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Proc Natl Acad Sci U S A. Aug 4, 2009; 106(31): 12885–12890.
Published online May 11, 2009. doi:  10.1073/pnas.0812530106
PMCID: PMC2722314
Immunology

IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells

Abstract

The development of T helper (TH)17 and regulatory T (Treg) cells is reciprocally regulated by cytokines. Transforming growth factor (TGF)-β alone induces FoxP3+ Treg cells, but together with IL-6 or IL-21 induces TH17 cells. Here we demonstrate that IL-9 is a key molecule that affects differentiation of TH17 cells and Treg function. IL-9 predominantly produced by TH17 cells, synergizes with TGF-β1 to differentiate naïve CD4+ T cells into TH17 cells, while IL-9 secretion by TH17 cells is regulated by IL-23. Interestingly, IL-9 enhances the suppressive functions of FoxP3+ CD4+ Treg cells in vitro, and absence of IL-9 signaling weakens the suppressive activity of nTregs in vivo, leading to an increase in effector cells and worsening of experimental autoimmune encephalomyelitis. The mechanism of IL-9 effects on TH17 and Tregs is through activation of STAT3 and STAT5 signaling. Our findings highlight a role of IL-9 as a regulator of pathogenic versus protective mechanisms of immune responses.

Keywords: autoimmunity, regulatory cells, tolerance

In the presence of antigen stimulation, naïve CD4+ T cells proliferate and differentiate into T helper type 1 (TH1) cells, TH2 cells, or interleukin-17 (IL-17)-producing T helper cells (TH17 cells) to exert specific effector functions. TH17 cells express the transcription factor retinoic acid orphan receptor gamma (ROR-γ)t (1), participate in the control of extracellular pathogens, and have an important role in human and experimental autoimmunity (2). TH17 cells have been identified as major inducers of tissue inflammation and autoimmunity. Since exaggerated responses of TH1, TH2, and TH17 cells can induce tissue inflammation, the maintenance of immune homeostasis and prevention of immunopathology is mediated by subsets of T cells called regulatory T cells (Tregs). Treg cell differentiation and function are driven by the transcription factor forkhead box P3 (FoxP3) (3, 4), and they are closely related to the generation of TH17 cells. Transforming growth factor (TGF)-β1 induces the differentiation of Treg cells (5), whereas TGF-β1 in combination with IL-6 (6, 7) or IL-21 (8) results in the differentiation of TH17 cells.

IL-9 is a T cell-derived factor preferentially expressed by TH2 cells (9), although a previous study suggested that regulatory T cells produce more IL-9 than TH2 cells (10); however this was not confirmed in induced (iTregs) or natural (nTregs) (11). It is a pleiotropic cytokine that targets cells of the lymphoid, myeloid, and mast cell lineages, as well as lung epithelial cells. IL-9 activities are mediated by a specific IL-9 receptor chain that forms a heterodimeric receptor with the common gamma chain (γc) also involved in IL-2, 4, 7, 15, and 21 signaling. The IL-9 receptor and γc associate with Janus kinase (JAK)1 and JAK3, respectively, and trigger the activation of STAT1, 3, and 5 (12, 13). Dysregulated IL-9 response in vitro can lead to autonomous cell growth and malignant transformation of lymphoid cells associated with constitutive activation of the JAK/STAT pathway (12). We have recently described a subset of T cells that predominantly produces IL-9 together with IL-10 and do not exhibit any regulatory properties despite producing large amounts of IL-10, but induce severe colitis and peripheral neuritis upon adoptive transfer into immune deficient hosts (14). Another recent report describes reprogramming of TH2 cells by TGF-β to cells producing IL-9 and IL-10 (11), and these cells were also shown to have effector and not regulatory functions. These data suggest that IL-9/IL-10-producing T cells are not regulatory T cells but effector T cells that induce tissue inflammation. However, whether these effector functions are mediated by IL-9 and what role does IL-9 play in pathogenicity of this unique subset of T cells has not been evaluated.

