WO1999018909A9 - Interferon-gamma regulatory factors - Google Patents

Abstract

The invention provides a novel mammalian T cell stimulatory factor, dendritic cell derived factor, which mediates the effects of GM-CSF on proliferation and production of IFN-η by T cells. The invention also provides mammalian T cell derived factors which inhibit IFN-η production.

Description

TNTERFERON-γ REGULATORY FACTORS

Field of the Invention

This invention relates to factors which regulate interferon-γ production, including dendritic cell derived factors which restores proliferation and interferon-γ production by T cells in GM-CSF -/- mice, and T cell derived factors which inhibit interferon-γ production.

Background of the Invention

Granulocyte/macrophage colony-stimulating factor (GM-CSF) has received much attention since the cloning of mouse and human GM-CSF and the availability of the recombinant product. Studies with recombinant GM-CSF (rGM-CSF) substantiated initial observations showing the effect of GM-CSF on the proliferation and maturation of myeloid progenitor cells in vitro and the increase in granulocytes and monocytes in peripheral blood following GM-CSF injection (Moore, Annu. Rev. Immunol. 9:159-191, 1991 ; Metcalf, Blood 67:257-267, 1986). GM-CSF has also been found to be critical for the in vitro differentiation and proliferation of dendritic cells (DC) from hematopoietic precursor cells (Witmer-Pack et al., J. Exp. Med. 166:1484-1498, 1987). Although the hematopoietic activities of GM-CSF have received most attention, there is growing interest in the immunological effects of this cytokine, particularly its use as an immunological adjuvant (Disis et al., Blood 88:202-210, 1996) and its ability to augment immune responses to tumor antigens. In a study of mouse B 16 melanoma, Dranoff et al. (Proc. Acad. Nat 'I Sci. USA 90:3539-3543, 1993) compared the relative immunogenicity of B16 cells transduced with retroviral vectors coding for various cytokines. In a comparison of 7 different cytokines, immunization with GM-CSF producing B16 cells showed the greatest protection against subsequent challenge with parental B16 cells, and this immunity was mediated by CD4⁺ and CD8⁺ T cells (Dranoff et al., 1993). Tao et al. reported that immunization with an idiotype/GM-CSF fusion protein produced higher levels of anti-idiotype antibodies and greater production against an idiotype positive B cell lymphoma than immunization with idiotype alone, idiotype mixed with GM-CSF or idiotype with adjuvant (Nature 362:755-758, 1993). Jager et al. have recently shown that rGM-CSF can enhance the generation of cytotoxic T lymphocytes (CTL) and development of hypersensitivity following immunization with peptides derived from melanoma differentiation antigens (Int. J. Cancer 67:54-62, 1996).

Several steps are required for the generation of a specific T cell response, starting with antigen processing and presentation of antigenic peptides by MHC on APC, followed by T cell priming from signals transmitted through the TCR and through co-stimulatory molecules, such as CD28 and CD40 ligand, resulting in the clonal expansion of T cells (Grewal et al., Nature 378:617-620, 1995; Bluestone, /mmwwty 2:555-559, 1995). Since the initial recognition of IFN-γ as an anti-viral agent (Wheelock, Science 149:310-

311, 1965), IFN-γ has emerged as one of the central mediators in inflammation and immunity (Farrar, and Schreiber, Ann. Rev. Immunol. 11 :571-611, 1993). For this reason, the regulation of IFN-γ biosynthesis has been of great interest, and the identification of IL-12 as a potent IFN-γ- inducing factor has made it possible to define more precisely the mechanism of IFN-γ production in innate and acquired immunity (Kobayashi et al., J. Exp. Med. 170:827-845, 1989). LPS is a potent activator of IL-12 production by macrophages, and IL-12 in turn activates T cells and NK cells; the two major cellular sources of IFN-γ to produce cytokine. For IFN-γ production by T cells, IL-2 is a major cofactor with IL-12 (Trinchieri, Ann. Rev. Immunol. 13:251-276, 1995), and TNF is a major IL-12 cofactor for IFN-γ production by NK cells (Tripp et al., Proc. Natl. Acad. Sci. USA 90:3725-3729, 1993). IL-10, a cytokine with anti- inflammatory activity mediated in part by downregulating IL-12 and IFN-γ production, is also a product of LPS-activated macrophages and is concomitantly produced with IL-12 (Berg et al., J. Clin. Invest. 96:2339-2347, 1995).

Consequently, it is desirable to obtain additional factors which regulate IFN-γ production as a means to modulate inflammation and immune responses.

Summary of the Invention

The availability of GM-CSF deficient mice makes it now possible to define more precisely the role of this cytokine in immune responses. In the present study, T and B cell functions in mice lacking GM-CSF were examined. Further, the basis for the poor IFN-γ response in LPS-injected GM-CSF-/- mice was analyzed.

The invention provides novel mammalian T cell stimulatory factors, termed dendritic cell derived factors, which mediate the effects of GM-CSF on CD4⁺ T lymphocyte proliferation and production of IFN-γ by CD4⁺ T cells. The dendritic cell derived factors also can restore the responsiveness of GM-CSF -/- CD4⁺ T cells to IL-2. In addition, the LPS-induced defect in IFN-γ production was corrected by a GM-CSF-induced dendritic cell derived factor. Methods for identification and isolation of the dendritic cell derived factors also are provided. The invention also provides factors which can inhibit the production of IFN-γ, termed T cell derived factors, and methods for identification and isolation of such factors.

The dendritic cell derived factor or T cell derived factor is from an mammal, including a human, a non-human primate, a sheep, a goat, a horse, a cow, a dog, a cat, and a rodent. The preferred dendritic cell derived factor is from an human.

According to one aspect of the invention, a dendritic cell derived factor is provided. The dendritic cell derived factor restores proliferation and interferon-γ production by CD4⁺ T cells when added to a mixture of CD4⁺ T cells from a GM-CSF -/- mammal immunized with an exogenous antigen such as KLH, non-dendritic cell antigen presenting cells from a GM-CSF -/- mammal, and the exogenous antigen. The restoration of proliferation and interferon-γ production is to levels substantially the same as those when the foregoing cells are derived from a wild type mammal, but which wild-type cells are free of exogenously added dendritic cell derived factor. Preferably the GM-CSF -/-mammal is a mouse. In some embodiments, the dendritic cell derived factor is present in a supernatant of a culture of dendritic cells derived from a GM-CSF +/+ mammal treated with LPS, which dendritic cells are cultured with GM-CSF. Preferably the GM-CSF +/+ mammal is a mouse. In other embodiments, the dendritic cell derived factor is present in a supernatant of a coculture of CD4⁺ T cells and dendritic cells pulsed with an exogenous antigen such as KLH. In the latter embodiments, the CD4⁺ T cells are derived from GM-CSF +/+ or GM-CSF -/- mammal, preferably mice, and the dendritic cells are derived from GM-CSF +/+ or GM-CSF -/-mammals, preferably mice. Functional variants of the foregoing dendritic cell derived factor also are provided.

According to another aspect of the invention, a dendritic cell isolate that has the characteristics of the foregoing dendritic cell derived factor is provided. Functional variants of the dendritic cell isolate also are provided. According to still another aspect of the invention, a dendritic cell derived factor of a mammal, preferably a human is provided. The dendritic cell derived factor restores interferon-γ production by CD8⁺ T cells when added to CD8⁺ T cells from GM-CSF '^" mammals, preferably mice, treated with lipopolysaccharide, the restoration being to levels substantially the same as those produced by CD8⁺ T cells from wild type mammals. In some embodiments the dendritic cell derived factor is present in a supernatant of a culture of dendritic cells derived from GM- CSF^{+ +} mammals, preferably mice, treated with LPS cultured with GM-CSF.

According to another aspect of the invention, methods for identifying an agent that has dendritic cell derived factor activity are provided. The methods include (a) preparing a mixture of (1) CD4⁺ T cells from a GM-CSF^"'^" mammal immunized with an exogenous antigen, (2) spleen cells from a GM-CSF -/- mammal, and (3) the exogenous antigen, and (b) applying a composition suspected of having the agent to the mixture of (a) and measuring the proliferation of the CD4⁺ T cells and the production of interferon-γ by the CD4⁺ T cells as a determination of the presence of the agent in the composition. Preferably the mammal is a mouse.

According to another aspect of the invention, methods for isolating a dendritic cell derived factor are provided. The methods include the steps of (a) preparing a mixture of (1) CD4⁺ T cells from a GM-CSF^"'^" mammal immunized with an exogenous antigen, (2) spleen cells from a GM-CSF^"7" mammal, and (3) the antigen, (b) preparing a culture of either (1) dendritic cells derived from a GM-CSF^+/+ mammal treated with lipopolysaccharide, the dendritic cells cultured with GM-CSF, or (2) the CD4⁺ T cells and dendritic cells pulsed with the antigen, (c) isolating a supernatant from the culture of step (b), (d) fractionating the supernatant into a plurality of fractions, and (e) applying one of the plurality of fractions to the mixture of (a) and measuring the proliferation of the CD4⁺ T cells and the production of interferon-γ by the CD4⁺ T cells as a determination of the presence of the dendritic cell derived factor in the fraction. Preferably the mammal is a mouse.

According to still another aspect of the invention, methods for identifying an agent that has dendritic cell derived factor activity are provided. The methods include (a) preparing a culture of CD8⁺ T cells from a GM-CSF^"7" mammal treated with lipopolysaccharide, and (b) applying a composition suspected of having the agent to the culture of (a) and measuring the production of interferon-γ by the CD8⁺ T cells as a determination of the presence of the agent in the composition. Preferably the mammal is a mouse.

According to another aspect of the invention, methods for isolating a dendritic cell derived factor are provided. The methods include the steps of (a) preparing a culture of CD8⁺ T cells from a GM-CSF^{" "} mammal treated with lipopolysaccharide, (b) preparing a culture of either (1) dendritic cells derived from a GM-CSF⁺⁷⁺ mammal treated with lipopolysaccharide, the dendritic cells cultured with GM-CSF, or (2) CD4⁺ T cells from a GM-CSF^"7" mammal immunized with an exogenous antigen and dendritic cells pulsed with the antigen, (c) isolating a supernatant from the culture of step (b), (d) fractionating the supernatant into a plurality of fractions, and (e) applying one of the plurality of fractions to the culture of (a) and measuring the production of interferon-γ by the CD8⁺ T cells as a determination of the presence of the dendritic cell derived factor in the fraction. Preferably the mammal is a mouse.

It believed that the dendritic cell derived factor is a protein encoded by a nucleic acid.

Non-protein factors can be isolated and characterized by procedures .known to one of ordinary skill in the art. A protein dendritic cell derived factor and the nucleic acids which encode such factors also are isolated and characterized by a host of protocols known to one of ordinaiy skill in the art. Thus, according to still another aspect of the invention, an isolated nucleic acid which encodes any of the foregoing dendritic cell derived factors or dendritic cell isolates, including the human homologs thereof is provided.

According to another aspect of the invention, an isolated nucleic acid which encodes a functional variant of any of the foregoing dendritic cell derived factors or dendritic cell isolates, including the human homologs thereof is provided.

The invention in another aspect provides an isolated polypeptide which selectively binds any of the foregoing dendritic cell derived factors or dendritic cell isolates, including the human homologs thereof. In some embodiments the isolated polypeptide is an Fab or F(ab) fragment of an antibody. In other embodiments the isolated polypeptide is a fragment of an antibody, the fragment including a CDR3 region selective for the protein. Instill other embodiments the isolated polypeptide is a monoclonal antibody, preferably a chimeric antibody or a humanized antibody.

According to yet another aspect of the invention, methods for increasing proliferation of a population of T cells are provided. The methods include administering to a subject in need of such treatment an amount of a dendritic cell derived factor effective to increase proliferation of the population of T cells.

According to another aspect of the invention, methods for increasing production of IFNγ by a population of T cells are provided. The methods include administering to a subject in need of such treatment an amount of a dendritic cell derived factor effective to increase production of

IFNγ.

