Although an inhibitory function of IL-4 in CD4 T cell IL-2 production has long been recognized, a mechanism mediating the inhibition remains unclear. In this study we demonstrate that IL-4 displays a potent suppressive function in IL-2 production of activated CD4 T cells through STAT6. IL-4-induced IL-2 suppression required IL-2 because IL-2 neutralization restored the production of IL-2 even in the presence of IL-4. In vivo, enhanced IL-2 production was found in nematode-infected IL-4- or STAT6-deficient animals, whereas immunization in the presence of IL-4 substantially diminished IL-2 production by Ag-specific CD4 T cells. IL-2 mRNA expression was reduced when T cells were stimulated in the presence of IL-4, whereas IL-2 mRNA decay was unaltered, suggesting that IL-4 mediates the suppression at a transcriptional level. Blimp-1 induced by IL-4 stimulation in activated CD4 T cells was found to be necessary to mediate the IL-2 inhibition as IL-4-mediated IL-2 suppression was less pronounced in activated CD4 T cells deficient in Blimp-1. Taken together, our results demonstrate a potential link with IL-4, Blimp-1, and IL-2 production, suggesting that Blimp-1 may play an important role in controlling IL-2 production in activated T cells and in adaptive T cell immunity.
Interleukin-4 has been primarily considered as a key differentiation factor that promotes the Th2 differentiation of naive CD4 T cells. However, inhibitory roles of IL-4 have been noticed. In macrophages or LPS-stimulated monocytes, IL-4 was shown to inhibit the production of TNF-α, IL-1β, and IL-12 (1, 2), and to suppress expression of FcγR and of metalloproteinase (2, 3). IL-4 was also demonstrated to inhibit expression of κ L chain and NF-κB activation in murine pre-B cells (4). In mast cells, IL-4 inhibited TNF-α production and FcεRI expression (5, 6). IL-4 also performs inhibitory roles in T cells. The most well-characterized inhibition elicited by IL-4 includes the inhibition of IFN-γ production during Th2 differentiation. A transcription factor, GATA-3, selectively expressed in Th2 cells by IL-4, plays crucial roles in down-regulating cytokine genes associated with Th1 differentiation (7). Th2 phenotype CD4 T clones are poor IL-2-producing cells (8), suggesting IL-4-mediated IL-2 suppression in CD4 Th cells. Indeed, IL-4 itself suppresses IL-2 production in primary CD4 T cells (9).
Mechanisms of IL-4-mediated suppression were examined. IL-4-mediated suppression of TNF-α in mast cells was mediated in part by inducing tristetraprolin (TTP),3 which binds to the AU-rich element located in the 3′ untranslated region (UTR) of the TNF-α mRNA and promotes mRNA degradation (6). In support of this observation, the inhibition of TTP expression by RNA interference prevented IL-4-mediated suppression of TNF-α production (6). IL-4-mediated suppression of IL-2 production in Jurkat cells was caused in part by inhibiting factors required for IL-2 transcription (10). Nonetheless, mechanisms involved in IL-4-mediated IL-2 suppression in primary T cells remain unexplored.
Blimp-1 (B lymphocyte-induced maturation protein-1) is a transcription repressor that plays a critical role in terminal differentiation of B cells into Ab-secreting plasma cells (11). Blimp-1 contains five consecutive Kruppel-like zinc finger domains through which Blimp-1 binds to target DNA sequences (12), and the proline-rich domain of Blimp-1 is believed to recruit other proteins involved in gene repression (13, 14). Ectopic expression of Blimp-1 was sufficient to induce phenotypic changes that are associated with plasmacytic differentiation (15, 16). Roles of Blimp-1 in T cells have recently been examined (17). Blimp-1 is highly expressed in a subset of effector/memory T cells of both CD4 and CD8 lineages. Studies using mice with a T cell specific deletion generated by a Cre-loxP recombination or by a fetal liver cell reconstitution demonstrated that Blimp-1 plays a crucial role in controlling T cell homeostasis (18, 19). Blimp-1 deficiency in T cells resulted in enhanced proliferation and in lethal multiorgan inflammatory disorders. Furthermore, cytokine production, particularly IL-2 and IFN-γ, of Blimp-1-deficient T cells was also significantly enhanced, suggesting that Blimp-1 may play an important role in controlling T cell effector functions (18, 19). In support of this suggestion, Blimp-1 was shown to directly suppress IL-2 production in T cells (20).
