BCL6 Controls Th9 Cell Development by Repressing Il9 Ribal Bassil, William Orent, Marta Olah, Ahmed T. Kurdi, Michael Frangieh, Thomas Buttrick, Samia J. Khoury and This information is current as Wassim Elyaman of September 29, 2021. J Immunol published online 30 May 2014 http://www.jimmunol.org/content/early/2014/05/29/jimmun ol.1303184 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 30, 2014, doi:10.4049/jimmunol.1303184 The Journal of Immunology

BCL6 Controls Th9 Cell Development by Repressing Il9 Transcription

Ribal Bassil,*,1 William Orent,*,1 Marta Olah,* Ahmed T. Kurdi,* Michael Frangieh,* Thomas Buttrick,* Samia J. Khoury,*,† and Wassim Elyaman*

The transcriptional repressor 6 (BCL6) is required for the development of Th follicular cells, and it has been shown to suppress Th2 cell differentiation. We demonstrate that BCL6 is a key regulator of Th9 cell development. BCL6 expression is transiently downregulated in polarized Th9 cells, and forced expression of BCL6 in Th9 cells impairs Th9 cell differentiation. In contrast, BCL6 knockdown upregulated IL-9 production in Th9 cells. The function of BCL6 in Th9 cells is under the control of IL-2/JAK3/STAT5 signaling pathway. Using chromatin immunoprecipitation, we show that, in Th9 cells, BCL6 and STAT5 bind to adjacent motifs in the Il9 promoter. Furthermore, we found that STAT5 binding was associated with the abundance of

a permissive histone mark at the Il9 promoter, whereas under conditions in which BCL6 binding was predominant, a re- Downloaded from pressive histone mark was prevalent. The effects of STAT5 and BCL6 on IL-9 transcription were further demonstrated using an IL-9 luciferase reporter assay in which BCL6 repressed STAT5-mediated Il9 transactivation. In experimental autoim-

mune encephalomyelitis, forced expression of BCL6 in myelin oligodendrocyte glycoprotein35–55-specific Th9 cells resulted in decreased IL-9 production and induction of IFN-g, causing an exacerbation of the clinical disease. Our findings demonstrate a novel role of BCL6 in the regulation of Th9 cell development and their encephalitogenicity. The Journal of Immunology,

2014, 193: 000–000. http://www.jimmunol.org/

ollowing Ag stimulation, naive CD4+ T cells differentiate Networks of cytokines and transcription factors are critical for into one of several functional classes of effector cells. In determining CD4+ T cell fates and effector cytokine production. F addition to the classical Th1 and Th2 lineages, Th17 cells Indeed, each subset utilizes a master regulatory transcription fac- have been described and extensively characterized. Recently, a tor and a particular STAT (10). The relationships are as follows: new subset of IL-9–producing Th cells induced in vitro by IL-4 Th2, GATA-binding 3/STAT5; Th1, T-box transcription and TGF-b1 has been identified (1, 2). Traditionally associated factor expressed in T cells (T-bet)/STAT4; Th17, retinoid orphan with the Th2 response, IL-9 is a pleiotropic cytokine that impacts gt/STAT3; inducible Treg, forkhead box protein 3 by guest on September 29, 2021 inflammation by exerting broad effects on a variety of cell types (Foxp3)/STAT5. Recent studies suggest that Th follicular cells such as CD4+ T cells, mast cells, and epithelial cells. Recent re- may also fit the paradigm with the factors being B cell lymphoma ports by our group and others demonstrated that IL-9 exerts pro- 6 (Bcl-6)/STAT3. Interestingly, in many instances, the STAT in- or anti-inflammatory properties depending on the inflammatory volved also plays a role in the induction of the master transcrip- milieu by regulating Th17 and CD4+Foxp3+ regulatory T cell tional regulator [reviewed in (11)]. The Il9 is responsive to (Treg) expansion and survival (3–6). Moreover, adoptive transfer multiple factors that bind and induce a conserved noncoding se- of Th9 cells has shown divergent functions from other transferred quence in reporter assays, including IRF4, PU.1, NF-kB, and Smad/ subsets in models of tumor immunity, autoimmune encephalo- Notch complexes (3, 12–14). Recently, transcription factors of the myelitis, and allergic airway disease (7–9). STAT family, STAT5 and STAT6, were shown to be critical for Th9 cell development (15, 16). The Bcl6 , originally identified as an oncogene for B cell lymphoma, encodes a transcriptional repressor *Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Med- protein that regulates T cell differentiation by repressing Th1 and † ical School, Boston, MA 02115; and Abu Haidar Neuroscience Institute, American Th2 cell development (17–19). BCL6 knockout mice exhibit sig- University of Beirut, Beirut 1107-2020, Lebanon nificant growth retardation and invariably die by 10 wk of age (20, 1R.B. and W.O. contributed equally to this work. 21). BCL6 knockout mice have multiple immunological defects, Received for publication November 27, 2013. Accepted for publication May 1, 2014. including lack of germinal center formation and spontaneous de- This work was supported by National Institutes of Health Grants AI071448 (to S.J.K.) velopment of severe Th2-type inflammatory disease, particularly and AI093838 (to S.J.K. and W.E.) and National Multiple Sclerosis Society Grants RG3945 (to S.J.K.) and PP1734 (to W.E.). affecting the heart and lungs (20, 21). The DNA motifs recognized Address correspondence and reprint requests to Dr. Wassim Elyaman and Dr. Samia by BCL6 are highly homologous to the core consensus-binding J. Khoury, Center for Neurologic Diseases, Brigham and Women’s Hospital, 77 sequence TTC-NNN-GAA (in which N is any nucleotide) of Avenue Louis Pasteur, Boston, MA 02115. E-mail addresses: [email protected]. STAT5 (20, 22), a positive regulator of Th9 cell development (16), harvard.edu (W.E.) and [email protected] (S.J.K.) which suggests that BCL6 may play a role in the transcriptional The online version of this article contains supplemental material. regulation of the Il9 locus and Th9 cell development. Abbreviations used in this article: BCL6, B cell lymphoma 6; ChIP, chromatin immunoprecipitation; EAE, experimental autoimmune encephalomyelitis; H3K4me1, In the current study, we analyzed the role of BCL6 in the regu- monomethyl histone [3H]lysine 4; MOG, myelin oligodendrocyte glycoprotein; MSCV, lation of Th9 cell development and encephalitogenicity. We dem- murine stem cell virus vector; siRNA, small interfering RNA; Treg, regulatory T cell; onstrate that BCL6 controls Th9 cell differentiation by direct binding WT, wild-type. and regulation of the Il9 locus. Furthermore, BCL6 function in Th9 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 cells is regulated by the IL-2/JAK3/STAT5 signaling pathway.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303184 2 BCL6 IS A NEGATIVE REGULATOR OF Th9 CELLS

