JP2002509861A - Therapeutic blockade of ICER synthesis to prevent ICIR-mediated inhibition of immune cell activity - Google Patents

Abstract

  (57) [Summary] The present invention relates to a reagent for regulating immune cell activity using a substance that regulates the activity of ICER, and a method thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Many human illnesses, such as those caused by cancer and infectious organisms, arise from the ability of those causative substances to escape destruction by immune cells. In particular, recent studies indicate that tumor rejection and destruction of infectious organisms by the immune system are critically dependent on proper antigen presenting cell (APC) function and T cell restimulation within tumor deposits. It was revealed. Many tumors and infectious agents probably escape immune recognition and rejection by local inhibition of APC and lymphocyte function.

For example, cancer cells may cause the production of prostaglandin E2 (PGE2) in nearby normal host cells such as macrophages. Allev, DG et al.,
Stand. J. Immunol. 39: 31-38, 1994. PGE2 inhibits T cell proliferation, thereby reducing the ability of the host immune system to destroy cancer cells.

[0003] Tumor cells and infectious substances may also produce other substances that directly or indirectly decrease the activity of the host immune response. Accordingly, there is a need for therapeutic strategies that prevent or reduce the reduction in host immune response.

[0004] The present invention relates to strategies for preventing or reducing the reduced immune response in people suffering from a disease in which reduced immune response contributes to disease progression. Such diseases include cancer or pathogen infection.

SUMMARY OF THE INVENTION [0005] One aspect of the present invention is a substance for decreasing the level of ICER expression for increasing immune cell activity. In some embodiments, the substance comprises a nucleic acid. In some embodiments, the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA. In another embodiment, the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA.

[0006] Another aspect of the present invention is the use of a substance that reduces the level of ICER expression in the manufacture of a medicament for increasing immune cell activity. In some embodiments, the substance comprises a nucleic acid.

[0007] In some embodiments, the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA. In another embodiment, the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. In some embodiments, the medicament is for treating cancer. In another embodiment, the medicament is for the treatment of a pathogenic infection. In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, NK cells, monocytes, dendritic cells, and antigen presenting cells. In another embodiment, the immune cells are T cells.

[0008] Another aspect of the invention is a ribozyme comprising at least one catalytic sequence having endonuclease activity and at least one targeting sequence,
At least one targeting sequence is one that is complementary to a sequence present in at least one isoform of ICER mRNA.

Another embodiment of the present invention is an immune cell into which a substance capable of reducing the expression of ICER has been introduced. In some embodiments, the substance comprises a nucleic acid. In some embodiments, the nucleic acid comprises a ribozyme comprising at least one catalytic sequence having endonuclease activity and at least one targeting sequence, wherein the at least one targeting sequence is an ICER in a cell.
It is complementary to a sequence present in at least one isoform of the mRNA. In another embodiment, the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. In another embodiment, the immune cells are selected from the group consisting of T cells, B cells, NK cells, monocytes, dendritic cells, and antigen presenting cells. In some embodiments, the immune cells are T cells.

[0010] Another aspect of the invention is a method of increasing the activity of an immune cell, comprising reducing the level of ICER expression in the immune cell. In some embodiments, the level of ICER expression is reduced by introducing into the immune cell a nucleic acid that inhibits ICER expression. In some embodiments, the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA. In another embodiment, the nucleic acid is at least one of the ICER mRNAs.
An antisense nucleic acid comprising a sequence complementary to at least a portion of one of the isoforms. In some embodiments, the immune cell is a T cell,
It is selected from the group consisting of B cells, NK cells, monocytes, dendritic cells, and antigen presenting cells.

[0011] Another embodiment of the present invention provides a method for detecting the activity of immune cells in an individual, comprising the steps of removing immune cells from the individual, introducing a substance that inhibits ICER expression into the immune cells, and reintroducing the immune cells into the individual. It is a way to increase. In some embodiments, the substance comprises a nucleic acid. In some embodiments, the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA.

[0012] In some embodiments, the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA.

[0013] In another embodiment, the immune cells are selected from the group consisting of T cells, B cells, NK cells, monocytes, dendritic cells, and antigen presenting cells. In yet another embodiment,
Immune cells are T cells. In some embodiments, the individual is in a condition that reduces immune cell activity.

[0014] In another embodiment, the individual is infected with a pathogenic organism. In another embodiment, the individual has cancer. Another embodiment of the present invention is a method of increasing the activity of an individual's immune cells, comprising introducing a substance that inhibits ICER expression into the individual's immune cells. In some embodiments, the substance comprises a nucleic acid. In some embodiments, the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA.

In another embodiment, the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, NK cells, and antigen presenting cells. In another embodiment, the immune cells are T cells. In some embodiments, the individual is in a condition that reduces immune cell activity. In another embodiment, the individual is infected with a pathogenic organism. In another embodiment, the individual has cancer.

Abbreviations IFN-γ, interferon γ; MIP-1α, macrophage inflammatory protein-1α; MIP-1β, macrophage inflammatory protein-1β; 8-Br-cAMP: 8-Br-adenosine X, 5′-cy Click monophosphate AICD, activity-induced cell death; AP-1: activating protein 1 bZIP, basic region / leucine zipper; BZTP: basic region / leucine zipper cAMP, 3 ′, 5′-cyclic adenosine monophosphate CBP, CREB binding protein CD2BRE, CD28 response element CRE: cAMP response element CREB: CRE binding protein HAT, histone-acetyl-transferase CREM: CRE modulator protein DNA binding region of NFAT, PBL, peripheral blood lymphocytes EMSA: motorized transfer assay FasL, Fas Ligand, GM-CSF, granulocyte-monocyte colony stimulating factor GM-CSF: granulocyte-monocyte colony stimulating factor HTLV-1: human T-cell leukemia virus ICER Inducible cAMP early repressor IFNγ: interferon γ IL-12, interleukin 12 IL-13, interleukin 13 IL-2, interleukin 2 IL-4, interleukin 4 NFAT DBD: NFAT DNA binding domain NFAT: activated T cell nuclear factor NFkB, nuclear factor kB PBL, peripheral blood lymphocyte PGE: prostaglandin E PGE ₂ : prostaglandin E ₂ PKA: protein kinase A TcR: T cell receptor PMA: 12-O-tetradecanoyl Horbol 13-acetate T helper 1, Th1 T helper 2, Th2.

Detailed Description of Preferred Embodiments I. Therapeutic blockade of ICER synthesis to prevent ICER-mediated inhibition of immune cell activity Down-regulation of the immune response plays an important role in evolving the cause of the misfortune of many humans. For example, cancer cells or infectious agents can avoid destruction by host immune cells by inhibiting or down-regulating the activity of such cells. In particular, the immune response is circumvented by preventing the expression of genes encoding cytokines essential for the initiation of the immune response. The present invention relates to therapeutic uses of antisense nucleic acids, comprising down-regulating the activity of a protein involved in inhibiting immune cell activity, the inducible cAMP early repressor (ICER) protein.

A group of factors has been identified that regulate gene transcription via a site on the promoter called the cAMP response element (CRE). They are therefore called CRE-binding proteins (CRE-BP). Most of the CRE-BP is constitutively expressed by cells and retains a P-box domain that allows its function to be switched by phosphorylation.

One of the subgroups of these factors is CREB protein as well as CREM-t
Glutamine-rich "flanking the P-box, which mediates nuclear transcription activation after the P-box is phosphorylated, represented by individual CREM proteins such as"
Q domain ”. Such P-box phosphorylation is achieved by several enzymes, including cAMP-activating enzyme protein kinase A (PKA). Thus, this “Q domain” containing CRE-BP subgroup includes constitutively expressed nuclear transcription activators that can switch on cAMP-induced phosphorylation.

Conversely, another subgroup of CREP-BP is CREM-, CREM-
And the constitutively expressed CREM protein, such as CREM-, lacks the activated glutamine-rich "Q" domain and therefore functions as a nuclear transcription repressor factor; its P-box via cAMP / PKA Phosphorylation reduces its activity.

Although an increase in cAMP induces phosphorylation of CRE-BP in the above two subgroups, it does not induce new synthesis, but the family of the third subgroup, the ICER isoform, is reversed. It was found to be induced by elevated cellular cAMP. Since all ICER isoforms lack a P-box, phosphorylation does not appear to play a significant role in its function as much as in a P-box containing CRE-BP. In addition, since all ICER isoforms lack a "Q" domain, they function as nuclear repressors. (Lelli, E. and P. Sassone-Corsi, 1994, Signal Transduction and Gene Regulation; Nuclear Response to cAMP, J. Biol Chem 269: 17359-17362; the disclosure of which is incorporated herein by reference. In).

[0022] ICER mRNA is transcribed from an internal promoter located in an intron within the CREM gene. The ICER promoter contains four CREs in tandem and various neuroendocrine cell lines such as AtT20 adrenocorticotropic hormone secreting cells, GH3
And GC somatotroph-producing pituitary / lactating cells, TSH thyroid cells, T3 gonadotropes, PC12 pheochromocytoma cells, and JEG-3 choriocarcinoma cells, and can be highly induced by cAMP.

There are four isoforms of ICER transcripts, termed ICER I, ICER I, ICER II, and ICE R II, each of which encodes a protein with a DNA binding domain. (Molina, CA et al., Cell 75: 875-888, 1993; the disclosure of which is incorporated herein by reference). These isoforms result from the second splicing of the ICER transcript. The sequence of full-length ICER is fractionally cut to produce four isoforms, which are disclosed in Biochemistry 119: 298-303 (1994), the disclosure of which is incorporated herein by reference. However, the sequence at that time is "ICER
Was not named. FIG. 1, which is derived from FIG. 2 of Biochemistry 119: 298-303 (1994), the disclosure of which is incorporated herein by reference, shows the transcript sequence of full-length ICER mRNA in various domains of the mRNA sequence. It is listed with. FIG. 2 taken from FIG. 8 of Gellersen et al., Molecular Endocrinology 11: 97-113 (1997), the disclosure of which is incorporated herein by reference, shows the structures of the four ICER isoforms. .

As illustrated in FIGS. 1 and 2, each isoform of the translated ICER protein comprises an isoform comprising a bZIP DNA binding domain (DBD I or DBD II), DB DI and ICER II / ICER II, Includes isoforms containing DBD II. The bZIP domain is present on many proteins that regulate transcription. Proteins containing bZIP domains can form heterodimers with other bZIP proteins that have unique DNA binding and transcription properties. That b
Due to the absence of the ZIP domain and the "Q" domain, ICER is believed to heterodimerize with other nuclear transcription activators, such as CREB, and block its activating properties. As described in more detail below, ICER is able to form a triple inhibitory complex with activated T cell nuclear factor (NF-AT).

[0025] The ICER protein binds to the CRE of the ICER promoter, thereby allowing autoinhibition of the ICER gene. In addition to its role as an autoinhibitor,
ICER has also been shown to affect a variety of corticotropin-secreting cell functions. Forskolin-treated cells expressing high levels of ICER are then blocked in the G2 / M phase of the cell cycle via ICER-mediated down-regulation of the cyclin A promoter that binds to the CRE. (Lamas, M. et al., Molecular Endocrinolgy 11: 1425-1434, 1997; the disclosure of which is incorporated herein by reference). Conversely, expression of the ICER antisense in these cells overcomes the G2 / M block.

The present invention is based on the observation that, in addition to its activity in neuroendocrine cells such as adrenocorticotropic hormone-secreting cells, ICER is involved in down-regulation of various gene activities involved in stimulating an immune response. . In particular, ICER binds to a number of recognition sites present on gene promoters that encode cytokines essential for the immune response. Therefore, a technique for reducing the expression level of ICER in immune cells is useful as a therapeutic means for preventing down-regulation of immune cell activity and for studying the mechanism of immune cell activity. In particular, IC
Decreased ER expression levels can be used to modulate immune cell activity by blocking the inhibition of cytokine expression and / or to modulate other functions of cells of the immune system.

Initial experiments demonstrated that ICER expression was up-regulated in medullary thymocytes or T cell lines treated with forskolin or PGE2, known agonists of the cAMP signaling pathway. (Bodor, J.
Natl. Acad. Sci. USA 93: 3536-3541, 1998, the disclosure of which is incorporated herein by reference). ICER mRNA levels were elevated in T cells treated with both forskolin and the protein synthesis inhibitor cycloheximidine, as compared to mRNA levels observed in cells receiving only forskolin, which indicates that in T cells And ICE in neuroendocrine cells
R indicates that the promoter is autoinhibited.

The following examples further describe the regulation of ICER expression and the types of immune cells in which ICER is expressed. Example 1 ICER expression on T cells can be induced by agents other than cAMP: Calcium mobilization stimulates cytokine release via stimulation of T cell receptors (TcR) or addition of calcium ionophore (CI) However, events that lead to proliferation on T lymphocytes can be initiated. To maximize T cell activation and avoid anergy, drugs (protein kinase C activators such as PMA / TPA), or cross-linking of T cell CD28 molecules with anti-CD28 antibodies or appropriate costimulatory molecules Any form, such as the addition of expressing antigen presenting cells, requires additional co-stimulation (2, 3).

T cell proliferation and cytokine secretion can be inhibited by various agents, such as cAMP agonists, IL-10 and glucocorticoids (2,3). In this example, ICERmR in the presence of CI or the cAMP agonist forskolin
The induction of NA was studied. In the present example, induction of ICER mRNA in the presence of CI or the cAMP agonist forskolin was tested as follows.

Lymphocytes were obtained from normal human donors by leukapheresis / elution (4), and CD4 ⁺ T cells were isolated using a commercially available negative immunoselection column (4). These CD4 ⁺ T cells were co-cultured with autologous mononuclear cells every two weeks, but the mononuclear cells were pulsed with tetanus protein (TET) for 24 hours prior to co-culture (4). rIL-2 (300 IU / ml) was added to the co-culture every 3-4 days to promote TET-specific CD4 ⁺ T cell proliferation. At the end of the growth cycle, CD4 ⁺ T cells were washed and rested overnight in IL-2 free media. The following day, they were cultured in the following groups: untreated; forskolin (100 micromolar); CI ionomycin (1 microgram / ml), ionomycin + forskolin. Normal anti-tetanus human CD4 ⁺ T cells were cultured without additives, forskolin (100 micromol), ionomycin (1 microgram), or ionomycin plus forskolin for 4 or 30 hours. Cells were harvested at the end of the 4 hour culture phase and RNAse protection assays were performed as described (5) by Bodor et al.

FIG. 3 shows the results. The expected ICER isoform band is identified on the right. In addition, the band being generated is such that the ICER sequence of the sample is
It is believed to be an artifact resulting from a polymorphism when different from T-cell leukemia (from Jalkat (5)).

As shown in FIG. 3, forskolin or ionomycin significantly induced the expression of ICER mRNA in CD4 ⁺ T cells within 4 hours (FIG. 3, lanes 1-4). After 4 hours of culture, ICER induction is prominent in the ionomycin, forskolin, and ionomycin + forskolin treatment groups. ICER I induction is prominent in both forskolin and ionomycin, whereas the other isoforms are prominent only in the presence of ionomycin. After 30 minutes of culture, ICER induction is significantly maintained only in the group treated with both forskolin and ionomycin.

The expression of ICER in mononuclear cells was examined as described in Example 2 below. Example 2 Expression of ICER in mononuclear cells: Calcium ionophore (CI) induces dendritic cell (DC) -like differentiation of mononuclear cells and includes new expression of costimulatory molecule B7.1 (4 ) (U.S. Pat. No. 5,643,
786 (issued July 1, 1997) and U.S. Patent Application Serial No. 08 / 885,853.
671 (filed June 30, 1997), the disclosure of which is incorporated herein by reference). This is elevated by the presence of certain cytokines such as GM-CSF (6). However, certain other agents inhibit CI-mediated differentiation of mononuclear cells and DC, including cAMP agonists such as PGE2 and forskolin, interleukin-10, and glucocorticoids (6-11). Each of these inhibitors suppresses the induction of B7.1 expression seen in mononuclear cells upon CI treatment (6)
. In this experiment, the ability of each of these agents to induce ICER mRNA in mononuclear cells ("inhibitory" or "stimulatory") was examined.

Mononuclear cells were obtained from normal human donors by leukocyte export / elution (4).
These were freshly cultured in several groups: untreated, forskolin (100 micromolar), prednisolone (10 micromolar), rIL-10 (1000 units / molar).
ml), GM-CSF (50 ng / ml), calcium ionophore (ionomycin, 1 microgram / ml). After 4 hours of culture, groups were harvested and ICERmR
RNAse protection assays for NA were performed as described in (5).

[0035] Fresh human mononuclear cells were treated with untreated, forskolin (100 micromolar), prednisolone (10 micromolar), rIL-10 (1000 units / ml), r
hGM-CSF (50 ng / ml), calcium ionophore (ionomycin,
(1 microgram / ml) for 4 hours. After 4 hours of culture, groups were harvested and an RNAase protection assay for ICER mRNA was performed as described in (5). The expected ICER isoform band is identified on the right. In addition, the bands generated are believed to be artifacts resulting from polymorphisms where the ICER sequence of the sample differs from the ICER template of the assay (from T-cell leukemia Jalkat (5)).

FIG. 4 shows the results. As shown in FIG. 4, after 4 hours of culture, induction of ICER is evident in all treatment groups (calcium ionophore >>IL-10>forskolin>
Prednisolone> GM-CSF). Although all isoforms are significantly induced by the calcium ionophore, induction of ICER I is the most prominent isoform.

[0037] The duration of ICER expression was studied as described in Example 3 below. Example 3 Sustained ICER Expression: cAMP agonists (forskolin, PGE2, dibutyl cAMP) have been used elsewhere to induce ICER expression in various cells. In such experiments, expression of ICER mRNA was transient and generally diminished after a few hours. The main mechanism explaining this attenuation is that the synthesized ICER protein binds to and inhibits its own promoter, thereby
It stops the transcription of R mRNA (12). The ICER protein itself has a half-life of several hours and appears to undergo rapid proteosomal degradation following ubiquitination (13). Because of these observed metabolic trends, there is no prediction that sustained ICER mRNA will be observed. The reason is that the presence of the ICER protein suppresses the
This is because it is expected that the synthesis of A is down-regulated.

Surprisingly, the data presented below demonstrate that sustained IC
Expression of ER mRNA occurs, indicating that cells under these circumstances experience a functionally inhibited situation. Sustained ICER expression was initially assessed in T lymphocytes.

Anti-TET CD4 ⁺ T cells were freshly prepared and expanded into culture as described above.
After resting overnight in medium without IL-2, no addition, forskolin (100 micromolar), ionomycin (1 microgram / ml) or ionomycin +
Cells cultured with forskolin were treated with ICER at 4 and 30 hours of culture.
Obtained for mRNA quantification (see above).

In addition, a microproliferation assay was performed as follows. After resting overnight in IL-2 free medium, anti-tetanus human CD4 ⁺ T cells were added without addition, ionomycin (1 microgram / ml), 50 u / ml rhIL-2, tetanus pulsed autologous mononuclear cells, Alternatively, stimulation was performed with immobilized anti-CD3 (100 ng / well). Each treatment condition was tested with and without forskolin (100 micromolar). In addition tritiated thymidine ⁽³ H-TdR) in 24 hours, and Shutoku cells to 40 hours, proliferation was quantified. Cells were counted by a scintillation counter, during which cell proliferation was quantified.

CD4 ⁺ T cells treated with forskolin, ionomycin, or ionomycin + forskolin each expressed high levels of ICER at 4 hours of culture, whereas only the ionomycin + forskolin group increased at 30 hours of culture. Level I
CER mRNA continued to be expressed (FIG. 3 above).

The results of the microproliferation assay are shown in FIG. As shown in FIG.
During the time, the ionomycin only group showed significant proliferation, probably driven by self-co-stimulation from B7.1 and B7.2 on T lymphocytes (not shown). In contrast, highly reproducible experiments showed that the ionomycin + forskolin group inhibited growth by 70-90% compared to the ionomycin alone group (FIG. 5). Such inhibition could be partially reversed in a dose-dependent manner when the PKA antagonist RPcAMPS was included in addition to forskolin and ionomycin (not disclosed), although the observed forskolin-induced inhibition was as expected. He supports at least PKA dependence.

The above results demonstrate that the cAMP agonist forskolin potently inhibited proliferation induced by each of the stimuli tested. Persistent ICER expression was also evaluated in mononuclear cells as described in Example 4 below.

Example 4 Persistent ICER expression in mononuclear cells: Fresh (uncultured) human mononuclear cells were added to unsupplemented, forskolin (10
0 micromolar), calcium ionophore (ionomycin, 1 microgram / ml) or forskolin + ionophore for 4 or 30 hours,
Subsequent cultures were performed in 24-well plates for FACS analysis or 8-well plates for ICER mRNA quantification. Cells were harvested at the end of the culture period and RNAse protection assays were performed as described (5) by Bodor et al. Cells were obtained at 4 and 30 hours of culture for ICER mRNA quantification (see above). At 30 hours, cells were harvested from a 24-well plate for quantification of expression of costimulatory molecule B7.1.

FIG. 6 shows the results. The expected ICER isoform band is identified on the right. In addition, the band being generated is such that the ICER sequence of the sample is
It is believed to be an artifact resulting from a polymorphism when different from T-cell leukemia (from Jalkat (5)).

As previously demonstrated (4), CI treatment induces only DC-like differentiation of mononuclear cells containing new B7.1 expression. Forskolin treatment, when added alone, has no detectable effect on mononuclear cell B7.1 expression, but B7.
1 significantly inhibited the up-regulation of expression (not shown). After 4 hours of culture, induction of ICER was induced by ionophore, forskolin, and ionophore plus.
It is remarkable in the forskolin treatment group. ICER1 induction appears to be significant for both forskolin and ionophore, while other isoforms are significantly induced only in the presence of ionophore.

After 30 hours of culture, induction of ICER is significantly maintained only in groups treated with both forskolin and ionophore. ICER I and ICER II are 3
It appears to be the two most prominent isoforms at 0 hours (FIG. 6).

The above results indicate that the transient transcription of the ICER gene was found in T lymphocytes and monocytes.
Demonstrating that such transient expression of ICER does not correlate with long-term functional inhibition, as can be evidenced by various agents that result in either functional inhibition or stimulation. I have. Alternatively, when long-term functional inhibition was observed, sustained high levels of ICER mRNA expression were also observed. It is believed that such persistent ICER mRNA expression may be present in the absence of high levels of ICER protein, in which case high levels of ICER mRNA expression would be associated with high levels of ICER protein translation.

Without being limited to a particular mechanism of action, such persistent ICER expression appears to require costimulation via one or more signaling pathways; eg, inhibition (cAMP agonist) And a stimulus (calcium ionophore) signal would be required. Thus, therapeutic strategies that reduce or prevent the persistent development of ICER, as described below, are particularly effective.

ICE when both cAMP- and calcium-dependent pathways are activated
The observation that sustained expression of R occurs, contrary to the purely cAMP-dependent ICER induction studies previously performed (12), suggests that the newly synthesized ICER protein negatively regulates the ICER mRNA promoter under circumstances. Suggests that the direction cannot be adjusted.

Recently, it has recently been demonstrated that sustained ICER expression occurs in human endometrial stromal cells treated with medroxyprogesterone acetate and relaxin (12 ×
). However, in their studies, it appears that ICER lacks the previously reported inhibitory effects of the decidual (dPRL) promoter, and that, even if ICER expression persists, (12x). The authors suggested that co-phosphorylation of competing activating proteins such as CREM-2 and CREB was blocking the suppressive effect of ICER in those cells (12x). While not being limited to a particular mechanism of action, we believe that such a competitive mechanism is not accounted for in this study because sustained ICER expression in lymphocytes and mononuclear cells correlates well with functional suppression of cells. Because you do. Instead, dual stimulation of a PKA agonist (forskolin) and a calcium activator (ionophore) not only induces the transcription and translation of ICER, but does not strongly confer ICER repressive effects on other gene promoters. It also appears to induce negative autoregulatory selective blockade of ICER. If such negative ICER autoregulatory selective blockade is due to the induction of a previously unidentified nuclear regulatory protein, blocking such hypothetical regulatory protein translation could be a potent therapeutic method. As can be demonstrated, it should be possible to block the persistent expression of ICER without interfering with cellular events that require transient ICER expression for homeostasis. Such regulatory proteins will function by interfering with negative ICER autoregulation without blocking the ability of ICER to bind and suppress other enhancers / promoters. For clarity, we refer to this as ICER's negative self-regulating repressor (RINSR).

