. BRIEF DESCRIPTION OF THE DRAWINGS

3. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods. 4. Figure 1 shows the physiological function of Sρi6. (A) Homologous recombination in ES cells from

C57BL/6 mice between the Spi6 targeting vector and chromosomal wild-type allele generated mutant Spi6 neo alleles in which the critical exon 7 was replaced with the G418 resistance marker (neo). Recombinants harbored restriction fragments of altered size were detected by probing Southern blots with 5 ' and 3 ' external probes. Transient transfection with the Cre recombinase gene induced recombination between loxp sites (filled arrows) and the excision of neo, which resulted in a restriction fragments of altered size detected by the 5' probe.

5. Figure 2 shows the protease specificity of Spi6. (A) Purification of Spi6 (43kD). (B) Inhibition of protease by Sρi6 in vitro. Proteases (2OnM) were incubated with rSpiό (20OnM) over time at 37°C then assayed against labeled peptide substrate. The activity was compared with that of protease alone controls (100% activity, 0% inhibition) and the % of inhibition determined. For serine proteases the following substrates were used at ImM (Al-Khunaizi et al., 2002b; Cooley et al., 1998): human cathepsin G , Suc- AAPF-pNA; human elastase, MeOSuc-AAPV-joNA; human PR-3, Boc-A-ONP; in assay buffer (2OmM Tris-HCl pH 7.4, 50OmM NaCl, 0.1% PEG), human granzyme B, ITED- pNA; human granzyme A, BLT- />NA; human granzyme K, BLT- pNA; in assay buffer (10OmM Tris-HCl). For cysteine proteases the following substrates (Calbiochem, San Diego, CA) were used: cathepsin B, cathepsin L, Z-FR- /?N A; in reaction buffer (20OmM KH₂PO₄, 2mM EDTA, pH6.1) caspase 1, (Ac-WEHD-^NA; caspase 3, Ac- DEVD-- pNA; in reaction buffer (5OmM HEPES pH 7.4, 10OmM NaCl, 1OmM DTT, ImM EDTA, 10% glycerol, 0.1% CHAPS).

6. Figure 3 shows intrasplenic injection of DCs primes CD8 T cell expansion. Immature C57BL/6 DCs unpulsed or pulsed with the LCMV GP33 peptide (10³) were injected into the spleen of C57BL/6 mice.

After 7 d the number of CD8 cells in PBLs staining with GP33-tetramers and FCM was determined. The eificacy of DC function and or tne injection procedure is evidenced by the expansion of GP33 specific CD8

T cells.

7. Figure 4 shows Spi6 KO BMDCs are susceptible to CTL-induced PCD. (A) DNA fragmentation in GP33-pulsed BMDCs (Target, T) was measured after 4 h incubation of P14 CTLs (Effector, E) over a range of E/T ratios, as described before (Matzinger, 1991). Each value is the mean of 12 determinations from

BMDCs from 3 mice ± SEM (A) BMDCs from B6 and Spiό KO mice. (B) BMDCs from B6 with or without CMA (35nM).

8. Figure 5 shows Spi6 KO BMDCs are no more susceptible to Fas-induced PCD than are B6 BMDCs. Cells were incubated with anti-Fas mAb (JO2) for 16h with cyclohexamide. 9. Figure 6A shows Spi6 KO BMDCs are susceptible to CTL-induced lysis. Increased CTL-induced

PCD of Spi6 KO gave rise to increased lysis as measured by ⁵¹Cr-release from GP33-pulsed BMDCs after 4 h incubation with P14 CTLs. Figure 6(B) show a lower number of LCMV-specific CTLs in Sρi6 KO mice. The absolute number of GP 33 tet⁺ CD8⁺ cells is indicated for B6 and Spi6 KO mice and was determined 8 d after infection with LCMV Armstrong (2 x 10⁵ PFU/mouse). 10. Figure 7 shows the occurence of more PCD of LCMV-specific CTLs in Spiό KO mice. Values are the mean absolute number of 5 determinations from individual mice ± SEM.

11. Figure 8 shows altered expansion and contraction of LCMV-specific CD8 T cells in Spi6 KO mice. Each value is the mean from 5 determinations from PBLs of individual mice ± SEM.

12. Figure 9 shows decreased IFN-γin Spi6 KO mice. Mice were infected with LCMV then IFN-γ measured in the serum over time by ELISA. Data is the mean from 4 mice ± SEM.

13. Figure 10 shows lower NK cell activity against Cr-labeled tumor cells from the spleen of Spiό KO mice compared to B6 controls. (A) The lysis of ⁵¹Cr-labeled YAC target cells by spleen cells was measured after 4 h. (B) Spleen cells were incubated with YAC targets for 4 h at which point they were stained with anti-DX5-PE to identify NK cells and the DNA dye YOPRO-I to identify apoptotic cells then by FCM (Opferman et al., 2001). In both parts the data is the mean from 4 separate mice ± SEM.

14. Figure 11 shows the clearance of LM from Spiό KO mice. Mice were infected i.p. with LM (10⁵ CFU) mice. Over time mice were killed and the spleens and livers removed and homogenized and 10-fold serial dilutions of organ homogenates plated on Trypticase-soy agar. Bacterial colonies were counted after incubation at 37° C for 24 hours. Each titer is the mean of 4 determinations on individual mice ± SEM. 15. Figure 12 shows decreased IFN-γin Spiό KO mice. Mice were infected with 10⁵ LM and cytokine levels determined by ELISA. The data are the mean ± SEM of 4 mice.

16. Figure 13 shows leukocyte numbers in Spiό KO mice. Mice were infected with 10^s LM then after 3 d the % of cells determined in the spleen by staining for phenotypic markers and FCM. The data are the mean ± SEM of 4 mice. 17. Figure ^"Ϊ4 snow^'s decreased LM-specific CD8 T cells in Spi6 KO mice. Mice were infected with

10^s LM and the % of LM-specific CD8 T cells measured in PBLs after staining with 0VA-H-2K^b tetramers and anti-CD8 then FCM. The data are the mean ± SEM of 4 mice.

18. Figure 15 shows granzyme B in P14 CTLs. The specific activity (units (U)/mg total protein) of enzymes was measured in GP33-specific CTLs from B6 and Grn B KO mice.

19. Figure 16 shows no granzyme B in granulocytes. The specific activity (units (U)/mg total protein) of enzymes was measured in peritoneal cells from B6 and Grn B KO mice.

20. Figure 17 shows increased uptake and killing of LM by Spi6 KO granulocytes. Granulocytes were elicited by glycogen and the mice infected i.p. with LM. (A )The titer in peritoneal cells was determined after lysis in 0.2% NP40 over time. (B) After 2h, the titer of intracellular and extracellular LM was determined and combined to give the total titer. All data is the mean of 4 mice ± SEM.

21. Figure 18 shows granulocyte function from mixed bone-marrow chimeras. Glycogen elicited granulocytes from mixed chimeras were purified by FACS (Spi6 KO CD45.1+, B6 CD45.2+) and mixed with LM in vitro. After 2h, the titer of extracellular and intracellular LM was determined and combined to give the total titer. All data is the mean of 4 mice ± SEM.

22. Figure 19 shows expression oϊSpiό in CD8⁺ T cells. B-D, Gene expression was determined using real-time PCR, and is reported as a ratio compared with the control, cyclophilin A. Histograms represent individual determinations of two independent isolates (black and white), which contained cells purified from the pooled splenocytes of 5-10 mice. The expression of Spi6 and granzyme B was significantly higher in effectors and memory cells compared with naive cells. Negligible expression of MHC class //in all populations indicated the absence of contamination by APCs. _*,p < 0.05, _**, p < 0.01, _***,p < 0.001.

23. Figure 20 shows that Spi6 protects T cells specifically from granzyme B-mediated apoptosis. A, Jurkat clones transfected with the Spi6 cDNA or CD2 expression cassette vector (V) were incubated in the presence of sublytic concentrations of perform with (M) or without (M) granzyme B, or (□) in medium alone (-). Apoptosis was significantly reduced in the Jurkat clones transfected with Spi6, compared with Jurkat clones transfected with vector alone. B, Transfected Jurkat clones were cultured in the presence of medium alone (-), Ab against Fas (anti-Fas), or exposed to gamma-irradiation (T-IR). Apoptosis initiated by these stimuli did not differ between Jurkat clones transfected with Spi6 compared with controls, indicating that Sρi6 protects specifically from granzyme B-mediated apoptosis. Histograms are the mean ± SEM from three determinations. _*, p < 0.05.

24. Figure 21 shows the expression of Spi6 in B6 and Spi6 mice. B, Cells were isolated from two to four B6 and Spi6 mice (C), pooled, and the expression level of Spι6 determined by real-time PCR. Histograms are the mean of three determinations.

25. Figure 22 shows enhanced memory cell development in Spi6 mice. Spi6 (D), B6 (■), and GrnBKO (JB) mice were infected with LCMV, and memory cells detected in the spleen >180 days later by measuring ex vivo IFN-! production. A, Spi6 and GrnBKO mice had significantly higher percentages of memory cells than Bt'mice."^'"^,^' Th^'e ab'soluϊe number of memory cells generated was higher in Spi6 mice than B6 mice

(GP33: 2.9 times more; NP396: 2.1 times more; GP276: 2.8 times more). GrnBKO mice also generated more memory cells than B6 mice (GP33: 2.2 times more; NP396: 1.8 times more; GP276: 2.3 times more). C, The percentages of Ag-specific cells in the blood of Spi6, B6, and GrnBKO mice, detected using H-2D^b tetramers loaded with GP33 peptide, were not significantly different 8, 15, or 30 days postinfection.

Histograms are the mean ± SEM from combined experiments (n - 7-9 mice per group). _*,p < 0.05, _**,p < 0.01, when compared with B6 mice.

26. Figure 23 shows expression of Spi6 in CD8⁺ T cells increases the level of memory cells. Naive CD8⁺ T cells from P14 and P14 x Spi6 mice, both Thyl.2⁺, were purified using magnetic beads and adoptively transferred to Thyl.l⁺ congenic recipients. Recipients were infected with LCMV and more than

50 days later the percentage of memory cells in recipient spleens determined by measuring ex vivo IFN-T production. B, The percentages of P14 memory cells (*, mean: 0.61 ± 0.15%) or P14 X Sρi6 memory cells (O, mean: 1.5 ± 0.16%) from individual mice are indicated for one experiment. C, Percentages of memory cells from four experiments were normalized to the percentage of P14 or P14 x Sρi6 cells (Thyl.2⁺) in the blood 7 days after infection with LCMV. The normalized percentage of Pl 4 X Spi6 memory cells was significantly higher than P14 memory cells. D, The percentage of resident memory cells (Thyl .2^") was no different between recipients receiving P14 cells or P14 x Spi6 cells (p > 0.05). E, There was no significant difference (p > 0.05) in the level of Thyl.2⁺ donor P14 or P14 x Spi6 cells in the blood 7, 14, or 28 days after infection of recipients with LCMV (p > 0.05). Histograms are the mean ± SEM from four pooled experiments (n = 12-14 mice per group). _**, p < 0.01.

27. Figure 24 shows increased survival of Sρi6 KO mice after bacterial infection. Wild-type C57BL/6 (WT) or C57BL/6 Spi6 deficient (Spi6-/-) mice( n=12) were infected intranasaly with P.aeruginosa (50ml; 0.05OD) and survival measured over time.

28. Figure 25 shows an increased bactericidal activity of Spi6 KO granulocytes. Thioglycolate elicited granulocytes were harvested from the peritoneum of C57BL/6 (B6) and Spi6-deficient (Spi6 KO) mice

(2x105) and incubated with E.coli (2x106) at 37° C in vitro. The mean percentage titer oϊE.coli (duplicate determinations from 3 mice) was determined over time with the titer at t=0 100%.

29. Figure 26 shows the survival of Spi6-/- mice relative to wild type mice after bacterial challenge.

30. Figure 27 shows Spi6 is a physiologic inhibitor of elastase in mouse granulocytes. Organelle and cytosolic fractions were obtained from glycogen-elicited granulocytes (Fig. 16) and the activity of elastase determined by measuring the hydrolysis of fluorescently labeled peptide substrate specific for elastase but not other related proteases (e.g. PR-3 or cathepsin G - Fig. 2B). A significant increase in specific activity from Spi6 KO mice compared to B6 mice (pO.001) ** was observed.

31. Figure 28 shows the development of neutrophils as presented in Borregaard and Cowland, 1997. 32. Figure 29 shows a method of producing Spi6 deficient mice, (a) Homologous recombination between the Spiό targeting vector and wild-type allele in ES cells as detected by altered restriction fragments [Hind Ul (H), Spel (bjj using :>'"and 3' external probes. Cre induced loxP (filled arrows) mediated excision of neo resulting in restriction fragments of altered size (kb).

33. Figure 30 shows impaired survival of Sρi6 KO CTLs in vivo, (a) Mean number gp33⁺ CD8⁺ cells ± standard error of the mean (s.e.m.) (n=5 individual mice), (b) Mean percentage YOPRO-I⁺ of gp33⁺ CDS⁺ ± s.e.m. (n=5 individual mice), (c) Mean number Of OVA⁺ CD8⁺ spleen cells ± s.e.m. (n=5 individual mice),

(d) Mean percentage of YOPRO-I ⁺ Of OVA⁺ CD8⁺± s.e.m. (n=5 individual mice).

34. Figure 31 shows impaired CTL-immunity in Spi6 KO mice, (a) Ex vivo anti-LCMV CTL activity (mean quadruplicate determination) from individual mice versus the ratio of splenic leukocytes (S) to (T) targets, (b) Mean titre of clone 13 LCMV ± s.e.m. (n=5 individual mice). 35. Figure 32 shows increased GrB activity and apoptosis in Spi6 KO CTLs. Mean specific activity

(SA) ± s.e.m. of GrB and caspase 3 (casp 3) from Spi6 KO (S) or GrB KO (G) P14 CTLs CTLs (n=4 individual mice).

36.Figure 33 shows Sρi6 ensures the integrity of lytic granules by suppressing GrB. Distribution of the number OfGrB⁺ granules per P14 CTL (n=300 cells). The mean values are indicated, (b) Mean SA of GrA in the granule fractions of Pl 4 CTLs ± s.e.m (n=4 individual mice).

37. Figure 34 shows Spi6 inhibits HNE. (a) Kinetics and (b) stocbiometry of inhibition of NE activity by rSpiό.

38. Figure 35 shows Spi6 expression in leukocytes. RNA was extracted from purified cell populations and cDNA synthesized. Neutrophils were elicited from the peritoneum by injection with glycogen. Cells were isolated from 3 B6 mice, pooled and real-time PCR for Spi6 and the cyclofilin A housekeeping gene performed. Histograms are the mean of 3 determinations.

39. Figure 36 shows increased NE activity in Sρi6 KO neutrophils. Neutrophils were harvested by lavage with PBS (ImI) of the peritoneum 4h after i.p. injection with 15% glycogen (Sigma Aldrich, St. Louis MO) then activated for 24 h with E. coli (2xlO⁶/ml) at 37⁰C . Cells were lysed by sonication in hypotonic buffer (50 mM PIPES, 5OmM KCL, 5mM EGTA, 2mM MgCl₂ 5mM DTT, pH 7.6) then centrifuged at 3,000 x g for 20 min to remove nuclei then 15,000 x g for 30 min to give cytosol (supernatant) and granule (pellet) fractions. The granule pellet was resuspended in 1% Triton X-IOO, 1OmM Tris.HCl, 15OmM NaCl, pH 7.6 for 30 min on ice. NE activity was determined. Mean specific activity (SA) of NE from neutrophils ± SEM (n= 4 mice). 40. Figure 37 shows Spi6 KO neutrophils are susceptible to death. (A) Death (mean % PI⁺) and (B) apoptosis (mean %Y0P0R0) ± SEM (n= 4 mice) after stimulation in vitro with E. coli. YORPRO-I detects the early onset of apoptosis be measuring changes in DNA.

41. Figure 38 shows Spi6 KO neutrophils are susceptible to lysis. Lysis of neutrophils (mean extracellular LDH activity) ± SEM (n= 4 mice) after stimulation in vitro with E. coli. LDH was assayed according to the manufacturers protocol (Promega). 42. FΪgure^"_Φ srϊ'ό'ws" increased extracellular NE activity from Spi6 KO neutrophils. Neutrophils were harvested and activated with E. coli as in Figure 40. Overtime, cells were centrifuged and NE activity determined in the supernatant. Mean activity of NE from neutrophils ± SEM (n= 4 mice).

43. Figure 40 shows Spi6 KO neutrophils possess increased bactericidal activity. Glycogen-elicited peritoneal neutrophils (2xlO⁵) were incubated with E. coli (2xlO⁶) at 37° C in vitro. The number of viable bacteria measured over time by titer on LB agar plates. Mean titers of E. coli are expressed as a % of titer at t=0 + SEM (n=4 mice).

44.Figure 41 shows the effects of Spi6 deficiency on bacterial immunity. Percentage survival of B6 and Sρi6 KO mice (n=12) and bacterial titer (± SEM) after infection with (a) P. aeruginosa (titer in bronchoalveolar lavage, (BAL), (b) E. coli (titer after 12 h) and (C) L. monocytogenes (titer after 48 h).

C57BL/6 wild-type or C57BL/6 Spi6 KO mice (6-8 w old; 16-18 g) were infected with lethal doses of either (i.p 50μl) E. coli (4.6 xlO⁶ cfu/mouse) (i.n. 50μl) P. aeruginosa ( 5 xlO⁷ cfu/mouse) or (i.v. 50μl) L. monocytogenes EGDe( 4 x 10⁴ cfu/mouse) and survival measured over time. All titers were determined on LB plates after incubation at 37° C for 18h. 45. Figure 42 shows Serum IFN-γ after infection of Spi6 KO mice with L. monocytogenes. Mice were infected, then the concentration of IFN-γ determined in the serum by ELISA (BD Pharmingen). Mean IFN-γ concentration ± SEM (n= 4 mice).

46. Figure 43 shows the effect of exogenous HNE on immunity to P. aeruginosa. B6 (n=12) and P. aeruginosa titer (± SEM) after i.n. dosing with HNE at doses shown (U/kg mouse). The titer over time in the BAL is shown after dosing with 1.8U/kg mouse.

47. Figure 44 shows anti-serum that detects Spi6 in neutrophils. FCM plot showing staining of glycogen-elicited neutrophils from B6 (blue histogram) or Sρi6 KO mice (red histogram), with anti-rSρi6 antiserum (4μl). The secondary antibody was goat anti-rabbit IgG-APC.

48. Figure 45 shows the N-terminal sequence of the ova-family of serpins. Bold residues are charged. The hydrophobic elements are boxed. The positions of the hA and hB α-helices are indicated. The Sρi6 mutants lacking hA and hB domains are indicated. Residues in lower case are those added during cloning.

49. Figure 46 shows the sequences of siRNAs. Only shown are the sense strands of RNA duplexes. Starting positions for PI9 gene (SERPINB9, accession No NM004155) is indicated. PI9 sequences were selected based on an aligorithim that optimize siRNA gene silencing based on 8 independent criteria (Reynolds et al., 2004).

50. Figure 47 shows impaired survival and function of Spi6 KO CTLs. (a) Percentage (upper right corner) of gp33⁺ CD8 ⁺ and YOPRO-I⁺ cells, (b) Mean number gp33⁺ CD8⁺ cells ± standard error of the mean (s.e.m.) (n=5 individual mice), (c) Mean percentage YOPRO-I⁺ of gp33⁺ CD8⁺± s.e.m. (n=5 individual mice), (d) Percentage OVA⁺ CD8 ⁺ and YOPRO-I ⁺ cells, (e) Mean number OfOVA⁺ CD8⁺ ^' spleen cells ^"± mice), (f) Mean percentage of YOPRO-I ⁺ OfOVA⁺ CD8⁺± s.e.m.

(n=5 individual mice).

51. Figure 48 shows the effect of Spi6 on CTLs is dependent on GrB. B6 and Spi6 KO mice were infected with LCMV Armstrong (2x10⁵ pfu/mouse i.p.) then after 8d the (a) mean number of gp33⁺ CDS⁺ spleen cells and (b) mean percentage YOPRO-I⁺ of gp33⁺ CD8⁺ cells determined. P14 CD8 T cells (10⁴) were purified from B6 or Sρi6 KO mice (CD45.2⁺) and adoptively transferred to B6 CD45.1⁺ congenic mice, then the (c) mean number of donor LCMV specific (CD45.2⁺ gp33⁺ CD8⁺) and (d) mean percentage OfYOPRO-I⁺ donor cells determined. Recipient mice infected and fed BrdU for 8d then the (e) mean percentage BrdU^4" donor cells determined. All means are ± s.e.m. (n=5 mice). 52. Figure 49 shows impaired CTL-immunity in Spi6 KO mice. B6 and Spi6 KO mice were infected with LCMV clone 13 (10⁶ pfu/mouse i.v.). (a) Ex vivo anti-LCMV CTL activity was measured by determining % specific lysis of ⁵¹Cr-labeled RMA target (T) cells (H-2^b) pulsed with LCMV gp33 peptide (mean of 4 determinations) by splenocytes (S) from infected mice. Data is the mean % specific Cr⁵¹ -release ± s.e.m. (n=5 mice) at a S/T ratio of 50:1. (b) Titer of clone 13 LCMV determined in spleen homogenates by plaque assay on Vero cells. Data is the mean titer expressed as pfu per mg spleen ± s.e.m. (n=5 mice), (c) Ex vivo anti-LCMV CTL activity determined as % specific lysis gp33 -pulsed targets on d8 after infection with LCMV Armstrong (2 x 10⁵ pfu/mouse i.p.). Data is the mean of 4 determinations of % specific Cr⁵¹-release from individual mice over a range of S/T ratios, (d) Ex vivo anti-LCMV CTL activity was measured as in (a) at various ratios of gp33⁺CD8⁺ (CTL) to targets (T). P14 CD8 T cells (10⁴) were purified from B6 or Spi6 KO mice and adoptively transferred to wild-type B6 mice, then infected with LCMV clone 13 (10⁶ pfu/mouse i.v.). (e) Ex vivo anti-LCMV CTL activity determined as % specific lysis gp33-pulsed targets on d6 after infection. Data is the mean % specific Cr⁵¹-release ± s.e.m. (n=5 mice) over a range of S/T ratios, (f) Titer of clone 13 LCMV from the spleens of recipient mice on d6 after infection. Data is the mean titer expressed as pfu per mg spleen ± s.e.m. (n=5 mice). 53. Figure 50 shows Spi6 deficiency increases cytoplasmic GrB and apoptosis in CTLs. Mean specific activity (SA) ± s.e.m. of GrB and caspase 3 (casp 3) from CTLs (n=4 individual mice).

54. Figure 51 shows Spi6 deficiency destabilizes lytic granules, (a) Distribution of the number OfGrB⁺ granules per P14 CTL (n=300 cells). The mean values are indicated, (b) Effect of Z-AAD (OMe)-CMK on the number Of GrB⁺ granules per P14 CTL ± s.e.m (n=300 cells), (c) Mean SA of GrA in the granule fractions of P14 CTLs ± s.e.m (D=4 individual mice).

55. Figure 52 shows survival and activity of Spi6 KO neutrophils. A) Death (mean % PI⁺), (B) apoptosis (mean %Y0P0R0) and (C) lysis (mean extracellular LDH activity) ± SEM (n= 4 mice) after stimulation in vitro with E. coli. Mean titers of E. coli are expressed as a % of titer at t=0 ± SEM (n= 4 mice). (E) Effect of HNE (0.12U) on E. coli titer measured after 240 min. 56. Figure 53 shows the effects of Spi6 deficiency and HNE on bacterial immunity. Percentage survival of B6 and Spi6 KO mice (n=12) and bacterial titer (+ SEM) after infection with (a) P. aeruginosa (titer in η BALX^""(βyE'^'όo1ι (titeϊafteϊ Vl h) and (C) L. monocytogenes (titer after 48 h). (D) Percentage survival of

B6 (n=12) and P. aeruginosa titer (+ SEM) after i.n. dosing with HNE (1.8U/kg mouse) 57. Table 3 shows cell numbers in B6 and Spi6 mice (Xl O⁶).

58. Table 4 shows the effect of Spi6 deficiency on NE and neutrophils in the lung after P. aerginousa infection. NE activity (mU = milliunit = 1 U x 10^"3; units of activity as defined previously by Liu et al.,

(2003)) and the number of segmented neutrophils were determined for ImI of BAL from B6 or Spi6 KO mice and are ± SEM (n = 3-7 mice). At certain time points mice had died or data was not determined (n.d.). There was a significant difference in the activity of NE (P = 0.03) and neutrophil number (P = 0.002) in B6 compared to Spi6 KO BAL after 12 h. 59. Table 5 shows the effect of exogenous HNE in the lung after P. aeruginosa infection. NE activity

(mU) and the number of segmented neutrophils were determined for 1ml of BAL from B6 mice and are ± SEM (n = 3-8 mice). Mice were infected than after 4h either dosed ,(U/kg mouse) i.n. with HNE (+) or not (- ). At certain time points mice had died or data was not determined (n.d.). There was a significant difference (P = 0.02) in NE activity after 6 h and neutrophil number (P = 0.02) after 12 h between un-dosed and dosed mice.

60. Table 6 shows the effect of Spi6 deficiency on NE and neutrophils in the lung after P. aerginousa infection. NE activity (mU) and the number of segmented neutrophils were determined for ImI of BAL from B6 or Spi6 KO mice and are ± SEM (n = 3-7 mice). At certain time points mice had died or data was not determined (n.d.). There was a significant difference in the activity of NE (P = 0.03) and neutrophil number (P = 0.002) in B6 compared to Spi6 BAL after 12 h.

61. Table 7 shows the effect of exogenous HNE in the lung after P. aerginousa infection. NE activity (mU) and the number of segmented neutrophils were determined for ImI of BAL from B6 mice and are ± SEM (n = 3-8 mice). Prior to infection (4 h), mice were either dosed .(U/kg mouse) i.n. with HNE (+) or not (-). At certain time points mice had died or data was not determined (n.d.). There was a significant difference (P = 0.02) in NE activity after 6 h and neutrophil number (P = 0.02) after 12 h between un-dosed and dosed mice.

IV. DETAILED DESCRIPTION

62. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

63. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like. O4.κanges can De expressed Herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

65. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

66. "Primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.

67. "Probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. 68. As used herein, "immunological memory" refers to the physiological condition characterized by antigen-specific lymphocytes with the ability to provide recall responses upon future antigen experience which remain after an initial antigen experience and that cause a quicker immune response to the same antigen than a response in a similar organism which had not previously been challenged by the antigen. It is understood and herein contemplated that the lymphocytes that provide this protection can be CD4 or CD8 T-cells specific for the antigen.

69. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more folly describe the stale oϊ^'ffi^'e art fo which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

70. "Memory" means a response of the pool of immune cells that are produced after antigen stimulation that will last for the life of an organism after antigen presentation.

71."Naϊve cell" or "naϊve cells" mean a cell that has not been exposed to antigen presentation.

72. It is understood that as Spi6 and PI9 are species homologs, where reference to one occurs, reference to the other is also disclosed, unless indicated otherwise. For example, if a Spi6 mouse is discussed, it is understood that a PI9 mouse is also disclosed. B. Compositions

73. The disclosed compositions and methods are related to Serpins, such as Sρi6 and PI9. Disclosed herein are compositions, such as specific mice which overexpress Spi6, and mice which have their native Spi6 disrupted producing a knockout phenotype. These mice, both the Spi6 overexpressing mice and the Spi6 knockout mice and numerouse experiments disclosed herein with these mice have identified a number of roles for Spi6 in the general inflammation process, as well as specific roles, such as its role in Granzyme

B regulation. These roles for Spi6 and experiments performed to confirm and further define the roles of the Spi6 class of Serpins has also identified a number of diseases which can also be regulated by either Spi6 (or the corresponding homologs, such as PI9) enhancement or inhibition. Thus, also provided herein are numerous compositions which either enhance Spi6 expression or activity or inhibit Sρi6 expression or activity, as well as the methods of using these types of compositions in methods of treatment or regulation of cellular states or disease states.

1. Serpins

74. Proteolysis is crucial to a wide variety of cellular processes including programmed cell death (PCD), necrosis and cell differentiation. Homeostatic regulation of serine proteases is mainly achieved through interaction with inhibitors belonging to the Serine Protease Inhibitor (serpin) superfamily

(Silverman et al., 2001). Inhibitory serpins have a common mode of action: each contains a variable C- terminal reactive center loop (RCL) resembling the substrate of its cognate protease. On protease binding, the (RCL) is cleaved between the two residues designated Pi and P'i and it undergoes a conformational change that distorts the protease and irreversibly locks the serpin-protease complex (Silverman et al., 2001). 75. A comparison of RCLs for Ova-serpins from Sun et al., (1997), shows the appropriate amino acids for the RCL and the P and P' sites for Spi6 and PI9, which is herein incorporated by reference at least for any material related to the RCL and active sites of Spi6 and PI9 as well as other Ova-serpins (Sun, et al., 1997). One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the Pl 4 position of the reactive center loop. 76. The best characterized serpins are those that regulate extracellular serine proteases in vertebrates, however, recent evidence suggests that some function intracellularly. Such "ovalbumin" serpins include the ^" "^""^' fiuman proleinase iMihhδτs"& (FL-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. For example, serpins of the family that resembles chicken ovalbumin (ova-serpins) inhibit both serine and cysteine proteases in the cytoplasm (Bird, 1998). As another example, the cowpox serpin Crm A inhibits both granzyme B and caspases 1 and 8 (Komiyama et al., 1994; Quan et al., 1995; Zhou et al., 1997). In addition, PI-9 is a potent granzyme B (graB) inhibitor that has an unusual

Pl GIu and is present primarily in lymphocytes.

77. Examples of other elastase inhibitors include, but are not limited to human secretory leukocyte proteinase inhibitor SLPI (SEQ ID NOS 30 and 31), mouse leukocyte proteinase inhibitor SLPI (SEQ ID NOS 36 and 37), human monocyte/neutrophils elastase inhibitor MNEI (SEQ ID NOS 41 and 42), mouse EIA (SEQ ID NOS 39 and 40), humanα_rantitrypsin(SEQ ID NOS 28 and 29), and mouse α_rantitrypsin

(SEQ ID NOS 32 and 33). Spi6 and PI9 are also elastase inhibitors, and so mice transgenic for elastases can also be used to screen for inhibitors of serpins.

78. Serpins characteristically act as "suicide substrates" and inactivate proteases through the formation of a 1: 1 complex (Silverman et al., 2001). However whether or not the inhibition of proteases by serpins occurs through this mechanism seems to depend on the serpin and the protease in question (Al-Khunaizi et al., 2002a; Annand-efe-al->l-999-)^«"^

79. The serpins act as called "suicide inhibitors," forming an SDS-stable complex with their target protease. They typically associate with proteases by presenting a "bait" residue, in their reactive center, that is thought to mimic the normal substrate of the enzyme. The bait amino acid is called the Pl residue, with the amino acids toward the amino-terminal side of the scissile reactive center bond labeled in order PI, P2,

P3, etc. and the amino acids on the carboxyl side labeled PF, P2', etc. (Lawrence, et al., 1994).

80. There are also serpin-like inhibitors of granzyme B that act in a similar fashion as serpin inhibitors. An example of a serpin-like inhibitor is Crm A (SEQ ID NO:24 and 25 for the gene and protein sequences respectively). CrmA is a serpin-like protease encoded by cowpox virus. CrmA has been shown to be an inhibitor of granzyme B (Smith et al., 1996).

81. Substrates for the disclosed serpins can include, but are not limited to: granzyme B, cathepsin G (P28293), PR-3 (Q61096), neutrophil elastase (NP031945), mouse mast cell protein (MMCP) -1 (P11034), MMCP-2 (P15119), MMCP-3 (P21843), MMCP-4 (P21812), MMCP-5 (P21844), MMCP-8 (P43430), MMCP-9 (035164), MMCP-IO (AAK51075), or caspase 1 (P29452). 2. Immune System a) Immune system function

82. Recognition of antigen peptide-class I major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs), by T cell receptors (TCRs) on naϊve CDS T cells triggers proliferation and differentiation into effector cytotoxic T lymphocytes (CTLs) (Zinkernagel and Doherty, 1974). Engagement of TCRs by pMHC causes a CTL to kill an infected cell through the induction of PCD. CTLs induce PCD by either the exocytosis of granules containing executioner proteases, or the ligation of death receptors on target cells (Kagi et al., 1994b). The granule pathway of cytolysis involves perforin, which pϊόmόiesthe¹' entry of gfarizyine B and other cytotoxins into the cytoplasm of target cells where they initiate

PCD (Kagi et al., 1994a; Russell and Ley, 2002). In addition to CTLs, natural killer (NK) cells also use the perforin/granzyme B pathway to kill tumor or virally infected cells. However, unlike CD8 T cells, NK cells can kill immediately upon encountering a target without the need for further differentiation. 83. Mast cells and basophils, which are activated by immunoglobulin E (IgE) and allergen, play a prominent role in anaphylaxis. However, they express at least three types of IgE receptor, including the high affinity IgE receptor (Fc epsilon RI). The relative contribution of these IgE receptors, and possibly other receptors such as Fc epsilon RII/CD23 and Mac-2, to the genesis of in vivo anaphylaxis was elucidated to by generating Fc epsilon Rl-deficient mice (Dombrowicz et al., 1993). These Fc epsilon RI- deficient mice appear normal and express a normal number of mast cells, but they are resistant to cutaneous and systemic anaphylaxis. Such data demonstrates that Fc epsilon RI is necessary for the initiation of IgE- dependent anaphylactic reactions. Therefore, interfering with its function should be an effective means of treating allergy, regardless of the allergen specificity (Dombrowicz et al., 1993). b) Serpin immune system relationships 84. Mammalian ova-serpins can protect both APCs and CTLs from PCD caused by granzyme B. In humans, Proteinase Inhibitor 9 (PI9) (SEQ ID NOs 1 and 2, for gene and protein sequences, respectively) is a potent inhibitor of granzyme B (Sun et al., 1996). In mice, Serine Protease Inhibitor 6 (Spi6) is a homologue of PI9, can also inhibit granzyme B (Sun et al., 1997a). SPI6 comprises a 1.8-kilobase cDNA (SEQ ID NO:3) encoding a 374-amino acid polypeptide (SEQ ID NO:4) that is 68% identical to PI-9 (SEQ ID NO:2). Although the reactive loop of SPI6 differs from PI-9, both contain a GIu in a region likely to contain the Pl-Pl' bond of PI9 (Sun et al., 1997a). However in Spi6 the GIu (single amino acid code -E) is off one place compared to PI9. Both Spi6 and PI9 have a Cys-Cys motif in the Pl' and P2 'positions and are 69% homologous in the RCL. As used herein, unless otherwise indicated Spi6 and PI9 refer to any protein having the functional activity of the Spi6 set forth in SEQ ID NO:4 and PI9 set forth in SEQ ID NO:2. PI9 is located in the cytoplasm of cytolytic lymphocytes (CTLs and Natural. Killer (NK) cells) and activated

APCs such as macrophages and dendritic cells (DCs)(Bladergroen et al., 2001; Hirst et al., 2003). Both PI9 and Spi6, when over expressed can protect cells from granule-mediated PCD through the inhibition of granzyme B (Medema et al., 2001a; Medema et al., 2001b ). Therefore it has been suggested that a physiological function of PI9 and Spi6 is to protect healthy cytolytic lymphocytes and DCs from "misdirected" granzyme B, which leaks into the cytoplasm during upon activation (Bird, 1999). Support for this view comes from the observation that over expression of PI9 in human CTLs increases survival and potency (Hirst et al., 2003).

85. In addition to the known substrate of Spi6, granzyme B, there are a variety of mouse chymotrypsin- like proteases that contain homologous sequences to granzyme B that may serve as targets of Spi6. Examples of such proteases include: cathepsin G (P28293), PR-3 (Q61096), neutrophils elastase

(NP031945), mouse mast cell protein (MMCP) '-1 (P11034), MMCP-2 (P15119), MMCP-3 (P21843), MMCP-4 (P21812), MMCP-5 (P21844), MMCP-8 (P43430), MMCP-9 (035164), MMCP-10 (AAK51075). ^" SS. Perforin Is iiόWnly'r^u'lred for killing but also induces the PCD of CTLs and maintains the size of the effector cell pool after infection (Badovanic et al, 2000; Kagi et al., 1994a; Spaner et al., 1999; Spaner et al., 1998). This implies that PCD of CTLs can be induced by 'misdirected' granzymes. It has been observed that selective inhibition of granule-mediated cytolysis not only protects targets from PCD but also the CTL itself, and that the extent of killing limits memory CD8 T cell development (Opferman et al., 2001).

Provided herein are methods of protecting T cells from the granule exocytosis pathway of programmed cell death, comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. Also provided are methods of protecting T cells from the granule exocytosis pathway of programmed cell death, comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof. It is understood that any means of overexpressing Spi6 or PI9 would be aid in protecting T cells from the granule exocytsis pathway of programmed cell death.

87. Cytolytic activity may also regulate the priming of CTL responses by DCs. Elimination of antigen- specific DCs by cognate CTLs and the suppression of secondary immune responses has been observed in several mouse models (Hermans et al., 2000; Loyer et al., 1999). It has been suggested that in perforin- deficient mice, DCs are protected from CTL activity and so are better able to prime the expansion of antigen-specific CD8 T cells (Badovanic et al., 2000; Loyer et al., 1999; Stepp et al., 1999). However the control of DC viability in anti-viral responses remains controversial in the light of the finding that elimination of DC by CTLs was largely independent of perforin or Fas (Ludewig et al., 2001). c) Memory cells 88. The differentiation of naϊve CD8 T cells into CTLs is accompanied by an expansion in cell number then a severe contraction in which 90-95% of the expanded cells die through PCD (Ahmed and Gray, 1996; Murali-Krishna et al., 1998; Sprent et al., 2002)). In spite of this considerable cell death, more antigen- specific cells remain at the end of the immune response than existed before the infection and comprise the population of memory CD8 T cells, which are responsible for host protection from re-infection. Those effectors that survive the contraction phase acquire the characteristics of memory cells gradually during a transition into the memory phase (Opferman et al., 1999; Kaech et al. 2002). The memory phase can extend for the lifetime of the host (Sprent et al., 2002), providing immunity as the result of both an increased precursor frequency of Ag-specific cells and improved function compared with naive cells (Berard et al., 2002). The potency of memory responses is due to an increase in the frequency of antigen-specific lymphocytes and also to the qualitatively more effective response of memory CD8 T cells (Ahmed and

Gray, 1996). The eventual magnitude and duration of T-cell immunity are the sum effect of changes occurring in all three phases of the T cell response, which are priming /expansion, death and memory. Studies support a linear differentiation model of memory CD8 T cell development, which predicts that memory cells are the direct progeny of effectors (Kaech et al., 2002; Opferman et al., 1999). The induction of PCD during the contraction phase is critically dependent on another effector molecule produced by cytolytic lymphocytes - interferon -γ (IFN-γ)(Badovanic et al., 2000; Badovinac et al., 2004). It has been recently shown that the escape of memory-cell precursors from PCD, seems to be dependent on the upregulation of so called protective factors that inhibit PCD caused by inflammatory mediators (Liu et al., 2004). Discϊδ^'se^'3 her^'ein^'it rϊas"^"Been demonstrated that transgenic Spi6 can increase the survival of long- term memory CD8 T cells, which retain granzyme B expression. Furthermore, unlike naive cells, CD8⁺ memory T cells (memory cells) can directly kill target cells because they retain the machinery necessary for the granule exocytosis pathway (Opferman et al., 1999; Kaech et al. 2002; Selin et al., 1997). Maintenance of immunological memory is an active process; the level of memory cells is preserved by slow division

(Tough et al., 1994; Bellier et al., 2003; Veiga-Fernandes et al., 2004) and can be influenced by subsequent infections (Selin et al., 2004).

89. Also provided herein are methods of increasing the number of CD8+ memory T cells, by inhibiting programmed cell death, comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. Further provided herein are methods of increasing the number of CD8+ memory T cells, by inhibiting programmed cell death, comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof. d) Neutrophil elastase and cathepsin G and granulocytes

90. The serine proteases neutrophil elastase (NE) and cathepsin G (CG) play a critical role in the ability of granulocytes to both digest extracellular matrix components, as they exit the circulation and migrate to the site of infection, and to their ability to digest bacteria within the phagolysosome (Lehrer and Ganz, 1990). Mutant mouse models have demonstrated overlapping roles for NE and CG in granulocyte immunity to microbial infection (Belaaouaj et al., 1998; Maclvor et al., 1999; Tkalcevic et al., 2000) Any of these mice can be crossed with the mice disclosed herein. A third serine protease stored along with NE and CG in the azurophilic granules of neutrophils, which shares similar substrate specificity, is Proteinase 3

(PR-3) (Rao et al., 1991). While playing a protective role in host immunity, neutrophils and their serine proteases have also been implicated in numerous inflammatory diseases (Malech and Gallin, 1987). In addition to circulating serpins, intracellular ova-serpins have been implicated in the control of azurophilic granule proteases. In humans Proteinase inhibitor 6 (PI6) is a potent inhibitor of CG in the cytoplasm of monocytes and neutrophils (Scott et al., 1999). In addition to granzyme B, PI9 also inhibits NE and PI9 derived peptide substrates are specifically recognized by PR-3 (Dahlen et al., 1999; Korkmaz et al., 2002).

91. Although granzyme B is a phenotypic marker for NK cells and CTLs, there have been reports of expression of both granzyme A and granzyme B in human granulocyte (Hochegger et al., 2004; Wagner et al., 2004). On the basis of these studies a role for granzyme B has been postulated in polymorphonuclear leukocyte (PMNL)-mediated antibody-dependent cellular cytotoxicity (ADCC), which is important for the elimination of tumor and virally-infected cells. In addition the presence of a peptide sequence with bactericidal activity in granzyme B (Shafer et al., 1991), raises the possibility of a role for granzyme B in bacterial defense within the scope of granulocyte function. However, the expression of perforin and granzymes in human granulocytes is controversial because others have failed to confirm initial findings (Grossman and Ley, 2004; Metkar and Froelich, 2004). Direct functional analysis in mouse models will hopefully clarify whether granzyme B plays a role in granulocyte defense against bacteria. Despite the fact bactericidal activity is critically dependent on proteases, until the disclosed results herein, it was still not known whether serpins play any part in the control of granulocyte or macrophage function. 91. TEe effector mecKaMsms that provide immunity to infection are tightly controlled. This is because immune responses to infection have the capacity to cause inflammatory and autoimmune disease. Disclosed herein endogenous serpins in mammals can provide a level of control of both the response of the innate and adaptive immune system to infection. 93. The encounter of (antigen = Ag) Ag-inexperienced CD8⁺ T cells (naive cells) with processed Ag triggers clonal expansion and differentiation into CTLs (effectors) (Sprent et al., 2002). Effectors kill infected or abnormal cells either by exocytosis of cytolytic granules or the engagement of Fas by Fas ligand (Kagi et al., 1994b). Perform is critical for the granule exocytosis pathway and facilitates the entry of proteases, such as granzyme B, into the target cell cytoplasm where granzyme B initiates programmed cell death (PCD) by proteolytic cleavage of caspases and Bid (Catalfamo et al., 2003; Trapani et al., 2003;

Lieberman, J.. 2003).

94. Factors regulating each stage of the immune response have been reported to impact the development of CD8⁺ T cell memory. (Seder, et al., 2003; Ahmed and Gray. 1996). Experiments with knockout mice indicate that the contraction phase requires the effector cytokine IFN-T (Badovinac et al., 2000). In addition, perform has been implicated in controlling both the expansion of activated Ag-specific cells (Badovinac et al., 2000; Badovinac et al., 2003) and the elimination of activated CD8⁺ T cells after viral infection (Kagi et al., 1999) in graft-vs-host disease (Spaner et al., 1999) and during chronic infection (Matloubian et al., 1999; Gallimore et al., 1998). Because perform is a vehicle for the delivery of granule toxins, thephenotypes observed in perforin-deficient mice could be due to ineffective administration of executioner proteases such as granzymes. Interestingly, observations in vitro indicate that cathepsin B on the membrane of CTLs can cleave exocytosed perform and so protect T cells from PCD (Balaji, et al., 2002). However, whether factors can protect CD8⁺ T cells from executioner granzymes during an in vivo immune response remains to be determined. e) Serpins and protection from inflammatory disease 95. Serpins play a critical role in suppressing neutrophil serine proteases and so protect against inflammatory disease. The serpin αl -antitrypsin (αl-AT) is a potent inhibitor of NE (k = 10⁷ IVT's^XBeatty et al., 1980) and when secreted into the serum and airways of the lung protects from chronic obstructive pulmonary diseases (COPD) (Gadek et al., 1981a). Deficiency in αl-AT leads to a pathological increase in NE activity and neutrophil influx into the lung in humans (Gadek et al., 1981b) and mice (Martorana et al., 1993) and causes about 1-2% of all cases of COPD. In addition, secretory leukoprotease inhibitor (SLPI) also inhibits extracellular NE (k = 3 x 10⁶ M^-1S^"1) and deficiency in mice increases susceptibility to endotoxin induced septic shock (Nakamura et al., 2003). f) Serpins and host defense from infection.

96. Members of the ovalbumin-like (ova-like) family of serpins lack a traditional secretion signal peptide and are located within the cytoplasm of cells (Remold-O'Donnell, 1993). However, some ova- serpins use a non-conventional internal signal sequence that targets either exclusively for secretion (ovalbumin)(Braell and Lodish, 1982) or for both the cytosol and secretion (plasminogen activator inhibitor ^" "^""X P^'AΪ-2 )'(βeϊm ¹Sf al.T56β4ffiϊe ^"ova-serpin, Proteinase inhibitor 9 (PI9) is widely expressed in the cytoplasm of both lymphoid and myeloid leukocytes (Hirst et al., 2003; Sun et al., 1996). PI9 inhibits the granzyme B (GrB) serine protease used by cytotoxic T cells (CTLs) and Natural Killer cells (NK) to kill infected cells (Sun et al., 1996). Therefore, it has been suggested that PI9 may promote the survival of leukocytes by protecting them from GrB. In addition to inhibiting granzyme B, PI9 also inhibits NE albeit weakly in vitro (k= 1 x 10⁵ M^-1S^"1) (Dahlen et al., 1999). g) Neutrophils

97. The neutrophil constitutes the first line of defense in protecting the host from invading bacterial and fungal pathogens. It is a highly potent cytotoxic cell and possesses an armory of antimicrobial proteins and biochemical pathways that can be used in this protective role (Bainton, 1999). Neutrophils have a relatively short half-life in the circulation of between 8-20 h and so to provide the first line of defense are produced in large numbers (40-65% of white blood cells) and are highly motile being able to leave the circulation and enter infected tissues.

(1) Neutrophil morphology and development 98. Microscopic examination of mature neutrophils reveals two striking features: a single multi-lobed nucleus and a dense granular cytoplasm. The acquisition of granules is determined by the timing of biosynthesis of their characteristic proteins during the development of neutrophils in the bone marrow (Borregaard and Cowland, 1997). The earliest identifiable cells of the neutrophil lineage are myeloblasts (MB), which are relatively undifferentiated, have a large nucleus and almost no cytoplasmic granules (Figure 28). Differentiation into promyelocytes (PMC) results in the acquisition of the earliest granule population - azurophil granules, which are characterized by myeloperoxidase (MPO) as well as an array of antimicrobial serine proteases (neutrophil elastase (NE), cathepsin G (Cat G), proteinase-3 (PR-3)), hydrolytic enzymes and lysozyme (Bainton and Farquhar, 1966). PMCs are capable of proliferation and cell line HL-60 was originally isolated from a patient with promyelocytic leukemia (Collins et al., 1977). Myelocytes (MC) accumulate large numbers of so-called specific granules (Borregaard and Cowland, 1997).

These are MPO-negative and contain stores of preformed membrane proteins, which when translocated to the plasma membrane during neutrophil activation enhance neutrophil responsiveness (Borregaard and Cowland, 1997). At this stage, the synthesis of azurophil granules ceases and the number per cell decreases during progressive divisions. Metamyelocytes (MMC), band cells (BC) and segmented cells (Segm) are more mature forms, which are incapable of division and can be identified by both the onset of multi-lobed nuclear morphology and increased granule content and glycogen particles in the cytoplasm. At these stages gelatinase-containing granules start to appear, which are functionally related to specific granules and are MPO-negative (Rjeldsen et al., 1994). In mature neutrophils secretory vesicles rapidly deliver receptors or effector molecules to the plasma membrane or intracellular vesicles (Borregaard et al., 1990). (2) Microbicidal activity of neutrophils

99. Phagocytic neutrophils confer immunity by engulfing invading microbes (Metchnikoff, 1905). It was supposed that killing was effected by the contents of cytoplasmic granules released into the phagocytic vacuole in which the microbe was encapsulated. This hypothesis was supplanted by the supposition that the killing agents were toxic"reaclϊve"oxygen species (ROS)(Mandell, 1974; Sbarra and Karnovsky, 1959), supported by the discovery of chronic granulomatous disease (CGD), a human condition characterized by profound susceptibility to bacterial and fungal infection (Gallin and Malech, 1990). The finding that phagocytes from these individuals are unable to generate ROS (Gallin and Malech, 1990) or to kill microbes efficiently was taken to imply that the respiratory burst promotes killing by generating superoxide (Babior et al., 1975; Babior et al., 1973) and H₂O₂ (Klebanoff, 1975). The H₂O₂ is thought also to exert an indirect effect, as a substrate for MPO-cataylsed halogenation (Klebanoff, 1975). The genetic defects in CGD lie in the multi-component reduced nicotinamide dinucleotide phosphate (NADPH) oxidase (Gallin and Malech, 1990). 100. Mice deficient in serine proteases from azurophil granules have confirmed the original supposition that the contents of cytoplasmic granules kill microbes. Mice deficient in NE (NE KO mice) were susceptible to lethal infection with the gram-negative bacteria Klebsiella pneumonaie and Escherichia coli and the fungus Candida albicans, because neutrophils from these mice failed to digest the microbes after encapsulation (Belaaouaj et al., 1998; Reeves et al., 2002). NE kills E. coli by digesting the cell wall protein Omp A (Belaaouaj et al., 2000) and can cleave Pseudomonas aeruginosa flagellin, implying that NE prevents infection by inhibiting attachment to host epithelium (Lopez-Boado et al., 2004). Cat G KO mice are susceptible to gram-positive Stapliloccocus aureus infection (Reeves et al., 2002) and NE KO x Cat G KO mice are susceptible to infection with the fungi Aspergillus fumigates (Tkalcevic et al., 2000). PR-3 shares similar substrate specificity to NE and Cat G and is also stored in azurophil granules (Rao et al., 1991). However the requirement for PR-3 in protecting against microbial infection has yet to be examined.