Materials and Methods inhibitor mixture (Sigma-Aldrich); 20 mg total protein was loaded into Mice and reagents each well of a SDS-PAGE gel for separation by electrophoresis and then transferred on nitrocellulose membrane. The resulting blots were blocked C57BL/6 and Rag22/2 mice were purchased from Jackson Laboratory, and for 1 h with TBS-Tween 20 containing 5% BSA and then probed for 3 h at myelin oligodendrocyte glycoprotein (MOG)35–55 TCR transgenic mice room temperature with primary Abs directed against the following: (2D2) were previously described (23). Mice were housed in the pathogen- phospho-STAT5 (Tyr694), phospho-JAK3 (Tyr980/981), STAT5, JAK3, or free animal facility at Harvard Medical School, New Research Building, in BCL6 (Cell Signaling Technology). b-actin mouse mAb (Sigma-Aldrich) accordance with the guidelines of the Committee of Animal Research at was used as the loading control. Blots were then washed five times and the Harvard Medical School and the National Institutes of Health animal probed for 1 h with the appropriate HRP-conjugated secondary Ab. research guidelines as set forth in the Guide for the Care and Use of Membranes were developed with Immobilon Western Chemiluminescent Laboratory Animals. The following fluorescent conjugated primary mAb HRP substrate (Millipore). were purchased from BD Biosciences (San Jose, CA): anti-mouse IL-9 PE, IL-17 allophycocyanin, and anti-mouse CD4-Pacific Blue. JAK3 inhibitor RNA interference VI (420126) and STAT5 inhibitor (573108) were obtained from Calbio- To knock down the expression of Stat5a and Bcl6, CD4+ T cells were chem (San Diego, CA). The 7-aminoactinomycin D stain for dead cell purified and nucleoporated with small interfering RNA (siRNA) specific exclusion was obtained from BD Biosciences. for Stat5a or Bcl6 (Santa Cruz Biotechnology). Scrambled siRNA was used In vitro T cell differentiation, retroviral cell transduction, and as control. Cells were transfected using an Amaxa nucleoporator system. Briefly, 5–10 3 106 CD4+ T cells were resuspended in 100 ml Nucleo- cytokine assay fector solution and transfected with 100 nM siRNA using Amaxa Nucle- Naive CD4+ T cells were purified from C57BL/6 wild-type (WT) mice ofector (Lonza, Basel, Switzerland). After transfection, the cells were using anti-CD4 beads (Miltenyi Biotec, Auburn, CA) and flow sorted into incubated for 6 h at 37˚C, followed by polarization under Th9 conditions. + high naive CD4 CD62L T cells by flow cytometry on a FACSAria T cell Passive experimental autoimmune encephalomyelitis model Downloaded from sorter (BD Biosciences). CD4+ T cells were stimulated with plate-bound anti-CD3 (4 mg/ml; 145-2C11; BD Biosciences) and soluble anti-CD28 For the generation of myelin-specific Th9 cells, CD4+CD62L+ cells were (2 mg/ml; BD Biosciences) for 4 d in a serum-free culture medium (X- prepared from spleens of MOG35–55 TCR transgenic mice (2D2) using VIVO-20; Lonza, Hopkinton, MA) supplemented with 50 mM 2-ME, 1 mM MACS beads. CD4+ cells were transduced with MSCV-BCL6-GFP or sodium pyruvate, nonessential amino acids, L-glutamine, and 100 U/ml control empty vector MSCV-GFP, followed by stimulation with MOG35–55 penicillin/100 mg/ml streptomycin in the presence of recombinant cyto- peptide (20 mg/ml) in the presence of IL-4 (20 ng/ml) and TGF-b1 (3 ng/ml) kines. Polarization of Th9 cells was in the presence of human TGF-b1 for 3 d in the presence of splenic CD11c+ dendritic cells isolated from 2D2 (3 ng/ml) plus mouse IL-4 (10 ng/ml). Cells were supplemented with rIL-2 mice. One million cells were transferred into Rag22/2 recipient mice via http://www.jimmunol.org/ (40 ng/ml), where indicated. Rat anti-mouse IL-2 Ab (BD Biosciences) i.p. injection, followed by immunization with the emulsion made of 50 mg was used for blocking of IL-2 activity in vitro. All recombinant MOG35–55 (MEVGWYRSPFSRVVHLYRNGK; New England Peptide) were from R&D Systems. JAK3 (250 mM) and STAT5 (50 mM) inhibitors and CFA. Each animal also received i.p. injections of 69 ng pertussis toxin were added along with the polarization medium at the start of differenti- on days 1 and 3 after transfer. The experimental autoimmune encephalo- ation. For intracellular flow cytometry staining, cells were restimulated myelitis (EAE) clinical score was determined as follows: 0, no disease; 0.5, with 12-O-tetradecanoylphorbol-13-acetate (20 ng/ml; Sigma-Aldrich, St. partial tail paralysis; 1, complete tail paralysis; 2, partial hind limb pa- Louis, MO), ionomycin (300 ng/ml; Sigma-Aldrich), and 2 mM monensin ralysis; 3, complete hind limb paralysis; 4, complete hind limb and partial (GolgiStop; BD Biosciences) for 4 h at 37˚C. Cells were washed, stained front limb paralysis; and 5, moribund or dead animals. for surface markers, fixed, and permeabilized, and fluochrome-conjugated

