IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1

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1 Eur. J. Immunol : Cellular immune response 1809 IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1 Clemens Neufert 1, Christoph Becker 1, Stefan Wirtz 1, Massimo C. Fantini 2, Benno Weigmann 1, Peter R. Galle 1 and Markus F. Neurath 1 1 Laboratory of Immunology, I. Medical Clinic, University of Mainz, Mainz, Germany 2 University of Tor Vergata Rome, Rome, Italy IL-27 is an IL-12-related cytokine frequently present at sites of inflammation that can promote both anti- and pro-inflammatory immune responses. Here, we have analyzed the mechanisms how IL-27 may drive such divergent immune responses. While IL-27 suppressed the development of proinflammatory Th17 cells, a novel role for this cytokine in inhibiting the development of anti-inflammatory, inducible regulatory T cells (itreg) was identified. In fact, IL-27 suppressed the development of adaptive, TGF-b-induced Forkhead box transcription factor p3-positive (Foxp3 + ) Treg. Whereas the blockade of Th17 development was dependent on the transcription factor STAT1, the suppression of itreg development was STAT1 independent, suggesting that IL-27 utilizes different signaling pathways to shape T cell-driven immune responses. Our data thus demonstrate that IL-27 controls the development of Th17 and itreg cells via differential effects on STAT1. Received 17/11/06 Revised 1/3/07 Accepted 27/4/07 [DOI /eji ] Key words: IL-27 STAT1 Th17 Treg Supporting information for this article is available at Introduction It is well known that naïve CD4 + T cells may polarize into different T helper cell lineages (Th1, Th2) which then control T effector cell responses. Recently, it has been shown that proinflammatory Th17 cells and inducible regulatory T cells (itreg) can be also generated de novo from the same precursors depending on the local cytokine milieu and other factors such as costimulatory signals from antigen-presenting cells (APC) [1 6]. Homeostasis of the immune system requires tight control mechanisms that regulate the balance between Correspondence: Markus F. Neurath, Laboratory of Immunology, I. Medical Clinic, University of Mainz, Langenbeckstrasse 1, Mainz, Germany Fax: neurath@1-med.klinik.uni-mainz.de Abbreviations: EBI3: Epstein-Barr virus-induced gene 3 Foxp3: Forkhead box transcription factor p3 itreg: inducible regulatory T cell pro- and anti-inflammatory T cell subsets. The relevance of this concept is underlined by the observation that changes in the relative number and function of T cell subsets have been linked to the pathogenesis of autoimmune diseases and tumor development [7 9]. However, the mechanisms that control these critical events are still incompletely understood. IL-27 is a type I cytokine composed of the Epstein- Barr virus-induced gene 3 (EBI3) and the p28 subunit [10]. Whereas EBI3 is related to the IL-12p40 protein and the soluble IL-6 receptor, IL-27p28 shows structural similarities to IL-12p35, IL-23p19 and IL-6. The heterodimeric receptor complex for IL-27 is formed by glycoprotein 130 (GP130) and WSX-1 (TCCR). GP130 is widely expressed and also part of the receptor complexes of IL-6 and other cytokines. In contrast, WSX-1 specifically transduces IL-27 signaling, and represents a homologue of the IL-12Rb2 subunit [11]. IL-27 is produced by dendritic cells, monocytes and endothelial cells [8, 12]. Its receptor has been found on various cell types such as T cell subsets, NK cells, NK