101. The respiratory burst and MPO activity were unaffected in NE KO and Cat G KO mice, arguing against the long held view that ROS directly kill microbes (Reeves et al., 2002). The activation of neutrophils provokes the influx of enormous concentrations of ROS into the endocytic vesicle, which is compensated for by a surge of K⁺ ions that cross the membrane in a pH-dependent manner via a large conductance Ca²⁺ activated channel (Ahluwalia et al., 2004). The consequent rise in ionic strength engenders the release of cationic granule NE and Cat G, from the anionic sulfated proteoglycan matrix. It is the serine proteases, thus activated, that are primary responsible for the destruction of the bacteria. Although it is well established that serine proteases digest engulfed bacteria in phagolysomes, recently it has been shown that secreted NE can kill bacteria trapped by neutrophils in webs of extracellular fibers (Brinkmann et al., 2004). Thus, activated extracellular as well as intracellular serine proteases are important for bacterial killing.

(3) Neutrophils as agents of inflammatory disease

102. While neutrophils are critical in host defense against microbes, excessive levels of inflammation during infection lead to tissue damage, organ dysfunction and disease. In septic shock, the normal beneficial response to bacteria becomes detrimental. In response to systemic infection, phagocytes move to the site of infection and into uninfected tissues and provoke tissue damage and multi-organ failure. Neutrophils have been implicated as major agents in the pathogenesis of chronic obstructive pulmonary diseases (COPD), which includes chronic bronchitis, peripheral airways disease and emphysema (Dar and ^lysiai, l^yyj. increased JNE, ana neutropnii activity causes empnysema in numans (Lraαek et al., 1981b;

Hubbard et al., 1991) and animal models (Janoff et al., 1977; Shapiro et al., 2003). Although NE is clearly a major cause of COPD, recent evidence suggests that it exerts its effects by activating macrophage elastase (macrophage metalloprotein 12, MMP 12), which is required for macrophage-mediated proteolysis and matrix invasion (Shipley et al., 1996). NE activates MMP12 by degrading tissue inhibitor of metalloproteinase (TIMP)-I (Okada et al., 1988), which is an inhibitor of MMP-12 (Shapiro et al., 2003). However this has only been shown in smoking induced emphysema and a role for NE activated MMP12 controlling acute bacterial infection is unclear.

103. Serine proteases such NE, stored in azurophilic granules, play a dominant role in the microbicidal activity of neutrophils by digesting phagocytosed microbes. However, serine protease inhibitors (serpins) protect against sepsis and other inflammatory diseases by suppressing NE activity. Thus, in addition to inflammatory macrophages and cytokines, it is assumed that neutrophils drive also the pathogenesis of sepsis by damaging tissue components through the action of microbicidal serine proteases. Conversely, since sepsis is caused by a failure to control bacteria at the site of infection, neutrophil activity may be protective rather than pathological.

104. Infection of the gastric mucosa with the gram-negative bacterium Helicobacter pylori occurs in half of the world's population (1994). In all cases, H. pylori induces gastritis and a subset of infections progress to peptic ulceration or gastric cancer (Allen, 2000; Allen, 2001). H. pylori stimulates a novel type of chronic inflammation that is characterized by a massive influx of neutrophils into the gastric mucosa and encounter bacteria in the mucus layer and at ulcer margins (Allen, 2000; Allen, 2001). The leukocytes of the innate immune system rather than lymphocytes seem to play the major role in controlling H. pylori infection (Blanchard et al., 1995). However, it has been suggested that inflammatory damage to mucosal tissue is a result of persistent infection caused by inefficient clearance by neutrophils (Allen et al., 2005). Thus, unresolved H. pylori infection may drive the infiltration of damaging neutrophils leading to disease. h) Granule biosynthesis

105. Proteins destined for export to granules contain N-terminal secretory signal sequences, which directs synthesis to the rough endoplasmic reticulum (RER)(Blobel and Dobberstein, 1975). Sorting in the trans-Golgi network (TGN) then segregates proteins destined for the constitutive exocytosis and insertion into the plasma membrane from those that will form storage granules capable of undergoing regulated exocytosis (Sossin et al., 1990). Storage granules are formed by aggregation of immature transport vesicles that bud off from TGN (Hartmann et al., 1995), but in neutrophils no common primary amino acid sequence structure has been identified that determines whether a protein is retained or constitutively secreted (Garwicz et al., 1995; Gullberg et al., 1995). The well known targeting of glycoproteins to lysosomes via the cation-dependent and cation-independent mannose 6-phosphate receptor (Dahms et al., 1989) is not required for sorting of proteins to azurophil granules (Castanon et al., 1988; Nauseef et al., 1992).

106. The sorting of proteins into the different storage granules in neutrophils seems to be dependent on the timing of expression rather than any specific targeting sequence (Borregaard et al., 1995). lhe expression windows of granule proteins throughout neutrophil differentiation are determined by specific transcription factors (Borregaard and Cowland, 1997). Thus, the differences in the protein content that define the different subsets of granules result from differences in the biosynthetic window of the various granule proteins relative to maturation (Fig.l). Direct evidence for this hypothesis comes from the observation that forced expression of Neutrophil gelatinase-associated lipocalin (NGAL), which is normally stored in specific granules, in HL-60 cells results in storage in azurophil granules (Le Cabec et al, 1996). In azurophil granules, proteolytic processing affects the maturation of microbidical enzymes as well as granule volume and morphology (Stromberg et al., 1986).

3. Animals and Vectors, and Cells a) Spi6 Knock out

107. Disclosed are methods and compositions related to vectors, cells, transgenic animals, and methods thereof that provide models to explore the functions of Spi6 as well as exploring the functions of PI9, such as the role of Sρi6 or PI9 in inflammatory diseases, including the role of Spi6 in the protection of cytotoxic T-cells from self-inflicted injury and the role of Spi6 in protecting against microbial infection. These cells, vectors, and animals have also elucidated new roles for SPI6 and PI9 and thus, have provide new therapeutic targets and activities.

108. For example, disclosed are transgenic non-human animals, transgenic animal cells, and vectors comprising a disrupted Spi6 gene. The transgenic non-human animals and transgenic animal cells can be a mammal or mammalian cells, respectively. For example, the transgenic non-human animal can be a mouse, a rabbit, a rat, a human, a pig, a hamster, a dog, a horse, a sheep, a goat, or any other mammal capable of transgenic manipulation. Further provided are cells, comprising the disclosed vectors, animals comprising the disclosed cells, and animals comprising the disclosed vectors.

109. The transgenic animal cells can be any cell including, an embryonic stem cell, an embryonic germ cell, a puripotent adult stem cell, a prostate cell, testis cell, bone cell, brain cell, or muscle cell. The transgenic animal cells can also comprise an immortal cell line such as a breast cell, a breast cancer cell, an ovary cell, or an ovary cancer cell.

(1) Disrupted Spi6 Gene

110. The disrupted Sρi6 gene can be any Sρi6 gene that has a different function than an unaltered Spi6 function. Optionally, the disrupted Spi6 gene can be a gene that encodes a non-functional SPI6 protein.

111. Optionally, the disrupted Spi6 gene can comprise a deleted exon, a point mutation, or a missense mutation. As such, the disrupted Spi6 gene can comprise a deleted exon wherein the deleted exon can be any Spi6 exon. The DNA sequence of Spi6 (Accession Number AL589871; SEQ ID NO: 73) and PI9 (Accession Number AL133351; SEQ ID NO: 74) are known as are the cDNA sequence of Spi6 and PI9 are provided as SEQ ID NOs: 3 and 1, respectively and the protein sequences of Spi6 and PI9 which are provided as SEQ ID NOs: 4 and 2, respectively. One of skill in the art would be able to surmise what sequences comprise the exons. For example, the disrupted Spi6 gene can comprise a deleted exon wherein the deleted exon Is exon 7. Exon^'7 contains the RCL and most of the coding region of Spi6, and thus provides a good candidate for disruption.

112. The disrupted Spi6 gene can also comprise other embodiments. For example, the disrupted Spi6 gene can also comprise a marker gene. Suitable marker genes are described below and include the E. coli lacZ gene, G418 resistance gene, HPRT, thymidine kinase, the green fluorescent protein (GFP), and the red fluorescent protein (RFP). The disrupted Spi6 gene can also comprise one or more loxP sites and one or more recombinase sites. The recombinase sites can flank some or all of the Spi6 exons. The recombinase can be a Cre or FIp recombinase.

(2) Transgenic non-human animals comprising a disrupted Spi6 gene 113. Provided herein are transgenic non-human animals comprising a disrupted Spi6 gene. The transgenic animal comprising a disrupted Spi6 gene can lack native SPI6 function or can lack native Spi6 expression.

114. The disruption of SPI6 function or Spi6 expression can lead to increased neutrophil elastase activity. The disrupted Spi6 expression can also increase immunity to sepsis causing bacteria without causing inflammatory disease as well as increasing neutrophil function. The neutrophil function of the transgenic animals can be greater than the neutrophil function in a non-transgenic animal. Optionally, the disrupted Spi6 expression can increase neutrophil recruitment to the site of infection. The increase in neutrophil recruitment to the site of infection can be 2, 3, 4, 5, 6, or 7 fold higher than an animal having non-disrupted Spi6 expression. 115. The disrupted Spi6 expression can also increase the survival of the transgenic animal from septic shock induced death after infection with bacteria. The bacteria can be either gram-negative or gram-positive bacteria. For example, a transgenic animal comprising a disrupted Spi6 gene can increase survival of the animal from septic shock induced death after infection with E. coli, P. aeruginosa, or L. monocytogenes.

116. Also provided are transgenic non-human animals comprising a disrupted Sρi6 gene wherein the amount of SPI6 produced in the animal is less than the amount of SPI6 produced in a non-transgenic animal.

117. Further provided are transgenic non-human animals comprising a disrupted Spi6 gene wherein the animal has increased immunity. For example, the transgenic non-human animal can have an increased immunity to bacteria including, but not limited to, E. coli, P. aeruginosa, and L. monocytogenes. 118. Also provided are transgenic non-human animals comprising a disrupted Sρi6 gene wherein the granulocytes of the animal have increased bactericidal activity. For example, provided herein are transgenic animals wherein infection of the transgenic animal with E. coli results in increased bactericidal activity of neutrophils than an animal not having a disrupted Spi6 gene.

(3) Transgenic animal cell comprising a disrupted Spi6 gene 119. Also provided herein are transgenic animal cells comprising a disrupted Spi6 gene. The transgenic animal cell can be a mammalian cell, including a human cell. Examples of other mammals are described above. F20. The §ans'genic"anϊmal cells comprising a disrupted Sρi6 gene can lack native SPI6 function or can lack native Spi6 expression. The disruption of SPI6 function or Spi6 expression can lead to increased neutrophil elastase activity. The disrupted Spi6 expression can also increase immunity to sepsis causing bacteria without causing inflammatory disease as well as increasing neutrophil function. The neutrophil function of the transgenic animal cells can be greater than the neutrophil function in a non- transgenic animal cell. Optionally, the disrupted Spi6 expression can increase neutrophil recruitment to the site of infection. The increase in neutrophil recruitment to the site of infection can be 2, 3, 4, 5, 6, or 7 fold higher than an animal cell having non-disrupted Spi6 expression.

121. The disrupted Sρi6 expression can also increase the survival of the transgenic animal cell from septic shock induced death after infection with bacteria. The bacteria can be either gram-negative or gram- positive bacteria. For example, a transgenic animal cell comprising a disrupted Spi6 gene can increase survival of the animal from septic shock induced death after infection with E. coli, P. aeruginosa, or L. monocytogenes.

122. Also provided are transgenic animal cells comprising a disrupted Spi6 gene wherein the amount of SPI6 produced in the animal is less than the amount of SPI6 produced in a non-transgenic animal cell.

123. Further provided are transgenic animal cells comprising a disrupted Spi6 gene wherein the animal has increased immunity. For example, the transgenic animal cell can have an increased immunity to bacteria including, but not limited to, E. coli, P. aeruginosa, and L. monocytogenes.

124. Also provided are transgenic non-human animals comprising a disrupted Spi6 gene wherein the granulocytes of the animal have increased bactericidal activity. For example, provided herein are transgenic animals wherein infection of the transgenic animal with E. coli results in increased bactericidal activity of neutrophils than an animal not having a disrupted Spi6 gene.

(4) Vectors comprising a disrupted Spi6 gene

125. Also provided herein are vectors comprising a portion of the Spi6 gene, wherein the portion of the Spi6 gene produces a disrupted Spi6 gene, and wherein the vector can homologously recombine with the Spi6 gene. For example, provided is a vector comprising the sequence of SEQ ID NO:72. Optionally, the vectors can comprise a selectable marker. Examples of suitable selectable markers include, but are not limited to, dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. The selectable marker can be a positive or negative selection marker. 126. Also provided are nucleic acid molecules produced by a process, the process comprising linking in an operative way a nucleic acid comprising the sequence of a Spi6 exon and sequence recognized by a recombinase enzyme. Further provided are cells produced by the process of transforming a cell with the nucleic acids produced by such a process.

(5) Methods of gene modification and gene disruption 127. The disclosed compositions and methods can be used for targeted gene disruption and modification in any animal that can undergo these events. 128. Gene modification and gene disruption refer to the methods, techniques, and compositions that surround the selective removal or alteration of a gene or stretch of chromosome in an animal, such as a mammal, in a way that propagates the modification through the germ line of the mammal. In general, a cell is transformed with a vector which is designed to homologously recombine with a region of a particular chromosome contained within the cell, as for example, described herein. This homologous recombination event can produce a chromosome which has exogenous DNA introduced, for example in frame, with the surrounding DNA. This type of protocol allows for very specific mutations, such as point mutations, to be introduced into the genome contained within the cell. Methods for performing this type of homologous recombination are disclosed herein. 129. One of the preferred characteristics of performing homologous recombination in mammalian cells is that the cells should be able to be cultured, because the desired recombination event occurs at a low frequency.

130. Once the cell is produced through the methods described herein, an animal can be produced from this cell through either stem cell technology or cloning technology. For example, if the cell into which the nucleic acid was transfected was a stem cell for the organism, then this cell, after transfection and culturing, can be used to produce an organism which will contain the gene modification or disruption in germ line cells, which can then in turn be used to produce another animal that possesses the gene modification or disruption in all of its cells. In other methods for production of an animal containing the gene modification or disruption in all of its cells, cloning technologies can be used. These technologies generally take the nucleus of the transfected cell and either through fusion or replacement fuse the transfected nucleus with an oocyte which can then be manipulated to produce an animal. The advantage of procedures that use cloning instead of ES technology is that cells other than ES cells can be transfected. For example, a fibroblast cell, which is very easy to culture can be used as the cell which is transfected and has a gene modification or disruption event take place, and then cells derived from this cell can be used to clone a whole animal.

131. Animals which are Spi6 or PI9 knockouts can be generated as discussed in the Examples. Optionally, the animals which are Spi6 or PI9 knockouts can be produced by a method comprising introducing into a non-human animal fertilized egg a recombinant nucleic acid molecule, which comprises a nucleic acid encoding a disrupted Spi6 gene whereby a transgenic animal expressing disrupted SPI6 is produced.

132. Also provided herein are method of producing animals which are Spi6 or PI9 knockouts. For example, provided herein is a method comprising administering a vector as described above, to an ES cell, culturing the cell, selecting a cell comprising the vector, fusing the selected cell with a blastocyst, thereby producing a chimera, incubating the chimera, and implanting the chimera into a surrogate mother to produce an offspring. Further disclosed are methods of producing an animal, the methods comprising fusing the chimera produced by the method above, with another chimera, and selecting live animals homozygous for vector DNA. As discussed ϊϊereϊn,^'"mere"are numerous implications that arise from the Spi6 knockout mouse including, for example, the use of inhibitors for Spi6 or PI9 which can reproduce the Spi6 knockout phenotype, along with the beneficial effects, of for example, increasing neutrophil elastase activity to increase immunity to bacterial infections. b) Overexpressing Spi6

(1) Trangenic Animals

133. Disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, which in certain embodiments overexpresses the serpin, such as Spi6 relative to a non-transgenic mouse. It is understood that any of the animals disclosed herein can be non-human animals.

134. Also disclosed is a transgenic animal or cell, wherein the serpin is Spi6 or PI9.

135. Also disclosed is a transgenic animal or cell, wherein the serpin is a serpin with at least 60% identity to Spi6, as set forth in SEQ ID NO:4.

136. Also disclosed is a transgenic animal or cell, wherein the serpin is Sρi6, wherein the Spi6 comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:4.

137. Also disclosed is a transgenic animal or cell, wherein the serpin is PI9, wherein the PI9 comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:2.

138. Also disclosed is a transgenic animal or cell, wherein the serpin is PI9, wherein the PI9 comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:4. 139. Also disclosed is ajransgenic animal or cell, wherein the serpin is Spi6, wherein the Spi6 comprises a sequence in which the complete sequence hybridizes to the sequence set forth in SEQ ID NO:3, wherein the hybridization takes place at 600 mM NaCl, 60 degrees Celcius, buffered to pH 7.6.

140. Also disclosed is a transgenic animal or cell, wherein the serpin is Spi6, wherein the Spi6 comprises a sequence in which the complete sequence hybridizes to the sequence set forth in SEQ ID NO: 3 after a wash of 15m M NaCl, 1.5m M Na3 citrate, 1% SDS, 65 degrees Celsius.

141. Also disclosed is a transgenic animal or cell, wherein the serpin is Spi6, wherein the Sρi6 comprises a sequence in which the complete sequence hybridizes to the sequence set forth in SEQ ID NO:3, wherein the hybridization takes place at 600 mM NaCl, 60 degrees Celcius, buffered to pH 7.6 and remains hybridized to the sequence set forth in SEQ ID NO:3 after a wash of 15 mM NaCl, 1.5 mM Na3 citrate, 1% SDS, at 65 degrees celius.

142. Also disclosed is a transgenic animal or cell, wherein the serpin is Spi6 or PI9, wherein the Spi6 comprises a sequence as set forth in SEQ ID NO:3.

143. Also disclosed is a transgenic animal or cell, wherein the serpin is Spi6 or PI9, wherein the PI9 comprises a sequence set forth in SEQ ID NO:1 144. Also disclosed is a transgenic non-human animal comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin is not Crm A (SEQ ID NO:24 and 25).

145. Also disclosed is a transgenic non-human animal comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the transgene comprises sequence encoding a serpin, a promoter operably linked to the serpin, and a selectable marker.

146. Also disclosed is a transgenic non-human animal comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin is a serpin having granzyme B, cathepsin G (P28293), PR-3 (Q61096), neutrophil elastase (NP031945), mouse mast cell protein (MMCP) -1 (Pl 1034), MMCP-2 (P15119), MMCP-3 (P21843), MMCP-4 (P21812), MMCP-5 (P21844), MMCP-8 (P43430), MMCP-9 (035164), MMCP-10 (AAK51075), or caspase 1 (P29452) as a substrate.

147. Also disclosed is a transgenic non-human animal comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin is a serpin having granzyme B, cathepsin G (P28293), PR-3 (Q61096), neutrophil elastase (NP031945), mouse mast cell protein (MMCP) -1 (P11034), MMCP-2 (P15119), MMCP-3 (P21843), MMCP-4 (P21812), MMCP-5 (P21844), MMCP-8 (P43430), MMCP-9 (035164), MMCP-10 (AAK51075), or caspase 1 (P29452) as a substrate, wherein the serpin is a serpin having Granzyme B as a substrate.

148. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the amount of serpin produced in the animal is more than the amount of serpin produced in a non-transgenic animal. 149. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the animal has increased protection against programmed cell death. In certain embodiments, protect or protection can be limited to protection that provides a means for greater than 20%, 30%, 50%, 60%, 70%, 80%, or 90% increase in the number of viable of cells after 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6, hours, 7 hours, 8 hours, 9 hours, 10, hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours 21 hours, 22 hours, 23 hours,

24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 60 hours, 72 hours, or 4 days.

150. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the transgenic non-human animal has a greater than 50% increase in number of viable of transgenic cells over the level of non-transgenic cells after a certain length of time.

151. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin is overexpressed, and wherein the overexpression of the serpin leads to an enhanced memory cell phenotype. For example, the memory cell phenotype of the transgenic non-human animals can be enhanced 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold higher than an animal not having the transgene 152. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein expression of the serpin is driven by the CD2 cassette.

153. Also disclosed are transgenic animals or cells, where CD2 drives expression in naϊve CD8 T cells but also all other T cells, and other white blood cells. Typically transgenic expression is restricted to blood cells unless a non-blood cell specific promoter is used.

154. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, where expression of the serpin is restricted to blood cells.

155. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin is overexpressed in blood cells, wherein the overexpression of the serpin leads to an enhanced memory cell phenotype. For.example, disclosed is a transgenic animal, where the serpin is overexpressed in naϊve CD8 T cells, NK cells, thymocytes, lymphocytes and phagocytic myeloid cells, wherein the overexpression of the serpin leads to an enhanced memory cell phenotype.

156. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin is overexpressed, and wherein the overexpression of the serpin leads to an enhanced memory cell phenotype, wherein the memory is enhanced 2, 3, 4, 5, 6, 7, 8, 9, 10, fold higher than an animal not having the transgene.

157. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin is expressed in naϊve cells at least 100, 75, 53, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or

2, fold, over the amount of the serpin expressed in the naϊve cells of an animal not having the transgene.

158. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the ratio of serpin to cyclophilin is at least 100, 75, 53, 50, 40, 31.6. 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

159. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the expression of the serpin was at least 100, 75, 53, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.8 5, 4, 3, or 2 fold higher in memory cells than in naϊve cells. 160. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin expression protects T cells. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin expression protects T cells from death initiated by granzyme B delivered perform. Further disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin expression protects T cells from granzyme B mediated apoptosis. IiSi; "^"" Also disc'losed'are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the serpin expression protects T cells from death initiated by granzyme B delivered perforin, wherein the protection is greater than the protection in a non-transgenic animal. 162. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein number of memory cells is enhanced when compared to an animal without the transgene. The number of memory cells can be enhanced 2, 3, 4, 5, or 6 fold higher than an animal having non transgenic Spi6 expression. For example, disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein number of memory cells is enhanced at least 2 fold when compared to an animal without the transgene.

163. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein infection of the transgenic mouse with (LCMV) results in higher numbers of memory cells than an animal not having the transgene. The animal not having the transgene can be a B6 mouse. 164. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein infection of the transgenic mouse with an experimental pathogen results in higher numbers of memory cells than an animal not having the transgene. Examples of experimental pathogens, other than LCMV, that give rise to memory cells in mice include, but are not limited to Listeria moncytogenes (bacterium), influenza (virus), and herpes simplex (virus). 165. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein infection of the transgenic mouse with an experimental pathogen results in higher numbers of memory cells than an animal not having the transgene, wherein the animal not expressing the transgene is a B6 mouse.

166. Disclosed are transgenic animals, wherein the expression pattern of Sρi6 correlates with granzyme B expression, wherein coexpression of Spi6 and granzyme B in anti-LCMV effectors is retained in resulting memory cells, wherein CD2 drives expression in hematopoietic cells, wherein the expression is highest in natural killer cells (NK cells), wherein the expression in the NK cells is is at least 10, 20, 30, 40, or 50 fold higher than in other cell types, and/or wherein the expression is highest in thymocytes, lymphocytes, or phagocytic myeloid cells. 167. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein Spi6 is coexpressed with granzyme B in anti-LCMV effectors and expression of both is retained in resulting memory cells.

168. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the animal was produced by a method comprising introducing into a non-human animal fertilized egg a recombinant nucleic acid molecule, which comprises a nucleic acid encoding a serpin whereby a transgenic animal expressing the serpin is produced. 169. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the transgenic animal has impaired neutrophil function.

170. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the transgenic non-human animals hasjmpaired in vivo responses to bacteria.

171. Also disclosed are transgenic non-human animals comprising a cell wherein the cell expresses a transgene coding for a serpin, wherein the transgenic non-human animals hasjmpaired in vivo responses to bacteria due to suppressed elastase activity in neutrophils. c) Other Mice 172. The disclosed mice and animals can be crossed with any other mouse or animal to produce both heterozygous and homozygous, through appropriate backcrossing and breeding techniques, mice.

173. Other mice can be produced by crossing either the heterozygous Spi6^+/~ Pm KO P14 CD8 T or Spi6^~'^~ homozygous mutant mice with another mouse, such that the newly generated mice have an identifiable phenotype. 174. Mice that can be crossed with either the heterozygous Spi6^+A Pm KO P14 CD8 T or Spi6^~'^~ homozygous mutant mice can be FcεRI KO mice (Dombrowicz et al., 1993) (Jackson Laboratory, Bar Harbor, ME)

175. The phenotypes of the crossed mice can have a combination of the phenotypes produced by each mouse individually. (1) Pfn KO P14 CD8 T Mice

176. The ability of GP33-pulsed CD8α dendritic cells to induce the proliferation of naϊve P14 CD8 T cells can also be examined in vitro. For these studies P14 mice can be crossed to perforin KO (Pfh KO) mice (Jackson Laboratory, Bar Harbor, ME) to generate Pfh KO P14 CD8 T cell responders.

(2) C57BL/6 IFN-γ KO mice 177. To determine whether IFN-γ is required to the licensing of DCs in Spi6 KO mice, C57BL/6

IFN-γ KO mice (Jackson Laboratory, Bar Harbor, ME) can be infected with LCMV and CD8α DCs can be purified and tested for their ability to induce the proliferation of P14 CD8 T cells. d) Compositions that modulate Spi6/PI9 activity

(1) Inhibitors of Spi6/PI9 178. Also provided herein are methods of inhibiting Spi6 expression comprising administering an inhibitor of Sρi6. Further provided herein are inhibitors of Spi6 or PI9. In certain emobodiments the inhibitors provided herein can inhibit Spi6 or PI9 expression. It is understood that the inhibition of SPI6 or PI9 with an inhibitor can be used in any of the methods disclosed herein related to SPI6 inhibition or Spi6 expression inhibition. It is also understood that the functional relationships between SPI6 and PI9 as discussed herein and provided by the sequence information provided herein and the knowledge that these molecules have cross reactivity and that molecules exist that can inhibit both and can be designed to inhibit one or the other indicates that there molecules disclosed for SPI6 inhibition can in certain embodiments, which can readily be determined as discussed herein, can also inhibit PI9 and vice versa. Inhibition of Spi6 expression can be achived in a variety of ways. For example, inhibition of Spi6 expression can be achieved by introducing a disrupted Sρi6 gene or a SPI6 inhibitor. SPI6 inhibitors include, but are not limited to, antibodies, siRNA, iRNA, aptamers, ribozymes, External guide sequences, small molecules and other compounds identified that have serpin inhibitory activity.

179. Compounds that can be used to inhibit serine protease inhibitors include, but are not limited to: WAY-140312 (See Crandall et al., 2004) XRl 853, XR334, Tiplaxtinin (See Elokdah et al., 2004), salicylic acid derivative HP129 (See Gils et al., 2002); anthram^'lic acid derivative AR-H029953XX (See

Bjoerquist et al., 1998); diketopiperazine XR5118 (See Charlton et al., 1997 and Friederich et al., 1997); butadiene derivative T-686 (See Ohtani et al., 1996), all of which are hereby incorporated by reference in their entirety for their teachings of compounds that can be used to inhibit serine protease inhibitors.

180. Optionally, peptide based strategies can be used to inhibit Spi6 expression. Peptide based strategies can inhibit Spi6 as described in Eitzman et al., (1995), which is hereby incorporated by reference in its entirety for its teaching of peptide based strategies can be used to inhibit expression of a target. For example, the tetradecapeptide, P1-P14 can be used to inhibit Sρi6 expression.

(a) Functional Nucleic Acids

181. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, siRNA and iRNA, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

182. Functional nucleic acid based approaches can be used to inhibit Spi6 expression. For example, siRNA, iRNA, aptamers, ribozymes, and external guide sequences directed towards PI9 or Spi6 can also be used. For example, ribozymes comprising the sequences of SEQ ID NOs: 46, 47 or 48 can be used. 183. Optionally, siRNA directed towards Spi6 can be used to silence Spi6. For example, siRNA molecules with a sequence selected from a group consisting of SEQ ID NOs: 49-68 can be used. siRNA molecules directed to kanamycin resistance protein, EGFP expression vector, 3' UTR of hepatitis C virus can be used as controls for comparisons. For example, siRNA molecules with a sequence selected from a group consisting of SEQ ID NOs: 69-71, respectively, can be used as controls. 184. Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA of Spi6 or r>^TQ ™. tøe genomic DNA of Spi6 or PI9 or they can interact with the polypeptide Spi6 PI9. Often functional nucleic acids^'are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

185. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (k_d)less than or equal to 10^"6, 10^"8, 10^~10, or 10^"12. A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non- limiting list of United States patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.

186. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293). Aptamers can bind very tightly with k_ds from the target molecule of less than 10^~12 M. It is preferred that the aptamers bind the target molecule with a kd less than 10^"6, 10^"s, 10^"10, or 10^"12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293).

It is preferred that the aptamer have a k_d with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the k_d with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. For example, when determining the specificity of Spi6 aptamers, the background protein could be serum albumin. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698. 18 /. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following United States patents: 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, but not limited to the following United States patents: 5,595,873 and 5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymes cleave KNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non-limiting list of United States patents:

5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

188. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k_d less than 10^"6, 10^"8, 10^"10, or 10^"12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773,

5,834,185, 5,869,246, 5,874,566, and 5,962,426.

189. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altaian, Science 238:407-409 (1990)). 190. Sϊmilarly;"^'eukSyo^'tic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells. (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altaian, EMBO J 14:159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)). Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in the following non-limiting list of United States patents: 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

191. It is also understood that the disclosed nucleic acids can be used for RNAi or RNA interference. It is thought that RNAi involves a two-step mechanism for RNA interference (RNAi): an initiation step and an effector step. For example, in the first step, input double-stranded (ds) RNA (siRNA) is processed into small fragments, such as 21-23-nucleotide 'guide sequences'. RNA amplification appears to be able to occur in whole animals. Typically then, the guide RNAs can be incorporated into a protein RNA complex which is cable of degrading RNA, the nuclease complex, which has been called the RNA- induced silencing complex (RISC). This RISC complex acts in the second effector step to destroy mRNAs that are recognized by the guide RNAs through base-pairing interactions. RNAi involves the introduction by any means of double stranded RNA into the cell which triggers events that cause the degradation of a target RNA. RNAi is a form of post-transcriptional gene silencing. Disclosed are RNA hairpins that can act in RNAi. For description of making and using RNAi molecules see See, e.g., Hammond et al., Nature Rev Gen 2: 110-119 (2001); Sharp, Genes Dev 15: 485-490 (2001), Waterhouse et al., Proc. Natl. Acad. Sci. USA 95(23): 13959-13964 (1998) all of which are incorporated herein by reference in their entireties and at least form material related to delivery and making of RNAi molecules.

192. RNAi has been shown to work in a number of cells, including mammalian cells. For work in mammalian cells it is preferred that the RNA molecules which will be used as targeting sequences within the RISC complex are shorter. For example, less than or equal to 50 or 40 or 30 or 29, 28, 27, 26, 25, 24, 23, ,22, 21, 20, 19, 18, 17, 16 , 15, 14, 13 , 12, 11, or 10 nucleotides in length. These RNA molecules can also have overhangs on the 3' or 5' ends relative to the target RNA which is to be cleaved. These overhangs can be at least or less than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleotides long. RNAi works in mammalian stem cells, such as mouse ES cells.

(2) Antibodies (a) Antibodies Generally

193. The term "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with Spi6 or PI9 such that Spi6 or PI9 are inhibited from interacting with there substrates, for example. Antibodies that bind the disclosed regions of Spi6 and PI9 involved in the interaction between Spi6 and PI9 and their substrates are also disclosed. The antibodies can be tested for their desired activity

. 3i using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.

194. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sd. USA, 81:6851-6855 (1984)).

195. The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4- co-receρtor complexes described herein. 196. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to

Barbas et al.

197. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

198. The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additiόnaTprop'erty, sucli^'"as'i:δ^"remove/add amino acids capable of disulfide bonding, to increase its bio- longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, MJ. Curr. Opin. BiotechnoL 3:348-354, 1992).

199. As used herein, the term "antibody" or "antibodies" can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

(b) Human antibodies

200. The disclosed human antibodies can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal

Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol., 147(l):86-95, 1991). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., /. MoI. Biol, 227:381, 1991; Marks et al., J. MoI. Biol, 222:581, 1991).

201. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sd. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol, 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.

(c) Humanized antibodies

202. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody. 203. To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (.e.g., a certain level ot specilicity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol, 2:593-596 (1992)). 204. Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No.

5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5, 939,598 (Kucherlapati et al.), U.S. Patent No. 6,130,364 (Jakobovits et al.), and U.S. Patent No. 6,180,377 (Morgan et al.).

(d) Administration of antibodies

205. Administration of the antibodies can be done as disclosed herein. Nucleic acid approaches for antibody delivery also exist. The broadly neutralizing anti Spi6 or PI9 antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of the nucleic acid can be by any means, as disclosed herein, for example. e) Vaccines

206. Spi6 and PI9 can be used in a vaccine. For example, disclosed herein are vaccines which decreases the number of boosters required to obtain memory cells comprising a SPI6 or PI9 protein, or a fragment thereof and a pharmaceutically acceptable excipient. Also disclosed are vaccines which decreases the number of boosters required to obtain memory cells comprising a SPI6 or PI9 protein, or a fragment thereof and a pharmaceutically acceptable excipient, further comprising a suitable adjuvant.

207. Also disclosed are vaccines which decrease the amount of time for full memory cell response, comprising a SPI6 or PI9 protein, or a fragment thereof and a pharmaceutically acceptable excipient. Also disclosed are vaccines which decrease the amount of time for full memory cell response, comprising a SPI6 or PI9 protein, or a fragment thereof and a pharmaceutically acceptable excipient, further comprising a suitable adjuvant.

208. The establishment of immunological memory is one of the goals of vaccine development. Yet, the establishment of immunological memory can take months to occur following the initial antigenic encounter. Additionally, the mere establishment of immunological memory is not necessarily sufficient to confer protection against future encounters with a pathogen or foreign antigen, as a small memory population may be overwhelmed by a pathogen. Therefore an additional goal is to establish a memory population large enough to provide the protection. For vaccine development, the sufficiency of the immunological memory can be improved through the administration of additional applications of the same or related antigens as the initial vaccine, referred to as a boost. However, multiple boosts may be required and current immunization regimens often require months between successive vaccine administrations. Thus, a continued problem plaguing vaccine development is the establishment of an effective means to rapidly establish protective immunity. 209. The establishment of a long-lived immune response to a target that is of a size large enough to protect the recipient and generated quickly enough to meet the needs of those receiving a vaccine is the continuing goal in the development of many vaccines. Vaccines refer to any composition that is administered to a subject with the goal of establishing an immune response to a particular target or targets. In certain embodiments the vaccines will produce an immune response that is a protective immune response. Vaccines can be, for example, prophylactic, that is, administered before a target is ever encountered, as is typically the case for Polio, measles, mumps, rubella, smallpox, chicken pox, and influenza vaccines, for example. Vaccines can also be therapeutic, providing an immune response to a target that is already within a subject, for example, a vaccine to a particular cancer. Typically vaccines are administered in a single or multiple doses called immunizations and are designed to generate memory T and B-cell populations. However, to date, no vaccine designed to generate memory T-cells has accomplished this task with a single dose, or immunization, of the vaccine. Often with vaccines directed to T-cell immunity, the initial immunization, or prime, generates a memory T-cell population that is insufficient to provide protection against future target encounter related to the antigen. Additionally, the few memory T- cells that are generated from the initial prime can take at least 2 months and can take years to finally transform from naϊve T-cells into memory T-cells. To overcome the problem of inadequate initial priming, additional immunizations, or boosts, comprising the same or related antigen are used to bolster the numbers of memory T-cells. However, for a boost to be effective, the memory T-cell population must be stabilized. That is, the target-specific T-cell population must have completed the transformation to memory cells and be in a steady-state. Thus, a prime-boost immunization regimen can require months between immunizations creating a tremendous lag in time between when immunity to a target is desired and when it is actually achieved. The methods disclosed herein overcome these problems.

210. Typically, memory T-cells can be characterized as long-lived antigen-specific T-cells having a combination of two or more of the following markers CD44⁺ (positive), CDl Ia⁺ (positive), CD43^1Bπ" (negative), CD62L^{m orLO}, CD127⁺(positive), and CD45RA^"(negative), CD27^hi, CD122^hi, IL-15R+. Memory T-cells can be divided into two major groups distinguished by the expression of CCR7 and CD62L. CCR7^'

, CD62L¹⁰ (negative) memory T-cells are referred to as "effector memory T-cells" (T_EM)- These cells generally are localized in the peripheral tissues such as the liver and lungs as well as the spleen, and produce rapid effector functions, such as IFN-γ production, upon stimulation. CCR7⁺ (positive) memory T-cells generally localize m the secondary lymphoid organs such as the thymus, bone marrow, and lymph nodes, although they can also be found in peripheral tissues. These cells are referred to as "central memory T- cells" (T_CM) and provide more effective protection to the host, against at least some pathogens, through increased proliferative capacity. It is understood that maintained within a population of memory T-cells is the potential for further expansion upon future antigen encounter. Thus, herein disclosed are methods of generating memory T-cells. The memory T-cells can be generated, for example, by mixing a target or antigen related to the target with dendritic cells and administering the mixture to a subject. It is understood that the disclosed methods can be used for the generation of, for example, central memory T-cells.

211. It is also contemplated that the booster immunization can comprise any antigen related to the target including, but not limited to, the same antigen supplied in the mixture provided in the prime comprising an antigen related to the target and a dendritic cell. Thus, it is understood that the antigen provided in the booster can be different from the antigen in the prime. It is also understood that the antigen provided in the booster can be different than Spi6 or PI9. It is further understood that the disclosed methods can comprise more than one boost. f) Compositions, characteristics, and relationships

212. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular Spi6 is disclosed and discussed and a number of modifications that can be made to a number of molecules including the Spi6 are discussed, specifically contemplated is each and every combination and permutation of Sρi6 and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

(1) Sequence similarities

213. It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non- natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

214. In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

215. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc.

Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

216. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710,

1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.

217. For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the

Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if ^'the" first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).

(2) Hybridization/selective hybridization 218. The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.

219. Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5⁰C to 20⁰C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular

Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids). A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art. 220. Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, ^{δ9 δ}\ 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the nbή-limitϊng nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed at under conditions where both the limiting and non- limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k_d, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k_d.

221. Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.

222. Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.

223. It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein. (3) Nucleic acids

224. There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example, Spi6 (SEQ ID NO:3) as well as any other proteins disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment. (a) Nucleotides and related molecules

225. A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an mternucleoside linkage, liie base moiety ot a nucleotide can De aαemn-y-yi (Aj, cytosm-i-yi ^Uj, guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. An non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). 226. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.

227. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.

228. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556),

229. A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.

230. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.

(b) Sequences

231. There are a variety of sequences related to, for example, SEQ ID NO:3 as well as any other protein disclosed herein that are disclosed on Genbank, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein. 232. A variety of sequences are provided herein and these and others can be found in Genbank, at www.pubmed.gov. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any sequence given the information disclosed herein and known in the art. (c) Primers and probes

233. Disclosed are compositions including primers and probes, which are capable of interacting with the genes disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the nucleic acid or region of the nucleic acid or they hybridize with the complement of the nucleic acid or complement of a region of the nucleic acid.

(4) Delivery of the compositions to cells

234. There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et al., Science,

247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modifed to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier. (a) Nucleic acid based delivery systems

235. Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

236. As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as SEQ ID NO: 3 into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. In some embodiments the vectors are derived from either a virus or a retrovirus. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia vims Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with ^'the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

237. Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans. (i) Retroviral Vectors

238. A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology-1985, American Society for Microbiology, pp. 229- 232, Washington, (1985), which is incorporated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference.

239. A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich ςeπnence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA syn1hesϊs;''ari'd''sp'eci^'βc^'sequeϊϊces' near the ends of the LTRs that enable the insertion ot me jj JN A state oi tne retrovirus to insert into the host genome. The removal of the gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

240. Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the

DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

(H) Adenoviral Vectors 241. The construction of replication-defective adenoviruses has been described (Berkner et al., J.

Virology 61:1213-1220 (1987); Massie et al., MoI. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993);

Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., MoL Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

242. A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome. (Hi) Adeno-asscociated viral vectors

243. Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

244. In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

245. Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No.

6,261,834 is herein incorproated by reference for material related to the AAV vector.

246. The disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.

247. The inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of

DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

(iv) Large payload viral vectors 248. Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter and Robertson,.Curr Opin MoI Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable. The maintenance of these episomes requires a specific EBV nuclear protein, EBNAl, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

249. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. (b) SSon-nucieic acia oasea systems

250. The disclosed compositions can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.

251. Thus, the compositions can comprise, in addition to the disclosed vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. MoI. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sd USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

252. In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec,

Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ). 253. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,

Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be used for a variety of other speciifc cell types. Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clatnrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). 254. Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral intergration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome. 255. Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art.

(c) In vivo/ex vivo

256. As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

257. If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

(5) Expression systems

258. The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required lor basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

(a) Viral Promoters and Enhancers

259. Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40

(SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ. et al., Gene

18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

260. Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, MX., et al., MoI. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself

(Osborne, T.F., et al., MoI. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes

(globin, elastase, albumin,O!-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. 261. The promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

262. In certain embodiments the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed.

In certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR. 263. It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin. 264! Expression' vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

(b) Markers

265. The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Marker genes include, but are not limited to the E. CoIi lacZ gene, which encodes β-galactosidase, the

G418 resistance gene, HPRT, thymidine kinase, the green fluorescent protein (GFP), and the red fluorescent protein (RFP).

266. In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR-cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

267. The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P.

Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug (J41S or neomycin (_geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

(6) Peptides

(a) Protein variants 268. As discussed herein there are numerous variants of the PI9 protein and Spi6 protein that are known and herein contemplated. In addition, to the known functional strain variants there are derivatives of the Spi6 or PI9 proteins which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross- linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions.

269. TABLE 1 :Amino Acid Abbreviations

270. Substantial changes in function or immunological identity are made by selecting that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation. ^'271^". For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

272. Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

273. Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o- amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.

274. It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology/identity to specific known sequences. For example, SEQ ID NO:2 sets forth a particular sequence of PI9 and SEQ ID NO:4 sets forth a particular sequence of a Spi6 protein. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

275. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP,

BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

276. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. ScL USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281 -306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. ¹I¹ T, . Iϊ is'un'def sϊό^'δ^'d that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

278. As mis specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. For example, one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO:4 is set forth in SEQ ID NO:3. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular organism from which that protein arises is also known and herein disclosed and described.

279. It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 1 and Table 2. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic Enginerring Reviews 13:197-216 (1995), Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs).

280. Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH₂NH-, — CH₂S-, -CH₂-CH₂ --, -CH=CH-- (cis and trans), -COCH₂ --, -CH(OH)CH₂-, and -CHH₂SO- (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and

Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (-CH₂NH-, CH₂CH₂-); Spatola et al. Life Sci 38:1243-1249 (1986) (-CH H₂-S); Harm J. Chem. Soc Perkin Trans. I 307-314 (1982) (-CH- CH-, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (-COCH₂-); Jennings-White et al.

Tetrahedron Lett 23:2533 (1982) (--COCH₂-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-CH(OH)CH₂-); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (-C(OH)CH₂- v c-nA Hruby Life Sci 31:189-199 (1982) (--CH₂-S-); each of which is incorporated herein by reference. A particularly preierred non-pepπαe linKage is --(_^JHbINu--. « is> uuueisiuuu mαi μcμuue an<uv^p L.OU nave more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

281. Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

282. D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together.

This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference).

(7) Pharmaceutical carriers/Delivery of pharamceutical products

283. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

284. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

285. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is mamMϊnecT''Se'e, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

286. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275- 281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). (a) Pharmaceutically Acceptable Carriers

287. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

288. Suitable carriers and their formulations are described in Remington: Tfie Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. . ,

2$".^'"" "^'"" KarniaceuficaTcaπriers are known to those skilled in the art. These most typically wouiα oe standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

290. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like. 291. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

292. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. 293. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

294. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

295. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. (b) Therapeutic Uses

296. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms and disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al, eds., Noges Publications, Park Ridge, NJ., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.

297. Following administration of a disclosed composition, such as an antibody, for treating, inhibiting, or preventing Spi6 or PI9 function, the efficacy of the therapeutic antibody can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a composition, such as an antibody, disclosed herein is efficacious in treating or inhibiting Spi6 or PI9 in a subject by observing that the composition reduces bacterial load, such as Listeria moncytogenes, or prevents a further increase in bacteria, such as Listeria moncytogenes. Bacteria loads, such as Listeria moncytogenes, can be measured by methods that are known in the art, for example, using polymerase chain reaction assays to detect the presence of the bacteria, such as Listeria moncytogenes, nucleic acid or antibody assays to detect the presence of bacteria, such as Listeria moncytogenes _\ protein in a sample (e.g., but not limited to, blood) from a subject or patient, or by measuring the level of circulating anti-bacteria antibody levels in the patient.

298. The compositions that inhibit Spi6 or PI9 interactions disclosed herein may be administered prophylactically to patients or subjects who are at risk for bacteria infection, such as being exosed to bacteria, such as Listeria moncytogenes.

299. Other molecules that interact with Spi6 or PI9 to inhibit Spi6 or PI9 interactions which do not have a specific pharmacuetical function, but which may be used for tracking changes within cellular chromosomes or for the delivery of diagnositc tools for example can be delivered in ways similar to those described for the pharmaceutical products. 3Od. "The dϊM'δg'S'STόiripositions and methods can also be used for example as tools to isolate and test new drug candidates for a variety of Spi6 and PI9 related diseases as well as disease related to inflammation and the immune system.

(8) Chips and micro arrays 301. Disclosed are chips where at least one address is the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Also disclosed are chips where at least one address is the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein.

302. Also disclosed are chips where at least one address is a variant of the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Also disclosed are chips where at least one address is a variant of the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein.

(9) Computer readable mediums

303. It is understood that the disclosed nucleic acids and proteins can be represented as a sequence consisting of the nucleotides of amino acids. There are a variety of ways to display these sequences, for example the nucleotide guanosine can be represented by G or g. Likewise the amino acid valine can be represented by VaI or V. Those of skill in the art understand how to display and express any nucleic acid or protein sequence in any of the variety of ways that exist, each of which is considered herein disclosed. Specifically contemplated herein is the display of these sequences on computer readable mediums, such as, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks, or other computer readable mediums. Also disclosed are the binary code representations of the disclosed sequences.

Those of skill in the art understand what computer readable mediums. Thus, computer readable mediums on which the nucleic acids or protein sequences are recorded, stored, or saved.

304. Disclosed are computer readable mediums comprising the sequences and information regarding the sequences set forth herein. Also disclosed are computer readable mediums comprising the sequences and information regarding the sequences set forth herein.

(lO)Compositions identified by screening with disclosed compositions / combinatorial chemistry

(a) Combinatorial chemistry

305. The disclosed compositions can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired way. The nucleic acids, peptides, and related molecules disclosed herein can be used as targets for the combinatorial approaches. Also disclosed are the compositions that are identified through combinatorial techniques or screening techniques in which the compositions disclosed in SEQ ID NOS: 1, 2, 3, and 4 or portions thereof, are used as the target in a combinatorial or screening protocol. 306. It is understood that when using the disclosed compositions in combinatorial techniques or screening methods, molecules, such as macromolecular molecules, will be identified that have particular desired properties such as inhibition or stimulation or the target molecule's function. The molecules identified^' WaϊsdlatM ¹WH¹Sn¹U¹SIiIg the disclosed compositions, such as, Spi6 or PI9, or animlas overexpressing, for example, are also disclosed. Thus, the products produced using the combinatorial or screening approaches that involve the disclosed compositions, such as, Spi6 or PI9, or animals overexpressing them, are also considered herein disclosed. 307. It is understood that the disclosed methods for identifying molecules that inhibit the interactions between, for example, Spi6 and granzyme B can be performed using high through put means. For example, putative inhibitors can be identified using Fluorescence Resonance Energy Transfer (FRET) to quickly identify interactions. The underlying theory of the techniques is that when two molecules are close in space, ie, interacting at a level beyond background, a signal is produced or a signal can be quenched. Then, a variety of experiments can be performed, including, for example, adding in a putative inhibitor. If the inhibitor competes with the interaction between the two signaling molecules, the signals will be removed from each other in space, and this will cause a decrease or an increase in the signal, depending on the type of signal used. This decrease or increasing signal can be correlated to the presence or absence of the putative inhibitor. Any signaling means can be used. For example, disclosed are methods of identifying an inhibitor of the interaction between any two of the disclosed molecules comprising, contacting a first molecule and a second molecule together in the presence of a putative inhibitor, wherein the first molecule or second molecule comprises a fluorescence donor, wherein the first or second molecule, typically the molecule not comprising the donor, comprises a fluorescence acceptor; and measuring Fluorescence Resonance Energy Transfer (FRET), in the presence of the putative inhibitor and the in absence of the putative inhibitor, wherein a decrease in FRET in the presence of the putative inhibitor as compared to

FRET measurement in its absence indicates the putative inhibitor inhibits binding between the two molecules. This type of method can be performed with a cell system as well.

308. Combinatorial chemistry includes but is not limited to all methods for isolating small • molecules or macromolecules that are capable of binding either a small molecule or another macromolecule, typically in an iterative process. Proteins, oligonucleotides, and sugars are examples of macromolecules.

For example, oligonucleotide molecules with a given function, catalytic or ligand-binding, can be isolated from a complex mixture of random oligonucleotides in what has been referred to as "in vitro genetics" (Szostak, TIBS 19:89, 1992). One synthesizes a large pool of molecules bearing random and defined sequences and subjects that complex mixture, for example, approximately 10¹⁵ individual sequences in 100 μg of a 100 nucleotide RNA, to some selection and enrichment process. Through repeated cycles of affinity chromatography and PCR amplification of the molecules bound to the ligand on the column, Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA molecules folded in such a way as to bind a small molecule dyes. DNA molecules with such ligand-binding behavior have been isolated as well (Ellington and Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goals exist for small organic molecules, proteins, antibodies and other macromolecules known to those of skill in the art. Screening sets of molecules for a desired activity whether based on small organic libraries, oligonucleotides, or antibodies is broadly referred to as combinatorial chemistry. Combinatorial techniques are particularly suited for defining binding mt^'eractions"betw^'een"nl'61ecures and for isolating molecules that have a specific binding activity, often called aptamers when the macromolecules are nucleic acids.