Abs were added. For the measurement of cytokines released in the culture Chromatin immunoprecipitation and quantitative PCR by guest on September 29, 2021 supernatants, cell culture supernatants were collected on day 4 after dif- + ferentiation, and the secreted cytokines were determined by fluorescent CD4 T cells were purified by MACS sorting and were polarized to bead-based Luminex technology (Luminex, Austin, TX) for the indicated Th9 phenotype. Chromatin immunoprecipitation (ChIP) was performed cytokines, in accordance with the manufacturers’ instructions. For intracellular according to the protocol described previously (25) with the following cytokine staining of infiltrated T cells, mice were sacrificed and perfused with modifications: chromatin was sheared using micrococcal nuclease (New PBS. Spinal cord tissues were digested with collagenase IV (Sigma-Aldrich) England Biolabs), and protein A magnetic beads (New England Biolabs) were used. Cell lysates were used for immunoprecipitation with anti- for 30 min at 37˚C, resuspended in 30% Percoll, and loaded onto 70% Percoll. 3 After centrifuge at 1300 3 g for 20 min, the CNS inflammatory cells were STAT5, anti-BCL6, anti-monomethyl histone [ H]lysine 4 (H3K4me1), retrieved from the 30/70% Percoll interface. Lymphocytes were prepared for and anti–pan-methyl-H3K9 (all from Cell Signaling Technology) and were intracellular flow cytometry staining, as described above, using anti–IL-9 PE- compared with control IgG. One region of the Il9 promoter containing and anti–IFN-g allophycocyanin-conjugated Abs. putative STAT5 and BCL6 binding sites as well as a region of the Bcl6 For forced expression of BCL6, naive CD4+ T cells were transduced promoter containing putative STAT5 binding sites was amplified by SYBR with a murine stem cell virus vector (MSCV)–based retroviral vector (24). Green quantitative PCR (Applied Biosystems) and quantified in duplicate Briefly, the retrovirus particles were produced by cotransfecting the plas- with the percentage of input method. The following primers were used: mids encoding for the ecotropic envelope gag-pol with the retroviral vector Il9 promoter: site 1 forward, 59-ACTGAGTTCCAGACTCCCGT-39, with Effectene (Qiagen) into the Phoenix packaging cells. Retrovirus su- and reverse, 59-GCCCAGCACAGAACTGAAGA-39; site 2 forward, 59- pernatant was harvested 48 h after transfection and centrifuged prior to GGATCCTCAAGGCCAATGCT-39, and reverse, 59-ACACCTCTGA- use. CD4+CD62L+ T cells isolated by MACS from WT mice were acti- GAAGTCGCTC-39; site 3 forward, 59-ACAGAAGTGTGCTGTCTGGT- vated with anti-CD3 and anti-CD28 for 24 h, retroviral supernatant was 39,andreverse,59-CCCCTTGAGCCACTGGATAC-39. Bcl6 promoter: added to the cells together with polybrene (8 mg/ml), and cells were in- site 1 forward, 59-CTGCGGAGCAATGGTAAAGC-39, and reverse, 59- cubated at 37˚C. After incubation, live cells were sorted with the Dead Cell ATAATCACCTGGTGTCCGGC-39; site 2 forward, 59-CGAGGAGCC- Removal Kit (Miltenyi Biotec), and cells were expanded for an additional GAGTTTATGGG-39, and reverse, 59-GAGAGTGCGCTTTGCTTTCC-39; 4 d under Th9 cell polarization condition. site 3 forward, 59-CGAATGACAGTCCCGACGAT-39, and reverse, 59- GCTTGGGATGCTCCTGTTGT-39. Expression analysis by real-time PCR Luciferase reporter assay RNA was purified using Stratagene RNA kit and transferred directly into Reporter vector coding for the firefly luciferase under the control of the Il9 the reverse-transcriptase reagent using the Applied Biosystems TaqMan 2 reverse-transcriptase reagents. Samples were subjected to real-time PCR promoter encompassing nucleotides 1310 to +32 bp was cloned into the analysis on PRISM 7000 Sequencer Detection System (Applied Bio- promoterless pGL3 Basic luciferase reporter gene vector (Promega). MSCV systems, Foster City, CA) under standard conditions. analyzed were expressing WT mouse Stat5a was previously described (26). Plasmid detected using commercially available assays (Applied Biosystems). Rel- encoding mouse Bcl6 (mBCL6/pCMV-SPORT6.1) was purchased from ative mRNA abundance was normalized against GAPDH. Open Biosystems. Reporter assays were carried out, as described previously (3). Briefly, 293T cells were transfected with 0.2 mg reporter vector coding Western blot for the firefly luciferase under the control of the Il9 promoter and with 0.2 mg STAT5A or BCL6 encoding plasmids. Cells were cultured for 24 h before Cells were lysed in radioimmunoprecipitation assay buffer (Thermo Sci- harvesting, and the relative Il9 promoter activity was measured using entific) with a protease inhibitor (Roche Diagnostics) and a phosphatase Promega kit in accordance with the manufacturer’s instructions. The Journal of Immunology 3