According to yet another aspect of the invention, methods of identifying an agent that has

T-cell derived factor activity are provided. The methods include preparing a GM-CSF -/- mammal depleted of CD4+ and CD8+ T-cells, inducing serum IFN-γ production in the mammal. administering to the mouse a composition suspected of having the agent, detecting serum IFN-γ. and comparing the serum IFN-γ with a control as a determination of the presence of the agent in the composition. In some embodiments the mammal is a mouse; preferably serum IFN-γ production is induced in the mouse by treating the mouse with lipopolysaccharide. In other embodiments, the composition is selected from the group consisting of a membrane fraction of a

T-cell, a cytosolic fraction of a T-cell, a secreted fraction of a T-cell, a size-selected fraction of a

T-cell homogenate, a protein fraction of a T-cell homogenate, and a non-protein fraction of a T- cell homogenate. Also provided are agents that have T-cell derived factor activity which are identified by the foregoing methods.

An isolated nucleic acid is provided according to another aspect of the invention. The nucleic acid encodes the T cell derived factor identified by the foregoing methods, or the human homolog thereof. In another aspect, the invention provides an isolated nucleic acid which encodes a functional variant of the T cell derived factor identified by the foregoing methods, or the human homolog thereof.

In still another aspect, the invention provides an isolated polypeptide which selectively binds a T cell derived factor identified by the foregoing methods, or the human homolog thereof. In some embodiments, the isolated polypeptide is an Fab or F(ab) fragment of an antibody or a fragment of an antibody including a CDR3 region selective for the protein, or is a monoclonal antibody. In preferred embodiments, the monoclonal antibody is a chimeric antibody or a humanized antibody.

According to another aspect of the invention, methods for decreasing serum IFNγ concentration in a subject are provided. The methods include administering to a subject in need of such treatment an amount of a T cell derived factor effective to decrease serum IFNγ concentration.

Use of the foregoing factors in the preparation of a medicament also is provided. These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.

Brief Description of the Figures

Figs. 1A and IB show the CTL and proliferative responses of naive GM-CSF +/+ and GM-CSF -/- spleen cells against BALB/c spleen cells. In Fig. 1 A, spleen cells from GM-CSF +/+ (+/+) or GM-CSF -/- (-/-) mice were stimulated in vitro with mitomycin C-treated BALB/c spleen cells. Cytotoxicity was assessed with ⁵¹Cr-labeled BALB/c RLc^l (O) and C57BL EL4 (•) target cells. In Fig. IB, spleen cells from GM-CSF +/+ or GM-CSF -/- mice were stimulated in vitro with mitomycin C-treated autologous (D) or BALB/c (D) spleen cells. Proliferation was determined by incorporation of [methyl-³H] thymidine.

Figs. 2A, 2B and 2C depict the results of generation of CTL against OVA peptide. Spleen cells of GM-CSF +/+ (Fig. 2A) or GM-CSF -/- mice (Fig. 2B, Fig. 2C) immunized with ONA peptide were stimulated with autologous spleen cells pulsed with OVA peptide and treated with mitomycin C. Cytotoxicity was assessed with ⁵¹Cr-labeled C57BL EL4 target cells pulsed with OVA peptide (O) or with no peptide (•). 5 ng rGM-CSF was injected with OVA peptide in the hind footpads of GM-CSF -/- mice followed by 10 ng/day rGM-CSF injected intraperitoneally for 5 days, resulting in the generation of OVA specific CTL (Fig. 2C). Fig. 3 A and B show the antibody responses to KLH. In Fig. 3 A. 100 μg KLH in CFA was injected in hind footpads of GM-CSF -/- (•) and GM-CSF +/+ (O) mice. In Fig. 3B, 100 μg KLH with 40 ng rGM-CSF in CFA were injected in hind footpads of GM-CSF -/- mice followed by 10 ng/day rGM-CSF injected intraperitoneally for 5 days a week until mice were sacrificed. Fig. 3B depicts antibody responses in GM-CSF -/- mice(#) and GM-CSF -/- mice with rGM-CSF (Δ). Each group had four mice. Individual mice were bled at 1, 2 and 4 weeks after immunization and sera were titered for anti-KLH antibody at double dilutions of 1 : 500 to 1 : 16000 using isotype-specific ELISA. The results of serum samples diluted 1 :4000 are plotted in this figure. Similar results were obtained with the other serum dilutions. Specificity of antibodies were confirmed using HEL as a negative control. Figs. 4 A and 4B depict CD4⁺ T cell proliferative responses to KLH, immobilized anti-

CD3 mAb and ConA following immunization with KLH in CFA. In Fig. 4A, purified CD4⁺ T cells from draining lymphnodes of immunized GM-CSF -/- or GM-CSF +/+ mice were stimulated with spleen cells pulsed with KLH (D) or without KLH (D) as indicated. Spleen cells were pulsed with 100 μg/ml KLH for 1 h. In Fig. 4B, purified CD4" T cells from immunized GM-CSF -/- (•) or GM-CSF +/+ (O) mice were stimulated with immobilized anti-CD3 mAb or Con A in vitro. Proliferation was determined by incorporation of [methyl-³H] thymidine. Figs. 5 A and 5B show that both DC and culture supernatants of DC stimulate the proliferative responses and IFΝ-γ production by CD4¹ T cells from KLH-immunized GM-CSF -/- mice. In Fig. 5A. CD4⁺ T cells from KLH-immunized or naive mice were stimulated with autologous spleen cells or DC pulsed with KLH as indicated. To analyze the effect of supernatants from DC. 100 μ\ supernatants were added to cultures of CD4⁺ T cells and I LH- pulsed spleen cells from immunized GM-CSF -/- mice at a final volume of 200 μ\ (shown in Fig. 5B). Two sources of DC supernatants were used; 1) co-cultures of immunized GM-CSF +/+ or GM-CSF -/- CD4⁺ T cells and KLH-puIsed autologous DC (mitomycin C-untreated) for two days, and 2) cultures of DC from LPS-treated GM-CSF +/+ or GM-CSF -/- mice with 100 ng/ml rGM-CSF for two days. For control purposes, the effects of rGM-CSF or IL-12 added to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from immunized GM-CSF -/- mice were also tested (Fig. 5B). Proliferation was determined by incorporation of [methyl-³H] thymidine and levels of IFNγ in the culture supernatants were measured by ELISA.

Fig. 6 shows serum levels of cytokines following LPS administration. GM-CSF +/+ mice (O) and GM-CSF -/- (•) mice were injected i.p. with 100 μg LPS and bled from the retroorbital plexus. The data are the mean ± S.D. of 4 mice.

Fig. 7 depicts the restoration of serum levels of IFN-γ in GM-CSF-/- mice injected with GM-CSF. GM-CSF -/- mice were injected i.p. with 100 μg LPS along with 200 ng (O), 100 ng (Δ), 50 ng (D), 25 ng GM-CSF (A) or no (+) GM-CSF and bled 7 hrs later. As controls, GM-CSF +/+ mice were injected i.p. with 100 μg LPS alone (•). Each symbol represents an individual mouse with each group having 5 mice.

Fig. 8 demonstrates low production of IFN-γ by T cells from LPS-injected GM-CSF -/- mice. GM-CSF+/+ mice (O) and GM-CSF-/- mice (•) were injected i.p. with 100 μg LPS and sacrificed 3 hrs later. Isolated 1 x 10⁵ CD4^~ or CD8⁺ T cells or 2 x 10⁵ CD16/32⁺ cells per well were cultured with IL-2 and IL-12 at the indicated concentrations. Supernatants were tested for IFN-γ by ELISA after a 3 day culture period. The data are the mean ± S.D. of 4 mice.

Figs. 9A and B shows the IFN-γ production and proliferative responses of T cells. Fig. 9A shows IFN-γ production and proliferative responses of T cells from LPS-injected and naive GM-CSF -/- and GM-CSF +/+ mice. Isolated CD8⁺ cells (1 x 10⁵ cells for IFN-γ assays and 2 x 10⁵ cells for proliferation assays) were cultured for 3 days with 10 pg/ml IL-12 and 100 U/ml (in the case of T cells from LPS-injected mice) or 10 pg/ml IL-12 and 1000 U/ml IL-2 (in the case of T cells from naive mice). Fig. 9B shows the effect of in vivo or in vitro supplementations with GM-CSF on IFN-γ production and proliferative response of CD8⁺ T cells. For in vivo supplementation with GM-CSF, mice were injected i.p. with 100 μg LPS alone, or 100 μg LPS with 100 ng GM-CSF: and spleens were harvested 3 hrs later. For in vitro supplementation, 25 ng/ml GM-CSF was added to cultures of CD8^" T cells from LPS-injected GM-CSF -/- mice. For IFN-γ assays, 1 x 10⁵ CD8⁺ T cells were cultured with 100 U/ml IL-2 and 100 pg/ml IL-12 for 3 days. For proliferation assays, 2 x 10⁵ T cells were cultured with 100 U/ml IL-2 for 3 days. IFN-γ in supernatants were measured by ELISA and proliferative responses were determined by incorporation of [methylJH] thymidine. The data are the mean ± S.D. of 4 mice.

Fig. 10 depicts the restoration of IFN-γ production from CD8⁺ T cells of LPS-injected GM-CSF -/- mice by supernatants from DC stimulated with GM-CSF and IL-4. 50 μl of DC culture supernatants with 100 U/ml IL-2 and 10 pg/ml IL-12 (total volume of 200 μl) and were added to cultures of 1 x 10⁵ CD8⁺ T cells from LPS-injected GM-CSF -/- mice. For proliferation assays, 2 x 10⁵ CD8" T cells were comparably cultured with 100 U/ml IL-2. For control purposes, effect of 2.5 ng/ml GM-CSF and 0.5 ng/ml IL-4 added to cultures of CD8⁺ T cells from LPS-injected GM-CSF -/- mice was tested. Following a 3 day culture period, supernatants were tested for IFN-γ by ELISA. Proliferative responses were determined by incorporation of [me hylJH] thymidine. The data are the mean ± S.D. of 3 mice.

Figs. 11 A and 1 IB shows the effects of supernatants from DC cultures. Fig. 11 A shows restoration of IFN-γ levels in LPS-injected GM-CSF -/- mice by DC culture supernatants. GM-CSF -/- mice were injected with 100 μg LPS along with 0.5 ml DC culture supernatant or 100 ng IL-18, or 5 ng GM-CSF and 1 ng IL-4. For control purposes, GM-CSF +/+ mice were injected with 100 μg LPS alone. All mice were bled 7 hrs later. The data are the mean ± S.D. of 4 mice. Fig. 1 IB depicts the effect of injection of supernatant from DC cultures on in vitro IFN-γ production and proliferative responses of CD8⁺ T cells. Mice were injected with LPS alone or LPS with other reagents as mentioned above and sacrificed 3 hrs later. T cell cultures were perfoimed as described for Fig. 9. The data are the mean ± S.D. of 4 mice.

Detailed Description of the Invention

The present invention relates in part to a novel dendritic cell derived factor which increases the responses of T cells to stimulatory factors such as antigens and lipopolysaccharide (LPS). As described in greater detail below, the dendritic cell derived factor is produced by dendritic cells activated by GM-CSF. The dendritic cell derived factor is also produced by dendritic cells which interact with CD4^* T cells in the absence or the presence of GM-CSF. The dendritic cell derived factor is not IL-12, IL-18 or GM-CSF.

The dendritic cell derived factor also can increase the production of IFNγ by CD4⁺ T cells derived from GM-CSF -/- mice. The dendritic cell derived factor also can restore the responsiveness of GM-CSF -/- CD4⁺ T cells to IL-2. Further, the dendritic cell derived factor can restore the production of IFNγ in CD8⁺ T cells from GM-CSF -/- mice treated with LPS. Therefore the dendritic cell derived factor is useful generally in conditions where increased proliferation of CD4⁺ T cells and increased production of IFN-γ by CD4⁺ and/or CD8⁺ T cells is desirable. These conditions include immunization of a mammal with an antigen (e.g. vaccination) to increase the mammal's immune response to such an antigen. A mammal as used herein means humans, non-human primates, dogs, cats, pigs, rodents, cows, sheep, horses, and goats.