As described in Example 5 below, in addition to being inducible in T lymphocytes and mononuclear cells, ICER is also induced in NK cells. Example 5 Induction of ICER in NK cells: Induction of cAMP in cultured NK lymphocytes inhibits NK-targeted lysis. This suggests that ICER is inducible in NK cells and inhibits various NK functions. ICER induction in NK cells was evaluated as follows.

The donor was subjected to a leukocyte export / elution method and observed a lymphocyte-rich fraction (4). The lymphocytes were cultured with anti-CD56 conjugated immunoparamagnetic beads, washed, and then loaded onto a magnetized column to positively select NK cells, which were then eluted from the magnet (4). As a result, the purity of the NK cells became 95 to 98%.
These cells were tested at different time points (time 0, 24 hours) for ICER mRNA and protein expression with and without cAMP-induced forskolin treatment.

Positively immunoselected NK cells initially expressed ICER; in other experiments, NK cells did not express ICER, although they were instead purified by negative immunoselection; It demonstrates that immunoselection itself induced ICER. However, such expression was transient. Within 24 hours of culture, ICER levels returned to baseline, but ICER expression could be induced again by forskolin treatment (not shown).

The above results indicate that the expression of ICER is inducible in NK cells and may explain the putative inhibitory effects of cAMP agonists. ICER expression can also be induced in B cells as described in Example 6 below.

Example 6 ICER Induction in B Cells: Expression of ICER has been shown to be inducible by forskolin in the transformed B cell lines X-50-7 and AKATA cell lines (5 ).
This suggests that ICER expression can also be induced in normal B cells, because various stimuli that increase BAMP in c lymphocytes show various inhibitory effects. (15, 16). In particular, c
Treatment of lymphocytes with both the AMP agonist PGE2 and the immune complex results in long-term B
It produces cell unresponsiveness, while not occurring with either treatment alone (15,16).

Although not limited to a particular mechanism of action, such inhibition seen in connection with dual stimuli persists in T lymphocytes and mononuclear cells after combination treatment with calcium ionophore and forskolin. Closely parallel to the inhibition seen when expression of sex ICER is observed.

The expression of ICER in B cells was examined as described in Example 7 below. Example 7 Expression of ICER in B cells: B lymphocytes were freshly prepared with> 95% purity from fresh human peripheral blood lymphocytes by positive selection with anti-CD20 conjugated immunoparamagnetic beads (see above). ). Insufficient numbers were obtained for culture, but freshly isolated B cells expressed the ICER protein, as was NK cells newly selected by positive immunoselection (see above). .

The above results show that ICER expression occurs in normal B cells as well as in transformed B cell lines, and in some circumstances can be described as a putative inhibitory effect of cAMP. The procedure of Example 8 was performed to confirm that ICER sustained expression of ICER protein occurs under conditions that cause sustained expression of ICER mRNA.

Example 8 Demonstration that ICER Sustained Expression of ICER Protein Occurs Under Conditions that Trigger Sustained Expression of ICER mRNA: Induces Sustained Expression of ICER mRNA and Potent Functions such as Forskolin + Ionomycin Exposure of cells to a combined stimulus that causes mechanical inhibition. A protein sample is obtained from the cells, and the level of ICER protein is quantified by an ICER protein-recognizing antibody. ICER of cells in which persistent ICER expression has been induced
The protein level is compared to that of non-induced cells. The results indicate that ICER sustained expression of ICER protein occurs under conditions that induce sustained expression of ICER mRNA.

II. Role of ICER in Cyclic AMP-Mediated Attenuation of Cytokine Gene Expression in Human Thymocytes It is well established that cAMP signaling is inhibitory for T cell proliferation and effector function. In particular, cAMP inhibits T helper-1 cytokine gene expression (30-32). Early studies on fibroblasts indicate that elevated levels of intracellular cAMP inhibit upstream signaling pathways involved in cell proliferation and differentiation. In fibroblasts, increased levels of intracellular cAMP are involved in the MAP kinase signaling pathway ERK1, ERK
Inhibit T2 and JNK kinases, but in contrast to fibroblasts, T cell ERK
1 and ERK2 are insensitive to elevated levels of intracellular cAMP (35)
. Furthermore, cAMP-mediated inhibition of JNK kinase in T cells shows a delayed kinetics, but the observation correlates with induction of the cAMP-induced early repressor ICER (36). Furthermore, NFAT overexpression achieved by transferring NFAT-encoding cDNA into lymphoma cells is associated with c-expression of IL-2 gene expression.
It eliminates the sensitivity of AMP-mediated inhibition (37, 38). Importantly, PK
Phosphorylation of the amino-terminal serine of NFAT by A, despite its ability to protect IL-2 gene expression, does not prevent NFAT from translocating to the nucleus calcineurin (39,40). The view that newly synthesized transcriptional repressors are involved in cAMP-mediated transcriptional attenuation of T helper-1 cytokine expression, rather than inhibiting signaling pathways upstream, suggests that cAMP is present in the presence of both RNA and protein synthesis inhibitors. This was further reinforced by reports that the inhibition of mediated IL-2 expression was reduced (35).

As discussed above, ICER is a transcriptional repressor that appears to act as a systemic negative regulator of the CREB and CREM families of transcription factors and other related bZIP family members (41-44). The ICER isoform represents the only cAMP-inducible CREM subfamily of transcription factors and contains a cAMP response element within the internal P2 promoter. Because of the autoregulation of the cAMP-inducible P2 promoter, the expression of ICER is intrinsically rhythmic. I
Rhythmic expression of CER was first described in the pineal gland and in the hypothalamus-pituitary-gonad axis (45, 46). However, the P2 promoter of ICER can also induce into organs other than the pineal gland and the hypothalamus-pituitary-gonad axis, such as in a specific subset of T lymphocytes, including human medullary thymocytes (36). Importantly, ectopically expressed ICER in the Jurkat T cell line can replace the inhibitory effect of cAMP on transcriptional attenuation of IL-2 promoter activity (36).
.

T cell activating nuclear factor (NFAT) and activating protein 1 (AP-1)
During T lymphocyte proliferation, they present two major transcription factor families involved in the transcription of the IL-2 promoter (47-49). To elucidate the possible mechanism by which ICER down-regulates the expression of the IL-2 gene, bacterially expressed ICER can be used alone or in the minimal DNA binding domain of NFAT (NFAT
Reported to be essential for the complete induction of the IL-2 gene in the presence of DBD) (50)
Experiments were performed to bind to all five NFAT motifs of the IL-2 promoter. The highest affinity ICER binding is the CD28-responsive element (C
E28RE; -160 NFAT / AP-1 complex site) and the IL-2 promoter motif with significant sequences homologous to the conserved proximal regions (-73 to -48 bp) of both the human and mouse promoters of the IFNγ gene (- 90) found at the site (50, 51).

In addition, certain NFAT / AP-1 composite sites present in the IL-4, GM-CSF, and TNF-α promoters resemble those located in the IL-2 promoter (52- 56). The mechanism underlying NFAT action is NFAT
It is believed that AT and / or NFAT / AP-1 are required to bind to the NFAT / AP-1 complex that binds to the motif as a ternary complex (47
). These complexes contain IL-2, IL-4, GM-CSF, and TNF-α
It is believed to be essential for the transcriptional expression of immunoregulatory cytokines during T cell proliferation (48).

As shown below, ICER is capable of expressing these NFAT / AP-1 complex D
It binds to the NA site directly or indirectly by forming a complex with the rel homologous region of NFAT (NFAT DBD). In addition, induction of the ICER-immunoreactive complex was detected in extracts prepared from human medullary thymocytes treated with forskolin and ionomycin. Ectopically expressed ICER includes IL-2, GM-CSF, and TN activated by ionomycin and phorbol ester.
Represses transcription from the F-α promoter, which is responsible for ICE response to cAMP.
The induction of R may explain the cAMP-mediated attenuation of transcription observed in the T helper-1 cytokine response.

The following techniques and techniques were used in the following experiments. Preparation of human medullary thymocytes: Human thymus was obtained from a child (3 months to 4 years) undergoing orthodontic cardiac surgery. Thymocytes were fractionated by discontinuous Parcoll gradient method (Pharmacia) (57). Cells with densities of 1.060 <o <1.070 and P> 1.070 were collected and large thymocytes (significantly enriched in medullary thymocytes) and small thymocytes (cortical) according to established protocols. (36). Isolated human thymocytes were maintained in culture for a short period in RPMI 1640 medium (10% calf serum, supplemented with 50 units / ml penicillin and 25 μg / ml streptomycin) and treated as indicated.

RNase protection analysis: RNA extraction was performed as described (Qiagen). RNA probe hCK1
And hCK3 were purchased from Pharmingen and labeled with [α- ³² P] CTP using reagents from the RNA probe kit (Ambion). These probes were used for RNase protection studies according to the protocol provided by Ambion (R
PAII ribonuclease protection assay kit).

Western blot analysis: Separation of total cell protein (50 μg) was determined by SDS-polyacrylamide gel electrophoresis (10%) using Tris-glycine buffer (25 mM Tris, 250
mM glycine, and 0.1% SDS) at room temperature at 40 mA for 2 hours.
The protein was dissolved in 10 mM Tris / glycine buffer containing 12% methanol,
Electromigration (0.4 mm) over Immobilon P membrane (Millipore) overnight at 0 ° C.
A). The membrane was blocked in a Tris buffered saline solution containing 0.05% Tween 20 (Sigma) and 5% skim milk powder (Bio-Rad) for 1 hour at room temperature with gentle stirring. For immunological detection, no milk powder is included but ICER or 1
: The same solution containing the CREM-specific antiserum (CS4) diluted to 10,000
Stir for one hour, then wash three times, then incubate with horseradish peroxidase conjugated to a secondary antibody (Amersham) diluted 1: 5,000 for one hour, then wash nine times, and finally ECL kit (Amersham) The color developed.

Expression and Purification of Recombinant Protein: Human ICERII cDNA was subcloned into the pGEXKG vector (Pharmacia) and expressed in bacteria as a GST-fusion protein. pGSTagCR
The EB construct has been described previously (58). Purification of both ICER and CREB was performed according to previously established protocols for CREB, with some modifications (58). NFATpXS (1-187), which encompasses the minimal NFATp DNA binding domain, was expressed in bacteria as a hexahistidine-tagged protein and purified as previously reported (59). Recombinant c-Fos
(Fos139-243) and c-Jun (Jun187-334) were purified from Escherichia coli overexpressing strains by nickel chelate affinity chromatography (60, 81).

Nuclear Extraction and Gel Mobility Shift Assay: Whole cell extracts were subjected to high salt extraction to 50 mM HEPES, 250 mM
Prepared as previously described using NaCl, 5 mM EDTA, 0.5 mM DTT and protease inhibitors (20 μM leupeptin, 10 μg / ml aprotinin, 2 mM phenylmethylsulfonyl fluoride) (59). The binding reaction was performed with 20 mM HEPES, 1 mM-MgCl2, 50 mM-KCl, 12% glycerol, 0.1 mM-EDTA, 0.5 mM-DTT, 0.2 [mu] g poly (dl-dC) as a non-specific competitor and recombinant protein. Alternatively, it was performed as indicated in a 15 μl reaction volume containing the whole cell extract. Add 32P-labeled oligonucleotide and, where indicated, excess unlabeled competitor oligonucleotide;
Incubate at room temperature for 10 minutes. The sample was pre-run at 4 ° C. for 2 hours and then run on a 4% polyacrylamide gel in 0.5 × TBE at 200 V for 2 hours. The dried gel was exposed overnight for autoradiography. Oligonucleotides containing the NFAT complex site of the following human promoter were used:

[0071]

[Table 1] Lower case letters are SpeI / XbaI recognition site (IL-2) or BamHI / BgII.
The overhang of the I recognition site (GM-CSF, IL-4, TNF-α) is shown. The following oligonucleotides were used as competitors: nf (mouse IL4N FAT, positions -69 to -79) SEQ ID NO: (SEQ ID NO: 12): 5'-ATAA AATTTTTCCAATGTAAAA-3 '; ap (human Metallothionein IIA
AP-1 site "MRE" positions -114 to -88) SEQ ID NO: 13: 5'-GA GCCGCAAGTGACTCAGCCGCGGGCG-3 '; and tre (mouse c-fos gene oligonucleotide, surrounding CRE site at position -60) SEQ ID NO: 14: 5'-gatccCAGTTCGCCCCAGTGACGTA GGAAGTCCATCa-3 '. Lower case letters indicate an overhang of the BamHI / BgII recognition site.

GST pull-down assay: GST-ICER (GST-CREB) Sepharose beads prepared as described above were subjected to 50 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM ED.
TA, 10 mM-Na3 (PO4) 2, 10 mM-NaF, 0.1% Triton X
The mixture was diluted 1:10 in a final volume of 250 μl containing 100, 2.5 mM leupeptin, 20 mM-PMSF, 100 μg / ml aprotinin and recombinant protein, and cultured on a rotator at 4 ° C. for 1.5 hours. The beads were then mixed with the same buffer for 3 times.
Washed once, resuspended directly in Laemli buffer, 10% SDS-PA
Loaded on GE gel. Retained NFAT DBD protein was visualized by Western blotting using R59 anti-NFAT DBD antiserum as described below.

Transient overexpression in Jurkat T cells: Transfection assays were performed by the DEAE-dextran technique. Typically, 2 μg of the reporter and the same amount of the ICER expression vector were transferred to 10 ⁷ Jurkat cells, and 18 hours after the transfer, phorbol ester (10 ⁻⁵ mg) was transferred.
/ Ml) and ionomycin (1 μg / ml) for 48 hours. Luciferase and chloramphenicol acetyltransferase assays and quantification are described separately (Promega (62)). The conversion of [ ¹⁴ C] chloramphenicol to its acetylated form was determined by ImageQuant.
(Molecular Dynamics).

Example 9 cAMP-Mediated Transcription Attenuation of T Helper-1 Responsive Cytokine Genes in Human Medullary Thymocytes
IL-4, IL-5, IL-9, IL-10, M-13, IL-14, IL-1
5, IFNβ, IFNγ, MIF, TNF-α, Ltβ, TGFβ1, TGFβ
2, and MIRN used to assess mRNA levels of TGFβ3)
It exhibits the properties of native T helper-O (ThO) and T helper-1 (Th1) with significant IL-2 and IFNγ inhibitory action, as evidenced by ase protection (FIG. 7). Stimulation of medullary thymocytes with a combination of phorbol ester and ionomycin significantly induced the synthesis of mRNA encoding IL-2, IFNγ and, to a lesser extent, the synthesis of TNF-α and Ltβ mRNA. Induction (FIG. 7A: lane 4; FIG. 7B: lane 4). At the same time, co-treatment with forskolin or 8-Br-cAMP also inhibited IL-2, IFNγ, TNFα and Lt
β intracellular mRNA levels were reduced (FIG. 7A: lanes 5 and 6; FIG. 7B: lanes 5 and 6). At least a portion of this transcriptional decay is due to the transcriptional repressor ICER c
NFAT / AP- essential for AMP-mediated expression and T helper 1 cytokine expression
It is believed to be based on the subsequent blockage of one complex DNA. Inhibition by ICER is DNA
Directly through binding to the element or the rel homology domain of NFAT (NFAT
It occurs indirectly via protein-protein interactions, such as for DBD).

Example 10 Cyclic AM of T Helper-1 Cytokine Transcription in Human Medullary Thymocytes
P-mediated decay correlates with cAMP-mediated induction of the transcriptional repressor ICER: To test the mechanisms involved in ICER-mediated inhibitors, the presence of ICER in human medullary thymocytes after forskolin treatment was examined, and ICER was IL- 2 and IFNγ promoters were explored to interact with key NFAT / AP-1 enhancer motifs. Western blotting using an ICER-specific antiserum (36) confirmed that the ICER protein was indeed detectable in human medullary thymocytes, but was absent in cortical thymocytes 3 hours after forskolin treatment (FIG. 7C). ). This finding is consistent with the previously observed delayed appearance and subsequent induction of ICER mRNA in medullary thymocytes after exposure to forskolin (36). These observations indicate that cytokine expression of cA
FIG. 4 shows that MP-mediated inhibition occurs in a stage-specific manner in cells of the T cell lineage.

Example 11 ICER Binds to the NFAT / AP-1 Complex Site of the IL-2 Promoter: To further address the mechanism by which ICER downregulates IL-2 gene expression, full induction of the IL-2 gene All of the five NFAT motifs of the IL-2 promoter reported to be essential for E. coli (50) were examined by binding ICER expressed by bacteria (FIG. 8A). Of the five NFAT sites, four at positions (-90), (-135), (-160) and (-280)
Due to the intrinsic ability to bind to the AT / AP-1 complex, NFAT / AP
-1 was previously characterized as a complex site (63). The fifth closest NFAT site, NFAT-45, did not bind to the NFAT / AP-1 complex and was determined to be an exclusive NFAT binding site (63). ICER expressed by bacteria
, And ICER expressed in Cos cells (data not disclosed), to varying degrees,
Binds to all five NFAT sites (FIG. 8B). The strongest bond is at position (-
90) and (-160) at the NFAT / AP-1 complex site, moderate binding to the (-135) NFAT / AP-1 complex site, and weaker binding to most distal and proximal NFAT. And the NFAT / AP-1 complex site, positions (-45) and (
-280). The binding specificity of the recombinant ICER was evaluated using a CREM-specific antiserum (CS4), which did not affect non-specific binding, but did “Supershift” (FIG. 8B, lanes 1-10); similarly, the binding specificity
(64) was evaluated by using a control oligonucleotide that encompassed the first 21 bp repeat (H21) of the HTLV-I LTR promoter containing the motif (64).
) (FIG. 213, lanes 11 and 12).

Example 12 Binding of ICER to the NFAT / AP-1 Complex Site in the Presence of NFAT DBD To better understand the role of the interaction of ICER with the NFAT / AP-1 complex DNA motif, the minimal DNA binding domain of NFAT The effectiveness of ICER in binding to DNA motifs in the presence of (NFAT DBD) was tested. This NFAT domain has the highest degree of conservation among NFAT family members (65) and is both necessary and sufficient for DNA binding and also for association of AP-1 with NFAT (59). . All NFAT motifs on the IL-2 promoter that were tested bound efficiently to NFAT, with the exception of the NFAT / AP-1 complex site below position (-90) (FIG. 8C). The most striking ability of NFAT to associate with ICER is at the site with the highest DNA binding efficiency for both proteins, especially at site (-160) (CD28RE) and to a lesser extent at position (-45).
It was observed at the FAT site. Interestingly, this NFAT-45 site is
R as compared to IC itself against NFAT in the NFAT / ICER complex
There is a tendency to make ER binding stronger (Figure 8C, lanes 1-3). Since NFAT-45 of the IL-2 promoter is the only one of the five sites tested that did not have an adjacent AP-1 site, the finding that this site forms a NFAT / ICER complex has been implicated in protein-DNA action. In addition, protein-protein interactions suggest that ICER itself may be linked to NFAT.

Example 13 ICER Can Interact Directly with the NFAT DBD: To test whether ICER and NFAT can interact directly in the absence of DNA, a truncated NFAT consisting of a DNA binding domain ( A GST-pull down assay was performed using a GST-ICER fusion protein linked to a Sepharose matrix in the presence of (NFAT DB D) (FIG. 9, lane 1).
). GST-ICER formed a complex with NFAT DBD as evidenced by the retention of NFAT DBD. Interestingly, GST-CREB failed to associate with the NFAT DBD under the same conditions (FIG. 9, lane 10). NFAT found on CD28RE (-160 motif) of IL-2 promoter
To examine the specificity of both components of the / ICER triple complex, a CREM-specific antiserum (
CS4) and antiserum prepared against the minimal DNA binding domain of NFAT (R5
Both of 9) were used. Both antisera prevented or reduced NFAT / ICER complex levels (FIG. 8D, lanes 4 and 5, respectively). Unlabeled containing NFAT (nf oligonucleotide spanning positions -69 to -79 of mouse IL-4NFAT) or AP-1 motif (ap oligonucleotide spanning positions -114 to -88 of human metallothionein IIA AP-1 site (MRE)). The competition reaction with the oligonucleotide removed the NFAT / ICER complex (FIG. 8D, lanes 6 and 7, respectively), suggesting that both ICER and NFAT are essential components of the observed complex. .

Example 14 The NFAT / ICER ternary complex has I in comparison with the NFAT / AP-1 complex.
The NFAT DBD shows distinct features for the various NFAT motifs: Similar experiments performed by NFAT DBD and truncated forms of Fos and Jun show that both NFAT / AP-1 and NFAT / ICER complexes. CD
It was confirmed in vitro that it exists in an environment of 28 RE (-160 NFAT / AP).
-1 complex site; FIG. 8D, lanes 8-14). Interestingly, in the presence of NFAT DBD, the complex of NFAT / ICER and NFAT / AP-1
Although it appears to exhibit unequal binding affinity to the / AP-1 motif, most I
The CER protein was retained in a complex with NFAT DBD. For example, IL known as a major site of NFAT / AP-1 complex binding from studies of NFAT
The (-280) motif of the -2 promoter efficiently binds to the NFAT / AP-1 complex (61, 66), but shows little or no detectable formation of NFAT / ICER (FIG. 8C, Lanes 13-15). In contrast, human I
The CD28RE (-160 NFAT / AP-1 complex site) of the L-2 promoter is NFAT / ICER with the same or slightly higher efficiency as the NFAT / AP-1 complex.
A complex is formed (FIG. 8D).

Example 15 ICER binds directly to a conserved proximal motif of the IFNγ promoter: CD28RE (480 showing equally high affinity for both ICER and NFAT
Motif), the NFAT / of the IL-2 promoter at the (-90) position.
The AP-1 motif has significant homology to the conserved proximal element of the IFNγ promoter, but does not interact with the NFAT DBID (FIG. 10). Studies performed on the conserved proximal motif of the human and mouse IFNγ promoters have
/ Deletion of high affinity ICER and NFAT binding in the presence of DBD, or NF
The formation of the AT / ICER complex was shown (FIG. 10); the situation is similar to that observed for the homology (-90) motif of the IL-2 promoter (FIG. 8).

Example 16 Relevant NFAT / GM-CSF, IL-4, and TNF-α Promoters
The AP-1 complex site binds ICER alone or in a complex: The NFAT / AP-1 binding site is already essential for efficient activation of the GM-CSF, IL-4, and TNF-α promoters. It is shown. Therefore, binding of ICER and NFAT was studied both as purified recombinant proteins and in extracts prepared from human medullary thymocytes treated with forskolin and ionomycin (FIG. 11). These studies show that ICER can express the GM-CSF, IL-4, and TNF-α promoters by themselves or as a complex with the NFAT DBD, similar to experiments using the binding site motifs of the IL-2 and IFNγ promoters. Can bind to these complex sites.

The GM-420 DNA motif bound strongly to the purified NFAT / ICER complex (FIG. 11B, lane 6), while the GM-330 and GM-550 motifs bound very weakly to the complex (FIG. 11B, lane 6). FIG. 11B, lanes 3 and 9). It should be noted that the GM-420 motif has been shown to constitute the essential enhancer core of the GM-CSF promoter (52). Similarly, NFA
T / ICER readily forms a complex with the −80 element of the IL-4 promoter and the −95 element of the TNF-α promoter (FIG. 11B, lanes 12 and 15, respectively). Interestingly, the κ3 motif of the TNFα promoter
It contains a "reverse CRE" motif adjacent to the FAT complex site (27), but IL-2
, IL-4, and a complex of electrophoretic mobility different from that observed in the GM-CSF promoter motif (FIGS. 8C and 11B). further,
NFAT DBD alone formed a very slow mobility complex (lane 14), suggesting that NFAT binds as an oligomer to the TNF-α motif.

When the isolated human medullary thymocytes were treated with forskolin and ionomycin,
Expression of ICER not found in uninduced thymocytes (FIG. 11C) (FIGS. 11D and 1)
1E) is easily induced. ICER binding to oligonucleotides containing the NFAT / AP-1 complex site was due to competition for binding with CRE-containing oligonucleotides (FIG. 11D, lanes 4, 7 and 10) or antiserum (C
-Inhibited by interference with Ab) (FIG. 11E, lanes 4, 7 and 10)
.

Example 17 Ectopically Expressed ICER Suppresses NFAT-Mediated Activation of IL-2, GM-CSF and TNF-α Promoters: Various Cytokines in Which Expression of ICER Is Observed in Medullary Thymocytes To determine if the effect of forskolin on transcriptional attenuation of the promoter could be replaced, ICER (isotype II) was expressed in Jurkat T cells in a transient transfection assay. ICER expression down-regulated IL-2, GM-CSF and TNF-α promoters activated by co-treatment of cells with PMA and ionomycin, while ectopic expression of any of the ICER isoforms was identical. VP of (3xGAL4) -CR-CAT under conditions
None proved to have any significant effect on 16-mediated transactivation (FIG. 12). Thus, when induced by PMA and ionomycin, ICER is induced by forskolin in the transcriptional downregulation of calcineurin-dependent NFAT / AP-1 mediated transactivation of the IL-2, GM-CSF and TNF-α promoters. , And can be substituted for it.

