RORC2 Is Involved in Polarization through Interaction with the FOXP3 Promoter

This information is current as Simone Burgler, Pierre-Yves Mantel, Claudio Bassin, Nadia of September 28, 2021. Ouaked, Cezmi A. Akdis and Carsten B. Schmidt-Weber J Immunol 2010; 184:6161-6169; Prepublished online 28 April 2010; doi: 10.4049/jimmunol.0903243

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

RORC2 Is Involved in T Cell Polarization through Interaction with the FOXP3 Promoter

Simone Burgler,* Pierre-Yves Mantel,*,† Claudio Bassin,* Nadia Ouaked,* Cezmi A. Akdis,* and Carsten B. Schmidt-Weber‡

The process of Th cell differentiation toward polarized effector T cells tailors specific against invading pathogens while allowing tolerance against commensal microorganisms, harmless allergens, or autologous Ags. Identification of the mechanisms underlying this polarization process is therefore central to understand how the confers immunity and tolerance. The present study demonstrates that retinoic acid -related orphan receptor C2 (RORC2), a key factor in Th17 cell development, inhibits FOXP3 expression in human T cells. Although overexpression of RORC2 in naive T cells reduces levels of FOXP3, small interfering RNA-mediated knockdown of RORC2 enhances its expression. RORC2 mediates this inhibition at least partially by binding to two out of four ROR-responsive elements on the FOXP3 promoter. Knockdown of RORC2 promotes high Downloaded from FOXP3 levels and decreased expression of proinflammatory b form of pro-IL-1, IL-6, IL-17A, IFN-g, and TNF-a in differentiating naive T cells, suggesting that the role of RORC2 in Th17 cell development involves not only induction of Th17- characteristic , but also suppression of -specific programs. Together, this study identifies RORC2 as a polarizing factor in transcriptional cross-regulation and provides novel viewpoints on the control of versus effector immune responses. The Journal of Immunology, 2010, 184: 6161–6169. http://www.jimmunol.org/

D4+ effector cells can be divided into different subsets neutrophil-attracting chemokines in epithelial or other tissue cells according to their and function. Each subset is (2). In contrast to the effector subsets, regulatory T cells (Tregs) C crucial for mounting an appropriate immune response to restrict the strength and duration of the response and establish a certain type of pathogen (1, 2). Differentiation of naive Th cells tolerance to harmless self- and non–self-Ags. Failure in either toward a specific effector subset is therefore an important process development or function of Tregs causes severe hyperproliferative in immune system homeostasis. T cell development is governed diseases and is assumed to underlie autoimmune and allergic by lineage-specific transcription factors that regulate expression of disorders. FOXP3 is the of Tregs. Retroviral characteristic surface receptors and cytokines. The Th1-specific transfer of FOXP3 is sufficient to convert naive T cells into a Treg by guest on September 28, 2021 T box (T-bet), for example, coordinates pro- phenotype (6) and confers suppressor function on peripheral CD4+ duction of IFN-g and TNF-a (3), whereas the GATA-binding CD252 T cells (7, 8). Expression of FOXP3 is regulated through 3 (GATA-3) regulates Th2 development including pro- interaction of several TCR-induced transcription factors, including duction of their characteristic cytokines IL-4, IL-5, IL-9, and NFAT, AP-1, and cAMP response element binding protein IL-13 (4). Development of Th17 cells and expression of their (CREB)/activating transcription factor, with the promoter or an specific cytokines IL-17A, IL-17F, and IL-26 is promoted by the enhancer element of the foxp3 gene (9–12). Although TCR -related orphan receptor gt/C2 (RORgt/ stimulation induces basal levels of FOXP3, maximal and sustained RORC2) (5). Along with Th1 and Th2 cells, Th17 cells promote FOXP3 expression requires additional stimulation by cytokines inflammatory reactions and are important for the induction of such as IL-2, TGF-b, and IL-27 (10, 13–16). Cross-inhibition of T cell subsets during differentiation is an important mechanism to ensure expansion of the polarized pop- *Swiss Institute of Allergy and Research, Davos, Switzerland; †Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA ulations. Cytokines produced by one subset inhibit differentiation of 02115; and ‡Centre of Allergy and Environment, Technical University and Helmholtz other subsets. This antagonism is mirrored on the level of tran- Centre Munich, Munich, Germany scription factors, which cross-regulate each other and finally imprint Received for publication October 6, 2009. Accepted for publication March 26, 2010. or unlock the transcriptional program on the chromatin. T-bet di- This work was supported by the Swiss National Science Foundation Grants 310000- rectly inhibits Th2 differentiation by binding to its master regulator 112329 and 3200B0-118226. GATA-3, preventing it from DNA binding and activation of Th2- Address correspondence and reprint requests to Prof. Dr. Carsten Schmidt-Weber, specific genes (17, 18). Likewise, GATA-3 can downregulate Director of the Centre of Allergy and Environment, Technical University and Helm- holtz Centre Munich, Biedersteiner Strasse 29, 80802 Munich, Germany. E-mail STAT4, a transcription factor important for Th1 differentiation address: [email protected] (19). By binding to GATA-binding sites in the FOXP3 promoter, The online version of this article contains supplemental material. GATA-3 also negatively regulates FOXP3 expression and thereby Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; DBD, DNA- inhibits Treg differentiation (20). The Th17-driving transcription binding domain; GATA-3, GATA-binding protein 3; IL-1b, b form of pro-IL-1; MFI, factor RORC2 is expressed by both Treg and Th17 cells, whereas median fluorescence intensity; mut, mutated; ROR, retinoic acid receptor-related orphan receptor; RORE, retinoic acid receptor-related orphan receptor response ele- only Tregs express FOXP3 (21–23). In Tregs, FOXP3 suppresses ment; siRNA, small interfering RNA; siScram, scrambled siRNA; T-bet, Th1-specific transcriptional activity of RORC2 by direct interaction, thereby T box transcription factor; Treg, regulatory T cell; TSS, transcription start site; wt, inhibiting Th17 differentiation (22, 24). wild-type. RORC2 belongs to the family of RORs that consists of three Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 members: RORA (NR1F1, RORa, RZRa), RORB (NF1F2, RORb, www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903243 6162 RORC2 INHIBITS Tregs THROUGH INTERACTION WITH FOXP3 PROMOTER