309. There are a number of methods for isolating proteins which either have de novo activity or a modified activity. For example, phage display libraries have been used to isolate numerous peptides that interact with a specific target. (See for example, United States Patent No. 6,031 ,071 ; 5,824,520; 5,596,079; and 5,565,332 which are herein incorporated by reference at least for their material related to phage display and methods relate to combinatorial chemistry)

310. A preferred method for isolating proteins that have a given function is described by Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorial chemistry method couples the functional power of proteins and the genetic power of nucleic acids. An RNA molecule is generated in which a puromycin molecule is covalently attached to the 3 '-end of the RNA molecule. An in vitro translation of this modified RNA molecule causes the correct protein, encoded by the RNA to be translated. In addition, because of the attachment of the puromycin, a peptdyl acceptor which cannot be extended, the growing peptide chain is attached to the puromycin which is attached to the RNA. Thus, the protein molecule is attached to the genetic material that encodes it. Normal in vitro selection procedures can now be done to isolate functional peptides. Once the selection procedure for peptide function is complete traditional nucleic acid manipulation procedures are performed to amplify the nucleic acid that codes for the selected functional peptides. After amplification of the genetic material, new RNA is transcribed with puromycin at the 3 '-end, new peptide is translated and another functional round of selection is performed. Thus, protein selection can be performed in an iterative manner just like nucleic acid selection techniques. The peptide which is translated is controlled by the sequence of the RNA attached to the puromycin. This sequence can be anything from a random sequence engineered for optimum translation (i.e. no stop codons etc.) or it can be a degenerate sequence of a known RNA molecule to look for improved or altered function of a known peptide. The conditions for nucleic acid amplification and in vitro translation are well known to those of ordinary skill in the art and are preferably performed as in

Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).

311. Another preferred method for combinatorial methods designed to isolate peptides is described in Cohen et al. (Cohen B.A.,et al, Proc. Natl. Acad. Sci. USA 95(24): 14272-7 (1998)). This method utilizes and modifies two-hybrid technology. Yeast two-hybrid systems are useful for the detection and analysis of protein-.protein interactions. The two-hybrid system, initially described in the yeast Saccharomyces cerevisiae, is a powerful molecular genetic technique for identifying new regulatory molecules, specific to the protein of interest (Fields and Song, Nature 340:245-6 (1989)). Cohen et al., modified this technology so that novel interactions between synthetic or engineered peptide sequences could be identified which bind a molecule of choice . The benefit of this type of technology is that the selection is done in an intracellular environment. The method utilizes a library of peptide molecules that attached to an acidic activation domain. A peptide of choice, for example an active portion of Spi6 or PI9, is attached to a DNA"Mriairtg'^raomaiiQ"cil"a'tratiscriptional activation protein, such as Gal 4. By performing the Two-hybrid technique on this type of system, molecules that bind the active portion of Spi6 or PI9 can be identified.

312. Using methodology well known to those of skill in the art, in combination with various combinatorial libraries, one can isolate and characterize those small molecules or macromolecules, which bind to or interact with the desired target. The relative binding affinity of these compounds can be compared and optimum compounds identified using competitive binding studies, which are well known to those of skill in the art.

313. Techniques for making combinatorial libraries and screening combinatorial libraries to isolate molecules which bind a desired target are well known to those of skill in the art. Representative techniques and methods can be found in but are not limited to United States patents 5,084,824, 5,288,514, 5,449,754,

5,506,337, 5,539,083, 5,545,568, 5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680, 5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899, 5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598, 5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014, 5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107, 5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972, 5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,

5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792, 5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356, 5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371, 6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

314. Combinatorial libraries can be made from a wide array of molecules using a number of different synthetic techniques. For example, libraries containing fused 2,4-pyrimidinediones (United States patent 6,025,371) dihydrobenzopyrans (United States Patent 6,017,768and 5,821,130), amide alcohols (United States Patent 5,976,894), hydroxy-amino acid amides (United States Patent 5,972,719) carbohydrates (United States patent 5,965,719), l,4-benzodiazepin-2,5-diones (United States patent 5,962,337), cyclics (United States patent 5,958,792), biaryl amino acid amides (United States patent 5,948,696), thiophenes (United States patent 5,942,387), tricyclic Tetrahydroquinolines (United States patent 5,925,527), benzofurans (United States patent 5,919,955), isoquinolines (United States patent 5,916,899), hydantoin and thiohydantoin (United States patent 5,859,190), indoles (United States patent 5,856,496), imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (United States patent 5,856,107) substituted 2-methylene-2, 3-dihydrothiazoles (United States patent 5,847,150), quinolines (United States patent 5,840,500), PNA (United States patent 5,831,014), containing tags (United States patent 5,721,099), polyketides (United States patent 5,712,146), morpholino-subunits (United States patent 5,698,685 and 5,506,337), sulfamides (United States patent 5,618,825), and benzodiazepines (United States patent 5,288,514).

315. Screening molecules similar to Sρi6 or PI9 substrates for inhibition of Spi6 activity is a method of isolating desired compounds.

316. As used herein combinatorial methods and libraries included traditional screening methods and libraries as well as methods and libraries used in interative processes. (b) Computer assisted drug design

317. The disclosed compositions can be used as targets for any molecular modeling technique to identify either the structure of the disclosed compositions or to identify potential or actual molecules, such as small molecules, which interact in a desired way with the disclosed compositions. The nucleic acids, peptides, and related molecules disclosed herein can be used as targets in any molecular modeling program or approach.

318. It is understood that when using the disclosed compositions in modeling techniques, molecules, such as macromolecular molecules, will be identified that have particular desired properties such as inhibition or stimulation or the target molecule's function. The molecules identified and isolated when using the disclosed compositions, such as, Spi6 and PI9, are also disclosed. Thus, the products produced using the molecular modeling approaches that involve the disclosed compositions, such as, Spi6 and PI9, are also considered herein disclosed.

319. Thus, one way to isolate molecules that bind a molecule of choice is through rational design. This is achieved through structural information and computer modeling. Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.

320. Examples of molecular modeling systems are the CHARMm and QUANTA programs, Polygen Corporation, Waltham, MA. CHARMm perforins the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

321. A number of articles review computer modeling of drugs interactive with specific proteins, such as Rotivinen, et al, 1988 Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57 (June

16, 1988); McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol Toxiciol. 29, 111-122; Perry and Davies, QSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect to a model enzyme for nucleic acid components, Askew, et al., 1989 J. Am. Chem. Soc. Ill, 1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign,

Inc., Pasadena, CA., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted ϊό ^"design of'MSieeuies Specifically interacting with specific regions of DNA or KiSfA, once that region is identified.

322. Although described above with reference to design and generation of compounds which could alter binding, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which alter substrate binding or enzymatic activity.

(ll)Kits

323. Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended. For example, disclosed is a kit for assessing whether a particular compound inhibits Spi6 function, comprising the oligonucleotides set forth in SEQ ID Nos: 3 and 1. (12) Compositions with similar functions

324. It is understood that the compositions disclosed herein have-certainriunctions, such as inhibiting Spi6 or PI9. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures which can perform the same function which are related to the disclosed structures, and that these structures will ultimately achieve the same result, for example stimulation or inhibition of Spi6 or PI9.

C. Methods

325. The disclosed compositions can be used in a variety of ways as research tools. For example, the disclosed compositions, such as the transgenic mice and knock-out mice can be used to study the mechanisms surrounding Spi6. 326. The compositions can be used for example as targets in combinatorial chemistry protocols or other screening protocols to isolate molecules that possess desired functional properties related to Sρi6.

327. The disclosed compositions can also be used as diagnostic tools related to diseases of the immune system, such as inflammatory diseases, as well as models of these diseases.

328. The disclosed compositions can be used as discussed herein as either reagents in micro arrays or as reagents to probe or analyze existing microarrays. The disclosed compositions can be used in any known method for isolating or identifying single nucleotide polymorphisms. The compositions can also be used in any method for determining allelic analysis of for example, particularly allelic analysis as it relates to Spi6 and functions. The compositions can also be used in any known method of screening assays, related to chip/micro arrays. The compositions can also be used in any known way of using the computer readable embodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions. 1 : Methods of Using Spi6 and PI9 a) Methods of Increasing Immunity to Viral Infection

329. Disclosed herein are methods of increasing immunity comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. For example, disclosed is a method of increasing immunity to viral infection comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. Also disclosed is a method of increasing immunity to viral infection comprising administering a vector comprising a nucleic acid capable of encoding Spi6 or PI9.

330. Further disclosed is a method of increasing immunity to viral infection comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof. 331. The virus can be Lymphocytic chorioimeningitis virus, Herpes simplex virus type-1 , Herpes simplex virus type-2, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus,

Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency cirus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1 , and Human Immunodeficiency virus type-2. b) Methods of Protecting the Integrity of Lytic Granules

332. As provided in the examples below, in addition to suppressing the activity of cytoplasmic GrB, Spi6 also ensured the integrity of lytic granules. As such, provided herein are methods of protecting the integrity of lytic granules comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. Also provides are methods of protecting the integrity of lytic granules comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof. c) Methods of Protecting Against Inflammatory Disease

333. Provided herein are methods of protecting against inflammatory disease comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. Also provided herein are methods of protecting against inflammatory disease comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof.

334. Inflammatory diseases include, but are not limited to inflammatory bowel disease (IBD), systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome, ulcerative colitis, Crohn's disease, autoimmune hemolytic anemia, sterility, myasthenia gravis, multiple sclerosis,

Basedow's disease, thrombopenia purpura, insulin-dependent diabetes mellitus, allergy; asthma, atopic disease, arteriosclerosis, myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia, as well as "gfaft-f erSus-Mst" aM Mst-Versu's^'-graft disease. For example, disclosed is a method of protecting against transplantation induced graft-versus-host and host-versus-graft disease comprising administering an effective amount of SPI6 or PI9 protein or a fragment thereof. Also disclosed is a method of protecting against transplantation induced graft-versus-host and host-versus-graft disease comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid.

335. Some inflammatory diseased are induced by Granzyme B activity. As such, protection against the inflammatory disease can be a result of suppression of Granzyme B activity. Examples of Granzyme B-induced inflammatory diseases include, but are not limited to achalasia, early rheumatoid factor positive rheumatoid arthritis, atherosclerosis, transplant vascular disease, childhood bronchiolititis, Crohn's Disease, Ulcerative Colitis, Rasmussen's encephalitis, early rheumatoid arthritis, graft-versus-host disease, systemic lupus erythematosus, lichen sclerosus et atrophicus, and systemic sclerosis.

336. The disclosed method of protecting against inflammatory disease can also be used to dampen inflammation due to chronic infection. For example, chronic infection in COPD patients can lead to deleterious inflammation responses in the respiratory system. As discussed herein, if the inflammation reaction is completely reduced, this can help with the inflammation effects, but can create other problems with respect to bacterial immunity and the patients' ability to fight off an infection. Disclosed herein, the use, of SPI6 or PI9 or active fragments, or their expression from genes can create a beneficial situation where the inflammation reaction, and for example, the neutrophil elastase activity can be dampened, but not eliminated because the SPI6 and PI9 do not completely reduce these responses and activities as discussed herein. Thus, the use of SPI6 or PI9 to treat these types of response can be used alone or in conjunction with other therapies to achieve the appropriate amount of inflammation reduction or neutrophil elastase activity reduction without increasing susceptibility to infection to a high level. d) Methods of Inhibiting Granzyme B Activity

337. As shown in the examples below, Spi6 is a potent inhibitor of Granzyme B. In addition, the examples below show that Granzyme B is active in the cytoplasm and Spi6 is also expressed in the cytoplasm. As such, provided are methods of inhibiting Granzyme B activity in the cytoplasm by administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid.

338. Also provided are methods of inhibiting the presence of Granzyme B in the cytoplasm by administering an effective amount of SPI6 or PI9 protein, or a fragment thereof. e) Methods of Inhibiting Neutrophil Elastase

339. As described above, while neutrophils are critical in host defense, excessive levels of inflammation during infection lead to tissue damage, organ dysfunction and disease. A direct role for neutrophils is indicated by the observation that NE KO mice undergo less endotoxin-mediated septic shock (Tkalcevic et al., 2000). As such, methods of inhibiting neutrophil elastase would be of particular use. As provided in the examples below, the disruption of SPI6 function or Spi6 expression can lead to increased neutrophil elastase activity, whereas overexpression of Spi6 can lead to inhibition of neutrophil elastase activity. 'ϋ4U'. "'"" "'"'iPfoMW Kf bin are methods of inhibiting neutrophil elastase by administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. Also provided herein are methods of inhibiting neutrophil elastase by administering an effective amount of SPI6 or PI9 protein, or a fragment thereof.

341. In addition, exogenous inhibitors of neutrophil elastase would be of particular use as well. Provided herein are methods of inhibiting neutrophil elastase by administering an exogenous inhibitor of neutrophil elastase. Exogenous inhibitors of neutrophil elastase include, but are not limited to αl-antitrysin, monocyte neutrophil elastase inhibitor (MNEI), or SLPl. These inhibitors can be added in combination with the compositions disclosed herein to decrease neutrophil elastase activity, but to not eliminate it. For example, less SLPl can be administered then what would have previously been used to achieve a reduced neutrophil elastase activity, but not to eliminate the activity. In certain embodiments, with any of the methods and compositions disclosed herein, it is preferred to reduce neutrophil elastase activity by 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 %. f) Methods of Using a Spi6 Overexpressing animal

(1) Identification of inhibitors of Spi6

342. The ability of small molecules to inhibit the activity of serpins has been shown previously. For example, a 14-amino acid peptide, corresponding to the plasminogen activator inhibitor-i (PAI-I) reactive center loop (residues 333-346), was shown to rapidly inhibit PAI-i function (Eitzman et al., 1995).

In addition, the structurally novel small molecule_^WAY-140312, was also shown to be a potent inactivator of PAI-I (Crandall et al., 2004). It is understood that these molecules can have cross reactivity with the other serpins, and as such these molecules can also function in certain embodiments as inhibitors of Sρi6 and or PI9. Other molecules having inhibitor activity of other serpins or elastase inhibitors are also disclosed and can be used as inhibitors of Spi6 or PI9. It is understood that these and other molecules can be tested for their efficacy of inhibition, by for example, testing them in the mouse and animal models disclosed herein.

(2) Testing the activity in vivo of known or potential inhibitors of Spi6 or PI9 343. The disclosed animals, such as mice, such as the transgenic animals, can be used to test, in vivo the activity of various molecules which either are known to have or could have Spi6 or PI9 inhibitory activity. These molecules can be tested as disclosed herein, but at least can be tested by administration of the molecule or molecules to the animal and assaying the effect the molecule has on the animal, and comparing that to, for example, a nontransgenic animal. Any of the assays for Spi6 and PI9 phenotypes of, for example, the transgenic animals discussed herein can be used to determine the effect of the molecule on

Spi6 or PI9. 2."^' iWfetB'ό'd's of Identifying, Screening, and Testing Compounds for Spi6/PI9 Modulation Activity a) Identifying inhibitors

344. Disclosed are methods of identifying a candidate inhibitor of Spi6 activity comprising (a) preparing a first cell culture that overexpresses Spiβ; (b) adding the candidate inhibitor to the cell culture (c) incubating the cell culture under conditions and for a time sufficient to detect an inhibitory effect by the candidate inhibitor; and (d) determining the effect of the candidate inhibitor on the Spi6 activity.

345. Also disclosed are methods of identifying a candidate inhibitor of PI9 activity comprising (a) preparing a first cell culture that overexpresses PI9; (b) adding the candidate inhibitor to the cell culture (c) incubating the cell culture under conditions and for a time sufficient to detect an inhibitory effect by the candidate inhibitor; and (d) determining the effect of the candidate inhibitor on the PI9 activity.

346. These methods as well as all the methods disclosed herein can be used in any combination to achieve the desired result. For example, due to the similarities between PI9 and Spi6 as discussed above, a candidate inhibitor of PI9 can be identified by a method comprising (a) preparing a first cell culture that overexpresses Spi6; (b) adding the candidate inhibitor to the cell culture (c) incubating the cell culture under conditions and for a time sufficient to detect an inhibitory effect by the candidate inhibitor; and (d) determining the effect of the candidate inhibitor on the Spi6 activity.

347. Also disclosed are methods, wherein the cell culture comprises a neutrophil or macrophage, and methods further comprising assaying for increased in vitro bactericidal activity of neutrophils or macrophages, and/or wherein the neutrophils or macrophages are from a Spiβ transgenic mice.

348. Also disclosed is a method of identifying a candidate inhibitor of Spiό activity comprising (a) preparing a first cell culture that overexpresses Spiό; (b) adding the candidate inhibitor to the cell culture (c) incubating the cell culture under conditions and for a time sufficient to detect an inhibitory effect by the candidate inhibitor; and (d) determining the effect of the candidate inhibitor on the Spiό activity and further comprising assaying the cell for increased in vitro bactericidal activity of neutrophils or macrophages from

Spiό transgenic mice.

349. Also disclosed is a method of identifying a candidate inhibitor of PI9 activity comprising (a) preparing a first cell culture that overexpresses PI9; (b) adding the candidate inhibitor to the cell culture (c) incubating the cell culture under conditions and for a time sufficient to detect an inhibitory effect by the candidate inhibitor; (d) determining the effect of the candidate inhibitor on the PI9 activity and assaying the cell for increased in vitro bactericidal activity of neutrophils or macrophages from PI9 transgenic mice.

350. Also disclosed is a method of identifying a candidate inhibitor of Spiό activity comprising (a) preparing a transgenic non-human animal that overexpresses Spiό; (b) administering the candidate inhibitor to the animal (c) determining the effect of the candidate inhibitor on the Spiό activity. Also disclosed is a method of identifying a candidate inhibitor of PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses PI9; (b) administering the candidate inhibitor to the animal (c) determining the -^,«^+ of the candidate inhibitor on the PI9 activity. 3'MT - "^■Al'so'tuscioseαis a method of identifying a candidate inhibitor of Spi6/PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses Spi6; (b) administering the candidate inhibitor to the animal (c) determining the effect of the candidate inhibitor on the Spi6 activity, wherein the transgenic non-human animal comprises a cell wherein the cell expresses a transgene coding for a serpin.

352. Also disclosed is a method of screening a set of candidate inhibitors of Spi6 activity comprising (a) preparing a transgenic non-human animal that overexpresses Spi6; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the Sρi6 activity. Further disclosed is a method of screening a set of candidate inhibitors of PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses PI9; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors n the PI9 activity.

353. Also disclosed is a method of screening a set of candidate inhibitors of Spi6 activity comprising (a) preparing a transgenic non-human animal that overexpresses Spi6; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the Spi6 activity, further comprising d) the step of subdividing the set of candidate inhibitors into subsets of candidate inhibitors, and e) testing each subset for inhibitory activity in the transgenic non-human animal that overexpresses Sρi6 until a subset having the inhibitory activity is identified. Also disclosed is a method of screening a set of candidate inhibitors of PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses PI9; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the PI9 activity, further comprising d) the step of subdividing the set of candidate inhibitors into subsets of candidate inhibitors, and e) testing each subset for inhibitory activity in the transgenic non-human animal that overexpresses PI9 until a subset having the inhibitory activity is identified.

354. Also disclosed is a method of screening a set of candidate inhibitors of Sρi6 activity comprising (a) preparing a transgenic non-human animal that overexpresses Sρi6; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the Spi6 activity, further comprising d) the step of subdividing the set of candidate inhibitors into subsets of candidate inhibitors, and e) testing each subset for inhibitory activity in the transgenic non-human animal that overexpresses Spi6 until a subset having the inhibitory activity is identified, further comprising f) the step of subdividing the identified subset of candidate inhibitors in a set of small subsets of candidate inhibitors, g) and testing each small subset of candidate inhibitors for inhibitory activity in the transgenic non-human animal that overepxresses Spi6 until a small subset having the activity is identified.

355. Also disclosed is a method of screening a set of candidate inhibitors of PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses PI9; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the PI9 activity, further comprising d) the step of subdividing the set of candidate inhibitors into subsets of candidate inhibitors, and e) testing each subset for inhibitory activity in the transgenic non-human animal tliat¹ ove'feipϊes's'es' PB¹ ttitiWsutfeet having the inhibitory activity is identified, further comprising f) the step of subdividing the identified subset of candidate inhibitors in a set of small subsets of candidate inhibitors, g) and testing each small subset of candidate inhibitors for inhibitory activity in the transgenic non-human animal that overepxresses PI9 until a small subset having the activity is identified. 356. Also disclosed is a method of screening a set of candidate inhibitors of Spi6 activity comprising (a) preparing a transgenic non-human animal that overexpresses Spi6; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the Spi6 activity, further comprising d) the step of subdividing the set of candidate inhibitors into subsets of candidate inhibitors, and e) testing each subset for inhibitory activity in the transgenic non-human animal that overexpresses Spi6 until a subset having the inhibitory activity is identified, further comprising f) the step of subdividing the identified subset of candidate inhibitors in a set of small subsets of candidate inhibitors, g) and testing each small subset of candidate inhibitors for inhibitory activity in the transgenic non-human animal that overepxresses Spi6 until a small subset having the activity is identified, further comprising repeating steps f and g until a single candidate inhibitor having inhibitory activity is identified. 357. Also disclosed is a method of screening a set of candidate inhibitors of PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses PI9; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the PI9 activity, further comprising d) the step of subdividing the set of candidate inhibitors into subsets of candidate inhibitors, and e) testing each subset for inhibitory activity in the transgenic non-human animal that overexpresses PI9 until a subset having the inhibitory activity is identified, further comprising f) the step of subdividing the identified subset of candidate inhibitors in a set of small subsets of candidate inhibitors, g) and testing each small subset of candidate inhibitors for inhibitory activity in the transgenic non-human animal that overepxresses PI9 until a small subset having the activity is identified, further comprising repeating steps f and g until a single candidate inhibitor having inhibitory activity is identified. 358. Disclosed is a method of testing an inhibitor of Spi6 activity comprising (a) preparing a transgenic nonhuman animal that overexpresses Spi6; (b) administering the inhibitor to the animal (c) determining the effect of the inhibitor on the Sρi6 activity. Also disclosed is a method of testing an inhibitor of PI9 activity comprising (a) preparing a transgenic nonhuman animal that overexpresses PI9; (b) administering the inhibitor to the animal (c) determining the effect of the inhibitor on the PI9 activity. 359. Also disclosed is a method of testing an inhibitor of Spi6 activity comprising (a) preparing a transgenic nonhuman animal that overexpresses Sρi6; (b) administering the inhibitor to the animal (c) determining the effect of the inhibitor on the Spi6 activity, further comprising comparing the activity of the inhibitor to the activity of a known standard.

360. Also disclosed is a method of testing an inhibitor of PI9 activity comprising (a) preparing a transgenic nonhuman animal that overexpresses PI9; (b) administering the inhibitor to the animal (c) determining the effect of the inhibitor on the PI9 activity, further comprising comparing the activity of the inhibitor to the activity of a known standard. 56 ϊv Also'yϊMσsfel-t'is a method of testing an inhibitor of Spi6 activity comprising (a) preparing a transgenic nonhuman animal that overexpresses Spi6; (b) administering the inhibitor to the animal (c) determining the effect of the inhibitor on the Spi6 activity, further comprising comparing the activity of the inhibitor to the activity of a known standard, further comprising comparing the activity of the inhibitor to the activity of the inhibitor that occurred in a previous test of the compound in the transgenic nonhuman animal.

362. Also disclosed is a method of testing an inhibitor of PI9 activity comprising (a) preparing a transgenic nonhuman animal that overexpresses PI9; (b) administering the inhibitor to the animal (c) determining the effect of the inhibitor on the PI9activity, further comprising comparing the activity of the inhibitor to the activity of a known standard, further comprising comparing the activity of the inhibitor to the activity of the inhibitor that occurred in a previous test of the compound in the transgenic nonhuman animal.

3. Method of Inhibiting Infections a) Increasing Spi6 expression

363. Disclosed is a method of treating a disease caused by excess granzyme B activity by inhibiting Granzyme B, comprising administering a composition, wherein the composition expresses Spi6. Also disclosed is a method of treating a disease caused by excess granzyme B activity by inhibiting

Granzyme B, comprising administering a composition, wherein the composition expresses PI9

364. Also disclosed is method of treating a disease caused by excess granzyme B activity by inhibiting Granzyme B, comprising administering a composition, wherein the composition expresses Sρi6 or PI9. Also disclosed is method of treating a disease caused by excess granzyme B activity by inhibiting Granzyme B, comprising comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid. Also disclosed is method of treating a disease caused by excess granzyme B activity by inhibiting Granzyme B, comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof. Examples of diseases caused by excess granzyme B activityjnclude, but are not limited to: Graft versus Host Disease, Rheumatoid Arthritis, Systemic Lupus Erythematosis, Transplant Vascular Disease, Athrelosclerosis, Exanthematous Pustulosis, Scleroderma, Ischemic digital loss, Rasmussens Encephalitis,

Goodpastures Disease, Primary Bilary Cirrhosis, Autoimmune Hepatitis, Cutaneous Lichen Sclerosus et Atrophecus, Achalasia, Crohn's Disease, Ulcerative Colits, Sjogren's Syndrome, Autoimmune myositis, and Paraneoplastic Cerebellar Degeneration. b) Inhibiting Spi6 activity 365. As discussed herein, by inhibiting Spi6 or PI9 activity an increase in immunity to various bacterial infections can take place because of an increase in an effective inflammatory response. The bacterial infection can be any bacterial infection, including those set forth herein. As described herein, by inhibiting Spi6 activity, animals exhibited an increased survival rate from pneumonia-induced death after infection with the gram-negative bacterium P. aeruginosa (See Example 3 and Figure 41(a)). In addition, by inhibiting Spi6 activity mice were resistant to lethal infection with gram-positive L. monocytogenes

(Figure 41(c)). The increased survival of Spi6 KO mice was due to increased clearance of bacteria, as evidenced by lower titers in the infected tissues (Figure 41(a)-(c)). In particular, by inhibiting Spi6 activity, Via kfrβctbuζ'Wce'lsuBjecifeαto 'bacterial infection exhibited around a 60% survival rate as opposed to a 0% survival rate for controls. The exact mechanism by which inhibiting Sρi6 activity results in increased survival can be in part due to Spiό's ability to regulate neutrophil elastase activity.

366. It is known that neutrophil elastase (NE) plays a critical role immunity to bacterial infection (Lehrer and Ganz, 1990). As shown herein, increased clearance of P. aeruginosa in Spi6 KO mice correlated with about a 2-fold increase in NE activity. In addition, doses of human NE (HNE) up to 1.8U/kg protected B6 mice from P. aeruginosa induced pneumonia (Figure 43). 1.8U/kg corresponds to 90μg /kg or 3 x 10^"9moles/kg. The relationship between Spi6 and NE and the effect on the immune system is in balance.

367. The discussion herein, as well as the figures and examples, provide a correlation between Sρi6 activity and NE activity. First, by inhibiting Spi6 activity, NE activity can increase, thereby resulting in increased protection from bacterial infection. Second, by dosing a mouse with 1.2 to 2.4 U/kg of HNE, survival rate can be increased between 15 and 30%. It is understood that Spi6 is not a complete inhibitor of HNE. Spi6 reduces HNE activity but it does not eliminate, and thus completely removal of Spi6 through a knockout, does not allow deleterious formation of HNE, but rather just enough that beneficial rather than detrimental effects of an over active immune response are seen in bacterial challenged mice. Bacterial infection is decreased without the effects of sepsis, for example.

368. Since complete ablation of Spi6 activity resulted in a less than optimum survival rate of 60% when mice were subjected to bacterial infection (Figure 41 (a)) and mice dosed with 1.2 to 2.4 U/kg of HNE, resulted in a less than optimum survival rate of 30%, a method employing a means of inhibiting Spi6 and adding exogenous NE to a mouse is disclosed herein to lead to an optimum survival rate.

369. The results provided herein show that there is an optimal range for increasing HNE or NE activity in a bacteria challenged organism, which is less than that which would be expected for a full dose of HNE or NE (or less than that which arises after prolonged infection and an abnormally high amount of NE is released causing deleterious inflammatory effects along with the infection, such as in COPD patients), but more than what would be naturally produced in the initial response. Thus, provided are methods which either 1) reduce Spi6 activity by administering a Spi6 inihibitor, 2) increase NE or HNE activity by an optimal amount which produces an increase in resistance or survival or immunity by administering a HNE or NE activator or, for example, administering HNE or NE directly, or 3) performing a combination of 1) and 2). 370. For example, a Spi6 inhibitor can be administered so that it reduces Spi6 activity to 0, 1, 2, 3,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent of the non-inhibited activity of Spi6. 371. Also, a Spi6 inhibitor can be administered so that it increases the HNE or NE activity by 0, 5,

10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 1125, 130, ¹ ^ ¹40, 150, 160, 170, 175, 180, 185, 190, 200, 225, or 250 percent, for example, or any percent in Detweeπ^'',""'cⁱofflpared"tθ"a wneπ Spi6 is not inhibited. For example, in Spi6 KO mice, after 6 hours of Sρi6 inhibition, NE activity increased 24%. After 12 hours of Spi6 inhibition, NE activity increased 156%, and after 24 hours, NE activity returned to the basal level. Another example, is at time = 6h the NE activity is increased by 24% over the B6 mouse. It is also understood that, for example, by 24 hours, the NE activity has decrease in the SPI6 KO to that of the B6 mouse.

372. Furthermore, HNE or NE can be administered at a concentration of 0.1 U/kg, 0.2 U/kg, 0.3 U/kg, 0.4 U/kg, 0.5 U/kg, 0.6 U/kg, 0.7 U/kg, 0.8 U/kg, 0.9 U/kg, 1.0 U/kg, 1.1 U/kg, 1.2 U/kg, 1.3 U/kg, 1.4 U/kg, 1.5 U/kg, 1.6 U/kg, 1.7 U/kg, 1.8 U/kg, 1.9 U/kg, 2.0 U/kg, 2.1 U/kg, 2.2 U/kg, 2.3 U/kg, 2.4 U/kg, 2.5 U/kg, 2.6 U/kg, 2.7 U/kg, 2.8 U/kg, 2.9 U/kg, 3.0 U/kg, 3.1 U/kg, 3.2 U/kg, 3.3 U/kg, 3.4 U/kg, or 3.5 U/kg. One unit is defined as the amount of enzyme that will hydrolyze 1.0 μmol of MeO-Suc-Ala-Ala-

Pro-Val-pNA per minute at 25⁰C, pH 8.0 or alternatively, one unit of NE releases 1 μmol 4-nitroanilide per minute from methoxysuccinyl-ala-ala-pro-val-4-mtroanilide at 25 ⁰C, pH 8.0.

373. Also, disclosed herein are methods of increasing immunity or resistance to a bacterial infection comprising administering NE and an inhibitor of Spi6. NE doses can be any disclosed herein in combination with any doses of a Sρi6 inhibitor disclosed herein.

(a) Types of bacteria

374. In certain embodiments, the bacteria can be a drug resistant bacterium. For example, drug resistant bacteria include but are not limited to: Methicillin-resistant S. aureus (MRSA), Vancomycin- resistant Enterococci (VRE), Amikacin- and β-lactam-resistant K. pneumoniae, Vancomycin-resistant Staphylococcus aureus (VRSA), Enterococcus, H. influenzae, M. tuberculosis, N. gonorrhoeae, P. falciparum, P. aeruginosa, S. dysenteriae, S. aureus, S. pneumoniae, K. pneumonia, E. coli, Salmonella.

375. In certain embodiments, the bacteria can be an extracellular bacterium. For example, extracellular bacteria include, but are not limited to: N. gonorrhoeae, N. meningitides, H. influenzae type b, Νontypeable H. influenzae, H. ducreyi, B. pertussis, P. aeruginosa, E. coli, V. cholera, H. pylori, T. pallidum, S. pneumoniae, S. aureus, S. pyogenes, S. agalactiae, C. diphtheria, C. tetani, C. perfringens.

4. Methods of Identifying Spi6 Substrates

376. Deficiency in Sρi6 enhances the function of granulocytes. Determination of the physiological relevant protease or proteases inhibited by Spi6 in mouse granulocytes as well as allowing examining the mechanism by which Spi6 affects granulocyte function will shed light on a potently new effector protease in bacterial immunity.

377. Disclosed is a method of identifying target proteases of Spi6. For example, disclosed are methods as set forth in the examples.

378. Also disclosed is a method of purifying protease-Spi6 complexes from granulocytes. For example, disclosed are methods as set forth in the examples. 5:' Method δf Identifying a Gene Regulated by Spib

379. Also, disclosed are methods of identifying a gene regulated by Spi6. For example, disclosed is a method of identifying a gene regulated by Spi6 comprising performing a microarray gene expression analysis of a Spi6 knockout mouse, wherein the gene expression analysis produces a first data set of expressed genes in the Spi6 KO mouse; performing a microarray gene expression analysis of a wild-type mouse, wherein the gene expression analysis produces a second data set of expressed genes in the wild-type mouse; comparing the first data set with the second data set; and identifying the genes in the Spi6 knockout mouse that are expressed differently than the wild-type mouse.

380. In addition, disclosed are methods of identifying a gene regulated by PI9. For example, disclosed is a method of identifying a gene regulated by PI9 comprising performing a microarray gene expression analysis of a PI9 knockout mouse, wherein the gene expression analysis produces a first data set of expressed genes in the PI9 KO mouse; performing a microarray gene expression analysis of a wild-type mouse, wherein the gene expression analysis produces a second data set of expressed genes in the wild-type mouse; comparing the first data set with the second data set; and identifying the genes in the PI9 knockout mouse that are expressed differently than the wild-type mouse.

6. Method of Drug Discovery

381. Identification of new drugs is an important aspect of medical innovation. As discussed above, Sρi6 and PI9 interact and regulate several molecules that are involved in the inflammatory system. Animals and humans alike, that lack native Spi6 or PI9 function can exhibit conditions that are characteristic of a lack of homeostatic regulation of serine proteases. As such, identifying drugs that can compensate for a lack of native Spi6 or PI9 function can be of great use. Provided herein are methods of drug discovery. For example, disclosed is a method of drug discovery comprising administering a candidate drug to the Spi6 KO mouse.

7. Method of Enhancing Immunity 382. The treatment of sepsis by neutralizing inflammatory cytokines (e.g. TNF-α, IL-I) or receptors (e.g. TNFR, TLR) has met with limited success, presumably because these therapies impair bacterial clearance. Targeting Spi6 increases immunity to sepsis causing bacteria without giving inflammatory disease. Thus, inhibition of similar serpins in humans, such as PI9, can lead to alternative strategies to treat sepsis through bacterial clearance by increasing rapid neutrophil function. The increased prevalence of antibiotic resistant bacteria would also increase the need to develop new therapies that rapidly and safely increase neutrophil bactericidal activity during acute infection.

383. Provided herein are methods of enhancing immunity by inhibiting Spi6 expression. Immunity can be immunity to bacterial infection. As discussed above, the bacteria causing the infection can be gram positive or gram negative. In addition, the bacterial infection can be in any tissue, including the lung, liver, blood, peritoneum, or spleen.

384. As described above, the serine protease neutrophil elastase (NE) plays a critical role in the ability of granulocytes to function properly. Increased NE activity resulted in hyperactive neutrophils from Spϊβ-β&fv&ibM'Αiύe^MbΕ^ΨόϊθBted from lethal sepsis through increased clearance of both gram negative and positive bacteria.

385. Also provided are methods of enhancing immunity. For example, provided is a method of enhancing immunity by increasing neutrophil elastase activity comprising administering an inhibitor of Spi6. Further provided is a method of increasing immunity to sepsis causing bacteria without giving inflammatory disease, comprising administering an inhibitor of Spi6. Inhibitors of Spi6 are discussed above. Also provided is a method of increasing neutrophil function comprising administering an inhibitor of Spi6.

386. In addition to the methods described above, provided are combination and cycling therapies to enhance immunity. For example, provided is a method of enhancing immunity by administering an inhibitor of Spi6 in combination with human neutrophil elastase (HNE). Dosages of HNE can be from

0.1U/kg to 3.5U/kg, and as discussed herein.

8. Method of Increasing Bactericidal Activity in Granulocytes

387. Granulocytes both digest extracellular matrix components, as they exit the circulation and migrate to the site of infection, and digest bacteria within the phagolysosome. As such, granulocytes play a critical role the inflammatory reponse process. Provided herein are methods of increasing the bactericidal activity in granulocytes comprising disrupting SPI6 function. Methods of disrupting function are provided above.

9. Methods of Inhibiting Neutrophil Elastase

388. Alternative to the methods described herein related to increasing neutrophil elastase activity, provided herein are methods of inhibiting neutrophil elastase. For example, provided is a method of inhibiting neutrophil elastase comprising administering a vector expressing Spi6. Also provided is a method of inhibiting neutrophil elastase comprising administering an effective amount of SPI6 protein or a fragment thereof.

10. Methods of Treatment 389. Provided herein are methods of treating bacterial infections and sepsis in a subject. For example, provided is a method of treating a bacterial infection in a subject comprising administering to the subject an effective amount of a SPI6 inhibitor.

390. Also provided is a method of treating a bacterial infection in a subject comprising introducing an effective amount of a disrupted Spi6 gene to the subject. 391. Further provided is a method of treating sepsis in a subject comprising administering to the subject an effective amount of a SPI6 inhibitor.

392. Also provided is a method of treating sepsis in a subject comprising introducing an effective amount of a disrupted Spi6 gene to the subject.

393. Serpins such as Spi6 have the propensity to form multimers, which inhibit function. One could introduce a mutant form of Spi6 defective in NE binding (i.e. mutated or deleted RCL) which would catalyze the formation of inactive multimers inhibiting the activity of endogenous protein. As such, provided iS a^" meihWOT'tfeiatmf 1'TOteϊiaϊ infection in a subject comprising administering to the subject an effective amount of a disfunctional SPI6 protein or a fragment thereof, wherein the dysfunctional SPI6 protein or a fragment thereof prevents native SPI6 from inhibiting neutrophil elastase function. a) Method of Treatment with Combination Therapy 394. Provided herein are methods of treating a bacterial infection and sepsis in a subject. For example, provided is a method of treating a bacterial infection in a subject comprising administering to the subject an effective amount if an inhibitor of Spi6 in combination with human neutrophil elastase, and wherein the concentration of human neutrophil elastase is less than 1.8U/kg.

395. Also provided is a method of treating sepsis in a subject comprising administering to the subject an effective amount if an inhibitor of Spi6 in combination with human neutrophil elastase, and wherein the concentration of human neutrophil elastase is less than 1.8U/kg

D. Methods of making the compositions

396. The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.

1. Nucleic acid synthesis

397. For example, the nucleic acids, such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd

Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et ah, Bioconjug. Cheni. 5:3-7 (1994).

2. Peptide synthesis 398. One method of producing the disclosed proteins, such as SEQ ID NO.4 is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposiπg"a'τernunai grbup'Wffiόrπs functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N. Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY (which is herein incorporated by reference at least for material related to peptide synthesis). Alternatively, the peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions.

399. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide— thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97- 101; Clark-Lewis I et al., J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

400. Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).

3. Process claims for making the compositions

401. Disclosed are processes for making the compositions as well as making the intermediates leading to the compositions. For example, disclosed are nucleic acids in SEQ ID NOs: 1 and 3. There are a variety of methods that can be used for making these compositions, such as synthetic chemical methods and standard molecular biology methods. It is understood that the methods of making these and the other disclosed compositions are specifically disclosed.

402. Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid comprising the sequence of Spi6 or PI9 and a sequence controlling the expression of the nucleic acid. 403. Also disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence having 80% identity to a sequence set forth in SEQ ID NO:3, for example, and a sequence controlling the expression of the nucleic acid. 404 Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence that hybridizes under stringent hybridization conditions to a sequence set forth SEQ ID NO 3, for example, and a sequence controlling the expression of the nucleic acid 405 Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide set forth m SEQ ID NO 4, for example, and a sequence controlling an expression of the nucleic acid molecule

406 Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide having 80% identity to a peptide set forth in SEQ ID NO 4, for example, and a sequence controlling an expression of the nucleic acid molecule

407 Disclosed are nucleic acids produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide having 80% identity to a peptide set forth in SEQ ID NO 4, for example, wherein any change from the SEQ ID NO 4, for example, are conservative changes and a sequence controlling an expression of the nucleic acid molecule

408 Disclosed are cells produced by the process of transforming the cell with any of the disclosed nucleic acids Disclosed are cells produced by the process of transforming the cell with any of the non- naturally occurring disclosed nucleic acids

409 Disclosed are any of the disclosed peptides produced by the process of expressing any of the disclosed nucleic acids Disclosed are any of the non-naturally occurring disclosed peptides produced by the process of expressing any of the disclosed nucleic acids Disclosed are any of the disclosed peptides produced by the process of expressing any of the non-naturally disclosed nucleic acids

410 Disclosed are animals produced by the process of transfecting a cell within the annnal with any of the nucleic acid molecules disclosed herein Disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the animal is a mammal Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate

411 Also disclose are animals produced by the process of adding to the animal any of the cells disclosed herein

E. Examples

412 The following examples are put forth so as to provide those of ordinary skill m the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure Efforts have been made to ensure accuracy with respect to numbers (e g , amounts, temperature, etc ), but some errors and deviations should be accounted for Unless indicated otherwise, pans axe parts oy weϊgnt, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1 Spi6 Knock-out mouse a) Methods (1) Vertebrate Animals

413. Animals can be housed in an approved animal care facility. Twenty-four hour-a-day veterinarian and veterinary technician consultants can be available for the time period of the project. Animals can be monitored for well being with the specifics of the animal protocol, which includes twice weekly monitoring. 414. Animals can be anesthetized by the injection of nembutal (30-70mg/kg i.p.) prior to euthanization by CO₂. This method of euthanasia is approved by the Panel on Euthanasia of the American Veterinary Medical Association.

415. Mice can be anesthetized after injection of ketamine (80 mg/kg and xylazine (10 mg/kg)). Animals can then be bled by retro-orbital bleeding. (2) Spiβ-Deficient Mice

416. To address the physiological function of Spi6, Spiό-άeFicϊeτή mice in the C57BL/6 background were generated through ES cell technology. Standard techniques were used to generate a mutant Spiό allele in C57BL/6 ES cells (See Fig. IA). Spiό mutant alleles lack exon 7, which as well as encoding most of Spiό encompasses the functionally essential reactive center loop. To avoid any effects of the G418 resistance gene (neo) on the transcription of genes such as Spil3, which are adjacent to Spiό and may perform similar functions, Cre recombinase was used to excise neo (Sun et al., 1997a). Southern Blot analysis was used to identify two ES cell clones harboring mutant Spiό neo alleles (69 and 389) using 5' and 3 ' probes. A representative example of a clone derived from 389 harboring a mutant SpiόΔneo allele in which neo has been excised. C57BL/6 ES cells harboring ύieSpiό Δneo allele were microinjected into blastocysts from BALB/c mice and the resulting chimeras backcrossed to C57BL/6 mice. Progeny derived from ES cell were identified by black coat color and screened for the mutant SpiόΔneo allele by Southern blots. The two lines of C57BL/6 ES cells harboring mutant Spiό alleles were used to generate Spiό ^+/~ mutant C57BL/6 mice after blastocyst microinjection using standard procedures. Chimeras were backcrossed to C57BL/6 mice and heterozygous Spiό^+/~ mice identified by Southern blot, which were then intercrossed to generate Spiό^'1' homozygous mutant mice.

(3) Recombinant Spiό

417. Recombinant Spi6 was generated in the pET expression system in E.coli as a fusion protein with glutathione transferase (GST), using standard procedures recommended by the manufacturer (Novagen). The GST tag was removed by factor X proteolysis and recombinant (r) Spi6 (43 kD) purified to homogeneity (Fig. 2A). Recombinant Spi6 was incubated with protease at al 0-fold molar excess for varying periods of time then the residual activity measured against labeled peptide substrates (Annand et al., 1999; Coeshott et al., 1999; Liu et al., 2003). (4) Generation of Bone Marrow Derived Dendritic Cells (BMDCs)

418. BMDCs were generated as described previously (Lutz et al., 1999). For example, bone marrow cells (10⁵/ml; 20ml) were cultured in a 10cm dish with GM-CSF for 6 days (200U/ml) then for a further 3 days ( 100U/ml), at which point the cell surface expression of CD lie and I-A^b was examined for evidence of a DC-like phenotype.

(5) Generation of Functional DCs

419. Functional DCs were generated and delivered recipient mice by intrasplenic injection, as described previously (Ludewig et al., 1998; Lutz et al., 1999).

(6) Detection of LCMV- Specific CTLs 420. LCMV-specific CTLs were detected in the spleen 8 days after infection by staining with H-

2D^b-tetramers loaded with the GP 33 peptide antigen from LCMV then flow cytometric analysis (FCM).

421. To examine the role of Spi6 in the homeostasis of CD8 T cells, mice (Mus musculus) (C57BL/6 wild-type, C57BL/6 thyl.l congenic, C57BL/6 Spiό KO) were infected i.p. with LCMV Armstrong (2 xlO⁵ PFU/ml) diluted from high titer frozen stocks in a 100-200μl volume in PBS using a 25- G needle. For secondary infections either mice were infected with either LCMV Armstrong (10⁶

PFU/mouse i.p) or LCMV Armstrong clone 13 (10^δ PFU/mouse i.v.). For i.v. injection, recipient mice were placed in a restrainer and the tail warmed with a heat lamp to allow visualization of the tail vein, then sterilized by washing with 70% ethanol then LCMV injected into the lumen of the tail vein in a 100-200μl volume in PBS using a 25-G needle. The spleen was removed from sacrificed mice to allow the measurement of the number of LCMV-specific CD8 T cells and LCMV using standard plaque assays on

Vero cells.

422. To examine the role of Spi6 in the development of virus-specific CTLs, Sρi6 KO mice were infected with LCMV Armstrong (2x10⁵ PFU/mouse). The level of LCMV-specific CD8 cells in the population of peripheral blood leukocytes (PBLs) was measured by staining with H-2D^b-tetramers loaded with the GP 33 peptide antigen from LCMV then FCM (Murali-Krishna et al., 1998).

(7) Listeria monocytogenes (LM) infection

423. To examine the role of different leukocytes or cytokines in the resistance of mice to LM, C57BL/6 wild-type and C57BL/6 Spi6 KO were infected i.v. with an attenuated strain of I. monocytogenes (strain DBL-1942), diluted from freshly grown cultures in a 100-200μl volume in PBS using a 25-G needle. Titers of LM were determined in homogenates of the spleen and liver after sacrifice. To measure recruitment of macrophages or neutrophils and obtain cells for ex vivo studies mice were infected i.p. (10⁴- 10⁵ CFU/mouse). For survival experiments mice were injected i.v. with LM of the EGD (wild-type) stain (10⁴-10⁶ CFU/mouse). Mice were observed in 12 h intervals for 3 d and those that show signs of acute disease (ruffled fur, hunched posture, immobility, and apparent weight loss) were immediately sacrificed. Samples of hearts blood, liver and spleen can then be taken post-mortem and subjected to histopathological analysis. (a) Examination of Key Indicators [from Enhanced immunity to LM in Spi6-deficient mice below]

424. As such, key indicators were examined to try to understand the increased resistance of Sρ6 KO mice to infection. IFN-γ levels were analyzed. Staining for OVA-peptide CD8 T cells with tetramers. (8) Determination of whether a substrate of Spi6 - granzyme B - is expressed in mouse granulocytes

425. B6 mice were injected i.p. with glycogen as described in Lopez-Boado et al., 2004 and after 4 h granulocytes (Gr-I⁺ CDl Ib⁺) harvested and purified by FACS. Isotype controls were included to ensure that the fluorescence of granulocytes was a result of specific marker-staining and not cross-reactivity with Fcγ receptors. As a positive control, the activity of granzyme B in CTLs was examined. Spleen cells from

P14 transgenic mice were incubated with GP33 peptide (10^"7M) and IL-2 (lOU/ml) for 3 d to give P14 CTLs (>90% P14TCR⁺ CD8⁺). Cells were lysed by sonication in hypotonic buffer (50 mM PIPES, 5OmM KCL, 5mM EGTA, 2mM MgCl₂ 5mM DTT, pH 7.6) then centrifuged at 15, 000 x g for 30 min to give cytosol (supernatant) and organelle (pellet) fractions. The organelle pellet was resuspended in 1% Triton X-100, 1OmM Tris.HCl, 15OmM NaCl, pH 7.6 for 30 min on ice.

426. To control for hydrolysis of the granzyme peptide substrate (Ac-IEPD-pNA) by other proteases (such as caspase 8 (Thornberry et al., 1997) ) the activity in P14 CTLs from granzyme (Grn B) KO P14 transgenic mice was measured (Heusel et al., 1994).

(9) Granulocyte function in Spi6 KO 427. Granulocytes (90% Gr-I⁺ CDl Ib⁺) were elicited in the peritoneum by glycogen injection as described previously (Lopez-Boado et al., 2004). Mice were then injected i.p. with LM (10⁶ CFU).

(lO)Production of Mixed Bone Marrow Chimeras

428. Bone marrow (3x 10⁶) from wild-type C57BL/6 CD45.2 congenic and C57BL/7 Spi6 KO (CD45.1) mice was mixed 1:1 and adoptively transferred into lethally irradiated (1200 rads) C57BL/6 mice. After 6 weeks, glycogen elicited granulocytes (97% Gr-I⁺ CDl Ib⁺) were stained and CD45.1⁺ and CD45.2⁺ cells purified by FACS. Granulocytes (10⁷/ 0.2ml) were mixed with LM (10^δ) and the number of surviving bacteria measured after 2 h.

(ll)Adoptive transfer

429. To examine the homeostasis of CD8 T cells after infection with LCMV, spleen cells from C57BL/6 P14 or C57BL/6 Spi6KO transgenic mice can be adoptively transferred by i.v. injection to

C57BL/6 thyl .1 congenic mice. To examine priming of CD8 T cell responses by dendritic cells (DCs), bone-marrow derived DCs can be adoptively transferred to recipient mice by i.v. injection. For i.v. injection, recipient mice can be placed in a restrainer and the tail warmed with a heat lamp to allow visualization of the tail vein, then the tail can be sterilized by washing with 70% ethanol, and then LCMV can be injected into the lumen of the tail vein in a 100-200μl volume in PBS using a 25-G needle. (12)Antibody depletion

430. To examine the role of different leukocytes or cytokines in the resistance of mice to LM, C57BL/6 wild-type, or C57BL/6 Spi6 KO mice can be injected i.v. with purified mAbs (0.2-1.0 mg) to neutralize cytokines or deplete cells. For i.v. injection, recipient mice can be placed in a restrainer and the tail warmed with a heat lamp to allow visualization of the tail vein, then sterilized by washing with 70% ethanol then LCMV injected into the lumen of the tail vein in a 100-200μl volume in PBS using a 25-G needle.

(13)LIP-CLOD depletion

431. To examine the role of macrophages in the resistance of mice to LM C57BL/6 wild-type, or C57BL/6 Spi6 KO mice can be injected i.v. with preparations of LIP-CLOD (20-80 μg CLOD/mouse; lOOμl/lOg body weight). For i.v. injection, recipient mice can be placed in a restrainer and the tail warmed with a heat lamp to allow visualization of the tail vein, then sterilized by washing with 70% ethanol then LCMV injected into the lumen of the tail vein in a 100-200μl volume in PBS using a 25-G needle.