The mechanism of cAMP-mediated inhibition of cytokine expression in T lymphocyte proliferation is
Attributable to cAMP-mediated inactivation of the upstream signaling pathway that directs T lymphocyte proliferation. While this hypothesis is supported by fibroblast studies (34), T
It was not confirmed by cell studies (35). Surprisingly, several protein kinases required for T cell proliferation have been found to be insensitive or delayed in response to high levels of intracellular cAMP (35). The above results indicate that expression of the transcription repressor ICER in human medullary thymocytes correlates with delayed cAMP-mediated transcriptional decay of the T helper-1 cytokine response.

[0086] Footprinting and electrophoretic mobility shift analysis for the IL-2 promoter revealed (63) that the AM site originally defined at position -150 of the IL-2 promoter (66, 67): The major CD28 response element (CD28RE) containing the upstream NFAT binding site (66, 67) is to present a new NFAT / AP-1 complex site at position -160. Although initial observations identified NFκB as a major component of the complex (70, 71), re-experimentation has determined that NFAT is a relevant component of the complex that binds to CD28RE in vivo ( 63, 72). These findings indicate that CD28RE (the -160 complex site of the IL-2 promoter) binds to ICER alone or as a NFAT / ICER complex. These findings are important to better understand the direct cAMP-mediated transcriptional decay of IL-2 expression. Furthermore, a potential indirect role of ICER was demonstrated in transgenic mice overexpressing dominant-negative CREB mutants (functional homologues of ICER), which impaired IL-2 expression in thymocytes ( 73). Since the induction of IL-2 and IFNγ expression depends on the activity conferred by each individual DNA motif (51, 63), direct binding of ICER and / or inhibitory NFAT / ICER Evidence for formation at any of the sites indicates that cAMP-mediated IL-2 and I
A description of the mechanisms involved in transcriptional attenuation of FNγ expression could be provided. These findings correlate with the observation that the conserved proximal motif of the IFNγ promoter was inhibited by forskolin in thymocyte proliferation in mice transgenic with the IFNγ promoter-luciferase reporter gene (74). . These findings further indicate that the NFAT / AP-1 motif transmits ICER-mediated transcriptional attenuation, whether it binds directly to ICER or forms a NFAT / ICER complex. Suggest that you can.

A number of NFAT / AP-1 complex sites that have been identified as essential determinants of their expression in relation to the GM-CSF, IL-4 and TNF-α promoters (52, 54, 56) are It seems that we can meet with. This property does not seem to be a universal feature shared by all of the NFAT / AP-1 complex sites tested, because relatively small differences in DNA sequence indicate that ICER binding and NFAT / I
This is because it has an important effect on the formation of the CER complex. In this case, GM-
The CSF promoter is shown, in which case the GM-420 element shows binding to the ICER and NFAT / ICER complexes, while the neighboring motif GM-3
30 and GM-550 bind loose ICER and / or NFAT / I
Only the formation of the CER complex is shown. Strong binding of ICER to the GM-420 element has already been demonstrated by deletion analysis as an essential core of the GM-CSF enhancer (54), which indicates that ICER plays a key role in the transcriptional attenuation of GM-CSF expression. Suggest that it plays. Similar binding studies performed on several NFAT / AP-1 complex sites important in relation to the IL-4 and TNF-α promoters have previously been shown to be essential for efficient expression. These sites (52, 54, 56), alone or as a complex, bind to ICER in the same manner as the IL-2 and IFNγ promoter motifs. Whether the induction of ICER can selectively regulate T helper-1 versus T helper-2 cytokine expression in peripheral lymphocytes has not yet been determined.

It has been reported that human medullary thymocytes synthesize ICER mRNA 3 hours after forskolin treatment, whereas cortical thymocytes do not (36). Western immunoblot with an ICER-specific antiserum showed that under these conditions ICERmRN
It was confirmed that A was efficiently translated into an ICER protein. Furthermore, human medullary thymocytes treated with forskolin and ionomycin using an oligonucleotide probe containing a NFAT / AP-1 DNA motif, where the endogenously expressed ICER protein can form a NFAT / ICER complex in vitro. Was detected in extracts prepared from In contrast to ICER expressed by bacteria, ICER expressed endogenously in medullary thymocytes shows altered mobility in gel shift assays, indicating the binding properties of ICER and / or degradation involved in proteolysis in vivo. This suggests that post-translational modifications are involved in the regulation of the pathway. IC
ICER-containing complexes that are immunoreactive with the ER supershift antisera compete effectively with oligonucleotides containing the CRE or NFAT motif. The NFAT antiserum, which is unable to recognize directly bound ICER, still affects the mobility of the complex containing ICER, suggesting the potential formation of the NFAT / ICER complex in vivo. The ambiguity in which DNA protein complexes are observed in thymocyte extracts on gel shift assays may be due to post-translational modifications of the proteins involved (data not disclosed) and / or their potential consequences for DNA binding. In this regard, it cannot be ruled out that proteins other than ICER and NFAT containing homologous bZIP or rel homology regions may also be involved in the formation of ICER-containing complexes. Finally, ICER in Jurkat cells
Ectopic expression of IL-2, GM-CSF and TN
It is possible to effectively inhibit NFAT-mediated phorbol ester / ionophore-induced expression of the F-α promoter.

Thus, inducible ICER expression in developing and differentiating human medullary thymocytes, as well as in certain subsets of human peripheral blood lymphocytes (36) and mononuclear cells (study in progress), It could significantly affect effector function. ICE
The proposed inhibitory effect on the effector function of the R-mediated immune system is
It may be related to the ability to bind to a wide range of CRE and AP-1 motifs and / or to inactivate certain transcriptional complexes via protein-protein interactions.

III. Inhibition of T cell effector function through ICER-mediated transcriptional attenuation of cytokine and chemokine expression. ) Is cAM
It is a potent regulator that suppresses the expression of specific cytokine genes in T lymphocytes in response to the P / PKA signaling pathway (76, 77). Depending on the nature of the costimulatory factors critical for the production of interleukin 2 (IL-2)
Expression of a number of regulatory cytokines, such as FN-γ, IL-4, IL-5, and IL-13, has been shown in human peripheral cells, where T helper 1 and T helper 2 polarize to a dominant phenotype in vitro. It can be either sensitive or resistant to inhibition by cAMP in blood T lymphocytes. In the absence of IL-2, forskolin, known as an agonist that enhances cAMP, causes a dramatic attenuation of transcription in the early expression of many immunoregulatory cytokines and chemokines. This cAMP-mediated attenuation of transcription strongly correlates with the induction of ICER. To prevent ICER itself from participating in the observed inhibitory effects, rather than forskolin-mediated elevation of intracellular cAMP, transgenic mice expressing ICER under the lymphocyte-specific lck promoter were used. Created. ICE at high level by stimulation
The transgenic thymocytes expressing R were MIP-1α and MIP-1α.
As in β, IL-2 and IFN-γ could not be expressed. In addition, T cells from spleen of transgenic mice show a conditional defect similar to anergy in the proliferative response caused by lack of allogeneic response in the mixed lymphocyte reaction, and ICER-mediated transcriptional attenuation of cytokines and chemokines. Has been suggested to play an important role in inactivating T cell effector functions.

CREB (78) and CRE, Basic Leucine Zipper Transcription Factors (80)
The ICER (inducible cAMP early repressor) belonging to the M family is a signaling pathway of the click AMP-dependent protein kinase A (cAMP / PKA) (
Acts as an overt inactive regulator in 81). ICER was first described in the pineal gland as a potent repressor of cAMP-induced transcription derived from rhythmic adrenergic signals originating from the biological clock (82), followed by several in the hypothalamus-pituitary gland axis Have been shown to regulate the physiology of
83)). Until the discovery in the immune system, which was proposed to act as a potent regulator of T cell effector function (76), ICER was initially thought to be exceptionally expressed in the hypothalamic-pituitary gland axis. .

Importantly, ICER is the only known cAMP-inducible subfamily of CREB / CREM transcription factors. ICER is composed of four isoforms resulting from alternative splicing of its transcript (ICERI
, ICERIγ, ICERII, ICERIIγ) (B1), CREB-like (IC
ERI) or CREM-like (ICERII) with alternating exons γ encoding DNA binding domains. After translation by alternative splicing, ubiquitously expressed CREB and / or related transcripts containing homologous basic leucine zipper (bZIP) domains such as CREM, ATF, Fos and Jun family members (84, 85) The ICER protein can bind indiscriminately to a wide variety of cyclic reactive elements (CREs) and CRE-like DNA motifs that are uniformly occupied by factors. However, the ICER subfamily spontaneously truncates full-length CREM to use an alternative P2 promoter located in the center of the CREM gene. Therefore, the transcription of ICER is
M, starting downstream of the transactivation domain of CREB-like or CREM-like DNA
Because of the binding domain, the ICER subfamily lacks the transactivation domain upstream of the CREM transcription factor (81). The lack of the CREM transactivation domain, which is highly homologous to the transactivation domain of CREB,
CREB binding protein / p300 (CBP / p) that predetermines ICER to act as a strong transcriptional repressor (BB) that blocks B-mediated transcription
300) (86, 87) are thought to be involved in the inability of ICER to recruit (81).

G showing specificity for the sequence of various ligands such as prostanoids and chemokines
There are over 1,200 protein-bound seven transmembrane receptors, many of which, including the hormone prostaglandin E ₂ (PGE ₂ ) receptor, are present on the surface of T cells (89). Such receptors often activate cAMP / PKA and many other signaling pathways and bind to CREs of various promoters via phosphorylation of CREB serine 133 residues (90-92). Phosphorylated CREB (or its homologues (93, 94)) can recruit CBR / p300 complexes involved in a variety of T cell responses including proliferation, differentiation, or hormonal responses (86, 95, 96). However, on the other hand, ICER
It can be induced in parallel via a CRE-like autoregulated responsive element (CARE) located at the central intron P2 promoter of the EM gene (97). Once expressed, the ICER protein converts the ICER protein at the P2 promoter.
CR for binding to a number of CRE-like DNA binding motifs, including its own CARE
It has the ability to compete with the EB, eventually leading to its own down regulation. A number of promoters are critically involved in growth, including, for example, a CRE motif such as the CRE motif of the c-fos promoter at position (-60) (88, 98). Thus, ICER is expressed before its expression ceases (97
), It is possible to first inhibit the genes involved in T cell activation and / or proliferation (99). Thus, the stability, binding and information exchange of the ICER protein, together with the intrinsic cyclical nature of its expression, may explain an elaborate balance toward the conditional features of cAMP-mediated transcriptional decay. There is.

Despite the large number of CREM splice variants initiated by the P1 promoter, ICER is known as the only CREM isoform expressed on cAMP-inducible T lymphocytes (76, 77). ). Since cAMP-mediated expression of CREM reaches extremely high levels in T cells, ICE
R can compete effectively, thereby repressing ubiquitously expressed CREB-mediated transcription. The isoform of (100-102) CREM, which is constitutively expressed in a stage-specific manner in many tissues of the hypothalamic pituitary gland axis, is T
Such competition is probably more effective in T cells, since they are not potentially abundantly expressed in cells. Thus, ICER does not have a transactivation domain, and at least in the context of the CREB / CREM family of transcription factors, without the transactivation domain, it is unlikely that the CBP / p300 complex will be recruited. ICER competition will ultimately lead to CRE
B will lead to dissociation of the recruitment of CBP / p300 mediated. As a result, the binding of ICER leads to the dissociation of CBP, which is the histone-associated CBP.
Lack of acetyltransferase (HAT) activity eliminates the early stages of transcription initiation and makes it impossible to maintain the structure required for chromatin transcription (103, 104).
). Furthermore, in the absence of CBP / p300, the interaction between NFAT and NFκB may also be affected, since full transcriptional activity appears to be dependent on the interaction with CBP (105, 106). . Thus, a CRE-like motif adjacent to the NFAT or NFκB binding motif will become
It is possible to bind to CBP to promote 1 and bZIP mediated transcription.

We have previously reported that cAMP-mediated transcriptional decline in IL-2 expression correlates strongly with induction of ICER in T cells (76, 77). In addition, we have shown that ICER binds directly or indirectly via a protein-protein interaction with a Rel homology domain of NFAT to a DNA motif critical for IL-2 expression (77). ). Interestingly, IL-2
This interaction between bZIP and the Rel homology domain, which may lead to the formation of an inhibitory NFAT / ICER three-dimensional complex under the promoter, is most prominent at the CD28-reactive element (CD28RE). . In addition, CD28RE
CD28RE and the adjacent CRE-like AP-1 motif (CD28RE-TRE
) Has the ability to bind to other members of the CREB / ATF family of transcription factors that can interact directly with (107). In order to achieve full activation of the CD28RE, which leads to recruitment of the CBP / p300 complex, induction via the (108) T cell receptor (TcR), which leads to phosphorylation of CREB, is induced by CD28.
Must be enabled by co-stimulation (109). Apparently, in the signaling pathway activated by CD28-mediated costimulation, the activated bZIP family of proteins represented by phosphorylated CREB and its close homologs (
110) can integrate CBP into cooperative interactions with NFAT or NFκB
It is necessary that Rel homologous proteins such as can regulate IL-2 expression in a highly cooperative manner (110-112).

Importantly, CD28RE, which has the highest affinity for ICER binding (77) under the IL-2 promoter, has recently been shown to be essential for conferring anergy in T lymphocytes (113). . In particular, the requirement for protein synthesis to induce a non-reactive state suggests that newly synthesized proteins are required to maintain a non-reactive state. Thus, IGE synthesized in response to PGE ₂ and many other ligands via the G protein-coupled seven transmembrane receptor.
CER may be critically involved in the induction and maintenance of anergy.

[0097] T helper cells are not a homogenous population and can be divided into at least two subsets named T helper 1 (Th1) and T helper 2 (Th2) based on cytokine expression. Th1 secretes IL-2 and IFN, while Th2 secretes IL-4, IL-5, IL-10 and IL-13. Th
There is now abundant evidence that the ratio of 1 to Th2 cells is strongly associated with the prognosis of a wide variety of syndromes, including autoimmune, allergic and infectious diseases (for review (117-119). Many cytokines have been identified as triggering various types of leukocytes at sites of infection and inflammation (120,
121). They are produced locally in tissues and act on leukocytes via selective receptors. Differential expression of cytokine receptors may largely determine Th1 and Th2 migration and homing to tissues (122, 123). It uses a different fusion co-receptor to Th to human immunodeficiency virus (HIV) strains.
It may also alter the sensitivity of 1 and Th2 (124, 125). Thus, chemokines are part of the effector and amplification mechanisms of the polarized Th1 and Th2 mediated immune responses, and their receptors are similarly targeted as targets of selective regulation of T cell-dependent immunity, as well as Th1 pairs. It has a role as a Th2 marker.

ICER was previously shown to down-regulate IL-2 expression in transient gene transfer assays (76, 77), but has been shown to be similar in vivo. . Analysis of ICER transgenic mice, but not elevated levels of intracellular cAMP. ICER itself is IL
It was confirmed that expression of -2 and many cytokine-induced chemokines may be directed at cAMP-mediated transcriptional attenuation. The results obtained herein show that serine 133 is not phosphorylated and therefore cannot be used to support CREB-mediated transcription, probably due to damage of CBP / p300 (126).
Correlates with the findings reported in transgenic mice of the apparently inactive CREB mutant created by a single amino acid substitution of alanine with alanine. Such transgenic mice, like ICER transgenic mice, cannot produce IL-2 and are defective in T cell proliferation (126). Mouse T cells expressing the overt inactive CREB mutant cannot proliferate even in the presence of exogenously added IL-2, whereas T cells overexpressing ICER are IL-2 Is capable of partially correcting growth defects (data not shown). Lack of IL-2 production with a defect in T cell proliferation is a hallmark of graft host disease and results in marked immune dysfunction (127,128). Importantly, splenocytes of ICER transgenic mice in an allogeneic mixed lymphocyte reaction
Showing a lack of proliferation, it has been suggested that ICER has the ability to suppress its ability to undergo activation. The following method was used in the experiments described below.

(Stable Polarization of Peripheral Blood T Cells to Th1 and Th2 Phenotypes after Polyclonal Activation) Polarization was performed according to Asselin et al. (132). Briefly, PBMCs of normal donors were isolated from leuco packs by density gradient centrifugation in Ficoll and cultured (L-glutamine, 5% heat-inactivated type AB serum (Sigma, St. Louis)).
RPMI, penicillin, streptomycin, and sodium pyruvate
1640). PBMC (5 × 10 ⁵ / ml) were sensitized with soluble sOKT3 (10 ng / ml), and for the phenotype in which IL-12-induced type 1 was dominant, recombinant human IL-12 (2 ng / ml) and rhIL-2 ( 10 ng / ml) combination,
For the phenotype in which IL-4 induction type 2 was dominant, the cells were cultured for 3 days in the presence of rIL-4 (10 ng / ml). Wash cells and add 3-4 more with fresh cytokines
Cultured for days. At the end of the first week, cells were washed, resuspended at 1 × 10 ⁶ and maintained with hrIL-2 (10 ng / ml) for another one or two weeks until re-irritation.
In the absence or presence of forskolin (0.1 mM), PHA (Gibco BRL) was used.
, MD) or phorbol ester (10 ng / ml) and ionomycin (1 g / ml) was added to restimulate the cells.

Preparation of Human Peripheral Blood Lymphocyte Fraction As previously detailed (157), suspension-separated PBLs were prepared and briefly described below. Informed consent was provided to healthy donors for leukocyte export and countercurrent centrifugal suspension. All recovery steps were performed with pyrogen-free reagents. Each donor was initially 5-7 liters of whole blood on a Fenwal CS3000 blood cell separator (Baxter. Healthcare Corp., Deefield, Ill.) Programmed to minimize neutrophil contamination. Was carried out. Leukocytes were enriched in a small volume collection chamber to reduce platelet contamination. This enrichment usually recovers 4 to ¹⁰ × 10 ⁹ peripheral blood mononuclear cells, which are immediately washed with a large volume of normal saline anticoagulated with citrate,
Contaminated platelets and plasma were removed. The washed cells do not contain Ca ²⁺ / Mg ²⁺
Pyrogen-free HBSS (Bio White Tucker, Walkersville, MD
) And a model J-6-M centrifuge (Beckman Instrument, Palo Alto, equipped with a JE 5.0 suspension separation rotor operating at 1725 g at 20 ° C.)
CA). The cells are loaded at a flow rate of 120 cc / min.
Fractions were collected at each step in the range of 140 cc / min, and then a fraction enriched in lymphocytes was obtained. Fractions were collected in life cell tissue culture vessels (Baxter, Healthcare) on ice to inhibit cell adhesion. Ficoll Hypak without pyrogen (
The red blood cells were further purified by density gradient centrifugation using a biowhicker. Each fraction separated by suspension was subjected to further separation as described below,
Then it was analyzed by flow cytometry and immediately used for experiments.

(Preparation of T Cells Selected Negatively from Peripheral Blood Lymphocytes Using Superparamagnetic Beads) Using a subparamagnetic microbead (Miltenyi Biotech, Auburn CA) to sub-populate the PBL Fractionated. Wash and incubation buffers were maintained at 4 ° C., Ca / Mg free, 0.5% bovine serum albumin (Sigma Chemical Co., St. Louis), and no EDTA. 10 ⁹ suspension-separated PBLs were resuspended in 4 ml of the wash incubation buffer described above, and cell surface markers (anti-CD11b, CD1
6, CD19, CD36 and CD56) were first incubated with 1 ml of a cocktail of hapten-conjugated mAbs, then washed and incubated with 1 ml of anti-hapten microbeads. All incubations are 8
C. for 15 minutes. After the incubation was completed, the cells were washed and resuspended in buffer. Indirectly labeled cells (negative selection of T cells) were applied to a CS 'column attached to a VirioMACS with a 19 G needle to flow resistance and 30
ml wash fractions were collected. Usually, the purity of negatively selected T cells is about 95% of CD3 ⁺ cells (data not shown), with CD4 ⁺ cells (75%) predominant over CD8 ⁺ cells (25%). After separation with paramagnetic beads (panT), 1%
Less than CD56 ⁺ CD16 ⁺ (NK cells), CD3 ⁺ CD56 ⁺ double positive cells expressing both T and NK cell markers, CD19 ⁺ B lymphocytes and CD14 ⁺ monocytes could be detected (Data not shown).

(Flow cytometry) PBL was analyzed before and after separation on a MACS column using fluorescence multicolor flow cytometry (FACSort, Becton Dickinson). For cell surface analysis, the following monoclonal antibodies were used: fluorescein isothiocyanate (FITC) conjugated mouse anti-human CD3 and phycoerythrin (PE) conjugated mouse anti-human CD16 purchased from Pharmingen (San Diego, CA).
PE-conjugated mouse anti-human CD19 purchased from Dako A / S (Denmark), FITC-conjugated mouse anti-human CD16, PE-conjugated mouse anti-human CD56, FITC-conjugated mouse anti-virus purchased from Medarex (Anandale, NJ). Human CD
4. TRI color (TC) conjugated mouse anti-human CD3, CD8, CD14, and F
ITC, PE and TC-conjugated IgG subclass matched control antibodies were purchased from Carthage Laboratories (Burlingham, CA). Cells were stained at 4 ° C using Ca ²⁺ / Mg ²⁺ -free DPBS containing 0.5% BSA and 0.025% sodium azide as a dilution / wash FACS buffer. 0.2mg
Non-specific binding to FcR was blocked by incubating with 10 / ml human IgG (Sigma Chemical Co.) for 10-15 minutes and cells were triple-stained with FITC, PE and TC bound Ab for 30 minutes. After washing with cold FACS buffer,
Cells were fixed with 1% paraformaldehyde in PBS. A three-color analysis was performed.

Immunoprecipitation Cells were metabolically labeled with ³⁵ S translabel (ICN Biochemicals, Calif.) According to established protocols, and RIPA buffer (0.15 M) supplemented with a complete protease inhibitor cocktail. NaCl, 50 mM Tris-Cl, p
H7.2, 1% Triton X-100, 1% sodium deoxycholate, 0%
. It was dissolved in 1% SDS), centrifuged at 14,000 rpm for 30 minutes at 4 ° C., and the supernatant was collected and precleared using protein A Sepharose 4B beads (Pharmacia, Uppsala, Sweden). The immune complexes were collected on Protein A Sepharose 4B beads pre-conjugated with CS4-CREM-specific antiserum and incubated at 4 ° C for 3 hours.
It was shaken for 0 minutes and washed three times with RIPA buffer. The immunocomplexes were removed from the beads with Lambli sample buffer and fractionated on 15% SDS-PAGE under reducing conditions. ^{The 35} S signal was sensitized by treating the gel with PPO (2,5-diphenylxazole, Sigma, St. Louis). Using an O-XAR film (Eastman, Kodak, MA), ³⁵ S-labeled protein was detected by exposing to light at -70 ° C for 1 to 10 days.

(Ribonuclease protection assay) RNA was extracted using RN Easy Kit (Qiagen). ICER
RNA probes for were prepared by Xhol or Xbal digestion of pJL5 corresponding to the full length cDNA of human ICERII described previously (76). R
NA probes hAPO3 and mAPO3 were purchased from Farmingen (San Diego, C
[Α ³² P] purchased from A) using the reagents of RNA probe kit (Ambion).
] UTP. The probe was used for ribonuclease protection testing according to the protocol provided by Ambion (RPAII ribonuclease protection assay kit).

Generation and Characterization of ICER Transgenic Mice A 0.36 kb fragment encompassing ICER encoding a sequence driven by the proximal lck promoter was introduced into the mouse germline by pronuclear microinjection (58).
). From several primary lines, three lines were selected for analysis based on the expression level of the transgene. For measurement of lymphocyte proliferation, freshly separated lymphocytes (2 ×
10 ⁵ ) were cultured in triplicate on a 96-well tissue culture plate (Falcon) with 200 μl of DMEM medium containing 10% FCS (Gibco-BRL).
PMA (10 ng / ml), ionomycin (1 μg / ml), anti-CD3 mAb
, 142-2C11 (10 μg / immobilized on plastic tissue culture plate)
lymph) was activated for 48 hours at 37 ° C. by treating with ConA (2 μg / ml). 48 hours after activation, cells were labeled by incubating for 18 hours in tissue culture medium (1 μCi / ml; specific activity 2 ci / mmol) containing ³ H-thymidine (Amersham). The cells were collected on a glass fiber filter, and the incorporation of ³ H-thymidine was measured using a scintillation counter. For allogeneic stimulation, spleen cells of ICER transgenic or non-transgenic mice were cultured for 48 hours with either allogeneic spleen cells obtained from C57BL / 6 or allogeneic spleen cells obtained from BALB / c. It was then labeled and counted as described above.

