Differentiation IL-27 Limits IL-2 Production During

IL-27 Limits IL-2 Production during Th1 Differentiation Alejandro V. Villarino, Jason S. Stumhofer, Christiaan J. M. Saris, Robert A. Kastelein, Frederic J. de Sauvage and This information is current as Christopher A. Hunter of September 28, 2021. J Immunol 2006; 176:237-247; ; doi: 10.4049/jimmunol.176.1.237 http://www.jimmunol.org/content/176/1/237 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-27 Limits IL-2 Production during Th1 Differentiation1

Alejandro V. Villarino,* Jason S. Stumhofer,* Christiaan J. M. Saris,‡ Robert A. Kastelein,§ Frederic J. de Sauvage,† and Christopher A. Hunter2*

Although the ability of IL-27 to promote T cell responses is well documented, the anti-inflammatory properties of this cytokine remain poorly understood. The current work demonstrates that during infection with Toxoplasma gondii, IL-27R-deficient mice generate aberrant IL-2 responses that are associated with the development of a lethal inflammatory disease. Because in vivo depletion of IL-2 prolongs the survival of infected IL-27R؊/؊ mice, these data suggest that IL-27 curbs the development of immunopathology by limiting parasite-induced IL-2 production. Consistent with this hypothesis, IL-27R؊/؊ CD4؉ T cells produce more IL-2 than wild-type counterparts during in vitro differentiation, and when rIL-27 is introduced, it can suppress the expression of IL-2 mRNA and protein by the latter group. Additionally, these studies reveal that, like IL-27, IL-12 can inhibit IL-2 production, and although each employs distinct mechanisms, they can synergize to enhance the effect. In contrast, this property is not shared by closely related cytokines IL-6 and IL-23. Thus, while traditionally viewed as proinflammatory agents, the present Downloaded from findings establish that IL-27 and IL-12 cooperate to limit the availability of IL-2, a potent T cell growth and survival factor. Moreover, because the current studies demonstrate that both can induce expression of suppressor of cytokine signaling 3, a protein that tempers cytokine receptor signaling, they also suggest that IL-27 and IL-12 share additionally inhibitory properties. The Journal of Immunology, 2006, 176: 237–247.

nterleukin-27 is a member of the IL-6/IL-12 family of cyto- tive (low), the importance of IL-27R expression is evidenced by http://www.jimmunol.org/ kines that, like IL-12 (IL-12p35/IL-12p40) and IL-23 (IL- the unique CD4ϩ T cell phenotypes noted in IL-27R-deficient (IL- I 23p19/IL-12p40), is secreted as a helical protein (IL-27p28) 27RϪ/Ϫ) mice and the heterogeneous Jak/STAT family signaling bound to a soluble receptor-like subunit (EBI3) (1). Produced by cascade that this receptor can propagate (Jak 1 and Jak 2; STAT1, APCs in response to host- and pathogen-derived inflammatory 3, 4, and 5) (9–11, 14). Moreovoer, IL-27 can be secreted by the cues (2), IL-27 mediates its cellular effects through a high affinity same APCs that provide the impetus for T cell activation and IL- receptor complex that includes a unique subunit (IL-27R, WSX-1, 27R expression (TCR ligation), thus placing this cytokine-receptor TCCR) and gp130 (3), a component shared by several cytokines pairing in an ideal position to influence Th cell responses. including IL-6, IL-11, OSM, G-CSF, and LIF (4). Although gp130 When present during primary stimulation of naive CD4ϩ T by guest on September 28, 2021 is present on a range of immune and nonimmune cells (5), the cells, IL-27 enhances proliferation and promotes differentiation highest levels of IL-27R are found on the surface of resting NK into type I (Th1) effector cells that secrete IFN-␥ (1). By inducing cells, resting NKT cells, regulatory T cells, effector T cells, and expression of T-bet, a transcription factor whose target genes in- memory T cells, suggesting that IL-27 is directed at key elements clude IFN-␥ and IL-12R␤2, IL-27 directly promotes effector cy- of innate and adaptive immunity (6). Accordingly, IL-27R-defi- tokine production (IFN-␥) and sensitizes activated CD4ϩ T cells to cient mice (IL-27RϪ/Ϫ) develop aberrant inflammatory responses IL-12, a dominant factor in the polarization of Th1 responses (9– following infection with various pathogens (7). 11). In turn, because CD4ϩ T cells from IL-27RϪ/Ϫ mice produce Although IL-27R mRNA can be detected in several immune less IFN-␥ than wild-type (WT)3 counterparts during acute infec- lineages (3, 8), initial studies have focused on the role of IL-27 in tion with Leishmania major (8), a consensus emerged that IL-27 is directing (CD4ϩ) Th cell responses (1, 9–11). Similar to the IL- critical for rapid induction of type I inflammation (15–18). How- 12R (IL-12R␤2), the ligand-specific component of the IL-27R is ever, during chronic leishmaniasis, IL-27RϪ/Ϫ or EBI3Ϫ/Ϫ mice present at low levels on the surface of naive CD4ϩ T cells, while are able to develop protective Th1 responses that, while delayed, the shared subunit (IL-12R␤1/gp130) is more abundant (6, 12, 13). are sufficient to control parasite replication (19, 20). Furthermore, Conversely, activated CD4ϩ T cells express high levels of IL-27R when challenged with an avirulent mycobacterium (bacillus in vivo and in vitro, an effect shown to be mediated through a Calmette-Guerin) (8) or during chronic infection with Mycobac- TCR-dependent process (6). Whether induced (high) or constitu- terium tuberculosis (21, 22), IL-27RϪ/Ϫ mice generate Th1 responses that are comparable to those of WT counterparts and, following infection with Trypanosoma cruzi, their production of *Department of Pathobiology, University of Pennsylvania School of Veterinary Med- ␥ icine, Philadelphia, PA 19104; †Department of Molecular Biology, Genentech, South IFN- is enhanced (23). Thus, while it can augment the production ϩ San Francisco, CA 94080; ‡Department of Inflammation Research, Amgen, Thousand of IFN-␥ by CD4 T cells, these in vivo findings demonstrate that § Oaks, CA 91320; and DNAX Research Institute, Palo Alto, CA 94304 IL-27 is not required for the development of type I immunity. Received for publication June 9, 2005. Accepted for publication October 12, 2005. Studies with IL-27R-deficient mice suggest that IL-27 does not The costs of publication of this article were defrayed in part by the payment of page determine the polarity (Th1 vs Th2) of CD4ϩ T cell responses, but, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. instead, regulates the intensity of pathogen-induced inflammation. 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 ad- 3 Abbreviations used in this paper: WT, wild type; BFA, brefeldin A; FasL, Fas dress: [email protected] ligand; SOCS, suppressor of cytokine signaling.