(14)Sterile recruitment and harvest of leukocytes 432. To examine the effect of Spi6 on the recruitment of leukocytes, C57BL/6 wild-type, or

C57BL/6 Spi6 KO mice can be injected with 3% thioglycollate (0.3ml/10g body weight) to induce sterile peritonitis then the recruitment of macrophages to the peritoneum measured over the course of 6 d. To generate macrophages and neutrophils as a source for in vitro studies, bone-marrow can be obtained from the femur after the sacrifice of C57BL/6 wild-type, or C57BL/6 Sρi6 KO mice. (15)Intrasplenic adoptive transfer

433. To transfer a small number of dendritic cells or NK cells intrasplenic injection after survival surgery can be performed. Mice can be anesthetized after injection of ketamine (80 mg/kg and xylazine (10 mg/kg)). With sterile dissecting scissors, a small incision can be made in the skin then the body wall on the side of the animal. Sterile, blunt forceps can then be used to pull out the spleen, which can be injected with cells 100-200μl volume in PBS using a 25-G needle. Once the injection is complete, the body wall can be sutured with 2 or 3 stitches and the skin closed with wound clips. Mice can then be monitored for 48 h for signs of pain or distress (excessive grooming of wound and cessation of feeding). b) Results

(1) Spi6-deficient mice 434. C57BL/6 Spi6 ^"Λmice were generated from both lines of independently targeted ES cells.

They were born with the expected Mendelian frequency and exhibit no obvious developmental abnormalities. Analysis of lymphocyte populations in naive C57BL/6 Spi6 ^~'^~ mice (referred to as Sρi6 KO mice) revealed that there was no difference in the number of thymocytes or mature T cells, B cells, monocytes/macrophages, granulocytes, DCs or NK cells compared to C57BL/6 controls. (2) Protease specificity of Spi6

435. As a result of recombinant Spi6 being incubated with protease at alO-fold molar excess for g periods of time then the residual activity measured against labeled peptide substrates it was ucLcmmieu uia_L opio imiiuiieu giaiizyme XJ o . icuc — J ΛI U ivj. a _j \r ig. AOJ, HI a raie very ciose to that originally described for yeast rSpiδ (4 xlO⁴ M^"1 s^"x)(Sun et al., 1997b). It was also determined that Spi6 could not inhibit other granzymes, such as granzyme A (grn A) or granzyme K (grn K) nor the lysosomal cathepsins B and L. Although the human homologue, PI9 inhibits caspase 1 (Annand et al., 1999), Spi6 could not. (Fig. 2B). Spiό exhibited minimal inhibition of the granulocyte proteases elastase and PR-3.

However the reaction was too slow for a rate constant to be determined and it very unlikely to be of physiological relevance.

(3) Spi6 is required for protection from granule-mediated PCD

436. Over expression of Spi6 can protect tumor cell lines from PCD induced by CTLs or purified granzyme B (Medema et al., 2001a). To test the physiological relevance of cytoprotection by Spi6 the sensitivity of bone marrow derived dendritic cells (BMDCs) from Spi6 KO mice to PCD induced by CTLs was examined. BMDCs were chosen because the upregulation of Spi6 in BMDCs after treatment with inflammatory stimulators has been proposed to protect from CTL killing (Medema et al., 2001b). After 9 days between 80-90% of cultured cells were CDl 1 C⁺ I-A^b+ (immature). BMDCs were "matured" after an additional day of culture in LPS (0.2μg/ml), as evidenced by the up-regulation of the CDlIc and I-A^b markers. The morphology of the cells was also consistent with that of DCs.

437. Low numbers (10³) of immature DCs that had been pulsed with the GP 33 peptide from LCMV could prime the expansion of antigen-specific CD8 T cells 7 d after injection into the spleens of C57BL/6 mice (See Fig. 3). Thus indicating the ability to make functionally competant DCs and to be able to use intrasplenic injection to induce CD8 T cell expansion by the delivery of very low numbers of cells.

BMDCs from Sρi6 KO mice were susceptible to PCD induced by P14 CTLs after pulsing with GP 33 (See Fig. 4A).

438. Treatment of CTLs with concanamycin A (CMA) inhibits granule-mediated cytolysis, but does not affect Fas-mediated cytotoxicity, and was used to determine the relative contribution of each pathway to the induction of target cell PCD (Kataoka et al., 1996). P14 CTLs treated with CMA did not induce the PCD of gp33-pulsed DCs, indicating that the increased PCD of Spi6 KO BMDCs to PCD was due to increased sensitivity to granule-mediated PCD (Fig. 4B). This conclusion is supported by Fig. 5, where it is shown that BMDCs from Spi6 KO mice are equally susceptible to PCD induced by anti-Fas antibody. Spi6 is a physiologically relevant inhibitor of the granule pathway but not of the Fas pathway of PCD. The increased CTL-induced PCD of Spi6 KO gave rise to increased lysis as measured by ⁵¹Cr-release

(See Fig. 6A). The extent of increased lysis is less than the induction of PCD in Spi6 KO targets because granzyme A, which is not inhibited by Spi6, induces lysis without PCD (Russell and Ley, 2002).

(4) Spi6-deficient CTLs are susceptible to PCD

439. Consistent with a role in protecting CTLs from self-inflicted damage, it has recently been demonstrated that Spi6 is upregulated during the differentiation of naive cells into CTLs and can protect cells from granzyme-mediated PCD (Phillips et al., 2004). To directly test whether Spi6 protects CTLs from PCD the viability of CTLs from Spi6 KO mice after infection with LCMV Armstrong was examined. LCMV-specilic C ill's Were detected in the spleen 8 days after infection by staining with H-2D^b-tetramers loaded with the GP 33 peptide antigen from LCMV then flow cytometric analysis (FCM) (Murali-Krishna et al., 1998). A lower percentage (from 8.3% in the B6 control and 1.6% in the Spi KO) and absolute number (Fig. 6B) of LCMV-specific CD8 T cells in Spi6 KO mice was observed. When splenocytes were counterstained with the YOPRO-I dye, which measures the onset of apoptosis by detecting condensation of nuclear DNA (Idziorek et al., 1995), an increased number of apoptotic LCMV-specific CD8 cells from Spi6 KO mice was observed (See Fig. 7). Cytoprotection by Spi6 is physiologically relevant mechanism that ensures the viability and survival of CTLs in vivo.

(5) Altered kinetics of anti-LCMV CD8 development in Spi6-deficient mice

440. To examine the role of Sρi6 in the development of virus-specific CTLs, Spi6 KO mice were infected with LCMV Armstrong (2x10⁵ PFU/mouse). The level of LCMV-specific CD8 cells in the population of peripheral blood leukocytes (PBLs) was measured by staining with H-2D^b-tetramers loaded with the GP 33 peptide antigen from LCMV then FCM (Murali-Krishna et al., 1998). In wild-type B6 controls an expansion in the number of LCMV-specific CD8 T cells to a peak after about 8 d, then a contraction over the next 2 weeks to a number in the memory phase that was stable for up to 140 days was observed (See Fig. 8).

441. However the kinetics of LCMV-specific CD8 T cells expansion and contraction was dramatically different in Spi6 KO mice. In mutant mice the expansion of LCMV-specific CD8 T cells was significantly impaired, as evidenced by a 5-fold decrease in cell number after d 8 (See Fig. 8). This is consistent with lower recovery of LCMV-specific CD8 T cells from the spleen (See Figs. 6B and C). In Sρi6 KO mice from the peak of the response the number of LCMV-specific CD8 T cells then remained constant for up until 140 days after infection. Thus, because the absence of a contraction phase in Spi6 KO mice, the level of LCMV-specific CD8 T cells during the memory phase was the same as in controls. (6) Impaired activated NK cell survival from Spi6 KO mice

442. NK cells provide immediate immunity through the lysis of tumor cells or infected cells and through the production of cytokines, notably IFN-γ. Although they are important mediators of innate immunity they use the same granzyme B/perforin effector mechanism as CTLs do to kill target cells. Given the role of Spi6 in protecting CTLs from granzyme B -induced PCD, NK cells in Sρi6 KO mice were examined.

443. In ex vivo assays, significantly lower NK cell activity against ⁵¹Cr-labeled tumor cells from the spleen of Spi6 KO mice compared to B6 controls was observed (See Fig. 10A). This was not due a lower number of NK cells because the number of DX5⁺ NK cells in the spleens of SpiόKO mice was unaffected. Rather, impaired cytolytic function correlated with an increase in the proportion of NK cells undergoing PCD during the killing assay (See Fig. 10B). Therefore in the absence of the granzyme B inhibitor Spi6, as for CTLs (See Fig. 7), activated NK cells are also susceptible to PCD. (7) Enhanced immunity to LM in Spi6-deficient mice

444. The host response against Listeria is characterized by the complex interplay between the innate and adaptive components of the immune system (Harty et al., 1996). Innate effectors such as granulocytes, macrophages and NK cells play a crucial role in control of bacterial growth during the initial stages of the infection, especially in the liver (Conlan and North, 1991; Conlan and North, 1994; Dunn and

North, 1991; Portnoy, 1992). To examine the role of Sρi6 immunity to bacteria, Spi6 KO mice were infected with the attenuated LM strain DPL-1942, (Brundage et al., 1993), which has been engineered to express the SIINFEKL peptide epitope from ovalbumin (OVA)presented by H-2K^b (Pope et al., 200I)(IO⁵ CFU/mouse). A substantial decrease in bacterial load in Sρi6 KO mice was observed (See Fig. 11). The highest level of control of LM occurred in the liver of Spi6 KO mice, reducing the load at the peak of bacterial growth on d 3 by about 100-fold compared to wild-type B6 mice.

445. It is well established that the clearance of LM is afforded by ThI -driven cellular immunity with early production of IFN-γ by NK cells, then later T cells, driving macrophage and CTL clearance of LM (Harty et al., 1996). As such, key indicators were examined to try to understand the increased resistance of Sρi6 KO mice to infection. It was observed that the serum level of IFN-γ was reduced in Spi6

KO mice (Fig. 12). At the peak of infection on d 3 there was no difference in the number macrophages in the spleen and the number of CD4 T cells was actually lower in Sρi6 KO mice (See Fig. 13). As observed with LCMV infection, the number of LM-specific CD8 T cells, as evidenced by staining for OVA-peptide CD8 T cells with tetramers, was lower in Spi6 KO mice (See Fig. 14). The increased clearance of LM did correlate with increased numbers of granulocytic (CDl Ib⁺ Gr-I⁺) myeloid cells in Spi6 KO mice.

Therefore, the 100-fold increase in LM protection afforded by Spi6 deficiency is not due to an increase in ThI -driven cellular immunity.

446. The decrease in IFN-γ levels may well be due to impaired survival of NK cells and CTLs (See Fig. 10). Increased clearance of LM did however correlate with increased number of granulocytic (CDl Ib⁺ Gr-I⁺) myeloid cells in Spi6 KO mice .This indicates that increased antimicrobial activity of granulocytes can play a role in the enhanced immunity of Spi6 KO mice to LM.

(8) Granulocytes do not express granzyme B

447. To examine the role of Spi6 in granulocyte function it was determined whether a substrate of Spi6 - granzyme B - is expressed in mouse granulocytes. The greatest granzyme B activity was detected in the organelle fraction. High activity was also observed in the cytosol (See Fig. 15). This may indicate that in fully activated CTLs the proportion of granzyme B outside of the granule maybe greater than is currently appreciated. Specific activity in Grn B KO cells was minimal. The specific activity of caspase 3 was greatest in the cytosol and was lower in Grn B KO cells. Thus, implying that granzyme B induces the PCD of CTLs, at least in vitro. In contrast there was no specific granzyme B activity in glycogen-elicited granulocytes, in either organelle or cytosolic fractions (See Fig. 16). The specific activity of PR-3 was significant in both fractions, presumably reflecting that fact that it is widely distributed throughout granulocytes (Rao et al., 1991). In contrast to the findings of investigators with human granulocytes (Hocneggefef'ai:, 2ϋϋ4; wagner et al., 2004), this study indicates mouse granulocytes do not express granzyme B.

(9) Increased bactericidal activity of Spi6 KO granulocytes

448. Granulocyte function in Spi6 KO mice was also examined. A dramatic increase in the uptake of LM in Spi6 KO granulocytes as soon as 5 min after injection was observed (See Fig. 17A). This correlated with an increase in the killing of LM by Spi6 KO granulocytes, which was measured after 2 h (See Fig. 17B). These data indicate that Spi6 KO granulocytes have increased bactericidal activity.

449. To examine the level at which Spi6-deficiency affects granulocyte function mixed bone marrow chimera experiments were performed. Compared to wild-type, significantly more killing of LM by Spi6 KO granulocytes (CD45.1⁺) was observed compared to B6 granulocytes (CD45.2⁺). Therefore Spi6 does not affect granulocyte activity through non-granulocyte leukocytes. When the experiment was repeated in Spi6 KO recipients, an increase in B6 granulocyte killing compared to Spi6 KO cells was not observed, ruling out the possibility that Sρi6 affects granulocyte activity indirectly through non- hematopoetic stromal cells. Therefore, Sρi6-deficiency affects granulocyte activity directly in a cell autonomous manner.

(10)Spi6's protective effect on CTLs from PCD in vivo

450. The development of purified anti-LCMV CD8 T cells from Spi6 KO mice after adoptive transfer can be examined by using Spi6 KO mice that have been crossed with C57BL/6 P14 TCR (Vα2, Vβδ.l) (Pircher et al., 1990) mice to generate Spiό ^+/+ PU ^+/" (B6 P14 mice) and Spiό ^''^'PU ^+/" (Spi6 KO P14 mice) in the C57BL/6 background. CD8 T cells can be purified from the spleens of B6 P14 and Spi6

KO P14 mice (both of which are thyl.2⁺, thyl.l^") by magnetic bead sorting (about 97% pure). In each batch of CD8 T cells the frequency of P14 CD8 T cells can be determined (typically 10-20% of CD8 T cells) and can adoptively equal numbers (i.v. injection) of P14 CD8 T cells (10⁵ P14 CD8 cells typically in a population of 1-2 x 10⁶ CDS T cells) from B6 P14 and Sρi6 KO P14 mice to female thyl.2^", thyl.l⁺ C57BL/6 mice. After 5 days, recipients can be infected with LCMV (2 x 10⁵PFU/mouse i.p.). At the peak of the response on d 8, LCMV-specific donor cells can be identified by staining with anti-thyl.2, anti-CD8 antibodies and gp33/H-2D^b tetramers then three color FCM on PBLs and spleen cells. This allows for a comparison of the number of LCMV-specific CD8 T cells from B6 and Spiό KO mice at the peak of the response to LCMV. To measure the proportion of donor LCMV-specific CTLs undergoing PCD on d 8, the donor LCMV-specific CTLs can be stained as before, but the YOPRO-I DNA-binding dye can be included.

Following the staining four color FCM can be performed. (Idziorek et al., 1995)(Fig. 7). The percentage of YOPRO-I⁺ cells of the thyl.l⁺, GP33⁺ CD8 T population can be compared to allow for a determination of whether Spiό protects CD8 T cells from PCD in a cell autonomous fashion. (ϊl)Granzyme B deficiency can rescue the clonal burst defect in Spi6 KO mice

(a) Physiological relevance ofgranzyme B.

451. One can examine whether granzyme B deficiency can rescue the clonal burst defect in Spi6 KO mice. C57BL/6 granzyme B KO (Gm B KO) mice (Heusel et al., 1994; Phillips et al., 2004 ), can be crossed to generate Spi6 KO P14 mice. CD8 T cells can then be purified from the spleens of Grn B KO Spi6 KO P14 mice and adoptively transferred to C57BL/6 thyl.l⁺ mice then infected with LCMV.

(b) The effect ofgranzyme B deficiency on PCD

452. Sρi6 is a physiologically relevant inhibitor of "misdirected" granzyme B in CTLs, and this can be confirmed by measuring the specific activity ofgranzyme B in the cytosol of P14 CTLs from wild- type and Spi6 KO mice. For example, cells can be lysed (60 min, on ice) in hypotonic buffer (5OmM PIPES, pH 7.6, 5OmM KCl, 5mM EGTA, 2mM MgCl₂, 5mM DTT) then the organelle pellet and cytosol supernatant recovered by centrifugation at 15,000 x g for 10 min. The organelle pellet can then be resuspended in 1% Triton X-IOO, 1OmM Tris.HCl, pH 7.6, 15OmM NaCl for 60 min on ice before enzyme assay. The activity in LCMV-specific CTLs from Grn B KO mice can be measured to control for substrate

(Ac-ITEP-^NA) hydrolysis from other proteases (Fig. 15).

(12)Role of Spi6 on priming anti-LCMV CD8 T cells by DCs

453. Immature BMDCs from B6 and Spi6 KO mice can be pulsed with GP 33 and adoptively transferred by intrasplenic injection (range 10³-10⁵ cells/mouse) to wild-type B6 responder mice (Fig. 3). After 7 d the number of LCMV-specific CD8 T cells can be determined in PBLs and the spleen by staining with GP33 tetramers and anti-CD8 antibody. Intrasplenic injection of un-pulsed BMDCs can be used as a negative control.

454. A role for Spi6 for DC priming, can also be determined by measuring the levels of BrdU incorportion using standard procedures. As described previously (Phillips et al., 2004), mice can be given BrdU in their drinking water (0.8 mg/ml) for 1 wk then 10⁶ splenocytes can be surface stained with GP33 tetramer and anti-CD8 and intracellularly stained with anti-BrdU or isotype control. The percentage of GP33 tetramer⁺ CD8⁺ BMU⁺ cells can be used as a read out for the number of LCMV-specific CD8 T cells proliferating in the spleens of Spi6 KO and B6 mice.

455. To determine if Spi6 protects DCs in priming secondary responses, wild-type B6 mice can be infected with LCMV then after 80 d primed with GP33-pulsed BMDCs from either B6 or Spi6 KO mice.

The expansion of secondary GP33-specific specific CD8 T cells can be monitored in the spleen and PBLs after 5 d with GP33 tetramers and anti-CD8 antibody.

(13)Requirement of Spi6 for licensing of DCs

456. Spi6 KO and control mice can be infected with LCMV then after 3-5 d CD8α⁺ DCs can be purified (BeIz et al., 2004). For example, cell suspensions can be generated from the spleen by protease digestion and non-DC cells depleted by magnetic bead sorting (Myenthi Biotech) with the following antibόdi'es:"anti-CD3;ⁱanH-tKyl.l, anti-CD19, anti-Gr-1, and anti-Ter-199. CD8α DCs (CD8α⁺ CD45RA^") can then be purified by antibody staining and FACS.

457. The ability of GP33-pulsed CD8α DCs to induce the proliferation of naϊve P14 CD8 T cells can also be examined in vitro. For these studies P14 mice can be crossed to perforin KO (Pm KO) mice (Jackson Laboratory, Bar Harbor, ME) to generate Pm KO P14 CD8 T cell responders.

458. This avoids any effects of the increase susceptibility of Spi6 KO CD8α DCs to granule- mediated PCD (Fig. 4). For example, CD8 T cells can be purified from the spleens of Pm KO P14 mice by magnetic bead sorting, labeled with CFSE (5xlO⁴) incubated with FACS-purified CD8α DCs (10⁴) in V- bottom 96 well plates. After 5 d, cultures can be analyzed for proliferation by FCM. The dose of GP33 used for DC pulsing can be titrated (10^~9-10^~s M) to improve the ability to resolve any differences in priming efficiency between control and Sρi6 KO CD8α DC.

(14)Requirement of IFN-γ for licensing of DC

459. To determine whether IFN-γ is required to the licensing of DCs in Spi6 KO mice, C57BL/6 IFN-γ KO mice (Jackson Laboratory, Bar Harbor, ME) can be infected with LCMV and CD8α DCs can be purified and tested for their ability to induce the proliferation of P14 CD8 T cells.

(15) Cellular defect responsible for reduced IFN-γ.

460. NK cells can be depleted from C57BL/6 mice by i.v. injection of anti-NK 1.1 niAb (300μg/mouse)( PK136 mAb ,eBiosource) which can be verified by staining with anti-NKl.l and anti-DX5 mAb and FCM (Brown et al., 2001). Depleted mice can be reconstituted with NKLl⁺ cells purified from either Spi6 KO or control mice. Spleen cell suspensions can be depleted of non-NK cells by magnetic bead sorting then positively-bead sorted for NKLl⁺ cells. Intrasplenic injection can be used to deliver relatively small numbers of cells to the site of CD8 T cell priming and expansion (10³-10⁵ per mouse).

461. To determine whether the defect in CD8 T cells survival is responsible for diminished IFN-γ production and priming, P14 CD8 T cells from P14 Spi6 KO or P14 control mice (10³-10⁴) can be adoptively transferred to Sρi6 KO recipients, which can then be infected with LCMV. IFN-γ levels can then be determined in the serum by ELISA (For example, see Fig. 9). Alternatively, other subsets of DCs such as conventional DCs (CD45RA^" CD8α^") or plasmacytoid DCs (CD45RA⁺ CDSa⁺) (BeIz et al., 2004) can be used to prime P14 CD8 T cells after LCMV infection. .

(16)Role of IFN-γ in the contraction phase of LCMV-specifϊc CD8 T cells.

462. Spi6 KO and wild-type B6 mice can be infected with LCMV and then injected with CpG oligonucleotide (ODN) 1826 (25-75 μg/mouse, i.p.), to induce the production of IL-12, IFN-γ and type I interferon (Krieg, 2003; Takeda et al., 2003). The increase in serum levels of IFN-γ can be verified by ELISA and the contraction in the numbers of LCMV-specific CD8 T cells can also be measured. To confirm that the effect of CpG ODN 1826 is due to IFN-γ, B6 and Spi6 KO mice can be crossed with IFN-γ

KO mice. If the effects of CpG ODN 1826 on the contraction phase are nullified in the IFN-γ KO 'backgrόiMi!; others (Badovinac et al., 2004), one can conclude that IFN-γ is the agent through which it affects CD8 T cell number contraction. If reduced levels of IFN-γ are responsible for the absence of LCMV-specific CD8 T cell number contraction one can expect injection with CpG ODN 1826 will restore the contraction phase. One can also measure the proportion of LCMV tetramef^1" CD8⁺ cells undergoing PCD can be measured by staining with YOPRO-I, which identifies cells at the early stage of apoptosis by measuring DNA condensation (Fig. 7) (Idziorek et al., 1995). This can allow one to not only examine the effect of CpG ODN 1826 on cell number but also on the induction of PCD during the contraction phase.

463. Whether the reduced levels of IFN-γ in Spi6 KO mice gives rise to an increase in the frequency of memory-precursors can be tested by measuring the expression of the IL-7R, which is a marker for memory CD8 T cell-precursors (Badovinac et al., 2004; Kaech et al., 2004; Liu et al., 2004; Madakamutil et al., 2004) , during the contraction phase (about d8-22). The expression of the IL-7R can be determined by staining with antibodies specific for IL-7R on GP33-specific CD8⁺ cells and FCM. The effect of reduced levels of IFN-γ in Spi6 KO mice on the frequency of memory-precursors can be tested can also be determined by injecting Spi6 KO mice with CpG ODN 1826 followed by observing the frequency of

IL-7R⁺ GP33-specifϊc CD8 T cells. If the increase in memory-cell precursors in Spi6 KO mice is due to rescued levels of IFN-γ, then one can expect that injection with CpG ODN 1826 will reduce the frequency of IL-7⁺ memory-cell precursors.

(17)Role of homeostatic proliferation of CD8 T cells in Spi6 KO mice 464. Spi6 KO and B6 mice can be infected with varying doses of LCMV. The expansion and contraction of LCMV-specific CD8 T cells in Sρi6 KO and B6 mice can then be measured. One expects the clonal burst size in both animals to vary with the dose of LCMV Armstrong (2xl0⁵-2xl0⁶ PFU/mouse). In wild-type B6 mice one expects to observe a contraction phase irrespective of the size of the effector pool, such that 90-95% of cells are eliminated before the memory phase. In Spi6 KO mice it is known that at 2x10⁵ PFU there is no contraction phase. If when the number of effector cells increase one observes that increased expansion of effectors at high doses of LCMV results in a contraction phase one will conclude that homeostatic proliferation in Spi6 KO mice is responsible for the absence of contraction at lower levels of effector expansion. Conversely, the absence of a contraction phase even with an increased effector pool can indicate that homeostatic proliferation is unlikely to explain the absence of contraction in Spi6 KO mice.

465. In addition, BrdU labeling as disclosed herein can be used to determine if there is an increase proliferation of LCMV-specific CD8 T from d 8 until d 22 in Spi6 KO mice

466. Another method to restore IFN-γ to wild-type levels during the contraction phase by CpG ODN 1826 injection can be achieved by adoptively transfering wild-type NKl . I⁺ cells by intrasplenic injection.

467. Ex vivo assays can also be used measure the amount of LCMV antigen available to CD8 T cells in Spi6 KO mice (Badovinac et al., 2002). For example, about 3 x 10⁴ P14 CD8 T cells labeled with CFSE''c'an't)fe'ltii'kea"Wltli"abOm 3x10⁶ splenocytes from LCMV infected mice. Cells can then be incubated for 12 h and for the last 6 h in the presence of brefeldin A. CFSE-labeled cells can be analyzed by intracellular staining for IFN-γ then FCM. P14 CD8 T cells incubated with naϊve splenocytes in the presence or absence of antigen peptide could serve as negative and positive controls respectively. The percentage of IFN-γ⁺ P14 CD8 T cells can be used as a read out for the level of LCMV antigen in the spleens of Spi6 KO and B6 mice.

468. LCMV-specific CD8 T cells can also be purified from Spi6 KO and B6 mice by FACS (>98% purity) and adoptively transfered to antigen-free B6 mice.

(18)Ex vivo IFN-γ production 469. Spi6 KO mice can be infected with LCMV Armstrong (2xlO⁵ PFU/mouse, i.p.) and the number of memory CD8 T cells can be measured in the spleen after 80 d. The number of memory CD8 T cells can be determined by quantitating the number of CD8 T cells that produce intracellular IFN-yafter ex vivo stimulation with LCMV peptide antigen (Murali-Krishna et al., 1998). Spleen cells (10⁶/0.2ml/well) can be incubated with either of 3 LCMV immunodominant H-2D^b-restricted antigen peptides (10^"7M), NP 396 [FQPQNGQFI], GP33 [KAVYNFATM] and GP 276[SGVENPGGYCL] and IL-2 (lOU/ml) and after 5 h cells can be stained with antibodies specific for intracellular IFN-γ and surface CD8 then FCM. The level of background staining can be determined by staining with an anti-rat IgGi isotype control for the anti-IFN-γ -PE antibody. In parallel, spleen cells (10⁶/0.2ml/well) can be stained with H-2D^b-tetramer loaded with each of the three LCMV peptide antigens. LCMV-specific CD8 T cells from Spi6 KO memory mice can be examined for their relative functional equivalency as described previously herein.

(19)Recall expansion

470. B6 and Spi6 KO mice can be infected with LCMV as above then after 80 d the mice can be reinfected with LCMV Armstrong at a higher dose (10⁶ PFU/mouse i.p.). After 5 d, the secondary CTL response to LCMV can be examined in the spleen by measuring the number of CD8 T cells specific for LCMV peptide antigens. This can be done by measuring the number of IFN-γ ⁺ CD8⁺ cells generated after 5 hours of ex vivo stimulation with LCMV peptide antigen.

(20)Clearance of LCMV clone 13

471. Unlike the parental Armstrong strain of LCMV, the clone 13 variant cannot be cleared by naϊve B6 mice (Ahmed et al., 1984; Matloubian et al., 1993). However memory B6 mice are resistant to infection with clone 13 because they harbor memory LCMV-specific CD8 T cells, which mount a vigorous recall response that clears the virus (Lau et al., 1994).

472. The functional competence of the CD8 T cell memory in Spi6 KO mice can be tested by examining their resistance to infection by clone 13. B6 and Spi6 KO mice can be infected with LCMV Armstrong as above. After 80 d, B6 and Spi6 KO mice can be reinfected with LCMV clone 13 (10⁶PFU/mouse i.v.). After 8 d, the titer of LCMV clone 13 can be determined in the spleen using standard plaque assays on Vero cells (Ahmed et al., 1984). For memory B6 and Spi6 KO mice the relative "" ' veness of LCMV-memory CD8 T cells can be indicated by the clearance of clone 13. Direct infection ol wrurriatve' dB6 anfl ¹SpTO' EiV mice with clone 13 allows one to set the upper limit of LCMV titer in each case, from which memory-specific clearance can then be determined. More sensitive real-time PCR assay can also be used to measure LCMV clone 13.

(21)Examine the cell responsible for increased immunity to LM in Spi6 KO mice

473. One can examine which leukocyte population is responsible for the dramatic increase in LM clearance in Sρi6 KO mice (Fig. 11). It is unlikely that NK cells and CTLs contribute to the increased resistance because the survival of both cell types is impaired in Spi6 KO mice (Figs. 6 and 10). Furthermore, the decrease in IFN-γ also argues against a role for CD4 ThI cells in the increased immunity (Fig. 12). Therefore granulocytes and macrophages because these cell types are important in containing LM early on are likely candidates.

(22)Effect of Neutrophils on Clearance of LM from Spi6 KO

474. Gr-I depletion studies can be performed with the RB6-8C5 mAb (eBioscience) to directly examine whether neutrophils (Gr-I⁺ CDlIb⁺) are responsible for the increased clearance of LM from Spi6 KO (Conlan and North, 1991). Mice can be injected with RB6-8C5 (200ug/mouse i.v.) 2 and 4 days before infection with sub-lethal doses of attenuated LM (DPL-1942) (105 -106CFU/mouse) {Brundage, 1993 #136; Conlan and North, 1994; Czuprynski et al., 1994). Depletion of granulocytic myeloid cells can be verified by staining spleen cells with anti- Gr-I and CDl Ib then FCM. From d 1- 5 after infection the titer of LM can be determined in the spleen and liver by plating 10-fold serial dilutions of organ homogenates on Trypticase-soy agar. Bacterial colonies can then be counted after incubation at 37° C for 24 hours (Fig. 11).

One expects that RB6-8C5 treatment of B6 control mice will increase the titer of LM. If increased neutrophilic clearance is responsible for the decreased titers of LM in Spi6 KO mice then one expects the titer of LM to increase to the level of RB6-8C5 treated B6 controls.

475. Depletion studies can also be used to examine the role of macrophages in the immunity of SpiόKO mice to LM. Macrophages can be selectively depleted by liposome-encapsulated clondrate (Lip-

CLOD) induced apoptosis (Van Rooijen, 1989) as described previously . Lip-CLOD-depletion has been broadly used in different experimental models to investigate splenic and hepatic macrophage function (Palermo et al., 1999; Van Rooijen and Sanders, 1994). Liposomes containing clodronate (dichlorormethylene) can be prepared using standard procedures and i.v. injected into mice (20-80 μg CLOD/mouse; lOOμl/lOg body weight)(Van Rooijen and Sanders, 1994; van Rooijen et al., 1996).

476. The selective depletion of macrophages by LIP-CLOD, can be verified by staining splenocytes for F4/80⁺ CDl Ib⁺ (macrophages) and control Gr-I⁺ CDl Ib⁺ cells (granulocytes) 4 d after injection and FCM. Depletion in the liver can be verified by immunohistological analysis. Mice can then be infected with LM and clearance can be measured in SpiόKO and control B6 animals. 477. One expects that LIP-CLOD treatment of B6 control mice will increase the titer of LM. If increased macrophage clearance is responsible for the decreased titers of LM in Sρi6 KO mice then one expects tne'titer oi uavrto increase to the level of LIP-CLOD treated B6 controls. These experiments will allow determination of whether the increased clearance of LM is attributable to macrophages.

478. Since the quality of preparations of LIP-CLOD is critical for the successful depletion of macrophages (Van Rooijen and Sanders, 1994; van Rooijen et al., 1996), quality can be ensured, clodronate liposomes and control liposomes (10ml, enough for 50 mice) can be obtained (from Dr. N. van

Rooijen of the Free University in the Netherlands (www. ClodronateLiposomes.org)).

479. In addition the scope of investigation can be widened to address the role of both NK cells. To do so NK 1.1 depletion studies with the PK136 mAb (eBioscience) can be performed to determine whether NK cells (NKLl⁺) are responsible for the increased clearance of LM from Sρi6 KO. (Brown et al., 2001).

(23)Effect of increased immunity to LM on pathology of Spi6 KO mice

480. To determine the effect of increased inflammation in Spi6 KO mice on the survival of LM infected Spi6 KO mice lethal dose 50 (LD50) analysis of Spi6 KO and B6 control mice infected with the widely used wild-type EGD strain of LM (Darji et al., 2003) can be performed. LM EGD can be grown from fresh dilutions of overnight cultures washed into PBS and then injected into the tail vein of B6 and

Spi6 KO mice over a range of doses (3 x 10³ - 10⁶ CFU) with at least 5 mice for each dose. Mice can be observed in 12 hour intervals for 3 days and those that show signs of acute disease (ruffled fur, hunched posture, immobility, and apparent weight loss) can be immediately sacrificed. After initial percentage survival is determined over a range of LM doses, additional experiments can be performed with a narrower range of bacterial doses to more accurately determine LD50 values for Spi6 KO mice. The Dunnet procedure for all possible pairwise contrasts of Spi6 KO and B6 mean percentage can be used to determine statistical significance (Neighbors et al., 2001). An increased LD50 for Spi6 KO compared to B6 mice would be indicative of an increased survival after LM infection. Samples of liver and spleen can be taken post-mortem. 481. The spleens and livers of Spi6 KO mice can be examined for signs of pathology after infection with LM EGD. Standard histochemical analysis of H & E spleen and liver sections can be performed to compare the amount of necrotic tissue damage after infection of B6 wild-type and Sρi6 KO mice. These studies can be followed by TUNEL assays on tissue sections or cells suspensions to measure the proportion of splenocytes or hepatocytes undergoing PCD (Koniaris et al., 2003). Serum levels of liver enzymes such as transaminases can be measured as an additional measure of hepatocyte damage (Koniaris et al., 2003). Since one could not detect any obvious difference in the number of granulocyte (Gr-I⁺ CDlIb⁺) subsets in the bone-marrow, blood or spleen (neutrophils, eosinophils, basophils) before or after LM infection, Spi6 does not inhibit any known protease specific to granulocytes (Fig. 2B) and mouse granulocytes do not express granzyme B, which is the only known substrate of Spi6 (Fig. 16). (24) Effect of Spi6 on sterile granulocyte recruitment

482. The effect of Spi6 on sterile granulocyte recruitment can be examined as described previously (Lopez-Boado et al., 2004). For example, Spi6 KO mice can be injected i.p with 15% glycogen " ""^•• UtniZffiOUsey ffiα ffi&'ttiffiOerorUiJl lt> ur-l ceils (typically, >yi% ot total cells) recruited to the peritoneum can be measured after 4 h.

483. This will allow one to focus on the cell autonomous effects of Spi6 and avoid the effect of differences in inflammatory cytokines that occur (Fig. 12) after the infection of Spi6 KO mice with LM. An increase in recruitment of granulocytes in Spi6 KO mice will imply that this activity at least in part accounts for increased bactericidal function. In addition it will suggest that the protease target inhibited by Spi6, which one would predict to have increased activity in Spi6 KO cells, facilitates recruitment by digesting extracellular matrix components, as has been observed for neutrophil elastase (Belaaouaj, et al., 1998).

484. Bone-marrow can be harvested into Hank's balanced salt solution containing 1% bovine serum albumin (BSA) and neutrophils can be purified (75-85% Gr-I⁺ CDl Ib⁺) on a discontinuous Ficoll gradient (Histopaque 1119; Sigma)(MacIvor et al., 1999). The effect of Sρi6 on phagoctosis by granulocytes can be directly examined as described previously (Maclvor et al., 1999). The phagocytic uptake of 0.9μm-diameter FITC-labeled latex beads (Poly Sciences, Inc) by bone marrow derived granulocytes from Spi6 KO mice can be measured by FCM as described previously (Maclvor et al., 1999). To directly measure bactericidal function, neutrophils from Sρi6 KO mice can be isolated (10⁶) and examined in vitro for the killing of mid-log-phase LM (10⁷) after 30 min (Belaaouaj et al., 1998). LM can be titered by plating on Trypticase-soy agar and bacterial colonies counted after incubation at 37° C for 24 h.

(25)ADCC function 485. ADCC function of granulocytes from Spi6 KO mice can be measured using classical hemolysis assays on sheep red blood cells (sRBCs) (Gagnon and Joshua, 1980). For example, sRBCs can be labeled with ⁵¹Cr (3 x 10⁴) and then incubated with mouse-anti-sRBC mAb (IgG_2a clone: UN-2 (Diamond et al., 1979) (ATTC). ADDC activity of purified macrophages or granulocytes can then be determined after 4 h by measuring the release Of⁵¹Cr. (26)Role of IgE-dependent ADCC of eosinophils in the increased clearance of LM from Spi6 KO mice

486. Spi6 KO Fc epsilon RI KO mice (Dombrowicz et al., 1993) (Jackson Laboratory, Bar Harbor, ME) can be generated and the clearance of LM can be compared with Spi6 KO mice as described previously (Takai et al., 1994).The UN-2 mouse-anti-sRBC mAb was selected because antibodies of this isorype are preferentially bound by Fcγ receptors expressed by neutrophils and macrophages in the mouse (Diamond et al., 1979). One can optimize the concentration of mouse-anti-sRBC mAb needed to achieve good ADDC activity and can control for non-specific lysis in the absence of effectors and anti-sRBC mAb. An increase in ADCC activity can be interpreted as evidence for Spi6 controlling ADCC function of myeloid cells through the inhibition of a target protease. 487. Eosinophils mediate a special type of ADCC, which could conceivably be responsible for increased ADCC of granulocytes from Spi6 KO mice. Aspects focusing on IgG-dependent ADCC of neutrophils could miss this activity. To address this one can perform a series of alternative experiments. For exampierone can IgE-dependent ADCC of eosinophils is responsible for the increased clearance of LM from Spi6 KO mice. Spi6 KO Fc epsilon RI KO mice (Dombrowicz et al., 1993) (Jackson Laboratory, Bar Harbor, ME) can be generated and the clearance of LM compared with Spi6 KO mice. If one observes that deficiency in FcεRl diminishes the clearance of LM one can conclude that eosinophils are responsible for the increased clearance of LM in Spi6 KO mice.

(27)Effect of Spi6 on macrophage activity and function

488. Mice can be injected with 3% thioglycollate (0.3ml/10g body weight) to induce sterile peritonitis then the recruitment of macrophages to the peritoneum can be measured over the course of 4 d by staining for F4/80⁺ CDl Ib⁺ cells and FCM (Tkalcevic et al., 2000). 489. For macrophages, bone-marrow can be plated overnight in DMEM-10% fetal calf serum

(FCS) then non-adherent cells can be cultured in 30% L929 conditioned medium for 5 d (90-95% F4/80⁺ CDl lb⁺)(Coligan et al., 1995). Phagocytic uptake of 0.9μm-diameter FITC-labeled latex beads (Poly Sciences, Inc) from Spi6 KO mice can then be measured by FCM.

490. For bactericidal function, macrophages (10⁶) can be examined in vitro for the killing of mid- log-phase LM (10⁷) after 30 min and the production of bactericidal superoxide can be measured using standard procedures (Maclvor et al., 1999).

491. For ADCC function, assays can be conducted on Sρi6 KO macrophages as described elsewhere herein for granulocytes.

(28)Identifying target proteases of Spi6 in granulocytes (a) Purification of protease-Spiό complexes from granulocytes.

492. Glycogen-elicited granulocytes can be harvested (10⁷ /mouse) from the peritoneum of C57BL/6 mice (99% Gr-I⁺ CDl Ib⁺). For example, B6 mice were injected i.p. with 15% glycogen (ImL) then after 4 h cells harvested and stained anti-CDl Ib-PE and Gr-I-APC mAbs and purified by FACS. The % PE⁺ APC⁺ after FACS was 1%. Staining before FACS with isotype control (IC) mAbs revealed a 99% PE⁺ APC⁺ after FACS.

493. Granulocytes can be resuspended in extraction buffer (1% Triton X-IOO, 1OmM Tris.HCl, pH 7.6, 15OmM NaCl, 1OmM DTT) by incubation on ice for 30 min (Fig. 16; 10⁶-2xl0⁶ cells; 50-100μg) and added to GST-Sρi6-coupled agarose beads (Sigma-Aldrich; Fig. 2A) or GST-coupled agarose beads (5- lOμl; 15-30μg coupled protein) for 90 min (250μl) at RT while rotating. Beads can be centrifuged and washed 4-6 times in extraction buffer then resuspended and boiled in SDS-PAGE loading buffer (10% glycerol, 2 % SDS, 10OmM DTT, 5OmM Tris.HCl pH 6.8, 0.1 % bromophenol blue). Supernatants can be resolved by SDS-PAGE. Total protein can be visualized by silver staining, and GST-containing proteins by Western blotting with goat-anti-GST antibody (Amersham).

494. Putative complexes can be verified first by their absence from the GST-alone lane by them containing GST-Spi6 by Western blotting. The procedure can be scaled down and bands corresponding to cahdi'cϊaf'e"G'ST-Spi6':'"pfόtease complexes from SDS-PAGE gels stained with Coomassie blue (>O.lμg) can be excised.

495. To optimize the binding of GST-Spi6 to target proteins in lysates, complexes (92kD) with granzyme B (25kD) can be generated in lysates from P14 CTLs or in granulocyte lysates spiked with purified granzyme B as described previously (Medema et al., 2001a; Sun et al., 1997a).

(b) Identification of protease target(s) ofSpiβ in granulocytes.

496. To identify the protein that forms an SDS-stable complex with GST-Spi6, the excised gel band containing the candidate GST-Spi6: protease complexes from SDS-PAGE gels stained with Coomassie blue can be subjected to protease digestion and peptide microsequencing. For example, alkylated gel protein can be digested with Lys-C (Sigma- Aldrich; enzyme protein ratio 1 :20) and then with trypsin

(Sigma-Aldrich; enzyme protein ratio 1:10) in 5OmM ammonium carbonate pH 8.9, 0.25mM CaCl₂. Peptides can be subjected to one dimensional reverse phase HPLC/mass spectrometry using the ABI Qstar Pulsar-i instrument. A MS/MS spectra can then be generated for each peptide (4 ions per precursor scan). Data analysis can be carried out using the Mascot platform (Matrix Science) and Spectrum Mill software platform (Agilent Technologies). The results for all proteins detected after comparison of peptide sequence data bases can then be listed by protein name and search score. The requirement for a positive identification can be that the library matches agree between search platforms and that at least 4 distinct peptides for the tentatively identified protein be present.

497. To facilitate detection of specific GST-Spi6: protease complexes the amount of GST-Spi6 bait or protein in lysates can be varied. In addition, the concentration of triton X-100 can be increased in the washing steps.

498. 2-D gels electrophoresis (SDS-PAGE isoelectric focusing) can also be used to resolve GST- Sρi6: protease complexes.

499. In addition, co-immunoprecipitation with epitope-tagged Sρi6 can be used to pull down target proteases as described previously(Liu et al., 2003). For example, immunoprecipitation with anti-FLAG mAb (Sigma-Aldrich) can be used to pull down complexes from detergents lysates between FLAG-Spi6 and target protease, which can then be purified as described elsewhere herein.

500. The cDNA for each identified protein can then be cloned by PCR and then E. coli GST- fusion proteins can be generated kinetics and stochiometry of interaction with Spi6 can then be generated. 501. Based on homology to granzyme B, which is the only known substrate of Spi6, several mouse chymotrypsin-like proteases from protein data bases were identified that are expressed in mouse granulocytes. These candidate proteases can be expressed as GST-fusion proteins and examined for their ability to form SDS-stable complexes with Spi6 in vitro. The candidate proteases (with accession numbers) identified are: cathepsin G (P28293), PR-3 (Q61096), neutrophils elastase (NP031945), mouse mast cell protein (MMCP) -1 (P11034), MMCP-2 (P15119), MMCP-3 (P21843), MMCP-4 (P21812), MMCP-5 X?2m4f, *bfiW0P-¥{Ψ434yQ), MMCP-9 (035164), MMCP-IO (AAK51075). From these experiments, it has been shown that Spi6 binds the human forms of PR-3 and elastase.

(c) Physiological relevance of the granulocyte protease target of Spi6. 502. One can examine the affect of Spi6-target proteases on granulocyte function. This can be done by generating chimeras. Chimeras can be generated after the transduction of bone-marrow with MIGRl retroviruses expressing candidate proteases on polycistronic mRNA with GFP (Liu et al., 2004). The recruitment of GFP-positive granulocytes after glycogen injection can be examined by measuring LM killing after FACS-purification. Using ES technology (Fig. 1) mice deficient for those Spi6-target ρrotease(s) can be generated, which increase granulocyte function.

503. To test the relevance of the target protease in Spiβ function, target protease KO mice can be crossed with Spi6 KO mice. If one observes elevated activity of the target protease in Spiβ KO granulocytes (Fig. 16) one can conclude that Spiβ is a physiologically relevant inhibitor in vivo. If the protease is a true determinant of granulocyte function one can predict that for knock-out mice one can observe diminished LM clearance and impaired granulocyte function (i.e. recruitment, phagocytosis and bactericidal activity) as measured in vitro and ex vivo assays).

504. The amount of GST-Spi6 bait or protein in lysates can be varied to enhance detection of GST-Spi6-protease complexes. One can also increase the concentration of triton X-IOO in washing steps if multiple high molecular weight bands are observed, which upon MS sequencing are revealed as false positive complexes. The controls for specificity allows the optimization of these parameters. It is likely that the amount of GST-Spiβ: protease ( > 69kD) can be significantly less than unbound GST-Spi6 (69kD). If this is the case one can perform 2-D gels electrophoresis (SDS-PAGE x isoelectric focusing) for resolution. Co-immunopreciρitation with epitope-tagged Spiβ can also be used to detected GST-Spi6-portoease complexes through pull down assays. As has been done for other serpins (Liu et al., 2003), a FLAG-tagged Spiβ from NIH3T3 cells can be produced. Immunoprecipitation with anti-FLAG mAb (Sigma-Aldrich) can be used to pull down complexes from detergents lysates between FLAG-Sρi6 and target protease, which can then be purified as above.

505. Based on homology to granzyme B, a known substrate of Spi6, several mouse chymotrypsin- like proteases from protein data bases that are expressed in mouse granulocytes were identified. These candidate proteases can be expressed as GST-fusion proteins and examined for their ability to form SDS- stable complexes with Spi6 in vitro. The candidate proteases (with accession numbers) are: cathepsin G (P28293), PR-3 (Q61096), neutrophils elastase (NP031945), mouse mast cell protein (MMCP) -1 (Pl 1034), MMCP-2 (P15119), MMCP-3 (P21843), MMCP-4 (P21812), MMCP-5 (P21844), MMCP-8 (P43430), MMCP-9 (035164), MMCP-IO (AAK51075). Disclosed herein Spiβ binds the human forms of PR-3 and elastase. Species variation (about 30% difference in amino acids between human and mouse) can account for the difference in binding between these proteases and Granzyme B, and the mouse homologs can be relevant targets for Spiβ in mouse cells. C) conclusion

506. Disclosed herein are Spi6 over expression models. To examine the physiological mechanism by which Sρi6 controls immunity to a model virus and model bacterium a knock-out mouse model was also generated. The studies described above elucidate how the inhibition of proteases by Spi6 controls two cell biological process that are central to immune function, namely the control of T lymphocyte survival and the function of phaogocytic leukocytes. Since Spi6 is the mouse homologue of the human serpin PI9, the Spi6 KO and transgenic mice can serve as useful models to examine the roles of this class of protein to immunity and disease as well as models for testing for modulators of Spi6 or PI9.

507. Overall these data point to Spi6 as a specific inhibitor of granzyme B. By creating Spi6 KO mice, the role of Spi6 in a variety of cells was able to be observed.The results obtained in the studies described herein indicate a critical role of Spi6 in protecting CTLs, as well as NK cells, from PCD.

508. Spcifically, the lack of cell contraction in Spi6 KO mice finding is particularly interesting because Spi6 KO CTLs are susceptible to PCD (Fig. 7). Spi6 can inhibit pathways of PCD that control CTL survival but do not affect the severity of the contraction phase and so does not conform to the definition of a protective factor and does not select for memory-cell precursors (Liu et al., 2004). In addition these data seem to challenge the accepted view that the size of the memory pool is proportional to the clonal burst size (Ahmed and Gray, 1996; Hou et al., 1994). Spiδ deficiency appears to alleviate rather than increase the severity of the contraction phase, thus Sρi6 KO mice were examined for decreases in pro-apoptotic factors, which are known to induce the contraction phase, such as IFN-γ (Badovanic et al., 2000; Badovinac et al., 2004). The serum levels of IFN-γ in Sρi6 KO mice were diminished after L. monocytogenes infection.. d) Spi6 Activity

509. Disclosed are results which show the role of Spi6 in myeloid function. The ability of bead- coupled GST-Spi6 to bind to and isolate proteases ("pull down") in cell extracts was demonstrated. To complement the pull down approach it was shown that elastase is a physiologically relevant target of Spi6 inhibition in mouse granulocytes. This can be involved in the mechanism by which Spi6 controls granulocyte bactericidal activity.

(1) GST-Spi6 pull down of a protease substrate in cell extracts

510. The feasibility of using bead-coupled GST-Spi6 to pull down target proteases from cell extracts was shown. As described in Example 1, agarose-coupled GST-Spi6 (69kD) was incubated in detergent extracts from P14 CTLs, washed and bead-associated proteins resolved by SDS-PAGE. GST-

Spi6-coupled agarose beads (7μg) were incubated either alone (-) or with cytosolic extracts from wild-type (WT) or granzyme B KO (GrnBKO) P14 CTLs (10⁶ cells), then boiled in SDS and DTT and resolved by SDS-PAGE. After Coomassie staining, GST-Spi6 was detected as a 69kD band.. A ~ 92kD protein from wild-type but not granzyme B-deficient CTLs was identified. Since the samples were boiled in SDS and reducing agent before loading it was concluded that the 92kD complex between Spi6 and granzyme B is the result of a true serpin: serine protease interaction. Consistent with the known specificity of Spi6, SDS- resistant complexes could be detected when agarose-coupled GST-Spi6 was incubated with purified 'granzyrϊϊe' B'TOt'hot'gfanzymes Λ OΓ K (data not shown). These data indicate the feasibility of the GST-Spi6 pull down approach to identify Spi6 target proteases from cell extracts.