Based on these results, we examined whether IL-4 stimulation in CD4 T cells induces expression of Blimp-1 and whether Blimp-1 is involved in IL-4-mediated suppression of IL-2. We found that IL-4 inhibits IL-2 production in activated naive CD4 T cells both in vitro and in vivo and that Blimp-1 was dramatically induced when T cells were stimulated in the presence of IL-4. IL-4-mediated IL-2 suppression was significantly abrogated in CD4 T cells deficient in Blimp-1, suggesting that Blimp-1 is an underlying mediator of IL-2 suppression when naive CD4 T cells are activated in the presence of IL-4.
Materials and Methods
B10.A 5C.C7 TCR transgenic (Tg) Rag2−/− mice and BALB/c G4/G4 mice (21) were obtained from Dr. W. E. Paul (National Institutes of Health, Bethesda, MD) and bred in the animal facility of the Lerner Research Institute (Cleveland, OH) and of Malaghan Institute of Medical Research (Wellington, New Zealand). STAT6−/− mice were obtained from Taconic Farms. Blimp1GFP/+ mice were obtained from Dr. S. Nutt (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia). BALB/c, OT-II TCR Tg C57BL/6, CD45.1 C57BL/6, and C57BL/6 mice were purchased from The Jackson Laboratory. All experimental procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee.
T cell stimulation
CD4 T cells from 5C.C7 TCR Tg Rag2−/− mice express αβ TCR-specific for cytochrome c peptide in association with I-Ek MHC class II molecule. Negative isolation of CD4 T cells were previously reported (22). In brief, lymph node cells were labeled with FITC-conjugated anti-CD8, anti-B220, anti-I-Ek, anti-FcγR, anti-NK1.1, and anti-CD24 Abs (all purchased from eBioscience). Subsequently, cells were incubated with anti-FITC microbeads (Miltenyi Biotec) and then passed through an LS column (Miltenyi Biotec). Purity after isolation was routinely >97%. P13.9 cells, fibroblast lines that stably express I-Ek, CD80, and CD54 were provided from Dr. R. Germain (National Institutes of Health, Bethesda, MD) (23). CD4 T cells (5 × 105 cells/well) were cultured with P13.9 cells (1 × 105 cells/well) plus 0.1 μM cytochrome c peptide in the presence or absence of IL-4 (1000 U/ml). In some experiments, 10 μg/ml neutralizing anti-IL-2 Ab (BD Pharmingen) was included in the culture. To measure cytokine production, activated T cells were harvested and restimulated with PMA (10 ng/ml) and ionomycin (1 μM) for 4 h. Monensin (2 μM) was added during the last 2 h of restimulation. Cells were then fixed, permeabilized, and stained for CD4 and intracellular cytokine IL-2. Stained cells were analyzed using a FACSCalibur with FlowJo software (Tree Star). In some experiments requiring naive CD4 T cells, lymph node CD4 T cells were further sorted based on CD44low phenotype using a FACSAria cell sorter. Naive CD4 T cells were then cocultured with irradiated T-depleted splenocytes together with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) Abs. Where indicated, cells were restimulated on immobilized anti-CD3/CD28 Abs for 24 h and cytokine production was determined by ELISA. All the Abs for the ELISA were purchased from BD Pharmingen.
Quantitative real-time PCR
RNA was obtained from harvested T cells using an RNeasy kit (Qiagen) and then subsequently treated with DNase. cDNA was further generated using random hexamers and a Superscript III reverse transcriptase (Invitrogen). Expression of IL-2 and Blimp-1 was determined using a 7500 real-time PCR system (Applied Biosystems). IL-2- and Blimp-1-specific primers and TaqMan probe were purchased from Applied Biosystems. In some experiments, 5 μg/ml actinomycin D (Sigma-Aldrich) was added in the culture. IL-2 expression, particularly primary IL-2 transcript, was measured as previously described (24, 25). Primers that span exon or intron junctions of the IL-2 primary transcript were designed as follows: exon 1/intron 1 (sense) 5′-AGCAGCTGTTGATGGACCTA-3′ and (antisense) 5′-TAAACACAGCCTTTGGCAAG-3′; intron 2/exon 3 or (sense) 5′-GGTGAGCTGAGCTGATGGT-3′ and (antisense) 5′-TGAGTCAAATCCAGAACATGC-3′; exon 4/3′ UTR (sense) 5′-TTGAGTGCCAATTCGATGAT-3′ and (antisense) 5′-GCCTTATGTGTTGTAAGCAGGA-3′. Amplification was determined using a SYBR Green Master mix as described.
