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* Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104;
Department of Molecular Biology, Genentech, South San Francisco, CA 94080;
Department of Inflammation Research, Amgen, Thousand Oaks, CA 91320; and
DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Although IL-27R mRNA can be detected in several immune lineages (3, 8), initial studies have focused on the role of IL-27 in directing (CD4+) Th cell responses (1, 9, 10, 11). Similar to the IL-12R (IL-12R
2), the ligand-specific component of the IL-27R is present at low levels on the surface of naive CD4+ T cells, while the shared subunit (IL-12R1/gp130) is more abundant (6, 12, 13). Conversely, activated CD4+ T cells express high levels of IL-27R in vivo and in vitro, an effect shown to be mediated through a TCR-dependent process (6). Whether induced (high) or constitutive (low), the importance of IL-27R expression is evidenced by the unique CD4+ T cell phenotypes noted in IL-27R-deficient (IL-27R–/–) mice and the heterogeneous Jak/STAT family signaling cascade that this receptor can propagate (Jak 1 and Jak 2; STAT1, 3, 4, and 5) (9, 10, 11, 14). Moreovoer, IL-27 can be secreted by the same APCs that provide the impetus for T cell activation and IL-27R expression (TCR ligation), thus placing this cytokine-receptor pairing in an ideal position to influence Th cell responses.
When present during primary stimulation of naive CD4+ T cells, IL-27 enhances proliferation and promotes differentiation into type I (Th1) effector cells that secrete IFN-
(1). By inducing expression of T-bet, a transcription factor whose target genes include IFN- and IL-12R2, IL-27 directly promotes effector cytokine production (IFN-) and sensitizes activated CD4+ T cells to IL-12, a dominant factor in the polarization of Th1 responses (9, 10, 11). In turn, because CD4+ T cells from IL-27R–/– mice produce less IFN- than wild-type (WT)3 counterparts during acute infection with Leishmania major (8), a consensus emerged that IL-27 is critical for rapid induction of type I inflammation (15, 16, 17, 18). However, during chronic leishmaniasis, IL-27R–/– or EBI3–/– mice are able to develop protective Th1 responses that, while delayed, are sufficient to control parasite replication (19, 20). Furthermore, when challenged with an avirulent mycobacterium (bacillus Calmette-Guerin) (8) or during chronic infection with Mycobacterium tuberculosis (21, 22), IL-27R–/– mice generate Th1 responses that are comparable to those of WT counterparts and, following infection with Trypanosoma cruzi, their production of IFN- is enhanced (23). Thus, while it can augment the production of IFN- by CD4+ T cells, these in vivo findings demonstrate that IL-27 is not required for the development of type I immunity.
Studies with IL-27R-deficient mice suggest that IL-27 does not determine the polarity (Th1 vs Th2) of CD4+ T cell responses, but, instead, regulates the intensity of pathogen-induced inflammation. During infection with the intestinal helminth Trichuris muris, IL-27R–/– mice display accelerated type II (Th2) responses that, when compared with WT cohorts, mediate enhanced worm expulsion (24). Similarly, after challenge with the intracellular eukaryote Toxoplasma gondii, IL-27R–/– mice develop appropriately polarized Th1 responses that limit parasite replication at the site of infection. However, although WT animals are able to contract T cell responses once acute infection is abated, IL-27R–/– mice develop a lethal inflammatory disease that is characterized by escalating CD4+ T cell proliferation and IFN- production (25). Therefore, because IL-27R is required to suppress pathological T cell responses during infection with T. gondii, it is clear that, aside from a role in promoting T cell responses, this receptor also delivers critical inhibitory cues.