RZRb), and RORC (NF1F3, RORg, RZRg). All ROR genes gen- to remove the cytokines used for differentiation. Supernatants were col- erate several isoforms, which differ only in their N termini (25–27). lected 48 h after restimulation, and concentrations of IL-1b, IL-2, IL-5, IL- of the ROR family display a typical do- 6, IL-13, IL-17A, IFN-g, and TNF-a were determined by cytometric bead array (Bio-Rad) according to the manufacturer’s protocol. main structure consisting of four domains: an N-terminal domain, a highly conserved DNA-binding domain (DBD), a hinge domain, Western blotting and a C-terminal ligand-binding domain. The DBD binds to DNA, Protein extraction was performed as follows: the cells were pelleted and containing a so-called ROR response element (RORE), which resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM consists of the DNA sequence GGTCA as a core motive, preceded MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Roche Diagnostic by an A/T-rich sequence (28, 29). In contrast to other nuclear re- Systems, Somerville, NJ], and 0.1% Nonidet P-40) and incubated at 4˚C with ceptors, ROR family proteins bind DNA as monomers (26, 28, 30). agitation for 15 min. Insoluble material was removed by centrifugation, and the supernatants were diluted one-third with buffer D (as buffer C, but without They can interact with both coactivators and corepressors to posi- NaCl). Total protein concentrations were determined by a colorimetric pro- tively or negatively regulate transcription of target genes and are tein assay (Bio-Rad). Samples were loaded next to a prestained protein-mass involved in a wide range of differentiation processes. RORC plays ladder (Invitrogen/Life Technologies) on a NuPAGE 4–12% bis-Tris gel a key role in the development of lymph nodes and Peyer’s patches (Invitrogen/Life Technologies). The proteins were electroblotted onto a poly- vinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ). and the regulation of thymopoiesis (27, 31–33). Unspecific binding was blocked with TBS-Tween 5% BSA. After blocking, In contrast to T-bet, GATA-3, and FOXP3, RORC2 has not been the membrane was incubated with an anti-RORg Ab (Santa Cruz Bio- described to participate in cross-regulation of T cell subsets. Based technology, Santa Cruz, CA) in a 1:500 dilution in blocking buffer overnight on our observation that GATA-3 inhibits FOXP3 expression (20), we at 4˚C. Next, the membrane was washed and incubated with an anti-rabbit hypothesized that the preference for Th17 cells over Tregs might be HRP-labeled Ab (1:3000; Cell Signaling Technology, Beverly, MA) at room Downloaded from temperature for 1 h. The blot was developed using an ECL plus Western blot mediated through restriction of the foxp3 gene by RORC2. The Detection System (GE Healthcare, Piscataway, NJ) and visualized with an present study describes a role for RORC2 in T cell cross-regulation LAS 1000 camera (Fujifilm, Valhalla, NY). To confirm equal sample loading by negatively regulating FOXP3 expression, which involves binding and transfer, membranes were stripped, reblocked and reprobed using an anti- of RORC2 to the FOXP3 promoter region. GAPDH Ab (6C5, Ambion, Austin, TX). Flow cytometry http://www.jimmunol.org/ Materials and Methods Cells were stained with the CD25 mAb (Beckman Coulter, Fullerton, CA) Isolation of naive T cells prior to FOXP3 intracellular staining, which was performed according to the manufacturer’s protocol using Alexa Fluor 488 anti-human FOXP3 Flow PBMCs were isolated from buffy coats of healthy donors using Ficoll kit (295D; Biolegend, San Diego, CA). Matched isotype controls were (Biochrom, Berlin, Germany) density gradient centrifugation. CD4+ T cells used at the same protein concentration as the respective Abs. were purified using anti-CD4 magnetic beads and Detach-a-Bead Abs Cell acquisition by flow cytometry was done on a four-color FACS (Dynal, Invitrogen, Carlsbad, CA). CD45RA+ cells were isolated using EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for MACS magnetic beads (Miltenyi Biotec, Auburn, CA). data acquisition and evaluation.