Statistical analysis that IL-2 is transiently induced in Th9 cells, and this expression The Mann–Whitney U test was used for clinical disease analysis. Data are inversely correlates with a transient decrease in BCL6 mRNA expressed as mean 6 SEM and were compared using the unpaired Student expression in these cells (Fig. 2A). t test for experiments with two groups by Prism software v5. Data were Given that IL-2 is required for Th9 cells polarized in vitro and is considered statistically significant at p , 0.05. abundantly produced by these cells as measured by Luminex bead- based assay (Supplemental Fig. 1C), and because IL-2/STAT5 Results pathway has been shown to regulate BCL6 signals in follicular BCL6 is a negative regulator of IL-9 expression in Th9 cells Th cells (31, 32), we sought to analyze the activity of the key transducers of IL-2 signaling, STAT5, and its upstream kinase The transcriptional repressor BCL6 has emerged as a multi- JAK3, in polarized Th9 cells. Using bead-based Luminex assay functional regulator of lymphocyte differentiation and immune for phosphorylated STAT5ab, we compared the activity of STAT5 responses. In this study, we investigated the possibility as to in different Th subsets differentiated in vitro for several time points whether BCL6 is involved in murine Th9 cell development. We between 0 and 120 min according to standard protocols in the absence measured BCL6 protein expression in Th9 cells polarized in vitro of exogenous IL-2. We found that STAT5ab activation is predomi- with IL-4 and TGF-b1 and found it decreased as early as 8 h nantly induced in Th9 cells as early as 15 min after polarization (Fig. following differentiation, as shown by Western blot. BCL6 2B). Furthermore, we confirmed that STAT5 signaling is activated in downregulation was transient, suggesting an intrinsic mechanism Th9 cells, by measuring STAT5ab phosphorylation and its upstream for the control of BCL6 expression in Th9 cells (Fig. 1A). To protein kinase, JAK3. Thus, naive CD4+ T cells were stimulated under understand the functional relevance of the regulation of BCL6 Th9 conditions in the presence or absence of rIL-2 (0–60 min), and Downloaded from expression in Th9 cells, we asked whether BCL6-forced expres- cells were lysed and used for immunoblot analysis using Abs specific sion influences Th9 cell development in vitro. To address this, we for phosphorylation-dependent and independent JAK3 and STAT5. We + low high transduced naive CD4 CD4 CD62L T cells with MSCV- found that the phosphorylation of both JAK3 and STAT5 was induced BCL6-GFP retrovirus (BCL6-RV-GFP), cultured the cells under in Th9 cells, and this was sustained in the presence of added rIL-2 (Fig. Th9 conditions for 4 d, and measured intracellular cytokine ex- 2C). Quantification of phospho-JAK3 and STAT5 by measurements of pression using flow cytometry. We found that the frequency of the OD of the corresponding bands is shown (Supplemental Fig. 1D). http://www.jimmunol.org/ IL-9–positive cells was altered by ∼60% after BCL6 (GFP+) 2 Altogether, these data suggest the JAK3/STAT5 signaling cascade overexpression compared with BCL6-negative (GFP ) Th9 cells, is activated in Th9 cells and is further enhanced by IL-2 signaling. suggesting that the BCL6 is a negative regu- To investigate whether IL-2 is involved in the downregulation of lator of Th9 cells (Fig. 1B). To ascertain the involvement of BCL6 BCL6 in polarized Th9 cells, we measured BCL6 protein ex- in Th9 cell generation, we used BCL6-specific siRNA to knock- pression in Th9 cells differentiated in vitro for several days in the + down BCL6 in Th9 cells. Naive CD4 T cells presence of rIL-2 or anti–IL-2 neutralizing Ab. We found that, were nucleoporated with BCL6-specific siRNA, subsequently pola- whereas BCL6 protein levels were transiently decreased during rized to the Th9 lineage, and harvested 4 d after transfection. Con- Th9 cell differentiation, supplementation with rIL-2 induced sistent with the data obtained with BCL6 overexpression, BCL6 sustained suppression of BCL6 protein expression (Fig. 2D). In- by guest on September 29, 2021 knockdown resulted in an upregulation of IL-9 expression at both terestingly, when Th9 cells were differentiated in the presence of the protein and gene levels, as measured by bead-based Luminex anti–IL-2 neutralizing Ab, BCL6 expression was maintained assay and TaqMan PCR, respectively (Fig. 1C, 1D). Moreover, to throughout the polarization, suggesting that IL-2 signaling is address the functional role of increased BCL6 level at later stage a negative regulator of BCL6 in Th9 cells (Fig. 2E). Quantification of Th9 differentiation, we treated Th9 cells with BCL6-specific of BCL6 levels by densitometry is shown (Supplemental Fig. 1D). siRNA or scrambled siRNA on day 3 after polarization. Cells were kept for another 3 d under Th9 conditions, followed by analysis IL-2/JAK3/STAT5 inhibition upregulates BCL6 expression and for IL-9 production by Luminex. We found that BCL6 knockdown suppresses Th9 cell differentiation did not alter IL-9 production, suggesting that BCL6 is primarily Recent reports demonstrated that STAT5 signaling promotes Th9 involved in the early development of Th9 cells (Fig. 1E). cell differentiation and that STAT5 and BCL6 are cross-regulated in Th1 cells (33, 34). Given that JAK3 is an upstream positive reg- Activation of IL-2/JAK3/STAT5 axis inversely correlates with ulator of STAT5 (35), we hypothesized that BCL6 may be regu- BCL6 downregulation in Th9 cells lated by STAT5 in Th9 cells. Thus, we analyzed the effects of the IL-2 is a major growth factor for activated T cells and plays an inhibition of IL-2/JAK3/STAT5 signaling on the outcome of Th9 important role in murine and human Th9 cell expansion and dif- cell differentiation and BCL6 expression. CD4+ T cells were dif- ferentiation in vitro (27, 28). Additionally, activation of STAT5 is ferentiated under Th9 conditions in the presence or absence of an important component of IL-2 signaling and has been recently JAK3 (250 mM)- or STAT5 (100 mM)-specific inhibitors for 4 d. proposed as a positive regulator of Th9 cells (16, 29). To measure We found that addition of either inhibitor upregulated BCL6 the IL-2 signaling pathway downstream components in Th cells, mRNA expression as measured by TaqMan PCR (Fig. 3A). More- we first extracted gene expression profiling from RNA-sequencing over, JAK3 and STAT5 inhibitors suppressed the production of (RNA-Seq) data deposited on Gene Expression Omnibus (30). IL-9 as measured by intracellular cytokine staining as well as Analysis of IL-2 signaling in murine Th1, Th2, Th9, Th17, and by Luminex bead-based assay (Fig. 3B, 3C). inducible CD4+Foxp3+ Tregs differentiated in vitro according to Next, we used STAT5A-specific siRNA to inhibit STAT5 sig- standard protocols shows that the expression of Il2ra, Stat5a, and naling at the gene level. Thus, naive CD4+ T cells were nucleo- Stat5b is upregulated in Th9 cells (Supplemental Fig. 1A). Using porated with STAT5A-specific siRNA or with scrambled siRNA TaqMan PCR to verify the transcriptome-sequencing results in and subsequently polarized to the Th9 lineage and harvested 4 d different Th subsets, we found that mRNA levels of Il2ra, Stat5a, after transfection. Silencing of STAT5A specifically inhibited IL-9 and Stat5b are indeed elevated in Th9 cells compared with other production but did not affect the production of other cytokines, cell phenotypes (Supplemental Fig. 1B). Moreover, temporal including IL-5, IL-17, and IFN-g (Fig. 3D). Quantitative RT-PCR analysis of IL-2 expression in polarized Th9 cells demonstrates confirmed that STAT5A siRNA reduced the expression of Il9 and 4 BCL6 IS A NEGATIVE REGULATOR OF Th9 CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. BCL6 is a negative regulator of Th9 cells. (A) BCL6 is transiently downregulated in polarized Th9 cells. Immunoblot of BCL6 in Th9 cells polarized for 0–72 h in the absence of exogenous rIL-2. b-actin was used as internal control for protein loading. (B) BCL6-forced expression reduced IL-9 expression. Naive CD4+ T cells were prepared from the spleens of WT mice, and cultures were transduced with BCL6-RV-GFP encoding for BCL6-GFP. Cells were differentiated under Th9 cell conditions for 4 d, followed by intracellular cytokine staining, and BCL6-GFP–positive cells were gated on the basis of the GFP expression. (C) BCL6 knockdown in Th9 cells upregulates IL-9 production. Naive CD4+ T cells were transfected with siRNA specific for BCL6 or with scrambled (Scr) siRNA using Amaxa, followed by polarization under Th9 cell conditions for 4 d. Cytokine analysis of IL-5, IL-9, IL-17, and IFN-g expression in the supernatants was assessed by Luminex. (D) A representative gene expression of Il9, Bcl6, and Stat5a in Th9 cells that were transfected with siRNA specific for BCL6 is shown. Transfection with scrambled siRNA was used as a control. (E) IL-9 production in Th9 cells. Th9 cells were differentiated for 3 d, followed by transfection with siRNA specific for BCL6 or with scrambled siRNA. IL-9 release in the culture supernatants was measured by Luminex. Data are representative of three experiments with similar results. **p , 0.005.