2 1810 Clemens Neufert et al. Eur. J. Immunol : T cells, dendritic cells, B cells and mast cells. IL-27 signaling in T cells results in recruitment of several Jak family kinases and activation of STAT family transcription factors including STAT1and STAT3 [8]. Functional studies showed that IL-27 controls development and activation of Th1 cells by the induction of T-bet and IL-12Rb2 chain expression, suggesting a potential proinflammatory role of IL-27 [13, 14]. However, subsequent studies showed a more complex role for IL-27, as it may also exert anti-inflammatory functions in vivo [8, 15 18]. Thus, a growing number of studies suggests a central role of IL-27 within the cytokine network. However, its precise role requires further investigation. Here, we have identified a crucial role of IL-27 in controlling the development of Th17 and itreg cells. Results and discussion IL-27 is a potent inhibitor for TGF-b-induced Treg and Th17 cells IL-27 is a cytokine frequently present at sites of inflammation that has been shown to promote both anti- and pro-inflammatory responses depending on the cellular context [8]. Here, we studied the effect of IL-27 on the pro-inflammatory Th17 lineage, which can be generated by an activation of CD4 + CD25 T cells in the presence of TGF-b and IL-6 [3, 4]. Consistent with two recent studies [15, 16], we observed that IL-27 suppresses the development of Th17 cells, indicating a potential anti-inflammatory function of this cytokine (Supporting Information Fig. 1). These findings are consistent with in vivo data [15, 16], which showed that IL-27 signaling could ameliorate Th17-driven inflammation in models of experimental autoimmune encephalitis (EAE) in vivo. However, IL-27 has also been shown to drive inflammation, and specific blocking antibodies have been successfully used for the amelioration of chronic inflammatory diseases, suggesting that IL-27 may also exert potent proinflammatory functions [19, 20]. As Treg can counter-regulate inflammation and express the IL-27 receptor complex [5, 12, 13], we subsequently investigated the effects of IL-27 on Treg generated by stimulation of CD4 + CD25 T cells with TGF-b. Interestingly, it was found that IL-27 is a potent inhibitor of the development of such itreg (Fig. 1A). Whereas polyclonal stimulation of T cells in the presence of TGF-b resulted in a high number of Forkhead box transcription factor p3-positive (Foxp3 + ) T cells (50 70%), IL-27 reduced that number by more than Figure 1. IL-27 controls the generation of itreg cells in a concentration- and time-dependent manner. Freshly isolated CD4 + CD25 T cells were polyclonally stimulated and grown with TGF-b (5 ng/ml), WSX-1-FC (8 lg/ml) and IL-27 (20 ng/ml) as indicated. Cells were analyzed for the expression of Foxp3 by flow cytometry after 4 days (A). CD4 + CD25 T cells were cultured as described with TGF-b and varying concentrations of IL-27 for 5 days (B) or in the presence or absence of IL-27 (20 ng/ml) for 1 5 days (C). The inhibitory effect of IL-27 (20 ng/ml) was diminished, but also present when IL-27 was added at later time points (12 48 h after addition of TGF-b) (D). Similar data were obtained in at least two independent experiments. Error bars represent SD; ** p<0.01.

3 Eur. J. Immunol : Cellular immune response %, which is in the range of the recently described suppressive effect of IL-6 on itreg development [6]. Since IL-27 is the only known ligand for the WSX-1- receptor, we performed experiments in which IL-27 function was blocked by a recently developed soluble WSX-1-FC as a decoy receptor [17]. Interestingly, addition of soluble WSX-1-FC resulted in suppression of the effects of IL-27 on itreg development, indicating that the inhibitory effect was indeed mediated by IL-27 (Fig. 1A). The reduction of Foxp3 + itreg by IL-27 was concentration dependent and reached a plateau at a concentration of 20 ng/ml (Fig. 1B). IL-27 reduced the number of Foxp3 + itreg even when added to the cell culture at later time points (Fig. 1D). However, this effect decreased in a time-dependent fashion (Fig. 1C), suggesting that IL-27 controls early