(2) Increased elastase activity in Spi6 KO granulocytes

511. To complement the purification of proteases from GST-Spi6 pull down experiments one can test whether Spi6 can inhibit candidate proteases, based on their similarity to granzyme B. To do this, Spi6

KO granulocytes were examined for increased activity of candidate proteases. Elastase is an important effector protease of granulocytes (Belaaouaj et al, 1998) and is structurally related to granzyme B (Sun et al., 1997). It was shown that elastase activity is about 3-times higher in the cytosol of Sρi6 KO granulocytes compared to B6 controls. The increase in elastase activity was only evident in the cytosol, which is consistent with the intracellular location of Spi6. There was no increase in the activity of the related proteases PR-3 or cathepsin G. It was shown that Spi6 is a very weak inhibitor of human elastase consistent with mouse neutrophil elastase (30% difference in amino acids between human and mouse) can be a direct target of Spi6. As detailed in Example 1, one can determine if Spi6 can directly inhibit mouse elastase after one expresses the candidate protease in E.coli. . If this proves to be the case one can conclude that Spi6 suppresses granulocyte activity through the inhibition of elastase.

2. Example 2 A Role for the Granzyme B Inhibitor Serine Protease Inhibitor 6 in CD8⁺ Memory Cell Homeostasis

512. Generation and maintenance of protective immunological memory is the goal of vaccination programs. CD8⁺ memory T cells are derived directly from CTLs. The mechanisms underlying this transformation and the subsequent survival of memory cells are not completely understood. However, some effector molecules required by CTLs to eliminate infected cells have also been shown to control the number of Ag-specific cells. Disclosed herein, memory cells express high levels of serine protease inhibitor (Spi) 6, an inhibitor of the effector molecule granzyme B, and that Spi6 can protect T cells from granzyme B- mediated apoptosis. In mouse models, both elevated expression of Sρi6 and the complete absence of granzyme B in CD8⁺ T cells led to an increase in memory cells after infection with lymphocytic choriomeningitis virus. This was not the result of increased levels of antilymphocytic choriomeningitis virus CD8⁺ T cells during the expansion or contraction phases, but rather transgenic Sρi6 directly influenced the survival of CD8⁺ memory T cells. These results are consistent with expression of protective molecules, like Spi6, serves to shield metabolically active CD8⁺ memory T cells from their own effector molecules. a) Methods and Materials

(1) Mice and virus

513. To generate a vector that overexpress Spi6, Spi6 cDNA was cloned into the VA CD2 expression cassette (Zhumabekov, et al., 1995) via the Sma 1 restriction site. Spi6 cDNA was then expressed as the minimal open reading frame (1.1kb) (SEQ ID NO:43) without untranslated 5' and 3' sequences.

514. Fertilized eggs from C57BL/6 (B6) mice were microinjected with the Spi6 cDNA subcloned into the human CD2 expression cassette (Zhumabekov et al., 1995) to generate two transgenic founders, With Spiό primers: forward, 5'-GAA TTC CGG GCT GGA TTGAGA AGC C-3' (SEQ ID NO:5) and reverse, 5'-GGA TAC TGA AGA GAG AAC TCT CCC-3' (SEQ ID NO:6). Each founder was backcrossed to B6 mice to generate colonies oϊSpiό Tg^+/~ mice. The progeny of the highest expressing Spiό 2g^+/~ mice were intercrossed to generate Spi6 ,7g^+/+mice. Homozygous status was verified by backcrossing to B6 mice and the identification of 20 consecutive Spi6 Tg^+/~ progeny by PCR. Spi6 Tg⁺'⁺ mice were crossed with P14 TCR transgenic mice (Pircher et al., 1990), which had been backcrossed onto the B6 background, to generate Pl 4 TCR^M~ Spi6 Tg^+1" mice, which were further used to generate Pl 4 TCR^+1" Spi6 Tg^H+ mice by crossing with Spiό Tg*¹* mice. Spi6 Tg^+f+ (Spi6 mice) and Pl 4 TCR^+/~ Spi6 Tg*¹* mice (P 14 x Spiό mice) were used for all experiments unless otherwise indicated. Additionally, the Spiό cDNA was cloned into the 3x-Flag vector (Sigma-Aldrich, St. Louis, MO) and transfected into 293 T cells.

Fibroblasts transfected with 3x-Flag-Sρi6 exhibited cytoplasmic staining using anti-Flag mAb, indicating that the cDNA used to generate the Spiό mice directs the production of a viable protein.

515. B6, Thyl.l⁺ congenic B6, and granzyme B cluster-deficient B6 (Heusel et al., 1994) mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice were infected with LCMV Armstrong by i.p. injection of 2 x 10⁵ PFU.

(2) FACS analysis

516. All fluorochrome-conjugated mAbs were purchased from BD Pharmingen (San Diego, CA). H-2D^b tetramers used to detect Ag-specific cells in the peripheral blood and spleen were generated as previously described (Ober et al., 2000). CD8⁺ T cell populations used for real-time PCR were sorted to >97% purity using a MoFIo (DAKOCytomation, Carpinteria, CA). Briefly, single cell suspensions were prepared, by depletion of erythrocytes with ammonium chloride and purification with Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada), from the pooled spleens (5-10 mice) of naive or LCMV Armstrong infected B6 mice either 8 days (effector) or 50-100 days (memory) after infection. Splenocytes from naive mice were FACS purified directly after staining with anti-CD8α' allophycocyanin and anti-CD44 PE mAbs. Splenocytes from effector and memory mice were first sorted using anti-Thyl .2 magnetic beads

(Miltenyi Biotec, Auburn, CA), then FACS purified after staining with anti-CD8n' allophycocyanin and PE- labeled H-2D^b tetramers loaded with the three immunodominant LCMV peptides (NP396: FQPQNGQFI (SEQ ID NO:20), GP33: KAVYNFATM (SEQ ID NO:19), and GP276: SGVENPGGYCL (SEQ ID NO:21)) (Butz et al., 1998). Detection of functional memory cells recognizing each of the three immunodominant LCMV peptides in the spleens of immune mice by intracellular cytokine staining for IFN-

T was performed as previously described (Murali-Krishna et al., 1998). Flow Jo (Tree Star, Ashland, OR) software was used for all analyses.

517. In other experiments, hemopoietic cell populations were purified (>95%) for real-time PCR analysis from splenocytes usingmagnetic beads conjugated to phenotypic markers (Miltenyi Biotec). Macrophages were generated from bone marrow cultured in medium containing macrophage CSF. DCs

(immature) were generated from bone marrow as previously described (Lutz et al., 1999). (3) Real-time PCR

518. RNA was extracted from purified cell populations using TRIzol Reagent (Invitrogen Life Technologies, San Diego, CA) and then cDNA was generated using Superscript First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies) (Medhurst et al., 2000). Primer and probe sequences for Spiό (Sun et al., 1997): forward 5'-GCC ATC CAT CTTTTG AAG ATG C-3' (SEQ ID NO:7), reverse 5'-

TGC ACC CAA GAG AAC CAT AGC-3' (SEQ ID NO:8), probe 5'-TCC AAA AAT GTA TGT TAT TCT CCT GCG AGC ATC T-3' (SEQ ID NO:9); granzyme B (Heusel et al., 1994): forward 5'-ACA AGG ACC AGC TCT GTC CTT G-3'(SEQ ID NO: 10), reverse 5'-TGT CAG TTG GGT TGT CAC AGC-3' (SEQ ID NO:11), probe 5'-CCA ATG GAA CAC CTC TTC TGC CAC CA-3¹ (SEQ ID NO: 12); I-Aβ" (Larhammar et al., 1985) (MHC class II): forward 5'-TGTTAG GAA TGG AGA CTG GAC CTT-3'(SEQ ID NO: 13), reverse 5'-CCA CGA GGC AGC TGTAGA TGT-3'(SEQ ID NO: 14), probe 5'-CAG ACA ACA GTA ATG CTG GAA ATG ATC CCA-3'(SEQ ID NO: 15); and cyclophilin A (Medhurst et al., 2000): forward 5'-CCA TCA AAC CAT TCC TTC TGT AGC-3'(SEQ ID NO: 16), reverse 5'-AGC AGA GAT TAC AGG ACA TTG CG-3'(SEQ ID NO: 17), probe 5'-CAG GAG AGC GTC CCT ACC CCA TCT G-3¹ (SEQ ID NO:18) were designed using Primer Express software (PE Applied Biosystems, Foster City, CA). The unique specificity of each set was verified by checking the sequences against GenBank database. Real-time PCR were conducted using TaqMan Universal PCR Master Mix (PE Applied Biosystems) and run on an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). Data were captured and analyzed using Sequence Detector software (PE Applied Biosystems). 519. In addition to duplicate reactions for gene expression in naive, effector, and memory cells, each real-time PCR plate contained reactions for generating standard curves (using serial dilutions of a known quantity of cDNA generated from unsorted B6 splenocytes) for each gene being analyzed. The slope of the standard curve describes the efficiency of the real-time PCR. Only reactions that ran at >90% efficiency were included. An estimate of the amount of RNA in each experimental reaction was calculated using the equations of the standard curves. The data are reported as the ratio of the calculated amount of candidate RNA in a given sample by the calculated amount of the housekeeping control gene cyclophilin A in that same sample. Cyclophilin A was chosen as the housekeeping control because its expression level does not appear to be different between populations of T cells at different stages of activation (Kaech et al. 2002). 520. Real-time PCR was also used to compare the amount of Spiό mRNA in clones of Jurkat cells transfected with Spi6 and in Spiδ transgenic memory cells FACS-purified from the pooled spleens of three LCMV-immune mice. 18S rRNA was used as the standard for these experiments as the Jurkat cell line is derived from a human thymoma and the standard housekeeping control primers/probe set was generated for murine cyclophilin A. (4) Northern blot analysis

521. Splenocytes &omP14 TCR⁺'^' Spiό Tg-'^' Q?U),P14 TCR^+I~ Spiό Tg^+μ (P14 x Spi6, heterozygous) and Pl 4 TCR⁺'^' Spiό Tg^+I+ (P 14 X Spiό, homozygous) mice were depleted of erythrocytes by nium chloride and cultured for 3 days in complete medium containing human IL-2 (10 U/ml) and ^lGP33"'pe'fιfMώ'tlO^"""My." "JPWSt S days, cultures (>90% activated Pl 4 TCR^÷/~ cells) were purified using Lympholyte-M (Cedarlane Laboratories) and the RNA extracted using TRIzol Reagent (Invitrogen Life Technologies). RNA was enriched for mRNA using MicroFastTrack mRNA isolation kit (Invitrogen Life Technologies). Equal amounts of mRNA were run on a 1.5% gel, transferred to a Hybond-N* membrane (Amersham Biosciences) and the membrane hybridized overnight with a ³²P-labeled Spi6 cDNA (SEQ ID

NO:3). The final wash was in O.lx SSC with 0.5% SDS at 65⁰C and the membrane was exposed to film for 6 h. The membrane was then stripped and reprobed with a ³²P-labeled GAPDH cDNA (SEQ ID NO:30). The final wash was in 0.5x SSC with 0.5% SDS at 65°C and the membrane was exposed to film for 4 h.

(5) Apoptosis assays 522. Jurkat cells (human thymoma) were cotransfected with 5 μg of either the Spi6 cDNA cloned into the CD2 expression cassette (Zhumabekov et al., 1995) or empty vector and PGK-Neo (5 μg) by electroporation (280 V, 975 μF). Transfectants were selected and cloned in 1 mg/ml G418 (Invitrogen Life Technologies) over a 3-wk period. Clones were treated for 2 h with human perforin at sublytic concentrations (0.2 U/ml) and human granzyme B (2 μg/ml) (Froelich et al., 1996, Bird et al., 1998), or cultured overnight with anti-human Fas IPO-4 mAb (0.12 μg/ml) (Rokhlin et al., 1997) or subjected to gamma-irradiation (4456 rads) and apoptosis measured after 20 h. The early onset of apoptosis was detected in non-necrotic cells (propidium iodide-negative) by staining with YOPRO-I, according to the manufacturer's instructions (Molecular Probes, Eugene, OR) (Idziorek et al., 1995).

(6) Adoptive transfers 523. Naive CD8⁺ T cells were purified (>90% pure) from the spleens of P14 or P14 x Spi6 mice

(Thyl .2⁺) by positively sorting with anti-CD8 magnetic beads (Miltenyi Biotec), then adoptively transferred (10⁵) by i.v. injection into Thy 1.I⁺ B6 mice. Recipients were rested for 2 days and then infected with LCMV. Thyl.2⁺ donor P14 or P14 x Sρi6 cells were followed in the peripheral blood of recipients by staining with anti-Thyl.2 mAbs. (7) BrdU incorporation

524. The level of memory cell turnover was determined by incorporation of BrdU. LCMV- immune Thyl. I⁺ mice that had received either P14 or P14 x Spi6 cells (Thyl.2⁺) were given BrdU (Sigma- Aldrich) for 1 wk in their drinking water (0.8 mg/ml) and then analyzed. Briefly, 10⁶ splenocytes were surface stained using anti-Thyl .2 PE mAbs and then fixed using Cytofix/Cytoperm solution (BD Pharmingen, San Jose, CA). Fixed cells were washed once in Perm/Wash solution (BD Pharmingen, San

Jose, CA) both before and after incubation with 100 U DNase I (Sigma-Aldrich) for 2 h at 37°C. Cells were then stained with anti-BrdU FITC (BD Pharmingen, San Jose, CA) or isotype control (BD Pharmingen, San Jose, CA), washed in Perm/Wash solution, and analyzed by FACS.

(8) Statistical analyses 525. All j? values were determined using t tests. Statistical analyses shown in Figure 19 were performed using all values obtained for all isolates. b) Results

(1) Spi6 is up-regulated in effector and memory cells

526. Before beginning to address a physiological role for Spi6 in the development of memory cells, the expression pattern of Sρi6 in CD8⁺ T cells after infection with LCMV was first examined. Anti- LCMV cells were purified (>97% pure) 8 days (effectors) or more than 50 days (memory cells) after infection by staining with anti-CD8a- mAbs (SEQ ID NO:32) and H-2D^b tetramers (SEQ ID NO:33) loaded with the dominant NP396(SEQ ID NO:20), GP33(SEQ ID NO: 19), and GP276(SEQ ID NO:21) Naive cells (CD8⁺CD44^l0W) were purified by FACS from the spleens of B6 mice. Effectors and memory cells were isolated from the spleens of B6 mice 8 days or more than 50 days after infection with LCMV by FACS of CD8⁺ tetramer⁺ cells. The initial percentages and final purity of naive or tetramer^4* CD8⁺ T cells for naϊve cells was 99% (from 7.9%), for effector cells was 98%(from 23%), and for memory cells was 97% (from 2.5%).

527. Real-time PCR using primers and probes specific for Spiό and the housekeeping gene cyclophilin A was performed on cDNA generated from RNA isolated from the purified cell populations (Medhurst et al., 2000). Two separate isolates each of naive, effector, and memory cells were assessed for

Spiό expression. One representative value for each isolate is expressed in Fig. 19B as the ratio of Spiό Xo cyclophilin. Naive cells did not express high levels oϊ Spiό (an. average ratio of 0.6), but 8 days after LCMV infection, Ag-specific effectors had significantly up-regulated Spi6 expression (an average ratio of 11.9, p < 0.001 compared with naive cells). Therefore, separate isolates of effector cells were found to express Spi6 28.6-fold and 11 -fold higher than naive cells. A third independent isolate of effector cells was also assessed for Spi6 expression and found to be 53-fold higher than naive cells (a Spi6 to cyclophilin ratio of 31.7). This up-regulation is consistent with the original identification of Spi6 in a CTL cell line (Sun et al., 1997). In memory cells, the expression of Spiό fell 3.4-fold from the level detected in effectors (an average ratio of 3.5, p < 0.05 compared with effectors), but remained, on average, 5.8-fold higher than naive cells (p < 0.001).

528. The expression pattern of Spiό in CD8⁺ T cells correlated with that of granzyme B (Fig. 19C), the granule protease inhibited by Sρi6 (Sun et al., 1997, Medema et al., 2001a). Granzyme B expression was hugely up-regulated in effector cells (>200-fold higher than naive cells, p < 0.001). The expression of granzyme B, although 24-fold lower than effectors (p < 0.001), was retained in memory cells, which expressed -*9-fbld higher levels of granzyme B than naive cells (p < 0.01). This result is consistent with previous reports of granzyme B transcript and protein expression in populations of CD8⁺ T cells (Kaech et al. 2002, Wherry et al., 2003, Hamann et al., 1997). The level of MHC class II expression in all populations of purified cells was negligible (Fig. 19D), indicating that no contaminating APCs were present. Real-time PCR analysis, therefore, indicated that Spiό is coexpressed with granzyme B in anti-LCMV effectors, and expression of both is retained in the resulting memory cells. (2) Spi6 protects T cells specifically from granzyme B-mediated apoptosis

529. The ability of Spi6 to protect T cells from granzyme B was also examined. Cloned transfectants of Jurkat cells that expressed Spiό under the control of a human CD2 expression cassette (Zhumabekov et al., 1995) (0.45 and 0.31 ng per ng of rRNA compared with O.001 ng of SpiόmRNA per nanogram of rRNA in untransfected controls) were used to test the ability of Spi6 to protect T cells from granzyme B-induced PCD. Granzyme B, in the presence of sublytic concentrations of perform, induced significantly less (p < 0.05) apoptosis in Spiό transfectants compared with controls (Fig. 20A) (Froelich et al., 1996). This protection was specific to granzyme B, because the percentage of apoptosis initiated by either ligation of the Fas death receptor (Rokhlin et al., 1997) or gamma-irradiation was no different between Spi6 transfectants and controls (Fig. 20B). Therefore, as with the human granzyme B inhibitor PI9 (Bird et al., 1998), expression of Spi6 can protect T cells specifically from death initiated by granzyme B delivered byperforin.

(S) Generation and characterization of Spi6 transgenic mice 530. To study the role of Spi6 in CD8⁺ T cell biology in vivo, transgenic mice expressing the 1.1 kb Spi6 cDNA under the control of the human CD2 promoter (Zhumabekov et al., 1995) were generated. Two founder mice were backcrossed to B6 mice to generate colonies of mice expressing heterozygous transgenic Spi6, and the offspring of these mice intercrossed to generate homozygous transgenic Spi6 mice (referred to hereafter as Spi6 mice). 531. The endogenous level of Spi6 in CTLs was significantly higher than that of naive CD8⁺ T cells (Fig. 19B). As such, the ability of transgene-driven expression of Spi6 in effectors to increase the total level of Spi6 mRNA over that of the endogenous level was examined. Spi6 mice were crossed to transgenic mice expressing theP14 TCR (SEQ ID NO:34), which recognizes the GP33 peptide from LCMV(SEQ ID NO:19) in the context of H-2D^b (Pircher et al., 1990). Splenocytes from P14 and P14 x Sρi6 mice were cultured with GP33 peptide and IL-2 for 3 days, at which time >90% of the cells have become activated P14 cells. Northern blot analysis of poly(A)⁺ RNA confirmed the expression of the endogenous Spi6 mRNA (2.4 kb) in all P14 CTLs. PoIy(A)⁺ RNA for Northern blots was isolated from CTLs generated from P14 mice and mice heterozygous (P 14 x Spi6 (het)) or homozygous (P 14 x Spi6 (homo)) for transgenic Spi6 expression. Hybridization with a ³²P-labeled Spi6 cDNA probe identified the endogenous (2.4 kb) and transgenic (-2.1 kb) Spi6 transcripts. Blots were stripped and reprobed with a ³²P-labeled GAPDH cDNA to reveal equal loading of mRNA (Sun et al., 1997). Effectors from P14 X Spi6 mice, however, expressed high levels of a smaller transgenic Spi6 transcript (^2.1 kb). The transgenic transcript lacks the 3' untranslated region of the endogenous transcript and terminates at a CD2-specific sequence (Zhumabekov et al., 1995). Overall, the level of Spiό was significantly higher in CTLs from Spi6 mice. 532. The CD2 expression cassette also drives the expression of genes in cells other than CD8⁺ T cells (Zhumabekov et al., 1995). Analysis of Spi6 expression was performed by real-time PCR on various hemopoietic cell populations. Endogenous expression of Spiό in B6 mice was found in all cell types tested, with MK"&gIIs"e5φressiϊ.g'tiie^""JUgnest levels (Fig.21B). The transgene increased expression of Spi6 at least 40-fold in all cell types, with the highest expression levels in thymocytes and other lymphocytes (Fig. 21C).

533. Increased expression of Spi6 in transgenic mice did not alter thymic development because the numbers of CD4 single- positive, CD8 single-positive, and CD4 CD8 double-positive thymocytes were no different in Spi6 and B6 mice (Table 3). Additionally, the number of B cells and T cells in the spleen and lymph nodes of Spi6 mice were no different from the numbers in B6 mice (Table 3). Although, on average, the percentage of T cells that were memory-phenotype (CD44high) in Spi6 mice was slightly higher than B6 mice (CD44high of CD4+ cells in lymph node: 13.9 ± 1.4% vs 10.7 ± 0.9% and in spleen: 23.1 ± 1.1% vs 17.5 ± 1.7%; CD44high of CD8+ cells in lymph node: 19.3 ± 1.0% vs 17 ± 1.6%, and in spleen: 24.9 ± 0.6% vs 22.9 ± 2.0%), this difference was not significant (p > 0.05).

Table 3 Cell numbers in B 6 and Spi6 mice

Tissue Cell Type B6 mice Spi6 mice

Thymus CD4⁺ 7.5 ± 0.8 6.9 ± 0.3

CD8⁺ 3.1 ± 0.3 3.3 ± 0.3

CD4⁺CD8⁺ 63.1 ± 7.1 53.2 ± 2.2

Lymph nodes B cells 3.9 ± 0.3 4.2 ± 0.4

CD4⁺ T cells 2.8 ± 0.16 2.9 ± 0.16

CD8⁺ T cells 1.9 ± 0.13 1.8 ± 0.14

Spleen B cells 27.2 ± 3.4 19.1 ± 1.6

CD4⁺ T cells 5.9 ± 0.5 5.6 ± 0.5

CD8⁺ T cells 2.8 ± 0.3 2.8 ± 0.2

(4) Enhanced development of memory cells in Spi6 mice after LCMV infection

534. Spi6 expression was highly up-regulated in effectors and the resulting memory cells (Fig. 19B). To assess whether Spiό plays a role in the development of CD8⁺ T cell memory, Spi6 mice were infected with LCMV and the number of memory cells that developed were quantitated. A hallmark of πicmυiy υcn& i& ine'SDffltyTσrespond immediately to Ag rechallenge (Berard et al., 2002). Therefore, 180 or more days after LCMV infection, the number of CD8⁺ memory T cells that produced IFN-Tf after ex vivo stimulation with Ag peptide were measured (Murali-Krishna et al., 1998). Infection of Spi6 mice resulted in significantly more memory cells compared with B6 mice, whether represented as a percentage of splenocytes (p < 0.05 or 0.01, Fig. 22A) or by absolute cell number (p < 0.05, Fig. 22B). Specifically, compared with B6 mice memory cell numbers in Spi6 mice were 2.9-fold higher for cells recognizing the GP33 peptide (SEQ ID NO:19), 2.1 -fold higher for cells recognizing the NP396 peptide (SEQ ID NO:20) and 2.8-fold higher for cells recognizing the GP276 peptide (SEQ ID NO:21).

535. If Spi6 increased the amount of memory cells through the inhibition of granzyme B-mediated PCD₅ the absence of granzyme B would be predicted to have a similar effect on memory cell development.

Therefore, the number of memory cells generated after LCMV infection of GrnBKO mice was determined (Heusel et al., 1994). The presence of granzyme B is not essential for resolution of an LCMV infection, so there is no persisting Ag and the kinetics of CD8⁺ T cell expansion and contraction can be followed in GrnBKO mice (Zajac et al., 2003). Similar to Sρi6 mice, GrnBKO mice also had elevated numbers of memory cells compared with B6 mice (Fig. 22, A and B). 2.2-fold more memory cells recognizing the GP33 peptide (SEQ ID NO: 19) (p < 0.05), 1.8-fold more memory cells recognizing the NP396 peptide (SEQ ID NO:20) and2.3-fold more memory cells recognizing the GP276 peptide(SEQ ID NO:21) (p< 0.05) in GrnBKO mice were detected.

536. The increase in the number of memory cells in Spi6 and GrnBKO mice was not the result of increased clonal burst size. Eight days after infection, the peak level of anti-GP33 CD8⁺ cells in Spi6 (7.3 ±

0.5%), B6 (7.1 ± 0.4%) and GrnBKO mice (8.2 ± 0.5%) was no different (Fig. 22C). In addition, the increase in memory cell numbers was also not attributable to an effect on the contraction phase, as 15 days postinfection the percentages of anti-GP33 CD8⁺ cells in Spi6 (5.0 ± 0.4%), B6 (5.0 ± 0.4%) and GrnBKO mice (5.5 ±0.3%) were also equivalent (Fig. 22C). Also, at 30 days postinfection the percentage of anti- GP33 CD8⁺ cells in Spi6 (4.4 ± 0.4%) and GrnBKO mice (5.0 ± 0.4%) was higher than B6 mice (3.5 ±

0.4%), although this was notyet significant (p > 0.05, Fig. 22C).

(5) Cell-autonomous Spi6 expression increases the frequency of memory cells

537. Spi6 mice have elevated expression levels oiSpiό in several types of hemopoietic cells (Fig. 21). Therefore, the increase in memory cell numbers detected in Spi6 mice could be the result of enhanced priming (Wong et al., 2003) by Sρi6 transgenic DCs (Medema et al., 2001b). To examine the role of Spi6 in CD8⁺ T cells during the development of memory, the anti-LCMV response of P14 X Spi6 cells after adoptive transfer, a system that has been well characterized, was examined (Kaech et al. 2002, Petschner et al., 1998, Butz et al., 1998). Naive CD8⁺ T cells were purified from P14 and P14 X Spi6 mice (both Thyl.2⁺) then adoptively transferred (10⁵) into Thyl.l⁺ congenic B6 recipients. After 2 days, recipients were infected with

LCMV and the levels of P14 memory cells determined after at least 50 days by ex vivo IFN-T production. "55K¹ Gating ¹On WI .2^{^} or Thyl.2⁺ cells during FACS analysis allowed for detection of the endogenous (Thyl .2^") and donor (Thyl .2⁺) memory cells (CDS⁺IFN-I^*1") in recipients. To do so, naive CD8⁺ T cells from P14 and P14 X Spi6 mice, both Thyl.2⁺, were purified using magnetic beads and adoptively transferred to Thyl. I⁺ congenic recipients. Recipients were infected with LCMV and more than 50 days later the percentage of memory cells in recipient spleens determined by measuring ex vivo IFN-T production. FACS scans from representative recipients given P14 or P14 X Spi6 cells. Total live cells did not stain with the isotype control (IC) when stimulated with GP33 peptide, nor did they stain for IFN-Ϊ production in the absence of stimulation (No peptide). Donor and recipient memory cells were CDS⁺IFN-T⁺ and Thyl.2⁺ or Thyl .2^", respectively. Stimulation with GP33 revealed that virtually all (91-98%) of the Thyl.2⁺ cells were functional CDS⁺IFN-T⁺ memory cells. Elevated levels of P14 x Spi6 memory cells in recipients compared with P 14 memory cell levels, an average of 1.5 ±0.16% vs 0.61 ± 0.15% (p < 0.01, Fig. 23B) was observed. When the data from several experiments were pooled and normalized there were twice as many P14 x Spi6 memory cells as P14 memory cells (2.0 ± 0.2% vs 1.1 ± 0.3%,/? < 0.01, Fig. 23C). The same was true for the actual percentage (not normalized) of P14 x Sρi6 memory cells compared with P14 memory cells (0.85 ± 0.13% vs 0.48 ± 0.12%,/? < 0.05). Normalized memory cell percentages were calculated proportionally by considering the percentage of Thyl.2⁺ cells in the peripheral blood of recipients at the peak of the response 7 days postinfection as 100%. The percentages of endogenous (Thyl .2^") memory cells generated in recipients receiving P14 cells did not differ from those recipients receiving P14 x Spi6 cells (Fig. 23D). 539. As in the experiments using intact animals (Fig. 22C), there was no difference in the level of

P14 and P14 x Spi6 cells at the peak of the response on day 7 (44.7 ± 1.7% vs 41.3 ± 1.8%, Fig. 23E) or during the contraction phase, measured on day 14 (15.4 ± 1.2% vs 15.0 ± 1.1%, Fig. 23E). In addition, after 28 days postinfection, more P14 X Spi6 cells could be detected in recipients than P14 cells (7.7 ± 0.9% vs 5.7 ± 1.2%, Fig. 23E). However, unlike the difference detected after at least 50 days (Fig. 23C), the difference at this early time point was not yet significant (p > 0.05). Transgenic Spi6 expression, therefore, led to an increased frequency of anti-LCMV memory cells by directly protecting CD8⁺ T cells, not during the expansion or contraction phases, but rather during the memory phase.

540. Formally, the increased frequency of Spi6 transgenic memory cells can be due to either enhanced proliferation of memory cells containing transgenic Spi6 or to increased long-term survival. To help distinguish between these possibilities, LCMV-immune Thyl . I⁺ mice that had received P14 orP14 x

Spi6 cells were given BrdU in their drinking water and 1 wk later the turnover of memory cells in the spleens of these animals determined by BrdU incorporation. The frequency of P 14 and P14 x Sρi6 memory cells that had divided (18 ± 0.5% vs 18.1 ± 0.4%) was the same, indicating that memory cells containing transgenic Spi6 appear to proliferate similarly to wild-type memory cells. This finding supports the idea that transgenic Spiό increases the long-term survival of memory cells rather than increasing proliferation.

541. In fact, FACS-purified Spi6 transgenic memory cells from LCMV-immune mice expressed levels of Spi6 (3.1 ngof S/?z^'(5 mRNA per nanogram of rRNA) higher than those capable of protection of ww clones from granzyme B in vitro (0.45 and 0.31 ng ofSpiό mRNA per nanogram of rRNA, Fig. 20). ' Sprø'expression can improve the survival of memory cells by protecting them in a cell-autonomous fashion, supporting the idea that granzyme B contributes to CD8⁺ memory T cell homeostasis. c) Conclusion 542. The level of CD8⁺ memory T cells is determined by events in the expansion, contraction and memory phases of the immune response (Sprent et al., 2002). Here, it is shown that CD8⁺ memory T cells express elevated levels of Spiδ, and that inhibition of granzyme B (by deficiency or expression of transgenic Spiβ) increases the number of CD8⁺ memory T cells

543. Previous studies have indicated that Spi6 can inhibit granzyme B and, importantly, protects cells from the granule exocytosis pathway of PCD (Sun et al., 1997, Medema et al., 2001b, Medema et al.,

2001a). These studies were extended here and it was demonstrated that Spi6 can protect T cells from granzyme B-mediated PCD (Fig. 20). An earlier study reported that GrnBKO mice have wild-type levels of anti-LCMV memory cells (Zajac et al., 2003). Other effector molecules, perform and IFN-T, have previously been shown to influence Ag-specific cells during the expansion and contraction phases (Badovinac et al., 2000, Kagi et al., 1999, Spaner et al., 1999, Matloubian et al., 1999, Gallimore et al.,

1998), so it would appear that effector molecules can regulate the levels of CD8⁺ T cells throughout the entire course of an immune response.

544. The enhanced memory cell phenotype shown in Spi6 mice (2- to 3-fold higher than B6 mice, Fig. 22) is about the same magnitude observed in other transgenic systems with higher memory cell levels, after over-expression of calcium/calmodulin kinase II and the Bcl-6 transcriptional repressor (Bui et al.,

2000, Ichii et al., 2002). Additionally, transgene driven expression of Bcl-2 and Bcl-x_L, two well known anti-apoptotic proteins, did not lead to increases in memory cell numbers after infection, even though over- expression of these proteins protected effector cells from apoptosis in vitro (Petschner et al., 1998). So, simply over-expressing an anti-apoptotic protein in CD8⁺ T cells does not account for the development of increased memory cell levels in Sρi6 mice.

545. Immunological memory not only requires the initial differentiation of CD8⁺ memory cells, but also their long-term maintenance (Sprent et al., 2002). The impact of some molecules, such as the IL-15 cytokine, on memory cells is, in fact, a result of their action well past the contraction phase of CD8⁺ memory T cell development (Schluns et al., 2003). Consistent with previous reports (Zajac et al., 2003), persistently elevated levels of effectors or an altered contraction phase after infection of GrnBKO mice with LCMV, a phenotype mimicked in Spi6 mice was not found (Fig. 22C). Therefore, there was an impact of inhibition of granzyme B that is specific to the memory phase of the response to LCMV and did not lead to autoimmunity or immunopathology (Figs. 22 and 23). This is in stark contrast to the phenotype of perform deficiency, which results in elevated levels of effectors, delayed contraction and pathology (Badovinac et al., 2000, Kagi et al., 1999). However, perforin-deficient mice have a defect in pathogen clearance that leads to persistence of Ag, which drives dysregulation of effectors in these animals. Neither GrnBKO (Zajac et al., 2003) nor Spi6 mice have a defect in viral clearance. Therefore, Ag did not persist to drive effector cell dysregulation. UήdeftlTdfeB'"cl5iiditi'<3nS"Ol'ϊιoπnai viral clearance, true memory cell development could be assessed in the GrnBKO and Spi6 mice, unlike in perforin-deficient mice. However, the massive increases in the numbers of activated CD8⁺ T cells in perforin-deficient mice and humans clearly demonstrate a role for the granule exocytosis pathway of PCD in the control of activated CD8⁺ T cells. Thus, granule proteases other than granzyme B can limit clonal burst size and eliminate CD8⁺ T cells after infection, or are able to compensate for granzyme B in its absence.

546. The finding that transgenic Spi6 expression in memory cells reciprocated the phenotype of granzyme B deficiency is consistent with Spi6 being an endogenous inhibitor of granzyme B in CD8⁺T cells (Fig. 20). Further, that the Spi6 and GrnBKO mouse memory phenotypes are similar suggests that the amount of Spi6 expressed in the Spi6 transgenic memory cells was sufficient to block all the granzyme B capable of initiating apoptosis. This explains why the massive up-regulation of Spi6 did not increase memory cell numbers higher than 3 -fold, as transgenic Spi6 can only inhibit all the granzyme B. Despite increasing the number of memory cells, transgenic Spi6 did not affect the level of CTLs (Figs. 22C and 23E). Further expressing Spi6 in the effectors did not have an additional protective effect during the expansion and contraction phases (Fig. 19B). However, once the endogenous level decreases somewhat to the level in memory cells, the additional Sρi6 in the Sρi6 mice protected the memory cells at a level they do not ordinarily achieve endogenously.

547. One of the salient qualities of memory cells is the ability to respond quickly to Ag, mediated, in part, because they have preformed granules containing toxins such as perform and granzyme B (Opferman et al., 1999, Wherry et al., 2003). It has recently been reported that this immediate response is the result of a specialized G₀/Gi cell cycle state predisposing memory cells to rapid division upon stimulation (Veiga- Fernandes et al., 2004). At any given time, a certain percentage of the memory cell population is in cell cycle (Tough et al., 1994), and the ability of memory cells to cycle is absolutely critical to an anamnestic response (Bellier et al., 2003). Long-lived memory cells, therefore, exist in a metabolically active state over a long period of time in the presence of cytotoxins, which suggests this makes them susceptible to death induced by those toxins. PI9, the human homologue of Spi6, can protect both NK cells and CTLs from PCD by inhibiting "misdirected" granzyme B that has leaked into the cytoplasm (Hirst et al., 2003, Ida et al., 2003). Activated CD8⁺ T cells appear to selectively leak granzyme B from their granules (and not granzyme A or perforin) (Bidere et al., 2002). 548. This data supports the idea that granzyme B is capable of initiating apoptosis in Ag-specific cells after the contraction phase as the level of endogenous Spi6 decreases. Transgenic Spi6 protected Ag- specific cells as they transitioned into memory cells, thus a slow increase in Ag-specific cell number from day 15 to day 50, when the difference had become large enough to be statistically significant was detected. Because they retain granzyme B expression, memory cells appear to be susceptible to granzyme B-initiated apoptosis. It is clear from the plateau in the difference in number of Spi6 and wild-type memory cells that is reached (day 50 and 180 frequencies are similar, Phillips et al. Figs. 22A and 23B) that the memory cells containing transgenic Spi6 do not continue to expand without control. 34y. aome vrπueiu suaius υf LCMV have been shown to persist in vivo instead of being cleared

(Ahmed et al., 1984). Under conditions of persistent Ag exposure, the CD8+ T cell response can clonally exhaust. During clonal exhaustion the Ag-specific cells that cannot clear the pathogen undergo progressive dysfunction and are eventually completely eliminated, possibly to avoid the induction of autoimmunity during prolonged activation (Moskophidis et al., 1993). The mechanism(s) responsible for clonal exhaustion are not clear. Although other molecules that can influence the numbers of activated Ag-specific CD8+ T cells, such as perforin, IFN-I and Fas, have been implicated in clonal exhaustion (Lohman et al., 1996, Zhou et al., 2002), there is no evidence to suggest that granzyme B plays a role in this process to date. 3. Example 3 a) Spi6 deficient mice.

550. To address the role of Sρi6 (Sun, J., et al., (1997) in leukocyte function, Spi6-deficient mice were generated. Using homologous recombination in ES cells from C57BL/6 mice (B6 mice), exon 7 of Sρi6 was deleted, which encodes 60% of Spi6 and includes the critical reactive center loop (RCL), which is required for target protease inhibition. Results were confirmed via Southern blot analysis of two ES cell clones with wild-type (WT: 37) or mutant (M: 69 and 389) Spi6 neo alleles. Using the 5' probe, the Southern blot analysis revealed a 6.9 kb band (mutant) and a 7.2 kb band (wild-type) for both the 68 and 389 clones. The 3' probe revealed a 9.8 kb band (wild-type) and a 5.8 kb band (mutant) for both the 68 and 389 clones. Using Cre-mediated recombination, the G418-resistance cassette was removed to avoid affecting the transcription of closely linked serpin genes (such as Spil 3), which may have similar functions to Spi6. These results were confirmed via Southern Blot analysis which revealed a 7.2 kb (wt) and 6.9kb (neo) band for the 389 clone and a 7.2kb (wild-type) and a 5.8 kb (Δneo) band for the 389Δ neo clone. Mice homozygous for Spi6 mutant alleles in the C57BL/6 (B6) background (Spi6 KO mice) were also generated and confirmed via Southern Blot analysis. Spi6 KO mice did not display any significant difference in the number of myeloid (neutrophils and monocytes) or lymphoid cells (T and B cells) in the thymus or blood. b) Impaired survival of Spi6-defϊcient CTLs.

551. Spi6 is up-regulated in CTLs and overexpression can inhibit GrB-mediated apoptosis in vitro (see above and see also Phillips et al., 2004). To examine the physiological role of Sρi6, the CTL response of Spi6 KO mice to infection was examined. Mice were infected with LCMV Armstrong (Phillips et al., 2004) and the attenuated DPL-1942 strain of LM, which had been engineered to express ovalbumin and generate an H-2K^b-restricted peptide antigen (OVA)(Pope et al., 2001). To avoid the effects of persistent antigen on interferon-driven apoptosis (Badovinac et al., 2000), doses of LCMV Armstrong (2xlO⁵ pfu) and LM DPL-1942 (10⁵ cfu) that result in complete clearance in both B6 and Spi6 KO mice after 8 d were used. Staining with gp 33/H-2D^b-tetramers revealed that the number of CTL specific for LCMV Armstrong was diminished in Sρi6 KO mice compared to B6 controls (5-fold lower) (Figure 30(a)). Furthermore, Spi6 KO mice harbored about 9-times less LM-specific CTLs after infection (Figure 30(c)). The lower number of CTLs in Spi6 KO mice correlated with an increase in the onset of apoptosis of both LCMV (Figure 30(b)) and LM-specific CD8 T cells (Figure 30(d)), as evidenced by increased staining with the DNA dye YOPRO- Y, whi^'αh (lefecfreariy'cMό'ffiό'Sbrilal changes in apoptosis (Opferman et al., 2001). Thus, Spi6 is required for the survival and protection of CTLs from apoptosis in vivo. Adoptive transfer experiments indicate that the defective survival of Spi6 KO CTLs is GrB-dependent and cell autonomous. c) Impaired CTL-immunity to virus in Spi6-deficient mice 552. The lower number of LCMV-specific CTLs in Spi6 KO mice resulted in significantly (P = 4 x 10^"5) impaired CTL-activity in Spi6 KO mice (8.7 ± 0.25 % specific lysis, n=5 mice) in ex vivo assays (Splenocyte/Target ratio = 5.5) compared to B6 mice (24.7 ± 1.2 % specific lysis, n=5 mice) (Figure 3 l(a)). Next, the requirement for Spi6 in immunity to high doses (10^δ pfu) of the clone 13 variant of LCMV Armstrong was examined. The clearance of the clone 13 LCMV from the spleen of Sρi6 KO mice was impaired, as evidenced by a 5-fold increase in titer 6 d after infection (Figure 31(b)). Therefore, the defect in

CTL survival leads to impaired immunity to virus in Spi6 KO mice. d) Spi6 protects CTLs from apoptosis by suppressing GrB in the cytoplasm.

553. To determine whether GrB from granules into the cytoplasm can lead to the activation induced cell death of cytolytic lymphocytes, LCMV-specific CTLs were generated by culturing spleen cells from P14 mice with gp33 peptide for 2 d in vitro (>90% of viable cells), then subjected to sub-cellular fractionation. Enzyme assays revealed significantly (P = 0.004) increased specific activity of GrB in the cytoplasm of Spi6 KO CTLs (Figure 32). Consistent with its specificity as a serpin specific for GrB, it can be concluded that Spi6 inhibits cytoplasmic GrB in CTLs.

554. Enzyme assays also revealed a lower specific activity of the caspase 3 executioner protease in the cytoplasm of GrB KO CTLs, indicating that endogenous GrB induces apoptosis of CTLs (Fig. 4). There was a corresponding increase in the specific activity of caspase 3 in the cytoplasm of Spi6 KO CTLs (Figure 32). Since the substrates cleaved by GrB that trigger apoptosis are located in the cytoplasm (Russell and Ley, 2002), it can be concluded that increased GrB activity in the cytoplasm leads to increased apoptosis in Spi6 KO CTLs. e) Spi6 ensures the integrity of lytic granules by suppressing GrB.

555. In addition to suppressing the activity of cytoplasmic GrB, Spi6 also ensured the integrity of lytic granules. In Spi6 KO CTLs, a decrease in the specific activity of GrB in the granule fraction was observed (Figure 32). Confocal immunofluorescence microscopy (CIM) showed a 2-fold decrease (P = 0.03) in the number OfGrB+ granules in CTLs from Spi6 KO compared to B6 mice (Fig. 5 a and b). 556. A loss of GrB⁺ Pm⁺ granules in Spi6 KO CTLs was also observed.. Unlike GrB, granzyme A

(GrA) does not cleave after aspartic acids but instead is a tiyptase and cleaves after basic amino acids (Odake et al., 1991). Measurement of GrA activity using a specific substrate (Odake et al., 1991) served as an additional marker for granule integrity independent of GrB. There was a 2-fold decrease (P = 9 x 10^"5) in granule-associated GrA specific activity in Spi6 KO CTLs compared to B6 control CTLs (Figure 33 (b)). The defect in Spi6 KO CTLs could be corrected in Sρi6 KO x GrB KO CTLs, as indicated by a complete rescue of GrA specific activity in granules (Figure 33(b)). As such, it can be concluded that the increased 'trfB'kctivity'ltfilie'fcyibipla'sft'Of Spi6 KO is the catalyst for granule breakdown and amplification of GrB- mediated apoptosis. f) Spi6 is an inhibitor of Neutrophil Elastase.

557. The human homologue of Spi6 - Proteinase inhibitor 9 (PI9)- inhibits GrB in CTLs (Sun et al., 1996) and can also inhibit NE (Dahlen et al, 1999) and is expressed in neutrophils (Hirst et al., 2003).

Recombinant Spi6 was generated in the pEX expression system in E. coli as a fusion protein with glutathione transferase (GST), using Standard procedures recommended by the manufacturer (Amersham, Piscataway, NJ). The GST tag was removed by factor X proteolysis and recombinant (r) Spi6 (43 kD) purified to homogeneity. The ability of recombinant Spi6 to inhibit purified human NE (HNE) was examined under pseudo-first order kinetic conditions. SDS-PAGE gel stained by Coomassie blue was used to detect rSpiό (43kD). NE activity was determined by measuring the hydrolysis of MeOSuc-AAPV-AMC (ImM) (Calbiochem, San Diego, CA) at 25 ⁰C in 2OmM Tris-HCl pH 7.4, 50OmMNaCl, 0.1% PEG. HNE activity (2OnM) decreased with time to an endpoint of zero activity in accordance with the exponential decay function [% activity = 100 x e (-k_ObS x t) (k_ObS = observed pseudo-first order rate constant, t = time)] after incubation with recombinant Spi6. HNE incubated alone showed insignificant losses in activity.

Reactions performed at different fixed concentrations (400, 600 and 80OnM) of Spi6 gave pseudo-first order rate constants (k_0bS), which increased in proportion to the Spi6 concentration.

558. An apparent second order inhibition rate constant of 1.0 ± 0.7 x 10⁴ M^-1S^"1 (Figure 34(a)) could be determined for the reaction using the equation: k_ObS = k_app x [Spi6]₀ (k_app = apparent second order rate constant for the inhibition reaction, [Spi6]₀ is the nominal concentration of Spi6).

559. To determine the stochiometry of the inhibition (SI) reaction, HNE (2OnM) was incubated with varying molar ratios of Spi6 (1.25-10-times HNE), insufficient to completely inhibit the enzyme, then inhibition of enzyme activity followed until an endpoint activity was reached. A stochiometry (SI) of ~15:1 was revealed (Figure 34(b)) giving a corrected rate of enzyme inhibition (k) of 1.5± 1.1 x 10⁵ M^-1S^"1. Compared to other serpins such as oi_l -antitrypsin (αi-AT)(k_app = 10⁷ MT¹S^"1, SI = 1:1) (Beatty et al., 1980) and secretory leukoprotease inhibitor (SLPI) (k_app = 2 x 10⁶ M^-1S^"1, SI = 1:1) (Ying et al., 1994).

g) Spi6 is a physiological inhibitor of NE in neutrophils.

560. Real-time PCR confirmed the expression of Spi6 in neutrophils from B6 mice at levels comparable to those in other myeloid and lymphoid cells (Figure 35). To determine this, RNA was extracted from purified cell populations and cDNA synthesized. Neutrophils were elicited from the peritoneum by injection with glycogen (Lopez-Boado et al., 2004) . Cells were isolated from 3 B6 mice, pooled, and realtime PCR for Spi6 and the cyclofilin A housekeeping gene was performed. Histograms are the mean of 3 determinations. 561. To determine NE activity in Sρi6 KO neutrophils, neutrophils were harvested by lavage with

PBS (ImI) of the peritoneum 4h after i.p. injection with 15% glycogen (Sigma Aldrich, St. Louis, MO) then activated for 24 h with E. coli (2xlO6/ml) at 37°C. Cells were lysed by sonication in hypotonic buffer (50 Ml PΪPESt"5UrriM ¹KCl^mM EGTA, 2mM MgC12 5mM DTT, pH 7.6) then centrifuged at 3,000 x g for 20 min to remove nuclei then 15,000 x g for 30 min to give cytosol (supernatant) and granule (pellet) fractions. The granule pellet was resuspended in 1% Triton X-100, 1OmM Tris.HCl, 15OmM NaCl, pH 7.6 for 30 min on ice. NE activity was determined as in Figure 34. Mean specific activity (SA) of NE from neutrophils ± SEM (n= 4 mice).

562. The enzyme assays revealed a 3-fold increase in NE specific activity within the granule and cytosolic compartments of Spi6 KO compared to C57BL/6 (B6) neutrophils after stimulation by E. coli (Figure 36). Thus, Spi6 acts to suppress NE in neutrophils. h) Spi6 is targeted to auzurophilic granules 563. To determine whether Spi6 localizes to auzurophilic granules, transfected HL 60 cells were seeded on wells of poly-L-Lysine coated slides then fixed and permeabilized in acetone/methanol and blocked with 10% normal horse serum in PBS for 60 min at 20⁰C. Cells were stained with anti-FLAG mAb (1:1000; IgGi Sigma Aldrich, St. Louis, MO) or anti-human CD63 mAb (1:1000, Calbiochem, San Diego, CA) or rabbit anti-HNE (1 : 1000, Calbiochem, San Diego, CA) then secondary antibodies conjugated to FITC or PE then DAPI.

564. The intracellular location of Spi6 was determined in HL-60 promyelocytes as described in (Bainton, 1999; Collins et al., 1977), which is hereby incorporated by reference for its teaching of determination of intracellular locations of a gene product. HL-60 cells were transiently transfected by electroporation (0.3kV; 500μF) with Spi6 cDNA cloned into the 3 x FLAG-CMV-14 expression vector (Sigma-Aldrich, St. Louis, MO). Specifically, transfected HL 60 cells were seeded on wells of poly-L-

Lysine coated slides then fixed and permeabilized in acetone/methanol and blocked with 10% normal horse serum in PBS for 60 min at 20⁰C. Cells were stained with anti-FLAG mAb (1:1000; IgG₁ Sigma Aldrich) or anti-human CD63 mAb (1:1000, Calbiochem) or rabbit anti-HNE (1:1000, Calbiochem) then secondary antibodies conjugated to FITC or PE then DAPI. Also, transfected HL 60 cells were seeded on wells of poly-L-Lysine coated slides then fixed and permeabilized in acetone/methanol and blocked with 10% normal horse serum in PBS for 60 min at 2O⁰C. Cells were stained with anti-FLAG mAb (1:1000; IgGi Sigma Aldrich) or anti-human CD63 mAb (1:1000, Calbiochem) or rabbit anti-HNE (1:1000, Calbiochem) then secondary antibodies conjugated to FITC or PE then DAPI. CIM revealed FLAG-tagged Sρi6 not only in the cytoplasm as expected but also with azurophilic granules as indicated by co-localization with CD63 (marker for azurophil granule membrane) and NE (marker for azurophil granule matrix). i) Increased death and lysis of Spi6 KO neutrophils.