Confocal microscope analysis
CD4 T cells stimulated with or without IL-4 were mounted on a polylysine-coated coverslides and fixed in 4% paraformaldehyde. Cells were permeabilized with PBS-3% BSA/0.3% Triton X-100 and stained with rat anti-mouse Blimp-1 (Santa Cruz Biotechnology) followed by Alexa Fluor 568 goat anti-rat IgG (Invitrogen), FITC anti-CD4, and DAPI (4′,6′-diamidino-2-phenylindole). Stained cells were visualized with a Leica TCS-SP spectral laser scanning confocal microscope. Reconstitutions of acquired images were performed by using Photoshop software (Adobe Systems).
Fetal liver cell reconstitution
Blimp1GFP/+ heterozygous mice were intercrossed and fetal liver cells were isolated from E14.5 embryos and genotyped as previously described (26). GFP homozygous (Blimp-1-deficient) fetal liver cells (3 × 106) were i.v. injected into lethally irradiated C57BL/6 recipients. Mice were analyzed for reconstitution 5 wk posttransfer. Before experiments, blood cells were analyzed for Blimp-1 genotype by PCR as previously described (26). Naive CD4 T cells were isolated by cell sorting and subsequently used for in vitro experiments.
Nippostrongylus brasiliensis infection
Mice were s.c. injected with 600 L3 third-stage larvae Nippostrongylus brasiliensis. Mesenteric lymph nodes were harvested 5 and 10 days after infection and stimulated on immobilized anti-CD3/CD28 Abs for 6 h. IL-2 production was determined by intracellular cytokine staining.
In vivo immunization
OT-II TCR Tg CD4 T cells specific for OVA peptide were adoptively transferred into CD45.1 B6 recipients (3 × 106 cells/recipient). The recipients were subsequently implanted with a miniosmotic pump (Durect) containing either OVA protein (100 μg) alone or OVA protein plus IL-4 (106 U). Mice were sacrificed at 7 days postimplantation. Lymph node cells were ex vivo stimulated with PMA plus ionomycin. IL-2-producing CD45.2+ donor CD4 T cells were then determined by FACS analysis.
IL-4-producing Th2 type CD4 T cells are poor IL-2 producers
We recently reported that naive CD4 T cells stimulated with Ag in coculture with basophils differentiate into IL-4-producing Th2 effector cells without addition of exogenous IL-4 (27). While investigating the phenotypes of the resulting Th2 type cells, we observed that the Th2 effector CD4 T cells were poor IL-2 producers while acquiring the capacity to produce Th2 effector cytokines such as IL-4, IL-10, and IL-13 upon restimulation (supplemental materials).4 In contrast, CD4 T cells stimulated without basophils or with IL-4-deficient basophils failed to produce Th2 cytokines but preserved IL-2-producing capacity (supplemental material). Given that IL-4, directly involved in Th2 differentiation, has long been known to suppress IL-2 production in activated T cells (9), these results prompted us to propose the hypothesis that IL-4 induces a factor that negatively control IL-2 production in activated CD4 T cells.