Differentiation of Treg and Th17 cells Amplification of FOXP3 promoter fragments by guest on September 28, 2021 CD45RA+ T cells were stimulated with soluble anti-CD3 and anti-CD28 FOXP3 promoter fragments were amplified by conventional PCR using Abs (4 mg/ml) in serum-free AIM-V medium (Life Technologies, Rock- a biotinylated reverse primer (59-bio-ACCTTACCTGGCTGGAATCACG- ville, MD) supplemented with IL-2 (25 U/ml; Proreo Pharma, Liestal, 39) situated 177 bp downstream of the transcription start site (TSS). Multiple Switzerland). Anti-CD3 and anti-CD28 Abs were produced using the hy- forward primers were designed to generate FOXP3 promoter fragments of bridoma cell line OKT3 (American Type Culture Collection, Manassas, decreasing length (Supplemental Table II). The position of the primers are 2 VA) and the hybridoma cell line 15E8 (CLB). Tregs were generated in the depicted in Fig. 3A. The plasmid pGL4 FOXP3 1210/+177 served as presence of TGF-b (5 ng/ml; R&D Systems, Minneapolis, MN) and a template. Site-directed mutagenesis in the FOXP3 promoter region was neutralizing anti–IL-12 (5 mg/ml; R&D Systems) and anti–IFN-g (1 mg/ introduced using the QuickChange kit (Stratagene, La Jolla, CA) according ml; R&D Systems) Abs. Th17 cells were differentiated as previously de- to the manufacturer’s instructions. Primers used for mutagenesis are listed in scribed (23) in the presence of TGF-b, IL-6 (20 ng/ml; PeproTech, Rocky Supplemental Table III. Reactions were conducted in 75 mM Tris HCl Hill, NJ), b form of pro-IL-1 (IL-1b, 10 ng/ml; PeproTech), IL-23 (10 ng/ (pH 8.8), 20 mM (NH4)2SO4, 2 mM MgCl2, 0.2 mM 29-deoxynucleoside ml; eBioscience, San Diego, CA), and neutralizing anti–IL-12 and anti– 59-triphosphate, 0.2 mM of each primer, 6 mg/ml template DNA (pGL4 2 IFN-g Abs. Dose titrations were performed and the optimal dose for each FOXP3 1210 /+177), and 1.25 U Taq DNA polymerase (Fermentas). The and Ab was determined in preliminary experiments. same PCR conditions were used for the amplification of all the products: initial denaturation step (2 min, 94˚C), 42 cycles of 94˚C for 30 s, 56˚C for Isolation of RNA, cDNA synthesis, and quantitative RT-PCR 30 s, 72˚C for 60 s, and final elongation step (7 min, 72˚C). PCR products were purified by ethanol precipitation. For this purpose, 1/10 volume 3 M Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA) sodium acetate and 3 volumes 100% ethanol were added to the PCR prod- according to the manufacturer’s protocol. Reverse transcription was per- ucts, and samples were mixed, precipitated for 1 h at 280˚C, and harvested formed with TaqMan reverse transcription reagents (Fermentas, Burling- by centrifugation (30 min, maximum speed, 4˚C). The supernatant was re- ton, Ontario, Canada) using random hexamer primers according to the moved, residual ethanol was allowed to evaporate, and the pellet was eluted manufacturer’s protocol. in blocking buffer (PBS, 0.1% BSA, 0.05% Tween 20). To quantify the PCR primers were designed based on the sequences reported in GenBank FOXP3 promoter fragments, samples were loaded next to a MassRuler DNA using the Primer Express software version 1.2 (Applied Biosystems, Foster Ladder Mix (Fermentas) on a 1% agarose gel and quantified using the AIDA City, CA). Primers used for relative quantification are listed in Supplemental image analyzer software (raytest, Straubenhardt, Germany). For fragment Table I. All primers are spanning - borders and have been verified F11 and competitor oligonucleotides, sense and antisense oligonucleotides for efficacy over a four-log concentration range. The prepared cDNAs were (Microsynth, Balgach, Switzerland; sequences in Supplemental Table II) amplified using iTaq SYBR Green Supermix with ROX (Bio-Rad, Hercules, were annealed for 10 min at 95˚C. CA) according to the manufacturer’s recommendations in an ABI Prism 7900 Sequence Detection System (Applied Biosystems). Relative quanti- FOXP3 promoter ELISA fication and calculation of the range of confidence was performed using the Protein extraction was performed as described for Western blotting, and DD a comparative CT method. EF-1 was used as endogenous control. All 384-well flat bottom plates were precoated with streptavidin (Pierce, amplifications were conducted in triplicates. Rockford, IL) and were washed three times with washing buffer (PBS, Determination of cytokine concentration 0.05% Tween 20). Biotinylated FOXP3 promoter fragments or consensus sequences were added (1 pmol/well; 50 fmol/ml) and incubated for 1 h at T cells were cultured for 5 d under Th17 conditions. Before restimulation room temperature. Upon three wash cycles, nuclear extracts were added at with soluble anti-CD3 and anti-CD28 Abs (4 mg/ml), the cells were washed concentration of 1 mg/ml and incubated overnight at 4˚C in the presence of The Journal of Immunology 6163