Stat5a mRNA (Fig. 3E). To check whether the reduction in IL-9 in that these transcription factors may have opposing roles in the STAT5A siRNA-treated cells was due to differential cell survival, regulation of the Il9 promoter in Th9 cells. To investigate this, we we performed 7-aminoactinomycin D flow staining of nucleopo- searched the Il9 promoter for potential binding sites for STAT5 rated cells, which revealed no changes between control and and BCL6 using Biobase database. We identified three over- STAT5A siRNA-treated cells (data not shown). lapping putative binding sites for STAT5 and BCL6 upstream of Further analysis of STAT5A-specific siRNA showed that the the transcription start site (TSS) (Fig. 4A). To determine the Il9 treatment was associated with an increase in Bcl6 mRNA expres- promoter occupancies by STAT5 and BCL6, binding motifs were sion (Fig. 3E), in agreement with the elevated Bcl6 expression in used to design ChIP experiments. Primer sets flanking the STAT5 Th9 cells treated with IL-2/JAK3/STAT5 inhibitors (Fig. 3A) and and BCL6 binding at three sites in the Il9 promoter were designed the decrease in BCL6 protein expression in Th9 cells exposed to to amplify the immunoprecipitated ChIP DNA by quantitative IL-2 (Fig. 2C). These findings suggest that BCL6, a transcriptional PCR (Fig. 4A). CD4+CD62Lhigh T cells were differentiated under repressor that has been shown to modulate STAT signaling in Th9 cell-polarizing conditions for 1–4 d and then analyzed by B cells (36, 37), plays a role in the regulation of Th9 cell differ- ChIP-PCR. We found that STAT5 binds significantly to sites 1, 2, entiation under the control of STAT5 signaling. and 3 in the Il9 promoter in Th9 cells 2 d after cell polarization (Fig. 4B, Supplemental Fig. 2). We next investigated whether Opposite roles of BCL6 and STAT5 in binding and regulating STAT5 binding to the Il9 promoter in Th9 cells is modulated by the Il9 promoter in Th9 cells IL-2 signaling. We observed that IL-2 blockade using IL-2 neu- Because IL-2 enhances the differentiation of Th9 cells and exerts tralizing Ab abolished STAT5 binding to the Il9 promoter. Simi- opposite effects on STAT5 and BCL6 expression, we hypothesized larly, pharmacological inhibition of both JAK3 and STAT5 The Journal of Immunology 5 Downloaded from