In the present study, we show that TH17 cells produce large quantities of IL-9 that act on both TH17 and Treg cells. In the presence of TGF-β, IL-9 differentiates naïve CD4+ T cells into TH17 cells. Paradoxically, IL-9 also acts on nTregs and enhances their suppressive function in vitro and in vivo. Mice lacking IL-9 receptor (IL-9R−/−) exhibit a more severe course of experimental autoimmune encephalomyelitis (EAE) and have a defect in the suppressive activity of Tregs.

Results

IL-9 Is Produced by TH17 Cells.

TH17 cells produce several cytokines including IL-17, IL-21, and IL-22 (2). We sought to determine the additional cytokines that are secreted by TH17 cells. FACS-sorted naïve CD4+CD62LhiFoxP3 T cells isolated from FoxP3.GFP “knock-in” (FoxP3.GFP.KI) reporter mice were differentiated into TH1, TH2, and TH17 according to established protocols (Fig. S1A). Inducible Tregs(iTregs) (7) were generated by activating CD4+CD62LhiFoxP3 T cells in the presence of TGF-β (Fig. S1B), and nTregs were sorted from naïve FoxP3.GFP.KI mice based on in vivo FoxP3 expression (Fig. S1C). Using multiplex bead-based Luminex technology, we identified IL-9 as a dominant cytokine produced both by TH2 and TH17 cells (Fig. 1A). These data were confirmed by quantitative Taqman PCR (Fig. 1A) and by flow cytometry (Fig. 1B). Moreover, IL-9 was not detected in the supernatants of nTregs or iTreg (Fig. 1A), but IL-9R mRNA was highly expressed by nTregs and not by iTregs (Fig. 1A).

Fig. 1.
TH17 cells produce IL-9. CD4+CD62LhiFoxP3/GFP T cells from FoxP3-GFP.KI mice were stimulated with anti-CD3 and anti-CD28 for 3–5 days in the presence of the corresponding cytokines. (A) IL-9 relative expression as determined by bead-based ...

Naïve CD4+ T cells activated with plate-bound anti-CD3 and anti-CD28 in the presence of recombinant mouse IL-9 had increased secretion of the TH2 (IL-4, IL-10, and IL-13), cytokines, and at the same time down-regulated IFN (IFN)-γ and granulocyte-macrophage colony stimulation factor (GM-CSF) production (Fig. 1C and Fig. S2). Interestingly, cells exposed to IL-9 produce significant amounts of TGF-β1 and this was confirmed by Taqman PCR (Fig. 1C). We also observed an increase in the IL-9 mRNA levels in response to IL-9 indicating an autocrine positive feedback (Fig. S2), by which IL-9 induces T cells that further produce IL-9, and thus setting up an autoamplification loop. In combination with TGF-β, IL-9 had a similar effect on T cell differentiation as IL-21 (Fig. 1C and Fig. S2). Unlike IL-6, which normally inhibits TGF-β induced FoxP3, IL-9 did not inhibit the TGF-β-mediated conversion of FoxP3 (GFP) naïve CD4+ T cells from the FoxP3.GFP.KI reporter mice into FoxP3+GFP+ Treg cells (Fig. S3).

IL-9 Synergizes with TGF-β1 to Differentiate Naïve CD4+ Cells into TH17 Cells.