IL-4 directly suppresses IL-2 production in CD4 T cells
To examine how IL-4 suppresses IL-2 production in CD4 T cells, we used in vitro culture system using 5C.C7 TCR Tg CD4 T cells specific for cytochrome c peptide in context of I-Ek (28). 5C.C7 TCR Tg mice are in Rag2−/− background so that the majority (>98%) of peripheral CD4 T cells express naive phenotype (CD44lowCD62Lhigh). Lymph node CD4 T cells were purified and stimulated for 3 days with cytochrome c peptide plus P13.9 fibroblast cells that are stably transfected with CD80 and I-Ek as surrogate APCs (29). IL-2 production was measured by intracellular cytokine staining following ex vivo stimulation with PMA plus ionomycin. As shown in Fig. 1,A, IL-2 production dramatically decreased as exogenous IL-4 added to the stimulation culture increased. An 80∼90% of CD4 T cells stimulated without IL-4 were IL-2+, whereas only 10∼20% of CD4 T cells were IL-2+ when 1000 U/ml IL-4 was added. When IL-2 production was daily determined by ELISA from the culture supernatant, IL-2 in the supernatant peaked at 48 h poststimulation and decreased thereafter (Fig. 1,B). When IL-4 was included in the culture, similar levels of IL-2 were initially found at 24 h after stimulation but IL-2 levels gradually decreased, and little IL-2 was found in the supernatant at 72 h poststimulation, suggesting that T cells may have consumed all the IL-2 produced earlier without further producing it (Fig. 1,B). This notion was better demonstrated when CD4 T cells activated for 3 days were restimulated for 24 h on immobilized anti-CD3/CD28 Abs, and IL-2 production was measured by ELISA. The presence of IL-4 during the primary stimulation completely abolished the capacity to produce IL-2 following restimulation (Fig. 1,C). Given that no IL-4 was included during the secondary restimulation, these results strongly suggest that IL-2-producing capacity of CD4 T cells appears to be determined by the presence of IL-4 during primary stimulation. Indeed, stimulation of naive CD4 T cells in the presence of IL-4 for 24 h was sufficient to down-regulate IL-2 production. Thus, these T cells displayed poor IL-2 production even after subsequent 3-day restimulation in the absence of IL-4 (Fig. 1,D). Approximately 82% of T cells prestimulated without IL-4 for 24 h became IL-2-producing cells after subsequent 3-day restimulation without IL-4. In contrast, fewer than 5% of CD4 T cells prestimulated with IL-4 for 24 h and restimulated without IL-4 for 3 days became IL-2-producing cells. Next, we tested whether STAT6 is required for IL-4 to mediate the inhibition. CD4 T cells isolated from either wild-type or STAT6 KO mice were stimulated for 3 days with soluble anti-CD3/CD28 Abs and irradiated T-depleted splenocytes. The presence of IL-4 during the stimulation significantly suppressed IL-2 production by wild-type CD4 T cells (Fig. 1 E). The IL-4-dependent suppression was not observed in STAT6 KO CD4 T cells, indicating that STAT6 is needed for IL-4 to mediate the inhibition of IL-2. Similar levels of inhibition were also found when CD8 T cells from either wild-type or STAT6 KO mice were stimulated with IL-4 (data not shown). Taken together, these results suggest that the presence of IL-4 during early stimulation is sufficient to subsequently program activated T cells not to produce IL-2.
IL-4 is a potent inhibitor of IL-2 production in CD4 T cells
Previous studies reported that other cytokines including IL-12 and IL-27 also limit IL-2 production during Th1 differentiation (30, 31). We thus examined whether these cytokines play inhibitory roles in the current experimental system. 5C.C7 naive CD4 T cells were stimulated with cytochrome c peptide and P13.9 cells in the presence of the indicated cytokines for 3 days and IL-2 production was examined. Consistent with the previous studies, both IL-12 and IL-27 displayed moderate inhibition in T cell IL-2 production (Fig. 2,A). Similarly, TGF-β inhibited IL-2 production but the inhibition was relatively marginal compared with other cytokines. However, none of these cytokines were as potent as IL-4 in down-regulating IL-2 production by CD4 T cells. No significant IL-2 suppression was found in response to IL-7, IL-13, and IFN-γ. Although IL-2 has been demonstrated to down-regulate its own production through a negative feedback mechanism (20, 30), we found no evidence of IL-2 inhibition when exogenous human or mouse IL-2 was included in the culture (Fig. 2,A and data not shown). As IL-2-induced IL-2 inhibition in vitro was observed when CD4 T cells were stimulated with anti-CD3 Ab (or together with anti-CD28 Ab) (20, 30), it is possible that the mode of stimulation might account for the discrepancy. In support of this possibility, when 5C.C7 CD4 T cells were stimulated with soluble anti-CD3/CD28 Abs with T-depleted splenocytes instead, both IL-4 and IL-2 became suppressive (Fig. 2,B). The inhibition mediated by IL-2 was comparable to that by IL-4. Furthermore, polyclonal CD4 T cells stimulated with soluble anti-CD3/CD28 Abs produced less IL-2 when either IL-4 or IL-2 was included (Fig. 2 C). Therefore, IL-2-mediated suppression appears to be influenced by the mode of activation, whereas IL-4-mediated suppression seems more potent regardless of the mode of activation.