During infection with the intestinal helminth Trichuris muris, IL- were then labeled with CFSE (5 ␮g/ml; Sigma-Aldrich), stimulated with 27RϪ/Ϫ mice display accelerated type II (Th2) responses that, soluble anti-CD3 Ab (1 ␮g/ml) and soluble anti-CD28 Ab (1 ␮g/ml), and when compared with WT cohorts, mediate enhanced worm expul- cultured in complete RPMI 1640 (10% heat-killed FBS, 100 U/ml peni- cillin, 1 mg/ml streptomycin, nonessential amino acids, and 2-ME). In all sion (24). Similarly, after challenge with the intracellular eu- experiments, cells were cultured at 2 ϫ 106 cells/ml in tissue culture- Ϫ/Ϫ karyote Toxoplasma gondii, IL-27R mice develop appropri- treated 96- or 48-well plates (200 or 500 ␮l/well). For Th1-polarizing ately polarized Th1 responses that limit parasite replication at the conditions, cultures were supplemented with rIL-12 (5 ng/ml; Genetics site of infection. However, although WT animals are able to con- Institute). IL-27 (200 ng/ml) was provided by F. DeSauvage (Genentech, Ϫ/Ϫ South San Francisco, CA), and IL-23 (100 ng/ml) by R. Kastelein (DNAX, tract T cell responses once acute infection is abated, IL-27R Palo Alto, CA). Recombinant IL-6 (10 ng/ml) and IFN-␥ (100 U/ml) were mice develop a lethal inflammatory disease that is characterized by purchased from eBioscience and BD Pharmingen, respectively. To assay ϩ escalating CD4 T cell proliferation and IFN-␥ production (25). the amount of IL-2 and IFN-␥ secreted during culture, supernatants were Therefore, because IL-27R is required to suppress pathological T harvested after 48 and 72 h (before PMA/ionomycin/BFA), respectively, cell responses during infection with T. gondii, it is clear that, aside and cytokine concentrations were determined by ELISA. from a role in promoting T cell responses, this receptor also de- RT-PCR livers critical inhibitory cues. To assay gene transcription, mRNA was isolated from cells using standard Previous work from this laboratory has shown that during in- procedures and converted to cDNA as described (25). PCR was then used Ϫ/Ϫ fection with T. gondii in IL-27R mice, acute mortality is asso- to quantify message levels, and ␤-actin expression was used as an internal ciated with exaggerated production of IL-2 (25). In turn, the cur- control to assure equal loading of every reaction. Primers: CIS, 5Ј-ACAT rent study demonstrates that the survival of these animals is GGTCCTCTGCGTACA-3Ј,5Ј-CAGCTGTCACATGCATGC-3Ј; suppressor of cytokine signaling 1 (SOCS1), 5Ј-CACTCACTTCCGCACCT prolonged when IL-2 is neutralized in vivo and that rIL-27 dra- Downloaded from TCC-3Ј,5Ј-CAGCCGGTCAGATCTGGAAG-3Ј; and SOCS3, 5Ј-TGCG matically reduces the expression of IL-2 mRNA and protein by CCATGGTCACCCACA-3Ј,5Ј-GCTCCTTAAAGTGGAGCATCATAC ϩ WT CD4 T cells. Together, these data imply that IL-27 curbs the TGA-3Ј. For real-time PCR, IL-2-specific primers and probes were pur- development of pathogenic inflammatory responses by limiting the chased from Applied Biosystems, and amplification was performed accord- production of IL-2, a potent T cell growth and survival factor. ing to the manufacturer’s specifications. mRNA levels were normalized with respect to ubiquitous 18S ribosomal mRNA, and expression of IL-2 is Additionally, the present findings establish that this property is represented as the fold induction over naive, WT CD4ϩ T cell controls.

shared by IL-12, but not fellow IL-6/IL-12 family members IL-6 http://www.jimmunol.org/ and IL-23. Thus, while contemporary dogma holds that IL-2 is a Statistics Th1-type cytokine, its production is actively suppressed by two Statistical differences between experimental groups were determined by cytokines that are associated with promoting such responses. paired Student’s t test. A star above the lower value of two experimental groups represents significant differences ( p Ͻ 0.05). Materials and Methods Mice and T. gondii infections Results IL-27RϪ/Ϫ CD4ϩ T cells display enhanced IL-2 responses in Mice deficient in IL-27R (WSX-1/TCCR) were generated, as described (8), vivo and in vitro and bred as homozygotes in a specific-pathogen free environment at the