FIGURE 2. Activation of IL-2/JAK3/STAT5 axis inversely correlates with BCL6 downregulation in Th9 cells. (A) Opposite regulation of IL-2 and BCL6 in polarized Th9 cells. Naive CD4+ T cells were cultured under Th9 conditions for 4 d in the absence of exogenous rIL-2 and BCL6 and IL-2 mRNA expression was assessed by TaqMan PCR. (B) Increased STAT5 activation in Th9 cells. Polarized Th and inducible Tregs were differentiated in vitro for http://www.jimmunol.org/ 0–120 min (min), and phospho-STAT5 (pSTAT5) was assessed using bead-based Luminex assay. (C) Representative immunoblot of phospho-dependent and -independent JAK3 and STAT5 in naive CD4+CD62Lhigh polarized under Th9 conditions for the indicated time points (0–60 min) with rIL-4 plus TGF-b1 in the presence or absence of exogenous rIL-2. Phospho-epitope of each protein kinase is indicated. b-actin was used as internal control for protein loading. (D) rIL-2 inhibited BCL6 expression in Th9 cells. Naive CD4+ T cells were polarized under Th9 cell conditions in the presence or absence of exogenous rIL-2, and BCL6 expression was assessed for up to 4 d. b-actin expression is shown. (E) Anti–IL-2 neutralizing Ab rescued the decrease in BCL6 expression in Th9 cells. Naive CD4+ T cells were polarized under Th9 cell conditions in the presence or absence of anti–IL-2 neutralizing Ab, and BCL6 expression was assessed on days 0, 1, and 2. b-actin expression is shown.

abolished STAT5 recruitment to the Il9 promoter in Th9 cells, significantly to the Bcl6 promoter and this process was abolished by guest on September 29, 2021 which correlates with the suppression of IL-9 expression (Fig. 4B, when cells were exposed to anti–IL-2 blocking Ab or to JAK3 and Supplemental Fig. 2). We confirmed the specificity of STAT5 STAT5 inhibitors (Fig. 4E), which correlates with the identified binding by amplifying a region of the Il9 promoter that does not role of STAT5 as a positive regulator of Th9 cell development. contain STAT5 binding sites (data not shown). To analyze the functional relevance of the binding of STAT5 and Given that STAT5 and BCL6 have opposite effects on IL-9 BCL6 to their target sequences in the Il9 locus, we investigated the expression in Th9 cells, we investigated whether the two tran- ability of STAT5 and BCL6 to regulate the activity of the Il9 scription factors compete for binding to the same binding motifs in promoter in reporter assays. We used the reporter construct pGL3- the Il9 promoter. Indeed, we found that BCL6 binding to the Il9 Il9 containing the firefly luciferase gene under the control of the promoter inversely correlates with STAT5 binding to site 2 and to Il9 promoter. First, we found that cotransfection of the pGL3-Il9 a lesser extent to sites 1 and 3. Strikingly, this process was under luciferase reporter construct with a plasmid encoding constitu- the control of IL-2 signaling because inhibition of either IL-2, tively active mutant of STAT5 (STAT5aS711F) (26) in 293T cells JAK3, or STAT5 suppressed STAT5 binding, whereas it facili- resulted in a significant increase in Il9 transcription (Fig. 4F). In tated BCL6 recruitment to the same motif, particularly to site 2 agreement with the function of BCL6 as a negative regulator of and to a lesser extent to sites 1 and 3 in the Il9 promoter (Fig. 4C, Th9 cell development, we found that BCL6 decreased Il9 pro- Supplemental Fig. 2). We next studied whether chromatin mod- moter activity significantly in a luciferase reporter assay. Strik- ifications at the STAT5- and BCL6-binding motifs within the Il9 ingly, cotransfection of BCL6 with STAT5 resulted in a very promoter could explain the regulation of IL-9 by IL-2 signaling. significant suppression of STAT5-positive activity (p , 0.0001), Analysis of histone 3 ([3H]) methylation in Th9 cells revealed suggesting that BCL6 is a dominant-negative regulator of the Il9 increased permissive H3K4me1 modifications at site 2 in the Il9 transcription in Th9 cells (Fig. 4F). STAT5A overexpression in promoter with decreased repressive [3H]lysine 9 pan-methylation 293T cells is comparable in the presence or absence of exogenous (H3K9-pan-me) chromatin modifications on day 2 after in vitro BCL6 (Supplemental Fig. 3). polarization. Interestingly, IL-2 neutralization increased the H3K9-pan repressive mark, whereas it has no significant influence BCL6 overexpression in myelin-specific Th9 cells regulates on the H3K4me1 permissive mark (Fig. 4D). their encephalitogenicity Previously, STAT5 has been shown to bind the Bcl6 promoter Previous studies demonstrated that adoptive transfer of myelin- and downregulate its transcription in B-lymphoma cells and other reactive Th9 cells induces EAE (7). To investigate whether hematopoietic cell lines (38). To investigate a similar outcome in BCL6-mediated IL-9 repression modulates EAE, we carried out Th9 cells, we analyzed STAT5 recruitment to the Bcl6 promoter in adoptive transfer experiments with MOG35–55-pulsed Th9 cells polarized Th9 cells exposed to IL-2/JAK3/STAT5 blocking agents. following BCL6 overexpression. To generate myelin-specific Th9 + Interestingly, we found that, under basal conditions, STAT5 binds cells overexpressing BCL6, MOG35–55-TCR transgenic CD4 6 BCL6 IS A NEGATIVE REGULATOR OF Th9 CELLS Downloaded from