Previous work from this laboratory has shown that during infection with T. gondii in IL-27R–/– mice, acute mortality is associated with exaggerated production of IL-2 (25). In turn, the current study demonstrates that the survival of these animals is prolonged when IL-2 is neutralized in vivo and that rIL-27 dramatically reduces the expression of IL-2 mRNA and protein by WT CD4+ T cells. Together, these data imply that IL-27 curbs the development of pathogenic inflammatory responses by limiting the production of IL-2, a potent T cell growth and survival factor. Additionally, the present findings establish that this property is shared by IL-12, but not fellow IL-6/IL-12 family members IL-6 and IL-23. Thus, while contemporary dogma holds that IL-2 is a Th1-type cytokine, its production is actively suppressed by two cytokines that are associated with promoting such responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Mice deficient in IL-27R (WSX-1/TCCR) were generated, as described (8), and bred as homozygotes in a specific-pathogen free environment at the University of Pennsylvania. Age-matched WT (C57BL/6) controls were purchased from The Jackson Laboratory. Mice deficient in STAT1, STAT4, T-bet, and the corresponding WT controls (SEJ129 or BALB/c) were purchased from The Jackson Laboratory. For infections, the ME49 strain of T. gondii was maintained in mice (Swiss Webster and CBA/CaJ; The Jackson Laboratory) and tissue cysts were prepared as described (25). At 5–8 wk of age, groups of three to five mice were infected with 20 cysts i.p. All experiments were conducted following the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee.
In vivo Ab treatments
Neutralizing anti-IL-2 (clone: S4B6) and anti-IFN- (clone: XMG1.2) mAbs were generated by transferring monoclonal rat B cell hybridoma cells into nude mice and precipitating Abs from the resulting ascites fluid (Harlan Bioproducts). Infected mice were treated with 2 mg of sterile rat Ig (control), anti-IL-2, or anti-IFN- on days 7, 9, and 11 postinfection, and survival was monitored. Before the last Ab treatment (day 10), serum samples were collected and levels of IFN- were determined by ELISA.
Flow cytometry
For ex vivo experiments, spleens were isolated from uninfected or T. gondii-infected mice (day 14), dissociated into a single cell suspension, and depleted of erythrocytes using 0.86% (w/v) ammonium chloride (Sigma-Aldrich). Splenocytes were then washed and immediately stained for surface protein expression using the following Abs: anti-CD4, anti-CD25, anti-CD44, anti-CD62L, and anti-CD95 (Fas ligand (FasL)) (eBioscience). To assay in vitro cytokine production, CD4+ T cells were stimulated for 24 or 48 h (see below) and then pulsed with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich). Two hours later, cells were treated with brefeldin A (BFA) (2 h of BFA at 10 µg/ml; Sigma-Aldrich) and then stained for intracellular IL-2 and IFN- in combination with surface CD4 (eBioscience).
In vitro CD4+ T cell differentiation
Splenocytes were isolated from uninfected mice, as above, and depleted of CD8+ and NK1.1+ cells by magnetic bead separation (Polysciences). Cells were then labeled with CFSE (5 µg/ml; Sigma-Aldrich), stimulated with soluble anti-CD3 Ab (1 µg/ml) and soluble anti-CD28 Ab (1 µg/ml), and cultured in complete RPMI 1640 (10% heat-killed FBS, 100 U/ml penicillin, 1 mg/ml streptomycin, nonessential amino acids, and 2-ME). In all experiments, cells were cultured at 2 x 106 cells/ml in tissue culture-treated 96- or 48-well plates (200 or 500 µl/well). For Th1-polarizing conditions, cultures were supplemented with rIL-12 (5 ng/ml; Genetics Institute). IL-27 (200 ng/ml) was provided by F. DeSauvage (Genentech, South San Francisco, CA), and IL-23 (100 ng/ml) by R. Kastelein (DNAX, Palo Alto, CA). Recombinant IL-6 (10 ng/ml) and IFN- (100 U/ml) were purchased from eBioscience and BD Pharmingen, respectively. To assay the amount of IL-2 and IFN- secreted during culture, supernatants were harvested after 48 and 72 h (before PMA/ionomycin/BFA), respectively, and cytokine concentrations were determined by ELISA.