565. The effect of increased NE activity on Spi6 KO neutrophils in vitro was also examined. Spi6 KO neutrophils were susceptible to necrosis, as evidenced by increased propidium iodide (PI) permeability (Figure 37(a)) in the absence of apoptosis (Figure 37(b)). This resulted in increased lysis as evidenced by an increase in the release of cytoplasmic lactate dehydrogenase (LDH) (Figure 38). Given the well described ability of NE to damage cells by the digestion protein components (Dar and Crystal, 1999), it was determined that Sρi6 is required to protect neutrophils from self-inflicted damage by NE. ¹J) Increased extracellular NE activity from cultured Spi6 KO neutrophils

566. To determine whether Spi6 KO neutrophils possess increased bactericidal activity, glycogen- elicited peritoneal neutrophils (2x10⁵) were incubated with E. coli (2x10⁶) at 37° C in vitro. The number of viable bacteria measured over time by titer on LB agar plates. Mean titers of E. coli are expressed as a % of titer at t=0 ± SEM (n= 4 mice). In addition to increased intracellular NE activity (Figure 36), increased NE activity in the tissue culture medium of cultured Spi6 KO neutrophils was also observed (Figure 40). k) Increased survival of Spi6 KO mice after acute infection with bacteria

567. The effect of increased neutrophil activity on survival of Spi6 KO mice after bacterial infection was also examined. To do so, the percentage survival of B6 and Spi6 KO mice (n=12) and bacterial titer (+ SEM) after infection with (a) P. aeruginosa (titer in bronchoalveolar lavage, (BAL), (b) E. coli (titer after 12 h) and (C) L. monocytogenes (titer after 48 h). C57BL/6 wild-type or C57BL/6 Spi6 KO mice (6-8 w old; 16-18 g) were infected with lethal doses of either (i.p 50μl) E. coli (4.6 xlO6 cfu/mouse), (i.n. 50μl) P. aeruginosa ( 5 xlO7 cfu/mouse) or (i.v. 50μl) L. monocytogenes EGDe (4 x 104 cfu/mouse) and survival measured over time. 568. Pseudonomas aeruginosa infection was performed by infecting C57BL/6 wild-type and hLP-

PI9-FLAG transgenic i.n. (50μl in PBS in PBS, 25 gauge needle) with P. aeruginosa (5 xlO⁷ cfu) and survival monitored every 12 h for 7 d. Mice that show signs of acute disease (ruffled fur, hunched posture, immobility, and apparent weight loss) were immediately sacrificed. Bacteria were titered from the liver and spleen of euthanized mice by standard overnight LB plate assay. In each survival experiment at least 20 mice were used to achieve statistical significance. In some experiments, mice are dosed with HNE (1.2 -

7U/kg; 50μl in PBS, 25 gauge needle) 4 h before infection. Mice were euthanized and PBS injected (ImI) into the trachea with a tied off blunt 25 gauge needle than the lunge lavaged twice and BAL recovered.

569. All titers were determined on LB plates after incubation at 37° C for 18h. In addition, the serum IFN-γ after infection of Sρi6 KO mice with L. monocytogenes was also examined. To determine serum IFN-γ after infection of Sρi6 KO mice with L. monocytogenes. Mice were infected as in Fig. 42 then the concentration of IFN-γ determined in the serum by ELISA (BD Pharmingen, San Jose, CA). Mean IFN-γ concentration ± SEM (n= 4 mice).

570. An increased survival of Spi6 KO from pneumonia-induced death after infection with the gram-negative bacterium P. aeruginosa (Figure 41(a)) and sepsis-induced death after infection with E. coli (Figure 41 (b)) was observed. In addition, Spi6 KO mice were resistant to lethal infection with gram- positive L. monocytogenes (Figure 41 (c)). The increased survival of Spi6 KO mice was due to increased clearance of bacteria, as evidenced by lower titers in the infected tissues (Figure 41(a)-(c)). NE is required for the clearance of gram-negative bacteria and so increased NE activity in neutrophils can account for the increased clearance in Spi6 KO mice. However, NE is not required for the killing of gram-positive bacteria. 571. L. monocytogenes is a facultative intracellular bacterium, which can escape neutrophil phagocytosis by residing in macrophages (Harty et al., 1996). Consequently, activation of macrophages by ^ττ~ ^τ produced in ThI immune reactions is critical to resolve L. monocytogenes infection. However, this mechanism is unlikely to" expla'm'tne increased clearance of L. monocytogenes in Spi6 KO mice because IFN-γ levels were actually lower than in controls (Fig. 16).

1) Protection of Spi6 KO mice from P. aeruginosa infection

572. To further examine inflammation in Spi6 KO mice the effect of Spi6 deficiency on NE and neutrophils in the lung after P. aerginousa infection was examined. NE activity (mU) and the number of segmented neutrophils were determined for 1ml of BAL from B6 or Spi6 KO mice and are + SEM (n = 3-7 mice). At certain time points mice had died or data was not determined (n.d.). There was a significant difference in the activity of NE (P = 0.03) and neutrophil number (P = 0.002) in B6 compared to Spi6 KO BAL after 12 h.

573. The increased clearance of P. aeruginosa in Spi6 KO mice correlated with about a 2-fold increase in NE activity and 7-fold increase in neutrophil recruitment into the lung alveolar spaces. Table 4 shows the effect of Spi6 deficiency on NE and neutrophils in the lung after P. aerginousa infection. NE activity (mU) and the number of segmented neutrophils were determined for ImI of BAL from B6 or Spi6 KO mice and are ± SEM (n = 3-7 mice). At certain time points mice had died or data was not determined (n.d.). There was a significant difference in the activity of NE (P = 0.03) and neutrophil number (P = 0.002) in B6 compared to Sρi6 KO BAL after 12 h.

Table 4.

574. The protection from P. aeruginosa induced pneumonia in Sρi6 KO mice is due to increased NE-mediated inflammation activity was also directly tested. B6 mice were dosed with HNE then P. aeruginosa immunity measured. In addition, NE activity (mU) and the number of segmented neutrophils were determined for ImI of BAL from B6 mice and are ± SEM (n = 3-8 mice). Mice were infected than after 4h either dosed .(U/kg mouse) i.n. with HNE (+) or not (-). At certain time points mice had died or data was not determined (n.d.). There was a significant difference (P = 0.02) in NE activity after 6 h and neutrophil number (P = 0.02) after 12 h between un-dosed and dosed mice.

575. Doses of HNE up to 1.8U/kg protected B6 mice fromP. aeruginosa induced pneumonia (Figure 43). The protective dose of HNE eliminated P. aeruginosa from the lung (Figure 43). The increase in lung NE activity and neutrophil recruitment associated with protection from P. aeruginosa was comparable to Spi6 KO mice after 6h (Table 5). Thus, the resistance of Sρi6 KO mice to P. aeruginosa- induced pneumonia is due to increased clearance mediated by NE. Table 5.

576. Lung sections from mice after H & E staining. B6 control mice were dosed with 5.9U/kg NE 24 h after infection or with 1.8U /kg HNE 27 d after infection and Sρi6 KO 50 d after infection were observed. Higher doses of HNE (2.4-5.9U/kg), were detrimental to the survival of B6 mice after infection with P. aeruginosa, resulting in considerable damage of lung cellular structure and an increase in alveolar spaces reminiscent of severe emphysema. In contrast, lungs from either SpiόKO mice or B6 mice that received the protective dose of NE (1.8U/kg) showed no gross signs of damage and were indistinguishable from the lungs of uninfected mice. Thus, Spi6 KO mice survive acute bacterial infection because NE activity is increased to a level within a narrow range that increases bacterial clearance without concomitant tissue damage. m) Detection of endogenous Spi6 in mature neutrophils

577. To generate anti-sera, rabbits were immunized with rSpiβ in Complete Freund's adjuvant (5mg) then boosted on d 14, 42 and 56 (O.lmg protein) in Incomplete Freund's adjuvant. After 10 weeks, serum was affinity purified against CnBr-immoblilized rSpiβ (2mg) using standard. To examine the effectiveness of anti-Spi6 anti-serum we performed intracellular staining (ICS) and flow-cytometry (FCM) on glycogen-elicited neutrophils from B6 and Sρi6 KO mice. A 10-fold more intense staining in B6 compared to Spi6 KO neutrophils was observed, which indicates probable efficacy for detecting Sρi6 by CIM. 4. Example 4. Cell biology of NE inhibition by Spi6 in neutrophils a) Examination of the mechanism by which Spi6 associates with neutrophil granules.

(1) Spi6 association with granules via a secretory pathway

578. To determine whether Spi6 associates with granules via a secretory pathway, the. biosynthesis of Spi6 can be examined in HL-60 cells stably transfected with Spi6-FLAG. Cells (2 x

10^s/ml)) can be starved then labeled with ³⁵S-methionine/³⁵S-cysteine (10-25μCi/ml) for 30 min (pulse labeling). In chase experiments, pulse labeled cells can be re-suspended in tissue culture medium (2xlO⁶/ml) and at timed intervals up to 4 h, cells (10⁷/ml) withdrawn and subjected to homogenization and sub-cellular fractionation. Briefly, cells (10^s) can be re-suspended in 0.34M sucrose, 1OmM HEPES pH 7.3, 0.3mM "^{1^} \ and homogenized with a Dounce homogenizer in the presence of a cocktail of protease inhibitors. Unbroken <S81IS"εfod"riUbϊert.ffl: be removed by centrifugation at 500 x g for 10 min then the supernatant fractionated by centrifugation at 32,000 x g for 60 min through a gradient of 20% Percoll containing 15mM HEPES pH 7.3, 0.25mM sucrose on saturated sucrose. About 10 fractions (0.5ml) can be collected from the bottom and the distribution of storage granules/lysosomes (fractions 1-3), golgi-complex (fractions 5-8) and the cytosol (fraction 10) can be confirmed by assaying for the respective markers enzymes - NE (Figure 36), galactosyltransferase and LDH (Figure 38). The distribution of Spi6 can be determined by immunoprecipitation (IP) with anti-FLAG mAb coupled to agarose beads, according to manufacturers protocol (Sigma-Aldrich, St. Louis, MO), then SDS-PAGE and fiuorography. As a positive control for our pulse-chase experiments, the egress of Cat G from the golgi to storage granules after IP with anti-human Cat G antibodies (Calbiochem, San Diego, CA) can be followed.

579. The kinetics of acquisition of endoglycosidase H (endo H) resistance can also be examined. Endo H resistance indicates the conversion of Spi6 into a complex form of the N-linked oligosaccharide unit, as a measure for movement through the golgi. Brefeldin A, which blocks egress from the lumen of the ER to the golgi, can also be used to confirm the progress of Spi6 through the secretory pathway (Nuchtern et al., 1989). To examine the findings in HL-60 promyelocytes, the same pulse-chase and sub-cellular fractionation studies on different cell lines transfected with Spi6-FLAG can be used. These will be the rat basophilic/mast cell line RBL (Gullberg et al., 1995) and the mouse myeloid cell line 32D(Garwicz et al., 1995), using β -hexosaminidase as a maker for the storage granule/lysosome fractions.

(2) Examine role of internal signal sequence in entering the secretory pathway.

580. To examine ther role of an internal signal sequence of Spi6 in entering the secretory pathway can also be examined. PAI-2, like OVA, has only the minimal size to fold as a serpin and both proteins are secreted without cleavage of their signal sequences. Two regions promote PAI-2 translocation (Belin et al., 1996; Belin et al., 2004): an N-terminal hydrophobic domain within the first α-helix and an internal hydrophobic domain spanning the second α-helix (hA and hB domains (Huber and Carrell, 1989).

Comparison of N-terminal peptide sequences revealed two putative hydrophobic domains in Spi6 and PI9 in addition to charged residues (Figure 45). Whether these N-terminal sequences are required for the translocation of Spi6 into the lumen of the ER can be examined using an in vitro microsome system. Linearized templates of Spi6 cDNA in the pSP65 vector (Melton et al., 1984) can be used to generate 32P- labeled Spi6 mRNA (Belin et al., 1989). Spi6 mRNA can then be translated in a wheat-germ extract

(Promega, Madison WI) with or without rabbit reticulocyte microsomes, according to the manufacturers protocol (Promega, Madison WI). Proteins can also be labeled by ³⁵S-methionine incorporation then resolved by SDS-PAGE and visualized by autoradiograpy.

581. Sρi6 has 3 putative N-glycosylation sites (Asn-X-Ser/Thr) and so higher molecular weight glycosylated forms (secreted S- form) indicates translocation across the microsomal membrane. The presence of the S-form of Spi6 can be verified by its resistance to trypsin digestion (lmg/ml; 30 min at O⁰C) in the absence of detergent. To control for the possibility that internal elements within Spi6 may interfere

^■anslocation, a fusion protein in which the yeast prepo-α-factor signal sequence precedes the complete ήpio coαing'-sequence carroe-anaiyzed. Efficient translocation of tlie chimeric protein, as judged by the relative intensities of N-glycosylated versus unmodified protein, can indicate the absence of inhibitory sequences within the Spi6 protein.

582. In addition, the role of the putative internal signal sequences in this process can also be assessed by generating deletion mutants of Spi6 cDNA that lack either the hA (SpiόhA^") or hB (SpiόhB^") domains or both (Spi6hA^"B^~)(Figure 45), but which retain the 3 N-glycosylation sites, using standard molecular biology techniques. Briefly, PCR products encoding deletion mutants can be generated from wild-type Sρi6 open reading frame template using specific primers then cloned into the pSP65 vector. After in vitro transcription/translation the proportion of S- to C-forms of Spi6 can be determined for the mutants, as described above. Furthermore, the binding of the signal recognition protein (SRP) to Spi6 signal peptide my measuring the inhibition of Spi6 translation by the interaction can also be examined. In short, Spi6 can be translated in vitro as described, except without microsomes, and the effect of recombinant human SRP (0-20OnM; Abnova Corp., Heidelberg, Germany) on Sρi6 synthesis tested. As a control, the effect of SRP on the translation of the cytosolic protein sea urchin cyclin, which should not be affected can also be tested. The the binding of SRP to the Spi6 hA/hB deletion mutants can be measured, which allows for determination as to which hydrophobic domain of the signal sequence binds SRP. The binding of Spi6 signal sequence to SRP can be compared with that of preprolactin, which binds with relatively high affinity.

(3) Determine whether Spi6 is a matrix or membrane protein of azurophil granules. 583. To determine if Spi6 is a peripheral membrane protein of azurophil granules an immuno- electron microscopy (IEM) can be used to localize FLAG-Spi6 within the azurophil granules of transfected HL-60 cells. Ultrathin cryosections can be prepared from cells fixed in 0.5% glutaraldeyde / 4% paraformaldehyde. The sections can then be double immunolabeled with rabbit anti-Spiό and mouse anti- human MPO mAb (clone MPO-7, Dako) then incubated with goat anti-rabbit IgG linked to lOnm gold and goat anti-mouse IgG linked to 5nm gold. Cryosections showing expression of Spi6 within the golgi and within MPO-positive granules indicate that Sρi6 is delivered to the matrix of azurophil granules through the secretory pathway. Alternatively, the absence of expression in the golgi and association with the outside of MPO-positive granules indicate that Spi6 is a cytoplasmic peripheral protein of azurophil granules.

(4) Examine the granule association of endogenous Spi6 in neutrophils 584. In neutrophils no common primary amino acid sequence has been identified that determines whether a protein is retained or constitutively secreted. Rather, it is believed that the timing of expression during neutrophil maturation determines destination of proteins to different granule subsets. Association with azurophil granules in HL-60 indicates that some Spi6 is targeted to storage granules.

(5) Determine intracellular localization of Spi6 in neutrophils 585. To determine intracellular localization of Spi6 in neutrophils, CIM with anti-Spiό antiserum can be used to localize Spiό to granules in mature glycogen-elicited neutrophils from B6 mice. Cells can be fixed and permeabilized then stained with anti-Spiό antiserum and anti-rabbit IgG fluorescently labeled SecorMary attfitMIyVTffi optimal' dilutions of primary and secondary antibodies can be determined empirically. Azurophil granules can be localized by counter staining with anti-CD63 (clone CLB-180, BD Pharmingen, San Jose, CA), specific granules with anti-CDllb (clone Ml/70, BD Pharmingen, San Jose, CA), gelatinase granules with anti-macrophage metalloproteinase 9 (rabbit anti-MMP9 antiserum, Abeam, Cambridge, MA ) and secretory vesicles with anti-CD35 (clone 7G6, BD Biosciences, San Jose, CA).

Stained cells can be coated on poly L-lysine slide and CIM used to determine the co-localization of Sρi6 with a given granule subset. In parallel, sub-cellular fractionation can be used to localize Spi6 in mature neutrophils as described in Kjeldsen et al., (1994). Isolation and characterization of gelatinase granules from human neutrophils. Blood 83, 1640-1649. Briefly, glycogen-elicited neutrophils can be harvested from about 10 B6 mice (10⁸/ml) and lysed by cavitation in hypotonic buffer using a nitrogen bomb (5 min at 380 psi, Parr Instruments). Post-nuclear supernatant can be applied to a 3-layer Percoll gradient of densities 1.050 g/ml, 1.090 g/ml and 1.12 g/ml and centrifuged at 37, 000 xg for 30 min at 4°C to resolve the expected protein bands α - (azurophil granules), βl- (specific), β2- (gelatinase) and γ- (secretory vesicle). Fractions can be assayed for marker proteins using antibodies and ELISA: anti-CD63 for azurophil, anti- CDl Ib for specific granules, anti-gelatinase B for gelatinase granules and anti-CD35 for secretory vesicles.

Spi6 will be assayed by IP followed by Western blotting with anti-Spiό antiserum (Figure 44). The presence of Spi6 in fractions positive for a given marker protein indicates the localization of Spi6 to that population of granules.

(6) Examine the expression of Spi6 during neutrophil development. 586. To examine the expression of Spi6 during neutrophil development, the targeting to different sub-sets of granules is determined by when a protein is expressed during neutrophil maturation. Therefore, the granule localization of Sρi6 by determining at which stage during neutrophil development in the bone marrow it is expressed can be determined. Bone marrow can be fractionated on a 2-layer Percoll gradient of densities 1.065 and 1.080 g/ml by centrifugation (1000 x g for 20 min 4°C) as described in Cowland and Borregaard, (1999). Isolation of neutrophil precursors from bone marrow for biochemical and transcriptional analysis. J Immunol Methods 232, 191-200, which is hereby incorporated by reference in its entirety for its teaching of fractionation of bone marrow cells. Cells from band 1 (band and segmented cells), band 2 (myelocytes and metamyelocytes) and band 3 (myeloblasts and promyelocytes) can then be recovered. The identity of neutrophil forms can be confirmed by CIM after staining with anti-CD63 to detect azurophil granules (promylocytes and later forms), anti-CDl Ib (to detect specific granules, anti-

MMP9 to detect gelatinase granules (myelocytes and later forms) and anti-CD35 to detect secretory vesicles. Bone-marrow cells from B6 and Spi6 KO mice will be examined by ICS/FCM using Sρi6 anti-serum (Figure 44). Staining intensity in a sub-set of B6 cells over that of Spi6 KO cells indicates Spi6 expression. Expression as early as in promyelocytes is indicative of Spi6 targeting to azurophil granules. Absence from promyelocytes and expression in myelocytes and later forms is indicative of targeting to specific/gelatinase granules. Expression of Spi6 in only mature segmented cells is consistent with localization to secretory granules. (7) Association of Spi6 with NE during bacterial killing

587. Whether during bacterial digestion, Spi6 is delivered to NE-positive phagolysosomes by the fusion of either specific granules, gelatinase granules or secretory vesicles with azurophil granules can also be examined. Neutrophils can be incubated with flouresecently-labeled E. coli as described in Belaaouaj et al., (1998), which is hereby incorporated by reference in its entirety for its teaching of incubating neutrophils with flouresecently-labeled E. coli. (Figure 36). Spi6 can then be localized with NE and other granule markers during the fusion of granules to form the phagolysosome using CIM as described elsewhere herein. The phagolysosome can be identified as the compartment containing labeled bacteria and NE.

(8) Role of NE in tethering Spi6 to granules 588. Whether Spi6 is tethered to the outside of granules by association with its target serine protease can also be examined. The intracellular localization of Spi6 can be determined in neutrophils from NE KO mice using anti-Spi6 antiserum (see above). The miss-location of Spi6 in NE KO cells indicates a requirement for NE in tethering Spi6 to the storage granules of neutrophils. In addition, whether Cat G or PR-3 also are required for the tethering of Spi6 to storage granules using Cat G KO and PR-3 KO mice can also be examined. In addition, deletion mutants of Spi6 lacking the RCL region (amino acids 328-351) can be generated, which is required for interaction with a target protease. The deletion mutants can be expressed in HL-60 cells and the effect on the targeting of Spi6 to azurophil granules determined as described above. b) Suppression of extracellular NE by Spi6

(1) Azurophil granule stability in Sp6 KO neutrophils. 589. To determine the effect of Spi6 on azurophil granule stability the number of granules in Spi6

KO neutrophils can be measured. Azurophil granules can be visualized by staining for anti-CD63 and CIM (see above). As an additional measure for azurophil granules the number of MPO-positive granules can be measured by cyto-chemical staining for peroxidase then CIM as described in Bainton et al., (1971), which is hereby incorporated by reference in its entirety for its teaching of measuring MPO-positive granules by cyto- chemical staining for peroxidase then CIM. In addition, the cytoplasm of the azurophil granules can be examined for increased NE activity by enzyme assay (Figure 36) on cytoplasmic fractions from Percoll density centrifugation (see elsewhere herein). A decrease in the level of azurophil granules in Spi6 KO neutrophils is indicative of a requirement for Spi6 in ensuring granule stability.

(2) Contribution of NE in granule stability 590. Whether suppression of NE by Spi6 protects azurophil granules from breakdown can also be determined. CIM and sub-cellular fractionation can be used to compare the number of CD63-ρositive granules in neutrophils from Spi6 KO x NE KO and Sρi6 KO mice. As an additional measure for intact azurophil granules the number of MPO-positive granules can be measured by cyto-chemical staining for peroxidase then CIM and by peroxidase assay after sub-cellular fractionation as described above. In addition NE KO neutrophils can be compared with wild-type B6 neutrophils. An increase in the number granules in

NE KO neutrophils indicates that NE-catalyzed breakdown of granules occurs in wild-type neutrophils. ^"(S) Role of NE in neutrophil lysis

591. To determine directly if NE is responsible for the increased lysis of Sρi6 KO neutrophils, the release of cytoplasmic LDH (Figure 38) from Spi6 KO x NE KO neutrophils can be measured.

(4) Role of secreted Spi6 in suppressing extracellular NE. 592. To determine if Spi6 is secreted, the concentration in tissue culture supernatant by IP and

Western with rabbit anti-Spiό serum or anti-FLAG inAb can be measured using the Spi6-FLAG transfectants of HL-60 cells. The results can be confirmed by the results of the experiments above, which determined if Sρi6 is synthesized via the secretory pathway. In addition, mutants of Spi6 can be expressed in HL-60 cells and whether the deletion of the signal peptide abolishes secretion can be determined. 5. Example 5. Spi6 and Immunity to Gram-Positive Bacteria a) Requirement for azurophil proteases the killing of gram-positive bacteria in vitro

593. Spi6 KO mice can be crossed to generate Spi6 KO x NE KO, Spi6 KO x Cat G KO and Spi6 KO x PR-3 KO. The killing of S. aurues in double KO mice can then be compared with B6 wild-type controls, as well as Spi6 KO and NE KO, Cat G KO and PR-3 KO. Glycogen-elicited neutrophils (2x10⁶) can be incubated with S. aurues (2x10⁵) and the number of viable bacteria measured for up to 6 h, by titering colony forming units (cm) overnight on LB plates. In addition, it can be determined whether Spi6 KO neutrophils exhibit increased S. aurues killing and whether it can be abolished by either Cat G or PR-3 deficiency. b) Requirement for azurophil proteases in the clearance of gram-positive bacteria in vivo.

594. The survival of Spi6 KO and azurophil serine proteases KO mice after infection with S. aureus can also be determined. Age, sex and weight match B6 and KO mice can be infected (i.v.) with S. aureus (2-6 x 10⁵ cfu/mouse) and the survival of mice and and titer in the spleen and liver measured every 12 h for up to 7 d.

(a) Staphloccocus aureus infection

595. C57BL/6 wild-type and C57BL/6 Spi6 KO, NE KO, Cat G KO, SpiδKO x NE KO, Spi6 KO x Cat G KO, Spi6 KO x PR-3 KO mice can be infected i.v. by tail vein injection (lOOμl in PBS, 25 gauge needle) with S. aureus (NCTC 12981) cells (2-6 x 105 cfu) and survival monitored every 12 h for 7 d. Mice that show signs of acute disease (ruffled fur, hunched posture, immobility, and apparent weight loss) are immediately sacrificed. For i.v. injection, recipient mice can be placed in a restrainer and the tail warmed with a heat lamp to allow visualization of the tail vein, then sterilized by washing with 70% ethanol. Bacteria were titered from the liver and spleen of euthanized mice by standard overnight LB plate assay. In each survival experiment at least 20 mice are used to achieve statistical significance. These experiments address the role of Spi6 and its target protease in neutrophil immunity to gram-positive bacteria. c; ""D'eter'mine if other azurophil proteases are also inhibited by Spi6.

596. To determine whether the specific activity of either Cat G or PR-3 will increase in activated Spi6 KO neutrophils if these serine proteases are physiological targets of Spi6, glycogen-elicited neutrophils can be activated with S. aureus for up to 6 h, then granule and cytoplasmic fractions obtained after hypotonic lysis (Figure 43). NE activity can be assayed using the labeled substrate MeOSuc-Ala-Ala-Pro-

VaUpNA (2μM) in 2OmM HEPES pH 7.5, 0.15M NaCl, 0.01% BSA, 2% DMSO, 1% DMF at 25° C (Fig 10). Cat G can be assayed using the labeled substrate MeOSuc-Ala-Ala-Pro-Phe-j>NA (ImM) in 0.16M Tris.HCl pH 7.4, 1.6MNaCl at 25° C (Rao et al, 1991). Preliminary experiments with purified enzymes have demonstrated that the substrates used at the concentrations indicated specifically detect either NE or Cat G activity. However, there is no substrate that can detect PR-3 activity without contaminating hydrolysis from NE and Cat G. So PR-3 in the presence of SLPI (0.2μM) can be assayed, which completely inhibits NE and Cat G activity, with Boc~AJ.a-O-.pNA (2μM) in 0.1M HEPES pH 7.5, 0.1M NaCl, 1OmM CaCl₂, 0.05% Triton x-100, 5% DMSO at 25° C (Rao et al., 1991). To determine whether Spi6 suppresses the specific activity of Cat G or PR-3, in vitro inhibition studies can be performed. The k_app and SI of inhibition of recombinant Spi6 can be measured with purified Cat G or PR-3 (Athens Research and Technology,

Athens, Georgia) as done for NE. Comparison with NE indicates whether Spi6 can directly inhibit Cat G or PR-3.

597. Which gram-positive bacteria or fungi PR-3 KO mice are susceptible to can also be determined as well as whether Spi6-deficiency increases resistance to those microbes. 6. Example 6. Requirement for azurophil proteases in immunity to L. monocytogenes

598. Even though L. monocytogenes is a facultative intracellular bacterium, neutrophils are critical for the early control of infection of hepatocytes by killing extracellular bacteria. Therefore the increased resistance of Spi6 KO to L. monocytogenes can be due to increased activity of NE or other putative substrates of Spi6. a) Requirement for azurophil proteases immunity to L .monocytogenes

599. Age, sex and weight match B6 and Spi6 KO x serine protease (NE, Cat G and PR-3) KO mice can be infected (i.v.) with L. monocytogenes (4 x 10⁴ cfu/mόuse) and the survival and titer in the liver and spleen measured every 12 h for up to 8 d (Figure 41(c)). Briefly, C57BL/6 wild-type and C57BL/6 Spi6 KO, MMP-12 KO and Spi6 KO x MMP-12 KO can be infected i.v. by tail vein injection (lOOμl in PBS₅ 25 gauge needle) with L. monocytogenes (EGDe) cells (3-5 x 10⁴ cfu) and survival monitored every 12 h for 7 d. Mice that show signs of acute disease (ruffled fur, hunched posture, immobility, and apparent weight loss) are immediately sacrificed. Bacteria were titered from the liver and spleen of euthanized mice by standard overnight LB plate assay. In each survival experiment at least 20 mice are used to achieve statistical significance. b) Killing of L.monocytogenes by Spi6 KO neutrophils.

600. Glycogen-elicited neutrophils (2xlO⁵) from B6 and Spi6 KO x serine protease (NE, Cat G and PR-3) KO mice can be incubated with L. monocytogenes (2 xlO⁶) and the number of viable bacteria "measure'd iδr "ύpi'tό b n"; Dy tiτermg overnight on brain-heart infusion agar plates (Figure 41(c)). In addition, the specific activity of NE, Cat G and PR-3 in neutrophils activated with L. monocytogenes, can be measured as described elsewhere herein.

7. Example 7. Macrophage function in Spi6 KO mice 601. L. monocytogenes is a facultative intracellular bacterium, which can escape neutrophil phagocytosis by residing in macrophages. Consequently, activation of macrophages by IFN-γ produced in ThI immune reactions is critical to resolve L. monocytogenes infection. This mechanism is unlikely to explain the increased clearance of L. monocytogenes in Sρi6 KO mice because IFN-γ levels were actually lower than controls (Figure 42). NE can degrade tissue inhibitor of metalloproteinase (TIMP)-I, which is an inhibitor of MMP-12. a) Requirement of macrophages for increased immunity of Spi6 KO mice to L. monocytogenes.

602. Macrophages can be depleted from Sρi6 KO mice by liposome-encapsulated clondrate (Lip- CLOD) induced apoptosis as described in Van Rooijen, (1989),which is hereby incorporated by reference in its entirety for its teaching of such.. Briefly, C57BL/6 wild-type and C57BL/6 Spi6 KO mice are injected i.v. with preparations of LIP-CLOD (20-80 μg CLOD/mouse; lOOμl/lOg body weight).

603. Lip-CLOD-depletion has been broadly used in different experimental models to investigate splenic and hepatic macrophage function. Liposomes containing clodronate (dichlorormethylene) can be prepared using standard procedures and i.v. injected into mice (20-80 μg CLOD/mouse; lOOμl/lOg body weight). Pilot experiments to optimize the selective depletion of macrophages by Lip-CLOD can be conducted, which can be verified by staining splenocytes for F4/80⁺ CDl Ib⁺ (macrophages) and control Gr-I⁺ CDl Ib⁺ cells (granulocytes) 4 d after injection and FCM. Depletion in the liver can be verified by immunohistological analysis. Mice can then be infected with L. monocytogenes and clearance measured in Spi6KO and control B6 animals (Figure 41 (c)). b) Role of MMP-12 in immunity of Spi6 KO mice to L.monocytogenes

604. Whether increased MMP-12 activity leads to increased recruitment of Spi6 KO macrophages can also be determined by infecting mice with L. monocytogenes then the number of macrophages (F4/80⁺ CDl Ib⁺) can be measured in the liver over time. Spi6 KO can then be crossed to MMP-12 KO mice to generate Spi6 KO x MMP-12 KO mice to test the role of MMP-12 in recruitment. c) Effect of NE on the recruitment of macrophages in Spi6 KO mice

605. The biochemical basis for the increased recruitment of macrophages in Spi6 KO mice can also be examined by infecting mice with L. monocytogenes and macrophages can be purified from perfused liver by magnetic bead sorting with anti-F4/60 beads (Miltenyi Biotech, Auburn, CA). MMP-12 activity can be determined in macrophages by measuring the degradation of elastin. Briefly, Macrophages (10⁶/ml) are cultured on plates coated with ³H-labeled elastin (Elastin Products, Owensville, Missouri) for 3 days and the amount of elastin degraded measured by determining the production of solubilized ³H-elastin in the medium. oυo. wnetner invir-i is degraded by increased NE activity can be examined by measuring TIMP- 1 activity by reverse zymography in macrophages after L. monocytogenes infection. Briefly, macrophage protein extracts are resolved by PAGE in gelatin (Elastin Products, Owensville, Missouri) (lmg/ml) then incubated in 2.5% Triton X-IOO for 30 min then overnight in 5OmM Tris.HCl pH 8.0, 5mM CaCi₂ ImM ZnCl₂ at 37⁰C with rat uterine explant conditioned medium as a source of MMPs. TIMP-I will protect gelatin from degradation by inhibiting MMP- 12 and so a band of gelatin at 27kD after staining with 0.125% Coomassie blue is present.

8. Example 8. Spi6 and Inflammatory Disease

607. It is widely accepted that deficiency in NE inhibitors such as αl-AT and SLPI exacerbates inflammatory diseases such as COPD. The increase in NE activity that results from Spi6 deficiency protects from pneumonia caused by P. aeruginosa (Figure 41 (a)) and sepsis caused by E. coli (Figure 41 (b)) through increased bacterial clearance. In the case of acute infection with P. aeruginosa a 2-fold increase in extracellular NE (Figure 43), but no higher (Figure 43) was protective. Using transgenic mice that express PI9 in neutrophils, whether increased or decreased expression of the human homologue of Spi6 results in a beneficial outcome after Helicobacter infection can be determined using the techniques described herein as well as techniques known to one of skill in the art. a) Chronic Helicobacter.pylori infection in Spi6 KO mice

608. Infection with the gram-negative bacterium Helicobacter pylori stimulates a novel form of chronic inflammation that is characterized by a massive influx of neutrophils into the gastric mucosa leading to gastritis, peptic ulceration and in some cases gastric cancer.

(1) H. felis model.

609. Mice (6-8 w old) can be infected with H. felis (0.5-1.0 x 10⁹) (ATCC 49179) by gastric intubation on 3 consecutive days as described in Blanchard et al., (1995); Mohammadi et al., (1996); and Walker et al., (2002), all of whih are hereby incorporated by reference for their teachings of such techniques.

610. Briefly C57BL/6 wild-type and C57BL/6 Spi6 KO, NE KO, Cat G KO, SpiόKO x NE KO, Spi6 KO x Cat G KO, Spi6 KO x PR-3 KO, hLP-PI9-FLAG transgenic, hLP-PI9-FLAG transgenic x NE KO mice can be infected with H. felis (0.5-1.0 x 10⁹) (ATCC 49179) in 0.5ml Columbia broth by gastric intubation on 3 consecutive days. Mice are anesthetized and a polypropylene tube inserted down the oesophegus into the stomach. An attached syringe is used to inject the bacteria. Mice are sacrificed from 2 w up until 1Ow after infection and the stomach subjected to histopathological analysis. Mice that show signs of acute disease (ruffled fur, hunched posture, immobility, and apparent weight loss) are immediately sacrificed.

611. After at least 8 weeks, the stomachs of mice are histologically evaluated for inflammation and the level of H. felis. Briefly, strips of the entire curvature of the stomach are cut, fixed in 10% buffered formalin and embedded in paraffin. Sections can then be stained with H&E and Giesma reagents. Gastric sections can be evaluated.in a blinded fashion according to the following criteria. The overall intensity of mllammatioή in' the "IVX microscope field showing the most inflammation can be scored on a scale of 0 to 5 based on the following criteria: 1 = rare inflammatory cells, 2 = multiple clusters of inflammatory cells, 3 = diffuse inflammation of variable intensity with architectural disruption, 4 = diffuse inflammation uniformly severe, without architectural disruption, 5 = diffuse inflammation uniformly severe, with architectural disruption. The extent of inflammation can be determined and expressed as the percentage of the mucosal surface involved in inflammation. In addition, the frequency of crypt abscesses can be determined as evidence for chronic gastritis. To evaluate the character of inflammation, the following cellular components of the inflammatory infiltrate can be graded from 0 to 3: mononuclear cells, polymorphonuclear cells and plasma cells. H.felis does not reliably form discrete colonies and so is difficult to titer by plating on culture dishes. The extent of infection by measuring the average number of H.felis positive glands/cm observed in

Giesma-stained histological sections can also be examined. H. felis can be identified as characteristically curved and confirmed by urease assays after culturing on Columbia agar (Difco) containing 7% horse blood under microaerophilic conditions for 4 d. Both fundus and antrum of the stomach can be scored and averaged to give one value for bacterial infection of each mouse. Sufficient numbers of mice (n > 10) from each group can be examined to give statistically significant results.

(2) Role of Spi6 in H. felis induced inflammation.

612. B6 and Spi6 KO mice can be infected with H. felis then from 1 week until at least 8 weeks evaluated for inflammation and the level of H. felis.

(3) Target protease inhibited by Spi6 in neutrophil immunity to H. felis 613. The role of NE in the phenotype of Spi6 KO mice after infection with H. felis can also be directly examined. Spi6 KO x NE KO mice can be infected with H. felis and onset of gastritis and level of bacteria measured.

614. The infection of Spi6 KO x Cat G KO and Sρi6 KO x PR-3 KO mice with H. felis can also be examined. b) Translation of Spi6/PI9 into therapies to cure chronic inflammatory disease caused by infection

615. Helicobacter pylori infects the gastric mucosa of half the adult population worldwide (1994) and the incidence of antibiotic resistant forms is on the rise. Therefore there is need for alternative cures for diseases such as gastritis and peptic ulcers that are caused by H. pylori. PI9 the human homologue of Spi6 also inhibits NE and is expressed in neutrophils.

(1) H.felis infection of PI9 transgenic mice

616. Whether suppression of NE activity can alleviate disease in PI9 transgenic mice can be determined by amplifying the PI9 open reading frame from PI9 cDNAand cloning the PI9 open reading frame into the 3 x FLAG-CMV-14 expression vector (Sigma Aldrich, St. Louis, MO). DNA encoding PI9 with a C-terminal 3 x FLAG epitope tag can then be cloned into the Eco RI-Bam HI sites of the human lysozyme promoter (hLP) expression cassette, which directs expression in activated neutrophils and macrophages. Tl^-Frøϋ 'transcription is then driven by the proximal promoter of the human lysozyme gene (3.5kB) and terminated by stop sequences provided by human growth hormone gene (2.5kB).

(a) Generation of hLP-PI9-FLAG transgenic mice

617. One-cell embryos from C57BL/6 mice will be microinjected with hLP- PI9-FLAG DNA and implanted into the oviducts of pseudo-pregnant recipients. Transgenic mice can be identified by Southern blots of DNA from tail biopsies probed with hLP-PI9- FLAG DNA. Independent lines of heterozygous hLP-PI9-FLAG mice can be generated by backcrossing to wild-type C57BL/6 mice.

(b) Harvesting of thioglycollate activated macrophages and neutrophils

618. Thioglycollate activated macrophages (CDl Ib⁺ F4/80⁺) and neutrophils (CDl Ib⁺ Gr-I⁺) can be harvested from the peritoneum of hLP-PI9-FLAG transgenic mice and PI9 expression confirmed by ICS/FCM with anti-PI9 antibody. Alternatively, staining for transgenic PI9 using anti-FLAG mAb (Sigma Aldrich, St. Louis, MO) can be performed. (c) Glycogen-elicited neutrophils

619. The effect of transgenic PI9 on NE activity can be examined in glycogen-elicited neutrophils by enzyme assay. C57BL/6 wild-type and C57BL/6 Spi6 KO, NE KO, Cat G KO, Spi6KO x NE KO, Spi6 KO x Cat G KO, Sρi6 KO x PR-3 KO mice can be injected i.p with 15% glycogen in PBS (lml/mouse) using a 20-gauge needle and neutrophils harvested by peritoneal lavage with HBBS after 4 h. These neutrophils can be used to examine the granule association of endogenous Spi6 in neutrophils, the suppression of extracellular NE by Spi6 and the role of Spi6 in the killing of gram-positive Staphloccocus aureus in vitro.

620. Next hLP-PI9-FLAG transgenic mice can be infected with H. felis and bacterial clearance and inflammatory disease can be observed. Additionally, hLP-PI9-FLAG transgenic x NE KO mice can be observed.

(2) Silencing PI9 gene expression in vitro.

621. Whether silencing of PI9 gene expression will alleviate gastritis through protective neutrophil immunity can also be examined by knocking down PI9 expression in transgenic neutrophils using small inhibitory (si) RNAs (Figure 46). In addition, the ability of candidate siRNAs (PI9 1-5) (Dharmacon) to knock down PI9 expression in glycogen-elicited neutrophils from hLP-PI9-FLAG transgenic mice can also be examined. Briefly, neutrophils (2.5 x 10⁵ cells) can be transfected with siRNAs (1-10OmM) using oligofectamine (Invitrogen, Carlsbad, California) and PI9 expression assayed after 24 h. To control for the specificity of PI9 gene silencing irrelevant control (1-3) siRNAs can also be tested (Figure 46).

(3) Effect of silencing PI9 gene expression on H. felis infection. 622. The efficacy of using systemic delivery of siRNA to knock-down gene expression in the lung, liver, and spleen of adult mice has been widely demonstrated. In addition siRNAs can modulate gene expression Ol neutroprms aπerαeiivery to adult mice. This technology can be exploited to knock-down PI9 expression in neutrophils infiltrating the gastric mucosa. As such, hLP-PI9-FLAG transgenic mice (8-10 w old, 20-25 g) can be infected with H.felis. PI9-specific siRNA (50μg) can then be delivered to mucosal surfaces by gastric intubation. The efficacy of delivery of FITC-labeled siRNA can be assessed by CIM analysis of mucosal neutrophils. The reduction of PI9 expression in infiltrating neutrophils can then be assessed by immunohistochemistry on stomach sections and ICS/FCM of disaggregated tissue, using either anti-PI9 antibody or anti-FLAG mAb. The specificity of PI9 gene silencing can be controlled using irrelevant control (1-3) siRNAs (Figure 46). To confirm that any affect of PI9 silencing is through increased NE activity the experiments in hLP-PI9-FLAG transgenic x NE KO mice can be repeated. 623. In addition, the 'hydrodynamic transfer method' can be used to deliver siRNA to the circulation (i.v. injection about 1ml PBS). Intravenous injection of siRNA with cationic liposomes can also be considered if it improves the efficiency of delivery. Alternatively, i.p. injection can also prove to be an effective means to deliver to circulation and stomach.

624. Alternatively, pre-optimized pooled siRNAs (Smart pool reagent, Dharmacon Inc., Chicago, IL) can be used, which reliably reduce target gene expression by at least 80% in 95% of cases

(www.dharmacon. com) .

9. Example 9. Translation of studies into cures for pneumonia caused by bacterial infection.

625. As discussed above, increasing NE activity in the lung within a narrow range, either in Spi6 KO mice or by direct dosing (Figure 43) increases the clearance of P. aeruginosa and alleviates death caused by pneumonia (Figures 41(a) and 43). As has been observed by others before, higher doses NE damaged the lung, resulting in death from an emphysema-like condition (Figure 43). a) Role of MMP12 in the effects of NE on the lung after infection.

626. B6 and MMP 12 KO mice can be infected with P. aeruginosa then after 4 h instilled with HNE (0-7U/kg) (Figure 43). Survival can then be examined every 12 h for 96 h. Bacterial titer, NE activity and leukocyte numbers can be measured in the BAL mice. The lungs of survivors can be measured for signs of gross pathology using standard histological techniques.

627. Additionally, whether increased resistance to HNE translates into higher NE activity in the BAL of surviving mice can be examined. b) Efficacy of increasing lung-neutrophil function by suppressing PI9.

628. hLP-PI9-FLAG transgenic mice are infected with P. aeruginosa then siRNA (50μg) is delivered by intranasal injection in PBS. Every 12 h for 96 h the expression of FLAG-PI9 is measured in neutrophils from the BAL to determine the optimal conditions to knock-down PI9 expression. The delivery of siRNA and ablation of gene expression in lung neutrophils has been achieved by others using this protocol, see for example Lomas-Neira et al., (2005), which is hereby incorporated by reference in its entirety for its teaching of delivery of siRNA and ablation of gene expression in lung neutrophils. 10. Example 10. Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of lytic granules a) Methods

(1) Mice 629. Spi6 cDNA (Phillips et al., 2004) was used to clone mouse genomic DNA containing the

Spi6 locus (Serpin b9) from a bacterial artificial chromosome (BAC) library (129/Sv strain RPCI-22, Res Gen). The 5 ' homology region (4.1 kb Sac //- Kpn I fragment) and the 3 ' homology region (3.6 kb Not I— Xlio I fragment) were cloned on either side of the neo gene (1.8 kb Kpn I- Not I fragment) flanked by loxP recombination sites (Kuhn et al., 1995). C57BL/6 ES cells (Ware et al., 2003) were transfected with targeting vector (50μg). DNA from G418 resistant clones was digested with Spe /and Hind III and hybridized with probes to detect wild-type (5'- 7.6kb, 3'- 9.8kb) and mutant alleles (5'-6.9 kb, 3'-5.8 kb). ES cells from targeted clones (2/398) were transfected (30μg) with Cre (pBS185, Invitrogen, Carlsbad, California) and excision of the neo after loxP site-specific recombination was detected by the presence of a 5.8kb band after blotting with 5'- probe (Kuhn et al., 1995). C57BL/6 ES cells were microinjected into BALB/c blastocysts to produce chimeric mice, which were then backcrossed against wild-type C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). C57BL/6 Spi6^+/~ mice (from 2 independently targeted ES cell clones) were intercrossed to generate C57BL/6 Spi6^~f~ mice (Sρi6 KO mice), which were born at a Mendelian frequency.

630. Sρi6 KO mice were crossed with GrB-/- C57BL/6 (GrB KO) (Jackson Laboratory, Bar Harbor, ME) to generate C57BL/6 Spi6-/- GrB-/- mice (Spi6 KO x GrB KO). C57BL/6 P14 TCR+/- transgenic mice (B6 P14 mice) (Pircher et al., 1990) were crossed with either Spi6 KO or Spi6 KO x GrB KO mice to produce C57BL/6 Spi6 KO P14 TCR+/- (Spi6 KO P14 mice) or C57BL/6 Spi6 KO x GrB KO P14 TCR+/- (Spi6 KO x GrB KO P14 mice) transgenic mice. C57BL/6 CD45.1 congenic mice were purchased from Taconic. AU mice were maintained and bred under standard specific pathogen free (SPF) conditions.

(2) CTLs

631. Spleen cells (10⁶/ml) from B6 P14 mice, Sρi6 KO P14 mice or Spi6 KO x GrB KO P14 mice were cultured with LCMV gp33 peptide [KAVYNFATM] (10^"6M) and IL-2 (lOU/ml) for 2d to generate CTLs as described by Phillips et al., (2004), which is hereby incorporated by reference in its entirety for its teaching of such. To inhibit GrB, Z-AAD (OMe)-CMK (12.5μM; Sigma Aldrich, St. Louis, MO) was added to cultures. To measure ex vivo CTL-activity, RMA targets were pulsed with gp33 and labeled with ⁵¹Cr- and incubated with viable splenic leukocytes over a range of ratios. The specific release was determined after 4 h as follows: % specific release = (specific release-spontaneous release)/(maximum release- spontaneous release) x 100. (3) Western blotting

632. Antiserum specific to a Spi6 peptide (amino acids 35-47), ([CjRKLNKPDRKYSLR) was raised in rabbits using standard procedures as described in Coligan et al., (1995). Current Protocols in intoύnόlbgy ,¹¹VoI Wiley and Sons), which is hereby incorporated by reference in its entirety for its teaching of raising antiserum specific to a peptide in rabbits. Briefly, two rabbits were immunized with the peptide conjugated to EXH and then boosted twice with immunogen over a period of 3 months. Anti-Spi6 antibodies were affinity purified on columns of immunizing peptide, eluted in 3M KSCN and then dialyzed against PBS.

633. CTLs were lysed by sonication in hypotonic buffer (50 mM PIPES, 5OmM KCL, 5mM EGTA, 2mM MgCl₂ 5mM DTT, pH 7.6) then centrifuged at 3,000 x g for 20 min to remove nuclei then 15,000 xg for 30 min to give cytosol (supernatant) and granule (pellet) fractions. The granule pellet was resuspended in 1% Triton X-100, 1OmM Tris.HCl, 15OmM NaCl, pH 7.6 for 30 min on ice. Protein (50μg) was resolved by reducing SDS-PAGE then immunoblotted and probed with anti-Spi6 antiserum (7μg/ml) and goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (2μg/ml) (Sigma-Aldrich) then visualized by chemiluminescence (ECL-kit, Amersham, Piscataway, NJ). Blots were also probed with goat anti-mouse GrB (0.2μg/ml) (R&D Systems) and anti-goat IgG HRP (2μg/ml) (Sigma-Aldrich, St. Louis, MO) or with anti-actin monoclonal antibody clone ACTN05 (0.5μg/ml ) (Sigma Aldrich, St. Louis, MO) and anti-mouse IgG-HRP(2μg/ml) (Sigma-Aldrich, St. Louis, MO).

(4) Protease assays

634. Colorimetric assays for GrB were performed in reaction buffer using Ac-IEPD-pNA at 0.2mM and GrA using BLT ( N^a - benzyloxycarbonyl-L-lysine thiobenzyl ester at 0.2mM and Ellman's reagent at 1.76 mM in reaction buffer (10OmM Tris-HCl ) at 30 °C. Assays for caspase 3 were performed in reaction buffer (1OmM PIPES pH 7.4, 8mM DTT, 2mM EDTA, 0.1% CHAPS) at 30°C using Ac-DEVD-

/7NA (Calbiochem, San Diego, CA) at 0.2mM. Specific activity was determined by normalizing for the amount of protein. For GrB and GrA, an activity unit (U) is defined as the amount of enzyme that will generate 1 nmol of />NA per min and for caspase 3 an activity unit (U) is defined as the amount of enzyme that will generate 1 pmol ofpNA per min. (5) Fluorescence microscopy

635. P14 CTLs were seeded on wells of poly-L-Lysine coated slides, then fixed and permeabilized in acetone/methanol and blocked with 10% normal horse serum in PBS for 60 min at 20⁰C. P14 CTLs were stained with anti-murine GrB Ab (1 : 200; R&D Systems, Minneapolis, MN) and anti-murine Pfn Pl-8 mAb (1 : 100; Kamiya Biomedical Co., Seattle, WA) for 60 min at 20⁰C in 2% BSA/PBS. Slides were washed twice with 1 mM MgC12 PBS and once with PBS. Cells were incubated with biotin-conjugated mouse anti-

Rat mAb (1: 100; BD Pharmingen, San Jose, CA) and FITC conjugated rabbit anti-goat antibody (1: 200; Molecular Probes, Carlsbad, CA) for 60 min at 20⁰C. After washing with PBS, cells were further incubated with streptavidin-PE (1 μg/ml; BD Pharmingen, San Jose, CA) together with DAPI (Sigma Aldrich, St. Louis MO) and then washed and mounted in Vectashield mounting medium (Vector Labs, Burlingame, CA). Stained cells were analyzed using a Leica SP2 AOBS spectral laser scanning confocal microscope operated with the software LCS 2.5vl347. The mean number of granules was determined from the counting of multiple cell layers (100 cells in 3 separate counts, n=300). (J6) Infections

636. Mice were infected by either i.p. injection ( 2xlO⁵ pfu) of LCMV Armstrong or i.v. injection (10^δ pfu) of the clone 13 variant of LCMV Armstrong. For LM, mice were infected by i.v. injection of DPL-1942,(10⁵ cfu), which has been engineered to express ovalbumin and in C57BL/6 mice generates the H-2K^b-restricted peptide epitope (SIINFEKL : OVA). LCMV was titered on monolayers of Vero cells.