10 mg poly(deoxyinosinic-deoxycytidylic) acid (Sigma-Aldrich, St. Louis, MO) to block unspecific binding. For competition experiments, the nuclear extracts were preincubated with competitor oligonucleotides for 2 h at 4˚C. The plate was washed three times with buffer C/D and then incubated with the primary Ab (rabbit anti-RORC, 1:200 in buffer C/D; Biolegend) at 4˚C for 2 h. After three wash cycles with buffer C/D, a secondary Ab (anti- rabbit IgG-HRP, 1:3000 in buffer C/D; Cell Signaling Technology) was added and the plate was incubated for 1 h at 4˚C. The wells were washed four times with buffer C/D before substrate reagent was added (R&D Systems). The colorimetric reaction was stopped after 2–5 min by adding 2MH2SO4. Absorbance at 450 nm was measured using a Mithras mi- croplate reader (Berthold Technologies, Bad Wildbad, Germany).

Transfections Naive T cells were preactivated overnight in serum-free AIM-V medium (Life Technologies) containing soluble anti-CD3 and anti-CD28 Abs (4 mg/ ml). A total of 6–10 3 106 T cells were transfected with 3 mg pcDNA3.1- empty, pcDNA3.1-RORC2FL, or pcDNA3.1-RORC2DDBD plasmids in 100 ml Nucleofector solution (Amaxa Biosystems, Cologne, Germany) using the T-23 program. Small interfering RNA (siRNA) oligos (Ambion) were transfected at a concentration of 30 nM. Optimal concentrations were

determined in preliminary experiments (Supplemental Fig. 1). Two dif- Downloaded from ferent siRNA oligos were tested, and oligo siRNA1 was used in subsequent FIGURE 1. Overexpression of RORC2 inhibits FOXP3 expression. A, experiments. siRNA sequences are listed in Supplemental Table IV. Eight Naive CD4+ T cells were transfected with RORC2DDBD and RORC2FL hours posttransfection with plasmids or siRNA, the medium was replaced and expression of the proteins was analyzed by Western blot. B and C, with fresh, serum-free AIM-V medium, and cells were stimulated with Naive CD4+ T cells were transfected with RORC2FL and RORC2DDBD. anti-CD3 and anti-CD28 Abs (4 mg/ml) or as indicated. Eight hours posttransfection, the cells were stimulated with anti-CD3 and HEK cells were plated in six-well dishes and transfected with pcDNA3.1- anti-CD28 Abs in the presence of IL-2 and TGF-b. B, FOXP3 mRNA empty, pcDNA3.1-RORC2FL, or pcDNA3.1-RORC2DDBD plasmids at

levels were determined by RT-PCR after 48 h and 5 d, and levels in un- http://www.jimmunol.org/ 90–95% confluence using the Lipofectamine 2000 reagent (Invitrogen) C according to the manufacturer’s instruction. treated cells were set to 100%. , FOXP3 protein expression was measured by FACS after 5 d. Bars/numbers indicate mean and SD of at least three Chromatin immunoprecipitation independent experiments. p # 0.005. CD4+ T cells were transfected with pcDNA3.1-RORC2FL and cultured under Th17 conditions for 48 h. Alternatively, naive T cells were differen- + tiated toward Th17 cells. The chromatin immunoprecipitation (ChIP) assay autologous CFSE-labeled CD4 T cells. Cells were stimulated with 2.5 mg/ was performed as described (34). Protein–DNA complexes were cross- ml anti-CD3 mAb and cultured in a 96-well plate, and proliferation of linked with formaldehyde at a final concentration of 1.42% for 15 min. effector cells was determined by analyzing the CFSE dilution by flow + + Formaldehyde was quenched with 125 mM glycine for 5 min, and cells were cytometry after 5 d of culture. Gating on the CD4 CFSE T cells enabled harvested. Cells were lysed with immunoprecipitation buffer (150 mM NaCl, the exclusion of APCs and Tregs. by guest on September 28, 2021 50 mM Tris-HCl [pH 7.5], 5 mM EDTA, Nonidet P-40 [0.5% v/v]) con- taining phosphatase and protease inhibitor mixtures (both Roche, Basel, Switzerland); the chromatin was sheared by sonication and incubated with anti-RORC2 Abs (H-190 X; Santa Cruz Biotechnology) and normal rabbit IgG isotype controls (Santa Cruz Biotechnology) overnight at 4˚C. As a positive control, anti-human RNA polymerase II Abs and corresponding control IgG (both from SA Biosciences, Frederick, MD) were used. The cleared chromatin was then incubated with protein A agarose beads, and the DNAwas isolated with a 10% (w/v) Chelex 100 resin. Samples were treated with proteinase K at 55˚C for 30 min. The proteinase K was then inactivated by boiling the samples for 10 min. Real-time PCR was perfomed using specific primers for the FOXP3 promoter (Supplemental Table V) and con- trol GAPDH primer (SA Biosciences) for the RNA polymerase II. A negative control PCR for each immunoprecipitation using IGX1A negative control primer targeting open reading frame-free intergenic DNA (SA Biosciences) was performed. The fold enrichment in site occupancy was calculated in- corporating IgG isotype control values and input DNA values using the ChampionChIP qPCR data analysis file (SA Biosciences).