Given the critical role of the transcription factor STAT3 in TH17 differentiation (15) and given that IL-9 has been reported to induce activation of STAT3 (16), we next examined whether IL-9 could promote TH17 differentiation. As shown in Fig. 2A, stimulation with IL-6 plus TGF-β induced large amounts of IL-17 (Fig. 2A), and replacement of IL-6 by IL-9 was also effective in inducing IL-17-producing cells. Like IL-6, IL-9 in the absence of TGF-β could not induce TH17 differentiation. The effects of IL-9 on TH17 cell induction were comparable to those of IL-21 (Fig. 2 A and B). Moreover, neutralizing IL-9 with anti-IL-9 blocking antibody (10 μg/mL) reduced the amounts of IL-17 induced by IL-6 plus TGF-β or IL-21 plus TGF-β, suggesting that IL-9 produced by differentiating TH17 cells further enhances the development of TH17 cells (Fig. 2A). Induction of IL-17 by TGF-β plus IL-9 was confirmed at the single cell level with naïve CD4+ T cells activated for 2 rounds of 4 days each in the presence of TGF-β plus IL-9 and co-stained for IL-17/IL-4 (Fig. 2B) or IL-17/IFN-γ (Fig. S4), indicating that the combination of IL-9 and TGF-β is as effective in inducing differentiation of TH17 cells (Fig. 2B and Fig. S4). TH17 cells were the highest producers of IL-9 among the T cell subsets, suggesting an amplification loop in which IL-9 produced by TH17 cells participates in enhancing further differentiation of TH17 cells. This observation was further supported by the finding that the frequency of TH17 cells induced in IL-9R−/− cells by IL-6 plus TGF-β was significantly reduced compared to WT CD4+ T cells (Fig. 2C), suggesting that IL-9 might normally contribute to TH17 differentiation even when TH17 cells were differentiated by other cytokine cocktails (TGF-β plus IL-6 or TGF-β plus IL-21). To rule out the possibility that IL-6 may contribute to the induction of TH17 differentiation by IL-9, we compared the differentiation of naïve wild-type (WT) and IL-6−/− T cells in vitro in the presence of TGF-β and IL-9. Our data showed that IL-9 plus TGF-β was sufficient to drive the differentiation of IL-6−/− T cells into TH17 cells, although less efficiently than for WT T cells (Fig. S5).

Fig. 2.
IL-9 induces TH17 cell differentiation. (A) IL-17 production by naïve CD4+CD62Lhi T cells cultured under optimal TH17 conditions or under conditions in which IL-6 was omitted and replaced with IL-9, as measured by bead-based Luminex assay. (B ...

IL-23 Is a Negative Regulator of IL-9.

IL-23 is crucial for maintenance/stabilization but not for the differentiation of TH17 cells. Given that IL-23 has been shown to enhance the pathogenic potential of myelin-specific T cells in the induction of autoimmune encephalomyelitis (17), we tested the effects of IL-23 on TH17 cells. TH17 cells exposed to IL-23 during a secondary stimulation had a significantly reduced production of IL-9, while IL-17 production was preserved (Fig. 3A). Furthermore, TH17 cells from IL-23R−/− mice produced significantly higher levels of IL-9 as compared to WT cells in a secondary stimulation with IL-6 and TGF-β (Fig. 3B). These data were confirmed by quantitative PCR (Fig. 3C). Our findings suggest that IL-23 is a negative regulator of IL-9 secretion by TH17 cells.

Fig. 3.
IL-23 is a negative regulator of IL-9. IL-17 production by naïve CD4+CD62Lhi T cells cultured under optimal TH17 conditions. (A) The upper figures show IL-9 and IL-17 production after primary stimulation of naïve CD4+ T cells by TGF-β ...

IL-9 Enhances the Suppressive Functions of Regulatory FoxP3 CD4+ T Cells.

Given that nTregs express IL-9R but IL-9 does not inhibit Treg induction in vitro (Fig. 1D), we investigated the effects of IL-9 on Treg function. Purified CD4+FoxP3/GFP+ T cells from naïve FoxP3.KI mice suppressed CD3 driven effector T cell responses. Blockade of IL-9 signaling using an anti-IL-9-neutralizing antibody reversed nTreg-mediated suppression allowing effector T cells to proliferate (Fig. 4A) and produce effector cytokines (Fig. 4B). In contrast, addition of rIL-9 (20 ng/mL) increases the suppressive capacity of nTregs (Fig. 4A) and further decreased the production of IL-2, IL-17, and IFN-γ (Fig. 4B) as well as IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-1β (Fig. S6A).

Fig. 4.
IL-9 enhances the suppressive functions of Tregs. (A) CD4+FoxP3/GFP+ Tregs isolated by FACS sorting from the spleens of naïve FoxP3 reporter mice were tested for their suppressive capacity in vitro. A 1:1 ratio of naïve responder T cells ...