(7) Flow cytometry

637. The following mAbs were used (BD-Pharmingen): anti-CD8α(allophycocyanin [APC]- labeled), anti-CD45.2-fluorescein isothiocynate [FITC] or R-phycoerythrin [PE], anti-BrdU-FITC (IgGi) and IgGi isotype control-FITC . H-2D^b-tetramers with gp 33 or H-2K^b -tetramers with OVA [SIINFEKL] were labeled with streptavidin-PE (Beckman Coulter, Fullerton, CA). Splenocytes were prepared and stained with tetramers and mAbs as before. Apoptosis of live cells was measured with YOPRO-I dye (green fluorescence) (Molecular Probes, Carlsbad, CA), as before. Intracellular staining with anti-BrdU mAb was used to determine BrdU incorporation in CD45.2⁺ gp33⁺ CD8⁺ cells, as before.

(8) Adoptive transfer 638. Naϊve CD8⁺ cells were purified (>90%) from the spleens of P14 mice (CD45.2⁺) by positively sorting with anti-CD8 magnetic beads (Miltenyi Biotec) and adoptively transferred (10⁴) by i.v. injection into C57BL/6 CD45.1 mice and after 1 d infected with LCMV. To measure P14 CD8 T cell division, recipients were given BrdU in their drinking water (0.8mg/ml) for 8 d after LCMV infection.

(9) Statistics 639. The significance of difference was measured using two-tailed Student's t-tests. b) Results

(1) Impaired survival of Spi6-deficient CTLs

640. To address the role of the GrB inhibitor - Spi6 - in the protection of CTLs from self-inflicted damage, mice were generated. Using homologous recombination in ES cells from C57BL/6 mice (B6 mice) exon 7 was deleted, which encodes 60% ofSpiό and includes the critical reactive center loop (RCL), which is required for target protease inhibition (Figure 27(a)). Using Cre-mediated recombination, the G418-resistance cassette was removed to avoid affecting the transcription of closely linked serpin genes (such as SpilS), which may have similar functions to Spi6. Mice homozygous for Spiό mutant alleles in the C57BL/6 (B6) background (Spi6 KO mice) were generated as described above. Spi6 KO mice did not display any significant difference in the number of myeloid or lymphoid cells in the thymus or blood.

641. Spi6 is up-regulated in CTLs and over expression can inhibit Grb-mediated apoptosis in vitro. To examine the physiological role of Spi6, the CTL response of Spi6 KO mice to infection was examined. Mice were infected with LCMV Armstrong and the attenuated DPL- 1942 strain of LM, which had been engineered to express ovalbumin and generate an H-2K^b-restricted peptide antigen (OVA). To avoid the effects of persistent antigen on interferon-driven apoptosis, doses of LCMV Armstrong (2x10⁵ pfu) and LM DPL-1942 (10⁵ cfu) that result in complete clearance in both B6 and Spi6 KO mice after 8 d were useα. staimng-witn gp 3J/n-2D^b-tetramers revealed that the number of CTL specific for LCMV Armstrong was diminished in Spi6 KO mice compared to B6 controls (5-fold lower) (Figure 47(a) and (b)). Furthermore, Spi6 KO mice harbored about 9-times less LM-specific CTLs after infection (Figure 47(d) and (e)). The lower number of CTLs in Spi6 KO mice correlated with an increase in the onset of apoptosis of both LCMV (Figure 47 (a) and (c)) and LM-specific CD8 T cells (Figure 47 (d) and (f)), as evidenced by increased staining with the DNA dye YOPRO-I, which detects early chromosomal changes in apoptosis. Thus, Spi6 is required for the survival and protection of CTLs from apoptosis in vivo.

(2) Defective survival of Spi6 KO CTLs is GrB-dependent and cell autonomous 642. Whether inhibition of GrB was a physiological mechanism by which Spi6 ensured the survival of CTLs by examining the effect of GrB deficiency in Spi6 KO mice was also examined. After infection, the number of LCMV-specific CTLs in GrB KO mice are the same as in wild-type mice, possibly because other homeostatic factors also act to maintain a normal clonal burst size. It was observed that there was a 4-fold decrease in the recovery (Figure 48 (a)) and a significant (JP = I x 10^"8) increase in apoptosis (Figure 48 (b)) of LCMV-specific CTLs in Spi6 KO mice compared to B6 controls. In Spi6 KO x GrB KO mice the number (Figure 48 (a)) and proportion of CTLs undergoing apoptosis (Figure 48 (b)) were the same as B6 controls. Thus, the absence of GrB corrected the deficit in CTL survival caused by Spi6 deficiency and returned the level of LCMV-specific CTLs to wild-type levels. GrB KO mice have partially reduced expression of the linked granzymes C and F, which may also contribute to the correction of CTL survival in Spi6 KO mice. However, Sρi6 directly inhibits purified GrB in vitro and protects perforin-loaded cells from purified GrB. Therefore, protection from GrB is at least in part a physiological mechanism by which Spi6 ensures the survival of CTLs after infection.

643. Spi6 can protect DCs from granule-mediated killing by CTLs, and so reduced expansion of CTLs caused by defective priming may occur in Sρi6 KO mice. Thus, it was determined whether the requirement for Spi6 for CTLs survival was cell autonomous. Sρi6 KO mice were crossed with C57BL/6

P 14 transgenic mice, which express a T cell receptor (TCR) specific for the gp33 peptide antigen of LCMV in the context of H-2D^b. Naϊve P14 CD8 T cells (>90% pure) from Spi6 KO and B6 mice (CD45.2⁺) were adoptively transferred to B6 CD45.1 congenic recipients, which were then infected with LCMV. After 8 d, the number of donor LCMV-specific CTLs (CD45.2⁺) from Spi6 KO mice was diminished (14-fold) compared to B6 P14 donor cells (Figure 48 (c)). Therefore, the requirement for Spi6 in CTLs is cell intrinsic. As observed in the whole animal experiments, a significant (P = 0.04) increase in the proportion of donor Spi6 KO CTLs undergoing apoptosis after adoptive transfer to wild-type recipients was observed (Figure 48 (d)). There was, however, no significant difference (P = 0.2) in the rate of cell division of donor Spi6 CD8 T cells compared to B6 controls over the 8 d expansion period after LCMV infection (Figure 48 (e)). Thus, the diminished recovery of donor Sρi6 KO CTLs is a result of impaired survival due to increased apoptosis rather than impaired expansion. (3) Impaired CTL-immunity to virus in Spi6-deficient mice

644. Cytotoxic T cells are critical for immunity to virus and so we examined the ability of Spi6 KO mice to mount a CTL response and clear LCMV. The lower number of LCMV-specific CTLs resulted in significantly (P = 4 x ICT⁵) impaired CTL-activity in Spi6 KO mice (8.7 ± 0.25 % specific lysis, n=5 mice) in ex vivo assays (Splenocyte/Target ratio = 5.5) compared to B6 mice (24.7 ± 1.2 % specific lysis, n=5 mice) (Figure 49(a)). The requirement for Spi6 in immunity to high doses (10⁶ pfu) of the clone 13 variant of LCMV Armstrong was examined, which has previously revealed deficiencies in CTL-immunity to virus. The clearance of the clone 13 LCMV from the spleen of Sρi6 KO mice was impaired, as evidenced by a 5-fold increase in titer 6 d after infection (Figure 49(b)). Therefore, the defect in CTL survival leads to impaired immunity to virus in Sρi6 KO mice.

(4) Spi6 suppresses cytoplasmic GrB in CTLs

645. It has been suggested that leakage of GrB from granules into the cytoplasm can lead to the activation induced cell death of cytolytic lymphocytes. Therefore whether Spi6 was required to protect against this pathway of death in CTLs was also examined. LCMV-specific CTLs were generated by culturing spleen cells from P14 mice with gp33 for 2 d in vitro (>90% of viable cells), then subjected to subcellular fractionation. Western blots after reducing SDS-PAGE revealed Spi6 in the cytoplasm but not granule fractions of B6 CTLs. The sub-cellular localization of Spi6 to the cytoplasm is consistent with the lack of a secretory signal, as has been observed for its human homologue PI9 in CTLs. In these experiments, Spi6 was absent from Spi6 KO CTLs. 646. A 67 kD form of Sρi6 was present in the cytosol of B6 CTLs but not GrB KO CTLs. Probing

Sρi6 KO CTLs with anti-GrB antiserum failed to detect the 67 kD form, confirming it as a SDS-stable complex between Spi6 and GrB. Enzyme assays revealed significantly (P = 0.004) increased specific activity of GrB in the cytoplasm of Sρi6 KO CTLs (Figure 50). Consistent with its specificity as serpin of GrB, it can be concluded that Spi6 inhibits cytoplasmic GrB through the formation of covalent complexes. Enzyme assays also revealed a lower specific activity of the caspase 3 executioner protease in the cytoplasm of GrB KO CTLs, indicating that endogenous GrB induces apoptosis of CTLs ((Figure 48 (b)). There was a corresponding increase in the specific activity of caspase 3 in the cytoplasm of Spi6 KO CTLs (Fig. 50). Since the substrates cleaved by GrB that trigger apoptosis are located in the cytoplasm, it can be concluded that increased GrB activity in the cytoplasm leads to increased apoptosis in Spi6 KO CTLs. (5) Inhibition of GrB by Spi6 ensures the integrity of lytic granules

647. In addition to suppressing the activity of cytoplasmic GrB, Spi6 also ensured the integrity of lytic granules. In Spi6 KO CTLs, a decrease in the amount and specific activity of GrB in the granule fraction (Figure 50) was observed. Immunofluorescence studies showed a 2-fold decrease (P = 0.03) in the number OfGrB⁺ granules in CTLs from Spi6 KO compared to B6 mice (Figure 51 (a) and (b)). A loss of GrB⁺ Pfii⁺ granules in Spi6 KO CTLs was also observed (Figure 51(c)), implying that Spi6 prevents GrB leakage into the cytoplasm by ensuring general granule integrity. The defect in granule integrity was GrB- dependent because incubation with the GrB-specific inhibitor Z-AAD-CMK gave a significant (P = 2 x 10^"6) increase in the number of granules in Spi6 KO CTLs (Figure 51(d)). GrA does not cleave after aspartic acids and M, uffiM^rB^sWtrypteetad cleaves after basic amino acids. Measurement of GrA activity using a specific substrate served as an additional marker for granule integrity independent of GrB. There was a 2- fold decrease (P = 9 x 10^~5) in granule-associated GrA specific activity in Spi6 KO CTLs compared to B6 control CTLs (Figure 51(e)). The defect in Spi6 KO CTLs could be corrected in Spi6 KO x GrB KO CTLs, as indicated by a complete rescue of GrA specific activity in granules (Figure 51 (e)). The significant (P =

0.004) increase in granule-specific GrA activity in GrB KO CTLs over that of the level in B6 CTLs (Figure 51(e)), implies that GrB-catalyzed breakdown of granules also occurs in wild-type CTLs. c) Conclusion

648. Studies that describe the expression of PI9 and Spi6 in leukocytes and inflammatory sites have lead to the suggestion that serpins protect against GrB during immune reactions. These observations have been complimented by ectopic over-expression studies that show the potential for Spi6 in protecting from GrB-mediated death. However, artificially high levels of Sρi6 expression were required for cyto- protection, and so the physiological requirement for Sρi6 was not tested. To address this issue, we generated Spi6 KO mice and examined the viability, function and structure of CTLs. 649. The experiments above show that Spi6 protects CTLs from their own GrB by suppressing activity in the cytoplasm. The potency of GrB as a CTL effector molecule likely explains why less than a 2- fold increase in cytoplasmic GrB activity (Figure 50) results in the near complete loss (10-20% of wild-type level) of Spi6 KO CTLs in vivo (Figure 47). GrB kills by triggering multiple apoptotic pathways involving caspases and mitochondrial dysfunction in addition to DNA fragmentation. By directly targeting GrB, Spi6 can potentially suppress all of the toxic effects of a CTL effector molecule. It has been suggested that inhibition of another CTL effector molecule - Pfn - by cathepsin B also protects CTLs from self-inflicted damage. However, in vivo studies indicate that cathepsin B induces the apoptosis of CTLs. Thus, Spi6 seems to be unique as a physiologically relevant inhibitor of self-inflicted damage by CTLs.

650. The expression of peptide antigen/MHC (pMHC) on CTLs after infection by virus or after re- expression of target cell pMHC after killing can lead to fratricide. However, these findings with Spi6 are consistent with the induction of suicide by endogenous GrB in the cytoplasm of CTLs and NK cells. In these studies, it was found that Spi6 KO CTLs undergo increased apoptosis because their own granules release GrB into the cytoplasm. In addition, GrB-mediated granule breakdown likely also occurs in wild- type CTLs. This was verified by visualization OfGrB⁺ granules in P14 CTLs by immunofluorescence microscopy. Nuclei were visualized by DAPI. Visualization revealed as Granzyme B was present in the cytoplasm of of Spi6 KO cells as well as B6 cells, Visualization OfGrB⁺PSi⁺ granules was also performed and revealed the presence OfGrB⁺ Pfti⁺ granules in the cytoplasm of B6 cells as well as Spi6 KO cells. (See also Figure 51). Although these studies do not rule out the possibility that fratricide of CTLs as a physiological mechanism for clearing CTLs in vivo, they do show that GrB-mediated death can occur in wild-type CTLs without fratricide. 65 h" "-^lt "-(EytotcH^ l^J!c"eHs 'from patients with Chediak-Higashi and Griscelli syndromes and from the relevant mouse models (Beige and Ashen) have defective granule killing. This is due to mutations that disrupt the secretion of granzymes but do not diminish the number of lytic granules.

11. Example 11. A protective inflammatory response against sepsis 652. Inflammation protects against microbial infection and yet in sepsis inflammation becomes detrimental. Thus, sepsis in mice deficient in a neutrophil elastase (NE) inhibitor called Serine Protease Inhibitor 6 (Spi6) was examined. Increased NE activity resulted in hyperactive neutrophils from Spi6- deficient mice, which protected from lethal sepsis through increased clearance of both gram negative and positive bacteria. a) Methods

(1) Mice

653. Sρi6 KO mice were generated in the C57BL/6 background by homologous recombination (1). Wild-type C57BL/6 mice (B6 mice) were purchased from Jackson Laboratory (Bar Harbor, ME). All mice were maintained and bred under standard specific pathogen free (SPF) conditions. (2) Neutrophil sub-cellular fractionation

654. Neutrophils were harvested as described by Belaaouaj et al, Science, 289, 1185 (2000) which is hereby incorporated by reference in its entirety for its teaching of harvesting neutrophils. Briefly, neutrophils were harvested by lavage with PBS (ImI) of the peritoneum 4h after i.p. injection with 15% glycogen (Sigma Aldrich) then activated for 24 h with LPS. Cells were lysed by sonication in hypotonic buffer (50 mM PIPES, 5OmM KCL, 5mM EGTA, 2mM MgCl₂ 5mM DTT, pH 7.6) then centrifuged at

3,000 x g for 20 min to remove nuclei then 15,000 x g for 30 min to give cytosol (supernatant) and granule (pellet) fractions. The granule pellet was resuspended in 1% Triton X-100, 1OmM Tris.HCl, 15OmM NaCl, pH 7.6 for 30 min on ice. Protein concentration was determined by Lowry assay (Biorad)

(3) Protease assays 655. Colorimetric assays for NE were performed on purified human (H) NE (Athens Research and

Technology, Athens, Georgia), neutrophil extracts or BAL using a labeled peptide substrate. NE activity was determined by measuring the hydrolysis of MeOSuc-AAPV-jpNA (ImM) (Calbiochem) at 25 ⁰C in 2OmM Tris-HCl pH 7.4, 50OmM NaCl, 0.1% PEG. Hydrolysis was measured at exitation 38OnM and emission 44OnM using a Hitachi F-200 flourescence spectrophometer. HNE activity (2OnM) decreased with time to an endpoint of zero activity in accordance with the exponential decay function after incubation with recombinant Spi6. % activity = 100 x e (-k_obs x t) (k_obs = observed pseudo-first order rate constant, t = time).

656. HNE incubated alone showed insignificant losses in activity. Reactions performed at different fixed concentrations (200, 400 and 60OnM) of Spi6 resulted in similar exponential losses in activity, yielding observed pseudo-first order rate constants, which increased in proportion to the Spi6 concentration. From the slope of the latter proportional dependence, an apparent second order inhibition rate constant of 1.8 ± 0.7 x 10⁴ M^-1S^"1 could be determined for the reaction k_obs = k_app x [Sρi6]₀ (k_app = apparent second order rate constant for the inhibition reaction, [Spi6]₀ is the nominal concentration of Spi6 in the reaction). 037." "' WdeMrϊiϊlne me stoichiometry of the inhibition (SI) reaction, HNE (2OnM) was incubated with varying molar ratios of Spi6 (1.25-10-times HNE)₅ insufficient to completely inhibit the enzyme, then inhibition of enzyme activity followed until an endpoint activity was reached. The percentage of endpoint activity (y-axis) was plotted against [Spi6]/[HNE (x-axis). The abscissa intercept of this plot indicated that complete inhibition of elastase activity required ~15 moles of Spi6 per mole of enzyme

658. To determine the specificity of the MeOSuc-AAPV-j>NA for NE in cell extracts we compared the rate of hydrolysis by HNE (0.1U) with the same amounts of the related serine proteases human cathepsin G and PR-3 (Athens Research and Technology, Athens, Georgia). The hydrolysis of MeOSuc-AAPV-^NA by cathepsin G and PR-3 was <10% of that for HNE. (4) Fluorescence microscopy

659. HL-60 cells were transfected by electroporation (0.3kV; 500μF) with Spi6-FLAG cloned the 3 x FLAG-CMV- 14 expression vector (Sigma-Aldrich, St. Louis, MO) then seeded on wells of poly-L- Lysine coated slides then fixed and permeabilized in acetone/methanol and blocked with 10% normal horse serum in PBS for 60 min at 20⁰C. Cells were stained with rabbit anti-HNE (1:1000, Calbiochem) or anti- FLAG mAb (1 : 1000; IgGi Sigma Aldrich, St. Louis, MO) for 60 min at 2O⁰C in 2% BS A/PBS. Cells were washed then stained with the appropriate secondary antibodies conjugated to FITC or PE (Molecular Probes, Eugene, OR) together with DAPI. Cells were mounted in Vectashield mounting medium (Vector Labs, Burlingame, CA). Stained cells were analyzed using a Leica SP2 AOBS spectral laser scanning confocal microscope operated with the software LCS 2.5vl347. (S) Neutrophil bactericidal activity in vitro

660. Glycogen-elicited peritoneal neutrophils (2xlO⁵) were incubated with E. coli ((4) (2xlO⁶) at 37° C in vitro. The number of viable bacteria measured over time by titer on LB agar plates. The mean percentage titer of E.coli (was determined over time with the titer at t=0 100%. HNE (0.12U) was added to E. coli (10⁶) in 0.1ml and the titer measured after 4 h. The viability of neutrophils (10⁶/ml in PBS) was determined by staining with PI (death) and YOPRO-I (apoptosis) as according to manufacturer's instructions (Molecular Probes, Eugene, OR). The lysis of cells was determined by assaying LDH activity in tissue culture supernatant according to the manufacturer's instructions (Promega, Madison WI).

(6) Bacterial infection

661. C57BL/6 wild-type or C57BL/6 Spi6 KO mice (6-8 w old; 16-18 g) were infected with lethal doses of either (i.p 50μl) E. coli (4.6 xlO⁶ cfu/mouse)(¥), (i.n. 50μl) P. aeruginosa ( 5 xlO⁷ cfu/mouse) or

(i.v. 50μl) L. monocytogenes EGDe( 4 x 10⁴ cfu/mouse) and survival measured over time. All titers were determined on LB plates after incubation at 37° C for 18h. Leukocyte numbers were determined in BAL using standard cytometric analysis after lethal infection with P. aeruginosa. Mice were dosed by i.n. injection with HNE (Athens Research and Technology, Athens, Georgia) 4 h prior to lethal P. aeruginosa infection. Lung histology was performed on PFA fixed sections stained with H and E. B) 'MesiHts

(1) Effect of increased NE activity on Spi6 KO neutrophils in vitro

662. The effect of increased NE activity on Spi6 KO neutrophils in vitro was examined. Spi6 KO neutrophils were found to be susceptible to necrosis, as evidenced by increased propidium iodide (PI) permeability (Figure 52(a)) and lactate dehydrogenase (LDH) release (Figure 52(b)), in the absence of apoptosis (Figure 52(c)). Also observed was an increased bactericidal activity of Spi6 KO neutrophils against E. coli (Figure 52(d)). The direct addition of HNE could kill E. coli (Figure 52(e)). Therefore, increased NE activity is at least in part responsible for the increased bactericidal activity of Spi6 KO neutrophils. (2) Effect of increased neutrophil activity on survival of Spi6 KO mice after bacterial infection

663. The effect of increased neutrophil activity on survival of Spi6 KO mice after bacterial infection was also examined. An increased survival of Spi6 KO from septic shock induced death after infection with either gram-negative bacteria E. coli (Figure 53(a)) and P. aeruginosa (Figure 53(b)J was observed. In addition, Sρi6 KO mice were resistant to septic shock induced death following infection with gram-positive L. monocytogenes (Figure 53 (c)). The increased survival of Spi6 KO mice was due to increased clearance of bacteria, as evidenced by lower titers in the infected tissues (Figure 53(a)-(c)). NE is required for the clearance of gram-negative bacteria therefore increased activity in neutrophils from Spi6 KO mice can account for the increased clearance and survival. However, NE is not required for the killing of gram-positive bacteria.

(3) Effect of P. aeruginosa infection in the lung of Spi6 KO mice and protective dosing of HNE

664. The increased clearance of P. aeruginosa in Spi6 KO mice correlated with about a 2-fold increase in NE activity and 7-fold increase in neutrophil recruitment into the lung interstitium (Table 6). Table 6

665. Histological examination confirmed the increase in influx of inflammatory neutrophils, macrophages and lymphocytes into alveolar spaces as early as 6 h after infection of Spi6 KO mice. Direct testing on whether the protection from P. aeruginosa induced sepsis in Spi6 KO mice is due to increased NE-mediated inflammation activity was also perfomed. B6 mice were dosed with HNE then P. aeruginosa Λ -i-i ϊrπmtmitymeasureα'r'jj'Θscs oϊ'ruNii up to 1.8U/kg protected B6 mice from P. aeruginosa induced septic- death (Figure 53(d)) and resulted in increased lung NE activity and neutrophil recruitment after 6 h at levels over those of controls comparable to Spi6 KO mice (Table 7).

Table 7

666. A protective dose of HNE was determined to eliminate P. aeruginosa from the lung (Figure 53(d)). Higher doses of HNE (2.4-5.9U/kg), were detrimental to the survival of B6 mice after infection with P. aeruginosa (Figure 53(d)). Histology on lung sections from mice after H & E staining was performed on B6 dosed with 5.9U/kg NE 24 h after infection, B6 dosed with 1.8U /kg HNE 27 d after infection and (III) Spi6 KO 5O d after infection. Histological results revealed considerable damage of lung interstitium and an increase in alveolar spaces of the B6 mice. In contrast, lungs from either Spi6KO mice or B6 mice that received the protective dose of NE (1.8U/kg), showed no gross signs of damage and were indistinguishable from the lungs of uninfected mice. c) Conclusions 667. The fact that increased NE activity leads to increased survival after infection with gram negative and positive bacteria supports the view that increased inflammation can protect against sepsis by preventing bacterial dissemination. The treatment of sepsis by neutralizing inflammatory cytokines (e.g. TNF-α, IL-I) or receptors (e.g. TNFR, TLR) has met with limited success, presumably because these therapies impair bacterial clearance. Targeting Spi6 increases immunity to sepsis causing bacteria without giving inflammatory disease. Thus, inhibition of similar serpins in humans, such as PI9, can lead to alternative strategies to treat sepsis through bacterial clearance by increasing rapid neutrophil function. F. References

668. (1994) NIH Consensus Conference. Helicobacter pylori in peptic ulcer disease. NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. Jama 272, 65-69. 669. AMuwalia, J., Tinker, A., Clapp, L. H., Duchen, M. R., Abramov, A. Y., Pope, S., Nobles,

M., and Segal, A. W. (2004). The large-conductance Ca2+-activated K+ channel is essential for innate immunity. Nature 427, 853-858.

670. Ahmed, R., A. Salmi, L. D. Butler, J. M. Chiller, M. B. A. Oldstone. 1984. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 60:521.

671. Ahmed, R., and Gray, D. (1996). Immunological Memory and Protective Immunity: Understanding Their Relationship. Science 272:54-60.

672. Ahmed, R., D. Gray. 1996. Immunological memory and protective immunity: understanding their relation. Science 272:54. 673. Ahmed, R., Salmi, A., Butler, L. D., Chiller, J. M., and Oldstone, M. B. A. (1984). Selection of Genetic Variants of Lymphocytic Choriomeningitis Virus in Spleens of Persistently Infected Mice Role in Suppression of Cytotoxic T Lymphocyte Response and Viral Persistence. Journal of Experimental Medicine 60:521-540.

674. Al-Khunaizi, M., Luke, C. J., Askew, Y. S., Pak, S. C, Askew, D. J., Cataltepe, S., Miller, D., Mills, D. R., Tsu, C, Bromme, D., et al. (2002a). The serpin SQN-5 is a dual mechanistic-class inhibitor of serine and cysteine proteinases. Biochemistry 41:3189-3199.

675. Al-Khunaizi, M., Luke, C. J., Askew, Y. S., Pak, S. C, Askew, D. J., Cataltepe, S., Miller, D., Mills, D. R., Tsu, C, Bromme, D., et al. (2002b). The serpin SQN-5 is a dual mechanistic-class inhibitor of serine and cysteine proteinases. Biochemistry 41:3189-3199. 676. Allen, L. A. (2000). Modulating phagocyte activation: the pros and cons of Helicobacter pylori virulence factors. J Exp Med 191, 1451-1454.

677. Allen, L. A. (2001). The role of the neutrophil and phagocytosis in infection caused by Helicobacter pylori. Curr Opin Infect Dis 14, 273-277.

678. Allen, L. A., Beecher, B. R., Lynch, J. T., Rohner, O. V., and Wittine, L. M. (2005). Helicobacter pylori disrupts NADPH oxidase targeting in human neutrophils to induce extracellular superoxide release. J Immunol 174, 3658-3667.

679. Annand, R. R., Dahlen, J. R., Sprecher, C. A., De Dreu, P., Foster, D. C, Mankovich, J. A., Talanian, R. V., Kisiel, W., and Giegel, D. A. (1999). Caspase 1 (interleukin-lbeta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9. Biochem J 342 Pt 3:655-665. t>oυ. Asqmui, JD., iviυsiey, A. J., Barfield, A., Marshall, S. E., Heaps, A., Goon, P., Hanon, E., Tanaka, Y., Taylor, G. P., and Bangham, C. R. (2005). A functional CD8+ cell assay reveals individual variation in CD8⁺ cell antiviral efficacy and explains differences in human T-lymphotropic virus type 1 proviral load. J Gen Virol 86, 1515-1523. 681. Ashton-Rickardt, P. G. (2004). A license to remember. Nat Immunol (in press).

682. Babior, B. M., Curnutte, J. T., and Kipnes, R. S. (1975). Biological defense mechanisms. Evidence for the participation of superoxide in bacterial killing by xanthine oxidase. J Lab Clin Med 55,

235-244.

683. Babior, B. M., Kipnes, R. S., and Curnutte, J. T. (1973). Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 52, IAl-IAA.

684. Badovanic, V. P., Tvinnereim, A. R., and Harty, J. T. (2000). Regulation of antigen-specific T cell homeostasis by perforin and interferon-g. Science 290:1354-1357.

685. Badovinac, V. P., Porter, B. B., and Harty, J. T. (2002). Programmed contraction of CD8(+) T cells after infection. Nat Immunol 3:619-626. 686. Badovinac, V. P., Porter, B. B., and Harty, J. T. (2004). CD8(+) T cell contraction is controlled by early inflammation. Nat Immunol 5:809-817.

687. Badovinac, V. P., S. E. Hamilton, J. T. Harty. 2003. Viral infection results in massive CD8⁺ T cell expansion and mortality in vaccinated perforin-deficient mice. Immunity 18:463.

688. Baetz, K., Isaaz, S., and Griffiths, G. M. (1995). Loss of cytotoxic T lymphocyte function in Chediak-Higashi syndrome arises from a secretory defect that prevents lytic granule exocytosis. J Immunol

/54, 6122-6131.

689. Bainton, D. Inflammation : Basic Principles and Clinical Correlates G. Gallin, I. Goldstien, R. Snyderman, Eds. (Raven Press, New York, 1999) pp. 13-34.

690. Bainton, D. (1999). Developmental Biology of Neutrophils and Eosinophils. In Inflammation : Basic Principles and Clinical Correlates, G. Gallin, I. Goldstien, and R. Snyderman, eds. (New York,

Raven Press), pp. 13-34.

691. Bainton, D. F., and Farquhar, M. G. (1966). Origin of granules in polymorphonuclear leukocytes. Two types derived from opposite faces of the Golgi complex in developing granulocytes. J Cell Biol 28, 277-301. 692. Bainton, D. F., Ullyot, J. L., and Farquhar, M. G. (1971). The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J Exp Med 134, 907-934.

693. Balaji, K. N., N. Schaschke, W. Machleidt, M. Catalfamo, P. A. Henkart. 2002. Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation. J. Exp. Med. 196:493. 'ⁱø9^j|? " SσarDosaym. u., iNgαyen, Q. A., Tchernev, V. T., Ashley, J. A., Defter, J. C, Blaydes, S. M., Brandt, S. J., Chotai, D., Hodgman, C, Solari, R. C, et al. (1996). Identification of the homologous beige and Chediak-Higashi syndrome genes. Nature 382, 262-265.

695. Beatty, K., Bieth, J., and Travis, J. (1980). Kinetics of association of serine proteinases with native and oxidized alpha- 1 -proteinase inhibitor and alpha- 1-antichymotrypsin. J Biol Chem 255, 3931-

3934.

696. Belaaouaj, A., McCarthy, R., Baumann, M., Gao, Z., Ley, T. J., Abraham, S. N., and Shapiro, S. D. (1998). Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med 4:615-618. 697. Belaaouaj, A., Kim, K. S., and Shapiro, S. D. (2000). Degradation of outer membrane protein

A in Escherichia coli killing by neutrophil elastase. Science 289, 1185-1188.

698. Belin, D., Bost, S., Vassalli, J. D., and Strub, K. (1996). A two-step recognition of signal sequences determines the translocation efficiency of proteins. Embo J 15, 468-478.

699. Belin, D., Guzman, L. M., Bost, S., Konakova, M., Silva, F., and Beckwith, J. (2004). Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors. J MoI Biol 335, 437-453.

700. Belin, D., Wohlwend, A., Schleuning, W. D., Kruithof, E. K., and Vassalli, J. D. (1989). Facultative polypeptide translocation allows a single mRNA to encode the secreted and cytosolic forms of plasminogen activators inhibitor 2. Embo J 8, 3287-3294. 701. Bellier, B., V. Thomas-Vaslin, M.-F. Saron, D. Klatzmann. 2003. Turning immunological memory into amnesia by depletion of dividing T cells. Proc. Natl. Acad. ScL USA 100:15017.

702. BeIz, G. T., Smith, C. M., Eichner, D., Shortman, K., Karupiah, G., Carbone, F. R., and Heath, W. R. (2004). Cutting edge: conventional CD8 alpha+ dendritic cells are generally involved in priming CTL immunity to viruses. J Immunol 172:1996-2000. 703. Berard, M., D. F. Tough. 2002. Qualitative differences between naive and memory T cells.

Immunology 106:127.

704. Berke, G., and Amos, D. B. (1973). Cytotoxic lymphocytes in the absence of detectable antibody. Nature 242, 237-239.

705. Bevan, M. J. (2004). Helping the CD8(+) T-cell response. Nat Rev Immunol 4:595-602. 706. Bidere, N., M. Briet, A. Durrbach, C. Dumont, J. Feldmann, B. Charpentier, G. de Saint-

Basile, A. Senik. 2002. Selective inhibition of dipeptidyl peptidase I, not caspases, prevents the partial processing ofprocaspase-3 in CD3 -activated human CD8⁺ T lymphocytes. J. Biol. Chem. 277:32339.

707. Bird, C. H., V. R. Sutton, J. Sun, C. E. Hirst, A. Novak, S. Kumar, J. A. Trapani, P. I. Bird. 1998. Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects ^■against igtmzyme tf-meαiateα apoptosis without perturbing the Fas cell death pathway. MoI. Cell. Biol. 18:6387.

708. Bird, P. I. (1998). Serpins and Regulation of Cell Death. Results Probl Cell Differ 24:63-89.

709. Bird, P. I. (1999). Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins. Immunology and Cell Biology 77:47-57.

710. Bjorquist P, Ehnebom J, Inghardt T, Hansson L, Lindberg M, Linschoten M, Stromqvist M, Deinum J. (1998 ) Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis.

Biochemistry. 37(5): 1227-34. 711. Bladergroen, B. A., Strik, M. C. M., Bovenschen, N., van Berkum, O., Scheffer, G. L.,

Meijer, C. J. L. M., Hack, C. E., and Kummer, J. A. (2001). The Granzyme B Inhibitor, Protease Inhibitor 9, Is Mainly Expressed by Dendritic Cells and at Immune-Privileged Sites. Journal of Immunology 166:3218-

3225.

712. Blanchard, T. G., Czinn, S. J., Nedrud, J. G., and Redline, R. W. (1995). Helicobacter- _^ associated gastritis in SCID mice. Infect Immun 63, 1113-1115.

713. Blobel, G., and Dobberstein, B. (1975). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67, 835-851.

714. Borregaard, N., Christensen, L., Bejerrum, O. W., Birgens, H. S., and Clemmensen, I. (1990). Identification of a highly mobilizable subset of human neutrophil intracellular vesicles that contains tetranectin and latent alkaline phosphatase. J Clin Invest 85, 408-416.

715. Borregaard, N., and Cowland, J. B. (1997). Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503-3521.

716. Borregaard, N., Sehested, M., Nielsen, B. S., Sengelov, H., and Kjeldsen, L. (1995). Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation. Blood 55, 812-817.

717. Braell, W. A., and Lodish, H. F. (1982). Ovalbumin utilizes an NH2 -terminal signal sequence. J Biol Chem 257, 4578-4582.

718. Bretz, R., and Staubli, W. (1977). Detergent influence on rat-liver galactosyltransferase activities towards different acceptors. Eur J Biochem 77, 181-192.

719. Brinkmann, V., Reichard, U., Goosmann, C, Fauler, B., Uhlemann, Y., Weiss, D. S., Weinrauch, Y., and Zychlinsky, A. (2004). Neutrophil extracellular traps kill bacteria. Science 303, 1532- 1535. Kd? ""* ^v*®io-$r&?Nt-W:rDblaxn, A. O., Heusel, J. W., Smith, H. R., Beckman, D. L., Blattenberger, E. A., Dubbelde, C. E., Stone, L. R., Scalzo, A. A., and Yokoyama, W. M. (2001). Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 292:934-937.

721. Brundage, R. A., Smith, G. A., Camilli, A., Theriot, J. A., and Portnoy, D. A. (1993). Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc Natl

Acad Sci U S A 90:11890-11894.

722. Budihardjo, L, Oliver, H., Lutter, M., Luo, X., and Wang, X. (1999). Biochemical pathways of caspase activation during apoptosis. AnnuRevCellDevBiol 75, 269-290.

723. Bui, J. D., S. Calbo, K. Hayden-Martinez, L. P. Kane, P. Gardner, S. M. Hedrick. 2000. A role for CaMKII in T cell memory. Cell 100:457.

724. Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⁺ T cells during an acute virus infection. Immunity 8:167.

725. Butz, E., M. J. Bevan. 1998. Dynamics of the CD8⁺ T cell response during acute LCMV infection. Adv. Exp. Med. Biol. 452:111. 726. Castanon, M. J., Spevak, W., Adolf, G. R., Chlebowicz-Sledziewska, E., and Sledziewski, A. ^■

(1988). Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae. Gene 66, 223-234.

727. Catalfamo, M., P. A. Henkart. 2003. Perform and the granule exocytosis cytotoxicity pathway. Curr. Opin. Immunol. 15:523. 728. Charlton et al, Fibrinolysis Proteolysis, 11(1), 51-56 (1997)

729. Chihu, L., Ayala, G., Mohar, A., Hernandez, A., Herrera-Goepfert, R., Fierros, G., Gonzalez- Marquez, H., and Silva, J. (2005). Antimicrobial resistance and characterization of Helicobacter pylori strains isolated from Mexican adults with clinical outcome. J Chemother 17, 270-276.

730. Clarke, S., Greaves, D. R., Chung, L. P., Tree, P., and Gordon, S. (1996). The human lysozyme promoter directs reporter gene expression to activated myelomonocytic cells in transgenic mice.

Proc Natl Acad Sci U S A 93, 1434-1438.

731. Coeshott, C, Ohnemus, C, Pilyavskaya, A., Ross, S., Wieczorek, M., Kroona, H., Leimer, A. H., and Cheronis, J. (1999). Converting enzyme-independent release of tumor necrosis factor alpha and IL- lbeta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proc Natl Acad Sci U S A 96:6261-6266.

732. Coligan, J. E., Kruisbeek, A. M., Margulis, D. H., Shevach, E. M., and Strober, W. (1995). Current Protocols in Immunology, VoI 1 (New York, John Wiley and Sons).

733. Collins, S. J., Gallo, R. C, and Gallagher, R. E. (1977). Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270, 347-349. '"73'4V "CoWiatfa;'J.'J3-., and Borregaard, N. (1999). Isolation of neutrophil precursors from bone marrow for biochemical and transcriptional analysis. J Immunol Methods 232, 191-200.

735. Conlan, J. W., and North, R. J. (1991). Neutrophil-mediated dissolution of infected host cells as a defense strategy against a facultative intracellular bacterium. J Exp Med 174:741-744. 736. Conlan, J. W., and North, R. J. (1994). Neutrophils are essential for early anti-Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed by a granulocyte-depleting monoclonal antibody. J Exp Med 179:259-268.

737. Cooley, J., Mathieu, B., Remold-O'Donnell, E., and Mandle, R. J. (1998). Production of recombinant human monocyte/neutrophil elastase inhibitor (rM/NEI). Protein Expr Purif 14:38-44. 738. Crandall DL, Elokdah H, Di L, Herman JK, Gorlatova NV, Lawrence DA. (2004)

Characterization and comparative evaluation of a structurally unique PAI-I inhibitor exhibiting oral in-vivo efficacy. J Thromb Haemost. (8): 1422-8.

739. Czuprynski, C. J., Brown, J. F., Wagner, R. D., and Steinberg, H. (1994). Administration of antigranulocyte monoclonal antibody RB6-8C5 prevents expression of acquired resistance to Listeria monocytogenes infection in previously immunized mice. Infect Immun 62:5161-5163.

740. Dahlen, J. R., D. C. Foster, W. Kisiel. 1999. Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9. Biochim. Biophys. Acta. 1451:233.

741. Dahms, N. M., Lobel, P., and Kornfeld, S. (1989). Mannose 6-phosphate receptors and lysosomal enzyme targeting. J Biol Chem 264, 12115-12118. 742. Dar, K. J., and Crystal, R. G. (1999). Inflammatory Lung Disease: Molecular Determinants of

Emphysema, Bronchitis, and Fibrosis. In Inflammation : Basic Principles and Clinical Correlates, G. Gallin, I. Goldstien, and R. Snyderman, eds. (New York, Raven Press), pp. 1061-1082.

743. Darji, A., Mohamed, W., Domann, E., and Chakraborty, T. (2003). Induction of immune responses by attenuated isogenic mutant strains of Listeria monocytogenes. Vaccine 2 Suppl 2:S102-109. 744. de Bont, E. S., Reilly, C. R., Lo, D., Glimcher, L. H., and Laufer, T. M. (1999). A minimal level of MHC class II expression is sufficient to abrogate autoreactivity. Int Immunol 11, 1295-1306.

745. Diamond, B., Birshtein, B. K., and Scharff, M. D. (1979). Site of binding of mouse IgG2b to the Fc receptor on mouse macrophages. J Exp Med 150:721-726.

746. Dombrowicz, D., Flamand, V., Brigman, K. K., Koller, B. H., and Kinet, J. P. (1993). Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor alpha chain gene. Cell 75:969-976.

747. Dunn, P. L., and North, R. J. (1991)- Early gamma interferon production by natural killer cells is important in defense against murine listeriosis. Infect Immun 59:2892-2900. ^■MIS': ^•'-"^■• ""^■tatzrαair'-J^lϊ,"ray vVP, Lawrence DA, Francis-Chmura AM, Shore JD, Olson ST, Ginsburg D. (1995) Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro. J Clin Invest. 95(5):2416-20.

749. Elokdah H, Abou-Gharbia M, Herman JK, McFarlane G, Mugford CP, Krishnamurthy G, Crandall DL. (2004) Tiplaxtinin, a novel, orally efficacious inhibitor of plasminogen activator inhibitor-1 : design, synthesis, and preclinical characterization. J. Med. Chem, 47(14):3491-4.

750. Elviss, N. C₃ Owen, R. J., Breathnach, A., Palmer, C₃ and Shetty, N. (2005). Helicobacter pylori antibiotic-resistance patterns and risk factors in adult dyspeptic patients from ethnically diverse populations in central and south London during 2000. J Med Microbiol 54, 561 -51 A. 751. Fox, J. G., Lee, A., Otto, G., Taylor, N. S., and Murphy, J. C. (1991). Helicobacter felis gastritis in gnotobiotic rats: an animal model of Helicobacter pylori gastritis. Infect Immun 59, 785-791.

752. Friederich PW, Levi M, Biemond BJ, Charlton P, Templeton D, van Zonneveld AJ, Bevan P, Pannekoek H, ten Cate JW. (1997 ) Novel low-molecular-weight inhibitor of PAI-I (XR5118) promotes endogenous fibrinolysis and reduces posttlirombolysis thrombus growth in rabbits. Circulation. 96(3):916-21.

753. Froelich, C. J., K. Orth, J. Turbov, P. Seth, R. Gottlieb, B. Babior, G. M. Shah, R. C. Bleackley, V. M. Dixit, W. Hanna. 1996. New paradigm for lymphocyte granule-mediated cytotoxicity. J. Biol. Chem. 271:29073.

754. Gadek, J. E., Fells, G. A., Zimmerman, R. L., Rennard, S. L₃ and Crystal, R. G. (1981a). Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema. J Clin Invest 68, 889-898.

755. Gadek, J. E., Klein, H. G., Holland, P. V., and Crystal, R. G. (1981b). Replacement therapy of alpha 1 -antitrypsin deficiency. Reversal of protease-antiprotease imbalance within the alveolar structures of PiZ subjects. J Clin Invest 68, 1158-1165. 756. Gagnon, R. F., and Joshua, D. E. (1980). Antibody-dependent cell-mediated cytotoxicity

(ADCC) assay for specific IgG antibody produced in vitro. J Immunol Methods 36:243-254.

757. Gallimore, A., A. Glithero, A. Godkin, A. C. Tissot, A. Pluckthun, T. Elliott, H. Hengartner, R. M. Zinkernagel. 1998. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T Lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J. Exp. Med. 187:1383.

758. Gallin, J. L₃ and Malech, H. L. (1990). Update on chronic granulomatous diseases of childhood. Immunotherapy and potential for gene therapy. Jama 263, 1533-1537.

759. Garwicz, D., Lindmark, A., and Gullberg, U. (1995). Human cathepsin G lacking functional glycosylation site is proteolytically processed and targeted for storage in granules after transfection to the rat basophilic/mast cell line RBL or the murine myeloid cell line 32D. J Biol Chem 270, 28413-28418. '7OW '^■"" ^■"^■■υiIs"Λ,¹"MtaSsεn JM, Nar H, Kley JT, Wienen W, Ries UJ, Declerck PJ. (2002 ) Characterization and comparative evaluation of a novel PAI-I inhibitor. Thromb Haemost. 88(l):137-43.

761. Gong, B., Chen, Q., Endlich, B., Mazumder, S., and Almasan, A. (1999). Ionizing radiation- induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases. Cell Growth

Differ /0, 491-502.

762. Grossman, W. J., and Ley, T. J. (2004). Granzymes A and B are not expressed in human neutrophils. Blood 104:906-907; author reply 907-908.

763. Grossman, W. J., P. A. Revell, Z. H. Lu, H. Johnson, A. J. Bredemeyer, T. J. Ley. 2003. The orphan granzymes of humans and mice. Curr. Opin. Immunol. 15:545.

164. Gullberg, U., Lindmark, A., Lindgren, G., Persson, A. M., Nilsson, E., and Olsson, I. (1995). Carboxyl-terminal prodomain-deleted human leukocyte elastase and cathepsin G are efficiently targeted to granules and enzymatically activated in the rat basophilic/mast cell line RBL. J Biol Chem 270, 12912- 12918. 765. Gullberg, U., Lindmark, A., Nilsson, E., Persson, A. M., and Olsson, I. (1994). Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL. J Biol Chem 269,

25219-25225.

766. Hamann, D., P. A. Baars, M. H. G. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, R. A. W. van Lier. 1997. Phenotypic and functional separation of memory and effector human CD8⁺ T cells. J. Exp. Med. 186:1407.

161. Hanon, E., Stinchcombe, J. C, Saito, M., Asquith, B. E., Taylor, G. P., Tanaka, Y., Weber, J. N., Griffiths, G. M., and Bangham, C. R. (2000). Fratricide among CD8(+) T lymphocytes naturally infected with human T cell lymphotropic virus type I. Immunity IS, 657-664.

768. Hartmann, J., Scepek, S., and Lindau, M. (1995). Regulation of granule size in human and horse eosinophils by number of fusion events among unit granules. J Physiol 483 (Pt 1), 201-209.

769. Harty, J. T., Lenz, L. L., and Bevan, M. J. (1996). Primary and secondary immune responses to Listeria monocytogenes. Curr Opin Immunol 8:526-530.

770. Hautamaki, R. D., Kobayashi, D. K., Senior, R. M., and Shapiro, S. D. (1997). Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277, 2002-2004. 771. Hermans, I. F., Ritchie, D. S., Yang, J., Roberts, J. M., and Ronchese, F. (2000). CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity. J Immunol 164:3095-3101.

772. Heusel, J. W., R. L. Wesselschmidt, S. Shresta, J. H. Russell, T. J. Ley. 1994. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic taiwt cells. Cell 76:977. ^■773': Hir≤t;"crfi^r."; ESttzia, M. S., Bird, C. H., Warren, H. S., Cameron, P. U., Zhang, M., Ashton-

Rickardt, P. G., and Bird, P. I. (2003). The Intracellular Granzyme B Inhibitor, Proteinase Inhibitor 9, Is Up- Regulated During Accessory Cell Maturation and Effector Cell Degranulation, and Its Overexpression Enhances CTL Potency. Journal of Immunology 170:805-815. 774. Hochegger, K., Eller, P., and Rosenkranz, A. R. (2004). Granzyme A: an additional weapon of human polymorphonuclear neutrophils (PMNs) in innate immunity? Blood 103:1176.

775. Hou, S., Hyland, L., Ryan, K. W., Portner, A., and Doherty, P. C. (1994). Virus-specific CD8+ T-cell memory determined by clonal burst size. Nature 369:652-654.

776. Hu, Q., Bazemore Walker, C. R., Girao, C, Opferman, J. T., Sun, J., Shabanowitz, J., Hunt, D. F., and Ashton-Rickardt, P. G. (1997). Specific recognition of thymic self-peptides induces the positive selection of cytotoxic T lymphocytes. Immunity 7:221-231.

777. Huang, J.-F., Yang, Y., Sepulveda, H., Shi, W., Hwang, L, Peterson, P. A., Jackson, M. R., Sprent, J., and Cai, Z. (1999). TCR-Mediated Internalization of Peptide-MHC Complexes Acquired by T Cells. Science 286, 952-954. 778. Hubbard, R. C, Fells, G., Gadek, J., Pacholok, S., Humes, J., and Crystal, R. G. (1991).

Neutrophil accumulation in the lung in alpha 1-antitrypsin deficiency. Spontaneous release of leukotriene B4 by alveolar macrophages. J Clin Invest 88, 891-897.

779. Huber, R., and Carrell, R. W. (1989). Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry 28, 8951-8966. 780. Ichii, H., A. Sakamoto, M. Hatano, S. Okada, H. Toyama, S. Taki, M. Arima, Y. Kuroda, T.

Tokuhisa. 2002. Role for Bcl-6 in the generation and maintenance of memory CD8⁺ T cells. Nat. Immunol 3:558.

781. Ida, J., T. Nakashima, N. L. Kedersha, S. Yamasaki, M. Huang, Y. Izumi, T. Miyashita, T. Origuchi, A. Kawakami, K. Migita, et al 2003. Granzyme B leakage-induced cell death: a new type of activation-induced natural killer cell death. Eur. J. Immunol 33:3284.

782. Idziorek, T., Estaquier, J., De BeIs, F., and Ameisen, J.-C. (1995). YOPRO-I permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. Journal of Immunological Methods 185:249-258.

783. Jameson, S. C. (2002). Maintaining the norm: T-cell homeostasis. Nat Rev Immunol 2:547- 556.

784. Janoff, A., Sloan, B., Weinbaum, G., Damiano, V., Sandhaus, R. A., Elias, J., and Kimbel, P. (1977). Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease. Am Rev Respir Dis 115, 461-478.

785. Kaech, S. M., Hemby, S., Kersh, E., and Ahmed, R. (2002). Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111:837-851. 7StJ. " iς.aecϊϊ',"'δ:^Mivi., ian, J. T., Wherry, E. J., Konieczny, B., T.,, Surh, C. D., and Ahmed, R.

(2004). Selective expression of the interferon 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat Immunol 4:1191-1198.

787. Kagi, D., Odermatt, B., and Mak, T. W. (1999). Homeostatic regulation of CD8⁺ T cells by perforin. Eur J Immunol 29, 3262-3272.

788. Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner, P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265:528-530.

789. Kagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkernagel, R. M., and Hengartner, H. (1994a). Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31-37.

790. Kagi, D., Ledermann, B., Burki, K., Zinkernagel, R. M., and Hengartner, H. (1996). Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu Rev Immunol 14, 207-232. 791. Kaiserman, D., S. Knaggs, K. L. Scarff, A. Gillard, G. Mirza, M. Cadman, R. McKeone, P.

Denny, J. Cooley, C. Benarafa, et al 2002. Comparison of human chromosome 6p25 with mouse chromosome 13 reveals a greatly expanded ov-serpin gene repertoire in the mouse. Genomics 79:349.

