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

Wassim Elyamana, Elizabeth M. Bradshawa, Catherine Uyttenhoveb, Vale´ rie Dardalhona, Amit Awasthia, Jaime Imitolaa, Estelle Bettellia, Mohamed Oukkaa, Jacques van Snickb, Jean-Christophe Renauldb, Vijay K. Kuchrooa, and Samia J. Khourya,1

aCenter for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 and bLudwig Institute for Cancer Research and Experimental Medicine Unit, Universite´Catholique de Louvain, Brussels, Belgium

Edited by Richard A. Flavell, Yale University School of Medicine, New Haven, CT, and approved March 31, 2009 (received for review December 11, 2008)

The development of T helper (TH)17 and regulatory T (Treg) cells is ing large amounts of IL-10, but induce severe colitis and peripheral reciprocally regulated by . Transforming neuritis upon adoptive transfer into immune deficient hosts (14). ؉ ␤ (TGF)- alone induces FoxP3 Treg cells, but together with IL-6 or IL-21 Another recent report describes reprogramming of TH2 cells by induces TH17 cells. Here we demonstrate that IL-9 is a key molecule TGF-␤ to cells producing IL-9 and IL-10 (11), and these cells were that affects differentiation of TH17 cells and Treg function. IL-9 pre- also shown to have effector and not regulatory functions. These ␤ dominantly produced by TH17 cells, synergizes with TGF- 1 to differ- data suggest that IL-9/IL-10-producing T cells are not regulatory T ؉ entiate naïve CD4 T cells into TH17 cells, while IL-9 secretion by TH17 cells but effector T cells that induce tissue inflammation. However, cells is regulated by IL-23. Interestingly, IL-9 enhances the suppressive whether these effector functions are mediated by IL-9 and what role ؉ ؉ functions of FoxP3 CD4 Treg cells in vitro, and absence of IL-9 does IL-9 play in pathogenicity of this unique subset of T cells has signaling weakens the suppressive activity of nTregs in vivo, leading not been evaluated. to an increase in effector cells and worsening of experimental auto- In the present study, we show that TH17 cells produce large

immune encephalomyelitis. The mechanism of IL-9 effects on TH17 IMMUNOLOGY quantities of IL-9 that act on both TH17 and Treg cells. In the and Tregs is through activation of STAT3 and STAT5 signaling. Our presence of TGF-␤, IL-9 differentiates naïve CD4ϩ T cells into findings highlight a role of IL-9 as a regulator of pathogenic versus TH17 cells. Paradoxically, IL-9 also acts on nTregs and enhances their protective mechanisms of immune responses. suppressive function in vitro and in vivo. Mice lacking IL-9 receptor (IL-9RϪ/Ϫ) exhibit a more severe course of experimental autoim- ͉ ͉ autoimmunity regulatory cells tolerance mune encephalomyelitis (EAE) and have a defect in the suppressive activity of Tregs. n the presence of antigen stimulation, naïve CD4ϩ T cells Iproliferate and differentiate into T helper type 1 (TH1) cells, TH2 Results cells, or -17 (IL-17)-producing T helper cells (TH17 cells) IL-9 Is Produced by TH17 Cells. TH17 cells produce several cytokines to exert specific effector functions. TH17 cells express the transcrip- including IL-17, IL-21, and IL-22 (2). We sought to determine the ␥ tion factor retinoic acid orphan receptor gamma (ROR- )t (1), additional cytokines that are secreted by TH17 cells. FACS-sorted participate in the control of extracellular pathogens, and have an naïve CD4ϩCD62LhiFoxP3Ϫ T cells isolated from FoxP3.GFP important role in human and experimental autoimmunity (2). TH17 ‘‘knock-in’’ (FoxP3.GFP.KI) reporter mice were differentiated into cells have been identified as major inducers of tissue inflammation TH1, TH2, and TH17 according to established protocols (Fig. S1A). and autoimmunity. Since exaggerated responses of TH1, TH2, and Inducible Tregs(iTregs) (7) were generated by activating TH17 cells can induce tissue inflammation, the maintenance of CD4ϩCD62LhiFoxP3Ϫ T cells in the presence of TGF-␤ (Fig. S1B), immune homeostasis and prevention of immunopathology is me- and nTregs were sorted from naïve FoxP3.GFP.KI mice based on in diated by subsets of T cells called regulatory T cells (Tregs). Treg cell vivo FoxP3 expression (Fig. S1C). Using multiplex bead-based differentiation and function are driven by the transcription factor Luminex technology, we identified IL-9 as a dominant forkhead box P3 (FoxP3) (3, 4), and they are closely related to the ␤ produced both by TH2 and TH17 cells (Fig. 1A). These data were generation of TH17 cells. Transforming growth factor (TGF)- 1 confirmed by quantitative Taqman PCR (Fig. 1A) and by flow induces the differentiation of T cells (5), whereas TGF-␤1in reg cytometry (Fig. 1B). Moreover, IL-9 was not detected in the combination with IL-6 (6, 7) or IL-21 (8) results in the differenti- supernatants of nT or iT (Fig. 1A), but IL-9R mRNA was ation of T 17 cells. regs reg H highly expressed by nT and not by iT (Fig. 1A). IL-9 is a -derived factor preferentially expressed by T 2 regs regs H Naïve CD4ϩ T cells activated with plate-bound anti-CD3 and cells (9), although a previous study suggested that regulatory T cells anti-CD28 in the presence of recombinant mouse IL-9 had in- produce more IL-9 than TH2 cells (10); however this was not creased secretion of the TH2 (IL-4, IL-10, and IL-13), cytokines, confirmed in induced (iTregs) or natural (nTregs) (11). It is a ␥ pleiotropic cytokine that targets cells of the lymphoid, myeloid, and and at the same time down-regulated IFN (IFN)- and granulocyte- lineages, as well as lung epithelial cells. IL-9 activities are macrophage colony stimulation factor (GM-CSF) production (Fig. mediated by a specific IL-9 receptor chain that forms a het- erodimeric receptor with the (␥c) also Author contributions: W.E., C.U., J.I., J.V.S., J.-C.R., and S.J.K. designed research; W.E., involved in IL-2, 4, 7, 15, and 21 signaling. The IL-9 receptor and E.M.B., C.U., V.A.D., A.A., and J.V.S. performed research; C.U., E.B., M.O., J.V.S., J.-C.R., and ␥c associate with (JAK)1 and JAK3, respectively, and V.K.K. contributed new reagents/analytic tools; W.E., E.M.B., C.U., V.A.D., and J.V.S. ana- trigger the activation of STAT1, 3, and 5 (12, 13). Dysregulated IL-9 lyzed data; and W.E. wrote the paper. response in vitro can lead to autonomous cell growth and malignant The authors declare no conflict of interest. transformation of lymphoid cells associated with constitutive acti- This article is a PNAS Direct Submission. vation of the JAK/STAT pathway (12). We have recently described 1To whom correspondence should be addressed. E-mail: [email protected]. a subset of T cells that predominantly produces IL-9 together with This article contains supporting information online at www.pnas.org/cgi/content/full/ IL-10 and do not exhibit any regulatory properties despite produc- 0812530106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812530106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 28, 2021 A B Th1 Th2 Th17 0.12 0.18 1.13 0.64 3.56 1.04 5 5 5 iTregs 10 10 10