IL-9 has been shown to protect T cells against apoptosis through a STAT3/STAT5-dependent mechanism (13, 16). On the other hand, the transcription factors STAT3 and STAT5 are crucial for the development of TH17 and nTreg cells, respectively (1, 15, 18). Purified nTreg cells isolated from naïve FoxP3.KI mice and cultured for 4 days die quickly in a serum-free medium (Fig. 4C). Interestingly, addition of IL-9 (20 ng/mL) partially rescued Treg cells from apoptotic cell death and significantly increased the number of viable cells indicating that IL-9 could act as a survival factor for nTregs (Fig. 4C).

Signaling by IL-9 to nTreg cells is mediated by STAT3 and STAT5 phosphorylation within 30 min of IL-9 addition, as shown by increased mean fluorescent intensity (MFI) (P < 0.05 for pSTAT3 and P < 0.01 for pSTAT5) (Fig. 4D). IL-9 also increased the phosphorylation of ribosomal protein S6 kinase (P70S6 kinase) and of inhibitor of kappa B-alpha (IκB-α), a negative regulator of nuclear factor-kappa B (NF-κB) signaling pathway, indicating an increase in the activation of NF-κB (Fig. S6B). IL-9 did not regulate the phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (p38), nor the one of extracellular signal-regulated kinase (ERK MAPK1/2) and Akt, suggesting that the effects of IL-9 on nTregs are not mediated by these signaling pathways (Fig. S6B). IL-9-treated nTregs showed no changes in β-catenin total protein level, a protein that was recently shown to have powerful effects on the survival of Tregs (19) (Fig. S6B).

Endogenous IL-9 Regulates Treg Activity and Inflammation In Vivo.

Our findings that IL-9 influences both TH17 cell differentiation and Treg suppressive activity suggest that endogenous levels of IL-9 might participate in immune regulation in vivo. Indeed, using EAE, we addressed the role of IL-9 production on the immune responses in EAE by taking advantage of IL-9R−/− mice that have deletion of exons 2 to 6, which encode the entire extracellular domain of the receptor (20). IL-9R−/− mice immunized with suboptimal doses of MOG35–55 (75 μg) developed earlier and more severe disease compared to WT mice (mean disease onset 10.8 ± 1.1 versus 15.8 ± 0.9, P = 0.009 by unpaired t test and mean maximal score 2.5 ± 0.2 versus 1.2 ± 0.3, P = 0.008 by Mann-Whitney, respectively) (Fig. 5A). We confirmed these findings using anti-IL-9 blocking antibody in EAE induced in SJL/J mice (Fig. S7). In the periphery, T cells from IL-9R−/− mice mounted a strong proliferative response to MOG35–55 stimulation compared to WT cells (Fig. 5B). Next, we studied the cytokine profile of lymph node (onset of the disease) and central nervous system (CNS)-infiltrated CD4+ T cells (peak of the disease) isolated from the immunized mice and analyzed by flow cytometry. In the peripheral compartment, we found that IL-9R−/− mice had a higher frequency of TH1 (CD4+IFN-γ+) cells compared to WT mice (10.6 ± 1.0 versus 4.5 ± 0.4, P < 0.005, by unpaired t test, respectively) and to lesser extent of TH17 (CD4+IL-17+) cells (6.2 ± 0.4 versus 4.4 ± 0.5, P < 0.05, by unpaired t test, respectively) (Fig. 5C). Similarly, in the CNS, there was also a higher frequency of TH1 cells (19 ± 3.1 versus 10.5 ± 1.6, P < 0.005) and TH17 cells (12 ± 1.4 versus 7.4 ± 0.8, P < 0.001) in the IL-9R−/− mice compared to WT mice.

Fig. 5.
IL-9 signaling regulates the outcome of EAE. (A) Disease scores of IL-9R−/− and WT mice immunized with 75 μg MOG35–55/CFA. IL-9R−/− mice developed significantly higher score during the priming phase when ...

Next, we analyzed the state of FoxP3+ Tregs in the periphery of immunized mice. Although the frequency between WT and IL-9R−/− mice (10.40 ± 0.8 versus 11.3 ± 1.7, respectively) and absolute numbers (955 ± 140.7 versus 1055 ± 187, respectively) of FoxP3+ Tregs were identical, Treg cells from IL-9R−/− mice were less suppressive compared to Treg cells from WT mice (Fig. 5D). While Tregs from WT mice still showed significant suppression at 1:5 Treg:Teff ratio, Tregs from IL-9R−/− mice lost their suppressive function at that ratio (Fig. 5D).