Ϫ Ϫ by guest on September 28, 2021 University of Pennsylvania. Age-matched WT (C57BL/6) controls were Previous reports indicate that splenocytes from IL-27R / mice purchased from The Jackson Laboratory. Mice deficient in STAT1, produce more IL-2 than WT counterparts during infection with T. STAT4, T-bet, and the corresponding WT controls (SEJ129 or BALB/c) gondii (25). Therefore, because CD4ϩ T cells are the primary were purchased from The Jackson Laboratory. For infections, the ME49 strain of T. gondii was maintained in mice (Swiss Webster and CBA/CaJ; source of IL-2 during acute toxoplasmosis (26), these cells were Ϫ/Ϫ The Jackson Laboratory) and tissue cysts were prepared as described (25). isolated from uninfected WT or IL-27R mice, and IL-2 pro- At 5–8 wk of age, groups of three to five mice were infected with 20 cysts duction was compared during in vitro differentiation. When acti- i.p. All experiments were conducted following the guidelines of the Uni- vated under nonpolarizing (anti-CD3/anti-CD28) or Th1-polariz- versity of Pennsylvania Institutional Animal Care and Use Committee. ing conditions (anti-CD3/anti-CD28 ϩ rIL-12), IL-27RϪ/Ϫ CD4ϩ In vivo Ab treatments 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 Neutralizing anti-IL-2 (clone: S4B6) and anti-IFN-␥ (clone: XMG1.2) ϩ mAbs were generated by transferring monoclonal rat B cell hybridoma findings imply that, during infection with T. gondii, CD4 T cells cells into nude mice and precipitating Abs from the resulting ascites fluid are responsible for the exaggerated IL-2 production noted in IL- (Harlan Bioproducts). Infected mice were treated with 2 mg of sterile rat Ig 27RϪ/Ϫ animals. Consistent with this hypothesis, the frequency of (control), anti-IL-2, or anti-IFN-␥ on days 7, 9, and 11 postinfection, and IL-2ϩ CD4ϩ T cells is 2–3 times higher in IL-27RϪ/Ϫ mice than survival was monitored. Before the last Ab treatment (day 10), serum sam- WT counterparts during acute toxoplasmosis (Fig. 1D). ples were collected and levels of IFN-␥ were determined by ELISA. Upon encounter with APCs that present cognate Ags and pro- Flow cytometry vide costimulation, CD4ϩ T cells produce IL-2. In turn, this cy- ␣ For ex vivo experiments, spleens were isolated from uninfected or T. gon- tokine induces its own high affinity receptor (CD25/IL-2R ) and dii-infected mice (day 14), dissociated into a single cell suspension, and FasL, a cell surface molecule that promotes apoptosis (27, 28). depleted of erythrocytes using 0.86% (w/v) ammonium chloride (Sigma- Several factors may influence the expression CD25 and FasL, but Aldrich). Splenocytes were then washed and immediately stained for sur- because IL-2 is a dominant inducer of these proteins, their appear- face protein expression using the following Abs: anti-CD4, anti-CD25, ance on the surface of CD4ϩ T cells can be indicative of IL-2 anti-CD44, anti-CD62L, and anti-CD95 (Fas ligand (FasL)) (eBioscience). To assay in vitro cytokine production, CD4ϩ T cells were stimulated for 24 production (27, 29). Accordingly, due to a lack of IL-2 production, ϩ Ϫ Ϫ or 48 h (see below) and then pulsed with PMA (50 ng/ml; Sigma-Aldrich) most CD4 T cells from WT and IL-27R / mice display a naive and ionomycin (500 ng/ml; Sigma-Aldrich). Two hours later, cells were phenotype (CD25low FasLlow) prior to challenge with T. gondii ␮ treated with brefeldin A (BFA) (2 h of BFA at 10 g/ml; Sigma-Aldrich) (Fig. 1, C, E, and G). In contrast, during the first week of infection, and then stained for intracellular IL-2 and IFN-␥ in combination with sur- ϩ face CD4 (eBioscience). CD4 T cells from both groups become activated and produce IL-2, and a similar induction of CD25 and FasL is observed (25) ϩ In vitro CD4 T cell differentiation (data not shown). Once parasite replication is controlled, WT an- ϩ Splenocytes were isolated from uninfected mice, as above, and depleted of imals are able to contract CD4 T cell responses and, at day 14 CD8ϩ and NK1.1ϩ cells by magnetic bead separation (Polysciences). Cells postinfection, IL-2 production and surface levels of CD25 and The Journal of Immunology 239

FIGURE 1. IL-27RϪ/Ϫ CD4ϩ T cells display enhanced IL-2 responses. A,WTandIL-27RϪ/Ϫ CD4ϩ T cells were cultured under nonpolarizing conditions (anti- CD3 ϩ anti-CD28). After 48 h, culture supernatants were collected, and secretion of IL-2 was measured by ELISA. Additionally, expression of IL-2 mRNA was quantified by real-time PCR and is represented as the fold increase over naive CD4ϩ T cell controls. B,WTandIL-27RϪ/Ϫ CD4ϩ T cells were cultured under Th1-polarizing conditions (anti- CD3/CD28 ϩ rIL-12) for 48 h, and production of IL-2 was measured as above. For A and B, results are pooled from four separate experiments and the SD is represented by error bars. C–H, Splenocytes were isolated from either uninfected WT and IL-27RϪ/Ϫ mice or those challenged with T. gondii for 14 days. C and D, Cells were stimulated with plate-bound anti-CD3 Ab overnight (18 h) and treated with BFA (2 h) before staining for surface CD4 and intracellular IL-2. E and F, Surface levels of CD4 and CD25 were assayed by flow cytometry directly ex vivo, and the percentage of CD25high CD4ϩ cells is noted at the upper right of each plot. G and H, Surface levels of FasL were Downloaded from measured directly ex vivo, and the percentage of FasLhigh CD4ϩ cells is noted at the upper right. For C–H, only CD4ϩ events are displayed and results are representative of three to five separate experiments (three mice per group). I,WTandIL-27RϪ/Ϫ mice were challenged with T. gondii. At days 7, 9, and 11 postinfection, IL-27RϪ/Ϫ mice were treated with either control rat Ig, anti-IL-2 mAb, http://www.jimmunol.org/ or anti-IFN-␥ mAb, and survival was monitored. Results are representative of three separate experiments with similar results (three mice/group). J,WTandIL-27RϪ/Ϫ were infected and treated as before (I). Before the third Ab in- jection (day 11), serum samples were collected and levels of IFN-␥ measured by ELISA. Data are representative of three separate experiments, and error bars designate the SD within that trial (three mice/group). K, Naive WT and IL- 27RϪ/Ϫ CD4ϩ T cells were stimulated under Th1-polariz- by guest on September 28, 2021 ing conditions, and, where noted, IL-2 was neutralized with anti-murine mAb (anti-IL-2; 5 ␮g/ml). After 72 h, IFN-␥ production was measured by ELISA. Results are pooled from three separate experiments, and error bars designate the SD.