RT-PCR
To assay gene transcription, mRNA was isolated from cells using standard procedures and converted to cDNA as described (25). PCR was then used to quantify message levels, and -actin expression was used as an internal control to assure equal loading of every reaction. Primers: CIS, 5'-ACATGGTCCTCTGCGTACA-3', 5'-CAGCTGTCACATGCATGC-3'; suppressor of cytokine signaling 1 (SOCS1), 5'-CACTCACTTCCGCACCTTCC-3', 5'-CAGCCGGTCAGATCTGGAAG-3'; and SOCS3, 5'-TGCGCCATGGTCACCCACA-3', 5'-GCTCCTTAAAGTGGAGCATCATACTGA-3'. For real-time PCR, IL-2-specific primers and probes were purchased from Applied Biosystems, and amplification was performed according to the manufacturer’s specifications. mRNA levels were normalized with respect to ubiquitous 18S ribosomal mRNA, and expression of IL-2 is represented as the fold induction over naive, WT CD4+ T cell controls.
Statistics
Statistical differences between experimental groups were determined by paired Student’s t test. A star above the lower value of two experimental groups represents significant differences (p < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Previous reports indicate that splenocytes from IL-27R–/– mice produce more IL-2 than WT counterparts during infection with T. gondii (25). Therefore, because CD4+ T cells are the primary source of IL-2 during acute toxoplasmosis (26), these cells were isolated from uninfected WT or IL-27R–/– mice, and IL-2 production was compared during in vitro differentiation. When activated under nonpolarizing (anti-CD3/anti-CD28) or Th1-polarizing conditions (anti-CD3/anti-CD28 + rIL-12), IL-27R–/– CD4+ T cells express higher levels of IL-2 mRNA and secrete more IL-2 protein than WT counterparts (Fig. 1, A and B). These in vitro findings imply that, during infection with T. gondii, CD4+ T cells are responsible for the exaggerated IL-2 production noted in IL-27R–/– animals. Consistent with this hypothesis, the frequency of IL-2+ CD4+ T cells is 2–3 times higher in IL-27R–/– mice than WT counterparts during acute toxoplasmosis (Fig. 1D).
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) and FasL, a cell surface molecule that promotes apoptosis (27, 28). Several factors may influence the expression CD25 and FasL, but because IL-2 is a dominant inducer of these proteins, their appearance on the surface of CD4+ T cells can be indicative of IL-2 production (27, 29). Accordingly, due to a lack of IL-2 production, most CD4+ T cells from WT and IL-27R–/– mice display a naive phenotype (CD25low FasLlow) prior to challenge with T. gondii (Fig. 1, C, E, and G). In contrast, during the first week of infection, CD4+ T cells from both groups become activated and produce IL-2, and a similar induction of CD25 and FasL is observed (25) (data not shown). Once parasite replication is controlled, WT animals are able to contract CD4+ T cell responses and, at day 14 postinfection, IL-2 production and surface levels of CD25 and FasL are only slightly higher than those noted before challenge with T. gondii (Fig. 1, D, F, and H) (25). However, at this later time point, IL-27R–/– CD4+ T cells produce more IL-2 than WT cohorts and, in turn, the frequency of CD25+ and FasL+ cells is also higher (Fig. 1, D, F, and H) (25). Given that surface levels of activation markers CD44 and CD62L are comparable between WT and IL-27R–/– CD4+ T cells throughout infection (data not shown), it is unlikely that this difference in CD25 and FasL is due, as previously proposed (25), to an accumulation of activated cells in the latter group. Instead, the current data suggest that increased expression of CD25 and FasL reflects the sustained production of IL-2 in IL-27R–/– mice.
Because the studies presented in this work suggest that dysregulated IL-2 production contributes to the development of lethal inflammation in IL-27R–/– mice, this cytokine was neutralized during infection with T. gondii and survival was monitored. However, because IL-2 is required for resistance to this parasite (30), anti-murine IL-2 mAb could be administered only after protective T cell responses were established and acute parasite replication was controlled (day 7) (25). To assess the immunological impact of this regime, serum was collected before the last treatment and levels of IFN- were measured. Compared with control animals (rat Ig), there is improved survival and a reduction in circulating IFN- levels when infected IL-27R–/– mice are treated with anti-IL-2 mAb (Fig. 1, I and J). Likewise, during in vitro Th1 differentiation, IL-27R–/– CD4+ T cells produce more IFN- than WT cohorts, but, when IL-2 is neutralized, IFN- production is comparable (Fig. 1K).