nTregs 4 10 104 104 Th17 9 3 - 10 103 103

Th2 L I

2 Th1 10 102 102 Th0 0 0 0 67.6 32.1 84.2 13.9 82.8 12.6 0 50 100 150 200 250 300 350 400 450 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 1 2 3 4 5 6 7 8 9 0 102 103 104 105 0102 103 104 105 0102 103 104 105 IL-9 (pg/ml) IL-9 relative expression IL-9R relative expression IFN-γ IL-10 IL-17

TGF-β1 TGF-β1 C IL-4 IFN-γ 200 TGF-β 500 *** IL-21 400 IL-9 IL-6 300 100 IL-6 + TGF-β 200 IL-21 + TGF-β IL9 + TGF-β 100 No cytokine 0 0 0 100 200 300 0 100 200 300 400 500 no cytokine IL-9 no cytokin IL-9 pg/ml pg/ml

ϩ hi Ϫ Fig. 1. TH17 cells produce IL-9. CD4 CD62L FoxP3/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 Luminex assay (left) and by quantitative RT-PCR (middle) and IL-9 ϩ hi receptor relative expression (right) in TH0, TH1, TH2, TH17, nTregs, and iTregs.(B) Intracellular staining of cytokines in naïve CD4 CD62L T cells from B6 mice sorted ϩ by flow cytometry and cultured for 4 days in TH1, TH2, or TH17 conditions. (C) Effects of IL-9 on the production of IL-4, IFN-␥, and TGF-␤1 by naïve CD4 T cells as measured by bead-based Luminex assay. For TGF-␤1 assay, supernatants were ‘‘acid-treated’’ followed by bead-based Luminex assay. Effect of IL-9 on TGF-␤1 expression in T cells was also measured by quantitative Taqman PCR.