Discussion

IL-9, initially called P40, was associated with T cell growth factor activity (21, 22) and was thought to be produced by TH2 cells. More recently, other functions of IL-9 have started to be uncovered. We have identified a population of effector CD4+ T cells induced by TGF-β and IL-4 that produce large quantities of IL-9 and IL-10, yet do not exhibit any regulatory properties (14). Exposure of TH2 cells to TGF-β leads to generation of IL-10- and IL-9-producing cells (11, 14). We now report that TH17 cells produce large amounts of IL-9 that acts in an autocrine fashion on TH17 cells but also on FoxP3+ nTregs, the predominant T cell population expressing IL-9 receptor. These in vitro observations were substantiated by in vivo findings showing that nTregs isolated from immunized IL-9R−/− mice have impaired suppressive function associated with strong T cell proliferation and severe autoimmune encephalomyelitis. Similarly, in a model of Rag−/− mice that received CD4+CD25+ and effector cells to study allograft survival, blocking IL-9 greatly accelerated graft rejection mediated by CD8+ T cells (10). The authors reported that nTreg and iTreg cells produce more IL-9 than TH2 cells (10), but in this study, Tregs were isolated based on CD25 expression. In our present study, we isolated Treg cells from FoxP3.GFP reporter mice based on the expression of GFP and found that these cells do not express IL-9 as shown at the gene and protein levels. Similarly, naïve CD4+ T cells converted into iTregs in the presence of TGF-β do not produce IL-9. Furthermore, in addition to the role of IL-9 on Treg function, we demonstrated that in vitro, IL-9 synergizes with TGF-β1 to differentiate naïve CD4+ T cells into TH17 cells, which is independent of IL-6 signaling.

The physiological functions of IL-9 are linked to TH2 cell response and anti-apoptosis activities (9). We found that TH17 cells produce large amounts of IL-9, which acts with TGF-β to differentiate naïve CD4+ T cells into TH17 cells and that the frequency of TH17 cells induced in IL-9R−/− T cells under TH17 polarizing conditions in vitro was significantly reduced compared to WT CD4+ T cells. Thus, like IL-21 (8), IL-9, which uses γc receptor, must further amplify TH17 cells. In vivo, mice lacking IL-9R exhibited an increase in TH17 cells in seeming contrast to the in vitro observations. However, this is most likely due to subdued Treg response observed in the IL-9R−/− mice resulting in expansion and hyperactivaiton of all effector T cells and not just TH17 cells. Thus, in the IL-9R−/− mice, the effects of IL-9 on Treg function are dominant over induction of TH17 cells.

Over the last few years, several studies have broadened our understanding of the involvement of TH17 cells in tissue inflammation (5, 23, 24). However, despite the involvement of TH17 cells in several autoimmune disease models, several studies suggested that TH17 are not required for the induction of autoimmunity. For instance, TH1 cells can induce disease in the absence of TH17 cells, while TH17 cells are not sufficient to induce disease in the absence of TH1 cells (25, 26), such as in mice deficient in T-box21 (T-bet) and signal transducers and activators of transcription (STAT)-4 (27, 28), suggesting that TH17 cells may have nonpathogenic roles in autoimmune diseases. Furthermore, TH17 cells have been shown to produce IL-10, which has potent anti-inflammatory activities in EAE (29). Now we show that IL-9, a TH17 cell-associated cytokine enhances Treg function and regulates autoimmunity.