Reporter gene assays A total of 6–10 3 106 naive T cells were cotransfected with of 3 mgpGL4- empty or the pGL4-FOXP3 promoter luciferase reporter vector and 2 mg pcDNA3.1-empty, pcDNA3.1-RORC2FL or pcDNA3.1-RORC2DDBD plas- mids in 100 ml Nucleofector solution (Amaxa Biosystems). Electroporation was performed using the T-23 program. After 8 h, the medium was replaced with fresh serum-free AIM-V medium, and cells were stimulated with anti- FIGURE 2. siRNA-mediated knockdown of RORC2 increases FOXP3 CD3 and anti-CD28 Abs (4 mg/ml) for another 12 h until luciferase activity in expression. Naive CD4+ T cells were transfected with siRNA oligonu- cell lysates was measured using the dual luciferase assay system (Promega, cleotides targeting RORC2. Eight hours posttransfection, the cells were Madison, WI) in a Berthold Lumat LB 9507 luminometer (Berthold Tech- stimulated with anti-CD3 and anti-CD28 Abs in the presence of IL-2 and nologies) according to the manufacturer’s instructions. TGF-b. A, mRNA levels of RORC2 (left panel) and FOXP3 (right panel) were determined by RT-PCR after 48 h and 5 d. Levels in untreated cells Suppression assays were set to 100% or to 1, respectively. Bars represent mean and SD of at Naive T cells were transfected with a scrambled siRNA or siRNA against least 3 independent experiments. B, FOXP3 protein expression was mea- RORC2. Cells were cultured under Treg conditions for 10 d and then mixed sured by FACS after 5 d. Numbers indicate mean and SD of at least three with 2 3 105 autologous irradiated PBMCs that were used as APCs and independent experiments. MFI, median fluorescence intensity. p # 0.001. 6164 RORC2 INHIBITS Tregs THROUGH INTERACTION WITH FOXP3 PROMOTER

Plasmids CD3 and anti-CD28 Abs in the presence of IL-2 and TGF-b,and The human FOXP3 promoter was cloned into the pGL4 vector (Promega) to FOXP3 levels were determined by RT-PCR (Fig. 1B). In cells generate the pGL4 FOXP3 21210/+177 as previously described (9). Hu- transfected with RORC2FL, FOXP3 mRNA levels decreased ∼60% man RORC2FL and RORC2DDBD were cloned into a pcDNA3.1 vector and 66 6 23% after 48 h and 61 6 14% after 5 d. For control, in cells (Invitrogen) using the primers listed in Supplemental Table VI. transfected with the DNA-binding–deficient RORC2DDBD mu- Bioinformatics tant, FOXP3 levels were comparable to mock-transfected cells. To investigate whether RORC2 can change FOXP3 protein levels, cells Alignments of the human FOXP3 promoter were performed using the transfected with the RORC2 constructs were analyzed by flow cy- Genomatix software (www.genomatix.de). tometry after 5 d (Fig. 1C). Consistent with the results obtained by RT-PCR, RORC2FL-transfected cells showed a markedly de- Results creased FOXP3 staining (14.0 6 3.2%) compared with mock- Overexpression of RORC2 reduces FOXP3 expression (22.0 6 1.9%) or RORC2DDBD-transfected cells (23.2 6 1.8%). Although RORC2 activity is inhibited by FOXP3 in Tregs, this Taken together, these results demonstrate an inhibitory role for inhibitory function appears to be abolished in Th17 cells. Consid- RORC2 on FOXP3 mRNA and protein expression by a DNA ering the key roles of lineage-specific transcription factors in mutual binding-dependent mechanism. inhibition of T cell subsets, RORC2 itself represents a candidate for this unidentified suppressor of FOXP3. To test whether RORC2 Knockdown of RORC2 increases FOXP3 levels has the capacity to suppress FOXP3 expression, RORC2 was cloned The observation that high RORC2 levels result in a downregulation as a full-length construct (RORC2FL) as well as a truncated version of FOXP3 led us to the hypothesis that a decrease in RORC2 might Downloaded from lacking the DBD (RORC2DDBD), which was used as negative enhance the expression of FOXP3. To test this, RORC2 was knocked control. Naive CD4+ T cells were transfected with these plasmids, down using siRNA oligonucleotides. Two different oligonucleotides and Western blot analysis was performed to confirm recombinant weretested,andadosetitrationaswellasatimekineticwasperformed expression of the proteins (Fig. 1A). The molecular mass of the to validate RORC2 knockdown (Supplemental Fig. 1). Cells were proteins observed on the gel were in accordance with the calculated transfected with siRNA against RORC2 or with a scrambled siRNA molecular mass of ∼56 kDa for RORC2FL and 48 kDa for as negative control and then cultured with anti-CD3 and anti-CD28 http://www.jimmunol.org/ RORC2DDBD. The additional faint band at ∼50 kDa was due to Abs in the presence of IL-2 and TGF-b. After 48 h, mRNA levels of unspecific binding of the Ab. To test the influence of RORC2 on RORC2 and FOXP3 were determined by RT-PCR (Fig. 2A). In cells FOXP3 expression, transfected T cells were stimulated with anti- transfected with siRNA against RORC2, levels of RORC2 were by guest on September 28, 2021