Example 18 Human Peripheral Blood T Lymphocytes Are Strong Inducers of ICER Protein ICER mRNA Increases cAMP in Immature and Mature T Lymphocytes
Can be induced by an agonist (eg, forskolin or PGE2)
(76). To determine if ICER mRNA is being translated effectively
After treatment with forskolin or PGE2, human peripheral blood T
The presence of the ICER protein was monitored by immunoprecipitation of lysates prepared from lymphocytes.
(FIG. 13). Immunoprecipitation of whole cell extracts allows for ICER protein
Stability depending on protein stability, effective labeling, and
Continuous accumulation was confirmed. Such findings are informed by our knowledge in human medullary thymocytes.
The appearance is very different, the latter being the ICER protein after forskolin treatment.
Induction of protein is not detectable by any detectable IC after 12 hours of forskolin treatment.
It is only transient (after 3 hours), lacking the ER-specific signal (77).
Surprisingly, the expression of ICER in mature T lymphocytes is forskolin or
It is significantly stable during the first 18 hours after PGE2 treatment (FIG. 13). ICER Tan
Our findings, based on immunoprecipitation of protein, suggest that
Yuru ICER isoforms are transcribed by ICER only (76, 77, 97)
Utilizes the P2 promoter of the CREM gene inducible cAMP
Using several different ICER and CREM probes (76)
Ribonuclease protection assay. Therefore, ICER and IC
ER-g proteins (ICERI, ICERII, ICERIγ and ICERI)
Iγ) is the only cAMP-inducible CRE detectable in human peripheral blood lymphocytes
He is a member of the M family (FIG. 13). Out of the ordinary, ICER has many cA
Peripheral blood T-lymph during separation, possibly via release of an agonist that raises MP
Can be easily induced with a sphere (data not shown). Nevertheless, suspended matter
With the combination of isolation and negative selection, we can determine the level of ICER prior to forskolin stimulation.
Isolate "untouched" peripheral blood T lymphocytes without raising the bell
(Fig. 13). In fact, in T lymphocytes separated by negative selection (see below)
Antibody prepared against full-length CREM (CREMα) (129) of whole cell lysate
In serum immunoprecipitation, I was constitutively expressed prior to forskolin treatment.
CER or CREM could not be detected (FIG. 13 shows normal rabbit control in lane 2).
See lane 1 for CREM specific antiserum in C against antiserum N.
Want to be). In contrast, forskolin or PGE_TwoAfter 3 hours of treatment (lanes 3 and 9 in FIG. 1, respectively), the ICERI and ICERII (ICER)
Distinct signals are exon γ, ICERI-γ and ICERII-γ (ICE
R-γ) was also detected for its counterpart lacking. Forskolin and
And PGE_TwoAccumulation of ICER protein after treatment continued over time, first reaching extremely high levels after forskolin treatment (12 hours), followed by PGE _Two Very high levels were reached after treatment (18 hours). Our results show that physiologically significant amounts of ICER are PGE_TwoHas been shown to be readily induced in newly isolated human peripheral blood T lymphocytes in response to relatively unstable ligands such as
Even transient activation in the signal transduction pathway is sufficient to dramatically upregulate ICER.
Signal may be relayed.

Example 19 Stable Polarization of Peripheral Blood T Cells towards T Helper 1 and T Helper 2 Implies a potentially powerful intervention in the mediated immune response. In an attempt to elucidate the molecular mechanism for cAMP-mediated inhibition of helper T effector functions under in vitro conditions, Asselin et al. (19)
(1997) (130) revealed that quiescent polyclonal human T cells divide into a stable T helper 1 (Th1) or T helper 2 (Th2) phenotype. When unselected human peripheral blood mononuclear cells were sensitized with a soluble anti-CD3 monoclonal antibody (sOKT3) in the presence of human recombinant cytokines, (Th1 dominant subsets were IL-12 and IL-2, and Th2 dominant subsets) Has IL-
4 (see below for details) Proliferate in the presence of IL-2. Th1 and Th2
Flow cytometric analysis was performed prior to PHA restimulation to compare the expression of cell surface markers in the population. By this analysis, T cell in vitro
o Polarization showed a relatively minimal change in the CD4 / CD8 ratio, confirming a trend towards a memory phenotype represented by a major shift to cells bearing the CD45RO ⁺ marker in both populations ( (FIG. 14A). Thus, the expression of cytokines and chemokines after restimulation is important for memory T cells, as determined by the fact that both Th1 and Th2 subsets acquire the ability to produce IFNγ, a fundamental property of memory T cells (FIG. 14B). Is most likely to be derived (131
).

Example 20 Cyclic AMP-mediated transcriptional decay of cytokines and chemokines expressed in Th1 and Th2 predominant subsets correlates with induction of ICER As demonstrated above, ICER is a direct protein-protein Through interaction or indirectly via DNA binding to the adjacent AP-1 motif, I
It has the ability to interact with NFAT under the CD28RE of the L-2 promoter (77). The idea that ICER could form an inactivated NFAT / ICER complex that could lead to inhibition of NFAT-driven cytokines such as IL-2 was previously examined in a transient gene transfer assay (76). , 77). Such experiments, monitoring the effects of ectopically expressed ICER on the transcription of cytokine promoters driven by NFAT in activated Jurkat cells, have been combined with CAT or Luc reporters, in which ICER A number of NFAT-driven cytokines have been suggested to be involved in cAMP-mediated transcriptional attenuation. To demonstrate that ICER can also inhibit endogenous expression of cytokines in activated human T lymphocytes, polarize human peripheral blood T cells to the Th1 and Th2 phenotypes (132), and then PHA was restimulated in the absence or presence of choline (FIG. 14B). Ribonuclease protection assays show that the Th1 and Th2 subsets express INFγ, while the Th2 dominant population is a hallmark of the Th2 phenotype, IL-4 and IL-5.
Was expressed at significantly higher levels (FIG. 14). Furthermore, Th1
CAMP-mediated inhibition of endogenously expressed cytokines, which is characteristic of Th2 and Th2 phenotypes, is strongly correlated with forskolin-mediated induction of ICER in both subsets, and ICER is mediated by cAMP of cytokine expression. It is suggested to be involved in the observed effects on transcription decay. Furthermore, helper T
Initial expression of chemokines, which are known to be specific, is also susceptible to cAMP-mediated inhibition of MIP1α and MIP1β, but not RANTES, and is inversely correlated with ICER induction (FIG. 14C).

Example 21 Cyclic Cytokine Expression in T Cells with a Th1 and Th2 Dominant Phenotype (cyc
lic) -AMP-mediated down-regulation is critically dependent on restimulation Sensitivity or resistance to cAMP-mediated inhibition in Th1 and Th2 dominant T cell subsets is best reflected in IL-2 expression Is critically dependent on It is thought that a defect in IL-2 expression due to the inability to send a costimulatory signal is essential for anergy induction and maintenance (133). In fact, the transmission of both signals is often mimicked by treatment with phorbol esters and ionomycin, leading to expression of IL-2 above physiological levels in both Th1 and Th2 dominant populations, Appear to promote the resistance of cytokine expression to cAMP-mediated inhibition by (Fig. 15). In contrast, mitogen stimulation via plant agglutinin (PHA), which relays signals primarily through TcR, significantly reduces IL-2 in the absence of costimulatory signals, which correlates with sensitivity to cAMP-mediated transcriptional attenuation. Guide to level (FIG. 15). IL
Clear difference in the ability of cAMP to mediate inhibition of cytokine expression in -2 absence, formerly PGE ₂ - were noted mediated IL-4 and IL-5 inhibitors (134). Physiological differences that differentiate productive growth resulting from anergy in T lymphocytes may also reflect the different susceptibility of these cells to suppression from external environmental stimuli in the presence of IL-2-mediated autocrine loops It is best characterized under or in the absence (135). Thus, a signal that causes T cells to proliferate and produce high levels of IL-2 may appear to be resistant to cAMP-mediated inhibition, as opposed to cells with low or significantly reduced IL-2 expression. is there. Indeed, our data show that in the absence or presence of suboptimal costimulations that lead to low levels of IL-2 expression, compared to when costimulation leads to active IL-2 expression, T
Both h1 and Th2 cells support the idea that T helper function can be more easily deprived. However, both treatments used for restimulation (phorbol ester and ionomycin, and PHA) are not compatible with other NFAT-like IL-4, IL-5, and IL-13, which are required for effector function of the Th2 phenotype. It is effective for the expression of driving cytokine. Importantly, only PHA restimulation, which cannot lead to active expression of IL-2, is an IL that is susceptible to cAMP-mediated inhibition.
-4, IL-5, and IL-13 expression were provided (FIG. 15).

Unlike rodents, different levels of methylation of the INF-promoter reflect different expression of INF-γ in Th1 versus Th2 subsets (136), in humans,
The indistinguishable expression of INF-γ after restimulation in both T helper subsets is due to the uniform hypomethylation of the INF-γ promoter in both subsets (hypometabolism).
hylation) (see (137)), which correlates with higher overall INF-γ expression. Nevertheless, after PHA-mediated restimulation, Th2 cells, in contrast to Th1 cells, which retain the ability to maintain INF-γ expression at least partially even in the presence of forskolin, -Γ expression of cAM
Be more susceptible to P-mediated inhibition. Because many motifs involved in the expression of INF-γ are hypomethylated in both subsets, the observed different susceptibility to cAMP-mediated inhibition is associated with the expression of INF-γ in Th1 but not Th2. May be involved in the signaling pathway of p38 MAP kinase (137).

Example 22 Due to Impaired T Cell Proliferation in Transgenic Mice Expressing ICER
Incomplete production of IL-2 and INF-γ and MIP-1α and MIP-1β ICER itself is responsible for the cAMP-mediated transcriptional attenuation observed in cytokine and chemokine expression independent of forskolin-mediated intracellular cAMP increase. To prove that it could be involved, we generated ICER transgenic mice. ICERI into lymphoid cells under the control of the proximal lck promoter
I which selectively expresses a human homolog of the I isoform
CER transgenic mice have high levels of ICER in the transgenic thymus as confirmed by immunoprecipitation with CREM-specific antisera.
(FIG. 17A). Thus, with the high level of ICER expressed under the control of the heterologous promoter in the transgenic thymus, we were able to transduce and express IL-2 as well as the expression of chemokines that are initially expressed in activated transgenic thymocytes. And the inhibitory role in the expression of INF-γ can be tested directly. When activated with phorbol ester and ionomycin, in contrast to thymocytes obtained from non-transgenic littermates expressing normal amounts of IL-2 and INF-γ, transactivation Nick thymocytes were unable to express IL-2 and INF-γ (FIG. 16A). In addition, the ICER transgenic mice showed MIP-1α and MI as well as lymphotactin (Ltn) and IP-10 during expression of RANTES.
Inability to express P-1β, but conservatively, suggests a high specificity of ICER-mediated inhibition in both induced and background cytokine and chemokine expression (FIG. 16B).

ICs for altering effector functions of developing or transgenic lymphocytes
ER expression ability was examined. The effect of ICER on the differentiation of cells with constitutive ICER expression in the lymphoid fraction of mice was examined. Analysis of transgenic animals showed that the total number of thymocytes and spleen cells were similar in ICER transgenic and non-transgenic control littermates (FIG. 17D). Both transgenic and non-transgenic thymocytes have normal levels of CD3 and Tc
The transgenic animals express Ra / b and have a normal number of double negative (C
D4 ⁻ / CD8 ⁻ ), double positive (CD4 ⁺ / CD8 ⁺ ), single positive (CD4 ⁺ and CD8 ⁺ ) thymocytes and spleen T cells were present (data not shown). Thus, expression of ICER did not significantly disrupt T cell development. On the other hand, IC
ER transgenic splenocytes and peripheral blood lymphocytes were ConA, immobilized anti-CD3
After activation with monoclonal antibody 2C11, or phorbol ester and ionomycin treatment, they showed varying degrees of growth impairment (FIG. 17B). More importantly, the most dramatic difference is that after mitogen ConA stimulation, phorbol ester and ionomycin treatment show a significant but not significant difference in proliferation in the spleen of ICER transgenic mice. Was seen (FIG. 17B)
.

Our findings in ICER transgenic mice have been correlated with the sensitivity of mitogen stimulation to cAMP-mediated inhibition in polarized human peripheral blood T lymphocytes, indicating that PHA stimulated expression of cytokines and chemokines was -Treatment with phorbol ester and ionomycin, which is sensitive to mediated inhibition, gives cells resistant to the inhibitory effects of forskolin (Fig. 15). Importantly, these data strongly suggest that IL-2 lack was analyzed by ribonuclease protection in ICER transgenic thymocytes (FIG. 16) and polarized human peripheral blood T lymphocytes restimulated with PHA (FIG. 14). ICE
R is suggested to play an important role in inhibiting cAMP-mediated cytokine and chemokine expression through regulation of IL-2 expression. In addition, ICER
Splenocytes obtained from transgenic or non-transgenic mice
Allogeneic stimulation in a mixed lymphocyte reaction using culturing with allogeneic splenocytes from 57BL / 6 or allogeneic splenocytes from BALB / c resulted in dramatically different incorporation of thymidine, resulting in ICER trans. It was suggested that the transgenic splenocytes differed from the non-transgenic splenocytes both phenotypically and functionally (FIG. 17C). In vitro by molecular mechanism by the action of ICER
ro effectively arrests the clonal expansion of T cells, but does not appear to be due to clonal depletion of the responding population. The inability to produce effective proliferation suggests that the ICER transgenic lymphocytes are functionally inactivated.

[0114] ICER is responsible for a number of NFAs that are essential for cytokine expression in vitro.
It can bind to the T / AP-1 motif, but this binding can also occur in vivo
It has also been shown to reflect a transcriptional decay of endogenous cytokine expression at o. Our conclusions on ICER transgenic mice strongly correlate with previously reported observations in transgenic mice with dominant negative mutants of CREB. The transgenic mice were not phosphorylated due to the replacement of the serine (Ser) 133 residue with alanine (Ala), and therefore were unable to produce IL-2, and were probably unable to produce the transcription integrator CBP / p300. With severe impairment in T cell proliferation due to impaired ability to recruit
126). In fact, CBP-deficient mice show a general proliferative disorder (96),
This supports the idea that ER (or a dominant negative mutant of CREB) can compete with endogenously expressed CREB and stop recruiting CBP / p300.

In contrast to the artificial nature of dominant-negative CREB variants, ICER is the only known cAMP-induced transcriptional repressor among T lymphocytes that is physiologically relevant (76, 77). . Furthermore, induction of ICER is usually associated with a lack of T cell proliferation.
Strongly correlates with cAMP-mediated transcriptional decay of L-2 expression. Only ICER is CB
Although unlikely to effectively compete with the activated complex containing phosphorylated-CREB associated with P / p300, after degradation of the complex in the absence of IL-2, the ICER is reduced to CREB alone. It is possible to compete and impair the transcriptional ability for the next transcriptional activation. However, it is clear that the most important determinant of whether anergy is induced does not produce IL-2 (113,133).
. Thus, the binding of ICER to CD28RE of the IL-2 promoter, followed by a translocation of TcR-mediated NFAT, indicates that inactive N cannot recruit CBP / p300.
It may lead to the formation of a FAT / ICER complex, which renders the CD28RE motif unresponsive, resulting in a lack of IL-2 expression. Furthermore, induction of I CER in response to PGE ₂ has been reported in the absence of IL-2 production can lead to PGE ₂ over-mediated inhibition of T cell effector function (77,134).

One of the major functions of TcRs in the presence of costimulatory signals is to initiate the IL-2-mediated autocrine loop, which is required for the activation of proliferation, and therefore IL-2 receptor-mediated PI3 Activation of the kinase (139, 140) is a growth factor-mediated CREB
Signals required for activation of p70 ^rsk kinase involved in the phosphorylation of (98, 141, 142) can be relayed. Subsequent induction of transcription is believed to proceed via CBP / p300-dependent recruitment of the RNA polymerase II complex. Thus, once associated, the IL-2-mediated autocrine loop induced by phorbol ester and ionomycin is still a part of the ICER and / or CBP / p300 activation complex that may be unrepressed by the action of ICER. Due to unidentified regulation, it is possible to confer T cell resistance to cAMP-mediated inhibition even in the presence of ICER (77, 80), which
In contrast to the situation before CBP supplementation where the CER is sufficient for effective competition with CREB. In fact, IL-2 does not help restore T cell proliferation in cells transgenic with the dominant negative CREB mutant (126)
In sharp contrast, the ICER-mediated defect in T cell proliferation is exogenously IL-
2 can be at least partially recovered (data not shown).

[0117] Despite the similarity of both transgenic models, one major difference is the IC
It stems from the fact that the ER appears to be tightly regulated by the cyclic nature of its expression, which corresponds to the actual physiological situation in the cell (143). This suggests that the ubiquitously expressed wild-type CREB (80) is extremely stable irrespective of the physiological context within the cell due to its inherently stable nature.
In sharp contrast to the REB mutant. Thus, ICER-mediated conditional defects in T cell proliferation are crucial to the nature of restimulation, reflecting differential activation of multiple signaling pathways involved in costimulation of T cells in ICER transgenic mice. Looks to depend on In addition, the conditional features of ICER-mediated defects in T cell proliferation are regulated by a sophisticated network of signaling pathways that relay their effects through post-translational modifications that affect the ability of ICER to function. Has been suggested to be a tightly controlled molecular target. One reason for close scrutiny is the ability of ICER to bind to the CD28RE DNA binding motif, which is important for becoming anergic under the IL-2 promoter (77, 113). Thus, ICER binding impairs IL-2 production and subsequently disrupts the IL-2-mediated autocrine loop, which is important for further modification of the CBP / p300 complex involved in cell cycle progression in T cells. (144, 148). Furthermore, CBP / p30 in the presence of IL-2
0 modification is markedly different from that in the absence of IL-2 production.
It may render T cells resistant to AMP-mediated inhibition.

It is well known that activating complexes containing NFAT or NFκB Rel homologous proteins play a major role in the transcriptional regulation of many cytokine and chemokine promoters (149, 150). NFAT and NFκB are similar to CREB, Fos and Jun, which are also involved in CBP / p300 recruitment.
Often associated with ZIP transcription factors. Therefore, the effects of the action of ICER are:
For example, by binding to a non-occupied CD28RE under key cytokine or chemokine promoters such as IL-2 (77) and RANTES (152), or the CRE located at the respective promoter. It is indirect by down-regulating Fos and Jun via like-like motifs (98, 153, 154). Thus, there are a variety of direct and indirect mechanisms by which ICER is conditionally implicated in the transcriptional attenuation of many cytokines and chemokines.

During our studies, we noticed that ICER transgenic lymphocytes, apart from the costimulatory nature, had increased instability in the observed defects in T cell proliferation. One possible explanation is that endogenous expression of activated leucine zipper family members has been induced that may be involved in the recovery of ICER-mediated defects. A similar situation in the case of CREB-deficient mice reportedly, over time, has led to the expression of an activated CREM isoform that completely compensated for the observed defect (155). However, in the case of ICER transgenic mice, we did not detect expression of the fully complementing CREM isoform, probably due to the inability of the CREM-specific P1 promoter to function in T lymphocytes (data shown). Zu). Nevertheless, there are a number of different CREB / ATF isoforms, including a bZIP domain associated with an active transactivation domain, such that complement expression restores the ICER-mediated defect. Are included. Thus, combining heterodimerization of ICER with complementary expression of the activating isoform creates a broad spectrum of conditions that can partially or even completely complement ICER-mediated inhibition. Can be done.

In an attempt to elucidate the role of ICER in the cAMP-mediated suppression of cytokines and chemokines, we developed in vitro polarized human peripheral blood T cells.
The correlation between cAMP-mediated inhibition and ICER induction in lymphocytes was analyzed. Th
Our analysis of polarized human peripheral blood T lymphocytes with a 1 or Th2 phenotype indicates that the ability of T cells to become resistant or susceptible to cAMP-mediated inhibition of cytokine or chemokine expression depends on IL-2 expression. Has an important role to play. The critical importance of costimulation, highlighted by the induction of the potent transcriptional repressor ICER, which has the ability to inhibit IL-2 expression itself, is e.g.
Activation, such as induction of anergy or clonal depletion, could lead to the regulation of a sophisticated network that controls various effector functions of T cells. Thus, the constitutive expression of ICE prior to activation of T cells will
The temporary constraint of ICER induction circumvented in R transgenic lymphocytes is an essential component of the transcriptional component (eg, the AP-1 complex), which is thought to be required for the assembly of the transcription complex by CBP / p300. T cells may be deprived of their ability to induce. In addition, the ability of ICER to cover the critical CD28RE motif, in turn, recruits CBP / p300 to influence a high degree of cooperative interaction at the IL-2 promoter, leading to IL-2-mediated disruption of the autocrine loop. It could lead to bankruptcy. Lack of production of IL-2 results in a lack of cytokine production associated with defects in T cell proliferation, as seen in ICER transgenic mice.

However, the conditional feature in this defect seems to be critically dependent on the nature of the restimulation, and therefore, the costimulatory signaling pathways mimicked by tumor promoters such as phorbol esters The importance of is emphasized. Phorbol esters previously reported (156) to be able to restore T cells from anergy, possibly through release of Ras blockade, are thought to interfere with the induction and / or activity of the AP-1 complex ( 152). Thus, ICER transgenic lymphocytes, which express ICER constitutively by non-uniform promoters, function at least in part through their enhanced ability to transact at the costimulatory-directed IL-2 promoter. This gives rise to a functional AP-1 complex (85) which is thought to aid in a very cooperative association of factors (149). CER-like DNA vacated by ICER instead of AP-1 complex during the absence of AP-1
-It is attractive to think that it binds to the binding site and at the same time inhibits the expression of the AP-1 component. Because ICER lacks the transactivation domain, complementation with ubiquitously expressed CREB or Jun family members prevents recruitment of CBP / p300 and, in the absence of CBP-associated HAT activity, the associated promoter May lead to transcriptional silencing of Thus, APs are directly or indirectly via the transcriptional decay of Fos and Jun (10).
Binding of ICER, which leads to impairment of -1 function, inhibits in vitro inhibitory NFAT /
As shown by the formation of the ICER complex, it favors an ICER-mediated cooperative interaction with the Rel homology domain of NFAT (77).

Although such events seem to be interdependent, they are
It also seems to reflect the ability to reverse with the conditional and temporal features in P-mediated inhibition. Thus, the nature of costimulation clearly demonstrates that it is a critical moment that directs a proliferative response that is regulated by ICER-mediated transcriptional decay and determines sensitivity or resistance to environmental inhibitory stimuli. . It is also conceivable that co-stimulatory signals by phorbol esters, known as tumor-promoting substances, may escape ICER-mediated inhibition as they activate events related to tumor formation.

IV. Differential inducibility of ICER and its role in the regulation of Fas ligand expression on T and NK lymphocytes Under certain conditions, antigen receptor association may lead to Ts through induced Fas ligand expression. Causes apoptosis of lymphocytes. Recent reports show that cAMP / PKA
Forskolin, an activator of the pathway, has been shown to suppress Fas-dependent expression of Fas ligand, resulting in antagonism of activation-induced cell death. Here we show that forskolin-mediated induction of ICER (an inducible cAMP early repressor) correlates with transcriptional attenuation of Fas ligand expression in models of activation-induced cell death, 2B4 T cell hybridomas and human peripheral blood T lymphocytes. Report what you are doing. Strikingly, freshly isolated human peripheral blood NK cells enriched for CD56 ⁺ cells express ICER prior to forskolin treatment, whereas peripheral blood CD3 ⁺ T cells are capable of inducing ICER, In ¹⁹⁺ B cells its expression appears to be resistant to forskolin treatment. In both T and NK lymphocytes, high expression of ICER correlated with high expression of Fas ligand. We report that 2 is essential for NFAT-mediated Fas ligand expression.
Figure 4 shows that ICER specifically binds to the proximal NFAT DNA binding site of the Fas ligand promoter represented by one of the two NFAT motifs. In the presence of minimal NFAT DNA binding domain, the proximal NFAT motif is
Enable ICER and NFAT to form an NFAT / ICER three-dimensional complex in vitro. Furthermore, in activated 2B4 T cell hybridomas, ICER-
The proximal NFAT motif adds to the down-regulation of the mediated Fas ligand promoter. Such findings provide a further perspective on the mechanisms involved in cAMP-mediated transcriptional attenuation of Fas ligand expression in human T and NK lymphocytes.