Given that IL-2 promotes expansion of Th1 cells (31) and that acute toxoplasmosis is associated with a dramatic accumulation of these cells in IL-27R–/– mice (25), it can be surmised that the delay in time to death achieved through anti-IL-2 treatment may be due to a tempering of pathogenic Th1 responses. However, despite greatly elevated levels of IFN- in the serum of infected IL-27R–/– mice (Fig. 1J), survival is not prolonged when this cytokine is depleted in vivo (Fig. 1I). Because anti-IFN- was administered only after Th1 responses were established (day 7) and these animals succumb to infection with similar kinetics as untreated controls, it is unlikely that a recrudescence of parasite replication is the cause of death in these experiments. Instead, these studies demonstrate that, during acute toxoplasmosis in IL-27R–/– mice, IL-2 is required for the development of pathogenic CD4+ T cell responses that are characterized by exaggerated IFN- production, but are not dependent on it. Consistent with this hypothesis, survival of IL-27R–/– mice can be prolonged by depleting CD4+ T cells that produce IFN- and IL-2 during infection, but not by depleting CD8+ T cells that produce IFN- and little IL-2 (25). Moreover, their survival is also improved when treated with CTLA-4 Ig, a polypeptide that can block CD28-dependent costimulation and thereby limits in vivo IL-2 production (E. Huang and C. Hunter, unpublished observation). Still, it must be noted that CTLA-4 Ig can deliver inhibitory signals to the APCs that it binds (32) and, as a result, this rescue phenotype may not be solely due to a decrease in IL-2 production.
IL-27 and IL-12 limit the production of IL-2 by CD4+ T cells
Because a lack of IL-27R is associated with enhanced IL-2 responses in vivo and in vitro (Fig. 1), studies were performed to determine whether IL-27 directly regulates the production of this cytokine. Upon ligation of the TCR and costimulatory receptor CD28, CD4+ T cells become activated, rapidly produce IL-2, and, given they must proliferate to express high levels of IL-27R (6), it is not surprising that rIL-27 has little effect on IL-2 production during the first 24 h in culture (Fig. 2A). However, as these cells begin to divide, expression of IL-27R is enhanced (6), and while there is a natural reduction in the percentage of IL-2-positive cells after 48 h in culture (56 vs 35%), the decline is more pronounced when exogenous IL-27 is introduced (52 vs 13%; Fig. 2B). Because this decrease is concurrent to significant reductions in secreted protein and IL-2 mRNA levels (Fig. 2C), the current data suggest that, either directly or indirectly, IL-27 mediates transcriptional regulation of the IL-2 gene.
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2) is not expressed at high levels until CD4+ T cells have proliferated (33), and, as a result, neither IL-27 nor IL-12 affects the production of IL-2 during the first 24 h of culture (Fig. 2, A and D). Surprisingly, after 48 h, the percentage of IL-2-positive CD4+ T cells is significantly reduced when cultures are supplemented with only IL-12 (35 vs 12%; Fig. 2, B and E), and when IL-27 is also added, IL-2 production is almost completely abolished (3%; Fig. 2E). Given the corresponding reductions in levels of mRNA and secreted protein (Fig. 2, F and G), these studies establish that IL-12 and IL-27 cooperate to limit IL-2 production when CD4+ T cells are activated in highly polarizing Th1 conditions. In turn, because both are evident during infection with T. gondii, it is likely that IL-12 and IL-27 contribute to the suppressed IL-2 responses that are a hallmark of acute toxoplasmosis in WT mice (34, 35).