FasL are only slightly higher than those noted before challenge mAb (Fig. 1, I and J). Likewise, during in vitro Th1 differentiation, with T. gondii (Fig. 1, D, F, and H) (25). However, at this later IL-27RϪ/Ϫ CD4ϩ T cells produce more IFN-␥ than WT cohorts, time point, IL-27RϪ/Ϫ CD4ϩ T cells produce more IL-2 than WT but, when IL-2 is neutralized, IFN-␥ production is comparable cohorts and, in turn, the frequency of CD25ϩ and FasLϩ cells is (Fig. 1K). also higher (Fig. 1, D, F, and H) (25). Given that surface levels of Given that IL-2 promotes expansion of Th1 cells (31) and that activation markers CD44 and CD62L are comparable between WT acute toxoplasmosis is associated with a dramatic accumulation of and IL-27RϪ/Ϫ CD4ϩ T cells throughout infection (data not these cells in IL-27RϪ/Ϫ mice (25), it can be surmised that the shown), it is unlikely that this difference in CD25 and FasL is due, delay in time to death achieved through anti-IL-2 treatment may be as previously proposed (25), to an accumulation of activated cells due to a tempering of pathogenic Th1 responses. However, despite in the latter group. Instead, the current data suggest that increased greatly elevated levels of IFN-␥ in the serum of infected IL- expression of CD25 and FasL reﬂects the sustained production of 27RϪ/Ϫ mice (Fig. 1J), survival is not prolonged when this cyto- IL-2 in IL-27RϪ/Ϫ mice. kine is depleted in vivo (Fig. 1I). Because anti-IFN-␥ was admin- Because the studies presented in this work suggest that dysregu- istered only after Th1 responses were established (day 7) and these lated IL-2 production contributes to the development of lethal in- animals succumb to infection with similar kinetics as untreated ﬂammation in IL-27RϪ/Ϫ mice, this cytokine was neutralized dur- controls, it is unlikely that a recrudescence of parasite replication ing infection with T. gondii and survival was monitored. However, is the cause of death in these experiments. Instead, these studies because IL-2 is required for resistance to this parasite (30), anti- demonstrate that, during acute toxoplasmosis in IL-27RϪ/Ϫ mice, murine IL-2 mAb could be administered only after protective T IL-2 is required for the development of pathogenic CD4ϩ T cell cell responses were established and acute parasite replication was responses that are characterized by exaggerated IFN-␥ production, controlled (day 7) (25). To assess the immunological impact of this but are not dependent on it. Consistent with this hypothesis, sur- regime, serum was collected before the last treatment and levels of vival of IL-27RϪ/Ϫ mice can be prolonged by depleting CD4ϩ T IFN-␥ were measured. Compared with control animals (rat Ig), cells that produce IFN-␥ and IL-2 during infection, but not by there is improved survival and a reduction in circulating IFN-␥ depleting CD8ϩ T cells that produce IFN-␥ and little IL-2 (25). levels when infected IL-27RϪ/Ϫ mice are treated with anti-IL-2 Moreover, their survival is also improved when treated with 240 IL-27 LIMITS Th CELL IL-2 PRODUCTION

CTLA-4 Ig, a polypeptide that can block CD28-dependent co- is not surprising that rIL-27 has little effect on IL-2 production stimulation and thereby limits in vivo IL-2 production (E. Huang during the ﬁrst 24 h in culture (Fig. 2A). However, as these cells and C. Hunter, unpublished observation). Still, it must be noted begin to divide, expression of IL-27R is enhanced (6), and while that CTLA-4 Ig can deliver inhibitory signals to the APCs that it there is a natural reduction in the percentage of IL-2-positive cells binds (32) and, as a result, this rescue phenotype may not be solely after 48 h in culture (56 vs 35%), the decline is more pronounced due to a decrease in IL-2 production. when exogenous IL-27 is introduced (52 vs 13%; Fig. 2B). Be-

ϩ cause this decrease is concurrent to signiﬁcant reductions in se- IL-27 and IL-12 limit the production of IL-2 by CD4 T cells creted protein and IL-2 mRNA levels (Fig. 2C), the current data Because a lack of IL-27R is associated with enhanced IL-2 re- suggest that, either directly or indirectly, IL-27 mediates transcrip- sponses in vivo and in vitro (Fig. 1), studies were performed to tional regulation of the IL-2 gene. determine whether IL-27 directly regulates the production of this When cultured under conditions that promote Th1 differentia- cytokine. Upon ligation of the TCR and costimulatory receptor tion (anti-CD3/ CD28 ϩ rIL-12), IL-27RϪ/Ϫ CD4ϩ T cells pro- CD28, CD4ϩ T cells become activated, rapidly produce IL-2, and, duce more IL-2 than WT cohorts (Fig. 1B). Thus, to conﬁrm that given they must proliferate to express high levels of IL-27R (6), it IL-27 can limit IL-2 production regardless of the polarizing Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. IL-27 and IL-12 can suppress the production of IL-2 by activated CD4ϩ T cells. A,WTCD4ϩ T cells were labeled with CFSE and stimulated under nonpolarizing conditions (nonpolarizing anti-CD3 ϩ anti-CD28) with or without rIL-27. After 24 h, cells were pulsed with PMA/ionomycin and treated with BFA before staining for surface CD4 and intracellular IL-2. Only CD4ϩ events are displayed, and the percentage of IL-2ϩ CD4ϩ cells is displayed within the corresponding gate (n ϭ 4). B,WTCD4ϩ T cells were cultured as in A for 48 h, and production of IL-2 was assayed by ﬂow cytometry (n ϭ 6). C,WTCD4ϩ T cells were stimulated for 48 h (nonpolarizing Ϯ rIL-27) before culture supernatants were collected and secretion of IL-2 was measured by ELISA. Additionally, expression of IL-2 mRNA was quantiﬁed by real-time PCR and is represented as the fold increase over naive CD4ϩ T cell controls. Results are pooled from four separate experiments and the SD is denoted by error bars. D and E. CFSE-labeled WT CD4ϩ T cells were cultured under Th1-polarizing conditions with or without rIL-27. Production of IL-2 was assayed after 24 (D)or48(E) h, and the percentage of IL-2ϩ CD4ϩ cells is noted within the appropriate gate (n ϭ 4–5). F,WTCD4ϩ T cells were cultured (Th1 Ϯ rIL-27) for 48 h before secretion of IL-2 and expression of IL-2 mRNA were assayed, as above. Results are pooled from four separate experiments, and the SD is represented by error bars. G, CD4ϩ T cells were stimulated under nonpolarizing conditions, and, where noted, cultures were supplemented with only rIL-12. After 48 h, secretion of IL-2 and expression of IL-2 mRNA were measured as above. Results are pooled from four separate experiments, and the SD is represented by error bars. The Journal of Immunology 241