T cell receptor (TcR) -mediated activation in T cells is a Fas-dependent pathway
Thus, programmed cell death can be induced (159). T lymphocytes
T cell hybridoma 2B4.1, one of the models for studying cell death in
1 is a monoclonal antibody against allogeneic antigen, CD3ε, or phorbol ester
Apoptosis occurs when stimulated with le-ionomycin treatment (160, 161)
. Recent reports indicate that forskolin, an activator of the cAMP / PKA pathway,
, With or without transgenic Bcl-2 (162)
Antagonism of cell death. In addition, Forskolin
Not only inhibits sex-induced cell death (AICD), but also its promoter region
(163) Fas ligand identified and cloned including (164)
It has been shown that expression of (FasL) (162) is also suppressed. FasL is type 1
It is a transmembrane protein and a member of the TNF superfamily. Generally treated
-Deficient gld mice with lymphoproliferative disease and Fas-deficient lpr
Mice are defective in antigen-stimulated apoptosis and have Fas-activated T cells.
It strongly supports the idea that it is involved in apoptosis (165).

Some of the signal molecules and transcription factors that regulate T cell proliferation and effector function may be important regulators of gene-controlled apoptosis in apoptosis.

[0126] cAMP signaling is inhibitory for T cell proliferation and effector function (166). Diterpene forskolin is known as an activator of the cAMP / PKA signaling pathway that acts via direct stimulation of adenyl cyclase. By substituting for the internal promoter of the CREM gene, ICER can be induced in T lymphocytes in response to forskolin-mediated elevation of intracellular cAMP (167, 168). ICER is a potent transcriptional repressor of cAMP-responsive gene transcription and appears to act as a negative regulator of the CREB and CREM family of transcription factors and other related bZIP family members. The importance of CREB in the development of fetal T cells was recently revealed by impaired development of the αβ, but not the γδ, in CREB-deficient mice (169). In contrast, the dominant-negative form of CREB, which is a functional homolog of ICER, does not disrupt T cell development when expressed under the CD2 promoter, but proliferates for thymocytes and IL- 2 Production was defective (
170). The isoform of ICER contains cA in its internal P2 promoter.
A homologous cAMP-inducible CREM subfamily transcription repressor containing MP-reactive elements is meant. Due to the autoregulation of the cAMP-inducible P2 promoter, the expression of the transcriptional repressor ICER can be periodic in nature. Cyclic expression of ICER was first described in the pineal gland and hypothalamus-pituitary gland axis (171, 172). However, the P2 promoter of ICER is differentially inducible in other organs, such as the pineal gland and the hypothalamic-pituitary gland axis, but not in specific subsets of human lymphocytes (167, 168). ). Thus, by differential expression in lymphocytes in response to cAMP / PKA signaling, IC
ER may be a candidate for modulating a molecule such as FasL.

A transcription factor that is beneficial for inducible expression of many cytokine genes, NFAT (Activated T Cell Nuclear Factor) also plays an important role in regulating TcR-mediated FasL expression (173, 174). Two proximal and distal sites (173) identified in the Fas promoter via the deoxyribonuclease I footprint are activated T
Binds to NFAT protein obtained from cells. Although both sites appear to be important for optimal expression of FasL in activated T cells, such sites appear to be dispensable for constitutive FasL expression in Sertoli cells and induce inducible FsL.
It has been suggested that the expression of asL and constitutive FasL are regulated separately. Nevertheless, disruption of FasL expression induction on T cells with splenomegaly and lymphoproliferative disorders in NFATp-deficient mice implicates NFATp as a key transcription factor involved in Fas-induced apoptosis in activated T cells. (175). In addition, the proximal NFAT motif of the FasL promoter contains an IL
-2 of the proximal NFAT motif of the promoter and IL-2, GM-CSF,
Numerous N previously identified as essential under the IL-4 and TNFα promoters
It can associate with ICER in a manner similar to the FAT / AP-1 hybridized DNA binding site (168, 176-179). Minimal NFAT DNA binding domain (NF
AT-DBD) in the presence of (22,23), by protein-protein interaction via NFAT-DBD or by direct protein-DNA interaction via ICER, to the NFAT DNA binding site of the cytokine and ICER.
In a manner similar to that of interacting, the proximal NFAT motif of the FasL promoter can form an NFAT / ICER three-dimensional complex. Such a concept is called ICE
Consistent with and supports the hypothesis that R acts to regulate FasL expression. In a more transient gene transfer assay, forskolin-mediated induction of ICER was achieved with the FasL promoter and IL-2, GM-CS.
F and closely correlates with the ability of ectopically expressed ICER to suppress the activity of other NFAT-driven cytokine promoters such as TNF (16
8).

To study the role of ICER in cAMP-mediated transcriptional attenuation of FasL expression, the differential expression of ICER in lymphocytes was assessed and forskolin-mediated with induction of ICER in activated 2B4 T hybridomas The time course of the down-regulation of FasL expression was monitored. Furthermore, Fas
Transcriptional attenuation of L expression was compared to induction of ICER in human peripheral blood T lymphocytes activated in the presence of forskolin. The results of our analysis on T lymphocytes
It has been shown that forskolin-mediated ICER expression correlates with FasL down-regulation. In contrast, human peripheral blood NK lymphocytes under the conditions used in our study show a pre-existing ICER at the mRNA and protein levels prior to forskolin stimulation. However, the release of transcriptional repression in FasL expression, reflected by the cessation of ICER expression in NK lymphocytes activated with phorbol ester, is directly or indirectly related to the regulation of FasL expression by ICER. Can be considered. The following techniques and methods were used in the experiments described below.

(Preparation of Human Peripheral Blood Lymphocyte Fraction) As previously detailed (157), suspension-separated PBLs were prepared and briefly described below. Informed consent was provided to normal donors for leukocyte export and countercurrent centrifugal suspension. All recovery steps were performed with pyrogen-free reagents. Each donor was first run on a Fenwal CS3000 blood cell separator (Baxter healthCare Corp. Deerfield, IL) programmed to minimize neutrophil contamination. Leukocyte export was performed from 5-7 liters of whole blood. Leukocytes were enriched in a small volume collection chamber to reduce platelet contamination. This concentration usually results in 4
〜１０10 × 10 ⁹ peripheral blood mononuclear cells were collected and immediately washed with a large volume of normal saline anticoagulated with citrate to remove contaminating platelets and plasma. The washed cells were pyrogen-free HBSS (BioWhittaker, Walkersville, MD) containing no Ca ²⁺ / Mg ^2+.
)) And a model J-6-M centrifuge (Beckman Instruments, Palo Alto, Calif.) Equipped with a JE 5.0 suspension separator operating at 1725 g at 20 ° C. )) To separate the suspension. 12
Cells were loaded at a flow rate of 0 cc / min, fractions were collected at each step in the range of 120-140 cc / min, and then lymphocyte enriched fractions were obtained. Fractions were collected in life cell tissue culture vessels (Baxter, Healthcare (BioWhittaker Walkersville)) on ice to inhibit cell adhesion. Further purification was carried out by density gradient centrifugation using a pyrogen-free Ficoll Hypaque (BioWhittaker) to remove red blood cells. Each fraction separated by suspension was subjected to further separation as described below, then analyzed by flow cytometry and immediately used in experiments.

(Preparation of T cells negatively selected from CD3 ⁺ , CD19 ⁺ , CD56 ⁺ and peripheral blood lymphocytes using superparamagnetic beads) For positive selected cells, mouse anti-human mAb against CD3, CD19, or CD56 was used. Pre-bound superparamagnetic beads (Milteni Biotec, Auburn, CA) were used to fractionate the sub-population of PBLs while expressing hapten-bound non-T cell populations. MAbs specific for the surface markers present (anti-CD11b, Cd16, CD19, CD36, and CD56)
Negatively selected T cells were prepared by subsequent incubation with superparamagnetic anti-hapten-conjugated beads using a cocktail of. The wash and incubation buffer contained 0.5% bovine serum albumin (Sigma Chemicals, Co., St. Louis) without Ca / Mg maintained at 4 ° C. and EDTA. Was contained in DPBS. For the preparation of positive selection cells, fresh PBLs were washed and resuspended at 1 × 10 ⁶ cells per 0.8 ml, with 0. 2 ml of antibody coated microbeads were added. For negative selection T cells, a 10 ⁹ suspension separated PBL were resuspended in washing and incubation buffer 4ml described above, first incubated with a cocktail of 1ml containing mAb bound hapten, then washed, Incubated with 1 ml anti-hapten microbeads. All incubations were performed at 8 ° C. for 15 minutes. After the incubation was completed, the PBLs were washed and resuspended in buffer. Directly labeled cells (CD3 ⁺ , CD19 ⁺ , or CD56 ⁺ ) were subjected to a VS ⁺ column attached to a MidiMACS magnet (2.5 x 10 ⁸ cells per column). After washing 4 times with 3 ml, the column was removed from the magnet and the positive selection cells were washed out with 5 ml of buffer per column. Cells were evaluated for expression of CD3 ⁺ , CD16 ⁺ and CD56 ⁺ cell surface molecules by multicolor flow cytometry. Usually, the purity of the CD3 ⁺ T cells, CD19 ⁺ B cells and CD56 ⁺ NK cell populations is higher than 95% (data not shown). In the case of CD 3 ⁺ T cells and CD56 ⁺ NK cells, the T and NK cell marker Significant (about 10%) of CD3 ⁺ CD56 ⁺ double positive cells expressing both are present. Less than 1% of CD14 ⁺ monocytes or CD19 ⁺ B cells are detected in such a cell population (
Data not shown). The indirectly labeled cells (negative selection of T cells) were applied to a CS column attached to a VarioMACS ⁺ with a 19G needle as a flow resistance and 30 ml of the washed fraction was collected. Usually about 90% of the negatively selected T cell population
Are CD3 ⁺ cells and CD4 ⁺ (80%) exceeds CD8 ⁺ (10%). After paramagnetic bead separation ^{(PanT), CD56 + CD16 +} (NK cells) of less than 1% double positive cells expressing both T and NK cell markers CD3 ⁺ CD5 6 ⁺ cells, CD19 ⁺ B lymphocytes Spheres and CD14 ⁺ monocytes are detected (data not shown).

(Flow cytometry) Fluorescent multicolor flow cytometry (FACSort, Becton Dickinson)
(FACSort, Becton Dickinson)) and PBL were analyzed before and after separation on a MACS column. For cell surface analysis, the following monoclonal antibodies were used: fluorescein isothiocyanate (FITC) -conjugated mouse anti-human CD3 and phycoerythrin purchased from Pharmingen (San Diego, CA). (PE) conjugated mouse anti-human CD16, PE-conjugated mouse anti-human CD19 purchased from Dako A / S (Denmark), Medarex (Anandale,
NJ (Annandale, NJ)) purchased FITC-conjugated mouse anti-human CD16, PE-conjugated mouse anti-human CD56, FITC-conjugated mouse anti-human CD4, TRI color (TC) conjugated mouse anti-human CD3, CD8, CD14, And FITC, PE
IgG subclass matched control antibodies with TC bound thereto were purchased from Carthage Laboratories (Burlingame, CA). Ca ²⁺ / M
DPB without g ²⁺ , 0.5% BSA and 0.025% sodium azide
Cells were stained at 4 ° C. using S as dilution / wash FACS buffer. 0.2
mg / ml human IgG (Sigma Chemical Co.)
Incubation for 15 minutes prevents non-specific binding to FcR,
Cells were triple-stained for 30 minutes with TC, PE and TC-bound Ab. Cooling FAC
After washing with S buffer, cells were fixed with 1% paraformaldehyde in PBS. Then a three-color analysis was performed.

(Cell line) For the NK3.3 human NK cell line (distributed by Dr. J. Kornbluth), 25 mM HEP without antibiotics and supplemented with IL-2 (15 U / ml)
ES and 15% heat-inactivated fetal bovine serum, 15% lymphocult (Biotest
Diagnostics (Biotest Diagnostics) were added and maintained in RPMI 1640. For the NK92 human NK cell line (distributed by Dr. E. Long), 1
Maintained in a myelocult (StemCell Technologies, Vancouver) in the presence of 00 U / ml IL-2. For 2B4.11 T cell hybridoma (provided by J. Ashwell), 10% heat-inactivated fetal calf serum, penicillin (Gibco BRL) and gentamicin (Gibco BRL)
) Together with RPMI 1640 (excluding phenol red for luciferase assays).

Immunoprecipitation Cells were metabolically labeled with ³⁵ S translabel (ICN Biochemicals, Calif.) According to established protocols and RIPA buffer (0.15 M) supplemented with a complete protease inhibitor cocktail. NaCl, 50 mM Tris-Cl, p
H7.2, 1% Triton X-100, 1% sodium deoxycholate, 0%
. It was dissolved in 1% SDS), centrifuged at 14,000 rpm for 30 minutes at 4 ° C., and the supernatant was collected and precleared using protein A Sepharose 4B beads (Pharmacia, Uppsala, Sweden). The immune complexes were collected on Protein A Sepharose 4B beads pre-conjugated with CS4-CREM-specific antiserum and incubated at 4 ° C for 3 hours.
It was shaken for 0 minutes and washed three times with RIPA buffer. The immunocomplexes were removed from the beads with Lambli sample buffer and fractionated on 15% SDS-PAGE under reducing conditions. ^{The 35} S signal was sensitized by treating the gel with PPO (2,5-diphenylxazole, Sigma, St. Louis). Using an O-XAR film (Eastman Kodak, MA), ³⁵ S-labeled protein was detected by exposing at −70 ° C. for 1 to 10 days.

(Ribonuclease protection assay) RNA was extracted using RN Easy Kit (Qiagen).
RNA probes for ICER were previously described (76) human ICERII.
Prepared by Xhol or Xbal digestion of pJL5 corresponding to the full length cDNA of RNA probes hAPO3 and mAPO3 were purchased from Farmingen (San Diego, CA) and RNA probe kits (Ambion (Ambion)
Ambion)) and [α ³² P] UTP. The probe was used for ribonuclease protection testing according to the protocol provided by Ambion (RPAI I ribonuclease protection assay kit).

(Expression and Purification of Recombinant Protein) Human ICERII cDNA was subcloned with the pGEXG vector (Pharmacia) and expressed in bacteria as a GST-fusion protein. Formerly CRE
According to the protocol established for B (187), the ICER
Was purified. NFATpXS (1-187), which contains a minimal DNA binding domain of NFATp (distributed by Dr. A. Rao), was expressed in bacteria as a hexahistidine-tagged protein, as described previously (180). Purified.

Electrophoretic Gel Mobility Shift Assay 20 mM HEPES, 1 mM MgCl ₂ , 50 mM KCl, 12% glycerol, 0.1 mM EDTA, 0.5 mM DTT, non-specific competitor Binding reactions were performed in a reaction volume of 15 μl containing 0.2 μg of poly (dl-dC) and the recombinant protein as indicated. An oligonucleotide labeled with ³² P was added and incubated at room temperature for 10 minutes. After 2 hours pre-run, 0.5x
Samples were run on a 4% polyacrylamide gel in BE at 200 V for 2 hours. Autoradiography was performed overnight on the dried gel. The following oligonucleotides containing the NFAT hybrid site of the human promoter were used: SEQ ID NO. 15: FasLprox5'-gatccTAGCTAT
GGAAACTCTATTAa-3 'SEQ ID NO. 16: FasLdist5'-gatccCTGGGGCG
GAAACTTCCAGGa-3 'SEQ ID NO. 17: IL-2 (-160) 5'-ctagaAAAGA
ATTCCAAAGAGTCATCAGAAa-3 'SEQ ID NO. 37: GM-CSF (−420) 5′-gatccCAT
CTTTCTCATGGGAAAGATGACATCAGGGAa-3 ′ Lowercase letters indicate Spel / Xbal recognition site (IL-2) or BamHI / BgII.
The overhang for the I recognition site (FasL, GM-CSF) is shown.

(Transient Overexpression in 2B4 T Cell Hybridoma) The DEAE dextran method was modified on a 96-well culture plate (Packard, NJ (Packard, NJ)) coated or uncoated with the anti-CD3 monoclonal antibody 2C11. Transfer assays were performed. Usually, 2 (.12～16 which was partitioned reporter and two wells not two wells and coat the coat in advance 2C11 10 ⁸ cells gene transfer in ICER expression vector of the same amount of g
After time, the luciferite assay (Packard,
NJ (Packard, NJ)) and top emission (Packard, NJ (Packar, NJ)
d, NJ)). Chloramphenicol acetyltransferase assays and quantification methods have been described elsewhere (188).

Example 23 Induction of ICER Observed Early After Forskolin Treatment in Freshly Isolated Human Peripheral Blood T Lymphocytes Is Suppressed in B Lymphocytes ICER plays a functionally important role in lymphocytes As expected, we first looked for characteristics in different populations of human peripheral blood lymphocytes as to whether lymphocyte subpopulations exhibited differential regulation of ICER. Immunoprecipitation with CREM-specific antisera for CD3 ⁺ and CD19 ⁺ lymphocyte subsets showed that only lymphocytes enriched for CD3 ⁺ T cells showed elevated background levels of ICER protein, including forskolin ICE after 3 hours of treatment
This shows that significant early induction of R was accompanied (FIG. 18). Importantly,
CRE based on previously reported (167) ribonuclease protection test data
No other CREM isoforms were detected, consistent with the absence of M mRNA (see analysis of Jurkat T cells below in Figure 18 and Figure 23B). Forskolin-mediated ICER in peripheral blood lymphocytes before separation
(Lanes 1 to 4 in FIG. 1) were significantly increased in the population enriched for CD3 ⁺ T cells (lanes 5 to 8 in FIG. 1), but negligible in the population passed through CD3. (Lanes 9 to 12 in FIG. 1) and the population involved in inducible forskolin-mediated ICER expression detectable in freshly prepared unseparated human peripheral blood lymphocytes 3 hours after treatment is CD3 ⁺ T Suggested that it was a lymphocyte. Furthermore, the lymphocyte population enriched for CD19 ⁺ B cells did not show a significant increase in ICER compared to unseparated PBL and was resistant to forskolin treatment (
Lanes 13 to 16 in FIG. 18). In contrast to new peripheral blood lymphocytes, ICER was not detectable in the leukemia cells, Jurkat T cell line, which served as a control before forskolin treatment (lanes 21 and 22 in FIG. 18), which is the data for ribonuclease protection (FIG. 18). Jerkat T cells showed only limited levels of ICER mRNA (see lane 2 in FIG. 23B) and protein after forskolin treatment (lanes 23 and 24 in FIG. 18). This indicated that the signal detected by immunoprecipitation was ICER-specific. The differential regulation of ICER expression found in these studies supports the idea that ICER acts as a potentially important molecule in T lymphocyte function.

Example 24 FasL in activated 2B4 T cell hybridomas and human peripheral blood T lymphocytes
Forskolin-mediated transcriptional decay of expression correlates with induction of ICER Previous experiments have shown that ICER is efficiently expressed in T cells.
To study the role of ICER expression in T cell lineages, we further investigated 2B4
T cell hybridomas were utilized. Forskolin induced apoptosis in FasL expression in 2B4 hybridoma T cells treated with phorbol ester and ionomycin, which in many ways mimic TcR-mediated activation and induce AICD by up-regulating FasL expression. It has been previously reported that it is effectively prevented by transcriptional attenuation (161, 162). To elucidate the mechanism of inhibition of forskolin-mediated FasL expression, we investigated whether forskolin could induce ICER in 2B4 T cells activated with phorbol esters and ionomycin (FIG. 19A). In the presence of forskolin, phorbol ester and ionomycin treatment can further induce ICER mRNA in activated 2B4T cells (FIG. 19A, lanes 1 to 6), and in the presence of ICER F
The asL message could not be expressed (lanes 1 to 6 in FIGS. 19A and B). In contrast, in the absence of forskolin, high levels of FasLmRN
A accumulated and little or no specific expression of ICER (FIG. 19A).
And B lanes 7 to 12). Time-course data for activated 2B4 T cell hybridomas show down-regulation of FasL expression and a strong transcriptional repressor ICE.
R showed a strong correlation with the induction of R, indicating that forskolin-mediated ICER expression was
He supported the possibility of causing transcriptional decay of expression.

To assess the relevance of our observations in mouse 2B4 T cell hybridomas to fresh human T cells, we isolated human peripheral blood T lymphocytes using an ablation strategy rather than a positive selection. In addition, CD3 ⁺ cells were isolated by removing the double positive CD56 ⁺ CD3 ⁺ lymphocyte population expressing the NK cell marker. Due to the background level of ICER expression seen before forskolin treatment in CD3 ⁺ selected T lymphocytes, including the CD56 ⁺ CD3 ⁺ lymphocyte population, non-T cells such as CD56 ⁺ expressing NK lymphocytes also Was generated (FIG. 18). In fact, T cells that were negatively selected and depleted of CD56 ⁺ NK cells had very low levels of ICER mRNA before forskolin treatment.
(FIG. 20B, lane 4), but only phorbol ester and ionomycin treatment increased ICER mRNA levels in such cells (FIG. 20B, lane 2). This was probably due to contaminated cells, as no contaminating cells were present in the 2B4T cell line (Figure 19A lanes 7 to 1) or Jurkat T cell line (data not shown) and no significant expression of ICER could be detected. It is. T cells activated with phorbol ester and ionomycin in the presence of forskolin (FIG. 2)
0, in lane 1), a low level FasL message indicates an increased level of IC
In the T cell population activated with phorbol ester and ionomycin in the absence of forskolin, which correlated with the ER message, increased FasL expression was due to ICE
This was correlated with a decrease in R mRNA (lane 2 in FIG. 20).

Example 25 Induction of FasL expression in human peripheral blood NK lymphocytes activated with phorbol ester was enriched for CD56 ⁺ to examine ICER expression in cells of the NK lineage with cessation of ICER expression The human lymphocyte population was evaluated. Interestingly, they showed elevated levels of ICER mRNA prior to forskolin treatment, with a dramatic decrease in ICER mRNA 3 hours after phorbol ester treatment, which included FasL,
FAP (PNP1, protein tyrosine phosphatase 1E) and TRADO
(TNF receptor associated with death domain protein) was consistent with induction of expression (FIG. 21). Individual mRNA levels are measured in human lymphoid cells by Fas and TNF.
Evaluated by the ribonuclease protection assay used to evaluate messages of multiple components of the pathway (FLICE, FasL, Fas, FADD, FAP, FA
F, Fas2L, TNFRp55, TRADO and RIP) (FIG. 21A). FasL messages increased dramatically after phorbol ester treatment, but ICERmRN
The level of A decreased to the background (FIG. 21B).

Determine whether elevated ICER messages observed in newly isolated human peripheral blood lymphocytes enriched for CD56 ⁺ NK cells correlate with high levels of ICER prior to forskolin treatment in human peripheral blood NK cells In order for us to
Immunoprecipitation was performed with an M-specific antiserum (FIG. 22A). The data confirmed that ICER mRNA in newly isolated NK lymphocytes prior to forskolin treatment was efficiently translated into ICER protein. In addition, ICERmR in two human NK cell lines, NK3.3 (185) and NK92 (186).
When comparing NA to the Jurkat leukemia T cell line, which serves as a control for the human T cell line, the ribonuclease protection assay shows NK3.3 and less NK92.
Were found to show high levels of ICER mRNA before forskolin treatment (lanes 5 and 3 in FIG. 22B). Jurkat as a T cell control
T cell lines (lanes 1 and 2 in FIG. 22B) were also examined previously by other T cell lines (MOLT
3, MOLT4, HUT78 and CEM) also did not express significant levels of ICER mRNA before forskolin treatment. Thus, these experiments show a different pattern of ICER expression in CD56 ⁺ NK cells as opposed to CD3 ⁺ T cells or CD19 ⁺ B cells, which regulates FasL expression by reciprocally balancing the two expressions. He further supported the hypothesis.