Dendritic cell derived factor can be isolated from several sources, including dendritic cells which are activated by GM-CSF, cultures of antigen-presenting dendritic cells and antigen- specific CD4" T cells in the presence of GM-CSF and cultures of antigen-presenting dendritic cells and antigen-specific CD4⁺ T cells in the absence of GM-CSF. The presence of the dendritic cell derived factor is detectable by the effect of the factor on the proliferation and/or IFN-γ production by CD4⁺ or CD8⁺ T cells. Dendritic cell isolates which have the characteristics of the dendritic cell derived factor also are provided.

The present invention relates in another part to a novel factor derived from or induced by T cells (termed "T cell derived factor") which decreases the IFN-γ response of animals to stimulatory factors such as lipopolysaccharide (LPS). As described in greater detail below, the T cell derived factor is produced by T cells, as shown by experiments in which GM-CSF +/+ and GM-CSF -/- mice depleted of T cells and exposed to LPS exhibited increased serum IFN-γ concentration. Therefore the T cell derived factor is useful generally in conditions where decreased production of IFN-γ is desirable. These conditions include reducing a mammal's immune response to an antigen, reducing inflammatory responses, etc.. A mammal as used herein means humans, non-human primates, dogs, cats, pigs, rodents, cows, sheep, horses, and goats.

T cell derived factor can be isolated from several sources, including T cells isolated from mammals, cultured T cells and other biological materials which may express the T cell derived factor. The presence of the T cell derived factor is detectable by the effect of the factor on the IFN-γ production by mammals which retain T cells. Thus mammals can be depleted of T cells, exposed to compositions suspected of containing T cell derived factor, challenged with LPS, and serum IFN-γ levels determined. A T cell derived factor reduces the production of serum IFN-γ in the absence of T cells. T cell isolates which have the characteristics of the T cell derived factor also are provided.

As used herein with respect to dendritic cell derived factor or T cell derived factor, "isolated" means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may, but need not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. Substantially pure polypeptides may be produced by techniques well lcnown in the art. Because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight of the preparation. The protein is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, i.e. isolated from other proteins.

The dendritic cell derived factor or T cell derived factor can be isolated from a non- homogenous proteinaceous solution such as a cell culture supernatant or cell homogenate. For example, dendritic cells isolated from an animal which has intact GM-CSF (i.e. a GM-CSF +/+ animal) can be cultured according to standard cell culture techniques. In small scale, the cultures can be contained in culture plates, flasks, and dishes. In larger scale, the cultures can be contained in roller bottles, spinner flasks and other large scale culture vessels such as feimenters. The dendritic cells or T cells preferably are isolated from animals which are pretreated with a mitogen such as LPS. Preferably, the dendritic cell culture is stimulated for one or more days with GM-CSF at approximately 100 ng/ml. Other concentrations of GM-CSF which stimulate the dendritic cell derived factor can also be used. The dendritic cell derived factor also can be isolated from a co-culture of CD4⁺ T cells and dendritic cells taken from an animal previously exposed to an antigen. The dendritic cells are further pulsed with the antigen prior to and/or during the co-culture to promote interaction of the dendritic cells and T cells.

Conveniently, the dendritic cell derived factor can be isolated from the supernatants of the above-described cell cultures, although the entire culture can be homogenized and subjected to the steps described below for isolation of the dendritic cell derived factor. Typically the supernatant is removed by aspiration or by centrifugation of the cell culture to remove the cells. The cultures can also be filtered to remove cells and cell debris.

The dendritic cell derived factor containing supernatant can be fractionated according to standard chromatographic procedures to facilitate isolation of the dendritic cell derived factor. One of ordinary skill in the art will be familiar with such procedures, including size exclusion chromatography, FPLC, HPLC, gel filtration chromatography, ion-exchange chromatography, hydrophobic chromatography, etc.

The T cell derived factor can be isolated from T cell homogenates or supernatants, or other biological materials, using the methodology described above for dendritic cell derived factor isolation.

In preferred embodiments, the fractions of dendritic cell derived factor containing supernatant then are used to stimulate a culture of exogenous antigen-specific GM-CSF -/- CD4 T cells to proliferate or produce IFN-γ. The CD4⁺ T cells are derived from a mammal which (1) is null for GM-CSF so that the proliferative response of the CD4⁺ T cells is impaired, and (2) is previously immunized with an exogenous antigen such as keyhole limpet hemocyanin (KLH). The culture contains in addition to the CD4⁺ T cells, antigen presenting cells and the antigen recognized by the CD4⁺ T cells. Preferably the antigen and non-dendritic cell antigen-presenting cell (e.g. spleen cell, B cell) are contacted prior to adding to the culture (i.e., the antigen- presenting cells are "pre-loaded" with antigen). Alternatively, the fractions can be used to stimulate IFN-γ production by CD8⁺ T cells from GM-CSF -/-mammals treated with lipopolysaccharide (LPS).

The response of the CD4⁺ T cells in the culture can be measured by determining the proliferation of the CD4⁺ T cells using standard methods, including uptake of ³H-thymidine. The production of IFN-γ by CD4⁺ or CD8⁺ T cells, or CD16/32⁺ NK cells, in vivo or in vitro can be determined by methods well .known in the art, e.g. ELISA, some of which are described in greater detail in the Examples. Other suitable methods will be known to one of ordinary skill in the art and can be employed using only routine experimentation.

The fractions which are positive for the dendritic cell derived factor or T cell derived factor can be subjected to additional rounds of screening using the foregoing methodology. The purity of the fraction can be assessed after each round of culture stimulation by subjecting an aliquot of the fraction to SDS-PAGE or other analytical method for visualizing the mixture of constituents in the fraction. The nature of the dendritic cell derived factor as a protein, nucleic acid, lipid, carbohydrate etc., can be confirmed at any time by treating an aliquot of a positive fraction with non-specific degradative enzymes for the foregoing classes of molecules and testing the treated fraction in the same assays detailed above.

The dendritic cell derived factor or T cell derived factor can then be further isolated if desired using immunological and molecular biological methods (see, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York). For example, a fraction positive for the dendritic cell derived factor which is sufficiently purified can be subjected to protein sequencing according to standard methods. For example, the fraction can be subjected to SDS- PAGE, transferred to a membrane such as polyvinylidene fluoride by electroblotting, and N- terminal amino sequence determined by Edman degradation. Any sequence information can be used to screen databases for homology to existing proteins and also to generate degenerate nucleic acids useful for screening a cDNA library by standard methods such as colony hybridization or polymerase chain reaction. Alternatively, the positive fraction can be used to generate antibodies which recognize the dendritic cell derived factor. Such antibodies can then be used in expression cloning protocols, Western blots, and other techniques useful in isolation of the dendritic cell derived factor. In the foregoing methods, any cDNA libraries, expression libraries etc. are preferably created from dendritic cells of a type known to express the dendritic cell derived factor (e.g. GM-CSF activated dendritic cells).

As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5^* and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques .known to those of ordinary skill in the art. An isolated nucleic acid as used herein is not a naturally occurring chromosome. The dendritic cell derived factor or T cell derived factor also can be isolated using methods which permit isolation of a nucleic acid encoding the dendritic cell derived factor. For example, the techniques of subtractive hybridization or differential display can be used to isolate nucleic acid(s) which are expressed differentially in cells described above which are sources for the dendritic cell derived factor. Thus, for example, nucleic acids isolated from dendritic cells from GM-CSF +/+ mice treated with LPS and cultured in GM-CSF can be compared with nucleic acids isolated from the same cells from mice untreated with LPS, or from GM-CSF -/- mice. Nucleic acids isolated from dendritic cells (from GM-CSF +/+ or -/- mice) presenting an exogenous antigen such as KLH and co-cultured with KLH-specific CD4+ T cells can be compared with nucleic acids from the same cells treated in the same way except that the dendritic cells do not present the KLH antigen. The pairs of dendritic cells will produce nucleic acids alike in many respects except that only one of the pair of dendritic cells will express the nucleic acid(s) encoding the dendritic cell derived factor(s). These methods can be applied to T cells for isolation of the T cell derived factor. Application of these standard techniques will require only routine experimentation by one of ordinary skill in the art.

Isolation of a nucleic acid which encodes dendritic cell derived factor or T cell derived factor by either of the foregoing approaches (isolation of protein followed by cloning or isolation of nucleic acid by subtractive hybridization or differential display) permits isolation of homologs and alleles of the nucleic acid and encoded factors.

The invention thus involves in one aspect dendritic cell derived factors, genes encoding those polypeptides, functional modifications and variants of the foregoing, useful fragments of the foregoing, as well as diagnostics and therapeutics relating thereto. Homologs and alleles of the dendritic cell derived factor or T cell derived factor nucleic acids of the invention can be identified by conventional techniques. Thus, an aspect of the invention is those nucleic acid sequences which code for dendritic cell derived factors. Preferably the nucleic acid sequences hybridize under stringent conditions to a nucleic acid molecule encoding the mouse dendritic cells factor. The term "stringent conditions" as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically, stringent conditions, as used herein, refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyirolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH₂PO₄(pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid. After hybridization, the membrane upon which the DNA is transferred is washed, for example, in 2 x SSC at room temperature and then at 0J - 0.5 x SSC/OJ x SDS at temperatures up to 68°C.

There are other conditions, reagents, and so forth which can be used, which result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of dendritic cell derived factor nucleic acids of the invention (e.g., by using lower stringency conditions). The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.

In general homologs and alleles typically will share at least 40% nucleotide identity and/or at least 50% amino acid identity to the sequences of dendritic cell derived factor nucleic acid and polypeptides. respectively, in some instances will share at least 50% nucleotide identity and/or at least 65%) amino acid identity and in still other instances will share at least 60% nucleotide identity and/or at least 75% amino acid identity. Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.

In screening for dendritic cell derived factor or T cell derived factor genes, a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film to detect the radioactive signal. In screening for the expression of dendritic cell derived factor or T cell derived factor nucleic acids, Northern blot hybridizations using the foregoing conditions can be performed on samples taken from dendritic cells, T cells, or other biological materials. .Amplification protocols such as polymerase chain reaction using primers which hybridize to the dendritic cell derived factor or T cell derived factor nucleic acid sequences also can be used for detection of the dendritic cell derived factor or T cell derived factor genes or expression thereof.

The invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus. in vitro or in vivo, to incorporate a serine residue into an elongating dendritic cell derived factor. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.

The invention also provides isolated unique fragments of dendritic cell derived factor or T cell derived factor nucleic acid sequences or complements thereof. A unique fragment is one that is a 'signature' for the larger nucleic acid. It, for example, is long enough to assure that its precise sequence is not found in molecules within the human genome outside of the dendritic cell derived factor nucleic acids defined above (and human alleles). Those of ordinary skill in the art may apply no more than routine procedures to determine if a fragment is unique within the human genome. Unique fragments, however, exclude fragments completely composed of the known nucleotide sequences of any of GenBank deposited EST or other previously published sequences which overlap a dendritic cell derived factor sequence or a T cell derived factor sequence.

A fragment which is completely composed of a sequence described in GenBank deposits is one which does not include any of the nucleotides unique to a dendritic cell derived factor or a T cell derived factor nucleic acid. Thus, a unique fragment must contain a nucleotide sequence other than the exact sequence of those in GenBank or fragments thereof. The difference may be an addition, deletion or substitution with respect to the GenBank sequence or it may be a sequence wholly separate from the GenBank sequence. Unique fragments can be used as probes in Southern and Northern blot assays to identify such nucleic acids, or can be used in amplification assays such as those employing PCR. As .known to those skilled in the art, large probes such as 200, 250, 300 or more nucleotides are preferred for certain uses such as Southern and Northern blots, while smaller fragments will be preferred for uses such as PCR. Unique fragments also can be used to produce fusion proteins for generating antibodies or determining binding of the polypeptide fragments, or for generating immunoassay components. Likewise, unique fragments can be employed to produce nonfused fragments of the dendritic cell derived factor or T cell derived factor, useful, for example, in the preparation of antibodies, and in immunoassays. Unique fragments further can be used as antisense molecules to inhibit the expression of dendritic cell derived factor or T cell derived factor nucleic acids and polypeptides, particularly for therapeutic purposes as described in greater detail below. As will be recognized by those skilled in the art, the size of the unique fragment will depend upon its conservancy in the genetic code. Thus, some regions of dendritic cell derived factor or T cell derived factor nucleic acid sequences and complements thereof will require longer segments to be unique (e.g., 50, 75, 100, 150, 200, 250, 300 bases and so on) while others will require only short segments, typically between 12 and 32 nucleotides (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bases long). Virtually any segment of the polypeptide coding region of novel dendritic cell derived factor or T cell derived factor nucleic acids, or complements thereof, that is 20 or more nucleotides in length will be unique. Those skilled in the art are well versed in methods for selecting such sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from non-dendritic cell derived factor or non-T cell derived factor nucleic acids. A comparison of the sequence of the fragment to those on .known databases typically is all that is necessary, although in vitro confirmatory hybridization and sequencing analysis may be performed.