1C and Fig. S2). Interestingly, cells exposed to IL-9 produce IL-9 might normally contribute to TH17 differentiation even when significant amounts of TGF-␤1 and this was confirmed by Taqman TH17 cells were differentiated by other cytokine cocktails (TGF-␤ PCR (Fig. 1C). We also observed an increase in the IL-9 mRNA plus IL-6 or TGF-␤ plus IL-21). To rule out the possibility that IL-6 levels in response to IL-9 indicating an autocrine positive feedback may contribute to the induction of TH17 differentiation by IL-9, we (Fig. S2), by which IL-9 induces T cells that further produce IL-9, compared the differentiation of naïve wild-type (WT) and IL-6Ϫ/Ϫ and thus setting up an autoamplification loop. In combination with T cells in vitro in the presence of TGF-␤ and IL-9. Our data showed TGF-␤, IL-9 had a similar effect on T cell differentiation as IL-21 that IL-9 plus TGF-␤ was sufficient to drive the differentiation of Ϫ/Ϫ (Fig. 1C and Fig. S2). Unlike IL-6, which normally inhibits TGF-␤ IL-6 T cells into TH17 cells, although less efficiently than for WT induced FoxP3, IL-9 did not inhibit the TGF-␤-mediated conver- T cells (Fig. S5). sion of FoxP3Ϫ (GFPϪ) naïve CD4ϩ T cells from the ϩ ϩ FoxP3.GFP.KI reporter mice into FoxP3 GFP Treg cells (Fig. S3). 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-9 Synergizes with TGF- 1 to Differentiate Naïve CD4 Cells into TH17 IL-23 has been shown to enhance the pathogenic potential of Cells. Given the critical role of the transcription factor STAT3 in myelin-specific T cells in the induction of autoimmune encephalo- TH17 differentiation (15) and given that IL-9 has been reported to myelitis (17), we tested the effects of IL-23 on TH17 cells. TH17 cells induce activation of STAT3 (16), we next examined whether IL-9 exposed to IL-23 during a secondary stimulation had a significantly could promote TH17 differentiation. As shown in Fig. 2A, stimu- reduced production of IL-9, while IL-17 production was preserved ␤ Ϫ/Ϫ lation with IL-6 plus TGF- induced large amounts of IL-17 (Fig. (Fig. 3A). Furthermore, TH17 cells from IL-23R mice produced 2A), and replacement of IL-6 by IL-9 was also effective in inducing significantly higher levels of IL-9 as compared to WT cells in a IL-17-producing cells. Like IL-6, IL-9 in the absence of TGF-␤ secondary stimulation with IL-6 and TGF-␤ (Fig. 3B). These data could not induce TH17 differentiation. The effects of IL-9 on TH17 were confirmed by quantitative PCR (Fig. 3C). Our findings suggest cell induction were comparable to those of IL-21 (Fig. 2A and B). that IL-23 is a negative regulator of IL-9 secretion by TH17 cells. Moreover, neutralizing IL-9 with anti-IL-9 blocking antibody (10 ␮g/mL) reduced the amounts of IL-17 induced by IL-6 plus TGF-␤ IL-9 Enhances the Suppressive Functions of Regulatory FoxP3 CD4؉ T ␤ or IL-21 plus TGF- , suggesting that IL-9 produced by differenti- Cells. Given that nTregs express IL-9R but IL-9 does not inhibit Treg ating TH17 cells further enhances the development of TH17 cells induction in vitro (Fig. 1D), we investigated the effects of IL-9 on ␤ ϩ ϩ (Fig. 2A). Induction of IL-17 by TGF- plus IL-9 was confirmed at Treg function. Purified CD4 FoxP3/GFP T cells from naïve the single cell level with naïve CD4ϩ T cells activated for 2 rounds FoxP3.KI mice suppressed CD3 driven effector T cell responses. of 4 days each in the presence of TGF-␤ plus IL-9 and co-stained Blockade of IL-9 signaling using an anti-IL-9-neutralizing antibody ␥ for IL-17/IL-4 (Fig. 2B) or IL-17/IFN- (Fig. S4), indicating that the reversed nTreg-mediated suppression allowing effector T cells to combination of IL-9 and TGF-␤ is as effective in inducing differ- proliferate (Fig. 4A) and produce effector cytokines (Fig. 4B). In entiation of TH17 cells (Fig. 2B and Fig. S4). TH17 cells were the contrast, addition of rIL-9 (20 ng/mL) increases the suppressive highest producers of IL-9 among the T cell subsets, suggesting an capacity of nTregs (Fig. 4A) and further decreased the production amplification loop in which IL-9 produced by TH17 cells participates of IL-2, IL-17, and IFN-␥ (Fig. 4B) as well as IL-6, tumor necrosis in enhancing further differentiation of TH17 cells. This observation factor (TNF)-␣, and macrophage inflammatory (MIP)-1␤ was further supported by the finding that the frequency of TH17 (Fig. S6A). cells induced in IL-9RϪ/Ϫ cells by IL-6 plus TGF-␤ was significantly IL-9 has been shown to protect T cells against reduced compared to WT CD4ϩ T cells (Fig. 2C), suggesting that through a STAT3/STAT5-dependent mechanism (13, 16). On

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812530106 Elyaman et al. Downloaded by guest on September 28, 2021 A IL-17 the effects of IL-9 on nTregs are not mediated by these signaling IL-21 + TGF- + anti-IL-9 pathways (Fig. S6B). IL-9-treated nTregs showed no changes in * IL-21 + TGF- ␤-catenin total protein level, a protein that was recently shown to IL-21 have powerful effects on the survival of Tregs (19) (Fig. S6B). IL-6 + TGF- + anti-IL-9 IL-6 + TGF- * IL-6 Endogenous IL-9 Regulates Treg Activity and Inflammation In Vivo. Our IL9 + TGF- findings that IL-9 influences both TH17 cell differentiation and Treg TGF- suppressive activity suggest that endogenous levels of IL-9 might IL-9 participate in immune regulation in vivo. Indeed, using EAE, we No cytokine addressed the role of IL-9 production on the immune responses in 0 500 1000 1500 2000 2500 EAE by taking advantage of IL-9RϪ/Ϫ mice that have deletion of pg/ml exons 2 to 6, which encode the entire extracellular domain of the receptor (20). IL-9RϪ/Ϫ mice immunized with suboptimal doses of No cytokine IL-9 +TGF- IL-21+TGF- IL-6+TGF- ␮ B 0.38 0.52 12.68 1.95 44.6 6.23 MOG (75 g) developed earlier and more severe disease 5 5 11.78 2.155 5 35–55 10 10 10 10 compared to WT mice (mean disease onset 10.8 Ϯ 1.1 versus 15.8 Ϯ 4 4 4 4 10 10 10 10 0.9, P ϭ 0.009 by unpaired t test and mean maximal score 2.5 Ϯ 0.2 103 3 3 103 APC-A APC-A APC-A 10 10 APC-A versus 1.2 Ϯ 0.3, P ϭ 0.008 by Mann-Whitney, respectively) (Fig.