FIGURE 3. Opposite role of STAT5A and BCL6 in the regulation of Th9 cell development. (A–C) Inhibition of IL-2/JAK3/STAT5 signaling decreases IL-9 production in Th9 cells. (A) Heatmap of gene regulated in Th9 cells treated with anti–IL-2 neutralizing Ab and JAK3 and STAT5 inhibitors. Naive CD4+ T cells from WT mice were differentiated into Th9 cells in the presence or absence of the indicated treatment, and gene expression was measured by http://www.jimmunol.org/ TaqMan PCR. (B and C) IL-9 protein expression by bead-based Luminex assay (B) as well as by intracellular flow cytometry (C) in treated Th9 cells is shown. (D and E) STAT5 knockdown reduces IL-9 and upregulated BCL6 expression in Th9 cells. Naive CD4+ T cells transfected using Amaxa with siRNA specific for STAT5A or scrambled (Scr) siRNA, followed for polarization under Th9 cell conditions for 4 d. (D) Cytokine analysis of IL-5, IL-9, IL-17, and IFN-g expression in the supernatants was assessed by Luminex. (E) A representative gene expression of Il9, Bcl6, Stat5a, and Il2ra in Th9 cells that were transfected with siRNA specific for STAT5A, or scrambled siRNA and measured by TaqMan PCR. Data are representative of three experiments with similar results. ***p , 0.0001.

CD62Lhigh naive cells were sorted from 2D2 mice and were Th2, and Th17 cells, has been reported by several groups (17, 18, transduced with MSCV-BCL6-GFP retrovirus or with control 21, 41–43). In this study, we provide evidence demonstrating that by guest on September 29, 2021 vector and subsequently polarized into Th9 cells for 3 d in the BCL6 is a repressor of Th9 cell development through direct reg- presence of MOG35–55 (20 mg/ml) peptide and splenic dendritic ulation of the Il9 promoter. We report that BCL6 expression is cells. Analysis of the cytokine profile of BCL6-GFP–positive 2D2 reduced early in polarized Th9 cells under IL-4 plus TGF-b1 CD4+ T cells demonstrates a decrease in IL-4 and IL-9 expression conditions in an IL-2–dependent manner. At the transcription associated with an increase in IFN-g expression compared with level, BCL6 binds and induces Il9 repression, and this process is control cells as shown by TaqMan PCR (Fig. 5A). No changes in competition with the STAT5 signaling, a positive regulator of were detected in IL-17 expression (data not shown). To analyze Th9 cell differentiation. In autoimmunity, silencing of BCL6 in the effects of BCL6 on the encephalitogenicity of Th9 2D2 cells, myelin-reactive Th9 cells increases IL-9 and IL-4 expression and cells were transferred into Rag22/2 lymphopenic mice, followed decreases IFN-g production, leading to a reduction of their by reactivation with MOG35–55/CFA immunization, and mice were encephalitogenicity upon adoptive transfer in lymphopenic hosts. monitored for clinical disease development. We found that EAE IL-2, a growth factor linked to human autoimmune diseases (44), was markedly enhanced in recipients of Th9 MSCV-BCL6 com- is a critical element for the development of Th1 (45) and Th2 (46) pared with Th9 MSCV-RV control mice (mean maximal score for cells while inhibiting Th17 (47) and Th follicular cells (48). The Th9 MSCV-BCL6–treated mice 3 6 0.3 compared with Th9 a-subunit of the IL-2R (IL-2Ra) plays a pivotal role in the reg- MSCV-RV–treated mice 1.4 6 0.2; p , 0.001 by Mann–Whitney ulation of T cell function. Levels of a soluble form of IL-2Ra U test) (Fig. 5B). To test whether the cytokine profile of trans- lacking the transmembrane and cytoplasmic domains were shown ferred cells is maintained in recipient mice, spinal cord tissues to be increased in several autoimmune diseases, including multi- were collected ∼10 d posttransfer, 2D2 cells were identified using ple sclerosis (49). In addition, IL-2Ra genetic variants correlate the surface markers Va3.2/Vb11, and cells were analyzed for with the levels of soluble IL-2Ra in subjects with type 1 diabetes cytokine expression, confirming that BCL6-forced expression and multiple sclerosis (50, 51). Addition of IL-2 to murine and converts Th9 cells into Th1-like phenotype, as shown by an in- human Th9 cells enhances their differentiation (27, 28, 52). crease in IFN-g and suppression of IL-9 production (Fig. 5C). However, the molecular mechanisms of IL-2–mediated amplifi- cation of Th9 cell differentiation are still unclear. IL-2 is produced Discussion mainly by activated T cells after engagement of the TCR and the Previous reports uncovered a key role of BCL6 as a transcriptional CD28 costimulatory molecule (53). Our data demonstrate that, in regulator in various cell types, including B cells and T lymphocyte addition to Th1 and Th2 cells, IL-2 is massively produced early subsets within and outside the germinal centers, including CD4+ after Th9 cell polarization, suggesting a role of IL-2 signaling in the follicular Th cells (39), T regulatory (40), and Th cells, and in the regulation of Th9 cell development. Three subunits constitute the IL- control of T cell–dependent inflammation and autoimmunity (17, 2R, as follows: the a-chain (IL-2Ra, also known as CD25), the 20). The role of BCL6 in the regulation of Th cells, including Th1, b-chain (IL-2Rb, also known as CD122, shared by the IL-15R), and The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. IL-2–mediated control of STAT5 and BCL6 differential binding to the Il9 promoter. (A) Three predicted binding sites for BCL6 (open box) and STAT5 (filled boxes) in the Il9 promoter. PCR primer sets for each site are shown. (B and C) ChIP analysis of BCL6 and STAT5 binding to the Il9 promoter. Regulation of BCL6 and STAT5 binding to site 2 within the Il9 promoter. Naive CD4+ T cells from WT mice were polarized under Th9 cell conditions for 2 d in the presence of anti–IL-2 neutralizing Ab, JAK3, or STAT5 inhibitor. ChIP-SYBR Green PCR was performed to determine (B)STAT5 and (C) BCL6 binding to the Il9 promoter. Abs used for inflammatory protein are anti-STAT5, anti-BCL6, and control IgG. Total input DNA before inflammatory protein was used for normalization of data. (D) ChIP analysis of H3K4 (left panel) and H3K9 (right panel) modifications normalized to input controls at the BCL6 and STAT5 binding site 2 within the Il9 promoter in Th9 cells differentiated in vitro for 2 d. (E) ChIP analysis of STAT5 binding to the Bcl6 promoter in Th9 cells differentiated in vitro for 2 d. The graphs represent quantitative PCR analysis of the ratio of enriched Il9 and Bcl6 promoters with transcription factor binding sites to the input DNA. Data represent mean 6 SE of a representative experiment each performed in triplicate. (F) HEK 293T cells were transfected with active mutant of STAT5 (STAT5aS711F), BCL6, or combination of STAT5aS711F and BCL6 together with a constant amount of Il9 promoter-luciferase vector. Cells were lysed 24 h later, and luminescence was measured. Representative of three independent experiments. *p , 0.01, **p , 0.005, ***p , 0.0001. 8 BCL6 IS A NEGATIVE REGULATOR OF Th9 CELLS Downloaded from