Because IL-12 and IL-27 can each suppress IL-2 production, experiments were performed to determine whether this property is mimicked by other IL-6/IL-12 family cytokines. Despite a common cytokine (IL-12p40) and receptor (IL-12R1) subunit (36), the current work demonstrates that IL-12 and IL-23 do not share the ability to suppress Th cell IL-2 production (Fig. 3A). Likewise, although gp130 is a component in the heterodimeric receptors for IL-27 and IL-6 (7), only the former can limit IL-2 production (Fig. 3B). Given that IL-23R and IL-6R mRNA can be readily detected in these activated CD4+ T cells (data not shown), the inability of IL-23 and IL-6 to limit IL-2 production is not due to a lack of responsiveness. Instead, it is clear that these cytokines do not suppress IL-2 production and, because they share receptor components (IL-12R1/gp130) with those which possess this capacity (IL-12/IL-27), it can now be proposed that the ligand-specific subunits of the IL-12R (IL-12R2) and IL-27R (IL-27R) deliver the pertinent inhibitory cues.
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(7, 18). However, while the current findings demonstrate that each can suppress IL-2 production with similar potency (Fig. 2), they also support the idea that IL-12 is the dominant factor in driving Th1 differentiation. When CD4+ T cells are cultured under nonpolarizing conditions, IL-27 prompts a small rise in the percentage of IFN--positive cells and only a modest increase in the amount of IFN- they secrete (Fig. 4). In contrast, when IL-12 is added to these cultures, there is a 3-fold expansion in the percentage of IFN--positive CD4+ T cells and the amount of cytokine produced increases dramatically (Fig. 4). Because exogenous IL-27 has little effect on IFN- production when polarizing concentrations of IL-12 are available, these studies confirm a central role for IL-12 in promoting Th1 differentiation, and they suggest that IL-27 alone is not sufficient or required to polarize effector T cell populations.
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IL-6/IL-12 family cytokines mediate cellular effects upon binding their cognate receptors and thereby inducing phosphorylation of Jak/STAT proteins that migrate to the nucleus and promote or suppress expression of target genes (4). Therefore, because IL-12 and IL-27 can activate common signaling components in CD4+ T cells (36), it is possible that they use analogous pathways to inhibit IL-2 production. Phosphorylation of STAT4 is the most characteristic signaling event for IL-12 (18), and because this transcription factor can also be activated by IL-27, albeit to a lesser extent (10), the ability of these cytokines to suppress IL-2 production was measured in STAT4–/– CD4+ T cells. As above (Fig. 2), IL-12 limits the production of IL-2 by WT CD4+ T cells (19 vs 8%; Fig. 5A), but, in the absence of STAT4, it prompts only a modest reduction in the percentage of IL-2-positive cells (45 vs 36%; Fig. 5B). In fact, even when exogenous IL-12 is not added (nonpolarizing), there is a greater frequency of IL-2-positive cells in STAT4-deficient cultures than in WT counterparts (45 vs 19%; Fig. 5, A and B). These findings demonstrate that, when present at the time of CD4+ T cell priming, IL-12 can suppress IL-2 production through STAT4-dependent mechanisms. However, because IL-27 is a potent inhibitor of IL-2 in either WT or STAT4–/– CD4+ T cell cultures (Fig. 5, A and B), it is also apparent that STAT4-independent mechanisms can mediate this effect.