As mentioned above, the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a dendritic cell derived factor or T cell derived factor, to reduce the expression of dendritic cell derived factor or T cell derived factor, respectively. This is desirable in virtually any medical condition wherein a reduction of expression of dendritic cell derived factors or T cell derived factors is desirable, e.g., in the treatment of excess T cell proliferation, and in the treatment of conditions involving deficient or excessive interferon-γ production, such as autoimmune diseases, inflammation, etc. This is also useful for in vitro or in vivo testing of the the effects of a reduction of expression of one or more dendritic cell derived factors, or T cell derived factors.

As used herein, the term "antisense oligonucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.

It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the sequences of nucleic acids encoding dendritic cell derived factor or T cell derived factor, or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3 '-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al. Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins are not expected to bind. Finally, although the foregoing primarily describes cDNA sequences, one of ordinary skill in the art may easily derive the genomic DNA corresponding to the cDNA of a dendritic cell derived factor or T cell derived factor. Thus, the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to nucleic acids encoding dendritic cell derived factor or T cell derived factor. Similarly, antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation. In one set of embodiments, the antisense oligonucleotides of the invention may be composed of "natural^" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors. In preferred embodiments, however, the antisense oligonucleotides of the invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.

The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.

The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified oligonucleotides may include a 2'-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding dendritic cell derived factor or T cell derived factor, together with pharmaceutically acceptable carriers.

.Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a phaimaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well .known in the art, as further described below.

As used herein, a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays .known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transfoimed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said to be "operably" joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non- transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

Expression vectors containing all the necessary elements for expression are commercially available and .known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding a dendritic cell derived factor or fragment or variant thereof. That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell. Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV

(available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor lcc, which stimulates efficiently transcription in vitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferred expression vector is an adenovirus, described by Stratford-Perricaudet, which is defective for El and E3 proteins (J. Clin. Invest. 90:626-630, 1992). The use of the adenovirus as an Adeno.Pl A recombinant for the expression of an antigen is disclosed by Wamier et al., in intradermal injection in mice for immunization against PI A (Int. J. Cancer, 67:303-310, 1996). Additional vectors for delivery of nucleic acid are provided below.

The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of a vector and one or more of the previously discussed dendritic cell derived factor nucleic acid molecules. Other components may be added, as desired, as long as the previously mentioned nucleic acid molecules, which are required, are included. The invention also includes kits for amplification of a dendritic cell derived factor or T cell derived factor nucleic acid, including at least one pair of amplification primers which hybridize to a dendritic cell derived factor or T cell derived factor nucleic acid. The primers preferably are 12-32 nucleotides in length and are non- overlapping to prevent formation of "primer-dimers". One of the primers will hybridize to one strand of the dendritic cell derived factor or T cell derived factor nucleic acid and the second primer will hybridize to the complementary strand of the dendritic cell derived factor or T cell derived factor nucleic acid, in an arrangement which permits amplification of the dendritic cell derived factor or T cell derived factor nucleic acid, respectively. Selection of appropriate primer pairs is standard in the art. For example, the selection can be made with assistance of a computer program designed for such a purpose, optionally followed by testing the primers for amplification specificity and efficiency. The invention also permits the construction of dendritic cell derived factor or T cell derived factor gene "knock-outs" in cells and in animals, providing materials for studying certain aspects of immune system regulation.

The invention also provides isolated polypeptides (including whole proteins and partial proteins) encoded by the foregoing dendritic cell derived factor or T cell derived factor nucleic acids. Such polypeptides are useful, for example, alone or as fusion proteins to generate antibodies, as components of an immunoassay or diagnostic assay or as therapeutics. Dendritic cell derived factors or T cell derived factors can be isolated from biological samples including tissue or cell homogenates, and can also be expressed recombinately in a variety of prokaryotic and eukaryotic expression systems by constructing an expression vector appropriate to the expression system, introducing the expression vector into the expression system, and isolating the recombinately expressed protein. Short polypeptides, including antigenic peptides (such as are presented by MHC molecules on the surface of a cell for immune recognition) also can be synthesized chemically using well-established methods of peptide synthesis.

A unique fragment of a dendritic cell derived factor or T cell derived factor, in general, has the features and characteristics of unique fragments as discussed above in connection with nucleic acids. As will be recognized by those skilled in the art, the size of the unique fragment will depend upon factors such as whether the fragment constitutes a portion of a conserved protein domain. Thus, some regions of dendritic cell derived factor or T cell derived factor will require longer segments to be unique while others will require only short segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long).

Unique fragments of a polypeptide preferably are those fragments which retain a distinct functional capability of the polypeptide. Functional capabilities which can be retained in a unique fragment of a polypeptide include interaction with antibodies, interaction with other polypeptides or fragments thereof, selective binding of nucleic acids or proteins, and enzymatic activity. Those skilled in the art are well versed in methods for selecting unique amino acid sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from non-family members. A comparison of the sequence of the fragment to those on known databases typically is all that is necessary.

The invention embraces variants of the dendritic cell derived factor described above. As used herein, a "variant" of a dendritic cell derived factor or T cell derived factor is a polypeptide which contains one or more modifications to the primary amino acid sequence of a dendritic cell derived factor or T cell derived factor. Modifications which create a dendritic cell derived factor variant can be made to a dendritic cell derived factor 1) to reduce or eliminate an activity of a dendritic cell derived factor; 2) to enhance a property of a dendritic cell derived factor, such as protein stability in an expression system or the stability of protein-protein binding; or 3) to provide a novel activity or property to a dendritic cell derived factor, such as addition of an antigenic epitope or addition of a detectable moiety. Modifications to a dendritic cell derived factor are typically made to the nucleic acid which encodes the dendritic cell derived factor, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the dendritic cell derived factor amino acid sequence. One of skill in the art will be familiar with methods for predicting the effect on protein conformation of a change in protein sequence, and can thus "design" a variant dendritic cell derived factor according to known methods. One example of such a method is described by Dahiyat and Mayo in Science 278:82-87, 1997, whereby proteins can be designed de novo. The method can be applied to a known protein to vary a only a portion of the polypeptide sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of a dendritic cell derived factor can be proposed and tested to determine whether the variant retains a desired conformation.

In general, variants include dendritic cell derived factors which are modified specifically to alter a feature of the polypeptide unrelated to its desired physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfde linkages. Similarly, certain amino acids can be changed to enhance expression of a dendritic cell derived factor by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encode a dendritic cell derived factor preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non- variant dendritic cell derived factors) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The prefeired codons for translation of a nucleic acid in, e.g., E. coli, are well .known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a dendritic cell derived factor gene or cDNA clone to enhance expression of the polypeptide. The activity of variants of dendritic cell derived factor can be tested by cloning the gene encoding the variant dendritic cell derived factor into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the variant dendritic cell derived factor, and testing for a functional capability of the dendritic cell derived factor as disclosed herein. For example, the variant dendritic cell derived factor can be tested for reaction with autologous or allogeneic sera as disclosed in the Examples. Preparation of other variant polypeptides may favor testing of other activities, as will be known to one of ordinary skill in the art.

Modifications can be made to T cell derived factor as described above for dendritic cell derived factor.

The skilled artisan will also realize that conservative amino acid substitutions may be made in dendritic cell derived factors to provide functionally equivalent variants of the foregoing polypeptides, i.e, the variants retain the functional capabilities of the dendritic cell derived factor. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence .known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel. et al.. eds., John Wiley & Sons, Inc., New York. Exemplary functionally equivalent variants of the dendritic cell derived factor include conservative amino acid substitutions of a dendritic cell derived factor amino acid sequences. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R. H; (d) A, G; (e) S, T; (f) Q, N; and (g) E₅ D.

Conservative amino-acid substitutions in the amino acid sequence of dendritic cell derived factors or T cell derived factors to produce functionally equivalent variants typically are made by alteration of a nucleic acid encoding such factors. Such substitutions can be made by a variety of methods .known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of a gene encoding a dendritic cell derived factor. Where amino acid substitutions are made to a small polypeptide, (e.g., a unique fragment of a dendritic cell derived factor, such as an antigenic epitope), the substitutions can be made by directly synthesizing the polypeptide. The activity of functionally equivalent fragments of dendritic cell derived factors can be tested by cloning the gene encoding the altered dendritic cell derived factor into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered dendritic cell derived factor, and testing for a functional capability of the dendritic cell derived factor as disclosed herein. Peptides which are chemically synthesized can be tested directly for function, e.g., for binding to antisera recognizing dendritic cell derived factor. Functional variants and fragments of T cell derived factor also can be prepared and tested as described above for dendritic cell derived factors.

The invention also provides, in certain embodiments, "dominant negative" polypeptides derived from the dendritic cell derived factor or T cell derived factor. A dominant negative polypeptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect of the active protein. For example, a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the ligand. Likewise, a dominant negative catalytically-inactive kinase which interacts normally with target proteins but does not phosphorylate the target proteins can reduce phosphorylation of the target proteins in response to a cellular signal. Similarly, a dominant negative transcription factor which binds to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in a cell is a reduction in function of active proteins. One of ordinary skill in the art can assess the potential for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative variant polypeptides. For example, given the teachings contained herein of a dendritic cell derived factor, one of ordinary skill in the art can modify the sequence of the dendritic cell derived factor by site-specific mutagenesis, scanning mutagenesis, partial gene deletion or truncation, and the like. See, e.g., U.S. Patent No. 5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled artisan then can test the population of mutagenized polypeptides for diminution in a selected activity and/or for retention of such an activity. Other similar methods for creating and testing dominant negative variants of a protein will be apparent to one of ordinary skill in the art.

The invention also makes it possible isolate proteins which bind to the dendritic cell derived factor or T cell derived factor as disclosed herein, including antibodies and cellular binding partners of the dendritic cell derived factor or T cell derived factor such as receptors. Once the dendritic cell derived factor or T cell derived factor is isolated according to standard methods known to one of ordinary skill in the art, the dendritic cell derived factor or T cell derived factor(or even a substantially purified cell supernatant or fraction) can be used to generate polyclonal or monoclonal antibodies according to standard methods (see e.g., Harlow and Lane, eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988)

The proteins which bind to the dendritic cell derived factor or T cell derived factor can be used, for example, in screening assays to detect the presence or absence of dendritic cell derived factor or T cell derived factor and complexes of dendritic cell derived factor or T cell derived factor and their respective binding partners and in purification protocols to isolate dendritic cell derived factor or T cell derived factor and complexes of dendritic cell derived factor or T cell derived factor and their respective binding partners. The binding proteins also can be used to block the effects of dendritic cell derived factor or T cell derived factor. Such assays can be used to confirm the specificity of binding.

The invention, therefore, embraces peptide binding agents which, for example, can be antibodies or fragments of antibodies having the ability to selectively bind to dendritic cell derived factor. Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology.

Significantly, as is well-lαiown in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology. 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')₂ fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope- binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDRl through CDR3). The CDRs. and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. Thus, for example. PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')₂, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')₂ fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non- human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies. Thus, the invention involves polypeptides of numerous size and type that bind specifically to dendritic cell derived factor or T cell derived factor, and complexes of both dendritic cell derived factor or T cell derived factor and their binding partners. These polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties.