IL-2 is necessary for IL-4 to inhibit IL-2 production
Despite that IL-2 itself is not suppressive when 5C.C7 CD4 T cells were stimulated with Ag presented by P13.9 cells (shown in earlier experiments and Fig. 3,A), IL-4-mediated suppression of IL-2 production requires IL-2 to mediate the inhibition. When both IL-4 and neutralizing anti-IL-2 Ab were added in the culture, IL-2 production was significantly restored (Fig. 3, A and B). Consistent with the previous report (32), neutralizing IL-2 did not alter proliferation pattern of CD4 T cells determined by CFSE (data not shown). Although exogenous addition of human IL-2 itself up to 100 U/ml failed to diminish IL-2 production, addition of human IL-2 into the culture in the presence of anti-mouse IL-2 Ab regained the suppression in IL-2 production in a dose-dependent manner (Fig. 3,A). Consistent with previous reports (30), IL-12 suppressed IL-2 production, and the suppression was also abolished by IL-2 neutralization (Fig. 3 C). Taken together, these results suggest that IL-2 synergizes IL-4 (or IL-12) to achieve a maximum inhibition in IL-2 production, although IL-2 itself fails to mediate the inhibition in the current system.
IL-4 suppresses IL-2 production in vivo
To test whether IL-4-mediated IL-2 suppression occurs in vivo, we conducted two in vivo experiments. First, mice were infected with N. brasiliensis, an intestinal nematode that induces a robust type 2 immunity. CD4 T cells of N. brasiliensis-infected mice display typical Th2 phenotypes associated with IL-4 production (33). IL-2-producing CD4 T cells in the draining lymph nodes were determined by FACS analysis at the peak of the responses (day 10 postinfection). Approximately 20% of CD4 T cells from infected BALB/c mice expressed intracellular IL-2, whereas IL-4 deficiency resulted in significant increases in IL-2-expressing CD4 T cells as demonstrated in N. brasiliensis-infected IL-4-deficient G4/G4 mice (Fig. 4,A). Similarly, infection of STAT6−/− mice also increased the proportion and total number of IL-2-producing CD4 T cells. Because STAT6−/− mice fail to expel the parasites (34), it may be possible that the high parasite Ag loads may elevate Ag specific IL-2-producing CD4 T cells. However, this possibility is unlikely because IL-4-deficient mice expel the parasites as wild-type mice (34) and increased IL-2 production is still found (Fig. 4,A). The total number of IL-2-producing CD4 T cells was subsequently enumerated at different time points following N. brasiliensis infection. As shown in Fig. 4,B, an ∼3- to 4-fold increase in the number of IL-2+ CD4 T cells was found in N. brasiliensis-infected IL-4-deficient (G4/G4) or in STAT6−/− mice. These results strongly suggest that endogenous production of IL-4 seems to negatively control IL-2 production by CD4 T cells. Second, CD4 T cells were in vivo primed in the presence of exogenous IL-4 to see whether exogenous IL-4 suppresses IL-2 production. OT-II CD4 T cells specific for OVA were adoptively transferred into naive CD45.1 B6 recipients. The recipients were subsequently immunized with OVA alone or OVA plus IL-4 using a s.c. miniosmotic pump. CD4 T cell IL-2 production was then examined at day 7 postimmunization. As shown in Fig. 4 C, CD4 T cells from OVA immunized mice expressed substantial IL-2 (∼20%). In contrast, CD4 T cells from mice immunized with OVA plus IL-4 showed significant reduction in IL-2 production (∼5%). Taken together, these results demonstrate that IL-4, either endogenous or exogenous, could suppress IL-2 production by activated CD4 T cells.
IL-4 does not alter IL-2 mRNA stability
We next sought a mechanism by which IL-4 elicits inhibition in IL-2 production. IL-2 mRNA expression in CD4 T cells stimulated in the presence or absence of IL-4 was determined. Fig. 5,A shows kinetics of IL-2 mRNA expression determined by real-time PCR analysis within 24 h after stimulation. IL-2 mRNA expression peaked at 4 h after stimulation, and the levels gradually decreased thereafter. IL-2 mRNA expression in the presence of IL-4 showed similar kinetics, although ∼2-fold reduction was found at the peak of the response. IL-2 expression in the presence of IL-4 was ∼5-fold lower when measured at 24 and 48 h after stimulation. The difference became ∼100-fold at 72 h after stimulation (Fig. 5 B).