2 2 2 2 10 10 10 10 5A). We confirmed these findings using anti-IL-9 blocking antibody 0 0 0 0 62.2 36.9 75.5 10.6 77.3 8.05 35.7 13.5 in EAE induced in SJL/J mice (Fig. S7). In the periphery, T cells 2 3 4 5 2 3 4 5 IL-17 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 Ϫ/Ϫ PE-A PE-A PE-A PE-A from IL-9R mice mounted a strong proliferative response to TGF- IL-9 IL-21 IL-6 1.14 1.18 0.2 1.87 0.88 2.15 0.46 1.18 MOG stimulation compared to WT cells (Fig. 5B). Next, we 5 5 5 5 35–55 10 10 10 10 studied the cytokine profile of lymph node (onset of the disease) 104 104 104 104 and central nervous system (CNS)-infiltrated CD4ϩ T cells (peak

103 103 103 103 APC-A APC-A APC-A APC-A of the disease) isolated from the immunized mice and analyzed by

102 102 102 102 flow cytometry. In the peripheral compartment, we found that 0 0 0 0 Ϫ/Ϫ ϩ ␥ϩ 70.8 26.9 65.6 32.3 59.2 37.8 80.5 17.8 IL-9R mice had a higher frequency of TH1 (CD4 IFN- ) cells 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 PE-A PE-A PE-A PE-A compared to WT mice (10.6 Ϯ 1.0 versus 4.5 Ϯ 0.4, P Ͻ 0.005, by ϩ IL-4 IMMUNOLOGY unpaired t test, respectively) and to lesser extent of TH17 (CD4 IL- 17ϩ) cells (6.2 Ϯ 0.4 versus 4.4 Ϯ 0.5, P Ͻ 0.05, by unpaired t test, WT IL9R-/- C respectively) (Fig. 5C). Similarly, in the CNS, there was also a higher Ϯ Ϯ Ͻ No cytokine TGF- + IL-6 No cytokine TGF- + IL-6 frequency of TH1 cells (19 3.1 versus 10.5 1.6, P 0.005) and Ϫ/Ϫ 1.91 0.575 14.4 0.07 5.9 0.53 T 17 cells (12 Ϯ 1.4 versus 7.4 Ϯ 0.8, P Ͻ 0.001) in the IL-9R 11.2 0.21 10 H 4 mice compared to WT mice. 10 ϩ