FIGURE 3. IL-23 and IL-6 do not inhibit the production of IL-2 by activated CD4ϩ T cells. A,WTCD4ϩ T cells were stimulated under nonpolarizing conditions (anti-CD3 ϩ anti-CD28) and, where noted, cultures were supplemented with rIL-23, IL-12, or both cytokines. After 48 h, cells were pulsed with PMA/ionomycin and treated with BFA before staining for surface CD4 and intracellular IL-2. B,WTCD4ϩ T cells were stimulated as in A, and, where noted, cultures were supplemented with rIL-6, IL-27, or both cytokines. A and B, Only CD4ϩ events are displayed, and the percentage of IL-2ϩ CD4ϩ cells is displayed within the corresponding gate (n ϭ 3–4).

environment present at the time of priming, CD4ϩ T cell cul- nents (IL-12R␤1/gp130) with those which possess this capacity tures were supplemented with IL-12 and the percentage of IL- (IL-12/IL-27), it can now be proposed that the ligand-specific Downloaded from 2-positive cells was assayed. Similar to IL-27R, the ligand- subunits of the IL-12R (IL-12R␤2) and IL-27R (IL-27R) deliver specific component of the IL-12R (IL-12R␤2) is not expressed the pertinent inhibitory cues. ϩ at high levels until CD4 T cells have proliferated (33), and, as The present studies reveal that IL-12 and IL-27 can synergize to a result, neither IL-27 nor IL-12 affects the production of IL-2 inhibit IL-2 production, but previous work has demonstrated sim- during the first 24 h of culture (Fig. 2, A and D). Surprisingly, ilar cooperation for the induction of IFN-␥ (7, 18). However, while ϩ after 48 h, the percentage of IL-2-positive CD4 T cells is the current findings demonstrate that each can suppress IL-2 pro- http://www.jimmunol.org/ significantly reduced when cultures are supplemented with only duction with similar potency (Fig. 2), they also support the idea IL-12 (35 vs 12%; Fig. 2, B and E), and when IL-27 is also that IL-12 is the dominant factor in driving Th1 differentiation. added, IL-2 production is almost completely abolished (3%; When CD4ϩ T cells are cultured under nonpolarizing conditions, Fig. 2E). Given the corresponding reductions in levels of IL-27 prompts a small rise in the percentage of IFN-␥-positive mRNA and secreted protein (Fig. 2, F and G), these studies cells and only a modest increase in the amount of IFN-␥ they establish that IL-12 and IL-27 cooperate to limit IL-2 produc- secrete (Fig. 4). In contrast, when IL-12 is added to these cultures, tion when CD4ϩ T cells are activated in highly polarizing Th1 there is a 3-fold expansion in the percentage of IFN-␥-positive conditions. In turn, because both are evident during infection ϩ CD4 T cells and the amount of cytokine produced increases dra- by guest on September 28, 2021 with T. gondii, it is likely that IL-12 and IL-27 contribute to the matically (Fig. 4). Because exogenous IL-27 has little effect on suppressed IL-2 responses that are a hallmark of acute toxo- IFN-␥ production when polarizing concentrations of IL-12 are plasmosis in WT mice (34, 35). available, these studies confirm a central role for IL-12 in promot- Because IL-12 and IL-27 can each suppress IL-2 production, ing Th1 differentiation, and they suggest that IL-27 alone is not experiments were performed to determine whether this property is sufficient or required to polarize effector T cell populations. mimicked by other IL-6/IL-12 family cytokines. Despite a common cytokine (IL-12p40) and receptor (IL-12R␤1) subunit (36), the current work demonstrates that IL-12 and IL-23 do not share The role of STAT4, STAT1, and T-bet in regulating IL-2 the ability to suppress Th cell IL-2 production (Fig. 3A). Likewise, production 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. IL-6/IL-12 family cytokines mediate cellular effects upon binding 3B). Given that IL-23R and IL-6R␣ mRNA can be readily detected their cognate receptors and thereby inducing phosphorylation of in these activated CD4ϩ T cells (data not shown), the inability of Jak/STAT proteins that migrate to the nucleus and promote or IL-23 and IL-6 to limit IL-2 production is not due to a lack of suppress expression of target genes (4). Therefore, because IL-12 responsiveness. Instead, it is clear that these cytokines do not sup- and IL-27 can activate common signaling components in CD4ϩ T press IL-2 production and, because they share receptor compo- cells (36), it is possible that they use analogous pathways to inhibit

FIGURE 4. Comparing the potency of IL-27 and IL-12 in promoting Th1 differentiation. A,WTCD4ϩ T cells were labeled with CFSE, stimulated under nonpolarizing conditions (anti-CD3 ϩ anti-CD28), and, where noted, cultures were supplemented with rIL-27, IL-12, or both cytokines. After 48 h, cells were pulsed with PMA/ionomycin and treated with BFA before staining for surface CD4 and intracellular IFN-␥. Only CD4ϩ events are displayed and the percentage of IFN-␥ϩ CD4ϩ cells is displayed within the corresponding gate (n ϭ 5). B,WTCD4ϩ T cells were cultured as in A for 72 h, and secretion of IFN-␥ was measured by ELISA. Results are pooled from four separate experiments, and the SD is denoted by error bars. 242 IL-27 LIMITS Th CELL IL-2 PRODUCTION