IL-23 is clearly not required for the initial induction of TH17 cells in vitro or in vivo. However, production of IL-17 by memory effector cells is clearly enhanced in the presence of IL-23 (30), and it was shown that IL-23 maintained expression of IL-17 in activated TH17 cells (31). Although IL-23 was described 9 years ago (30), little is known about the role of IL-23 for TH17 cells in vivo. It has been suggested that full acquisition of pathogenic function by effector TH17 cells is mediated by IL-23 rather than by TGF-β and IL-6 (29). Recently, we have shown that induction of Tregs by retinoic acid is associated with the inhibition of IL-23R expression that impairs the stabilization and further maturation of the pathogenic TH17 phenotype (32). We now show that IL-9, a potent enhancer of Treg suppressive activity, is negatively regulated by IL-23. Thus, our data suggest a mechanism by which IL-23 down-regulates IL-9 production by TH17 cells leading to an enhancement of the pathogenicity of TH17 cells and at the same time IL-9 is not available to potentiate Treg function.

The mechanism of IL-9 effects on TH17 and Tregs is through activation of STAT3 and STAT5 signaling. The IL-9 receptor and γc associate with JAK1 and JAK3, respectively, and trigger the STAT1, 3, and 5 pathways in mouse lymphoid cell lines (12, 13, 33). An accumulating body of evidence indicates that STAT3 and STAT5 signaling pathways are key regulators of TH17/Treg development (1, 18, 34). STAT5 binds directly to the FoxP3 gene (18) and is required for optimal induction of Foxp3 in vitro, while STAT3 is required for IL-6-dependent down-regulation of FoxP3 (18). Furthermore, STAT3 and STAT5 play opposing roles in TH17 differentiation; STAT3 mediates the development of TH17 cells, while STAT5 negatively regulates TH17 differentiation in vitro and in vivo (31). Thus, by simultaneously activating STAT3 and STAT5 pathways, IL-9 influences the balance between TH17/Tregs and the development of the immune responses in vivo. A hallmark feature of the STAT family of transcription factors is their regulation of cell survival. Indeed, STAT3 and STAT5 have been shown to induce pro-survival signals mainly through the regulation of apoptosis-associated genes (35). Here, we demonstrate that IL-9 enhances the survival of nTregs and induces the activation of STAT3 and STAT5 signaling in nTregs.

In conclusion, our results highlight a role of IL-9 as a regulator of pathogenic versus protective mechanisms of immune responses. Thus, TH17 cells could enhance regulatory function by secreting IL-9 and hence tip the balance toward regulation of immunity.

Materials and Methods

Mice, EAE Induction, and Treatment.

Female C57BL/6 WT mice were purchased from The Jackson Laboratory. FoxP3-GFP.KI reporter mice, IL9R−/− mice, and MOG-TCR mice (2D2), have been previously described (7, 20, 36). IL-23R−/− mice were generated in V. Kuchroo's laboratory (Harvard Medical School) as follows. Briefly, an IRES-EGFP derived from pMSCV-IRES-EGFP was subcloned into the ClaI site and BamHI site of the TKPbs-LoxP-Neo cassette. An SV40 polyadenylation sequence derived from pTRE vector (Clontech) was subsequently cloned into the BamHI site. A BAC clone (RP23–204M15) containing C57BL/6 IL-23R genomic DNA was used as a template for PCR amplification to generate 5.1-kb and 1.8-kb arms. The targeting construct was electroporated into Bruce4 ES cells. Targeted ES cells were injected into BALB/c blastocysts, and male chimeras were bred with female C57BL/6. Germ line transmitted mice were bred with EIIA-Cre transgenic mice to remove the neomycin resistance gene. Homozygous mice that are deficient in IL-23R expression were used. Mice were housed in conventional, pathogen-free facilities at the New Research Building, Harvard Medical School (Boston, MA). All animal experiments were done with the approval of the Harvard Medical Area Standing Committee on Animals. EAE was induced in C57BL/6 WT as described previously (26). MOG peptide 35–55 (M-E-V-G-W-Y-R-S-P-F-S-R-O-V-H-L-Y-R-N-G-K) corresponding to the mouse sequence was synthesized in the Biopolymer Laboratory (University of California, Los Angeles, CA) and purified to greater than 99% by HPLC. C57BL/6 WT were immunized s.c. in the flank with 75 μg MOG35–55 peptide in 0.1 mL PBS and 0.1 mL CFA containing 0.4 mg Myobacterium tuberculosis (H37Ra; Difco Laboratories) and injected i.p. (i.p.) with 200 ng pertussis toxin (PT) (List Biological Laboratories) on the day of immunization and 2 days later. Animals were scored as follow: grade 1, limp tail or isolated weakness of gait without limp tail; grade 2, partial hind and front leg paralysis; grade 3, total hind leg paralysis; grade 4, total hind leg and partial front leg paralysis; grade 5, moribund or dead animal.