A FIGURE 5. BCL6 regulates Th9 cell pathogenicity in EAE mice. ( ) A representative gene expression of IL-4, IL-9, IL-17A, IFN-g, and BCL6 in Th9 http://www.jimmunol.org/ + overexpressing BCL6 and control cells. MOG35–55-specific CD4 T cells were isolated from 2D2 mice and were transduced with MSCV-BCL6-GFP retrovirus (RV) or with control empty vector (MSCV-RV), followed by in vitro polarization under Th9 conditions for 4 d in the presence of MOG35–55 peptide (20 mg/ml) and irradiated APCs. Cells were lysed and analyzed for gene expression by TaqMan. (B) A representative EAE experiment showing clinical scores (means 6 SEM) in mice after adoptive transfer of MOG35–55-stimulated 2D2 polarized under Th9 cell conditions with or without BCL6 2/2 overexpression (10 mice/group). Rag2 mice received MSCV-BCL6–transduced Th9 cells or control cells, followed by immunization with MOG35–55 peptide (100 mg/ml). (C) Intracellular staining of transferred 2D2 cells. Infiltrated cells were prepared from the spinal cords of recipient mice (3 mice/ group), followed by flow cytometry intracellular staining of IFN-g and IL-9. Data are representative of two experiments with similar results. **p , 0.01, ***p , 0.001. by guest on September 29, 2021 the common cytokine receptor g-chain (also known as CD132, factors and BCL6 directly compete for DNA binding (20, 58). shared by IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R). The IL-2/ BCL6 repression could thus involve direct competition for STAT5 IL-2R complex mediates downstream signaling through IL-2Rb binding, as well as epigenetic modifications induced by BCL6- and common cytokine receptor g-chain by inducing the associa- associated factors. Indeed, we observed an increase in the re- tion and phosphorylation of the tyrosine kinases JAK1 and JAK3 pressive H3K9 pan-methylation mark surrounding BCL6 binding (54, 55), leading to the activation of STAT5 pathway (56). In this site in Th9 cells cultured with anti–IL-2 neutralizing Ab. It should study, we provide novel evidence characterizing a regulatory be noted that the described interplay between STAT5 and BCL6 is network in which STAT5 functions as an upstream regulator of the most likely modulated for the long-term because STAT5 was expression and activity of BCL6 and promoting the development found to act as a transcriptional repressor on the BCL6 gene itself of Th9 cells. These findings are in agreement with a recent report (38). We also found that, in Th9 cells, STAT5 is recruited to the describing involvement of STAT5 signaling in Th9 cell polariza- BCL6 promoter and this was regulated by IL-2 signaling. tion in animal models of allergic airway inflammation (16, 29). Th cells exhibit various degrees of plasticity, namely, the ca- The molecular dissection of BCL6-mediated Th9 cell differ- pacity to change their phenotype when exposed to a new inflam- entiation inhibition indicates that BCL6 directly represses the Il9 matory milieu or upon regulation of transcription factors. An locus. We have previously reported that Th9 cell differentiation is increasing body of evidence demonstrates that the regulation of defective in GATA3-deficient mice, suggesting that GATA3 is Th9 cell development is a complex process requiring multiple cis- required for IL-9 transcription (1). Although our present report regulatory elements and specific trans-activating transcription provides direct evidence demonstrating that BCL6 directly reg- factors, including PU.1, IRF4, and RBPJ, which are in common ulates the Il9 promoter, it remains possible that GATA3, a known with other CD4+ T cell lineages, particularly Th2 cells (12, 14, 59, target of BCL6 (17), may provide an additional mechanism 60). Our identification of BCL6 transcriptional program in Th9 linking BCL6 to the regulation of Th9 cell differentiation. cell differentiation provides additional evidence of the plasticity of The BCL6 consensus-binding site resembles the IFN-g–acti- Th9 cells in which BCL6-forced expression resulted in a down- vated sequence motif recognized by the STAT family of tran- regulation of Th9/Th1 ratio, as shown by a decrease in IL-4/IL-9 scription factors, raising speculation that BCL6 may repress some expression and an increase in IFN-g production, leading to EAE cytokine response genes (57). Our data demonstrate that STAT5 exacerbation following adoptive transfer. It is worth noting that not only binds and activates the Il9 promoter transactivation but other transcription factors such as STAT1 have been reported to also competes with BCL6-binding activity to the Il9 promoter in modulate Th9 cell plasticity as shown by genetic deletion of an IL-2–dependent manner. These findings support previous STAT1 in these cells increased the Th9/Th1 ratio, whereas IL-27 studies demonstrating that STAT5 and BCL6 bind to distinct but suppressed IL-9 production and upregulated IFN-g expression in overlapping sequence motifs, supporting the proposal that STAT Th9 cells in a STAT1-dependent manner (61). The Journal of Immunology 9

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