Example 26 Fas Ligand Proximal NFAT Binding Site Binds ICER Alone or in Complex with NFTA DBD To further investigate the possible role of ICER in Fas ligand gene expression, We have reported that two Nases of the Fas ligand promoter, which have been reported to be essential for the complete induction of the Fas ligand gene, in bacterially expressed ICER.
The binding to the FAT motif was examined (FIG. 23A) (173, 174). IL- at the proximal and distal NFAT motifs and position (-160) of the Fas ligand promoter
Two promoters and two controls N in the GM-CSF promoter at position (-42)
ICER to specific oligonucleotides individually containing the FAT / AP-1 motif
The specificity of the recombinant ICER was evaluated using a CREM-specific antiserum (CS4) (168) that "supershifts" the binding of (Figure 23A and B). NFAT DBD and ICER are necessary and sufficient for DNA binding of NFAT and / or formation of a complex with ICER (168) or AP-1 (181), directly via an AP-1-like sequence adjacent to the NFAT motif. This control motif can both bind to ICER, indirectly through interaction with. ICER and ICER containing complexes can compete with unlabeled oligonucleotides containing the CRE motif (168). As with the proximal NFAT motif of the IL-2 promoter, we observed the specific binding of ICER to the proximal NFAT motif of the FasL promoter, creating a three-dimensional NFAT / ICER complex in the presence of NFAT DBD. Was observed. Unlike the proximal NFAT motif of the Fas ligand promoter, the distal NFAT motif cannot retain significant levels of ICER binding and / or the NFAT / ICER three-dimensional complex (FIG. 23C, lanes 4-6). Instead, the distal NFAT motif is NFAT in vitro.
Maintains high affinity with DBD, which involves dimerization of NFAT DBD,
Consistent with a proposed role for the distal NFAT motif in activation of Fas ligand expression (15,16). These experiments provide further evidence supporting ICER as a regulatory molecule in FasL expression.

Example 27 Ectopically Expressed ICER Suppresses the Transcription of Activated Human FasL from the Luciferase Reporter [0144] ICER reduces transcription of the FasL promoter found in 2B4 T cell hybridomas and peripheral blood T and NK cells. To determine if expression of E. coli mediates the effects of forskolin, different I
CER isoforms (ICERI, ICERIγ, ICERII, and ICE
RIIγ) was expressed ectopically in 2B4 T cell hybridomas. Expression of ICER down-regulated the human FasL promoter in the presence or absence of the distal NFAT motif (FIG. 24A) activated by monoclonal antibody 2C11 (specific for the CD3ε subunit of the CD3 complex). (FIG. 24B
). Ectopic expression of any ICER isoform is VP16-mediated (3 × GAL4
) -CR-CAT (168) had no significant effect on transactivation (data not shown). In addition, the FasL reporter, containing only the proximal NFAT motif, was shown to inhibit ICER-mediated repression following the high affinity of the proximal NFAT motif for ICER binding and complex formation as shown in gel shift assays (FIGS. 23 and 23B). It was found to be even more susceptible. Thus, in the down-regulation of FasL reporter transcription induced by anti-CD3ε stimulation in 2B4 T cells, ICER can be induced by and replace forskolin.

The mechanism of cAMP-mediated inhibition of FasL expression on activated T lymphocytes has been correlated with induction of cAMP-mediated potent transcriptional repressor ICER. By footprinting and electrophoretic mobility shift assays, two NFAT sites within the FasL promoter, proximal (126-144) and distal (263-2).
83) has been shown to be essential for high levels of FasL expression in T lymphocytes (173, 174). The role of NFAT in the activation of the FasL promoter was explained by the finding that activated T lymphocytes of NFATp knockout mice (175) cannot express FasL in T lymphocytes and the gld mouse model (1).
65) further enhances the finding of typical signs of congenital splenomegaly, suggesting that NFAT plays an important role in FasL activation in the T cell environment. Our observations suggest that the proximal NFA in the IL-2 promoter
Similar to the T motif (16), the proximal NFAT site of the FasL promoter
It shows that it has the ability to associate with AT DBD (181) and ICER and form an inhibitory NFAT / ICER complex. In addition, the FasL reporter, which contains only the proximal NFAT motif, can be used in transient gene transfer assays in I.
Shows strong sensitivity to CER-mediated suppression, with ICER alone or NFAT DB
Supports the ability to bind proximal NFAT in complex with D. Expression of ICER, but not high levels of intracellular cAMP elicited by forskolin
To demonstrate involvement in the observed inhibitory effects of the sL promoter, both the ICER and FasL reporters were expressed in 2B4T cells activated with anti-CD3ε antibody. Due to this transient gene transfer, a significant down-regulation of the activated FasL promoter in 2B4 T cell hybridomas resulted in different isoforms of ICER (ICERI, ICERII,
And ICERIIγ) and / or FasL reporter expression. Thus, even in the absence of high levels of intracellular cAMP, the transcriptional repressor ICER does not inhibit anti-CD in 2B4 T cell hybridomas.
Sufficient for transcriptional attenuation of FasL promoter activated by 3ε stimulation.

We have previously demonstrated that ex vivo cAMP- in contrast to immature cortical thymocytes.
Human medullary thymocytes were identified as a forskolin responsive population capable of inducing mediated ICER (167, 168). Here we selected for CD19 ^+, while the ability of forskolin to mediate early ICER induction was also maintained in a newly isolated subpopulation of human peripheral blood lymphocytes enriched for CD3 ⁺ mature T lymphocytes. It is revealed that B lymphocytes or CD56 ⁺ NK lymphocytes are present to a small extent. Despite the observation that neither NK nor B lymphocytes induce significant forskolin-mediated induction of ICER, they differ dramatically in the level of pre-existing ICER prior to forskolin treatment. T lymphocytes are mainly forskolin reactive and I
Although the CER-inducible lymphocyte population is composed, the background level of ICER in the positively selected CD3 ⁺ T lymphocytes is usually higher than the T
It seems to be higher than lymphocytes. Apart from the induction of ICER induced by CD3 ^+, another possible explanation for this observation is that large-scale expression of both T (CD3 ⁺ ) and NK (CD56 ⁺ ) cell surface markers. It is associated with a granulated lymphocyte population (189), which is present in CD3 ⁺ positively selected T cells and contributes to high background levels of ICER. Such double positive cells, which are excluded by negative selection of T cells, usually show high background levels of ICER mRNA. Nevertheless, the positively and negatively selected T cell populations contain a small number of contaminating cells, which may indirectly contribute to high levels of ICER after treatment with phorbol esters and ionomycin. (Eg, release of cAMP agonists such as prostaglandins). In contrast, phorbol esters and ionomycin do not recover ICER mRNA in T cell lines such as the 2B4 T cell hybridoma [FIG. 19A] or the Jurkat T cell line (data not shown), and the induction of ICER in such T cell lines Requires forskolin.

Unlike B cells or medullary thymocytes (167), freshly prepared human CD5
6 ⁺ NK cells showed ICER expression of significant levels prior to the forskolin treatment, at least detectable ICER specific signal in peripheral blood lymphocytes were suspended separated is observed prior to the separation of the above Some may be involved.
Importantly, the NK cell line NK3.3 and low amounts of NK92 express CD56 on the surface as well as newly isolated human NK cells (185, 186).
Although they have not been exposed to the CD56-specific antibodies used to isolate fresh human NK cells, they show high levels of ICER mRNA before forskolin treatment.

For the purposes of this study, treatment with human N
Mimics the association of stimuli involved in the Fas ligand-Fas pathway in K cells;
Leading to increased as-ligand expression, accompanied by an increased ability of NK cells in the reported cytotoxicity assay (190). Phorbol ester-mediated expression of the Fas ligand appears to be distinct from expression by NFAT.
Stoppage of CER expression correlates with derepression of Fas ligand expression. We are C
Although other factors, such as positive selection for D56 ⁺ NK cells, may contribute to the ICER-specific signal, cessation of ICER expression and subsequent up-regulation of FasL expression is due to the absence of NFAT function. Even below is consistent with the hypothesized inhibitory role of ICER in NK cells. Therefore, ICER is
Acts as a transcriptional repressor of s ligand expression, and transcription factors other than NFAT
It is considered to be effective even in an environment that is involved in a ligand expression. IC
ER can also be involved in the transcriptional regulation of an as yet unidentified transcription factor that acts in trans at the FasL promoter.

The role of ICER in cAMP-mediated transcriptional decay of the FasL promoter has been implicated in dexamethasone-mediated transcriptional decay of FasL (191) and has been demonstrated with a recently cloned glucocorticoid-inducible leucine zipper (GZ).
Very similar to (IP). This transcriptional repressor, which shows very high homology to the leucine zipper of ICER, is induced in lymphoid tissues by dexamethasone. In addition, induction of glucocorticoid-inducible leucine zipper-GZIP correlates with dexamethasone-induced transcriptional attenuation of FasL expression, and IC
Just as induction of the ER led to inhibition of forskolin-mediated AICD, A
Induced inhibition of ICD (191). Thus, signaling through a variety of agonists such as cAMP and dexamethasone, which ultimately leads to attenuated transcription of the FsaL promoter, is transmitted to T lymphocytes by induction of at least two independent transcriptional repressors -ICER and GILZ. Can be

V. Use of Substances That Modulate ICER Activity as Therapeutics or Reagents to Study Immune Cell Activity The above results demonstrate that ICER is involved in modulating immune cell activity. As discussed above, inhibition of ICER-mediated immune cell activity may be involved in the development of various clinical conditions. In particular, inhibition of the activity of ICER-mediated immune cells may contribute to the progression of symptoms such as cancer or disease caused by pathogen infection.

Thus, agents that reduce the level of ICER-mediated immune cell activity inhibition are meant to be attractive therapeutic tools for treating conditions caused or exacerbated by immune cell activity inhibition. I do.

In addition, substances that increase ICER activity mean that they are attractive therapeutic tools for treating conditions caused or exacerbated by the overactivity of immune cells. further,
Agents that reduce or enhance the level of ICER-mediated immune cell activity inhibition may be used to control the molecular mechanisms that govern immune cell activity, and the progression of disease caused or exacerbated by inhibition of immune cell activity, in vivo or in vitro. It is a useful reagent for studying or capturing features using a model.

As discussed above, tumors may have reduced immune cell activity, such as IL
-10, secretes PGE2 and other factors (17, 18), and has been shown to secrete such factors into stake cells (19-21). In addition to the experiments described above, which demonstrated the involvement of ICER in inhibiting the activity of immune cells, the involvement of ICER in inhibiting the activity of tumor-mediated immune cells may be further elucidated, as described below. . In particular, the involvement of ICER in tumor-mediated inhibition of immune cells such as, for example, T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells and antigen presenting cells has been demonstrated using the following methods. Is also good.

Example 28 Characterization of the involvement of ICER in inhibiting the activity of tumor-mediated immune cells Substance obtained from or secreted from living tumors, or dead tumor cells, substances obtained from tumor cell lysates, Alternatively, the supernatant of the tumor cells is cultured with immune system cells such as, for example, T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells, and antigen presenting cells. The immune activity of the cells is measured using standard methods such as, for example, cytokine assays (measuring secreted cytokines), proliferation assays, stimulation assays and target lysis assays.

[0155] ICER mRNA and protein are measured in cells suppressed in the above tumors to determine whether sustained ICER expression has occurred during such inhibition. In particular, the expression of ICER is measured in cells that are suppressed by the tumor in a situation where PGE2 is secreted or induced by the tumor. Various methods are used to determine whether specific factors are involved in such inhibition or sustained expression of ICER, for example, inhibitory antibodies, inhibitors of the PGE2 receptor.

The above experiments are performed in vitro or in vivo and characterize the mechanisms and factors involved in immune cell activity. The involvement of ICER in inhibiting the activity of immune cells caused by infectious agents,
The check may be performed using the method described above. As discussed above, infectious agents are known to produce factors that inhibit the activity of immune cells, such as, for example, IL-10, or cAMP stimulators (22, 23).

In such a method, the material obtained from or secreted by cells infected with the pathogen, or the material obtained from infected cells, infected cell lysate, or the supernatant of infected cells is used, for example, for T lymphocytes, Incubate with normal immune system cells such as B lymphocytes, NK cells, monocytes, dendritic cells and antigen presenting cells. The immune activity of the cells is measured using standard methods such as, for example, cytokine assays (measuring secreted cytokines), proliferation assays, stimulation assays and target lysis assays.

[0158] Immune cells whose activity is suppressed by mediated infection of ICER
MRNA and protein are determined to determine if sustained ICER expression has occurred during such inhibition. Using various methods, such as, for example, inhibitory antibodies, inhibitors of cytokine receptors, and other methods well known to those of skill in the art, which particular factor is involved in such inhibition and sustained expression of ICER Clarify if you are.

The above experiments are performed in vitro or in vivo and characterize the mechanisms and factors involved in immune cell activity. As discussed above, agents that reduce or enhance the level of ICER in immune cells are examined for mechanisms and factors involved in controlling the activity of immune cells using in vitro or in vivo model systems. It may also be used to treat conditions characterized or exacerbated by inhibition or stimulation of the activity of immune cells.

For example, various agents may be used to reduce or increase the level of ICER in immune cells such as T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells and antigen presenting cells. . Such agents include ribozymes, antisense nucleic acids, triple helices forming nucleic acids, and other factors that reduce or increase transcription from the ICER promoter.

[0161] Ribozymes are RNA molecules that have endonuclease activity. Ribozymes used to reduce ICER activity contain one or more catalytic regions capable of cleaving one or more target sites in mRNA encoding one or more ICER isoforms. Further, to provide specificity to ICER mRNA,
Ribozymes include one or more target sites that are complementary to one or more sequences in ICER mRNA.

[0162] Any type of ribozyme known to those of skill in the art may be used to reduce the level of activity of ICER. These include a nine base catalytic center, a double-stranded step loop structure, and two regions complementary to the target mRNA on either side of the catalytic center.
Hammerhead "ribozymes (Haseloff and Gerl
ach (1988) Nature 334: 585-591). Alternatively,
Ribozymes such as those described in US Pat. No. 5,116,742 may be used.

In some embodiments, a ribozyme for reducing the level of activity of ICER may have multiple catalytic domains as described in US Pat. No. 5,635,385. In a further embodiment, a ribozyme is disclosed in US Pat. No. 5,679,5.
One or more immobilized linkers may be included as described in US Pat. No. 55 and 5,650,502 to increase its stability in vivo. In another embodiment, one or more 2′-O as described in US Pat. No. 5,545,729.
-Alkylated nucleotides may be included to increase their stability in vivo.

[0164] The ability of various ribozymes to reduce the level of activity of ICER, as described below, may be In various types of immune system cells. Example 29 describes the analysis of the ability of various ribozymes to inhibit T cell activity.

Example 29 Analysis of Ability of Various Ribozymes to Inhibit T Cell Activity As discussed above, normal human T cells (such as freshly isolated peripheral blood CD4 ⁺ T cells) can Exposure to the agonist Forskolin causes ICER mRN
Expression of A and protein and treatment with a combination of ionomycin and forskolin results in sustained expression of ICER mRNA. When such T cells are treated with a cAMP stimulating substance or agonist such as forskolin or PGE2, T cells can be stimulated regardless of whether proliferation is stimulated by calcium ionophores or restimulation by specific antigens. A long block of growth occurs. Thus, the ability of a ribozyme to reduce the inhibition of ICER-mediated T cell activity can be measured by determining its ability to block the inhibition of T cells treated with forskolin, PGE2 or ionomycin and forskolin.

A series of ribozymes having different catalytic sequences capable of recognizing different cleavage sites and / or different target sequences capable of directing the ribozyme to different sites of the ICER mRNA encoding one or more ICER isoforms are known in the art. Design using technology. An IC such as a ribozyme having a marker sequence identical to one or more of the ICER mRNA type sequences so that they do not form ICER mRNA duplexes.
A ribozyme capable of cleaving ER mRNA may be used as a control. Alternatively, the control ribozyme may be a ribozyme that targets mRNA other than ICER mRNA.

[0167] The ribozyme is prepared using any technique known to those of skill in the art. In particular, ribozymes may be synthesized by conventional chemical synthesis methods. Alternatively, the ribozyme may be prepared by performing an in vitro transcription reaction with a linearized vector containing a ribozyme-encoding nucleotide sequence operably linked to a promoter. For example, a promoter suitable for an in vitro transcription reaction is a T7 promoter or an SP6 promoter.

Next, each ribozyme is introduced into T cells in a range of concentrations. The ribozyme may be introduced by any technique known to those skilled in the art. For example, a ribozyme may be introduced into a T cell with an unmodified molecule. Alternatively, using lipofection to T
Ribozymes may be introduced into cells.

The effect of each ribozyme on T cell ICER activity may be assessed using a variety of systems. One assay measures the effect of each ribozyme on T cell ICER activity by quantifying the ability of forskolin to reduce or disrupt the inhibition of T cell proliferation. In such assays, CD4 ⁺ T cells are sensitized with the ribozyme or combination of ribozymes to be screened prior to exposure to a cAMP-stimulating substance or agonist, such as forskolin. Immunoprecipitation and / or ribonuclease protection assays are performed to identify ribozymes that can potently block forskolin-induced ICER expression in such T cells. The results obtained for each screened ribozyme are compared with those of cells into which a control ribozyme has been introduced. A ribozyme that reduces the expression level or mRNA level of ICER to a statistically significant degree may be used as a therapeutic measure or as a reagent to study the molecular mechanisms governing the activity of immune cells. Preferably, the IC
Ribozymes that have reduced ER expression to a maximum are used as such therapeutics or reagents.

Inhibition of enhancer / promoter function of IL-2 gene (5) and cyclic AM
Based on the ability of ICER to inhibit diverse impairment of T cell function by P agonists (27), inhibition of ICER / cAMP-mediated T cell cytokine secretion and cAM
A ribozyme of ICER may be used to reduce or prevent inhibition of P-mediated T cell signaling. In yet another approach, standard assays for measuring T cell proliferation, cytokine release assays, signaling and target lysis assays quantify the ability to reduce or disrupt cAMP-induced suppression of T cell function. The effect of each ribozyme on the ICER activity of T cells is determined by comparison with the results obtained with a control ribozyme. ICER
Ribozymes that have reduced the expression or mRNA levels of to a statistically significant level may be used as therapeutics or as reagents to study the molecular mechanisms that govern immune cell activity. Preferably, a ribozyme with a maximally reduced expression of ICER is used as such a therapeutic measure or reagent.

Alternatively, a vector containing a nucleotide sequence encoding each test ribozyme operably linked to a promoter is introduced into freshly cultured T cells, such as CD4 ⁺ anti-TET ATUS cells. Vectors for expressing nucleic acids in immune cells include retroviral vectors (28, 29), episomal vectors, vectors that are stably integrated into the genome of the host cell, or vectors that are transiently present in the host cell. And may include any vector commonly used for DNA. That is ICER
A ribozyme having a target sequence identical to one or more ICER mRNA-type sequences so as not to form a duplex with mRNA, or a vector encoding a ribozyme targeting mRNA other than ICER mRNA is introduced into T cells as a control. IC
Immunoprecipitation or ribonuclease protection assays using ER antibodies are performed to determine the levels of ICER protein or intact ICER mRNA present in T cells containing the vector encoding each test ribozyme. The results are compared with levels in T cells containing control ribozymes. ICER expression level or mRNA
Ribozymes whose levels have been reduced to a statistically significant degree may be used as therapeutics or as reagents to study the molecular mechanisms governing the activity of immune cells. Preferably, a ribozyme with a maximally reduced expression of ICER is used as such a therapeutic measure or reagent.

In another approach, the ability of a ribozyme to inhibit the activity of T cells in vivo is measured as follows. Test and control ribozymes are introduced into T cells using any of the above methods (ie, bare nucleic acid, lipofection, and stable or transient expression vectors). T cells are then administered to tumor-bearing animals of the same line and the ability of the ribozyme to block the inhibition of the ICER-mediated immune response is measured. The results obtained for each test ribozyme are compared to the results for the control ribozyme. Ribozymes that have reduced the inhibition of the ICER-mediated immune response to a statistically significant degree may be used as therapeutics or as reagents to study the molecular mechanisms that govern the activity of immune cells. Preferably, a ribozyme that minimizes the inhibition of the ICER-mediated immune response is used as such a treatment or reagent.

For example, ribozymes may be used to reduce inhibition of ICER-mediated immune cell activity in immune cells other than T cells, such as NK cells, dendritic cells, or antigen presenting cells. A method for identifying a ribozyme that effectively reduces the inhibition of the activity of an ICER-mediated antigen presenting cell is described below.

Example 30 Method for Identifying a Ribozyme that Effectively Reduces Inhibition of ICER-Mediated Antigen Presenting Cell Activity Using the methods described above for T cells, ICER of antigen presenting cells such as monocytes or dendritic cells -Screen various ICER ribozymes to reduce or prevent mediated inhibition. Substances or agonists that stimulate cAMP, such as PGE2 or forskolin, exert a variety of inhibitory effects on antigen presenting cells (APCs), including inhibition of IL-12 secretion and inhibition of co-stimulatory molecule B7.1 upregulation. (See above). APCs, such as monocytes and dendritic cells (DCs), are stimulated with a substance that stimulates cAMP and ICE
Treat with R ribozyme or control ribozyme.

In one approach, ICER-mediated inhibition was measured by measuring PGE2 / forskolin-induced inhibition of calcium ionophore-stimulated CD80 (B7.1) expression on peripheral blood human monocytes (4 ). A ribozyme that has reduced the activity inhibition of antigen presenting cells to a statistically significant degree may be used as a therapeutic means or as a reagent for studying the molecular mechanisms governing the activity of immune cells. Preferably, a ribozyme having the lowest possible inhibition of the activity of antigen presenting cells is used as such a therapeutic means or reagent.

The ability of an ICER ribozyme to reduce or prevent ICER-mediated B lymphocyte activity inhibition may also be confirmed as follows. Example 31 Evaluation of the Ability of Various ICER Ribozymes to Reduce and Prevent ICER-Mediated Inhibition of B Lymphocyte Activity of Various ICER Ribozymes Similar experiments as described above for T cells and APCs were performed on B lymphocytes. . Induction of cAMP in B lymphocytes by counterstimulation has been shown to have a variety of inhibitory effects, including inducing B cell unresponsiveness during synchronous antigen stimulation (see above). The ICER ribozyme and control ribozyme are transiently or stably introduced into B lymphocytes treated with a cAMP agonist as described above.

Ribozymes that reduce inhibition of B cell activity to a statistically significant degree may be used in therapy or as reagents to study the molecular mechanisms that define immune cell activity. Preferably, ribozymes that reduce the inhibition of B cell activity to the greatest extent are used as therapies or reagents.

The ability of the ICER ribozyme to reduce and prevent ICER-mediated inhibition of NK cell activity may also be confirmed as follows. Example 32 Evaluation of the ability of various ICER ribozymes to reduce and prevent ICER-mediated inhibition of NK cell activity. Induction of cAMP in cultured NK lymphocytes inhibits NK targeted lysis (see above). ICER ribozyme and control ribozyme are transiently or stably introduced into NK cells treated with a cAMP agonist as described above. The degree of inhibition of targeted lysis in NK cells by cAMP agonists was
It is measured in NK cells having R ribozyme and control ribozyme.
Ribozymes that reduce the inhibition of NK cell activity to a statistically significant degree may be used in therapy or as reagents to study the molecular mechanisms that define immune cell activity. Preferably, ribozymes which reduce the suppression of NK cell activity to the greatest extent are used as treatments or reagents.

Antisense nucleic acids may also be used to reduce the level of ICER activity of immune cells. ICER antisense to various ICER isoforms has been stably transfected into various cell lines. (18, 24, 25). It has been reported that sorted cell lines can be stably transfected with constructs that result in permanent intracellular overexpression of ICER antisense (18), thereby reducing forskolin-transduction in transfected cells. Inducible ICER synthesis is blocked. Such cell lines do not die or exhibit toxicity due to ICER-antisense expression itself, but block the ICER-dependent effects of cAMP-stimulators or agonists. Thus, normal human T cells can be stably transfected with a vector that overexpresses an ICER antisense to reduce or prevent the inhibitory effect of ICER induced by cancer or other pathogens. Since some constructs block endogenous ICER induction in some cell lines (25), but not others (18), various antisense sequences may be used to reduce or prevent endogenous induction. Selection is performed to identify those that are sufficient and to determine whether each isoform has a different inhibitory capacity with certain antisense sequences.

ICER antisense nucleic acids that reduce cAMP-induced inhibition of immune cell activity of T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells and antigen presenting cells are identified as follows.

Example 33 Identification of ICER Antisense Molecules That Reduce ICER-Mediated Inhibition of T Cell Activity As described above, normal human T cells (such as freshly isolated peripheral blood CD4 ⁺ T cells) Following exposure of the agonist to forskolin, it expresses ICER mRNA and protein and preserved ICER mRNA expression appears after mixed treatment with ionomycin and forskolin. Treatment of such T cells with a cAMP-stimulator or agonist such as forskolin or PGE2 also results in proliferation being stimulated either by calcium ionophores or by specific antigenic restimulation. Significantly prolonged blockade of T cell proliferation. Thus, the ICE of the T cell activity of the antisense nucleic acid
The ability to reduce R-mediated inhibition may be measured by determining the ability to block the inhibition of T cells treated with forskolin, PGE2, or ionomycin and forskolin.