development rather than late differentiation of itreg. Suppression of itreg is a direct and specific effect of IL-27 In subsequent experiments, we addressed potential indirect effects by which IL-27 might inhibit itreg development. To rule out a major contribution of contaminating memory T cells, we performed experiments with naïve CD4 + CD25 CD62L + T cells, which confirmed the strong inhibitory effect of IL-27 on the development of itreg (Supporting Information Fig. 2). To further exclude an important role of any remaining antigen experienced cells, we took advantage of hemagglutinin TCR transgenic mice on a RAG-deficient background. These mice have only naïve T cells as described [21]. Antigen-specific stimulation of such cells with hemagglutinin in the presence of TGF-b resulted in a clear induction of itreg. Consistent with our previous findings, we observed an about 50% suppression of Foxp3 induction by IL-27 (Fig. 2A). Next, we tested various other pro-inflammatory cytokines including IL-1-b, IL-12, IL-17, IL-18, IL-23, TNF-a and IFN-c, which had no marked effect on itreg development, and additional experiments including blocking antibodies against IFN-c, IL-4, and IL-6Ra did not change the itreg inhibition mediated by IL-27 (Supporting Information Fig. 2), suggesting a direct rather than indirect effect by which IL-27 inhibits itreg development. For a better characterization of the cell populations developing in the presence of IL-27, we performed double staining for IFN-c and Foxp3 (Fig. 2B). Consistent with previous publications [10, 13], we observed an increase of IFN-c-producing cells upon treatment with IL-27. However, the increase of IFN-c + cells was lower than the decrease of Foxp3 + cells, arguing against a direct IL-27-mediated conversion of itreg cells into Th1 cells. Next, we tested the supernatants of the above cell cultures for various cytokines by cytometric bead assay. Cytokine production in the presence of TGF-b was low, and IL-27 had little or no effects on TGF-b-mediated suppression of cytokine levels (Fig. 2C). Thus, IL-27 promotes the development of IFN-c-producing cells, but had limited influence on the production of the other cytokines tested. As IL-27 can increase the proliferation of T cells [10], we analyzed the total cell numbers and the proliferation patterns. We observed a similar total cell number for cells treated with TGF-b and TGF-b plus IL-27, respectively (Fig. 3A). To rule out differential effects of IL-27 on the proliferation of Foxp3 + versus Foxp3 cells, we also performed co-staining for CFSE and Foxp3 (Fig. 3B). A detailed analysis revealed very similar cell numbers per division in the Foxp3 + versus Foxp3 cell fractions (Fig. 3C). Since differences in cell survival might affect the ratio of subpopulations in cell cultures, we performed apoptosis studies. Accordingly, we performed staining for Foxp3 and active Caspase 3, a marker for apoptosis that allows co-labeling with intracellular antigens. The rate of apoptotic cells in the Foxp3 + fraction was low Figure 2. Characterization of the effects of IL-27 on itreg development. Naïve T cells from HA TCR transgenic mice on a RAG-deficient background were stimulated in an antigenspecific fashion with HA (100 ng/ml) and grown in the presence of TGF-b (5 ng/ml) and IL-27 (20 ng/ml). Flow cytometric analysis for Foxp3 was performed at day 4 (A). CD4 + CD25 T cells from wild-type mice were polyclonally stimulated with plate-bound anti-cd3 and soluble anti-cd28 (1.5 lg/ml) plus TGF-b (5 ng/ml) and IL-27 (20 ng/ml). Cells were stained for IFN-c and Foxp3 and analyzed by flow cytometry at day 4 (B). Cells were cultured as above, and supernatants were harvested and analyzed by flow cytometric bead assay at day 5 (C). Similar data were obtained at least in two independent experiments.