The invention as described herein has a number of uses, some of which are described elsewhere herein. First, the invention permits isolation of the dendritic cell derived molecules or T cell derived factor molecules. A variety of methodologies well-known to the skilled practitioner can be utilized to obtain isolated dendritic cell derived factor or T cell derived factor molecules. The polypeptide may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition. Alternatively, an expression vector may be introduced into cells to cause production of the polypeptide. In another method, mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide. Translation of mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce polypeptide. Those skilled in the art also can readily follow known methods for isolating dendritic cell derived factor. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.

As detailed herein, the foregoing antibodies and other binding molecules may be used for example to identify tissues expressing protein or to purify protein. Antibodies also may be coupled to specific diagnostic labeling agents for imaging of cells and tissues that express a dendritic cell derived factor.

The dendritic cell derived factor or T cell derived factor also are useful for both in vitro and in vivo applications. In vitro, the dendritic cell derived factor or T cell derived factor can be used to investigate the mechanism of GM-CSF stimulated proliferation of CD4⁺ T cells, IFN-γ production by T cells or NK cells, etc. In vivo, the dendritic cell derived factor can be used in compositions for vaccination or immunization to boost the immune response to an antigen, or to boost proliferation of CD4⁺ T cells, or to increase IFNγ production by T cells. In vivo, the T cell derived factor can be used to reduce IFN-γ production, to suppress immune response, to decrease inflammation, etc.

As part of the foregoing compositions, the dendritic cell derived factor can be administered with one or more antigens and adjuvants to induce an immune response or to increase an immune response. An adjuvant is a substance incorporated into or administered with antigen which potentiates the immune response. Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes (thus the dendritic cell derived factor can be considered an adjuvant). Adjuvants of many kinds are well known in the art. Specific examples of adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract; DQS21, described in PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18, and QS-L1 (So et al., Mol. Cells 7:178-186, 1997); incomplete Freund's adjuvant; complete Freund's adjuvant; montanide; vitamin E; and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol. Preferably, the factor(s) are administered mixed with a combination of DQS21/MPL. The ratio of DQS21 to MPL typically will be about 1:10 to 10:1, preferably about 1:5 to 5:1 and more preferably about 1 : 1. Typically for human administration, DQS21 and MPL will be present in a vaccine formulation in the range of about 1 μg to about 100 μg. Other adjuvants are known in the art and can be used in the invention (see, e.g. Goding, Monoclonal Antibodies: Principles and Practice, 2nd Ed., 1986).

Other agents which stimulate the immune response of the subject can also be administered to the subject. For example, other cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties. Many other cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin-12 (IL-12), which has been shown to enhance the protective effects of vaccines (see, e.g., Science 268: 1432- 1434, 1995), IL-18, GM-CSF, IL-7 etc. Methods for the preparation of mixtures or emulsions of peptide and adjuvant are well .known to those of skill in the art of vaccination.

There are a number of immune response potentiating compounds that can be used in vaccination protocols. These include costimulatory molecules provided in either protein or nucleic acid form. Such costimulatory molecules include the B7-1 and B7-2 (CD80 and CD86 respectively) molecules which are expressed on dendritic cells (DC) and interact with the CD28 molecule expressed on the T cell. This interaction provides costimulation (signal 2) to an antigen/MHC/TCR stimulated (signal 1) T cell, increasing T cell proliferation and effector function. B7 also interacts with CTLA4 (CD 152) on T cells and studies involving CTLA4 and B7 ligands indicate that the B7-CTLA4 interaction can enhance antitumor immunity and CTL proliferation (Zheng P., et al. Proc. Natl. Acad. Sci. USA 95 (11):6284-6289 (1998)).

B7 typically is not expressed on tumor cells so they are not efficient antigen presenting cells (APCs) for T cells. Induction of B7 expression would enable the tumor cells to stimulate more efficiently CTL proliferation and effector function. A combination of B7/IL-6/IL-12 costimulation has been shown to induce IFN-gamma and a Thl cytokine profile in the T cell population leading to further enhanced T cell activity (Gajewski et al., J. Immunol, 154:5637- 5648 (1995)). Tumor cell transfection with B7 has ben discussed in relation to in vitro CTL expansion for adoptive transfer immunotherapy by Wang et al., (J. Immunol., 19:1-8 (1986)). Other delivery mechanisms for the B7 molecule would include nucleic acid (naked DNA) immunization (Kim J.. et al. Nat Biotechnol., 15:7:641-646 (1997)) and recombinant viruses such as adeno and pox (Wendtner et al., Gene Ther., 4:7:726-735 (1997)). These systems are all amenable to the construction and use of expression cassettes for the coexpression of B7 with other molecules of choice such as the antigens or fragment(s) of antigens discussed herein (including polytopes) or cytokines. These delivery systems can be used for induction of the appropriate molecules in vitro and for in vivo vaccination situations. The use of anti-CD28 antibodies to directly stimulate T cells in vitro and in vivo could also be considered.

Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCs and some tumor cells and interacts with CD2 expressed on T cells. This interaction induces T cell IL-2 and IFN-gamma production and can thus complement but not substitute, the B7/CD28 costimulatory interaction (Parra et al., J. Immunol., 158:637-642 (1997), Fenton et al.. J. Immunother., 21 :2:95- 108 (1998)).

Lymphocyte function associated antigen- 1 (LFA-1) is expressed on leukocytes and interacts with ICAM-1 expressed on APCs and some tumor cells. This interaction induces T cell IL-2 and IFN-gamma production and can thus complement but not substitute, the B7/CD28 costimulatory interaction (Fenton et al, J. Immunother. , 21 :2:95-108 (1998)). LFA-1 is thus a further example of a costimulatory molecule that could be provided in a vaccination protocol in the various ways discussed above for B7.

Complete CTL activation and effector function requires Th cell help through the interaction between the Th cell CD40L (CD40 ligand) molecule and the CD40 molecule expressed by DCs (Ridge et al, Nature, 393:474 (1998), Bennett et al., Nature, 393:478 (1998), Schoenberger et al., Nature, 393:480 (1998)). This mechanism of this costimulatory signal is likely to involve upregulation of B7 and associated IL-6/IL-12 production by the DC (APC). The CD40-CD40L interaction thus complements the signal 1 (antigen MHC-TCR) and signal 2 (B7-CD28) interactions.

The use of anti-CD40 antibodies to stimulate DC cells directly, would be expected to enhance a response to tumor antigens which are normally encountered outside of a inflammatory context or are presented by non-professional APCs (tumor cells). In these situations Th help and B7 costimulation signals are not provided. This mechanism might be used in the context of antigen pulsed DC based therapies or in situations where Th epitopes have not been defined within known TRA precursors.

Protein and peptide antigens can be delivered by a variety of methods .known to those of skill in the art. Methods of delivery include inhalation, transfer into antigen presenting cells in vitro for ex vivo delivery, injection and the like. Preferably antigens are administered by injection in combination with one or more adjuvants, as described herein, particularly by intradermal or subcutaneous injection. Dendritic cell derived factor or T cell derived factor can be delivered by similar methods, including as part of a composition containing antigen, adjuvant and dendritic cell derived factor or T cell derived factor. Dendritic cell derived factor or T cell derived factor can also be delivered intravenously.

In general, a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular components selected for combination with the dendritic cell derived factor or T cell derived factor, the severity of the condition being treated and the dosage required for therapeutic efficacy. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intradermal or infusion. The foregoing methods of therapy preferably include co-administration of an antigen with the dendritic cell derived factor to potentiate the immune response to the expressed antigen. According to some embodiments, dendritic cell derived factor is administered substantially simultaneously with an antigen (peptides, nucleic acids, cells presenting antigen, and the like) to increase a subject's immune response to the antigen. By "substantially simultaneously", it is meant that the dendritic cell derived factor is administered to the subject close enough in time with the administration of the antigen and adjuvant, whereby the dendritic cell derived factor

5 (or its metabolites) may exert a potentiating effect on the immune response activity of the antigen and adjuvant. Thus, by substantially simultaneously it is meant that the dendritic cell derived factor is administered before, at the same time, and/or after the administration of the antigen and adjuvant. The dendritic cell derived factor can be administered as a polypeptide, nucleic acid which expresses dendritic cell derived factor, and even as part of a nucleic acid o which expresses the antigen. T cell derived factor is administered in like fashion.

The invention is not limited in utility to human immunotherapy, but also provides a method for assessing the effects of therapeutic agents in mammalian models such as primates, pigs, sheep, dogs, rodents, and cows. The invention also provides a method for testing the effects of the dendritic cell derived factor in a mammal, including its effects on CD4⁺ T cells 5 alone or in combination with other agents, antigens, and the like.

The compositions of the invention are administered in effective amounts. Thus dendritic cell derived factor is administered in an amount effective to alter favorably the proliferation and/or IFN-γ production and/or IL-2 response of CD4⁺ T cells. T cell derived factor is administered in an amount effective to alter favorably the production of IFN-γ. An "effective 0 amount" is that amount of a composition necessary to increase any of the foregoing responses. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. 5 These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological 0 reasons or for virtually any other reasons.

When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

Dendritic cell derived factors or T cell derived factors useful according to the invention, optionally in combination with one or more antigens and adjuvants, may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of dendritic cell derived factor, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co. , Easton, PA.

The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of dendritic cell derived factor for increasing a CD4⁺ T cell response in a unit of weight or volume suitable for administration to a patient. Dendritic cell derived factor can be administered with an antigen and adjuvant, if so desired and optionally dendritic cell derived factor can be administered as one or more supplementary injections during the immunization method to further increase the immune response. The immune response can, for example, be measured by determining the activity of cytotoxic T lymphocytes. Methods for measuring cytotoxic T lymphocyte activity include measurement of tumor necrosis factor release by the cytotoxic T lymphocytes and measurement of chromium release as exemplified in the examples. Other assays will be .known to one of ordinary skill in the art and can be employed for measuring the level of the immune response. Specific assays for measuring CD4⁺ T cell activities also are provided herein. Other pharmaceutical compositions preferably contain an effective amount of T cell derived factor for reduction of IFN-γ production. The doses of dendritic cell derived factor or T cell derived factor administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the particular antigen, if any, used for immunization and the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

In general, doses of dendritic cell derived factor or T cell derived factor can be administered by injection in one or more sites in the subject. The administration of dendritic cell derived factor or T cell derived factor generally is carried out via injection intradermally (i.d.), subcutaneously (s.c). intravenously (i.v.) or intramuscularly (i.m.). Other protocols for the administration of dendritic cell derived factor will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration (e.g., intra-tumoral) and the like vary from the foregoing.

In another aspect of the invention, dendritic cell derived factor or T cell derived factor is used in the manufacture of a medicament for modulating an immune response. The medicament can be placed in a vial and be incorporated into a kit to be used for modulating a subject's immune response to an antigen. In one embodiment, an antigen placed in a vial (optionally with an adjuvant) can also be included in the same kit. An adjuvant can be placed in a separate vial and included in the foregoing kits. The kits can include instructions or other printed material on how to administer the dendritic cell derived factor or T cell derived factor and other components of the kits. In certain embodiments the dendritic cell derived factor can be part of a kit that does not include an antigen, but includes instructions or other printed material on how to combine the dendritic cell derived factor with an antigen (with or without an adjuvant).

Examples Materials and Methods Mice. GM-CSF deficient mice (GM-CSF -/- mice) on a C57BL/6 x 129 background were generated at the Melbourne Branch of Ludwig Institute for Cancer Research (Stanley et al., Proc. Natl. Acad. Sci. USA 91 :5592-5596, 1994) and breeding stocks were transferred to the New York Branch. (C57BL/6 x 129) F2 mice were used as controls for GM-CSF -/- mice. BALB/c mice were obtained from the breeding facility at Memorial Sloan-Kettering Cancer Center.

Monoclonal Antibodies (mAbs). Anti-L3T4 (CD4) and anti-Lyt2.2 (CD8) mAb were kindly provided by Drs. F. Fitch. (University of Chicago, Chicago, IL) and U. Hammerling (Memorial Sloan-Kettering Cancer Center, New York, NY), respectively. For immunodepletion of T cells, 50 μl anti-L3T4 and 25 μl anti-L3T4 and 25 μl anti-Lyt-2.2 mAbs were injected intravenously in a total volume of 200 μl. Other mAbs used in this study were purchased from Pharmingen (San Diego, CA).