IFN- Next, we analyzed the state of FoxP3 Tregs in the periphery of Pe-Cy7-A 3 10Pe-Cy7-A immunized mice. Although the frequency between WT and IL- Ϫ Ϫ 102 9R / mice (10.40 Ϯ 0.8 versus 11.3 Ϯ 1.7, respectively) and 88.2 16.5 0.35 85.2 8.38 0.41 81 0 85.2 absolute numbers (955 Ϯ 140.7 versus 1055 Ϯ 187, respectively) of FoxP3ϩ T were identical, T cells from IL-9RϪ/Ϫ mice were less IL-17 regs reg suppressive compared to Treg cells from WT mice (Fig. 5D). While Fig. 2. IL-9 induces TH17 cell differentiation. (A) IL-17 production by naïve Tregs from WT mice still showed significant suppression at 1:5 ϩ hi Ϫ/Ϫ CD4 CD62L T cells cultured under optimal TH17 conditions or under condi- Treg:Teff ratio, Tregs from IL-9R mice lost their suppressive tions in which IL-6 was omitted and replaced with IL-9, as measured by function at that ratio (Fig. 5D). bead-based Luminex assay. (B) Intracellular expression of IL-4 (x-axis) and IL-17 (y-axis) in WT CD4ϩ T cells was measured by flow cytometry following 2 rounds Discussion of activation in the presence of the cytokines listed on each histogram. (C) Intracellular expression of IFN-␥ and IL-17 in WT and IL-9RϪ/Ϫ CD4ϩ T cells after IL-9, initially called P40, was associated with T cell growth factor activation with or without TGF-␤ plus IL-6. Data are representative of 4 activity (21, 22) and was thought to be produced by TH2 cells. More independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.01. recently, other functions of IL-9 have started to be uncovered. We have identified a population of effector CD4ϩ T cells induced by TGF-␤ and IL-4 that produce large quantities of IL-9 and IL-10, yet the other hand, the transcription factors STAT3 and STAT5 are do not exhibit any regulatory properties (14). Exposure of TH2 cells crucial for the development of TH17 and nTreg cells, respectively to TGF-␤ leads to generation of IL-10- and IL-9-producing cells (1, 15, 18). Purified nTreg cells isolated from naïve FoxP3.KI mice (11, 14). We now report that TH17 cells produce large amounts of and cultured for 4 days die quickly in a serum-free medium (Fig. IL-9 that acts in an autocrine fashion on TH17 cells but also on ϩ 4C). Interestingly, addition of IL-9 (20 ng/mL) partially rescued FoxP3 nTregs, the predominant T cell population expressing IL-9 Treg cells from apoptotic cell death and significantly increased receptor. These in vitro observations were substantiated by in vivo Ϫ/Ϫ the number of viable cells indicating that IL-9 could act as a findings showing that nTregs isolated from immunized IL-9R survival factor for nTregs (Fig. 4C). mice have impaired suppressive function associated with strong T SignalingbyIL-9tonTreg cells is mediated by STAT3 and STAT5 cell proliferation and severe autoimmune encephalomyelitis. Sim- phosphorylation within 30 min of IL-9 addition, as shown by ilarly, in a model of RagϪ/Ϫ mice that received CD4ϩCD25ϩ and increased mean fluorescent intensity (MFI) (P Ͻ 0.05 for pSTAT3 effector cells to study allograft survival, blocking IL-9 greatly and P Ͻ 0.01 for pSTAT5) (Fig. 4D). IL-9 also increased the accelerated graft rejection mediated by CD8ϩ T cells (10). The phosphorylation of ribosomal protein S6 kinase (P70S6 kinase) and authors reported that nTreg and iTreg cells produce more IL-9 than of inhibitor of kappa B-alpha (I␬B-␣), a negative regulator of TH2 cells (10), but in this study, Tregs were isolated based on CD25 nuclear factor-kappa B (NF-␬B) signaling pathway, indicating an expression. In our present study, we isolated Treg cells from increase in the activation of NF-␬B(Fig. S6B). IL-9 did not regulate FoxP3.GFP reporter mice based on the expression of GFP and the phosphorylation of c-Jun N-terminal kinase (JNK), p38 mito- found that these cells do not express IL-9 as shown at the and ϩ gen-activated protein kinases (p38), nor the one of extracellular protein levels. Similarly, naïve CD4 T cells converted into iTregs in signal-regulated kinase (ERK MAPK1/2) and Akt, suggesting that the presence of TGF-␤ do not produce IL-9. Furthermore, in

Elyaman et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 28, 2021 A 1st stimulation 1st stimulation C 2nd stimulation IL-9 IL-17 P=0.002 IL-6 + TGF-β 35 WT 30 IL-23R-/- No cytokine 25

0 100 200 300 400 500 600 0 500 1000 1500 20 P=0.0002 pg/ml pg/ml o 2o stimulation 2 stimulation 15 IL-9 IL-17 10 IL-6 + TGF-β + IL-23 P=0.008 ** 5 IL-6 + TGF-β 0 no cytokine

0 75 150 225 300 375 0 500 1000 1500 2000 pg/ml pg/ml

WT 1st stimulation 1st stimulation 2nd stimulation nd 2 stimulation IL23R-/- B (IL-9) * 2500 80 (IL-9) 700 (IL-17) 700 (IL-17)

70 600 600 2000 60 500 500 50 1500 400 400 40 300 300 1000 30 200 200 20 500 10 100 100 0 0 0 0 No cytokine IL-6+TGF-β No cytokine IL-6+TGF-β No cytokine IL-6+TGF-β No cytokine IL-6+TGF-β

ϩ hi Fig. 3. IL-23 is a negative regulator of IL-9. IL-17 production by naïve CD4 CD62L T cells cultured under optimal TH17 conditions. (A) The upper figures show IL-9 and IL-17 production after primary stimulation of naïve CD4ϩ T cells by TGF-␤ plus IL-6. The lower figures show IL-9 and IL-17 after secondary stimulation ϩ Ϫ/Ϫ with IL-23 or TGF-␤ plus IL-6. (B) Naïve CD4 T cells from IL-23R or WT mice were purified and differentiated into TH17 cells with IL-6 plus TGF-␤ for 4 days, then cells were rested and stimulated again for another round with or without IL-6 and TGF-␤. IL-9 and IL-17 production on day 3 (first stimulation) and day 7 (second stimulation) in culture supernatants were measured by bead-based Luminex assay. (C) CD4ϩCD62Lhi naïve T cells from IL-23RϪ/Ϫ and WT mice were stimulated with anti-CD3 and anti-CD28 for 4 days under TH17 polarization conditions and then stimulated for a second round in the presence of the corresponding cytokines. IL-9 relative expression as determined by quantitative RT-PCR. Data are expressed as mean of triplicate wells (ϩ SD). Data are representative of 3 (A)or2(B and C) independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.01.