IL-2 production. Phosphorylation of STAT4 is the most charac- counterparts, there is a pronounced reduction in the percentage of teristic signaling event for IL-12 (18), and because this transcrip- IL-2-positive cells when STAT1-deficient cultures are supple- tion factor can also be activated by IL-27, albeit to a lesser extent mented with IL-27 (Fig. 5C). Moreover, although it induces IFN-␥ (10), the ability of these cytokines to suppress IL-2 production was by promoting expression of T-bet, a transcription factor that has measured in STAT4Ϫ/Ϫ CD4ϩ T cells. As above (Fig. 2), IL-12 been shown to suppress IL-2 production (37), IL-27 can also me- limits the production of IL-2 by WT CD4ϩ T cells (19 vs 8%; Fig. diate this effect in T-bet-deficient CD4ϩ T cells (Fig. 5D). Con- 5A), but, in the absence of STAT4, it prompts only a modest re- sistent with these findings, IFN-␣ and IFN-␥ both activate STAT1 duction in the percentage of IL-2-positive cells (45 vs 36%; Fig. and induce expression of T-bet, but neither affects the frequency of 5B). In fact, even when exogenous IL-12 is not added (nonpolar- IL-2-positive CD4ϩ T cells in nonpolarizing cultures (data not izing), there is a greater frequency of IL-2-positive cells in shown and Fig. 5E) (9, 38). Together, these studies demonstrate STAT4-deficient cultures than in WT counterparts (45 vs 19%; that IL-12 can limit IL-2 production through largely STAT4-de- Fig. 5, A and B). These findings demonstrate that, when present at pendent mechanisms, and that IL-27 does so independently of the time of CD4ϩ T cell priming, IL-12 can suppress IL-2 pro- STAT4, STAT1, and T-bet. duction through STAT4-dependent mechanisms. However, be- Ϫ/Ϫ ϩ cause IL-27 is a potent inhibitor of IL-2 in either WT or STAT4Ϫ/Ϫ IL-27R CD4 T cells display enhanced proliferation in the CD4ϩ T cell cultures (Fig. 5, A and B), it is also apparent that absence of IL-2 STAT4-independent mechanisms can mediate this effect. Because IL-2 is a potent growth and survival factor, the availabil- Although the current findings demonstrate that IL-12 and IL-27 ity of this cytokine has significant influence on the proliferation of ϩ Ϫ Ϫ ϩ use different signaling pathways to suppress IL-2 production, it is activated CD4 T cells (39). In turn, IL-27R / CD4 T cells Downloaded from well established that they promote IFN-␥ production through dis- produce more IL-2 than WT counterparts, this cytokine may be tinct means (36). Still, given that IL-12 employs STAT4 to inhibit responsible for the enhanced proliferation that has been noted in IL-2 (Fig. 5B) and to promote IFN-␥ (18), it is possible that IL-27 the former group (8, 25, 40). To determine whether enhanced IL-2 achieves the former (IL-2 suppression) by activating STAT1, the production is indeed responsible for this hyperproliferative pheno- same transcription factor that it employs to induce the latter (IFN-␥ type, WT and IL-27RϪ/Ϫ CD4ϩ T cells were labeled with CFSE

production) (9–11, 14). Consequently, the ability of IL-27 to sup- and then cultured with or without (anti-IL-2) endogenous cytokine. http://www.jimmunol.org/ press IL-2 was examined in STAT1Ϫ/Ϫ CD4ϩ T cells. As in WT After 72 h, dilution of CFSE was visualized by ﬂow cytometry and by guest on September 28, 2021

FIGURE 5. The role of STAT4 and STAT1 in regulating IL-2 production during CD4ϩ T cell differentiation. A–D, CD4ϩ T cells from WT (A), STAT4Ϫ/Ϫ (B), STAT1Ϫ/Ϫ (C), or T-betϪ/Ϫ (D) mice were stimulated under nonpolarizing conditions (anti-CD3 ϩ anti- CD28), and, where noted, cultures were supplemented with rIL-27, IL-12, or both cytokines. After 48 h, cells were pulsed with PMA/ionomycin and treated with BFA before staining for surface CD4 and intracellular IL-2. Only CD4ϩ events are displayed, and the percentage of IL-2ϩ CD4ϩ cells is displayed at the upper right corner of each plot (n ϭ 3). E,WTCD4ϩ T cells were cultured under nonpolarizing conditions with or without rIFN-␥ for 48 h. Only CD4ϩ events are displayed, and the percentage of IL-2ϩ CD4ϩ cells is displayed at the upper right (n ϭ 3). The Journal of Immunology 243

FIGURE 6. IL-27RϪ/Ϫ CD4ϩ T cells display enhanced proliferation in the absence of IL-2. A,WTandIL-27RϪ/Ϫ CD4ϩ T cells were labeled with CFSE Downloaded from and cultured under nonpolarizing or Th1 conditions with or without anti-IL-2 mAb. After 72 h, dilution of CFSE was quantified by flow cytometry, and the number of cells in each proliferative generation was annotated. CFSE profiles and cell numbers from one representative experiment are shown here. B, These values were then used to calculate the proliferative capacity and divisions per cell for each group. Individual experiments have been assigned distinct, corresponding symbols, and f represents the mean of all trials (n ϭ 6–7). A two-tailed, paired t test (p ϭ) was used to determine statistical difference between WT and IL-27RϪ/Ϫ cells. http://www.jimmunol.org/ the number of cells in each proliferative generation was used, as Discussion described previously (41), to determine the absolute number of Positive and negative regulation of IL-2 production during mitotic events in each culture (Fig. 6A). The proliferative capacity CD4ϩ T cell differentiation and number of divisions per cell were then extrapolated from these Ϫ Ϫ ϩ Upon encounter with pathogens and inflammatory stimuli in pe- calculations and compared between WT and IL-27R / CD4 T ripheral tissues, professional APCs migrate to primary lymphoid cells. Similar to prior observations (25), the proliferative capacity Ϫ Ϫ ϩ

organs, where they present Ags in the context of class II MHC by guest on September 28, 2021 and divisions per cell are higher for IL-27R / CD4 T cells than molecules and provide costimulation for the activation of naive WT cohorts in nonpolarizing or Th1-polarizing cultures (Fig. 6B). CD4ϩ T cells (46). Although the interaction with APCs provides Additionally, consistent with the findings of others (A. Wells, un- all of the signals necessary to induce proliferation, ligation of the published observations) and with studies in which CD28-depen- TCR and CD28 also prompts CD4ϩ T cells to quickly secrete IL-2, dent, IL-2-independent signals are sufficient to induce T cell ex- a cytokine that further enhances their proliferation and survival pansion (42), neutralization of IL-2 results in a modest, but (47). Given the paucity of Ag-specific CD4ϩ T cells at the onset of significant ( p Ͻ 0.05), decrease in the proliferation of WT and Ϫ Ϫ adaptive immunity, the immediate production of IL-2 ensures that IL-27R / T cells. Even so, the proliferative capacity and daugh- ters per cell are still higher for IL-27RϪ/Ϫ CD4ϩ 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-27RϪ/Ϫ CD4ϩ T cells (Fig. 6B).