Tumor Cell Lines. EL4 is a chemically induced leukemia cell line of C57BL origin. .RLc? 1 is a BALB/c radiation-induced leukemia.

Peptide. The ovalbumin (OVA) peptide spanning amino acid residues 257 to 264 of albumin (SIINFEKL; SEQ ID NOJ) was synthesized and purified by Bio-Synthesis Inc. (Lewisville, TX) (Dyall et al, Int. Immunol. 7:1205-1212, 1995).

OVA Peptide Immunization. 5 μg OVA peptide in TiterMax (CytRx, Norcross, GA) was injected in the hind footpads. In mice receiving rGM-CSF, 5 ng rGM-CSF (Pharmingen) was injected in the hind footpads with peptide followed by 10 ng rGM-CSF injected intraperitoneally for 5 days.

Keyhole Limpet Hemocyanin Immunization. Mice were immunized with 100 μg keyhole limpet hemocyanin (KLH) (Pierce, Rockford, IL) in complete Freund's adjuvant (CFA) (Sigma, St. Louis, MO) in the hind footpads. In mice receiving rGM-CSF, 40 ng rGM-CSF was injected in the hind footpads with KLH followed by 10 ng rGM-CSF injected intraperitoneally 5 days a week until mice were sacrificed.

Generation of CTL Specific to OVA Peptide. CTLs were generated as described previously (Dyall et al, 1995).

Mixed Lymphocyte Reaction. For proliferation assays, 3 x 10⁵ responding spleen cells (H-2^b background) were cultured with 2 x 10⁵ mitomycin C (Sigma)-treated BALB/c mice spleen cells for 4 days at 37 °C in a 5% CO₂ atmosphere and proliferation was determined by incorporation of [methy H] thymidine. For generation of H-2^d specific CTL, 3 x 10⁷ responding spleen cells were cultured with 2 x 10⁷ mitomycin C-treated BALB/c mice spleen cells for 5 days at 37 °C in a 5% CO₂ atmosphere. Cytotoxicity was assessed in ⁵¹Cr-release assays using BALB/c RLcf 1 as target cells.

T cell Response to KLH. Seven to ten days after immunization, mice were sacrificed and the popliteal lymphnodes were removed. To obtain purified CD4⁺ T cells, lymphnode cells were treated with anti-Lyt 2.2 mAb and rabbit serum as complement and then passed through a nylon wool column (>90% cells were Thy-1⁺CD4⁺CD8^"). Using spleen cells or purified B-cells as antigen presenting cells (APC), the cells were pulsed with 100 or 10 μg/ml KLH and 50 μg/ml mitomycin C for 1 hr. washed, and 1 x 10⁸ APC were then cultured with 1 x 10⁵ CD4⁺ T cells at 37 °C in a 5% CO² atmosphere for 3 days. Using dendritic cells (DC) as APC, the cells were pulsed with 10 g/ml KLH overnight and treated with 50 μg/ml mitomycin C for 1 hour before washing. 1 x 10⁵ DC were then cultured with 1 x 10⁵ CD4⁺ T cells at 37°C in a 5% CO₂ atmosphere for 3 days. To analyze T cell responses to non-specific stimulants, 1 x 10⁵ CD4⁺ T cells were cultured with immobilized anti-mouse CD3 mAb or concanavalin A (Con A) (Sigma) for 3 days. For experiments involving supernatants, 100 μl culture supernatant from co-cultures of CD4⁺ T cells and KLH-pulsed DC (mitomycin C-untreated) for two days or from cultures of 5 x 10⁵ DC stimulated with 100 ng/ml rGM-CSF in 96 well plate for two days were transferred to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from GM-CSF -/- mice at a final volume of200 μl.

Cytokine Assays. Culture supernatants were assayed by ELISA for IFN-γ and IL-4 using reagents from Pharmingen, and for IL-2 using the DuoSet reagent from Genzyme (Cambridge, MA). Serum levels of cytokines were measured by ELISA with reagents from the following sources; IFN-γ, TNFα, IL-12 p70 (Genzyme); GM-CSF, IL-lβ, IL-6, IL-10, TGF-βl (R&D Systems, Minneapolis, MN); IL-12 p40 (Biosource International, Camarillo, CA).

Cytokines and injections of cytokines to mice. Recombinant mouse GM-CSF (GM- CSF) and recombinant mouse IL-4 (IL-4) were purchased from Pharmingen. Recombinant human IL-2 (IL-2) and recombinant mouse IL-18 (IL-18) were purchased from Genzyme (Cambridge, MA) and Peprotech (Rocky Hill, NJ), respectively. Recombinant mouse IL-12 (IL- 12) was a kind gift of Dr. S. Wolf at Genetics Institute (Cambridge, MA). GM-CSF, IL-18, and IL-12 were diluted or dissolved in PBS (containing 1% syngeneic mouse serum in the case of IL- 12) and injected i.p. in a total volume of 200 μl.

LPS preparation and treatment. 100 μg LPS (from E. coli; serotype O127:B8, Sigma

(St. Louis, MO) was dispersed in PBS and injected i.p. in a total volume of 200 μl. Three hours after LPS injections, spleens were removed to prepare cell suspensions. Additional mice were bled from the retroorbital plexus at various times after LPS injection for serum harvest.

ELISA for detection of antibodies against KLH. KLH was dissolved in 0.1 M sodium bicarbonate at pH 8.3 at a concentration of 1 μg/ml. 100 μl KLH solution was added to each well of 96 well plates and incubated at 4°C for overnight. After extensive washing with phosphate buffered saline (PBS), 100 μl 2% bovine serum albumin in PBS was added and incubated overnight. After washing, serially diluted serum samples were added and incubated at room temperature for 2 hr. 100 μl of alkaline phosphatase-labeled mouse IgM-specific or mouse IgG subclass-specific Abs (diluted at 1 :5000 with PBS) was added after washing and incubated at room temperature for 1 hr. 100 μl substrate solution (JBL Scientific Inc., San Luis Obispo, CA) was added after the final wash and incubated at 37 °C for 20 minutes, and plates were then read by CytoFluor (Millipore, Bedford, MA) with 450 nm wavelength at excitation and 580 urn wavelength at emission. Specificity of Abs was confirmed using hen egg lysozyme (HEL) (Sigma) as a negative control.

Isolation and culture of T cells and CD16/32⁺ cells. To isolate defined cell populations spleen cells were incubated with biotinylated mAbs against mouse CD4, CD8, or CD16/32⁺ at 4°C for 30 min. Purity of isolated cells (>90%) was confirmed by flow cytometry. 1 x 10⁵ CD4^{^} or CD8⁺ cells or 2 x 10⁵ CD16/32⁺ cells per well were cultured in complete media (RPMI with 10% fetal bovine serum, 5 x 10^"5 M 2-mercaptoethanol and glutamine) at various concentrations of IL-2 and IL-12 in 96-well plates at a total volume of 200 μl for 3 days at 37 °C in a 5% CO, atmosphere. Culture supernatants were tested for IFN-γ. For proliferation assays, 2 x 10⁵ CD8 cells were cultured with IL-2 alone or IL-2 and IL-12 for 3 days at 37 °C in a 5% CO₂ atmosphere, and the proliferative response was determined by uptake of [methyl-³H] thymidine.

Isolation of DC and generation of DC-derived factor. For DC purification, collagenase-treated spleen cells were centrifuged over 55% percoll after T cell depletion using anti-Thyl mAb and complement. B-220⁺ cells at the interface were eliminated using biotinylated anti-B220 mAb and avidin-coated magnetic beads. The remaining cells were cultured in 60 mm petri dishes at 37 °C in a 5% CO₂ atmosphere overnight and floating cells were then used as enriched DC (> 45% CD1 lc⁺ IAb⁺⁺ CD16/32^", <2.0% Mac-1⁺⁺ CD16/32⁺) (Guery et al., 1996).

DC were isolated from spleen cells of LPS-injected (100 μg) (C57BL/6 x 129)F2 mice using biotinylated anti-mouse CD1 lc mAb and avidin-coated magnetic beads. 5 x 10⁵ CD1 lc^" cells were cultured in serum-free media (Sigma) with 10 ng/ml GM-CSF and 2 ng/ml IL-4 in 96 well plates for 4 days at 37 °C in a 5% CO₂ atmosphere. 50 μl culture supernatants were transferred to the cultures of CD4⁺ or CD8⁺ T cells from LPS-injected GM-CSF-/- mice with IL-2 and/or IL-12 at a total volume of 200 μl for 3 days at 37°C in a 5% CO₂ atmosphere. For proliferation assays, isolated CD4⁺ or CD8⁺ T cells were cultured with 100 U/ml IL-2 and 50 μl culture supernatants in a total volume of 200 μl for 3 days. For in vivo studies, 0.5 ml DC supernatants were injected i.p.

Purification of B cells. For B cell proliferation, spleen cells were centrifuged over 55% percoll (Sigma) after depletion of T cells using anti-L3T4 mAb, anti-Lyt 2.2 mAb and rabbit serum as complement. Mac-1⁺ and CD1 lc⁺ cells at the interface between media and 55% percoll were eliminated using biotinylated anti-Mac-1 and CD1 lc mAb and avidin-coated magnetic beads. The remaining cells were used as enriched B cells (>80% B220⁺, <1% Mac-1⁺, <0.6% CD1 lc⁺) (Guery et al., J. Exp. Med. 183:751-757, 1996).

Example 1: GM-CSF -/- mice respond normally to alloantigens. As a first step, the generation of allo-specific CTL from naive GM-CSF -/- and GM-CSF +/+ spleen cells after co- culture with BALB/c spleen cells was analyzed. As shown in Fig. 1 A, CTL from GM-CSF -/- mice lysed BALB/c target cells as effectively as those from GM -CSF +/+ mice. Fig. IB shows the proliferative responses of spleen cells from GM-CSF -/- and GM-CSF +/+ mice against BALB/c stimulatory cells. Cells from GM-CSF -/- mice proliferated as well as those from GM- CSF +/+ mice. The proliferative response of splenic T cells as well as isolated CD4⁺ T cells from GM-CSF -/- and GM-CSF +/+ mice to anti-mouse CD3 mAb, ConA and IL-2 was also comparable.

Example 2: Failure of CTL response to OVA peptide. Next, CD8⁺ T cell responses of GM-CSF -/- mice against an exogenous peptide were evaluated. OVA peptide spanning residues 257 to 264 (SIINFE.KL; SEQ ID NO: 1) is recognized by CTL in a K^b-restricted manner, and CTL can be generated without CD4⁺ T cell help (Dyall et al., 1995). GM-CSF -/- and GM-CSF +/+ mice were immunized with OVA peptide in adjuvant. Seven days after immunization, mice were sacrificed and spleen cells were cultured for 5 days with mitomycin C-treated autologous spleen cells pulsed with OVA peptide. Effector cells were assayed for cytotoxicity in ⁵¹Cr- release assay using El-4 cells (H-2^b) pulsed with OVA peptide. As shown in Fig. 2A, highly reactive CTL specific to OVA peptide were elicited from GM-CSF +/+ mice but not from GM- CSF -/- mice (Fig. 2B). When GM-CSF -/- mice were injected with rGM-CSF during the immunization period, reactive CTL against OVA peptide were generated (Fig. 2C).

Example 3: Delayed Ab production against KLH. It next was determined whether GM-CSF -/- mice respond normally to exogenous soluble proteins. For this purpose, GM-CSF -/- and GM-CSF +/+ mice were immunized with 100 μg KLH in CFA and bled at 1 week, 2 weeks and 4 weeks after immunization. IgM, IgGl, IgG2a, and IgG2b titers against KLH were quantitated by ELISA. As shown in Fig. 3a, GM-CSF -/- mice produced lower levels of IgG, particularly IgG2a, during the first two weeks following immunization. However, by 4 weeks, only IgG2a levels remained suppressed in GM-CSF -/- mice. IgM production was not compromised in GM-CSF -/- mice throughout the observation period, indicating that the T cell- independent pathway of Ab production was intact. Injection of rGM-CSF in vivo restored IgG production in GM-CSF -/- mice (Fig. 3b). These observations were confirmed using lower levels of immunization (10 μg and 1 μg .KLH).