addition to the role of IL-9 on Treg function, we demonstrated that EAE (29). Now we show that IL-9, a TH17 cell-associated cytokine ϩ in vitro, IL-9 synergizes with TGF-␤1 to differentiate naïve CD4 enhances Treg function and regulates autoimmunity. T cells into TH17 cells, which is independent of IL-6 signaling. IL-23 is clearly not required for the initial induction of TH17 cells The physiological functions of IL-9 are linked to TH2 cell in vitro or in vivo. However, production of IL-17 by memory response and anti-apoptosis activities (9). We found that TH17 cells effector cells is clearly enhanced in the presence of IL-23 (30), and produce large amounts of IL-9, which acts with TGF-␤ to differ- it was shown that IL-23 maintained expression of IL-17 in activated ϩ entiate naïve CD4 T cells into TH17 cells and that the frequency TH17 cells (31). Although IL-23 was described 9 years ago (30), little Ϫ/Ϫ of TH17 cells induced in IL-9R T cells under TH17 polarizing is known about the role of IL-23 for TH17 cells in vivo. It has been conditions in vitro was significantly reduced compared to WT suggested that full acquisition of pathogenic function by effector ϩ CD4 T cells. Thus, like IL-21 (8), IL-9, which uses ␥c receptor, TH17 cells is mediated by IL-23 rather than by TGF-␤ and IL-6 (29). must further amplify TH17 cells. In vivo, mice lacking IL-9R Recently, we have shown that induction of Tregs by retinoic acid is exhibited an increase in TH17 cells in seeming contrast to the in vitro associated with the inhibition of IL-23R expression that impairs the observations. However, this is most likely due to subdued Treg stabilization and further maturation of the pathogenic TH17 phe- Ϫ/Ϫ response observed in the IL-9R mice resulting in expansion and notype (32). We now show that IL-9, a potent enhancer of Treg hyperactivaiton of all effector T cells and not just TH17 cells. Thus, suppressive activity, is negatively regulated by IL-23. Thus, our data Ϫ/Ϫ in the IL-9R mice, the effects of IL-9 on Treg function are suggest a mechanism by which IL-23 down-regulates IL-9 produc- dominant over induction of TH17 cells. tion by TH17 cells leading to an enhancement of the pathogenicity Over the last few years, several studies have broadened our of TH17 cells and at the same time IL-9 is not available to potentiate understanding of the involvement of TH17 cells in tissue inflam- Treg function. mation (5, 23, 24). However, despite the involvement of TH17 cells The mechanism of IL-9 effects on TH17 and Tregs is through in several autoimmune disease models, several studies suggested activation of STAT3 and STAT5 signaling. The IL-9 receptor and that TH17 are not required for the induction of autoimmunity. For ␥c associate with JAK1 and JAK3, respectively, and trigger the instance, TH1 cells can induce disease in the absence of TH17 cells, STAT1, 3, and 5 pathways in mouse lymphoid cell lines (12, 13, 33). while TH17 cells are not sufficient to induce disease in the absence An accumulating body of evidence indicates that STAT3 and of TH1 cells (25, 26), such as in mice deficient in T-box21 (T-bet) STAT5 signaling pathways are key regulators of TH17/Treg devel- and signal transducers and activators of transcription (STAT)-4 (27, opment (1, 18, 34). STAT5 binds directly to the FoxP3 gene (18) and 28), suggesting that TH17 cells may have nonpathogenic roles in is required for optimal induction of Foxp3 in vitro, while STAT3 is autoimmune diseases. Furthermore, TH17 cells have been shown to required for IL-6-dependent down-regulation of FoxP3 (18). Fur- produce IL-10, which has potent anti-inflammatory activities in thermore, STAT3 and STAT5 play opposing roles in TH17 differ-

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812530106 Elyaman et al. Downloaded by guest on September 28, 2021 A B Effector Effector + Tregs 15000 p < 0.01 Effector + Tregs + IL-9 Fig. 4. IL-9 enhances the suppressive functions of ϩ ϩ Effector + Tregs + anti-IL-9 Tregs.(A) CD4 FoxP3/GFP Tregs isolated by FACS sorting 10000 2000 from the spleens of naïve FoxP3 reporter mice were p < 0.005 * tested for their suppressive capacity in vitro. A 1:1 ratio of naïve responder T cells (CD4ϩCD62Lϩ) cultured with 5000 1500 Tregs in the presence of irradiated syngeneic spleno- * cytes as APCs and anti-CD3 in a serum-free media. 1000 3 0 * * Mean H-thymidine incorporation indicated as c.p.m. * (ϩ SD) from triplicate wells. Recombinant IL-9 was IL-9 - - - + - * anti-IL9 - - - - + 500 added at a concentration of 20 ng/mL, and anti-IL-9 T-reg - - + + + neutralizing antibody was used at 10 ␮g/mL. (B) Super- T-eff + + + + + natants were collected after 48 h, and the cytokines Stim - 0 ␥ CD3 IL-2 IL-17 IFN- IL-2, IL-17, and IFN- were analyzed by bead-based Luminex assay. (C)nTregs were isolated from FoxP3 reporter mice and were cultured for 4 days in the C D presence or absence of IL-9 (20 ng/mL), and the number 600 Control IL-9 ** ** of nTregs undergoing apoptosis in each condition was 60 pSTAT3 500 counted using annexin V staining. (D) IL-9 induces pSTAT5 / 600 ** 400 STAT3 and STAT5␣␤ activation in nTregs. Freshly sorted 40 * nTreg cells were exposed to IL-9 (20 ng/mL) for the times 400 300 indicated on the x-axis followed by assessment of phos- Count 88.3 20 62.7 200 200 phorylated STAT3 and STAT5␣␤ using bead-based Lu- minex assay as described in Materials and Methods. 100 0 0 2 3 4 5 010 10 10 10 0102 103 104 105 Positive and negative controls of each phosphorylated Annexin V 0 substrate are shown. Data are representative of 3 (A and B)or2(C and D) independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.001. IMMUNOLOGY