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production through distinct means (36). Still, given that IL-12 employs STAT4 to inhibit IL-2 (Fig. 5B) and to promote IFN- (18), it is possible that IL-27 achieves the former (IL-2 suppression) by activating STAT1, the same transcription factor that it employs to induce the latter (IFN- production) (9, 10, 11, 14). Consequently, the ability of IL-27 to suppress IL-2 was examined in STAT1–/– CD4+ T cells. As in WT counterparts, there is a pronounced reduction in the percentage of IL-2-positive cells when STAT1-deficient cultures are supplemented with IL-27 (Fig. 5C). Moreover, although it induces IFN- by promoting expression of T-bet, a transcription factor that has been shown to suppress IL-2 production (37), IL-27 can also mediate this effect in T-bet-deficient CD4+ T cells (Fig. 5D). Consistent with these findings, IFN- and IFN- both activate STAT1 and induce expression of T-bet, but neither affects the frequency of IL-2-positive CD4+ T cells in nonpolarizing cultures (data not shown and Fig. 5E) (9, 38). Together, these studies demonstrate that IL-12 can limit IL-2 production through largely STAT4-dependent mechanisms, and that IL-27 does so independently of STAT4, STAT1, and T-bet. IL-27R–/– CD4+ T cells display enhanced proliferation in the absence of IL-2
Because IL-2 is a potent growth and survival factor, the availability of this cytokine has significant influence on the proliferation of activated CD4+ T cells (39). In turn, IL-27R–/– CD4+ T cells produce more IL-2 than WT counterparts, this cytokine may be responsible for the enhanced proliferation that has been noted in the former group (8, 25, 40). To determine whether enhanced IL-2 production is indeed responsible for this hyperproliferative phenotype, WT and IL-27R–/– CD4+ T cells were labeled with CFSE and then cultured with or without (anti-IL-2) endogenous cytokine. After 72 h, dilution of CFSE was visualized by flow cytometry and the number of cells in each proliferative generation was used, as described previously (41), to determine the absolute number of mitotic events in each culture (Fig. 6A). The proliferative capacity and number of divisions per cell were then extrapolated from these calculations and compared between WT and IL-27R–/– CD4+ T cells. Similar to prior observations (25), the proliferative capacity and divisions per cell are higher for IL-27R–/– CD4+ T cells than WT cohorts in nonpolarizing or Th1-polarizing cultures (Fig. 6B). Additionally, consistent with the findings of others (A. Wells, unpublished observations) and with studies in which CD28-dependent, IL-2-independent signals are sufficient to induce T cell expansion (42), neutralization of IL-2 results in a modest, but significant (p < 0.05), decrease in the proliferation of WT and IL-27R–/– T cells. Even so, the proliferative capacity and daughters per cell are still higher for IL-27R–/– CD4+ T cells than WT counterparts in the IL-2-deficient (anti-IL-2), nonpolarizing, or Th1-polarizing cultures, suggesting that enhanced IL-2 production is not wholly responsible for the hyperproliferative phenotype of IL-27R–/– CD4+ T cells (Fig. 6B).
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When cultured in the absence of IL-2, IL-27R–/– CD4+ T cells proliferate more than WT counterparts. Therefore, aside from its role in regulating IL-2 production, the IL-27R delivers additional inhibitory signals that directly regulate cellular proliferation. Given that SOCS proteins are induced by IL-6/IL-12 family cytokines and can suppress T cell responses (43), it is likely that they contribute to the antiproliferative effects of IL-27. Consistent with this hypothesis, IL-27 can induce expression of SOCS3 (Fig. 7), an inhibitory factor that has been shown to limit effector T cell proliferation (44, 45). Furthermore, like IL-27, IL-12 also promotes expression of SOCS3, but neither cytokine affects mRNA levels for CIS, SOCS1, and SOCS2 (Fig. 7 and data not shown). Thus, despite being viewed as proinflammatory agents, the current data establish that IL-27 and IL-12 share two analogous inhibitory properties, the ability to suppress IL-2 production and to induce expression of SOCS3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Upon encounter with pathogens and inflammatory stimuli in peripheral tissues, professional APCs migrate to primary lymphoid organs, where they present Ags in the context of class II MHC molecules and provide costimulation for the activation of naive CD4+ T cells (46). Although the interaction with APCs provides all of the signals necessary to induce proliferation, ligation of the TCR and CD28 also prompts CD4+ T cells to quickly secrete IL-2, a cytokine that further enhances their proliferation and survival (47). Given the paucity of Ag-specific CD4+ T cells at the onset of adaptive immunity, the immediate production of IL-2 ensures that a potent growth factor is available to support the initial expansion of clonotypic T cells. In fact, because activated CD4+ T cells can produce IL-2 before G1 phase of the cell cycle while they must cross this developmental checkpoint to become effector Th1 or Th2 cells capable of secreting IFN- and IL-4, respectively, IL-2 production precedes differentiation (48). Consistent with this idea, CD4+ T cell activation induces rapid epigenetic changes that promote IL-2 gene accessibility and transcription prior to cell cycle entry (49).