Example 4: Impaired proliferative response of CD4⁺ T cells to KLH. Fig. 4 shows the proliferative responses of CD4⁺ T cells from GM-CSF -/- and GM-CSF +/+ mice immunized with KLH. With spleen cells as APC, the proliferative response of CD4⁺ T cells from GM-CSF -/- mice was clearly impaired (Fig. 4a). To distinguish whether this defect is at the level of CD4⁺ T cells or APC, CD4⁺ T cells from GM-CSF +/+ mice and KLH-pulsed spleen cells from GM- CSF -/- mice or CD4⁺ T cells from GM-CSF -/- mice and KLH-pulsed spleen cells from GM- CSF +/+ mice were co-cultured. As shown in Fig. 4A, CD4⁺ T cells from GM-CSF +/+ mice cultured with APC from GM-CSF -/- mice proliferated as well as those cultured with APC from GM-CSF +/+ whereas CD4⁺ T cells from GM-CSF -/- cultured with APC from GM-CSF +/+ mice showed impaired proliferation, indicating that CD4⁺ T cells rather than APC are responsible for the low proliferative response against KLH in GM-CSF -/- mice. This defect is antigen specific, because CD4⁺ T cells from immunized GM-CSF -/- mice showed a normal response to anti-mouse CD3 mAb or Con A (Fig. 4B). With regard to cytokine production, the pattern of cytokines produced by CD4⁺ T cells from immunized GM-CSF -/- and GM-CSF +/+ mice could be distinguished. As shown in Table 1, CD4⁺ T cells from GM-CSF +/+ mice co-cultured with APC showed high production of IFN-γ and IL-4 and low production of IL-2. In contrast, CD4⁺ T cells from GM-CSF -/- mice produced low IFN-γ and IL-4 and high IL-2. Proliferative responses against KLH and IFN-γ and IL-4 production were partially restored by injection of rGM-CSF (Table 1).

Table 1. Proliferation and cytokine production of CD4⁺ T cells from GM-CSF -/- and +/+ mice in the response to KLH.

'Autologous spleen cells or B cells were used as APC. ^t Mean ± s.d. of triplicate determinations using CD4⁺ T cells and APC from the same mouse.

¹ CD4⁺ T cells and B cells were from GM-CSF -/- mice supplemented with rGM-CSF in vivo.

The results shown in Table 1 came from the analysis of individual mice. Comparable results were obtained in repeated experiments.

Example 5: Normalization of CD4⁺ T cell responses by DC or by transfer of supernatants from the DC cultures. It next was determined whether DC, which are known to be highly effective APC, could restore the proliferative response and IFN-γ production of CD4" T cells from GM-CSF -/- mice. As shown in Fig. 5A, CD4⁺ T cells from GM-CSF -/- mice showed vigorous proliferation against KLH and produced high levels of IFN-γ when DC from GM-CSF +/+ or GM-CSF -/- mice were used as APC. To analyze this rescue effect of DC, supernatants were harvested from cultures of CD4⁺ T cells and KLH-pulsed DC from KLH immunized GM-CSF -/- or GM-CSF +/+ mice. These supernatants were transfeired to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from KLH-immunized GM-CSF -/- mice. Fig. 5B shows that the proliferative response and IFN-γ production of CD4⁺ T cells from GM-CSF -/- mice were completely restored by supernatants from GM-CSF -/- or GM-CSF +/+ DC cultures. For further analysis, CD1 lc⁺ cells were isolated from GM-CSF +/+ mice injected with 100 μg LPS and cultured with 100 ng/ml rGM-CSF for 3 days. The supernatants from these isolated CD1 lc⁺ cells were added to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from KLH- immunized GM-CSF -/- mice. Under these conditions, CD4⁺ T cells proliferated vigorously and produced high amounts of IFN-γ, suggesting that these activated DC produce a factor or factors which can rescue T cell responses. Supernatants from DC isolated from GM-CSF +/+ mice not injected with LPS did not restore proliferative activity or cytokine production, nor could these activities be restored by supernatants of DC from LPS-injected GM-CSF -/- mice cultured with rGM-CSF. GM-CSF and IL-12 could not duplicate the effect of DC supernatants on the proliferative response and the cytokine production of GM-CSF -/- CD4⁺ T cells (Fig. 5B).

Example 6: Normalization of CD8⁺ T cell responses by transfer of supernatants from the DC cultures. In initial studies, GM-CSF -/- and GM-CSF +/+ mice were injected i.p. with 100 μg LPS and serum levels of cytokines were measured. Fig. 6 shows the kinetics of cytokine production release after LPS administration. The prominent finding was low serum levels of IFN-γ in GM-CSF-/- mice. The production of other cytokines, with the exception of GM-CSF, was comparable in GM-CSF -/- and GM-CSF +/+ mice.

Example 7: Restoration of serum IFN-γ in LPS-injected GM-CSF-/- mice by GM-CSF. To investigate whether IFN-γ production could be restored by GM-CSF, 100 μg LPS plus GM-CSF at various dose levels was injected i.p. into GM-CSF -/- mice and the mice were bled 7 hrs. later. As shown in Fig. 2, GM-CSF -/- mice injected with 100 ng GM-CSF and LPS produced levels of serum IFN-γ comparable to LPS-injected GM-CSF +/+ mice. Lower doses of GM-CSF had less or no restorative activity, and a higher dose appeared to have a suppressive effect on IFN-γ levels. GM-CSF exerted no significant effect on serum levels of other cytokines, such as TNFα, IL-10 and IL-12 p40, in LPS-injected GM-CSF-/- mice.

Example 8: Low IFN-γ production by T cells from GM-CSF-/- mice.

As T cells and NK cells are the two major sources of IFN-γ, CD4⁺ T cells, CD8⁺ T cells and CD16/32⁺ (NK) cells were isolated from LPS-injected mice and cultured with IL-2 and IL- 12 at concentrations shown in Fig. 8. Following a 3-day incubation period, supernatants from these cultures were tested for IFN-γ by ELISA. Lower levels of IFN-γ were observed in cultures of both CD4⁺ and CD8⁺ T cells from LPS-injected GM-CSF-/- mice. In contrast, IFN-γ production by CD 16/32" cells was not compromised (Fig. 8). These in vitro studies suggest that low serum levels of IFN-γ after LPS is due to reduced IFN-γ production by GM-CSF-/- T cells.

Example 9: Serum levels of IFN-γ in T cell-depleted mice.

GM-CSF-/- and +/+ mice were immunodepleted of CD4⁺ and CD8⁺ T cells and injected with 100 μg LPS. In contrast to the low levels of serum IFN-γ in non-depleted LPS-injected GM-CSF-/- mice, T cell-depleted GM-CSF-/- mice produced levels of IFN-γ comparable to GM-CSF+/+ mice (Table 2). This suggests that T cells in LPS-injected mice have an inhibitory activity on IFN-γ production by NK cells.

Table 2: Serum levels of IFN-γ in T cell-depleted GM-CSF-/- and +/+ mice after LPS injections

T cell immunodepletion Serum IFN-γ (pg/ml ± S.D.)

GM-CSF-/- GM-CSF+/+

No 18.8 ± 10.7 313.1 ± 66.6

Yes 489.7 ± 144.4 526.5 ± 128.3

For immunodepletion of T cells, mice were injected intravenously with 50 ml anti-CD4 mAb and

25 ml anti-CD8 mAb. All mice were injected i.p. with 100 ug LPS and bled 7 hrs. later. The data are the mean ± S.D. of 4 mice.

Example 10: Normal IL-2 and IL-12 response of T cells from naive GM-CSF-/- mice.

IL-12 has been reported to mediate the upregulation of IFN-γ production induced by LPS

(Magram, et al. Immunity 4:471-481, 1996). Therefore the IFN-γ response of GM-CSF-/- mice to IL-12 was tested. Mice were injected i.p. with different dose levels of IL-12 once a day for 4 days and bled 2 hrs after the last injections. Serum IFN-γ levels were then determined by ELISA assay. Table 3: Serum levels of IFN-γ in GM-CSF-/- and +/+ mice after injection of IL-12

Doses of IL-12 Serum IFN-γ (pg/ml ± S.D.) per injection

GM-CSF -/- GM-CSF +/+

5 μg 1655.3 ± 871.0 1735.0 ± 474.4

1 μg 506.8 ± 246.7 504.5 ± 194.2 ( 125.6 ± 24.8 ) ( 110.9 ± 12.6 )

0.1 μg 45.4 ± 23.5 37.6 ± 8.0

Data shown in parentheses are serum IFN-γ levels in T cell-depleted mice. The data are the mean ± S.D. of 4 mice.

Table 3 shows that IL-12-induced serum IFN-γ levels in GM-CSF-/- mice were comparable to levels in GM-CSF+/+ mice. T cell-depletion resulted in an equal reduction in serum levels of IFN-γ after injections of IL-12 to GM-CSF-/- and +/+ mice.

To analyze the basis for low IFN-γ induction by LPS and high IFN-γ induction by IL-12 in GM-CSF-/- mice, in vitro T cell responses to IL-2 and IL-12 were examined in naive and LPS-injected mice. CD8⁺ T cells from naive or LPS-injected mice were cultured with IL-2 and IL-12 for 3 days and IFN-γ production and proliferative response were measured. As shown in Fig. 9A, T cells from LPS-injected GM-CSF -/- mice showed suppressed proliferative responses as well as inhibited IFN-γ production in comparison to LPS-injected GM-CSF+/+ mice. In contrast, the response of T cells from naive GM-CSF -/- mice to IL-2 and IL-12 were comparable to the response to naive GM-CSF +/+ mice. Administration of GM-CSF to LPS-injected GM- CSF -/- mice restored the proliferative responses and IFN-γ production by T cells, whereas in vitro supplementation with GM-CSF had no effect (Fig. 9B). These results indicate that T cells from GM-CSF -/- mice are endowed with the capacity to respond normally to IL-2 and IL-12: LPS induces an inhibitory effect on these responses and GM-CSF, in an indirect fashion, abrogates this inhibitory activity.

Example 11: A DC derived factor(s) that restores IFN-γ production by T cells from LPS- injected GM-CSF -/- mice.

To analyze the indirect effect of GM-CSF on T cells, DC were isolated from LPS- injected GM-CSF +/+ mice and cultured with 10 ng/ml GM-CSF and 2 ng/ml IL-4 for 4 days. Supernatants from these cultures were transferred to cultures of CD8⁺ T cells from LPS-injected GM-CSF-/- mice. As shown in Fig. 10, supernatant from DC cultures restored IFN-γ production by CD8⁺ cells, but did not restore the defective proliferative response. Supernatant from comparably cultured DC from LPS-injected GM-CSF-/- mice or naive GM-CSF+/+ mice had no effect on IFN-γ production by CD8⁺ T cells from LPS-injected GM-CSF-/- mice, indicating that in vivo priming of DC with LPS is necessary to induce DC-derived factor and that dendritic cells from GM-CSF-/- mice may not be in the appropriate maturation or activation stage to produce factor.

To compare DC supernatant with IL-18, CD8⁺ cells from LPS-injected GM-CSF-/- mice were cultured with the following cytokine combinations: (1) 20 ng/ml IL-18 plus 100 pg/ml IL-2 or 1000 U/ml IL-12 and 2) 50 μl culture supernatant plus 100 pg/ml IL-2. For control purposes, effects of 2.5 ng/ml GM-CSF and 0.5 ng/ml IL-4 on IFN-γ production were tested. The results are shown in Table 4. Supernatant from DC cultures upregulated IFN-γ production by CD8⁺ cells cultured with either IL-12 or IL-2, whereas IL-18 was effective in augmenting IFN-γ production only in the presence of IL-12.