A set of nucleic acids complementary to all or a portion of one or more types of ICER mRNA is provided. In some embodiments, the antisense nucleic acid is an oligonucleotide that contains 50 or fewer contiguous nucleotides that are complementary to one or more types of ICER mRNA. In other embodiments, the antisense nucleic acids comprise at least 75 contiguous nucleotides complementary to one or more types of ICER mRNA. In a further embodiment, the antisense nucleic acid comprises one or more types of ICE
It comprises at least 100 contiguous nucleotides complementary to the R mRNA. In other embodiments, the antisense nucleic acids comprise at least 150 contiguous nucleotides complementary to one or more types of ICER mRNA. In a further embodiment, the antisense nucleic acids comprise at least 200 contiguous nucleotides complementary to one or more types of ICER mRNA. In additional embodiments, antisense nucleic acids include nucleic acids that are complementary to the entire sequence of one or more types of ICER mRNA.

[0183] In some embodiments, the antisense nucleic acid is a start codon common to all four ICER isoforms, a stop codon present in each isoform, or any present present in one or more ICER isoforms. Oligonucleotides that constitute "antisense" to various portions of the ICER gene, including other sequences. Such oligonucleotides may be synthesized using conventional techniques. In another embodiment, all isoforms of ICER consisting of a region of about 60 oligonucleotides upstream of the initiation codon are common to FER
ujimoto et al. (26) on the ICER sequence published in FIG.
Use the antisense sequence that follows through the first translated 24 oligonucleotides of the 60 to +24 ICER specific domain.

If the antisense sequence is longer than 50 bases in length, the nucleic acid encoding the antisense sequence may be linked in vitro to a vector operably linked to a promoter.
Ro may be synthesized by performing a transcription reaction. Alternatively, a vector encoding an antisense nucleic acid may be transiently or stably introduced into a T cell as described above.

The effect of each antisense nucleic acid on ICER activity on T cells may be assessed using a variety of systems. In one assay, ICER of T cells
The effect of each antisense nucleic acid on activity is determined by quantifying its ability to reduce and disrupt forskolin-induced inhibition of T cell proliferation. In such an assay, CD4 ⁺ T cells are pre-pulsed with an antisense nucleic acid or a combination of antisense nucleic acids prior to exposure to a cAMP-stimulator or antagonist such as forskolin. Immunoprecipitation and / or RN
Ase protection assays are performed to identify those antisense oligonucleotide sequences that can strongly block forskolin-induced ICER expression in these T cells. The results obtained for each selected antisense nucleic acid are compared to the results for cells with the control antisense nucleic acid. Antisense nucleic acids that reduce ICER expression levels or mRNA levels to a statistically significant degree may be used for therapy or as a reagent to study the molecular mechanisms that define immune cell activity. Preferably, an antisense nucleic acid that reduces ICER expression to the greatest extent is used for therapy or as a sample.

Similar to the reverse deficiency of cyclic AMP agonists of other T cell functions (27), IL-2
Based on the ability of ICER to inhibit enhancer / promoter function of genes (5
), ICER antisense also reduces ICER / cAMP-mediated inhibition of T cell cytokine secretion, as well as cAMP-mediated inhibition of T cell signaling;
Or it may be used to prevent it. In yet another approach, the effect of each antisense nucleic acid on T cell ICER activity is determined using standard assays to measure T cell proliferation, cytokine release assays, signaling and target lysis assays, and The results obtained with antisense nucleic acids are compared and determined by quantifying their ability to reduce and disrupt cAMP-induced suppression of T cell function. Antisense nucleic acids that reduce ICER expression levels or mRNA levels to these statistically significant degrees
It may be used for therapy or as a reagent to study the molecular mechanisms that define immune cell activity. Preferably, an antisense nucleic acid that reduces ICER expression to the greatest extent is used for therapy or as a sample.

Alternatively, a vector containing the nucleotide sequence encoding the respective test antisense nucleic acid operably linked to a promoter may be prepared using freshly cultured C
Introduce into T cells such as D4 ⁺ anti-TETANUS T cells. Vectors express nucleic acids in immune cells, including retroviral vectors (28, 29), episomal vectors, vectors stably linked into the genome of the host cell, or vectors transiently present in the host cell. Any vector conventionally used to do so may be included. As a control, a vector encoding a sense nucleic acid or an antisense nucleic acid targeting an mRNA other than the ICER mRNA is introduced into T cells. Immunoprecipitation with an ICER antibody or RNAse protection assay is performed to determine the level of ICER protein or intact ICER mRNA present in T cells containing the vector encoding the respective test antisense nucleic acid. . The results are compared to levels in T cells with control antisense nucleic acids. ICER expression levels, or mRNs, to a statistically significant degree
Antisense nucleic acids that reduce A levels may be used for therapy or as a reagent to study the molecular mechanisms that define immune cell activity. Preferably, an antisense nucleic acid that reduces ICEG expression to the greatest extent is used for therapy or as a sample.

In another approach, the ability of an antisense nucleic acid to suppress T cell activity in vivo is determined as follows. Test and control antisense nucleic acids are introduced into T cells using any of the methods described above (ie, intact nucleic acids, lipofection, and stable or transient expression vectors). T
The cells are now administered to syngeneic mice with cancer to determine the ability of the antisense nucleic acid to inhibit ICER-mediated inhibition of the immune response. The results obtained from each test antisense nucleic acid are compared to the results obtained with the control. Antisense nucleic acids that reduce ICER-mediated inhibition of the immune response to a statistically significant degree may be used for therapy or as a reagent to study the molecular mechanisms that define immune cell activity. Preferably, an antisense nucleic acid that reduces ICER-mediated suppression of the immune response to the greatest extent is used for therapy or as a sample.

Antisense nucleic acids can also be used to reduce ICER-mediated suppression of immune cell activity in immune cells other than T cells, such as B cells, NK cells, dendritic cells, or antigen presenting cells. Methods for identifying antisense nucleic acids that effectively reduce ICER-mediated inhibition of antigen presenting cell activity are described below.

Example 34 Identification of ICER Antisense Molecules That Decrease ICER-Mediated Inhibition of Antigen Presenting Cell Activity Various ICER antisense nucleic acids can be isolated from monocytes or dendritic cells using the procedures described above for T cells. Reduce ICER-mediated inhibition of antigen presenting cells of
Screened for ability to prevent. CAM such as PGE2 and Forskolin
P-stimulants or antagonists exert various inhibitory effects on antigen presenting cells (APCs), including inhibition of IL-12 secretion and inhibition of co-stimulatory molecule B7.1 up-regulation (see above). It was shown to have. APCs such as monocytes and dendritic cells (DC) are stimulated with a cAMP stimulant and treated with an ICER antisense nucleic acid or antisense nucleic acids as described in the T cell assay above.

In one approach, ICER-mediated inhibition may be measured by measuring PGE2 / forskolin-induced inhibition of calcium-ionophore-stimulated CD80 (B7.1) expression on peripheral blood human monocytes. (4). Antisense nucleic acids that reduce the inhibition of antigen presenting cell activity to a statistically significant degree may be used for therapy or as a reagent to study the molecular mechanisms that define immune cell activity.
Preferably, an antisense nucleic acid that reduces the inhibition of antigen presenting cell activity to the greatest extent is used for therapy or as a sample.

The ability of an ICER antisense nucleic acid to reduce or prevent ICER-mediated suppression of B lymphocyte activity may also be confirmed as follows. Example 36 ICE reducing or preventing ICER-mediated inhibition of B lymphocyte activity
R Evaluation of Activity of Various ICER Antisense Nucleic Acids The same experiment as described above for T cells and APCs is performed on B lymphocytes. Induction of cAMP in B lymphocytes by counterstimulation is exemplified by others with various inhibitory effects, including induction of B cell unresponsiveness during co-stimulation (see above). Have been. ICER antisense and control nucleic acids are transiently or stably introduced into B lymphocytes treated with a cAMP antagonist as described above.

Antisense nucleic acids that reduce the inhibition of B cell activity to a statistically significant degree may be used for therapy or as a reagent to study the molecular mechanisms that define immune cell activity. Preferably, an antisense nucleic acid that reduces the inhibition of B cell activity to the greatest extent is used for therapy or as a sample.

The ability of an ICER antisense nucleic acid to reduce and prevent ICER-mediated inhibition of NK cell activity may be confirmed as follows. Example 37 Evaluation of the ability of ICER various ICER antisense to reduce or prevent ICER-mediated inhibition of NK cell activity Induction of cAMP in cultured NK lymphocytes inhibits NK targeted lysis (see above) . ICER antisense nucleic acids and control nucleic acids were transiently or stably introduced into NK cells treated with a cAMP agonist as described above. The degree of inhibition of targeted lysis in NK cells by cAMP is determined in NK cells with ICER antisense and control nucleic acids. Antisense nucleic acids that reduce the inhibition of NK cell activity to a statistically significant degree may be used for therapy or as a reagent to study the molecular mechanisms that define immune cell activity. Preferably, an antisense nucleic acid that reduces the inhibition of NK cell activity to the greatest extent is used for therapy or as a sample.

As discussed above, agents that reduce the level of ICER activity in immune cells include
It may be used as a treatment to ameliorate or cure conditions that are the result of, or worsened by, ICER-mediated inhibition of immune cell activity. For example, agents that reduce the level of ICER activity of immune cells may be used to treat individuals who are infected with a tumor or pathogen. Alternatively, agents that increase the level of ICER activity may be used therapeutically to ameliorate or cure conditions that result from, or are exacerbated by, increased levels of immune cell activity. Such conditions include arthritis and lupus.

[0196] In some of the above therapeutic embodiments, the immune cells are obtained from an individual suffering from the condition to be treated. Agents that reduce or increase the level of ICER activity in immune cells are introduced into immune cells using any gene therapy technique known to those of skill in the art. For example, substances can be introduced using techniques that introduce nucleic acids themselves into cells, lipofection techniques, transfection techniques, or using vectors that encode substances that are transiently or stably contained in host cells. I do. The substance can be a ribozyme, an antisense nucleic acid, a composition that up-regulates or down-regulates the transcription of the ICER gene, or a composition that regulates the level of ICER protein in immune cells. After introducing the substance into the immune cells, the cells are reintroduced into the individual.

In another embodiment of the above treatment, the substance is introduced into the immune cells while the immune cells are in the individual suffering from the condition to be treated. The substance may be introduced as an intact nucleic acid, in the form of lipid vesicles, or via a vector encoding a substance that is stably or transiently contained in the host cell. The substance may be a composition that up-regulates or down-regulates the transcription of ribozymes, antisense nucleic acids, ICER genes, or a composition that regulates the level of ICER protein in immune cells.

Example 38 describes infection of tumors or pathogenic microorganisms, or ICE of T cell activity.
IC for reducing ICER-mediated inhibition of T cell activity in an individual suffering from or otherwise exacerbated by R-mediated inhibition
It describes the use of ER ribozymes or antisense nucleic acids.

Example 38 Use of Antisense to Reduce or Prevent ICER-Mediated Inhibition of T Cell Activity in Patients With Tumors T lymphocytes are infected with tumors or pathogenic microorganisms, or T cell activity ICER
-Isolate from patients suffering from any other condition that is the result of or worsened by mediated inhibition. T lymphocytes are either CD4 ⁺ T cells, CD8 ⁺ T cells, or both (eg, because the patient has already received a cancer vaccine to activate and increase the development of such subpopulations), Subpopulations with anti-cancer specificity and function may be included. T cells are grown in culture for several days or weeks,
Induced by various stimuli such as superantigen or autoantigen pulsed dendritic cells (DC) or by the addition of cytokines such as rhIL-2. During this culture, T cells are transfected with a retroviral vector containing sequences that produce ICER antisense, ICER ribozyme, or other substances that, when expressed in transduced cells, reduce ICER activity. Recent advances in transfection technology have allowed gene insertion success rates of up to 50% of the total lymphocyte population (28, 29). Proliferate lymphocytes to an appropriate number to ensure stable gene transfer, and administer lymphocytes as adoptive therapy to patients with tumors. Transformed lymphocytes are resistant to inducing sustained ICER protein synthesis in the tumor environment, and thus have distinctly improved anti-cancer functions.

A “suicide gene” such as the herpes simplex-thymidine kinase (HS-tk) gene may be co-transfected with an ICER antisense or ICER ribozyme vector to allow for the elimination of subsequently transduced lymphocytes. . Suicide genes can kill transduced T cells containing HS-tk by treating patients with ganciclovir (28).

Example 39 shows that in an individual suffering from a tumor or pathogenic microbial infection, or any other condition resulting from or exacerbated by ICER-mediated inhibition of monocyte or dendritic cell activity. , Monocyte or dendritic cell activity ICER
-Describes the use of ICER ribozymes or ICER antisense nucleic acids to reduce mediated inhibition.

Example 39 Use of Antisense to Reduce or Prevent ICER-Mediated Inhibition of Monocyte and Dendritic Cell Activity in Patients with Tumors Peripheral blood monocytes (that of both macrophages and dendritic cells Culturing monocytes, macrophages and DCs (such as CD34 ⁺ bone marrow cells) or infecting tumors or pathogenic microorganisms, or ICER-
Recover from patients with any other condition that is the result of or worsened by mediated inhibition and produces ICER antisense, ICER ribozyme, or a substance that reduces ICER activity when expressed in transduced cells Culture for a short time to achieve stable transfection with the retroviral vector containing the desired sequence. "Suicide genes", such as the herpes simplex-thymidine kinase (HS-tk) gene, to allow removal of later transduced bone marrow cells (28)
May be co-transfected (see above). The cells are re-administered to the patient, potentially providing a self-sustained precursor storage of antigen presenting cells that resists sustained inhibition of ICER protein synthesis in the cancer environment, and thus its potential for effective antigen presentation Increase power.

The above procedure is also the result of ICER-mediated inhibition of immune cell activity, or otherwise in patients suffering from an exacerbated condition, of other immune cells including B cells, NK cells and antigen presenting cells. This may be done to introduce into the mold a substance that reduces ICER-mediated inhibition.

In addition, the above procedures may be used to treat conditions that are the result of, or have been exacerbated by, increased levels of immune cell activity. In such a procedure, a vector encoding one or more ICER isoforms or a substance that increases ICER activity is introduced into an individual suffering from a condition as described above.

[0205] Agents that decrease or increase ICER activity may also be used as reagents to study molecular mechanisms and pathways that define immune cell activity. For example, in addition to cAMP agonists, many substances, including IL-10 and glucocorticoids, inhibit cells of the immune system (see above). In addition, cancer cells and infectious agents can (1) produce putative inhibitors such as prostaglandins and gastric L-10 (see above), and (2) induce putative inhibitors in host cells (see above). See), (3) the production of currently unidentified factors, and (4) the induction of currently unidentified factors, which may inhibit cells of the immune system. Therefore, I
A substance that decreases the activity of CER may be introduced into immune cells to determine whether the substance's ability to inhibit immune cell activity is reduced or eliminated. Alternatively, agents that increase ICER activity may be introduced into immune cells and their ability to reduce or prevent further inhibition of immune cell activity may be measured.

For example, in vitro or in vivo analysis as described above is performed using r
The inhibitory effects of substances such as IL-10, glucocorticoids, or cancer-derived substances on cells of the immune system, such as T cells, B cells, NK cells and APCs, are also ICER
This may be done to determine if it is blocked by an antisense nucleic acid or an ICER ribozyme. Cells treated with rIL-10, glucocorticoid, or a substance derived from cancer are designed to add an ICER antisense nucleic acid or ribozyme, or to stably express the ICER antisense nucleic acid or ribozyme as described above. The level of inhibition of immune cell activity is measured in cells with the substance and compared to that observed in cells with control nucleic acids to identify factors that inhibit immune cell activity by ICER.

A protein susceptible to sustained ICER expression under conditions where cytokine expression is inhibited by ICER, hereinafter referred to as RINSR (repressor of ICER negative autoregulation (RINSR)) Or R
The nucleic acid encoding the INSR protein (s) is isolated as follows.

Example 40 Detection and Isolation of RINSR A standardized technique such as differential display, continuous gene expression analysis (SAGE), and / or Gene Calling ™ (Curagen Corp.) can be used to generate multiple stimulations that result in sustained expression of ICER. For example, in the reaction to forskolin and ionomycin) to identify uniquely synthesized mRNAs / proteins. mRNAs or proteins are
-It is synthesized in response to such a combined stimulus rather than any stimulus that may constitute a factor that is responsive to suppress negative autoregulation. The gene encoding such a protein is cloned using standard procedures and the protein and mRNA sequence are compared to whether they are identical to the predicted mRNAs / protein, or if they are not yet identified regulatory molecules. Test to determine if you are presenting RINSR antisense sustained RINS
The antisense intervention therapy used to block R expression is now designed.

Example 41 Reduction of ICER activity using RINSR antisense As described above, ICER autoregulation inhibits its self-
It may be inhibited by RINSR, a protein that protects the ER. Subsequent to the identification and cloning of RINSR as described above, cells such as T cells, B cells, NK cells, monocytes, dendritic cells or other APCs may be treated with cAMP agonists and RINSR antisense or control as described above. Treat with any of the ribose sense nucleic acids. ICER-mediated inhibition of immune cell activity was reduced or prevented in cells with RINSR antisense, but not in cells with control nucleic acids.

[0210] ICER antisense may be used to reduce or prevent ICER-mediated suppression of immune cell activity in patients where immune cell activity is suppressed by cancer or pathogens. Representative examples of the use of ICER antisense as a therapy to reduce or prevent ICER-mediated inhibition of immune cell activity are described below. However, the ICER antisense has an immune cell activity of I
It will be appreciated that it may be used as a therapy to treat any condition that is inhibited by CER.

[0211] It will be appreciated that strategies other than ICER antisense or ICER ribozyme may be employed to reduce and prevent ICER-mediated suppression of immune cell activity. Such strategies include ICER gene transcription, ICER mRN
Any method of inhibiting or reducing the translation of A or the activity of the ICER protein is included. For example, in one such approach, peptides that block or reduce the activity of ICER or RINSR may be used.

Although the present invention has been described in terms of certain preferred embodiments, other embodiments that will be apparent to those of skill in the art in light of the present disclosure are also within the contemplation of the present invention. All text cited herein, including the references listed below, are hereby incorporated by reference in their entirety.

[0213]

[Table 2]

[0214]

[Table 3]

[0215]

[Table 4]

[0216]

[Table 5]

[0219]

[Table 6]

[0218]

[Table 7]

[0219]

[Table 8]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the ICER gene (SEQ ID NO: 1).

FIG. 2 shows the structures of four ICER transcription isoforms.

FIG. 3 shows ICER expression patterns in T cells treated with ionomycin, forskolin, or ionomycin plus forskolin.

FIG. 4 shows ICER expression patterns in monocytes treated with forskolin, prednisolone, IL-10, GM-CSF, and ionomycin.

FIG. 5 shows the inhibitory effect of forskolin treatment on the proliferation of T cells in contact with various substances that normally cause proliferation.

FIG. 6 shows ICER expression patterns in monocytes whose ionophore-induced differentiation is inhibited by forskolin.

FIG. 7. Cyclic AMP-mediated attenuation of T helper 1 cytokine transcription in human medullary thymocytes correlates with cAMP-mediated induction of the transcriptional repressor ICER. Unprocessed (U)
RNA from medullary thymocytes and human medullary thymocytes treated with forskolin (F) or 8-bromo-cAMP (Br) contains phorbol ester and ionomycin (PMA +
lono) was assessed in the absence (lanes 2 and 3) or presence (lanes 5 and 6) of cytokine expression in a ribonuclease protection assay using RIBOQUANT probes hCKJ (A) and hCK3 (B). When ribonuclease is present (+
The corresponding ribonuclease protected lobes after hybridization with yeast tRNA in () (lane 7) or in the absence (-) (lane 8) are also shown. Templates for analysis of hL32 and hGAPDH housekeeping genes were included to allow assessment of total RNA levels (Pharmingen)
. Each probe (lane B) moves at a slower speed than the protected band. This is due to the side sequence of the probe not protected by the mRNA. (C) Western immunoblot analysis was performed using an ICER-specific antiserum raised against a peptide surrounding the ICER-specific exon (38), which was induced in medullary thymocytes 3 hours later. 12 for forskolin treated
This indicates that it was not induced after hours. There is no detectable ICER protein in forskolin-treated human cortical thymocytes corresponding to the previously reported loss of detectable ICER mRNA in pre-treatment or cortical thymocytes (36).

FIG. 8. ICER binding and forming NFAT / ICER complex with NFAT / AP-1 is IL
-2 Synthesize the DNA binding site of the promoter. (A) IL-2 expression used in EMSA analysis (63)
List of NFAT / AP-1 complex sites of the IL-2 promoter previously shown to be essential for E. coli. N
FAT and AP-1 (top) are the NFAT / AP-1 complex and NFAT in the human IL-2 promoter
And the regions of homology between the consensus sequences of AP-1 and AP-1, respectively. The number on the left corresponds to the relative distance of the indicated DNA binding motif from the TATA box of the IL-2 promoter. (B) ICER expressed by bacteria binds specifically to (-90) (lanes 3 and 4) and (-160) CD28RE motifs (lanes 7 and 8) in boiled whole bacterial lysates, and the rest of the motif To a limited extent. The binding of ICER to these motifs is specific, as it is recognized by the CS4 CRME-specific antiserum (CS4), which causes a specific supershift (sICER), while the non-specific complex (NS) binds to CS4 ( It remains undisturbed in the presence of both (+) and normal rabbit antiserum (-). The control CRE consisted of an oligonucleotide containing a 21 bp repeat of the HTLV-1 LTR (H21CRE) (64). (C) In vitro binding of purified recombinant ICER and NFAT DBD (NFAT) protein was performed using CD28RE motif (460) (lane
12) NFAT on and to a lesser extent on the NFAT-45 motif (lane 3)
/ ICER produces a ternary complex (NF / IC). ((D) ICER and truncated Fos and Jun proteins (AP) form a similar complex (NF-IC vs. NF / AP) in the presence of NFAT DBD (NFAT) on the (-160) motif of the IL-2 promoter. Formed (lanes 3 and 9), which are recognized and restricted by CS4 anti-CREM (C) (lane 4) and DX anti-Fos (D) (lane 10) or K25 anti-Jun (K) (lane 11). To a lesser extent, they are also recognized by the R59NFAT (R) antiserum (lane 12.) The unlabeled oligonucleotide NFAT (nf) (lane 12)
6 and 13) and AP-1 (ap) (lanes 7 and 14) both compete effectively for the complex.

FIG. 9. ICER protein can interact directly with NFAT DBD.
Decreasing amounts of NFAT DBD protein retained on a Sepharose matrix
GST-linked ICER (GST-ICER, lanes 1,3,6) and a specific interaction in a GST pull-down assay with a negative control represented by an equal amount of GST matrix alone (GST, lanes 2,4,7) Works. Lane 5 shows the G held in the GST pull-down in lane 3.
Represents the input of NFAT DBD equal to protein added to ST-ICER beads. In contrast, GST-CREB (lane 10) does not interact with comparable amounts of NFAT DBD protein (input, lane 8). Lane 9 represents only GST-CREB beads. NFAT D
BD was retained on GST-ICER Sepharose beads, but was separated by SDS-PAGE and visualized by Western blot analysis using NFAT-specific antibody R59.

FIG. 10. ICER binds to a conserved proximal element of the IFNγ promoter homologous to the (-90) motif of the IL-2 promoter. (A) ICER binds to both human and mouse motifs of the IFNγ promoter (lanes 2 and 9), but these motifs are similar to the IL-2 promoter (-90) motif, NFATDBD (NFAT). (Lanes 3 and 10) cannot be combined. No complex formation between ICER and NFAT was detected by EMSA (lanes 4 and 11). ICER bound to a proximal element of the IFNγ promoter has been specifically “supershifted” (sICER) by the CS4CREM-specific antibody (C) (lanes 5 and 12). The mobility of the ICER complex is unlabeled CRE oligonucleotide (cre)
(Unlabeled NF) altered using competition with (lanes 8 and 13)
It cannot be changed in the presence of AT oligonucleotide (nf) (lanes 7 and 14). (B) IFN
The sequences of the human and mouse proximal elements of the gamma promoter show significant homology to the (-90) motif of the human IL-2 promoter.