792. Kaneko, F., Suzuki, H., Hasegawa, N., Kurabayshi, K., Saito, H., Otani, S., Nakamizo, H., Kawata, K., Miyairi, M., Ishii, K., and Ishii, H. (2004). High prevalence rate of Helicobacter pylori resistance to clarithromycin during long-term multiple antibiotic therapy for chronic respiratory disease caused by non-tuberculous mycobacteria. Aliment Pharmacol Ther 20 Suppl 1, 62-67.

793. Kassiotis, G., Garcia, S., Simpson, E., and Stockinger, B. (2002). Impairment of immunological memory in the absence of MHC despite survival of memory T cells. Nat Immunol 3:244- 250. 794. Kataoka, T., Shinohara, N., Takayama, H., Takaku, K., Kondo, S., Yonehara, S., and Nagai,

K. (1996). Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J Immunol 156:3678-3686.

795. Khamri, W., Moran, A. P., Worku, M. L., Karim, Q. N., Walker, M. M., Annuk, H., Ferris, J. A., Appelmelk, B. J., Eggleton, P., Reid, K. B., and Thursz, M. R. (2005). Variations in Helicobacter pylori Lipopolysaccharide To Evade the Innate Immune Component Surfactant Protein D. Infect Immun 73, 7677-

7686.

796. Kinoshita, T., Takeda, J., Hong, K., Kozono, H., Sakai, H., and Inoue, K. (1988). Monoclonal antibodies to mouse complement receptor type 1 (CRl). Their use in a distribution study showing that mouse erythrocytes and platelets are CRl -negative. J Immunol 140, 3066-3072. "^•w: "^• "jsgeiαsenvE'-raeugelov, H., Lollike, K., Nielsen, M. H., and Borregaard, N. (1994). Isolation and characterization of gelatinase granules from human neutrophils. Blood 83, 1640-1649.

798. Klebanoff, S. J. (1975). Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin Hematol 12, 117-142. 799. Komiyama, T., Ray, C. A., Pickup DJ., Howard, A. D., Thornberry, N. A., Peterson, E. P., and Salversen, G. (1994). Inhibition of interleukin-1 beta coverting enzyme by the cowpox virus serpin Crm A. An example of cross-class inhibition. JBiolChem 269:19331-19337.

800. Koniaris, L. G., McKillop, I. H., Schwartz, S. L, and Zimmers, T. A. (2003). Liver regeneration. J Am Coll Surg 197:634-659. 801. Korkmaz, B., Attucci, S., Hazouard, E., Ferrandiere, M., Jourdan, M. L., Brillard-Bourdet,

M., Juliano, L., and Gauthier, F. (2002). Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates. J Biol Chem 277:39074-39081.

802. Krieg, A. M. (2003). CpG motifs: the active ingredient in bacterial extracts? Nat Med 9:831- 835. 803. Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995). Inducible gene targeting in mice. Science 269, 1427-1429.

804. Kupfer, A., Singer, S. J., and Dennert, G. (1986). On the mechanism of unidirectional killing in mixtures of two cytotoxic T lymphocytes. Unidirectional polarization of cytoplasmic organelles and the membrane-associated cytoskeleton in the effector cell. J Exp Med 163, 489-498. 805. Larhammar, D., U. Hammerling, L. Rask, P. A. Peterson. 1985. Sequence of gene and cDNA encoding murine major histocompatibility complex class II gene Ar2. J. Biol. Chem. 260:14111.

806. Lawrence, DA, Olson ST, Palaniappan S, Ginsburg D., 1994. Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. J. Biol. Chem., 269(44): 27657- 27662. 807. Lau, L. L., Jamieson, B. D., Somasundaram, T., and Ahmed, R. (1994). Cytotoxic T-cell memory without antigen. Nature 369:648-652.

808. Le Cabec, V., Cowland, J. B., Calafat, J., and Borregaard, N. (1996). Targeting of proteins to granule subsets is determined by timing and not by sorting: The specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells. Proc Natl Acad Sci U S A 93, 6454-6457. 809. Lehrer, R. L, and Ganz, T. (1990). Antimicrobial polypeptides of human neutrophils. Blood

76:2169-2181.

810. Lieberman, J.. 2003. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat. Rev. Immunol. 3:361. WW '"^l ""Lm/M'.i'-Fmilϊps, ¹I., Zhang, M., Wang, Y., Opferman, J. T., Shah, R., and Ashton-Rickardt, P. G. (2004). Serine protease inhibitor 2 A is a protective factor for memory T cell development. Nat Immunol 5, 919-926.

812. Liu, N., Raja, S. M., Zazzeroni, F., Metkar, S. S., Shah, R., Zhang, M., Wang, Y., Bromme, D., Russin, W. A., Lee, J. C, et al. (2003). NF-kappa B protects from the lysosomal pathway of cell death.

EMBO J 22:5313-5322.

813. Lohman, B. L., E. S. Razvi, R. M. Welsh. 1996. T-lymphocyte downregulation after acute viral infection is not dependent on CD95 (Fas) receptor-ligand interactions. J. Virol. 70:8199.

814. Lomas-Neira, J. L., Chung, C. S., Wesche, D. E., Perl, M., and Ayala, A. (2005). In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury. J Leukoc Biol 77, 846-853.

815. Lopez-Boado, Y. S., Espinola, M., Bahr, S., and Belaaouaj, A. (2004). Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity. J Immunol 172:509-

515. 816. Loyer, V., Fontaine, P., Pion, S., Hetu, F., Roy, D. C, and Perreault, C. (1999). The in vivo fate of APCs displaying minor H antigen and/or MHC differences is regulated by CTLs specific for immunodominant class I-associated epitopes. J Immunol 163:6462-6467.

817. Ludewig, B., Bonilla, W. V., Dumrese, T., Odermatt, B., Zinkernagel, R. M., and Hengartner, H. (2001). Perforin-independent regulation of dendritic cell homeostasis by CD8(+) T cells in vivo: implications for adaptive immunotherapy. Eur J Immunol 31 : 1772-1779.

818. Ludewig, B., EhI, S., Karrer, U., Odermatt, B., Hengartner, H., and Zinkernagel, R. M. (1998). Dendritic Cells Efficiently Induce Protective Antiviral Immunity. Journal of Virology 72:3812- 3818.

819. Lutz, M. B., Kukutsch, N., Ogilvie, A. L. J., Robner, S., Koch, F., Romani, N., and Schuler, G. (1999). An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. Journal of Immunological Methods 223:77-92.

820. Lutz, M. B., N. Kukutsch, A. L. J. Ogilvie, S. Robner, F. Koch, N. Romani, G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77. 821. Maclvor, D. M., Shapiro, S. D., Pham, C. T., Belaaouaj, A., Abraham, S. N., and Ley, T. J.

(1999). Normal neutrophil function in cathepsin G-deficient mice. Blood 94:4282-4293.

822. Madakamutil, L. T., Christen, U., Lena, C. J., Wang-Zhu, Y., Attinger, A., Sundarrajan, M., Ellmeier, W., von Herrath, M. G., Jensen, P., Littman, D. R., and Cheroutre, H. (2004). CDδalphaalpha- mediated survival and differentiation of CD8 memory T cell precursors. Science 304:590-593. %3? "-" Ciallin, J. I. (1987). Current concepts: immunology. Neutrophils in human diseases. N Engl J Med 317:687-694.

824. Mandell, G. L. (1974). Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils. Infect Immun 9, 337-341. 825. Martorana, P. A., Brand, T., Gardi, C₅ van Even, P., de Santi, M. M., Calzoni, P.,

Marcolongo, P., and Lungarella, G. (1993). The pallid mouse. A model of genetic alpha 1 -antitrypsin deficiency. Lab Invest 68, 233-241.

826. Matloubian, M., Kolhekar, S. R., Somasundaram, T., and Ahmed, R. (1993). Molecular Determinants of Macrophage Tropism and Viral Persistence: Importance of Single Amino Acid Changes in the Polymerase and Glycoprotein of Lymphocytic Choriomeningitis Virus. Journal of Virology 67:7340-

7349.

827. Matloubian, M., Suresh, M., Glass, A., Galvan, M., Chow, K., Whitmire, J. K., Walsh, C. M., Clark, W. R., and Ahmed, R. (1999). A role for perform in downregulating T-cell responses during chronic viral infection. J Virol 73, 2527-2536. 828. Matzinger, P. (1991). The JAM test A simple assay for DNA fragmentation and cell death.

Journal of Immunological Methods 145:185-192.

829. McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., and Kay, M. A. (2002). RNA interference in adult mice. Nature 418, 38-39.

830. McMichael, A. (1998). Preparing for HIV vaccines that induce cytotoxic T lymphocytes. Curr Opin Immunol 10, 379-381.

831. Medema, J. P., Schuurhuis, D. H., Rea, D., van Tongeren, J., de Jong, J., Bres, S. A., Laban, S., Toes, R. E. M., Toebes, M., Schumacher, T. N. M., et al. (2001). Expression of the Serpin Serine Protease Inhibitor 6 Protects Dendritic Cells from Cytotoxic T Lymphocyte-induced Apoptosis: Differential Modulation by T Helper Type 1 and Type 2 Cells. J Exp Med 194, 657-667. 832. Medema, J. P., de Jong, J., Peltenburg, L. T. C₅ Verdegaal, E. M. E., Gorter, A., Bres, S. A.,

Franken, K. L. M. C, Hahne, M., Albar, J. P., Melief, C. J. M., and Offringa, R. (2001a). Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors. Proc Natl Acad Sci 98:11515-11520.

833. Medhurst, A. D., D. C. Harrison, S. J. Read, C. A. Campbell, M. J. Robbins, M. N. Pangalos. 2000. The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J- Neurosci. Methods 98:9.

834. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12, 7035-7056. '83¹S!¹ "^■■■' MeiikgdHbrG'.'yPd'stural, E., Feldmann, J., Certain, S., Ersoy, F., Dupuis, S., Wulffraat, N.,

Bianchi, D., Fischer, A., Le Deist, F., and de Saint Basile, G. (2000). Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet 25, 173-176.

836. Metchnikoff, B. (1905). Immunity in Infective Disease, Cambridge University Press). 837. Metkar, S. S., and Froelich, C. J. (2004). Human neutrophils lack granzyme A, granzyme B, and perform. Blood 104:905-906; author reply 907-908.

838. Millard, P. J., Henkart, M. P., Reynolds, C. W., and Henkart, P. A. (1984). Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors. J Immunol 132, 3197-3204.

839. Mohammadi, M., Czinn, S., Redline, R., andNedrud, J. (1996). Helicobacter-specific cell- mediated immune responses display a predominant ThI phenotype and promote a delayed-type hypersensitivity response in the stomachs of mice. J Immunol 156, 4729-4738.

840. Moskophidis, D., F. Lechner, H. Pircher, R. M. Zinkernagel. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature 362:758. 841. Murali-Krishna, K., Airman, J. D., Suresh, M., Sourdive, D. J. D., Zajac, A. J., Miller, J. D.,

Slansky, J., and Ahmed., R. (1998). Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177-187.

842. Murali-Krishna, K., J. D. Airman, M. Suresh, D. J. D. Sourdive, A. J. Zajac, J. D. Miller, J. Slanksy, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8: 177.

843. Nakamura, A., Mori, Y., Hagiwara, K., Suzuki, T., Sakakibara, T., Kikuchi, T., Igarashi, T., Ebina, M., Abe, T., Miyazaki, J., et al. (2003). Increased susceptibility to LPS-induced endotoxin shock in secretory leukoprotease inhibitor (SLPI)-deficient mice. J Exp Med 197, 669-61 A.

844. Nauseef, W. M., McCormick, S., and Yi, H. (1992). Roles of heme insertion and the mannose-6-ρhosphate receptor in processing of the human myeloid lysosomal enzyme, myeloperoxidase.

Blood SO, 2622-2633.

845. Neighbors, M., Xu, X., Barrat, F. J., Ruuls, S. R., Churakova, T., Debets, R., Bazan, J. F., Kastelein, R. A., Abrams, J. S., and O'Garra, A. (2001). A critical role for interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extends beyond its effects on Interferon gamma production. J Exp Med 194:343-354.

846. Nuchtern, J. G., Bonifacino, J. S., Biddison, W. E., and Klausner, R. D. (1989). Brefeldin A implicates egress from endoplasmic reticulum in class I restricted antigen presentation. Nature 339, 223- 226. WW'-i^f oObef^WT?; (?: Hu, J. T. Opferman, S. Hagevik, N. Chiu, C-R. Wang, P. G. Ashton-Rickardt

2000. Affinity of thymic self-peptides for the TCR determines the selection of CDS⁺ T lymphocytes in the thymus. Int. Immunol. 12:1353.

848. Ocker, M., Neureiter, D., Lueders, M., Zopf, S., Ganslmayer, M., Hahn, E. G., Herold, C, and Schuppan, D. (2005). Variants of bcl-2 specific siRNA for silencing antiapoptotic bcl-2 in pancreatic cancer. Gut 54, 1298-1308.

849. Odake, S., Kam, C. M., Narasimhan, L., Poe, M., Blake, J. T., Krahenbuhl, O., Tschopp, J., and Powers, J. C. (1991). Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry 30, 2217-2227.

850. Ohtani A, Takagi T, Hirano A, Murakami J, Sasaki Y. (1996 ) Inhibitory effect of a new butadiene derivative on the production of plasminogen activator inhibitor- 1 in cultured bovine endothelial cells. J Biochem (Tokyo). 120(6):1203-8.

851. Okada, Y., Watanabe, S., Nakanishi, L, Kishi, J., Hayakawa, T., Watorek, W., Travis, J., and Nagase, H. (1988). Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases. FEBS Lett 229, 157-160.

852. Opferman, J. T., Ober, B. T., and Ashton-Rickardt, P. G. (1999). Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 283:1745-1748.

853. Opferman, J. T., Ober, B. T., Narayanan, R., and Ashton-Rickardt, P. G. (2001). Suicide induced by cytolytic activity controls the differentiation of memory CD8+ T lymphocytes. International

Immunology 13:411-419.

854. Perelson, A. S., Neumann, A. U., Markowitz, M., Leonard, J. M., and Ho, D. D. (1996). HIV- 1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271, 1582-1586. 855. Palermo, M. S., Alves Rosa, M. F., Van Rooy^'en, N., and Isturiz, M. A. (1999). Depletion of liver and splenic macrophages reduces the lethality of Shiga toxin-2 in a mouse model. Clin Exp Immunol 116:462-467.

856. Perou, C. M., Justice, M. J., Pryor, R. J., and Kaplan, J. (1996a). Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes. Proc Natl Acad Sci U S A 93, 5905-5909.

857. Perou, C. M., Moore, K. J., Nagle, D. L., Misumi, D. J., Woolf, E. A., McGrail, S. H., Holmgren, L., Brody, T. H., Dussault, B. J., Jr., Monroe, C. A., et at (1996b). Identification of the murine beige gene by YAC complementation and positional cloning. Nat Genet 13, 303-308. »«".' '"'"fetseϋϊl&r,-*¹."^. £immermann, A. Strasser, D. Grillot, G. Nunez, H. Pircher. 1998.

Constitutive expression of Bcl-x_L or Bcl-2 prevents peptide antigen-induced T cell deletion but does not influence T cell homeostasis after a viral infection. Eur. J. Immunol. 28:560.

859. Phillips, T., Opferman, J. T., Shall, R., Liu, N., Froelich, C. J., and Ashton-Rickardt, P. G. (2004). A role for the granzyme B inhibitor serine protease inhibitor 6 in CD8+ memory cell homeostasis. J

Immunol 173, 3801-3809.

860. Pircher, H., Moskophidis, D., Rohrer, U., Burki, K., Hengartner, H., and Zinkernagel, R. M. (1990). Viral escape by selection of cytotoxic T cell-resistant virus variants in vivo. Nature 346:629-633.

861. Podack, E. R., Young, J. D., and Cohn, Z. A. (1985). Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc Natl Acad Sci U S A 82, 8629-

8633.

862. Pope, C, Earn, S. K., Marzo, A., Masopust, D., Williams, K., Jiang, J., Shen, H., and Lefrancois, L. (2001). Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J Immunol 166:3402-3409. 863. Portnoy, D. A. (1992). Innate immunity to a facultative intracellular bacterial pathogen. Curr

Opin Immunol 4:20-24.

864. Quan, L. T., Caputo, A., Bleackley, R. C, Pickup, D. J., and Salvesen, G. S. (1995). Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A. JBiolChem 270:10377- 10379. ' 865. Rao, N. V., Wehner, N. G., Marshall, B. C, Gray, W. R., Gray, B. H., and Hoidal, J. R.

(1991). Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties. J Biol Chem 266:9540-9548.

866. Reeves, E. P., Lu, H., Jacobs, H. L., Messina, C. G., Bolsover, S., Gabella, G., Potma, E. O., Warley, A., Roes, J., and Segal, A. W. (2002). Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416, 291-297.

867. Remold-O'Donnell, E. (1993). The ovalbumin family of serpin proteins. FEBS Lett 315, 105- 108.

868. Revell, P. A., Grossman, W. J., Thomas, D. A., Cao, X., Behl, R., Ratner, J. A., Lu, Z. H., and Ley, T. J. (2005). Granzyme B and the downstream granzymes C and/or F are important for cytotoxic lymphocyte functions. J Immunol 174, 2124-2131.

869. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., and Khvorova, A. (2004). Rational siRNA design for RNA interference. Nat Biotechnol 22, 326-330.

870. Rokhlin, O. W., G. A. Bishop, B. S. Hostager, T. J. Waldschmidt, S. P. Sidorenko, N. Pavloff, M. C. Kiefer, S. R. Umansky, R. A. Glover, M. B. Cohen. 1997. Fas-mediated apoptosis in human nrnstatic carcinoma cell lines. Cancer Res. 57:1758. 87-ϊ". ^•'^■■■'^{• •}''"'RustmfJ' rΑ'γaxia. Ley, T. J. (2002). Lymphocyte-mediated cytotoxicity. Annu Rev Immunol

20:323-370.

872. Sbarra, A. J., and Karπovsky, M. L. (1959). The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J Biol Chem 234, 1355-1362.

873. Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat Rev. Immunol. 3:269.

874. Scott, F. L., Hirst, C. E., Sun, J., Bird, C. H., Bottomley, S. P., and Bird, P. I. (1999). The intracellular serpin proteinase inhibitor 6 is expressed in monocytes and granulocytes and is a potent inhibitor of the azurophilic granule protease, cathepsin G. Blood 93:2089-2097.

875. Seder, R. A., R. Ahmed. 2003. Similarities and differences in CD4⁺ and CD8⁺ effector and memory T cell generation. Nat. Immunol. 4:835.

876. Selin, L. K., R. M. Welsh. 1997. Cytolytically active memory CTL present in lymphocytic choriomeningitis virus-immune mice after clearance of virus infection. J. Immunol. 158:5366. 877. Selin, L. K., R. M. Welsh. 2004. Plasticity of T cell memory responses to viruses. Immunity

20:5.

878. Sengelov, H., Kjeldsen, L., Kroeze, W., Berger, M., and Borregaard, N. (1994). Secretory vesicles are the intracellular reservoir of complement receptor 1 in human neutrophils. J Immunol 153, 804- 810. 879. Shafer, W. M., Pohl, J., Onunka, V. C, Bangalore, N., and Travis, J. (1991). Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide. J Biol Chem 266:112-116.

880. Shapiro, S. D., Goldstein, N. M., Houghton, A. M., Kobayashi, D. K., Kelley, D., and Belaaouaj, A. (2003). Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. Am J Pathol 163, 2329-2335.

881. Shi, L., Kraut, R. P., Aebersold, R., and Greenberg, A. H. (1992). Anatural killer cell granule protein that induces DNA fragmentation and apoptosis. J Exp Med 175, 553-566.

882. Shipley, J. M., Wesselschmidt, R. L., Kobayashi, D. K., Ley, T. J., and Shapiro, S. D. (1996). Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc Natl Acad Sci U S A 93, 3942-3946.

883. Silverman, G. A., Bird, P. L, Carrell,R.W.,, Church, F. C, Coughlin, P. B., Gettins, P. G. W., Irving, J. A., Lomas, D. A., Luke, C. J., Moyer, R. W., Pemberton, P. A., et al. (2001). The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. JBiolChem 276:33293- 33296. ⁽giδ-p »•«» 'i-KiMiI'Mr-Strassfter A, Vaux DL. (1996) CrmA expression in T lymphocytes of transgenic mice inhibits CD95 (Fas/APO-l)-transduced apoptosis, but does not cause lymphadenopathy or autoimmune disease. EMBO J.; 15(19):5167-76.

885. Song, E., Lee, S. K., Wang, J., Ince, N., Ouyang, N., Min, J., Chen, J., Shankar, P., and Lieberman, J. (2003). RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9,

347-351.

886. Sorensen, D. R., Leirdal, M., and Sioud, M. (2003). Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J MoI Biol 327, 761-766.

887. Sossin, W. S., Fisher, J. M., and Scheller, R. H. (1990). Sorting within the regulated secretory pathway occurs in the trans-Golgi network. J Cell Biol 110, 1-12.

Spaner, D., Raju, K., Rabinovich, B., and Miller, R. G. (1999). A Role for Perform in Activation-Induced T Cell Death In Vivo: Increased Expansion of Allogeneic Perforin-Deficient T Cells in SCID mice. Journal of Immunology 162:1192-1199.

889. Spaner, D., Raju, K., Rudvanyi, L., Lin, Y., and Miller, R. G. (1998). A Role for Perform in Activation-Induced Cell Death. Journal of Immunology 160:2655-2664.

890. Sprecher, C. A., K. A. Morgenstern, S. Mathewes, J. R. Dahlen, S. K. Schrader, D. C. Foster, W. Kisiel. 1995. Molecular cloning, expression, and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors. /. Biol. Chem. 270:29854.

891. Sprent, J., C. D. Surh. 2002. T cell memory. Aram. Rev. Immunol. 20:551. 892. Stepp, S. E., Dufourcq-Lagelouse, R., Le Deist, F., Bhawan, S., Certain, S., Mathew, P. A.,

Henter, J.-I., Bennett, M., Fischer, A., de Saint Basile, G., and Kumar, V. (1999). Perform Gene Defects in Familial Hemophagocytic Lymphohistiocytosis. Science 286:1957-1959.

893. Stinchcombe, J. C, Page, L. J., and Griffiths, G. M. (2000). Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak Higashi syndrome patients. Traffic 1, 435-444. 894. Stromberg, K., Persson, A. M., and Olsson, I. (1986). The processing and intracellular transport of myeloperoxidase. Modulation by lysosomotropic agents and monensin. Eur J Cell Biol 39, 424- 431.

895. Sun, J., Bird, C. H., Sutton, V., McDonald, L., Coughlin, P. B., De Jong, T. A., Trapani, J. A., and Bird, P. I. (1996). A Cytosolic Granzyme B Inhibitor Related to the Viral Apoptotic Regulator Cytokine Response Modifier A Is Present in Cytotoxic Lymphocytes. Journal of Biological Chemistry 271:27802-27809.

896. Sun, J., Ooms, L., Bird, C. H., Sutton, V. R., Trapani, J. A., and Bird, P. I. (1997). A New Family of 10 Murine Ovalbumin Serpins Includes Two Homologs of Proteinase Inhibitor 8 and Two Homologs of the Granzyme B Inhibitor (Proteinase Inhibitor 9). Journal of Biological Chemistry 272:15434-15441. ^■tyrø^t u '!»MbS.,"!H?Hi6g;'F:, Strachan, R., Jackson, D., Wallis, E., and Colman, A. (1984). Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes. Identification of an ovalbumin signal sequence. J MoI Biol 180, 645-666.

898. Takai, T., Li, M., Sylvestre, D., Clynes, R., and Ravetch, J. V. (1994). FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell 76:519-529.

899. Takeda, K., Kaisho, T., and Akira, S. (2003). Toll-like receptors. Annu Rev Immunol 21:335- 376.

900. Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., et al. (1997). A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem 272:17907-17911.

901. Tkalcevic, J., Novelli, M., Phylactides, M., Iredale, J. P., Segal, A. W., and Roes, J. (2000). Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12:201-210. 902. Tough, D. F., J. Sprent. 1994. Turnover of naive- and memory-phenotype T cells. J. Exp.

Med. 179:1127.

903. Trambas, C. M., and Griffiths, G. M. (2003). Delivering the kiss of death. Nat Immunol 4, 399-403.

904. Trapani, J. A., V. R. Sutton. 2003. Granzyme B: pro-apoptotic, antiviral and antitumor functions. Curr. Opin. Immunol. 15:534.

905. Van Rooijen, N. (1989). The liposome-mediated macrophage 'suicide' technique. J Immunol Methods 124:1-6.

906. Van Rooijen, N., and Sanders, A. (1994). Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 174:83-93. 907. Van Rooijen, N., Sanders, A., and van den Berg, T. K. (1996). Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine. J Immunol Methods 193:93-99.

908. Veiga-Fernandes, H., B. Rocha. 2004. High expression of active CDK6 in the cytoplasm of CD8 memory cells favors rapid division. Nat. Immunol. 5:31. 909. Wagner, C, Iking-Konert, C, Denefieh, B., Stegmaier, S., Hug, F., and Hansch, G. M.

(2004). Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils. Blood 103:1099-1104.

910. Walden, P. R., and Eisen, H. N. (1990). Cognate peptides induce self-destruction of CD8⁺ cytolytic T lymphocytes. Proc Natl Acad Sci USA 87, 9015-9019. '9TIf ^IU1 ""Waffidt^MrMr; Worku, M., Goggle, S., and Thursz, M. R. (2002). A novel method for assessing gastritis in the murine model demonstrates genetically determined variation in response to Helicobacter felis infection. Helicobacter 7, 265-268.

912. Ward, D. M., Griffiths, G. M., Stinchcombe, J. C, and Kaplan, J. (2000). Analysis of the lysosomal storage disease Chediak-Higashi syndrome. Traffic 1, 816-822.

913. Ware, C. B., Siverts, L. A., Nelson, A. M., Morton, J. F., and Ladiges, W. C. (2003). Utility of a C57BL/6 ES line versus 129 ES lines for targeted mutations in mice. Transgenic Res 12, 743-746.

914. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4:225.

915. Wilson, S. M., Yip, R., Swing, D. A., O'Sullivan, T. N., Zhang, Y., Novak, E. K., Swank, R. T., Russell, L. B., Copeland, N. G., and Jenkins, N. A. (2000). A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proc Natl Acad Sci U S A 97, 7933-7938.

916. Wong, P., E. G. Pamer. 2003. Feedback regulation of pathogen-specific T cell priming. Immunity 18:499.

917. Ye, R. D., Wun, T. C, and Sadler, J. E. (1988). Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells. J Biol Chem 263, 4869-

4875.

918. Ying, Q. L., Kemme, M., and Simon, S. R. (1994). Functions of the N-terminal domain of secretory leukoprotease inhibitor. Biochemistry 33, 5445-5450.

919. Zagury, D., Bernard, J., Thierness, N., Feldman, M., and Berke, G. (1975). Isolation and charcaterization of individual functionally recative cytotoxic T lymphocytes. Conjugation, killing and recycling at the single cell level. Eur J Immunol 5, 812-818.

920. Zajac, A. J., J. M. Dye, D. G. Quinn. 2003. Control of lymphocytic choriomeningitis virus infection in granzyme B deficient mice. Virology 305:1.

921. Zhang, G., Budker, V., and Wolff, J. A. (1999). High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 10, 1735-1737.

922. Zhou, Q., Snipas, S., Orth, K., Muzio, M., Dixit, V. M., and Salvesen, G. S. (1997). Target protease specificity of the viral serpin CrmA: analysis of five caspases. JBiolChem 272:7797-7800. 923. Zhou, S., R. Ou, L. Huang, D. Moskophidis. 2002. Critical role for perforin-, Fas/FasL-, and

TNFRl -mediated cytotoxic pathways in down-regulation of antigen-specific T cells during persistent infection. J. Virol. 76:829.

924. Zhumabekov, T., P. Corbella, M. Tolaini, D. Kioussis. 1995. Improved version of a human CD2 minigene based vector for T cell-specific expression in transgenic mice. J. Immunol. Methods 185:133. ^■ _ΨZ ^<3t "•••^{» ■}'^■■lOnJternagei¹; rcv M., and Doherty, P- C. (1974). Restriction of in vitro T cell mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semi-allogeneic system. Nature 248:701- 702.

G. Sequences 1. SEQ ID NO:l PI9 cDNA

2. SEQ ID NO:2 PI9 protein

3. SEQ ID NO:3 Spi6 cDNA

4. SEQ ID NO:4 Spi6 protein

5. SEQ ID NO:5 Spi6 forward primer to identify transgenic founder 6. SEQ ID NO: 6 Spi6 reverse primer to identify transgenic founder

7. SEQ ID NO:7 (Sun et al., 1997) Real-time PCR fwd primer of Spi6

8. SEQ ID NO:8 (Sun et al., 1997) Real-time PCR rev primer of Sρi6:

9. SEQ ID NO:9 (Sun et al., 1997) Real-time PCR probe of Spi6:

10. SEQ ID NO:10 Real-time PCR granzymeB (Heusel et al., 1994) forward primer: 11. SEQ ID NO:11 Real-time PCR granzymeB (Heusel et al., 1994) reverse primer:

12. SEQ ID NO: 12 Real-time PCR granzymeB (Heusel et al., 1994) probe:

13. SEQ ID NO: 13 Real-time PCR I-A/3^b (Larhammar et al., 1985) (MHC class II) forward primer:

14. SEQ ID NO: 14 Real-time PCRI-A/3^b (Larhammar et al., 1985) (MHC class II) reverse primer:

15. SEQ ID NO: 15 Real-time PCR I-A/3^b (Larhammar et al., 1985) (MHC class II) probe

16. SEQ ID NO:16 Real-time PCR cyclophilin A (Medhurst et al., 2000) forward primer:

17. SEQ ID NO: 17 Real-time PCR cyclophilin A (Medhurst et al., 2000) reverse primer

18. SEQ ID NO: 18 Real-time PCR cyclophilin A (Medhurst et al., 2000) probe: 19. SEQ ID NO: 19 GP33 peptide sequence from LCMV

20. SEQ ID NO:20 NP396 peptide sequence from LCMV

21. SEQ ID NO:21 GP276 peptide sequence from LCMV

'22."3¹E^ WΪKSΪΪ2 CD2 LCR (NT 019273)

23. SEQ ID NO:23 D2 Promoter (NT 019273)

24. SEQ ID NO:24 Cowpox virus white-pock variant (CPV- W2) (crmA) gene, complete cds (M14217) 25. SEQ ID NO:25 Cowpox virus white-pock variant (CPV-W2) (crmA) protein (M14217)

26. SEQ ID NO:26 Human alpha- 1 -antitrypsin (hal-AT) mRNA, complete cds (K01396)

27. SEQ ID NO:27 Human alpha-1 -antitrypsin (hal-AT) complete protein (K01396)

28. SEQ ID NO:28 Human plasminogen activator inhibitor-1 (PAI-I) mRNA, complete cds. (Ml 6006) 29. SEQ ID NO:29 Human plasminogen activator inhibitor-1 (PAI-I) complete protein

(Ml 6006)

30. SEQ ID NO: 30 Homo sapiens secretory leukocyte protease inhibitor (hSLPI)

(antileukoproteinase), mRNA (cDNA clone MGC:22479 IMAGE:4733996), complete cds.

(BC020708) 31. SEQ ID NO: 31 Homo sapiens secretory leukocyte protease inhibitor (hSLPI)

(antileukoproteinase), (cDNA clone MGC:22479 IMAGE:4733996), complete protein

(BC020708)

32. SEQ ID NO:32 Mus musculus serine (or cysteine) proteinase inhibitor, clade A, member

Ia (Serpinala) (mal-AT), mRNA. (NM_009245) mouse antitrypsin 33. SEQ ID NO:33 Mus musculus serine (or cysteine) proteinase inhibitor, clade A, member

Ia (Serpinala) (mal-AT), protein (NM_009245) mouse antitrypsin

34. SEQ ID NO:34 Mus musculus serine (or cysteine) proteinase inhibitor, clade E, member 1 (Serpinel) (mPAI-1), mRNA. (NM_008871)

35. SEQ ID NO:35 Mus musculus serine (or cysteine) proteinase inhibitor, clade E, member 1 (Serpinel) (mPAI-1), mRNA. (NM_008871)

36. SEQ ID NO:36 Mus musculus secretory leukocyte protease inhibitor (Slpi) (mSlpi), mRNA.(NM 0114141

37. SEQ ID NO:37 Mus musculus secretory leukocyte protease inhibitor (Slpi) (mSlpi), protein (NM_0114141 38. SEQ ID NO:38 pBluescript II SK(-), 2961 bp version 122001

39. SEQ ID NO:39 Mus musculus serine (or cysteine) proteinase inhibitor, clade B, member Ib (Serpinblb) (EIA), mRNA. (NMJ 73052)

40. SEQ ID NO:40 Mus musculus serine (or cysteine) proteinase inhibitor, clade B, member Ib (Serpinblb) (EIA), protein (NM_173052) 41. SEQ ID NO:41 Homo sapiens serine (or cysteine) proteinase inhibitor, clade B

(ovalbumin), member 1 (SERPINBl) (MNEI), mRNA.(NM_030666) Ψ2. S¹E¹Q¹ID JSIU:42 iiomo sapiens serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1 (SERPINBl) (MNEI), protein (NM_030666)

43. SEQ ID NO:43 Sυi6 ORF

44. SEQ ID NO:44 PI9 ORF 45. SEQ ID NO:45 PI9-3xFLAG transgenic DNA

46. SEQ ID NO:46 PI9 Ribozyme Hammerhead (Start: 1885)

47. SEQ ID NO:47 PI9 Ribozyme Hammerhead (Start: 276)

48. SEQ ID NO:48 PI9 Ribozyme Hammerhead (Start: 896)

49. SEQ ID NO:49 PI9 siRNA (Start: 1197) 50. SEQ ID NO:50 PI9 siRNA (Start: 1608)

51. SEQ ID NO:51 PI9 siRNA (Start: 379)

52. SEQ ID NO:52 PI9 siRNA (Start: 599)

53. SEQ ID NO:53 PI9 siRNA (Start: 679)

54. SEQ ID NO:54 PI9 siRNA (Start: 249) 55. SEQ ID NO:55 PI9 siRNA (Start: 1381)

56. SEQ ID NO:56 PI9 siRNA (Start: 378)

57. SEQ ID NO:57 PI9 siRNA (Start: 1609)

58. SEQ ID NO:58 PI9 siRNA (Start: 1610)

59. SEQ ID NO:59 PI9 siRNA (Start: 2150) 60. SEQ ID NO:60 PI9 siRNA (Start: 2201)

61. SEQ ID NO:61 PI9 siRNA (Start: 2370)

62. SEQ ID NO:62 PI9 siRNA (Start: 2458)

63. SEQ ID NO:63 PI9 siRNA (Start: 2460)

64. SEQ ID NO:64 PI9 siRNA (Start: 1197) 65. SEQ ID NO:65 PI9 siRNA (Start: 1608)

66. SEQ ID NO:66 PI9 siRNA (Start: 379)

67. SEQ ID NO:67 PI9 siRNA (Start: 599)

68. SEQ ID NO:68 PI9 siRNA (Start: 679)

69. SEQ ID NO:69 kanamycin resistance protein siRNAs 70. SEQ ID NO:70 EGFP expression vector siRNAs

71. SEQ ID NO:71 3' UTR of hepatitis C virus siRNAs

72. SEQ ID NO:72 Spi6 KO Sequence

73. SEQ ID NO:73 Spi6 DNA Sequence

74. SEQ ID NO:73 PI9 DNA Sequence

Claims

V. Claims

We claim:

I . A transgenic non-human animal comprising a cell wherein the cell expresses a transgene coding for a serpin.

2. The transgenic animal of claim 1 , wherein the serpin is a serpin with at least 60% identity to Sρi6, as set forth in SEQ ID NO:4.

3. The transgenic animal of claim 2, wherein the serpin is Spi6 or PI9.

4. The transgenic animal of claim 3, wherein the Spi6 comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:4.

5. The transgenic animal of claim 3, wherein the PI9 comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:2.

6. The transgenic animal of claim 2, wherein the Spi6 comprises a sequence in which the complete sequence hybridizes to the sequence set forth in SEQ ID NO:3, wherein the hybridization takes place at 600 mM NaCl, 60 degrees Celcius, buffered to pH 7.6.

7. The transgenic animal of claim 6, wherein the sequence remains hybridized to the sequence set forth in SEQ ID NO: 3 after a wash of 15mM NaCl, 1.5mM Na3 citrate, 1% SDS, 65 degrees celcius.

8. The transgenic animal of claim 2, wherein the Spi6 comprises a sequence as set forth in SEQ ID NO:3.

9. The transgenic animal of claim 2, wherein the PI9 comprises a sequence set forth in SEQ ID

NO:1.

10. The transgenic animal of claim 1, wherein the transgene comprises sequence encoding a serpin, a promoter operably linked to the serpin, and a selectable marker.

I I. The transgenic animal of claim 1, wherein the serpin is a serpin having granzyme B, cathepsin G (P28293), PR-3 (Q61096), neutrophil elastase (NPJB 1945), mouse mast cell protein (MMCP) -1

(Pl 1034), MMCP-2 (P15119), MMCP-3 (P21843), MMCP-4 (P21812), MMCP-5 (P21844), MMCP-8 (P43430), MMCP-9 (035164), MMCP-10 (AAK51075), or caspase 1 (P29452)_as a substrate.

12. The transgenic animal of claim 11, wherein the serpin is a serpin having Granzyme B as a substrate. ¹HP 'i.lrø!tr'anSgffliC'.;-uπ£naii' of claim 1, wherein the amount of serpin produced in the animal is more than the amount of serpin produced in a non-transgenic animal.

14. The transgenic animal of claim 1, wherein the animal has increased protection against programmed cell death.

15. The transgenic animal of claim 1, wherein the serpin is overexpressed, and wherein the overexpression of the serpin leads to an enhanced memory cell phenotype.

16. The transgenic animal of claim 15, wherein the memory is enhanced 2, 3, 4, 5, 6, 7, 8, 9, 10, fold higher than an animal not having the transgene.

17. The transgenic animal of claim 1, wherein the serpin is expressed in naive cells at least 100, 75, 53, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,

6, 5, 4, 3, or 2, fold, over the amount of the serpin expressed in the naive cells of an animal not having the transgene.

18. The transgenic animal of claim 1, wherein the ratio of serpin to cyclophilin is at least 100, 75, 53, 50, 40, 31.6. 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

19. The transgenic animal of claim 1, wherein the expression of the serpin was at least 100, 75, 53, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.8 5, 4, 3, or 2 fold higher in memory cells than in naϊve cells.

20. The transgenic animal of claim 1, wherein the serpin expression protects T cells from death initiated by granzyme B delivered perform.

21. The transgenic mouse of claim 20, wherein the protection is greater than the protection in a non- transgenic animal.

22. The transgenic animal of claim 1, wherein infection of the transgenic mouse with LCMV results in higher numbers of memory cells than an animal not having the transgene.

23. The transgenic animal of claim 22, wherein the animal not expressing the transgene is a B6 mouse.

24. The transgenic animal of cliam 1, wherein the expression pattern of Spi6 correlates with granzyme B expression.

25. The transgenic animal of claim 1, wherein coexpression of Spi6 and granzyme B in anti-LCMV effectors is retained in resulting memory cells. 2&H^ri^fπWalisg&¥ϊ<ϊ! aftitøaTOl claim 1, wherein CD2 drives expression in hematopoietic cells.

27. The transgenic animal of claim 1, wherein the expression is higherst in natural killer cells (NK cells)

28. The transgenic animal of claim 1, wherein the expression in the NK cells is at least 10, 20, 30, 40, or 50 fold higher than in other cell types.

29. The transgenic animal of claim 1, wherein the expression is highest in thymocytes, lymphocytes, or phagocytic myeloid cells.

30. The transgenic animal of claim 1 , wherein the animal was produced by a method comprising introducing into a non-human animal fertilized egg a recombinant nucleic acid molecule, which comprises a nucleic acid encoding a serpin whereby a transgenic animal expressing the serpin is produced.

31. A method of protecting T cells from the granule exocytosis pathway of programmed cell death, comprising administering a vector expressing Spi6.

32. A method of increasing the number of CD8+ memory T cells, by inhibiting programmed cell death, comprising administering a vector expressing Spi6.

33. A method of identifying a candidate inhibitor of Spi6/PI9 activity comprising (a) preparing a first cell culture that overexpresses Spi6; (b) adding the candidate inhibitor to the cell culture (c) incubating the cell culture under conditions and for a time sufficient to detect an inhibitory effect by the candidate inhibitor; and (d) determining the effect of the candidate inhibitor on the Spi6 activity.

34. The method of claim 33, wherein the cell culture comprises a neutrophil or macrophage.

35. The method of claim 33, further comprising assaying for increased in vitro bactericidal activity of neutrophils or macrophages.

36. The method of claim 34, wherein the neutrophils or macrophages are from a Sρi6 transgenic mouse.

37. A method of identifying a candidate inhibitor of Spi6/PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses Spi6; (b) administering the candidate inhibitor to the animal (c) determining the effect of the candidate inhibitor on the Spi6 activity.

38. The method of claim 37, wherein the transgenic animal comprises the transgenic animal of claim 1. 3ST- A"rϊiettioQ"θi_"δef€©ning a set of candidate inhibitors of Spi6/PI9 activity comprising (a) preparing a transgenic non-human animal that overexpresses Spi6; (b) administering the set of candidate inhibitors to the animal (c) determining the effect of the set of candidate inhibitors on the Spi6 activity.

40. The method of claim 39, further comprising d) the step of subdividing the set of candidate inhibitors into subsets of candidate inhibitors, and e) testing each subset for inhibitory activity in the transgenic non-human animal that overexpresses Spi6 until a subset having the inhibitory activity is identified.

41. The method of claim 40, further comprising f) the step of subdividing the identified subset of candidate inhibitors in a set of small subsets of candidate inhibitors, g) and testing each small subset of candidate inhibitors for inhibitory activity in the transgenic non-human animal that overexpresses Spi6 until a small subset having the activity is identified.

42. The method of clam 41, further comprising repeating steps f and g until a single candidate inhibitor having inhibitory activity is identified.

43. A method of testing an inhibitor of Spi6/PI9 activity comprising (a) preparing a transgenic nonhuman animal that overexpresses Spi6; (b) administering the inhibitor to the animal (c) determining the effect of the inhibitor on the Spi6 activity.

44. The method of claim 43, further comprising comparing the activity of the inhibitor to the activity of a known standard.

45. The method of claim 43, further comprising comparing the activity of the inhibitor to the activity of the inhibitor that occurred in a previous test of the compound in the transgenic nonhuman animal.

46. A method of treating a disease caused by excess granzyme B activity by inhibiting Granzyme B, comprising administering a composition, wherein the composition expresses Spi6.

47. The transgenic animal of claim 1, wherein the number of memory cells is enhanced when compared to an animal without the transgene.

48. The transgenic animal of claim 47, wherein the number of memory cells is enhanced at least 2 fold when compared to an animal without the transgene.

49. The transgenic animal of claim 47, wherein the number of memory cells is enhanced between 2 and 3 fold higher than an animal having non-disrupted Spi6 expression.

50. A vaccine which decreases the number of boosters required to obtain memory cells comprising a

SPI6 or PI9 protein, or a fragment thereof and a pharmaceutically acceptable excipient. ^•3T:""meΥaecffie"øreiamα JU, further comprising a suitable adjuvant

52. A vaccine which decreases the amount of time for full memory cell response comprising a SPI6 or PI9 protein, or a fragment thereof and a pharmaceutically acceptable excipient.

53. The vaccine of claim 52, further comprising a suitable adjuvant

54. A method of increasing immunity to viral infection comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid.

55. A method of increasing immunity to viral infection comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof.

56. A method of protecting the integrity of lytic granules comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid.

57. A method of protecting the integrity of lytic granules comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof.

58. A method of protecting against inflammatory disease comprising administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid.

59. A method of protecting against inflammatory disease comprising administering an effective amount of SPI6 or PI9 protein, or a fragment thereof.

60. A method of inhibiting Granzyme B activity in the cytoplasm by administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid.

61. A method of inhibiting the presence of Granzyme B in the cytoplasm by administering an effective amount of SPI6 or PI9 protein, or a fragment thereof.

62. A method of inhibiting neutrophil elastase by administering a vector expressing a Spi6 nucleic acid or a PI9 nucleic acid.

63. A transgenic non-human animal comprising a disrupted Sρi6 gene.

64. The transgenic animal of claim 63, wherein the amount of SPI6 produced in the animal is less than the amount of SPI6 produced in a non-transgenic animal.

65. The transgenic animal of claim 63, wherein the animal has increased immunity.

66. The transgenic animal of claim 63, wherein the granulocytes of the animal have increased bactericidal activity. ^■67? ^M^"tάa§φWύ"MimaTof claim 63, wherein the disrupted Spi6 gene lacks native SPI6 function.

68. The transgenic animal of claim 63, wherein the disrupted Spi6 gene lacks native Spi6 expression.

69. The transgenic animal of claim 67, wherein the disruption of SPI6 function leads to increased neutrophil elastase activity.

70. The transgenic animal of claim 68, wherein the disruption of Spi6 expression leads to increased neutrophil elastase activity.

71. The transgenic animal of claim 63, wherein the disrupted Spi6 expression increases immunity to sepsis causing bacteria without causing inflammatory disease.

72. The transgenic animal of claim 63, wherein the disrupted Spi6 expression increases neutrophil function.

73. The transgenic animal of claim 63, wherein the disrupted Spi6 expression increases neutrophil recruitment to the site of infection.

74. The transgenic animal of claim 73, wherein the amount of neutrophil recruitment is at least 2 fold higher than an animal having non-disrupted Spi6 expression.

75. The transgenic animal of claim 74, wherein the amount of neutrophil recruitment is 2, 3, 4, 5, 6, or 7 fold higher than an animal having non-disrupted Spi6 expression.

76. The transgenic animal of claim 63, wherein the disrupted Spi6 expression increases the survival of the animal from septic shock induced death after infection with bacteria

77. The transgenic mouse of claim 63, wherein the neutrophil function is greater than the neutrophil function in a non-transgenic animal.

78. The transgenic animal of claim 63, wherein infection of the transgenic animal with E. coli results in increased bactericidal activity of neutrophils than an animal not having a disrupted Spi6 gene.

79. The transgenic animal of claim 63, wherein the animal was produced by a method comprising introducing into a non-human animal fertilized egg a recombinant nucleic acid molecule, which comprises a nucleic acid encoding a disrupted Spi6 gene whereby a transgenic animal expressing disrupted SPI6 is produced.

80. A transgenic animal cell comprising a disrupted Spi6 gene.

81. A vector, the vector comprising a portion of the Sρi6 gene, wherein the portion of the Spi6 gene produces a disrupted Spi6 gene, and wherein the vector can homologously recombine with the Spi6 gene. 82'.'"ΩtfWctoi"'ό¹fclaitti^l"8r,^ιrcomprisingthe sequence of SEQ ID NO:72.

83. A cell comprising the vector of claim 81.

84. An animal comprising the cell of claim 83.

85. An animal comprising the vector of claim 81.

86. A nucleic acid molecule produced by a process, the process comprising linking in an operative way a nucleic acid comprising the sequence of a Spi6 exon and sequence recognized by a recombinase enzyme.

87. A cell produced by the process of transforming a cell with the nucleic acid of claim 86.

88. A method of identifying a gene regulated by Sρi6 comprising:

(a) performing a microarray gene expression analysis of a Spi6 knockout mouse, wherein the gene expression analysis produces a first data set of expressed genes in the Spi6 KO mouse;

(b) performing a microarray gene expression analysis of a wild-type mouse, wherein the gene expression analysis produces a second data set of expressed genes in the wild-type pmose;

(c) comparing the first data set with the second data set; and

(d) identifying the genes in the Spi6 knockout mouse that are expressed differently than the wild-type mouse.

89. A method of drug discovery comprising administering a candidate drug to the Spi6 KO mouse.

90. A method of producing an animal, the method comprising administering the vector of claim 81 to an ES cell, culturing the cell, selecting a cell comprising the vector, fusing the selected cell with a blastocyst, thereby producing a chimera, incubating the chimera, and implanting the chimera into a surrogate mother to produce an offspring .

91. A method of producing an animal, the method comprising fusing the chimera of 90, with another chimera, and selecting live animals homozygous for vector DNA.

92. A method of enhancing immunity by inhibiting Spi6 expression.

93. The method of claim 92, wherein a disrupted Spi6 gene is introduced.

94. The method of claim 92, wherein a SPI6 inhibitor is introduced.

95. A method of increasing bactericidal activity in granulocytes comprising disrupting SPI6 function. yo. me rnetnocrorciarm y5, wherein SPI6 function is disrupted by introducing a disrupted Spi6 gene.

97. The method of claim 95, wherein SPI6 function is disrupted by administering an inhibitor of Spi6.

98. A method of enhancing immunity by increasing neutrophil elastase activity comprising administering an inhibitor of Spi6.

99. A method of increasing immunity to sepsis causing bacteria without giving inflammatory disease, comprising administering an inhibitor of Sρi6.

100. A method of increasing neutrophil function comprising administering an inhibitor of Spi6.

101. A method of treating a bacterial infection in a subject comprising administering to the subject an effective amount of a SPI6 inhibitor.

102. A method of treating a bacterial infection in a subject comprising introducing an effective amount of a disrupted Spiό gene to the subject.

103. A method of treating sepsis in a subject comprising administering to the subject an effective amount of a SPI6 inhibitor.

104. A method of treating sepsis in a subject comprising introducing an effective amount of a disrupted Spi6 gene to the subject.

105. A method of inhibiting Spi6 expression comprising administering an inhibitor of Spi6.

106. A method of inhibiting neutrophil elastase comprising administering a vector expressing Spi6.

107. A method of inhibiting neutrophil elastase comprising administering an effective amount of SPI6 protein or a fragment thereof.

108. A method of enhancing immunity by administering an inhibitor of Spiό in combination with human neutrophil elastase.

109. A method of treating a bacterial infection in a subject comprising administering to the subject an effective amount if an inhibitor of Spi6 in combination with human neutrophil elastase, and wherein the concentration of human neutrophil elastase is less than 1.8U/kg.

110. A method of treating sepsis in a subject comprising administering to the subject an effective amount if an inhibitor of Spi6 in combination with human neutrophil elastase, and wherein the concentration of human neutrophil elastase is less than 1.8U/kg HT. ' "^■A'ϊnetnϋα OI treating a bacterial infection in a subject comprising administering to the subject an effective amount of a disfunctional SPI6 protein or a fragment thereof, wherein the dysfunctional SPI6 protein or a fragment thereof prevents native SPI6 from inhibiting neutrophil elastase function.

112. A method of treating a bacterial infection in a subject comprising administering to the subject an effective amount of a disfunctional PI9 protein or a fragment thereof, wherein the dysfunctional PI9 protein or a fragment thereof prevents native PI9 from inhibiting neutrophil elastase function.