FIGURE 3. The FOXP3 promoter contains con- served ROREs. A, Sequence of the FOXP3 promoter from bp 21246 to bp +223. ROREs are marked in blue; NFATbinding sites are marked in green. CAAT, GC, and TATA boxes are indicated with orange boxes. Primers used for generation of the promoter fragments are marked with black arrows. B, FOXP3 promoters of different species were aligned using the Genomatix software. Conserved bases are high- lighted in bold; RORE core sequences are marked with blue boxes. The Journal of Immunology 6165 knocked down by 76.1 6 14% compared with untransfected cells, whereas no effect was observed with scrambled siRNA, confirming the specificity of the assay. In cells with reduced RORC2 levels due to siRNA treatment, a 3-fold increase in FOXP3 was observed. To an- alyze FOXP3 expression on protein level, cells were cultured for 5 d under the same conditions and then subjected to FACS analysis (Fig. 2B). Consistent with the results obtained by RT-PCR, numbers of FOXP3-positive cells increased in the presence of siRNA against RORC2 compared with scrambled siRNA-transfected cells (from 20.1 6 2.0% to 28.5 6 4.0%). The median fluorescence intensities of FOXP3 were also slightly higher, suggesting that levels of FOXP3 increased on a per-cell basis as well. Together, these data reveal that downregulation of RORC2 increases FOXP3 expression and are in agreement with the results above showing reduced FOXP3 expres- sion after RORC2 overexpression. RORC2 binds to the FOXP3 promoter at RORE 2942 and RORE +115

Transcription factors of the nuclear receptor family like RORC2 Downloaded from control by binding to the promoter of their target genes. To investigate the mechanism by which RORC2 controls FOXP3 expression, we first analyzed the FOXP3 promoter sequence for the presence of ROREs, putative binding sites for RORC2 (Fig. 3A). The sequence from bp 21242 until bp +223 contains four

ROREs: three upstream (at 21096, 2942, and 2847) and one http://www.jimmunol.org/ downstream of the TSS (at +115). These putative binding sites for FIGURE 4. RORC2 binds to the FOXP3 promoter. A, Nuclear extracts ROR family members are conserved among the FOXP3 promoters of HEK cells transfected with either RORC2 or an empty vector were of different species (Fig. 3B). Direct binding of RORC2 to the incubated with a wild-type or mutated RORE consensus sequence or the FOXP3 promoter was assessed using a FOXP3 promoter ELISA FOXP3 promoter in the presence or absense of competitor oligonucleo- system (Fig. 4A). In this assay, either consensus sequences of 30 bp tides. Binding of RORC2 was quantified using the FOXP3 promoter containing one RORE or promoter fragments spanning bp 21116 ELISA assay. Bars show mean and standard deviations of triplicates rep- to +177 were coated on ELISA plates. Plates were incubated with resentative of three independent experiments. B, Nuclear extracts of HEK extracts of HEK cells transfected with RORC2 or an empty vector cells transfected with RORC2 were incubated with FOXP3 promoter