FIG. 11. ICER shows several Ns in GM-CSF, IL-4 and TNF-a promoters.
Form a complex on the FAT / AP-1 complex site. (A) NFAT / AP-1 complex previously identified as essential for expression of GM-CSF (54), IIL-4 (52), and TNF-α (55,56) promoters used in EMSA analysis List of parts. (B) Purified ICER and truncated NFATDBD protein are expressed in vitro with the enhancer core of the GM-CSF promoter GM-420 (lane 6), which has an affinity comparable to the CD28RE motif (-160) of the IL-2 promoter (FIG. 8). Form an NFAT / ICER complex on the proximal NFAT / AP-1 motif of the IL-4 promoter (IL4 (-80) (lane 12) and the κ3 motif of TNF-α (κTNF-α) (lane 15) . (CE) GM-CS
The NFAT / AP-1 complex site of the F, IL-4 and TNF-α promoters binds to ICER and forms an ICER-containing complex in whole cell extracts obtained from freshly prepared human medullary thymocytes. Purified ICERII, expressed by bacteria, and full-length NFATLL protein expressed out of place in Cos cells were obtained from untreated human medullary thymocytes (C
) Or extracts prepared from human medullary thymocytes treated with forskolin (3 hours) and ionomycin (15 minutes) (D, E) (lanes 3-11)
Serve as lanes 1 and 2) respectively. Complexes containing ICER and ICER were expressed by CRE motif (cre) (lanes 4, 7, 10) or NFAT motif (nf) (lanes 5, 8, 11) (panel C).
And D), and were immunoreactive with the CS4CREM-specific antiserum (C) (panels E, lanes 4, 7, 10), while the R59NFAT-specific antiserum (R) (lanes) 5,8,11) affected the mobility of a number of complexes but had little effect on ICER (panel E) binding.

FIG. 12. ICER isoform II represses transcription from NFAT / AP-1 activating cytokine promoter stimulated by PMA and ionomycin (P + 1) treatment. (
A) Promoter reporter, IL-2-CAT (human interleukin-2 (61) CD28RE (-160A
P-Luc); GM-CSF-CAT (54) (human granulocyte-macrophage colony stimulating factor), TN
F-α-Luc (having human tumor necrosis factor-a sequence from -614 to +20 in pGL2)
. Control (3 × GAL4) -CR-CAT (HTLV-1 LTR, three GAs replacing the (64) 21 bp repeat)
(With L-4 binding site) is transactivated by GAL4VP16 (73) and is unaffected by the isoforms of ICER (ICER II, ICER IIy, ICER 1) in Jurkat cells. (B) The amount of each cytokine reporter and ICER expression construct in transient transfection was kept constant (2 μg). The error bars represent standard deviations calculated from three or more experiments.

FIG. 13. Peripheral blood T lymphocytes can induce stable ICER protein by treatment with forskolin (F) or PGE ₂ (P). Immunoprecipitation of all ³⁵ S-labeled cell lysates using CREM-specific antiserum (C) and normal rabbit serum (N) was performed using forskolin (0.1 mM final).
) (F3, F12, F18) or PGE ₂ (500ng / ml final) treatment (P3, P12, P18) for 3 hours, after performing 12 hours and 18 hours, the ICER protein in peripheral blood T lymphocytes accumulation It indicates that you have done. ICER protein is hardly detectable in untreated (U) negatively selected T cells (lanes 1 and 2), but both treatments (lane 3 respectively)
And 4 and lanes 9 and 10) are clearly detectable 3 hours after, first after 12 hours in the presence of forskolin (lanes 5 and 6) and then after treatment in the presence of PGE ₂
Reached the maximum in 18 hours.

FIG. 14. Forskolin-mediated attenuation of cytokine and hemokine expression in human peripheral blood T lymphocytes such as IL-12-induced Th1- and IL-4-induced Th2 correlates with induction of the strong transcriptional repressor ICER. is there. In vitro polarized T cell populations with a dominant Th1 or Th2 phenotype were evaluated by flow cytometric analysis (A). T lymphocytes (containing 95% or more CD3 ⁺ cells) are shown, which are typical of those obtained after priming in vitro and conferring a Th1 or Th2 dominant phenotype. Was something. Non-CD3 ⁺ cells are CD14 ⁺ (Th1
1%, <1% for Th2), CD19 ⁺ (1% for Th1 and 2% for Th2), and CD16 ⁺ (2% for Th1 and 5% for Th2). Primed PBMC as described previously were maintained in IL-2 containing media until restimulation. In the experiments shown, cells were cultured for a total of 2 weeks before restimulation. The Th1 and Th2 populations shifted significantly toward the memory phenotype represented by the CD45RO marker. In the Th1 population, 57% of CD4 ⁺ and 35% of CD8 ⁺ cells maintained the same proportion of CD4 ⁺ versus CD8 ⁺ after polarization as before the polarization (data not shown). Th2 dominant culture is 60% CD
4 ⁺ and it contained 14% of the CD8 ⁺ cells. RNAs from the dominant populations of IL-12-induced Th1 and IL-4-induced Th2 are in the absence (−) or presence (
+) Restimulated with PHA in any of the above, human cytokine (B) and hemokine expression
(D) was simultaneously evaluated using a ribonuclease protection assay in parallel with ICER mRNA (C and E). For evaluation of human cytokine (b) expression, the hCK1 probe (hIL-5, hIL-10, hIL-14, hIL-15, hIL-9, hIL-2, hIL5) of the riboquant set was used.
IL-13 and hIFN-γ) were used, but for evaluation of human hemokine (D) expression, the hCK5 probe of the riboquant set (hLth, hRANTES, hIP10, hMIP1β, hMIP1α, hMCP1, hIL- B, hI-309) (Pharmingen) was used. The level of cytokine or hemokine expression of activated Th1 and Th2 lymphocytes was inversely correlated with ICER mRNA (C and E). Templates for analysis of hL32 and hGAPDH housekeeping genes were included to assay total RNA levels (Pharmingen). Note that each probe moves at a slower speed than the protected band. This is due to sequences located laterally in the probe that are not protected by the mRNA.

FIG. 15. Differential sensitivity of cytokine expression in a predominant subset of Th1 and Th2 to cAMP-mediated inhibition after restimulation with phorbol esters and either ionophore (P + I) or phytohemagglutinin (PHA). Human peripheral blood T-lymphocytes primed with OKT-3 and polarized in the presence of IL-12 (Th1) or IL-4 (Th2) have a P + without or with forskolin. Restimulated with I or PHA for 6 hours. After restimulation, the RNAs were probed for the hCK1 probe (hIL-5, hIL-4, hIL-10, hIL-14, hIL-15, hIL-9, hIL-2,
hIL-13, and hIFN-) (Pharmingen) and the JL5 probe (74), respectively, were evaluated for cytokine and ICER expression in a ribonuclease protection assay. In addition, the corresponding ribonuclease-protected probe was used in the presence of ribonuclease (lane 13) or without ribonuclease (lane 14).
The state after hybridization with RNA is shown. Includes templates for analysis of hL32 and hGAPDH housekeeping genes and can be used to assay total RNA levels (Pharmingen, San Diego, CA). Note that each probe moves at a slower speed than the protected band. This is due to sequences located laterally in the probe that are not protected by the mRNA.

FIG. 16: ICER-mediated transcriptional attenuation of cytokine and hemokine expression in activated lymphocytes from ICER transgenic mice. Freshly isolated
ICER transgenic (+) or control non-transgenic (-) thymocytes were activated with PMA + ionomycin for 3 hours. RNAs were isolated and the respective mCK1 probes (mIL-4, mIL-5, mIL-10, mIL-13, mIL-15, mIL-9, mIL-2, mIL1)
IL-6, mINF-γ) or mCK5 probe (mLtN, mRANTES, mEotaxin, mMIP-1β, mMI
Not only ICER expression but also cytokine (A) or hemokine production (B) using the riboquant set of P-1α, mMIP-2, mIP-10, mMCP-1, mTCA-3) (Pharmingen)
Were simultaneously analyzed in a ribonuclease protection assay. Also shown are the corresponding ribonuclease protection probes after hybridization with yeast RNA in the presence of ribonuclease (lane 4) or without ribonuclease (lane 5). Templates for analysis of hL32 and hGAPDH housekeeping genes are included and can be assayed for total RNA levels (Pharmingen, San Diego, CA). Note that each probe moves at a slower speed than the protected band. This is due to flanking sequences in the probe that are not protected by the mRNA.

FIG. 17. Proliferative deficiency in lymphocytes from ICER transgenic mice
Fall. (A) Freshly isolated thymocytes from ICER transgenic littermates or control non-transgenic littermates are isolated,³⁶S-labeled whole cell lysate was
Uptake by immunoprecipitation using CREM-specific antiserum (c) with normal rabbit serum (n).
I was killed. Proliferation of spleen cells (B) activated for 48 hours with ConA DE, PMA + ionomycin (PMA + ionopha) or immobilized anti-CD3 monoclonal antibody (2C11) ^Three Measured by H-thymidine incorporation. (C) ICER transgenic or non-
Response of allogeneic genotype to BALB / c spleen cells of allogeneic genotype in the reaction of mixed lymphocytes of C57BL / 8 spleen cells from transgenic control mice.
Irradiated, unsorted spleen cells from genetically syngeneic C57BL / 6 or allogeneic genotype BALB / c spleen cells can be converted to unsorted ICER transgenic or
Was added to the medium containing non-transgenic C57BL / 6 spleen cells. After 48 hours of co-culture, thymidine incorporation was measured as described. (D) of cells obtained from the thymus and spleen of transgenic or non-transgenic animals.
yield.

FIG. 18. The ICER protein is induced in freshly isolated human peripheral blood CD3 ⁺ T lymphocytes in an early response to forskolin, but to a lesser extent in CD19 ⁺ B lymphocytes. Immunoprecipitation using CREM-specific antiserum (C) (116) and normal rabbit antiserum (N) was separated after 3 hours of forskolin (F) treatment (0.1 Mm final) (lanes 1-4). This indicates that ICER protein (17-20 kDa) was induced in lymphocytes that did not exist. Untreated (U) actively selected C with various isoforms of ICER protein
It is detectable in D3 ⁺ T cells (lanes 5-8), indicating that the positively selected CD1
9 ⁺ B cells (lanes 13-16) and / or their respective passages (CD3 ⁺ PT, CD19 ⁺ PT)
(Lanes 9-12 and 17-20, respectively), indicating a higher background level. Purification of freshly isolated CD3 ⁺ T cells and CD19 ⁺ B cells was assessed by subsequent flow cytometric analysis. The background level of the ICER protein was specific, indicating that control immunoprecipitation of untreated Jurkat T cells (lanes 21 and 22) yielded any detectable ICER protein, consistent with the lack of detectable ICER mRNA. Although not released, ICER mRNA (FIG. 22B, lane 2) was observed after forskolin treatment.
(See lanes 23 and 24) as well as the ICER protein (lanes 23 and 24). Similarly, not only B cells (CD19 ⁺ ) (lanes 13-16) but also their passing population (CD19 ⁺ PT)
The immunoprecipitations in (lanes 17-20) show that after forskolin treatment, they induce moderate but detectable ICER protein compared to untreated cells.

FIG. 19. Forskolin-mediated down-regulation of FasL correlates with inducing ICER in activated 2B4 T cell hybridomas. Phorbol ester treated in the presence of forskolin (0.1 Mm final) (P + 1 + F) or without forskolin (P + I) or in the presence of forskolin alone (F) for the indicated time ( RNA from a 2B4 T cell hybridoma activated with 105 mg / ml) and ionomycin (1 g / ml) was tested for ICER expression (A) or FasL expression (B) in a ribonuclease protection assay using ICER or mAPO3 riboquant probe. Either was evaluated in parallel. Under these conditions, 2B4 T cells (FLICE, FaasL, Fas,
(FADD, FAP, FAF, Fas2L, TNFRp55, TRADD, and RIP) FasL expression as assayed by ribonuclease protection used for the evaluation of mRNAs of various components of the TNF pathway (B) After that it was changed dramatically. Activated phorbol ester-ionomycin treated 2B4 T cells were treated with forskolin alone (F) (lane 18) after 3 hours (P + I + F) (lane 6) in the presence of forskolin. Induces ICER to a similar or somewhat higher level compared to. Only phorbol ester ionomycin treatment (P + I) (lanes 7-12) induced minimal detectable ICER mRNA. B, shows that forskolin-mediated ICER induction is inversely correlated with FasL expression. The same RNAs evaluated for ICER induction in panel A underwent ribonuclease protection in panel B using the mouse riboquant probe mAPO3. After 3 hours of combined treatment (P + I + F), F
The minimum amount of asL message was detected (lane 8). Although forskolin-mediated attenuation of FasL expression in activated 2B4 cells (panel B; lane 6) is strongly correlated with forskolin-mediated induction of the transcriptional repressor ICER (panel A, lane 6), FasL expression is ICER (P + I) in activated 2B4 cells (panel B, lane
In the absence of 12), it remains undisturbed. Also, the appearance after hybridization of the corresponding ribonuclease protection probe with yeast tRNA in the presence of ribonuclease (+) (lane 19) or without (-) (lane 20) without ribonuclease is shown. Includes templates for analysis of mL32 and mGAPDH housekeeping genes, allowing for assay of total RNA levels (Pharmingen, San Diego, CA). Note that each probe moves at a slower speed than the protected band. This is due to flanking sequences in the probe that are not protected by the mRNA.

FIG. 20. Downregulation of FasL correlates with increased ICER expression in human peripheral blood T lymphocytes activated after forskolin treatment. The purity of negatively selected human peripheral blood T cells (PanT) was assessed by flow cytometry analysis. Freshly prepared RNAs from human peripheral blood T lymphocytes were treated with phorbol ester and ionomycin (P + I) in the presence of forskolin (F) or without forskolin (U) for 3 hours and FasL Alternatively, evaluation was performed in parallel using a ribonuclease protection assay using the hAPO3 riboquant probe or the ICER probe for evaluation of ICER mRNA, respectively. Activated human peripheral blood T cells have reduced levels of FasL message in the presence of forskolin (P + I / F) (top, lane 1) after phorbol ester and ionomycin treatment (P + I). , ICER (middle, lane 1). Conversely, in the absence of forskolin (P + I / U), T cells activated by phorbol esters and ionomycin increase the level of FasL message (A, lane 2) and ICERm
It was revealed that the level of RNA (middle, lane 2) was decreased. Untreated cells (U) (lane 4) have no Fasl and very few ICER messages. Templates for analysis of hL32 and hGAPDH housekeeping genes (bottom) are included and total RNA levels can be assessed (Pharmingen).

FIG. 21 Induces FasL expression in phorbol ester-activated human peripheral blood NK lymphocytes.
Doing so involves cessation of ICER expression. Eluted peripheral blood lymphocytes were used and CD58 ⁺ The NK cell population was isolated (Miltenyl Biotec, Auburn, CA). CD58⁺The purity of the NK cell population was assessed by flow cytometry analysis (see Experimental Methods). RNAs obtained from either untreated (U) and 1-hour (P1) or 3-hour (P3) phorbol ester-treated cells were obtained using the ribopent probe hAPO3 (Pharmingen, San Diego, CA) (A) or ICER ( It was evaluated using B). Also, mRNA levels were elevated for hFAP and hTRADD, and the corresponding ribonuclease-protected hAPO3 probe was hybridized with yeast tRNA in the presence of ribonuclease (+) (lane 3) or without (-) (lane 4) ribonuclease.
The state after turning on is shown. hL32 and hGAPDH housekeeping genes
Includes template for analysis, allowing assay of total RNA levels. Each probe (lanes 1 and 2) was obtained from its protected band (lane 4).
Move at a slow speed. This is in the probe not protected by mRNA
In the array located on the side of the.

FIG. 22. High levels of ICER can be detected in human peripheral blood CD56 ⁺ NK as well as human NK cell lines NK3.3 and NK92 prior to forskolin treatment. A. CREM identification Immunoprecipitation using antiserum (C) or normal rabbit antiserum (N) indicated the presence of ICER protein in untreated CD56 ⁺ NK cells (U) (lane 2) and a 3 hr folder. Shows a moderate increase after scolin (F) (lane 4) treatment. ICER protein is detectable in untreated CD56 ⁺ NK cells prior to forskolin treatment. B. RNAs from the human NK cell lines NK92 and NK3.3 were evaluated in a ribonuclease protection assay using an ICER probe for ICER mRNA before and after 3 hours of forskolin treatment (lanes 3-6). NK3.3 and, to a lesser extent, the NK92 human NK cell line also express high levels of ICER mRNA before forskolin treatment in contrast to the JurkatT cell line, which does not express detectable amounts of ICER mRNA before forskolin treatment. Indicated (lane
1)

FIG. 23. I of NFAT / ICER complex with NFAT motif proximal to FasL promoter.
CER binding and formation. A. The list of NFAT motifs of the FasL promoter, pre-drawn to be essential for NFAT-induced FasL expression in T lymphocytes (171,172), is IL-2
Control NFAT / AP-1 complex sites and GM-CS that can bind ICER either directly or indirectly
Used in electromigration assay analysis with the F promoter (166). NEAT and
AP-1 (top) is homologous between the NFAT / AP-1 site in human Fas ligand and sequences matching NFAT, and between NFAT and AP-1 sites in IL-2, and in each of the GM-CSF promoters The area of the relationship is shown. The numbers on the left correspond to the relative distance of the delineated DNA binding motif from the transcription start site. B. Purified, bacterially expressed ICER specifically binds to the proximal NFAT motif of the Fas ligand promoter (lanes 1 and 2) and to a lesser extent to the distal NFAT motif (lanes 3 and 4). There are but combine. The binding of ICER to these motifs is specific, it is CS4CRE
It is recognized by the M antiserum (CS4) and causes a specific "supershift" of ICER (sICER). C. In vitro binding of the purified recombinant ICER to the NFAT DBD (NFAT) protein produces an NFAT / ICER3 complex (NF / IC) on the proximal NFAT motif of the FasL promoter (lane 3) but a distal motif. It does not produce above (lane 8). The control NFAT / AP-1 complex is the oligonucleotide surrounding the (-160) position of the human IL-2 promoter (lanes 7-9) and the (-420) position of the human GM-CSF promoter (lanes 10-12). That bind ICER directly and / or NFAT / ICER3 complex (NF / I
C) (166). Free indicates that no probe is included.

FIG. 24. ICER inhibits transcription of a human FasL promoter activated by a 2B4 T cell hybridoma anti-CO3ε antibody (clone 2C11) from a luciferase reporter. The hFASl (-225) Luc reporter contains a proximal NFAT binding site (from -144 to -126). The hFasL (-511) Luc reporter is proximal (-144 to -126) and distal (-144).
-283 to -263) contains both NFAT sites (panel A). Both the mock transfection and the control pG13 empty Luc reporter (pG13 empty Luc) transfection alone show significant luciferase activity in 2C11 stimulated 2B4 T cells (panel B). Various isoforms of human ICER (ICERII, ICERIIγICERI and ICE
RIγ) has a differential inhibitory effect depending on the FasL reporter used (Panel B)
. The amount of each FasL reporter and ICER expression component in transient transfections was kept constant (2 μg). Error bars represent SD values calculated from three or more experiments. Control reporter (3xGAL4) -CR-CAT transactivated by GAL4VP16 (35) (having three GAL-4 binding sites replacing the 21 base pair repeat in the human T-cell lymphotropic virus type 1 long terminal repeat) (34) was not affected by ICER (10) (data not shown).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/04 A61P 37/04 C12N 5/10 C12N 5/00 B 15/09 ZNA 15/00 ZNAA (81 ) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA ( AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, U, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Bodol, Joseph United States of America Maryland 20852 Rockville College Parkway 443 (72) Inventor Weng, David, E . United States Ohio 44116 Rocky River Yacht Club Drive 221 (72) Inventor Koski, Gary, K. United States Maryland 20814 Bethesda Battery Lane 4918 Apartment No. 2 (72) Inventor Chernicky, Brian, J. United States New Jersey 08033 Haddenfield Tues Landing Road 77 (72) Inventor Bodlova, Jana Maryland, USA 20852 Rockville College Parkway 443 F-term (reference) 4B024 AA01 BA07 CA04 DA02 DA06 EA04 GA11 GA19 HA01 HA12 4B065 AA26X AA92X AA93Y AB01 AC14 BA02 BD35 CA24 CA27 CA44 4C084 AA13 AA17 ZB092 ZB262 ZB322 4C086 AA01 AA02 EA16 MA01 MA04 ZB09 ZB26 ZB32

Claims (42)

[Claims] 1. A substance for decreasing the expression level of ICER for increasing the activity of immune cells. 2. The substance according to claim 1, comprising a nucleic acid. 3. The substance according to claim 1, wherein the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA. 4. The substance of claim 1, wherein the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. 5. Use of a substance that reduces the expression level of ICER in the manufacture of a medicament for increasing immune cell activity. 6. Use according to claim 5, wherein the substance comprises a nucleic acid. 7. The use according to claim 5, wherein the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA. 8. The use according to claim 5, wherein the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a part of at least one isoform of ICER mRNA. 9. Use according to claim 5, wherein the medicament is for treating cancer. 10. Use according to claim 5, wherein the medicament is for the treatment of an infection by a pathogenic organism. 11. The use according to claim 5, wherein the immune cells are selected from the group consisting of T cells, B cells, NK cells, monocytes, dendritic cells, and antigen presenting cells. 12. Use according to claim 5, wherein the immune cells are T cells. 13. A ribozyme comprising at least one catalytic sequence having endonuclease activity and at least one targeting sequence, wherein at least one targeting sequence is present in at least one isoform of ICER mRNA. A ribozyme that is complementary to a sequence. 14. An immune cell into which a substance capable of reducing the expression of ICER has been introduced. 15. The immune cell according to claim 14, wherein the substance comprises a nucleic acid. 16. The nucleic acid is a ribozyme comprising at least one catalytic sequence having endonuclease activity and at least one targeting sequence, wherein at least one targeting sequence is at least one of ICERmRNA in an immune cell. 16. The immune cell of claim 15, comprising a ribozyme that is complementary to a sequence present in one isoform. 17. The immune cell of claim 15, wherein said nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. 18. The immune cell according to claim 14, wherein the immune cell is selected from the group consisting of a T cell, a B cell, an NK cell, a monocyte, a dendritic cell, and an antigen presenting cell. 19. The immune cell according to claim 18, wherein the immune cell is a T cell. 20. A method for increasing immune cell activity, comprising reducing ICER expression levels in immune cells. 21. The method according to claim 20, wherein the level of ICER expression is reduced by introducing a nucleic acid that inhibits the expression of ICER into immune cells. 22. The nucleic acid according to claim 21, wherein the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA.
The described method. 23. The method of claim 21, wherein the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. 24. The method according to claim 21, wherein the immune cells are selected from the group consisting of T cells, B cells, NK cells, monocytes, dendritic cells, and antigen presenting cells. 25. A method for increasing the immune cell activity of an individual, comprising the steps of removing immune cells from the individual, introducing a substance that inhibits ICER expression into the immune cells, and reintroducing the immune cells into the individual. A method that includes 26. The method according to claim 25, wherein the substance comprises a nucleic acid. 27. The nucleic acid according to claim 26, wherein the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA.
The described method. 28. The method of claim 26, wherein the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. 29. The method according to claim 25, wherein the immune cells are selected from the group consisting of T cells, B cells, NK cells, and antigen presenting cells. 30. The method according to claim 29, wherein the immune cells are T cells. 31. The method of claim 25, wherein the individual is in a condition where immune cell activity is reduced. 32. The method of claim 25, wherein the individual is infected with a pathogenic organism. 33. The method of claim 25, wherein said individual has cancer. 34. A method for increasing the immune cell activity of an individual, comprising introducing a substance that inhibits the expression of ICER into immune cells of the individual. 35. The method according to claim 34, wherein the substance comprises a nucleic acid. 36. The nucleic acid according to claim 35, wherein the nucleic acid comprises a ribozyme capable of cleaving at least one site in at least one isoform of ICER mRNA.
The described method. 37. The method of claim 35, wherein the nucleic acid comprises an antisense nucleic acid comprising a sequence complementary to at least a portion of at least one isoform of ICER mRNA. 38. The method according to claim 34, wherein the immune cells are selected from the group consisting of T cells, B cells, NK cells, and antigen presenting cells. 39. The method according to claim 38, wherein the immune cells are T cells. 40. The method of claim 34, wherein the individual is in a condition in which immune cell activity is reduced. 41. The method of claim 34, wherein said individual is infected with a pathogenic organism. 42. The method of claim 34, wherein said individual has cancer.