Aside from providing the impetus for CD4+ T cell activation and IL-2 production, APCs can produce IL-12 and IL-27, two cytokines that promote Th1 differentiation (18, 36). Still, although IL-12 and IL-27 may be present at the time of Ag encounter, naive CD4+ T cells express low levels of IL-12R2 (12) and IL-27R (6). Consequently, these cytokines have little influence on the events that induce IL-2 production, and, in turn, they do not significantly enhance IFN- production until after CD4+ T cells have passed through G1 phase of the cell cycle (Fig. 8A) (48). However, once these cells begin to proliferate, IL-12 becomes the dominant factor in promoting differentiation into Th1 effectors that secrete large amounts of IFN-, and, when concentrations of IL-12 are limiting, IL-27 can mediate this effect. These proinflammatory properties are well understood, but the data presented in this work reveal that IL-12 and IL-27 are also potent inhibitors of IL-2 production (Fig. 9). Therefore, as they determine the polarity of effector T cell responses, IL-12 and IL-27 also limit the availability of a key growth and survival factor (Fig. 9). Still, IL-12 and IL-27 cannot suppress IL-2 production until CD4+ T cells have crossed through G1 phase of the cell cycle (Fig. 8B), thus guaranteeing the rapid burst of IL-2 that is induced by activation and the initial nonpolar expansion of Ag-specific CD4+ T cells. This model is consistent with the idea that IL-2 is required to expand Ag-specific CD4+ T cell populations during a nascent immune response (31, 50) and with the finding that exaggerated IL-2 production can mediate severe pathology, as during acute toxoplasmosis in IL-27R–/– mice. In fact, because IL-27 does not appear to determine the polarity of in vivo T cell responses (24, 25), it can now be proposed that limiting IL-2 production is a primary function for this cytokine, while enhancing IFN- production is secondary. Moreover, because IL-27 is induced by pathogens that elicit Th1 or Th2 responses (24, 25), it may occupy a central role in suppressing IL-2 production regardless of the polarizing cytokine environment present during T cell activation. Accordingly, IL-27 can also inhibit IL-2 production when CD4+ T cells are cultured under Th2-polarizing conditions (data not shown).
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The importance of IL-2 as a T cell growth factor has been appreciated for over two decades (51). Still, although the cellular and molecular mechanisms that induce this cytokine are well understood (52), the processes that temper IL-2 production during acute inflammatory responses remain obscure. Various pharmacological agents, including cyclosporine A and FK506 (53, 54), can inhibit IL-2 production, and several host-derived factors have been shown to mediate this effect. Two examples are the inhibitory receptor CTLA-4 and the regulatory cytokine TGF-; the former can suppress IL-2 production by preventing CD28-dependent costimulation, and the latter by activating Smad3, a transcription factor that binds the IL-2 gene promoter and hinders transcription (55, 56). Along with others, these observations have established that IL-2 production can be regulated by factors that are classically associated with the suppression of Th cell responses. In contrast, the data presented in this work demonstrate that IL-2 production is also limited by two cytokines that promote the generation of type I (Th1) inflammation. A role for IL-12 in regulating IL-2 production has been noted previously (57), but the current work elaborates by demonstrating that this effect is largely mediated by the activation of STAT4. Furthermore, the present study establishes that IL-12 shares this property with IL-27, but not fellow IL-6/IL-12 family members IL-6 and IL-23. Together, IL-12 and IL-27 can effectively shut down the production of IL-2 by CD4+ T cells in vitro (Fig. 2), and because exaggerated IL-2 production has been noted during inflammatory responses in mice deficient for either cytokine, this effect is also apparent in vivo (25, 58).