Because IL-2 mRNA contains the AU-rich elements located within the 3′ UTR, and is rapidly degraded through the action of AU-rich element binding proteins such as TTP (35), we next examined whether IL-4 precipitates IL-2 mRNA degradation. Indeed, it was previously shown that IL-4 suppresses TNF-α production in murine mast cells by promoting mRNA degradation via TTP (6). It is possible that IL-4 induces TTP, which then rapidly enhances mRNA instability and degradation. To test this possibility, we measured IL-2 mRNA level by real-time PCR following the addition of actinomycin D to block a new transcription. The presence of IL-4 did not alter the kinetics of IL-2 mRNA decay when measured 44 h poststimulation, indicating that IL-4 does not appear to contribute to the IL-2 mRNA degradation (Fig. 5,C). Similar results were also found when measured 20 and 68 h poststimulation (data not shown). Consistent with these findings, we found no differences of TTP expression in the presence of IL-4 (data not shown). To further assess levels of IL-2 transcription we measured primary IL-2 transcript. The primary transcript is an early intermediate RNA transcribed from genomic DNA including sequences of both exons and introns and is rapidly processed into mature mRNA through multiple processes involving capping and splicing. Measuring the primary transcripts has been successfully used to evaluate transcriptional activation (24, 25). Thus, we designed primers that span exon or intron junctions so that the primary transcripts but not matured mRNA be amplified. RNA samples were also treated with DNase before cDNA synthesis. As shown in Fig. 5 D, PCR products that span exon 1/intron 1 and intron 2/exon 3 regions rapidly disappeared following the actinomycin D treatment. By contrast, PCR products that span exon 4/3′ UTR were relatively stable. Taken together, the presence of IL-4 did not alter the kinetics of degradation of the primary IL-2 transcript, strongly suggesting that IL-4-mediated inhibition of IL-2 production in CD4 T cells is not the result of enhanced mRNA decay.
IL-4 treatment induces expression of transcription repressor Blimp-1
Recently, it was demonstrated that expression of Blimp-1 is induced by IL-2 signaling in activated T cells and inhibits IL-2 production (20). This result prompted us to test whether Blimp-1 is induced in CD4 T cells stimulated with IL-4 and whether it plays a role in IL-2 inhibition. To test this possibility, CD4 T cells stimulated with Ag in the presence or absence of IL-4 were tested for Blimp-1 expression by real-time PCR. Consistent with previous reports (18, 19), Ag stimulation induced Blimp-1 expression compared with nonstimulated CD4 T cells (Fig. 6,A). The expression level was dramatically elevated when IL-4 was included (Fig. 6,A). Given that IL-2 can induce Blimp-1 (20) and that anti-IL-2 Ab restores IL-2 production even in the presence of IL-4 (Fig. 3,A), it is possible that Blimp-1 expression by IL-4 requires IL-2. Indeed, Blimp-1 expression was significantly diminished when IL-4 plus anti-IL-2 Ab was included in the culture (Fig. 6,B). However, it should be noted that IL-4 itself induces ∼5-fold increases in Blimp-1 expression even in the presence of anti-IL-2 Ab (Fig. 6,B). IL-4-induced Blimp-1 expression was further confirmed by confocal microscopic examination (Fig. 6 C). These results suggest that Blimp-1 induced in collaboration of IL-4 and IL-2 may be involved in suppressing IL-2 production in CD4 T cells.