fragments of different length. Binding of RORC2 was quantified using the by guest on September 28, 2021 as a control, and binding of RORC2 was quantified by chemi- FOXP3 promoter ELISA assay. Bars show mean and standard deviations luminescence following incubation with the appropriate Abs. of triplicates representative of three independent experiments. C, Nuclear Binding of RORC2 to the wild-type but not to the mutated con- extracts of HEK cells transfected with RORC2 were incubated with sensus sequence was observed, validating the specificity of the FOXP3 promoter fragments containing mutations in different ROREs. assay. Absorbance was even slightly higher when extracts of HEK Binding of RORC2 was quantified using the FOXP3 promoter ELISA cells transfected with RORC2 were incubated with the FOXP3 assay. Bars show mean and standard deviations of triplicates representative promoter sequence, whereas incubation with extracts from empty of three independent experiments. mut, mutated; wt, wild-type. vector-transfected cells produced only background levels of ab- sorbance. These results suggest that RORC2 binds to one or more of for RORC2 binding (Fig. 4C). A decreased absorbance in the ELISA the ROREs on the FOXP3 promoter. The sequence specificity of the was observed when analyzing constructs containing a mutation in binding was further confirmed by addition of soluble oligonu- RORE 2942 or RORE +115, whereas binding of RORC2 to the cleotides, which abolished binding by competition when they promoter was not affected by mutations in RORE 21096 and contained the wild-type but not a mutated consensus sequence. RORE 2847. These results confirm that RORE 2942 and RORE To investigate which of the four ROREs are important for binding +115 are involved in the binding of RORC2 to the FOXP3 promoter. of RORC2 to the FOXP3 promoter, serial deletions of the FOXP3 Human RORC2 seems not to recognize the corresponding mouse promoter were constructed, and fragments were coated on an ELISA ROREs, as only human but not mouse site-specific competitor oli- plate (Fig. 4B). Binding of RORC2 to the promoter fragment lacking gonucleotides reduced binding of RORC2 to the FOXP3 promoter RORE 21096 was equal to binding observed using the fragment (Supplemental Fig. 2). This suggests that binding of RORC2 to the containing all four ROREs. However, when RORC2 was incubated FOXP3 promoter is species specific. with a construct lacking RORE 2942, a drop in absorbance was To assess whether RORC2 binds to the FOXP3 promoter in living detected, suggesting that RORE 2942 is relevant for binding of T cells, a ChIP assay was performed (Fig. 5A). T cells were transfected RORC2 to the promoter. Deletion of RORE 2847 did not sub- with RORC2 or alternatively differentiated toward Th17 cells. Using stantially alter the absorbance of the promoter ELISA, whereas anti-RORC2 Abs, the FOXP3 promoter was immunoprecipitated, a clear drop in absorbance was found in the fragment also lacking confirming that RORC2 binding to the FOXP3 promoter occurs in RORE +115. RORC2 has been described to compete with NFAT for T cells. In contrast, no significant changes in site occupancy were DNA binding (35). As three putative NFAT-binding sites are located observed when negative control primer targeting open reading frame- on the FOXP3 promoter (Fig. 3A), a construct lacking these sites free intergenic DNA (IGX1A) were used. was analyzed for RORC2 binding (Fig. 4B). However, no difference To find out whether binding to the FOXP3 promoter is required in binding was observed. for RORC2-mediated inhibition, a FOXP3 promoter luciferase To confirm these results, all ROREs were individually mutated, assay was performed (Fig. 5B). Cotransfection of RORC2FL and and promoter constructs carrying these mutations were investigated a luciferase plasmid containing the FOXP3 promoter resulted in 6166 RORC2 INHIBITS Tregs THROUGH INTERACTION WITH FOXP3 PROMOTER

FIGURE 5. RORC2 binds to the FOXP3 promoter in vivo and controls its activity. A, Binding of RORC2 to the FOXP3 promoter in T cells tranfected Downloaded from with RORC2 and in Th17 cells was assessed by ChIP. Primers for IGX1A were used as a negative control in all immunoprecipitations. The results are normalized to inputs and isotype control Abs. Bars show mean and SD offour independent experiments. Statistical differences were verified by paired Student t test. B, Naive CD4+ T cells were cotransfected as indicated. Eight hours posttransfection, the cells were stimulated with anti-CD3 and anti- http://www.jimmunol.org/ CD28 Abs for 12 h until luciferase activity was measured. Bars represent mean and SD of three independent experiments. pp # 0.05. FIGURE 6. Downregulation of RORC2 shifts Th17 cells toward less in- flammatory phenotype. Naive CD4+ T cells were transfected with siRNA against RORC2. Eight hours posttransfection, the cells were cultured under Treg or Th17 conditions for 5 d. A, After 6 h of restimulation with anti-CD3 a 67% reduced luciferase activity compared with the luciferase and anti-CD28 Abs, FOXP3 mRNA levels were measured by RT-PCR. activity in cells transfected with the luciferase vector and an empty Levels in scrambled siRNA-transfected Tregs were set to 1. Bars represent vector (26.4 6 17-fold versus 79.3 6 13-fold increase to the basic mean and SD of three independent experiments. B, After 6 h of restimulation luciferase plasmid). In contrast, cotransfection with RORC2DDBD with PMA and ionomycin, FOXP3 expression was analyzed by FACS.