4 1812 Clemens Neufert et al. Eur. J. Immunol : and no significant changes were noted after addition of IL-27 (Fig. 3D). Taken together, these findings suggest that IL-27 prevents T cells from converting into Foxp3 + itreg rather than changing the ratio between effector T cells and itreg by indirect effects such as outgrowth of non-treg or promotion of apoptosis of itreg. IL-27-mediated suppression of itreg was confirmed by co-culture proliferation studies, where IL-27-treated cells displayed a diminished capacity to suppress the proliferation of CD4 + CD25 T cells (Fig. 4A). The inhibition of suppression is in accordance with the reduced number of itreg in cell populations treated with IL-27. In contrast to itreg, we did not observe any effect of IL-27 in cell culture on the number of naturally occurring Foxp3 + Treg (data not shown). Inhibition of itreg by IL-27 occurs via IL-2 independent mechanism IL-2 has been shown to be an important cytokine for the survival, homeostasis and suppressive function of Treg [22 24]. Whereas IL-2 does not seem to be necessary for the development of naturally occurring Treg [22, 23], we found here that IL-2 is mandatory for the generation of itreg (Fig. 4B), which is consistent with recent reports by others [24, 25]. IL-27 has been identified as a suppressor of IL-2 production in T cells [26]. Furthermore, IL-27 can increase the threshold level of IL-2 responsiveness of T cells, resulting in a relative IL-2 deprivation [27]. Taken together, IL-2 is an important player in the cell biology of itreg, and IL-27 influences the IL-2 production and responsiveness of T cells. Therefore, we analyzed the role of IL-2 in our experimental system. Diminished levels of IL-2 in the supernatants of cell cultures were found upon treatment with TGF-b, and IL-2 production was even more down-regulated in the presence of IL-27 and TGF-b (Fig. 2D). However, high doses of recombinant IL-2 could not rescue the IL-27- mediated suppression of itreg development, suggesting an IL-2-independent mechanism for the effects of IL-27 on itreg development (Fig. 4C). Suppression of itreg and Th17 cells by IL-27 differentially involves STAT1 The generation of itreg in our studies was dependent on TGF-b signaling, and itreg are known to show a diminished capacity to up-regulate SMAD7, the endogenous inhibitor of TGF-b signaling [1, 28, 29]. However, IL-27 had no effects on expression of SMAD7 in our experimental system (data not shown). We next focused on the STAT family of transcription factors, as IL-27 can signal via different STAT molecules. Experiments with T cells from STAT4-deficient animals revealed no major role of that transcription factor in Figure 3. Similar proliferation and apoptosis patterns in cells treated with TGF-b or TGF-b plus IL-27. CD4 + CD25 T cells were labeled with CFSE, and stimulated polyclonally in the presence of TGF-b (5 ng/ml) and IL-27 (20 ng/ml) for 4 days. Total cell numbers from wells of 48-well plates were analyzed by microscopy (A). Flow cytometric analysis was performed for CFSE and Foxp3, and is shown using dot plots (B). CFSE-proliferation profiles of the Foxp3 + and Foxp3 subpopulations are displayed separately as histograms (B). Proliferation patterns from histogram plots were used for calculation of cell numbers per division (C). Apoptosis analysis was done by co-staining of active caspase-3 and Foxp3 (D). Similar data were obtained at least in three independent experiments. Error bars represent SD.

5 Eur. J. Immunol : Cellular immune response 1813 Figure 4. Induction of itreg depends on IL-2, but the inhibition of itreg by IL-27 is independent of IL-2. For co-culture proliferation assays (A), CD4 + CD25 cells (target cells, T) were labeled with CFSE and stimulated with soluble anti-cd3 (1 lg/ml) and autologous APC. Cells from itreg cultures (60% Foxp3 + with TGF-b, 29% Foxp3 + with TGF-b plus IL-27) were added as suppressor cells (S) ( cells for 1:1.5 ratio; for 1:3 ratio; for 1:6 ratio). Cell proliferation was analyzed by FACS at day 4. For studies on the role of IL-2 (B), CD4 + CD25 T cells were polyclonally stimulated and grown with TGF-b (5 ng/ml), and neutralizing anti-il-2. Cells were analyzed for Foxp3 by FACS at day 4. For rescue studies with IL-2 (C), cells were cultured as described above with IL-27 (20 ng/ml) and IL-2. Foxp3 was measured by FACS at day 4. Each experiment was performed at least twice with similar