IL-4 fails to suppress IL-2 production in CD4 T cells-deficient in Blimp-1
We next determined whether Blimp-1 is directly involved in IL-4-mediated IL-2 suppression. As Blimp-1 deficiency is embryonic lethal, we reconstituted lethally irradiated B6 mice with fetal liver cells from Blimp1GFP/GFP (Blimp-1-deficient) mice as previously reported (26). The fetal liver reconstitution approach allows us to obtain Blimp1GFP/GFP T lymphocytes. Reconstitution was confirmed by PCR analysis before experiments. Because Blimp-1 deficiency results in substantial expansion of effector and memory phenotype T cells (18, 19), FACS sorted CD44low naive CD4 T cells were used. As shown in Fig. 6, D and E, stimulation of naive CD4 T cells from Blimp1+/− fetal liver cell reconstituted mice in the presence of IL-4 significantly reduced IL-2 production as determined by intracellular staining. In contrast, the reduction of IL-2 production by IL-4 was less pronounced in naive CD4 T cells isolated from Blimp1GFP/GFP reconstituted mice. Unlike previous studies showing that Blimp-1 deficiency in CD4 T cells cause elevated production of IL-2 and IFN-γ (19), we reproducibly found that IL-2 production of Blimp1+/− and Blimp1GFP/GFP CD4 T cells was comparable when stimulated in the absence of IL-4 (Fig. 6 D). Despite that the difference between these studies is unclear, these results strongly suggest that Blimp-1 is responsible for the IL-4 induced IL-2 suppression in activated CD4 T cells.
The present study presents a novel mechanism by which IL-4 mediates suppression in CD4 T cell IL-2 production by enhancing expression of Blimp-1, a transcriptional repressor that targets IL-2. IL-4/Blimp-1-dependent suppression requires IL-2 to achieve the inhibition as IL-2 neutralization significantly abolishes the suppression elicited by IL-4. Therefore, IL-2 initially produced from activated CD4 T cells binds to IL-2R on activated T cells, which subsequently induces Blimp-1 expression and then suppresses IL-2 as recently demonstrated by Gong and Malek (20). Our results indicate that the presence of IL-4 during the stimulation dramatically enhances Blimp-1 expression and potentiates IL-2 suppression.
IL-2 suppression through Blimp-1 appears to operate at a transcription level, namely Blimp-1 induced by TCR/IL-4/IL-2 stimulation translocates into the nucleus where it binds to the IL-2 promoter and represses the promoter activity. It is also possible that Blimp-1 represses a factor that positively enhances IL-2 transcription such as fos (17). In support of this idea, fos mRNA was elevated in Blimp-1-deficient CD4 T cells following stimulation (17).
Targets of Blimp-1 have been extensively investigated particularly in B cells. In the B cell lineage, Blimp-1 is highly expressed at the plasma cell stage in which Blimp-1 is believed to actively suppress genes involved in B cell functions and proliferation, including c-myc, cIIta, pax5, spiB, and id3 (15, 36, 37, 38). A CREB/ATF family transcription factor, XBP-1, has also been demonstrated to be a downstream factor of Blimp-1, directly regulating plasma cell functions by increasing cell size, lysosomal content, ribosome numbers, and total protein synthesis (37). In contrast, target genes of Blimp-1 in T cells are less well defined. In addition to IL-2, Blimp-1 is believed to inhibit tbet and bcl-6, both of which are induced by IFN-γ (17). Bcl-6 mRNA was found elevated in Blimp-1 -deficient T cells (17). As Bcl-6 inhibits GATA-3 expression and represses Th2 differentiation (39) and T-bet promotes Th1 differentiation, Blimp-1 might be a transcription factor highly associated with Th2 differentiation that cooperates with GATA-3 (40). Indeed, elevation in IFN-γ production and Th1-mediated pathology observed in Blimp-1-deficient animal models supports this notion (18, 19). However, in vitro differentiation of Blimp-1-deficient CD4 T cells into Th2 cells was not impaired (18). Therefore, in vivo roles of Blimp-1 in Th2 type immunity need to be better examined.
A study by Villarino et al. (30) showed that exogenous IL-2 significantly suppresses IL-2 production by activated splenic CD4 T cells through activation of STAT5. Similarly, study by Gong and Malek (20) demonstrated that IL-2 neutralization dramatically enhances IL-2 production. When naive 5C.C7 CD4 T cells were stimulated with P13.9, surrogate APCs, addition of recombinant human (or mouse) IL-2 itself had little effects on IL-2 production. Paradoxically, adding human IL-2 in the presence of anti-mouse IL-2 Ab into the same culture efficiently suppresses IL-2 production, indicating that IL-2 used in this study is functionally active. Given that P13.9 cells express high levels of MHC class II as well as costimulatory molecules such as CD80 (29), the magnitude of activation signals delivered from APCs may account for the differences from these studies. In support of this possibility, IL-2 production by naive 5C.C7 CD4 T cells (or polyclonal CD4 T cells) was dramatically inhibited by exogenous IL-2 when stimulated with polyclonal anti-CD3/CD28 Ab. Because Blimp-1 expression is substantially higher in effector/memory phenotype CD4 T cells or in Foxp3+ regulatory T cells (Tregs) (19), phenotypes of CD4 T cells may also be important for the sensitivity to IL-2-mediated inhibition.