entiation; STAT3 mediates the development of TH17 cells, while In conclusion, our results highlight a role of IL-9 as a regulator STAT5 negatively regulates TH17 differentiation in vitro and in vivo of pathogenic versus protective mechanisms of immune responses. (31). Thus, by simultaneously activating STAT3 and STAT5 path- Thus, TH17 cells could enhance regulatory function by secreting ways, IL-9 influences the balance between TH17/Tregs and the IL-9 and hence tip the balance toward regulation of immunity. development of the immune responses in vivo. A hallmark feature of the STAT family of transcription factors is their regulation of cell Materials and Methods survival. Indeed, STAT3 and STAT5 have been shown to induce Mice, EAE Induction, and Treatment. Female C57BL/6 WT mice were purchased pro-survival signals mainly through the regulation of apoptosis- from The Jackson Laboratory. FoxP3-GFP.KI reporter mice, IL9RϪ/Ϫ mice, and associated (35). Here, we demonstrate that IL-9 enhances the MOG-TCR mice (2D2), have been previously described (7, 20, 36). IL-23RϪ/Ϫ mice survival of nTregs and induces the activation of STAT3 and STAT5 were generated in V. Kuchroo’s laboratory (Harvard Medical School) as follows. signaling in nTregs. Briefly, an IRES-EGFP derived from pMSCV-IRES-EGFP was subcloned into the ClaI

Fig. 5. IL-9 signaling regulates the out- LN CNS come of EAE. (A) Disease scores of IL-9RϪ/Ϫ A C 105 3.8 0.55 105 8.26 2.99 ␮ 3 ****** *** and WT mice immunized with 75 g MOG35– ** 4 4 Ϫ Ϫ * 10 10 55/CFA. IL-9R / mice developed signifi- 103 103 cantly higher score during the priming * WT IL-9R-/- 2 2 102 10 phase when compared to control WT mice. 0 * 0 93.4 2.26 83 5.78 2 3 4 5 (B) On day 10 post-immunization, draining WT 0102 103 104 105 010 10 10 10 Ϫ/Ϫ * IFN- lymph nodes of IL-9R and WT were har- 105 9.6 2.39 105 16.8 2.46 1 vested and tested for MOG35–55-specific pro- * 104 104 3 liferation by H-thymidine incorporation re- 3 -/- 103 10 ported as c.p.m. (ϩ SD) from triplicate wells. IL-9R 102 102 (C) Lymph node (LN) cells and central ner- 0 0 83.1 4.93 0 71.8 8.95 ϩ vous system (CNS) infiltrated CD4 T cells 0 10 20 30 40 0102 103 104 105 0102 103 104 105 from IL-9RϪ/Ϫ and WT mice isolated on day Days post-immunization IL-17 12 and day 20, respectively, following im- munization and were tested for IL-17 and WT ␥ B D 15000 IFN- production by intracellular cytokine IL-9R-/- 25000 *** *** * staining using flow cytometry. The percent- WT *** * ages of positive cells are presented in each -/- 10000 ϩ ϩ 20000 IL-9R quadrant. (D) CD4 CD25 Tregs isolated by ** MACS beads from the spleens and lymph 15000 Ϫ Ϫ nodes of MOG -immunized IL-9R / ,or 5000 35–55 ** WT mice were tested for their suppressive 10000 capacity in vitro. MOG-TCR Tg responder T 5000 5 0 cells (Teff)at2ϫ 10 cells/well were cultured - - -1:11:21:5-1:11:21:5 Treg + - - + + + + + + + with titrated numbers of Tregs at Treg:Teff 0 Teff - + + + + + - + + + ratios indicated below the x-axis in the pres- 021050 MOG - - + + + + - + + + (MOG µg/ml) WT IL-9R-/- ence of irradiated syngeneic APCs (10 ϫ 105) 3 and MOG35–55 (20 ␮g/mL). Mean H-thymidine incorporation indicated as c.p.m. (ϩ SD) from triplicate wells. P values of unpaired t test comparison of the proliferation response in (B) and (D) are shown. Data are representative of 2 (B–D)or5(A) independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.0001.