Table 4: Effect of DC culture supernatant of IL-18 on IFN-γ production of CD8⁺ T cells

IFN-γ levels (U/ml) in supernatant from CD8⁺ T cells cultured with:

Addition of: 1000 U/ml IL-2 100 pg/ml IL- 12

None 24.1 22.9

GM-CSF and IL-4 34.0 38.2

DC culture supernatant 90.2 97.8

IL-18 24.1 111.1 To extend this comparison, LPS plus 0.5 ml DC culture supernatant or LPS plus 100 ng IL-18 were injected into GM-CSF-/- mice. As shown in Fig. 11A, the administration of DC culture supernatant or IL-18 upregulated serum IFN-γ levels in GM-CSF-/- mice coinjected with 100 μg LPS. Injection of 5 ng GM-CSF and 1 ng IL-4 (the two cytokines used in the DC cultures) exerted no effect on serum IFN-γ levels. Fig. 1 IB shows the in vitro IFN-γ production and proliferative response of CD8⁺ T cells from GM-CSF-/- mice comparably injected with LPS along with DC supernatant or IL-18. CD8⁺ cells from GM-CSF-/- mice injected with LPS and DC culture supernatant produced similar levels of IFN-γ and proliferated as well as those from LPS-injected GM-CSF+/+ mice, whereas administration of IL-18 did not reverse LPS-induced inhibition of T cell proliferation and IFN-γ production, indicating that factors other than, or in addition to, IL-18 in supernatant from DC cultures were responsible for upregulating the IL-2 and IL-12 responsiveness of T cells from LPS-injected GM-CSF-/- mice.

EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in their entirety.

We claim:

Claims

1. A dendritic cell derived factor that restores proliferation and interferon-╬│ production by CD4⁺ T cells when added to a mixture of:

(1) CD4⁺ T cells from a GM-CSF -/- mammal immunized with an exogenous antigen, (2) non-dendritic cell antigen presenting cells from a GM-CSF -/- mammal, and

(3) the exogenous antigen, restoration being to levels substantially the same as those when wild type mammals are used in (1) and (2), but free of exogenously added dendritic cell derived factor, and functional variants thereof.

2. The dendritic cell derived factor of claim 1, wherein the GM-CSF -/- mammals and wild type mammals are mice, and wherein the exogenous antigen is KLH.

3. The dendritic cell derived factor of claim 2, present in a supernatant of a culture of dendritic cells derived from GM-CSF +/+ mice treated with LPS cultured with GM-CSF.

4. The dendritic cell derived factor of claim 2, present in a supernatant of a coculture of CD4⁺ T cells and dendritic cells pulsed with KLH.

5. The dendritic cell derived factor of claim 4, wherein the CD4⁺ T cells are derived from

GM-CSF +/+ mice.

6. The dendritic cell derived factor of claim 4, wherein the CD4⁺ T cells are derived from GM-CSF -/- mice.

7. The dendritic cell derived factor of claim 4, wherein the dendritic cells are derived from GM-CSF -/-mice.

8. The dendritic cell derived factor of claim 4, wherein the dendritic cells are derived from GM-CSF +/+ mice.

9. A dendritic cell isolate that restores proliferation and interferon-╬│ production by CD4⁺ T cells when added to a mixture of

(1) CD4⁺ T cells from a GM-CSF -/- mammal immunized with an exogenous antigen,

(2) spleen cells from a GM-CSF -/- mammal, and

(3) an exogenous antigen, restoration being to levels substantially the same as those when wild type mammals are used in steps (1) and (2), but free of exogenously added dendritic cell isolate, and functional variants thereof.

10. The dendritic cell isolate of claim 9, wherein the GM-CSF -/- mammals and wild type mammals are mice, and wherein the exogenous antigen is KLH.

11. The dendritic cell isolate of claim 10, present in a supernatant of a culture of dendritic cells derived from GM-CSF +/+ mice treated with LPS cultured with GM-CSF.

12. The dendritic cell isolate of claim 10, present in a supernatant of a coculture of CD4⁺ T cells and dendritic cells pulsed with KLH.

13. The dendritic cell isolate of claim 12, wherein the CD4⁺ T cells are derived from GM-CSF +/+ mice.

14. The dendritic cell isolate of claim 12, wherein the CD4⁺ T cells are derived from GM-CSF -/- mice.

15. The dendritic cell isolate of claim 12, wherein the dendritic cells are derived from GM-CSF -/- mice.

16. The dendritic cell isolate of claim 12, wherein the dendritic cells are derived from GM-CSF +/+ mice.

17. A dendritic cell derived factor that restores interferon-╬│ production by CD8⁺ T cells when added to CD8⁺ T cells from GM-CSF -/- mammals treated with lipopolysaccharide, restoration being to levels substantially the same as those produced by CD8⁺ T cells from wild type mammals, and functional variants thereof.

18. The dendritic cell derived factor of claim 17, wherein the GM-CSF -/- mammals and wild type mammals are mice.

19. The dendritic cell derived factor of claim 17, present in a supernatant of a culture of dendritic cells derived from GM-CSF +/+ mice treated with LPS cultured with GM-CSF.

20. A dendritic cell derived factor that restores interferon-╬│ production and proliferation by CD8⁺ T cells in vitro when added to GM-CSF -/- mammals treated with lipopolysaccharide, restoration being to levels substantially the same as those produced by CD8⁺ T cells from wild type mammals, and functional variants thereof.

21. The dendritic cell derived factor of claim 20, wherein the GM-CSF -/- mammals and wild type mammals are mice.

22. The dendritic cell derived factor of claim 21 , present in a supernatant of a culture of dendritic cells derived from GM-CSF +/+ mice treated with LPS cultured with GM-CSF.

23. A dendritic cell isolate that restores interferon-╬│ production by CD8⁺ T cells when added to CD8⁺ T cells from GM-CSF -/- mammals treated with lipopolysaccharide, restoration being to levels substantially the same as those produced by CD8⁺ T cells from wild type mammals, and functional variants thereof.

24. The dendritic cell isolate of claim 23, wherein the GM-CSF -/- mammals and wild type mammals are mice.

25. The dendritic cell isolate of claim 23 , present in a supernatant of a culture of dendritic cells derived from GM-CSF +/+ mice treated with LPS cultured with GM-CSF.

26. A dendritic cell isolate that restores interferon-╬│ production and proliferation by CD8⁺ T cells in vitro when added to GM-CSF -/- mammals treated with lipopolysaccharide, restoration being to levels substantially the same as those produced by CD8⁺ T cells from wild type mammals, and functional variants thereof.

27. The dendritic cell derived factor of claim 26, wherein the GM-CSF -/- mammals and wild type mammals are mice.

28. The dendritic cell derived factor of claim 26, present in a supernatant of a culture of dendritic cells derived from GM-CSF +/+ mice treated with LPS cultured with GM-CSF.

29. A method for identifying an agent that has dendritic cell derived factor activity, comprising

(a) preparing a mixture of

(1) CD4⁺ T cells from GM-CSF -/- mice immunized with an exogenous antigen,

(2) spleen cells from GM-CSF -/- mice, and (3) the exogenous antigen, and

(b) applying a composition suspected of having the agent to the mixture of (a) and measuring the proliferation of the CD4⁺ T cells and the production of interferon-╬│ by the CD4⁺ T cells as a determination of the presence of the agent in the composition.

30. A method for isolating a dendritic cell derived factor, comprising (a) preparing a mixture of

(1) CD4⁺ T cells from GM-CSF -/- mice immunized with an exogenous antigen,

(2) spleen cells from GM-CSF -/- mice, and

(3) the antigen, (b) preparing a culture of either

(1) dendritic cells derived from GM-CSF +/+ mice treated with lipopolysaccharide, the dendritic cells cultured with GM-CSF, or

(2) the CD4⁺ T cells and dendritic cells pulsed with the antigen,

(c) isolating a supernatant from the culture of step (b) (d) fractionating the supernatant into a plurality of fractions, and

(e) applying one of the plurality of fractions to the mixture of (a) and measuring the proliferation of the CD4⁺ T cells and the production of interferon-╬│ by the CD4⁺ T cells as a determination of the presence of the dendritic cell derived factor in the fraction.

31. A method for identifying an agent that has dendritic cell derived factor activity, comprising

(a) preparing a culture of CD8⁺ T cells from GM-CSF -/- mice treated with lipopolysaccharide, and

(b) applying a composition suspected of having the agent to the culture of (a) and measuring the production of interferon-╬│ by the CD8⁺ T cells as a determination of the presence of the agent in the composition.

32. A method for isolating a dendritic cell derived factor, comprising

(a) preparing a culture of CD8⁺ T cells from GM-CSF -/- mice treated with lipopolysaccharide

(b) preparing a culture of either (1) dendritic cells derived from GM-CSF +/+ mice treated with lipopolysaccharide, the dendritic cells cultured with GM-CSF, or (2) CD4⁺ T cells from GM-CSF -/- mice immunized with an exogenous antigen and dendritic cells pulsed with the antigen,

(c) isolating a supernatant from the culture of step (b) (d) fractionating the supernatant into a plurality of fractions, and

(e) applying one of the plurality of fractions to the culture of (a) and measuring the the production of interferon-╬│ by the CD8⁺ T cells as a determination of the presence of the dendritic cell derived factor in the fraction.

33. A isolated nucleic acid which encodes the dendritic cell derived factor or isolate of any of claims 1, 9, 17, 20, 23 or 26, or the human homolog thereof.

34. A isolated nucleic acid which encodes a functional variant of the dendritic cell derived factor of any of claims 1, 9, 17, 20, 23 or 26, including the human homolog thereof.

35. An isolated polypeptide which selectively binds a dendritic cell derived factor of any of claims 1, 9, 17, 20, 23 or 26, including the human homolog thereof.

36. The isolated polypeptide of claim 35, wherein the isolated polypeptide is an Fab or F(ab) fragment of an antibody.

37. The isolated polypeptide of claim 35, wherein the isolated polypeptide is a fragment of an antibody, the fragment including a CDR3 region selective for the protein.

38. The isolated polypeptide of claim 35, wherein the isolated polypeptide is a monoclonal antibody.

39. The isolated polypeptide of claim 38, wherein the monoclonal antibody is a chimeric antibody or a humanized antibody.

40. A method for increasing proliferation of a population of T cells, comprising administering to a subject in need of such treatment an amount of a dendritic cell derived factor effective to increase proliferation of the population of T cells.

41. A method for increasing production of IFN╬│ by a population of T cells, comprising administering to a subject in need of such treatment an amount of a dendritic cell derived factor effective to increase production of IFN╬│.

42. A method of identifying an agent that has T-cell derived factor activity, comprising preparing a GM-CSF -/- mammal depleted of CD4+ and CD8+ T-cells, inducing serum IFN-╬│ production in the mammal, administering to the mouse a composition suspected of having the agent, detecting serum IFN-╬│, and comparing the serum IFN-╬│ with a control as a determination of the presence of the agent in the composition.

43. The method of claim 42, wherein the mammal is a mouse.

44. The method of claim 43, wherein serum IFN-╬│ production is induced in the mouse by treating the mouse with lipopolysaccharide.

45. The method of claim 42, wherein the composition is selected from the group consisting of a membrane fraction of a T-cell, a cytosolic fraction of a T-cell, a secreted fraction of a T-cell, a size-selected fraction of a T-cell homogenate, a protein fraction of a T-cell homogenate, and a non-protein fraction of a T-cell homogenate.

46. A isolated nucleic acid which encodes the T cell derived factor identified by the method of claim 42, or the human homolog thereof.

47. A isolated nucleic acid which encodes a functional variant of the T cell derived factor identified by the method of claim 42, or the human homolog thereof.

48. An isolated polypeptide which selectively binds a T cell derived factor identified by the method of claim 42, or the human homolog thereof.

49. The isolated polypeptide of claim 48, wherein the isolated polypeptide is an Fab or F(ab) fragment of an antibody.

50. The isolated polypeptide of claim 48, wherein the isolated polypeptide is a fragment of an antibody, the fragment including a CDR3 region selective for the protein.

51. The isolated polypeptide of claim 48, wherein the isolated polypeptide is a monoclonal antibody.

52. The isolated polypeptide of claim 51 , wherein the monoclonal antibody is a chimeric antibody or a humanized antibody.

53. A method for decreasing serum IFN╬│ concentration in a subject, comprising administering to a subject in need of such treatment an amount of a T cell derived factor effective to decrease serum IFN╬│ concentration.

54. An agent that has T-cell derived factor activity identified by the method of claim 42.