results. Error bars represent SD. IL-27-mediated suppression of Th17 cells or itreg (data not shown). Next, we analyzed the potential role of STAT1, as previous studies have demonstrated several STAT1-dependent pathways of IL-27 signaling. Consistent with very recent reports [15, 16], we found using STAT1-deficient mice that IL-27-mediated suppression of the Th17 lineage is dependent on the presence of STAT1 (Fig. 5A, B). However, we also demonstrated that the control of itreg development by IL-27 is independent of STAT1, since IL-27 diminished the number of Foxp3 + cells strikingly even in the absence of STAT1, as shown with T cells from STAT1 knockout animals (Fig. 5C). Thus, we suggest a model in which IL-27 modulates pro- and anti-inflammatory immune responses by suppressing both Th17 and itreg development (Fig. 6). A neutralizing effect of IL-27 on IL-6 signaling has been suggested for the inhibition of Th17 development [15]. However, direct evidence is lacking, and a major role of the candidate protein SOCS3 was ruled out by others [16]. Whereas IL-27 and IL-6 exert opposing roles in regard to the development of Th17 cells, both cytokines share the inhibitory function on the development of itreg. It is tempting to speculate about the molecular mechanisms involved. In contrast to the STAT1-mediated inhibition of Th17 cells, the suppression of itreg could occur via other STAT molecules, such as STAT3, which is activated by IL-27 and IL-6 [14, 30]. This concept is in accordance with recent data suggesting elevated Foxp3 expression in STAT3-deficient cells [31]. Figure 5. IL-27 suppresses the generation of Th17 cells in a STAT1-dependent fashion, whereas the IL-27-mediated inhibition of itreg development is independent of STAT1. CD4 + CD25 T cells were polyclonally stimulated, and TGF-b (5 ng/ml), IL-27 (20 ng/ml) and hil-6 (100 ng/ml) were added as indicated. For studies with Th17 cells, neutralizing antibodies against IFN-c (10 lg/ml) and IL-4 (10 lg/ml) were used. Cells from wild-type or STAT1-knockout animals were harvested at day 4, stained for the intracellular expression of IL-17 (A) or Foxp3 (C), and analyzed by flow cytometry. IL-17 from supernatants was quantified by cytometric bead assays at day 4 (B). Similar data were obtained in four independent experiments.

6 1814 Clemens Neufert et al. Eur. J. Immunol : Materials and methods Cells and cell purification Figure 6. IL-27 controls the development of itreg and Th17 cells. Hypothetical model of the effects of IL-27 on itreg and Th17 cell development. IL-27 modulates immune responses by suppressing development of both TGF-b-inducible cell lines. Whereas suppression of Th17 development is STAT1 dependent, inhibition of itreg development is STAT1 independent. Our data support a pivotal role of STAT1 in determining the effects of IL-27 function in T cells. Whereas the effects of IL-27 on Th17 cells are mediated via STAT1, the IL-27-mediated suppression of itreg development is STAT1 independent. This observation on itreg is in marked contrast to a recent report in which it was found that the development of naturally occurring Treg is diminished in the absence of STAT1 [32], and suggests that IL-27 exerts differential effects on itreg as compared to naturally occurring Treg via STAT1. Finally, our data suggest a model in which STAT1 controls IL-27-mediated immune responses by modulating the development of Th17 cells or itreg, respectively. Concluding remarks It is well known that IL-27 can promote both anti- and pro-inflammatory immune responses. Here, we provide evidence how IL-27 may exert such antagonistic effector functions, demonstrating that IL-27 can suppress the development of both pro-inflammatory Th17 cells as well as anti-inflammatory itreg. Whereas the inhibition of Th17 development was dependent on the transcription factor STAT1, the suppression of itreg induction was STAT1 independent. Utilizing different signaling pathways for the blockade of Th17 and itreg cells, IL-27 could shape T cell-driven immune responses and might play opposing roles in a way that goes beyond a counterregulation of the cell type predominantly present at a site of immune reaction. In summary, our data demonstrate that IL-27 controls the development of Th17 and itreg cells via differential effects on STAT1. Six- to ten-week-old mice (BALB/c, C57BL/6) were obtained from the specific pathogen-free animal facility of the Gutenberg