Elyaman et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 28, 2021 site and BamHI site of the TKPbs-LoxP-Neo cassette. An SV40 polyadenylation U/mL) at day 2 and 4. Monoclonal antibodies against mouse IL-4 and mouse IL-12 sequence derived from pTRE vector (Clontech) was subsequently cloned into the were purified from the supernatants of hybridomas obtained from the American BamHI site. A BAC clone (RP23–204M15) containing C57BL/6 IL-23R genomic DNA Type Culture Collection (ATCC). Recombinant mouse IL-9 was used at 20 ng/mL. was used as a template for PCR amplification to generate 5.1-kb and 1.8-kb arms. Blocking of IL-9 activity in vitro was performed by the addition of a rat anti-mouse The targeting construct was electroporated into Bruce4 ES cells. Targeted ES cells IL-9 antibody (10 ␮g/mL, clone 222622; R&D Systems). All recombinant were injected into BALB/c blastocysts, and male chimeras were bred with female were from R&D Systems. 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 Measurement of Cytokines. Cytokines were measured by intracellular cytokine IL-23R expression were used. Mice were housed in conventional, pathogen-free staining, bead-based Luminex cytokine assay and real-time PCR (see SI Methods). facilities at the New Research Building, Harvard Medical School (Boston, MA). All animal experiments were done with the approval of the Harvard Medical Area In Vitro T Cell Proliferation and Suppression Assay. Proliferation was determined Standing Committee on Animals. EAE was induced in C57BL/6 WT as described by incorporation of [3H]thymidine (see SI Methods). 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- ϩ ϩ G-K) corresponding to the mouse sequence was synthesized in the Biopolymer Multiplex Bead-Based Luminex Assays. CD4 FoxP3/GFP Treg cells Laboratory (University of California, Los Angeles, CA) and purified to greater than were isolated from spleens of naïve FoxP3.GFP reporter mice by FACS sorting and 99% by HPLC. C57BL/6 WT were immunized s.c. in the flank with 75 ␮g MOG35–55 incubated for different time points in serum-free media in the presence of peptide in 0.1 mL PBS and 0.1 mL CFA containing 0.4 mg Myobacterium tuber- recombinant rIL-9 (20 ng/mL). Following each incubation time, cells were washed culosis (H37Ra; Difco Laboratories) and injected i.p. (i.p.) with 200 ng pertussis and lysed, and equal amounts of total protein lysates were used for the detection toxin (PT) (List Biological Laboratories) on the day of immunization and 2 days of active phosphorylated proteins according to the manufacturer’s protocol later. Animals were scored as follow: grade 1, limp tail or isolated weakness of (Millipore). HeLa mixed cell lysates stimulated with epidermal growth factor gait without limp tail; grade 2, partial hind and front leg paralysis; grade 3, total (EGF), TNF-␣, or heat shocked were provided by the manufacturer and used as a hind leg paralysis; grade 4, total hind leg and partial front leg paralysis; grade 5, positive control. Bead assay in the absence of cell lysates was used as a negative moribund or dead animal. control. Samples were acquired using Luminex 100 System instrument (Luminex Corporation), and mean fluorescence intensity (MFI) was calculated. In Vitro T Cell Differentiation. Naïve CD4ϩ T cells were purified from FoxP3.GFP reporter mice using anti-CD4 beads (Miltenyi) and sorted into naïve Statistical Analysis. Difference between WT and IL-9RϪ/Ϫ immunized mice was CD4ϩCD62LhiFoxP3Ϫ T cells by flow cytometry on a FACSAria T cell sorter (BD analyzed by Mann-Whitney. The unpaired t test was used to compare cell pro- Biosciences). CD4ϩ T cells were stimulated with plate-bound anti-CD3 (4 ␮g/mL) liferation and cytokine production. (145–2C11; PharMingen) and soluble anti-CD28 (2 ␮g/mL; PharMingen) for 3–5 days in a serum-free media (X-VIVO-20; Lonza) supplemented with 50 ␮M 2-mer- ACKNOWLEDGMENTS. We thank D. Kozoriz for cell sorting and M.J. Bradshaw captoethanol, 1 mM sodium pyruvate, nonessential amino acids, L-glutamine, for technical assistance. This work was supported by research grants from the and 100 U/mL penicillin/100 ␮g/mL streptomycin in the presence of recombinant National Institutes of Health (R01AI067472, AI058680, and AI043496 to S.J.K.), cytokines. Polarization of T cells was in the presence of recombinant mouse IL-12 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 fellow- (10 ng/mL; R&D Systems) plus anti-IL-4 (11B.11; 10 g/mL) for TH1, mouse IL-4 (10 ships from the National Institute of Neurological Disorders and Stroke, and the ␮ ␤ ng/mL; R&D Systems) plus anti-IL-12 (C17.8; 10 g/mL) for TH2, and human TGF- 1 National Institute of Allergy and Infectious Diseases, respectively. C.U. and J.V.S. (3 ng/mL) plus IL-6 (30 ng/mL) or IL-21 (100 ng/mL) for TH17. Mouse recombinant research was supported by grants from the Fonds National de la Recherche IL-23 was used at 10 ng/mL. Cells were supplemented with recombinant IL-2 (50 Me´dicale and from the Fonds Charcot pour la Scle´rose en Plaques (Belgium).

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