IL-27 can induce expression of SOCS3 in CD4ϩ T cells When cultured in the absence of IL-2, IL-27RϪ/Ϫ CD4ϩ 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 FIGURE 7. IL-27 can promote expression of SOCS3. Expression of ϩ inhibitory factor that has been shown to limit effector T cell pro- SOCS proteins was compared between unstimulated WT CD4 T cells liferation (44, 45). Furthermore, like IL-27, IL-12 also promotes (Un.) and those cultured under nonpolarizing or Th1 conditions. All cultures contained neutralizing anti-IL-2 mAb, and, where noted, they were expression of SOCS3, but neither cytokine affects mRNA levels supplemented with rIL-27, IL-12, IL-6, or a combination of these cyto- for CIS, SOCS1, and SOCS2 (Fig. 7 and data not shown). Thus, kines. After 48 h, mRNA was isolated, and expression of SOCS1, SOCS3, despite being viewed as proinﬂammatory agents, the current data and ␤-actin was detected by RT-PCR. Additionally, cells were stimulated establish that IL-27 and IL-12 share two analogous inhibitory in the presence of anti-IL-2 and anti-IFN-␥ mAb before measuring mRNA properties, the ability to suppress IL-2 production and to induce levels for SOCS3 and ␤-actin. Results are representative of four separate expression of SOCS3. experiments. 244 IL-27 LIMITS Th CELL IL-2 PRODUCTION

FIGURE 8. A cell cycle requirement for IL-27 and IL-12 to mediate pro- and anti-inﬂammatory effects on CD4ϩ T cells. WT CD4ϩ T cells were labeled with CFSE and stimulated under nonpolarizing conditions, and, where noted, cultures were supplemented with rIL-

27, IL-12, or both cytokines. Cells were arrested in G1 phase of the cell cycle by treating with L-mimosine (300 ␮M) before and during culture. After 48 h, cells were pulsed with PMA/ionomycin and treated with BFA before staining for surface CD4 and intracellular IL-2 or IFN-␥. Only CD4ϩ events are displayed, and the percentage of IL-2ϩ or IFN-␥ϩ CD4ϩ cells is displayed within the corresponding gate. Results are representative of four separate experiments.

a potent growth factor is available to support the initial expansion in promoting differentiation into Th1 effectors that secrete large of clonotypic T cells. In fact, because activated CD4ϩ T cells can amounts of IFN-␥, and, when concentrations of IL-12 are limiting, Downloaded from produce IL-2 before G1 phase of the cell cycle while they must IL-27 can mediate this effect. These proinflammatory properties cross this developmental checkpoint to become effector Th1 or are well understood, but the data presented in this work reveal that Th2 cells capable of secreting IFN-␥ and IL-4, respectively, IL-2 IL-12 and IL-27 are also potent inhibitors of IL-2 production (Fig. production precedes differentiation (48). Consistent with this idea, 9). Therefore, as they determine the polarity of effector T cell CD4ϩ T cell activation induces rapid epigenetic changes that pro- responses, IL-12 and IL-27 also limit the availability of a key mote IL-2 gene accessibility and transcription prior to cell cycle growth and survival factor (Fig. 9). Still, IL-12 and IL-27 cannot http://www.jimmunol.org/ entry (49). suppress IL-2 production until CD4ϩ T cells have crossed through ϩ Aside from providing the impetus for CD4 T cell activation G1 phase of the cell cycle (Fig. 8B), thus guaranteeing the rapid and IL-2 production, APCs can produce IL-12 and IL-27, two burst of IL-2 that is induced by activation and the initial nonpolar cytokines that promote Th1 differentiation (18, 36). Still, although expansion of Ag-specific CD4ϩ T cells. This model is consistent IL-12 and IL-27 may be present at the time of Ag encounter, naive with the idea that IL-2 is required to expand Ag-specific CD4ϩ T CD4ϩ T cells express low levels of IL-12R␤2 (12) and IL-27R (6). cell populations during a nascent immune response (31, 50) and Consequently, these cytokines have little influence on the events with the finding that exaggerated IL-2 production can mediate se- that induce IL-2 production, and, in turn, they do not significantly vere pathology, as during acute toxoplasmosis in IL-27RϪ/Ϫ mice. enhance IFN-␥ production until after CD4ϩ T cells have passed In fact, because IL-27 does not appear to determine the polarity of by guest on September 28, 2021 through G1 phase of the cell cycle (Fig. 8A) (48). However, once in vivo T cell responses (24, 25), it can now be proposed that these cells begin to proliferate, IL-12 becomes the dominant factor limiting IL-2 production is a primary function for this cytokine,