University (Mainz, Germany). STAT1-knockout mice on the SV129/Ev background were purchased from Taconic (Hudson, NY). Hemagglutinin TCR transgenic mice on a RAG-deficient background and murine lymphoma cells (A20) were kindly provided by U. Hartwig (University of Mainz). Total splenocytes were harvested, and erythrocytes were lysed by hypotonic lysis. CD4 + CD25 and CD4 + CD25 CD62L + were purified by MACS (Miltenyi, Bergisch Gladbach, Germany). CD4 + T cell purity was consistently at 95%, as evaluated by FACS analysis. PBS and RPMI 1640 supplemented with 10% fetal bovine serum (Perbio Science, Bonn, Germany), 2 mm glutamine (Biochrom, Terre Haute, IN) and penicillin/streptomycin (Biochrom) were used for all cell isolation procedures. Tissue culture Tissue culture plates were obtained from Corning (Wiesbaden, Germany). Cells were grown in serum-free medium X-vivo15 (BioWhittaker, Heidelberg, Germany) without antibiotics in the presence or absence of human recombinant TGF-b (R&D, Wiesbaden, Germany) plus IL-27 (R&D) and other cytokines as indicated. Except for kinetic studies, cells were harvested after 4 5 days. For polyclonal T cell stimulation, anti-cd3 antibody (clone 145 2C11) -coated wells (10 lg/ml in PBS for 4 h at 37 C) were used in combination with soluble anti-cd28 (clone37.51) (1.5 lg/ml). Hemagglutinin (HA) peptide for antigen-specific CD4 stimulation was purchased from JPT Peptide Technologies (Berlin, Germany). Anti-mouse WSX-1-FC, recombinant murine IL-2, and anti-mouse IL-2 were purchased from R&D. Hyper IL-6 (hil-6), a designer molecule comprised of the extracellular part of the IL-6R fused to IL-6 for induction of IL-6 trans-signaling, was kindly provided by S. Rose-John (University of Kiel, Germany). Flow cytometry Intranuclear staining of Foxp3 was performed according to the manufacturer's protocol with PE-conjugated rat anti-mouse Foxp3 antibody (clone FJK-16s) from Ebioscience (San Diego, CA). Intracellular labeling for IL-17 (clone TC11 18H10.1, BioLegend, San Diego, CA) and IFN-c (clone XMG1.2, Caltag- Invitrogen, Hamburg, Germany) were performed with PE- or FITC-conjugated rat anti-mouse antibodies. Cells were stimulated with PMA (50 ng/ml; Calbiochem Merck, Darmstadt, Germany), ionomycin (500 ng/ml; Sigma-Aldrich), and Brefeldin A (5 lg/ml; Sigma-Aldrich) prior to staining, and Fix and Perm cell permeabilization reagents (Caltag-Invitrogen) were used according to the manufacturer's instructions. Surface staining with FITC- or TC-labeled rat anti-mouse CD4 antibodies (clone RM4 5, Caltag-Invitrogen) were performed before cells were processed for labeling of intracellular

7 Eur. J. Immunol : Cellular immune response 1815 antigens. Directly conjugated isotype-matched rat anti-mouse antibodies (Caltag-Invitrogen) were used as controls for unspecific staining. Apoptosis studies were performed with the Active Caspase 3 apoptosis kit (BD, Heidelberg, Germany) according to the manufacturer's protocol. The proliferation of cells was studied with CFSE (Molecular Probes Invitrogen, Karlsruhe, Germany), which was applied to single-cell suspensions at 0.25 lm in prewarmed PBS for 20 min. Next, cells were washed, and incubated with complete RPMI at 37 C for an additional 30 min. After two more washes, cells were seeded and stimulated as described above. Cell acquisition by flow cytometry was done on a FACSCalibur from BD. At least events were acquired and gated on intact CD4 + cells. FACS analysis was done using Summit software (DakoCytomation, Fort Collins, CO). Cytokine quantification Supernatants of cells were analyzed for cytokines by FlowCytomix assays according to the manufacturer's protocol (Bender MedSystems, Vienna, Austria). Co-culture proliferation studies For co-culture proliferation studies, CD4 + CD25 single-cell suspensions (target cells) were incubated with CFSE (Molecular Probes Invitrogen) at 1.25 lm in prewarmed PBS for 20 min. Next, cells were washed, and incubated with complete RPMI at 37 C for an additional 30 min. After two more washes, cells were resuspended in X-vivo, and seeded at cells with anti-cd3 antibody (1 lg/ml). Induced Treg (generated in the absence or presence of IL-27 and harvested plus washed at day 5) were added to the target cells at different ratios. Furthermore, APC treated with mitomycin C (100 lg/ ml) were added per well. Cells were analyzed by flow cytometry at day 4. 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