The data presented in this work demonstrate that IL-27 can suppress IL-2 in the absence of STAT4, STAT1, and T-bet. Moreover, because IL-6 induces phosphorylation of STAT3 (4), but does not inhibit IL-2 production, it is unlikely that IL-27 employs this signaling pathway to do so. Although direct evidence is still required to confirm that IL-27 can suppress IL-2 production without STAT3, the fact that IL-2 production is similar between WT and STAT3-deficient CD4+ T cells is consistent with this notion (59). Therefore, given that IL-2 mRNA levels are reduced when an activated form of STAT5 is expressed in CD4+ T cells (60), it is tempting to speculate that IL-27 uses this transcription factor to propagate a similar effect. Accordingly, IL-27 is a potent inducer of STAT5 phosphorylation (10, 14, 25), and because IL-12 can also induce some STAT5 activation (18), this hypothesis accounts for the limited ability of IL-12 to inhibit IL-2 production in STAT4–/– CD4+ T cells. Nevertheless, while the current studies have excluded several candidate pathways (STAT1/3/4), the molecular mechanisms that IL-27 employs to suppress IL-2 production remain uncertain.
Because IL-12 and IL-27 can prompt dramatic reductions in IL-2 mRNA levels, it is likely that they mediate transcriptional inhibition. Still, although several negative regulatory elements are present in the IL-2 promoter (61), there is no evidence that activated STATs translocate to the nucleus and directly obstruct transcription of this gene. Instead, it is likely that STAT4, and perhaps STAT5, each induce expression of additional factors that inhibit this process. In turn, it has long been known that blocking protein synthesis results in the superinduction of IL-2 mRNA (62), and several nuclear proteins have been shown to suppress IL-2 production when CD4+ T cells become anergic (63). Nevertheless, IL-2 production can be regulated by posttranscriptional modifications, and it is also possible that IL-27 and IL-12 inhibit cytokine production without directly affecting IL-2 gene transcription (64).
Additional inhibitory properties for IL-27
The current study demonstrates that, by neutralizing IL-2 during infection with T. gondii, the survival of IL-27R–/– mice can be prolonged. However, while these data suggest that exaggerated IL-2 production plays a central role in the development of pathogenic T cell responses during infection, it is possible that treatment with anti-IL-2 mAb acts as a general immunosuppressant and that dysregulated IL-2 production may not be causative in the development of this parasite-induced inflammatory disease. These interpretations are not mutually exclusive, and because anti-IL-2 mAb-treated mice eventually succumb to infection, it is likely that the IL-27R delivers inhibitory signals that are distinct from those regulating the production of IL-2. Consistent with this idea, previous reports have established that the production of various inflammatory cytokines, including IL-4, IL-6, IL-12, TNF-, and GM-CSF, is exaggerated during parasitic infections in IL-27R–/– mice, suggesting that IL-2 alone is not the cause for immunopathology during toxoplasmosis. Moreover, the current work demonstrates that IL-27 can also induce expression of SOCS3, an inhibitory protein that has been shown to limit the inflammatory responses associated with colitis and asthma (65, 66). Even so, further studies are needed to assess the in vivo relevance of IL-27-dependent SOCS3 induction, and, because depletion of IL-2 was only transient in the current experiments, it is possible that continued administration of anti-IL-2 mAb would further prolong the survival of IL-27R–/– mice, again, suggesting a central role for IL-2 in the immunopathology that develops in those animals. Still, it should be noted that because SOCS3 tempers signaling by gp130 (67), the current findings support the idea that SOCS proteins are part of a classic negative feedback loop that regulates IL-6/IL-12 family cytokines (43). Moreover, these studies are the first to describe cellular mechanisms for the anti-inflammatory effects of IL-27, and, as such, they build on mounting evidence that IL-27 is critical for limiting the intensity of adaptive immune responses.
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1 This work was supported by the State of Pennsylvania, Grant NIH42334 (AI41158 with a minority supplement to A.V.V.; A10662). 
2 Address correspondence and reprint requests to Dr. Christopher A. Hunter, 3800 Spruce Street (Rosenthal Building), Room 226, Philadelphia, PA 19104. E-mail address: chunter{at}vet.upenn.edu
3 Abbreviations used in this paper: WT, wild type; BFA, brefeldin A; FasL, Fas ligand; SOCS, suppressor of cytokine signaling.
Received for publication June 9, 2005. Accepted for publication October 12, 2005.
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represents the mean of all trials (n = 6–7). A two-tailed, paired t test (p =) was used to determine statistical difference between WT and IL-27R–/– cells.