It should be noted that IL-4 is not the only cytokine that suppresses IL-2 production in CD4 T cells. Our study confirms a suppressive function of IL-12 in IL-2 production (30). IL-12 is similar to IL-4 in that the suppression is also dependent on IL-2, although IL-12 is less potent suppressor compared with IL-4. Both IL-12 and IL-4 have been shown to induce Blimp-1, but IL-4 seems to be a stronger Blimp-1 inducer cytokine than IL-12 (20). In agreement, Blimp-1 expression level in Th1 CD4 T cells is lower in Th2 CD4 T cells (40). IL-27 is another cytokine that limits IL-2 production by CD4 T cells (30). Although IL-2 inhibition by IL-27 seems less pronounced compared with IL-12 or IL-4, whether IL-27 inhibits IL-2 via Blimp-1-dependent pathway remains to be determined.
It is interesting to note that IL-13, although activating STAT6 as IL-4, failed to display suppression in IL-2 production, indicating that a downstream signaling pathway leading to Blimp-1 expression seems different between the two cytokines. A potential STAT binding DNA sequence has been found in the human Blimp-1 promoter region (41). However, there might be additional factors necessary for Blimp-1 induction induced by IL-4 but not by IL-13. Molecular mechanisms involved in cytokine-mediated Blimp-1 expression in activated T cells will require further investigation. In addition, IL-4 elicits different inhibitory mechanisms in different cell types. Whereas IL-4 mediates suppression of TNF-α production mainly via a TTP-dependent mechanism in mast cells (6), TTP expression is not particularly altered by IL-4 in activated CD4 T cells. Signaling pathways of both IL-4 downstream and Blimp-1 upstream in these cells need to be defined to better understand the differences.
What is a biologic importance of Blimp-1-mediated IL-2 control by IL-4? IL-2 produced by activated CD4 T cells may be involved in controlling Treg functions because IL-2 is a critical factor for Treg homeostasis (42). In support of this reason, it was recently shown that IL-4 inhibits CD4+CD25+ Treg cell-mediated suppression (43). IL-4 stimulation of Tregs induced proliferation without altering their suppressive functions, whereas IL-4 stimulation of CD4+CD25− responder cells inhibited Treg-mediated suppression (43). These results are consistent with our results, suggesting that IL-2 inhibition may be related to the inhibition of Treg-mediated suppression. In addition, human Th2 cells were also shown to be less susceptible than Th1 cells to the suppressive activity of CD25+ regulatory thymocytes (44). It is possible that higher Blimp-1 level in Tregs is responsible for anergic phenotypes of Tregs, namely no IL-2 production and no proliferation (45). Tregs may depend on IL-2 produced by activated non-Tregs to maintain Blimp-1 expression and anergic phenotypes. Foxp3 was shown to directly induce Blimp-1 expression in Tregs (46).
In summary, our study demonstrates that suppression of IL-2 production in the course of IL-4-mediated CD4 T cell stimulation is achieved through enhancing Blimp-1 expression and that Blimp-1 is involved in suppressing IL-2 in activated CD4 T cells. Experiments to better define in vivo roles of Blimp-1 during adaptive immunity are underway.
We thank Drs. William Paul (National Institutes of Health), Stephen Nutt (Walter and Eliza Hall Institute of Medical Research), and Ronald Germain (National Institutes of Health) for providing 5C.C7 TCR Tg mice, Blimp-1GFP/+ mice, and P13.9 cell lines, respectively. We also thank Dr. Thomas Hamilton (Lerner Research Institute) for advice in carrying out RNA degradation experiment. Patrick Finnegan (Avon Lake High School, Science Internship Program, Cleveland Clinic Foundation) should be acknowledged for excellent technical assistance in confocal microscope analysis.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by startup funds from the Cleveland Clinic Foundation and in part by a Scientist Development grant from the American Heart Associations (to B.M.).
Abbreviations used in this paper: TTP, tristetraprolin; Tg, transgenic; UTR, untranslated region; Treg, regulatory T cell.
The online version of this article contains supplemental material.