FIGURE 9. A model for the positive and negative regulation of IL-2 production during CD4ϩ T cell differentiation. After they encounter pathogens and inflammatory stimuli, professional APCs enter primary lymphoid organs, where they present Ags in the context of MCHII molecules and provide the costimulation necessary for CD4ϩ T cell priming. Through their interactions with APCs in the spleen and lymph nodes, naive CD4ϩ T cells quickly produce large amounts of IL-2 and begin to proliferate. Because APCs can also secrete IL-12 and IL-27, they may promote the differentiation of activated CD4ϩ T cells into Th1 effector cells that secrete large amounts of IFN-␥, but relatively little IL-2. Committed Th1 cells then migrate to sites of inflammation, where IFN-␥ can induce a range of antimicrobial responses. In these periph- eral tissues, IL-12 and IL-27 further promote type I (Th1) immunity by enhancing survival and IFN-␥ production by Th1 cells that express high levels of IL- 12R␤2 and IL-27R. Additionally, these cytokines may curb extralymphoid proliferation by inhibiting IL-2 production. The Journal of Immunology 245 while enhancing IFN-␥ production is secondary. Moreover, be- mRNA (62), and several nuclear proteins have been shown to cause IL-27 is induced by pathogens that elicit Th1 or Th2 re- suppress IL-2 production when CD4ϩ T cells become anergic sponses (24, 25), it may occupy a central role in suppressing IL-2 (63). Nevertheless, IL-2 production can be regulated by post- production regardless of the polarizing cytokine environment transcriptional modifications, and it is also possible that IL-27 present during T cell activation. Accordingly, IL-27 can also in- and IL-12 inhibit cytokine production without directly affecting hibit IL-2 production when CD4ϩ T cells are cultured under Th2- IL-2 gene transcription (64). polarizing conditions (data not shown). Additional inhibitory properties for IL-27 Cellular factors that limit Th cell IL-2 production The current study demonstrates that, by neutralizing IL-2 during The importance of IL-2 as a T cell growth factor has been appre- infection with T. gondii, the survival of IL-27RϪ/Ϫ mice can be ciated for over two decades (51). Still, although the cellular and prolonged. However, while these data suggest that exaggerated molecular mechanisms that induce this cytokine are well under- IL-2 production plays a central role in the development of patho- stood (52), the processes that temper IL-2 production during acute genic T cell responses during infection, it is possible that treatment inflammatory responses remain obscure. Various pharmacological with anti-IL-2 mAb acts as a general immunosuppressant and that agents, including cyclosporine A and FK506 (53, 54), can inhibit dysregulated IL-2 production may not be causative in the devel- IL-2 production, and several host-derived factors have been shown opment of this parasite-induced inflammatory disease. These in- to mediate this effect. Two examples are the inhibitory receptor terpretations are not mutually exclusive, and because anti-IL-2 CTLA-4 and the regulatory cytokine TGF-␤; the former can sup- mAb-treated mice eventually succumb to infection, it is likely that press IL-2 production by preventing CD28-dependent costimula- the IL-27R delivers inhibitory signals that are distinct from those Downloaded from tion, and the latter by activating Smad3, a transcription factor that regulating the production of IL-2. Consistent with this idea, pre- binds the IL-2 gene promoter and hinders transcription (55, 56). vious reports have established that the production of various in- Along with others, these observations have established that IL-2 flammatory cytokines, including IL-4, IL-6, IL-12, TNF-␣, and production can be regulated by factors that are classically associ- GM-CSF, is exaggerated during parasitic infections in IL-27RϪ/Ϫ ated with the suppression of Th cell responses. In contrast, the data mice, suggesting that IL-2 alone is not the cause for immunopa- presented in this work demonstrate that IL-2 production is also thology during toxoplasmosis. Moreover, the current work dem- http://www.jimmunol.org/ limited by two cytokines that promote the generation of type I onstrates that IL-27 can also induce expression of SOCS3, an in- (Th1) inflammation. A role for IL-12 in regulating IL-2 production hibitory protein that has been shown to limit the inflammatory has been noted previously (57), but the current work elaborates by responses associated with colitis and asthma (65, 66). Even so, demonstrating that this effect is largely mediated by the activation further studies are needed to assess the in vivo relevance of of STAT4. Furthermore, the present study establishes that IL-12 IL-27-dependent SOCS3 induction, and, because depletion of shares this property with IL-27, but not fellow IL-6/IL-12 family IL-2 was only transient in the current experiments, it is possible members IL-6 and IL-23. Together, IL-12 and IL-27 can effec- that continued administration of anti-IL-2 mAb would further ϩ tively shut down the production of IL-2 by CD4 T cells in vitro prolong the survival of IL-27RϪ/Ϫ mice, again, suggesting a (Fig. 2), and because exaggerated IL-2 production has been noted central role for IL-2 in the immunopathology that develops in by guest on September 28, 2021 during inflammatory responses in mice deficient for either cyto- those animals. Still, it should be noted that because SOCS3 kine, this effect is also apparent in vivo (25, 58). tempers signaling by gp130 (67), the current findings support The data presented in this work demonstrate that IL-27 can sup- the idea that SOCS proteins are part of a classic negative feed- press IL-2 in the absence of STAT4, STAT1, and T-bet. Moreover, back loop that regulates IL-6/IL-12 family cytokines (43). because IL-6 induces phosphorylation of STAT3 (4), but does not Moreover, these studies are the first to describe cellular mech- inhibit IL-2 production, it is unlikely that IL-27 employs this sig- anisms for the anti-inflammatory effects of IL-27, and, as such, naling pathway to do so. Although direct evidence is still required they build on mounting evidence that IL-27 is critical for lim- to confirm that IL-27 can suppress IL-2 production without iting the intensity of adaptive immune responses. STAT3, the fact that IL-2 production is similar between WT and STAT3-deficient CD4ϩ T cells is consistent with this notion (59). Acknowledgments Therefore, given that IL-2 mRNA levels are reduced when an ac- We thank members of the Hunter laboratory for intellectual and experi- ϩ tivated form of STAT5 is expressed in CD4 T cells (60), it is mental input during the course of these studies. We also thank tempting to speculate that IL-27 uses this transcription factor to Drs. David Artis, Ed Pearce, Phil Scott, Robyn Starr, and Andrew Wells for propagate a similar effect. Accordingly, IL-27 is a potent inducer providing useful comments during the preparation of this manuscript. of STAT5 phosphorylation (10, 14, 25), and because IL-12 can also induce some STAT5 activation (18), this hypothesis accounts Disclosures for the limited ability of IL-12 to inhibit IL-2 production in C. A. Hunter and A. V. Villarino have a patent pending on the anti-in- STAT4Ϫ/Ϫ CD4ϩ T cells. Nevertheless, while the current studies flammatory properties of IL-27. have excluded several candidate pathways (STAT1/3/4), the molecular mechanisms that IL-27 employs to suppress IL-2 produc- References 1. Pflanz, S., J. C. Timans, J. Cheung, R. Rosales, H. Kanzler, J. Gilbert, L. Hibbert, tion remain uncertain. T. Churakova, M. Travis, E. Vaisberg, et al. 2002. 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