In Vitro T Cell Differentiation.

Naïve CD4+ T cells were purified from FoxP3.GFP reporter mice using anti-CD4 beads (Miltenyi) and sorted into naïve CD4+CD62LhiFoxP3 T cells by flow cytometry on a FACSAria T cell sorter (BD Biosciences). CD4+ T cells were stimulated with plate-bound anti-CD3 (4 μg/mL) (145–2C11; PharMingen) and soluble anti-CD28 (2 μg/mL; PharMingen) for 3–5 days in a serum-free media (X-VIVO-20; Lonza) supplemented with 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, nonessential amino acids, L-glutamine, and 100 U/mL penicillin/100 μg/mL streptomycin in the presence of recombinant cytokines. Polarization of T cells was in the presence of recombinant mouse IL-12 (10 ng/mL; R&D Systems) plus anti-IL-4 (11B.11; 10 μg/mL) for TH1, mouse IL-4 (10 ng/mL; R&D Systems) plus anti-IL-12 (C17.8; 10 μg/mL) for TH2, and human TGF-β1 (3 ng/mL) plus IL-6 (30 ng/mL) or IL-21 (100 ng/mL) for TH17. Mouse recombinant IL-23 was used at 10 ng/mL. Cells were supplemented with recombinant IL-2 (50 U/mL) at day 2 and 4. Monoclonal antibodies against mouse IL-4 and mouse IL-12 were purified from the supernatants of hybridomas obtained from the American Type Culture Collection (ATCC). Recombinant mouse IL-9 was used at 20 ng/mL. Blocking of IL-9 activity in vitro was performed by the addition of a rat anti-mouse IL-9 antibody (10 μg/mL, clone 222622; R&D Systems). All recombinant proteins were from R&D Systems.

Measurement of Cytokines.

Cytokines were measured by intracellular cytokine staining, bead-based Luminex cytokine assay and real-time PCR (see SI Methods).

In Vitro T Cell Proliferation and Suppression Assay.

Proliferation was determined by incorporation of [3H]thymidine (see SI Methods).

Multiplex Cell Signaling Bead-Based Luminex Assays.

CD4+FoxP3/GFP+ Treg cells were isolated from spleens of naïve FoxP3.GFP reporter mice by FACS sorting and incubated for different time points in serum-free media in the presence of recombinant rIL-9 (20 ng/mL). Following each incubation time, cells were washed and lysed, and equal amounts of total protein lysates were used for the detection of active phosphorylated proteins according to the manufacturer's protocol (Millipore). HeLa mixed cell lysates stimulated with epidermal growth factor (EGF), TNF-α, or heat shocked were provided by the manufacturer and used as a positive control. Bead assay in the absence of cell lysates was used as a negative control. Samples were acquired using Luminex 100 System instrument (Luminex Corporation), and mean fluorescence intensity (MFI) was calculated.

Statistical Analysis.

Difference between WT and IL-9R−/− immunized mice was analyzed by Mann-Whitney. The unpaired t test was used to compare cell proliferation and cytokine production.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank D. Kozoriz for cell sorting and M.J. Bradshaw for technical assistance. This work was supported by research grants from the National Institutes of Health (R01AI067472, AI058680, and AI043496 to S.J.K.), and the National Multiple Sclerosis Society (RG3666, RG2988, and RG3504 to S.J.K.). W.E. and E.M.B. are recipients of National Research Service Award fellowships from the National Institute of Neurological Disorders and Stroke, and the National Institute of Allergy and Infectious Diseases, respectively. C.U. and J.V.S. research was supported by grants from the Fonds National de la Recherche Médicale and from the Fonds Charcot pour la Sclérose en Plaques (Belgium).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812530106/DCSupplemental.

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