generated a luciferase activity comparable to empty vector co- Numbers indicate mean and SD of three independent experiments. C, After by guest on September 28, 2021 transfection. Taken together, these results suggest that RORC2 in- 48 h of restimulation with anti-CD3 and anti-CD28 Abs, cytokine concen- hibits FOXP3 expression by binding to its promoter at RORE 2942 tration was analyzed by cytometric bead array. Bars represent SEs of du- and RORE +115. plicates. Data shown are representative of five independent experiments. p # 0.005. siScram, scrambled siRNA. Knockdown of RORC2 shifts Th17 cells toward T cells with a less proinflammatory but more regulatory phenotype T cells were transfected with a scrambled siRNA or RORC2-specific To investigate whether these findings have an implication in T cell siRNA. The cells were then cultured under Treg conditions, and their differentiation, especially in the balance between Treg and Th17 capacity to suppress autologous effector cells was analyzed (Fig. 7). cells, we analyzed whether lack of RORC2 shifts Th17 cells toward Cells with decreased RORC2 levels and thus higher FOXP3 levels a Treg phenotype. Therefore, RORC2 was downregulated in naive were slightly more suppressive than scrambled siRNA-treated cells. T cells using siRNA, and the cells were cultured under either Treg The results, however, did not reach statistical significance. Taken or Th17 differentiation conditions. As expected, in control settings together, these results indicate that Th17 cell differentiation is im- (i.e.,scrambledsiRNA-transfectedcells),FOXP3mRNAlevelswere paired in RORC2-depleted cells and that these cells instead differ- clearly higher in Treg than in Th17 (Fig. 6A). However, in Th17 cells entiate toward a less proinflammatory but more tolerating subset. with downregulated RORC2 levels, FOXP3 expression was signif- icantly increased, almost reaching levels in siRNA-treated Tregs. Discussion Similarly, frequencies of FOXP3-positive cells in siRNA-trans- The current study demonstrates repression of FOXP3 by the Th17 fected Th17 cells were comparable to frequencies in cells cultured cell-promoting transcription factor RORC2 and thus identifies under Treg conditions, whereas in cells transfected with control a novel role for RORC2 in T cell polarization. siRNA, a considerably smaller fraction of Th17 cells was FOXP3 Overexpression of RORC2 decreased the levels of FOXP3 on positive (Fig. 6B). We further analyzed the cytokine profile of Th17 both the mRNA and protein levels. This relationship was confirmed cells carrying reduced RORC2 levels during differentiation due to by siRNA-mediated knockdown of RORC2, which increased FOXP3 siRNA treatment (Fig. 6C). As expected and previously reported expression, indicating an inverse correlation of the two factors. Binding (36, 37), IL-17A levels were markedly reduced in these cells. to the promoter of FOXP3 is necessary for RORC2-mediated repres- Moreover, these cells also showed a decreased expression of other sion, as overexpression of RORC2 lacking the DBD did not inhibit proinflammatory cytokines like IL-1b, IL-6, IFN-g, and TNF-a FOXP3 expression. Moreover, no decrease in luciferase activity was (Fig. 6C, left panel), whereas no changes in IL-5 and IL-13 levels observed upon cotransfection with the DBD-deficient construct. To were observed (Fig. 6C, right panel), suggesting a specific effect. further investigate the mechanism of repression, we analyzed the Finally, we investigated whether siRNA-mediated RORC2 FOXP3 promoter sequence from bp 21242 until bp +223 and identified knockdown leads to a higher suppressive capacity of Tregs. Naive four putative RORC2-binding sites. In agreement with the fact that The Journal of Immunology 6167 Downloaded from http://www.jimmunol.org/

FIGURE 7. Suppressive capacity of Tregs after siRNA-mediated RORC2 knockdown. Naive T cells were transfected with siRNA against RORC2 or a scrambled siRNA and cultured under Treg conditions. After 10 d, Tregs were cocultured with autologous irradiated PBMCs and CFSE-labeled CD4+ responder T cells in ratios as indicated in the presence of soluble anti-CD3. A, The proliferation of CD4+ responder T cells was assessed by analyzing the CFSE dilution by flow cytometry after 5 d. Data are representative of four independent experiments. B, Percentage of the maximal proliferation. Proliferation of stimulated CD4+ responder T cells without Tregs is set as 100%. Bars show mean and SD of four independent experiments. siScram, scrambled siRNA.

ROR transcription factors bind DNA as monomers (26, 28, 30), all Beside corepressor-mediated suppression, RORC2 might also inhibit ROREs identified consist of a single independent motif. Three of FOXP3 expression by competition with NFAT, which is important for by guest on September 28, 2021 these ROREs are located in a poorly characterized part of the FOXP3 TCR-induced foxp3 gene activation (9), as RORC2 has been shown to gene, directly upstream of the core promoter and the region, which we bind to NFAT binding sites and inhibit its transcriptional activity (35). previously identified as an inductive area of the promoter containing We did not detect binding of RORC2 to NFAT binding sites, suggesting TCR-inducible signals such as NFAT and AP-1 (9, 38). A fourth an alternate mechanism of repression in context of the foxp3 gene. RORE is situated downstream of the TSS. Using an ELISA-based Because IL-2 plays a major role in FOXP3 induction (13, 14), and system, we detected binding of RORC2 to RORE 2942 and to RORC2 has been shown to suppress IL-2 production (27), it may exert a lesser extent to RORE +115. ROREs consist of the DNA se- its inhibitory function through negative regulation of autocrine IL-2 quence GGTCA as a core motive and are often preceded by an secretion. In fact, levels of IL-2 dropped significantly upon siRNA- A/T-rich sequence (28, 29). This A/T-rich sequence of RORC2 mediated RORC2 knockdown (data not shown). binding sites is also found upstream of RORE 2942. Using a ChIP The siRNA-induced RORC2 knockdown during differentiation assay, we demonstrate that interaction of RORC2 and the pro- of Th17 cells led to a phenotype characterized not only by high moter region of FOXP3 occurs in vivo. Altogether, these experi- FOXP3 expression, but also by low levels of proinflammatory Th17 ments reveal that repression of FOXP3 by RORC2 involves and Th1 cytokines such as IL-1b, IL-6, and IL-17A as well as IFN-g physical binding of RORC2 to two ROREs on its promoter, a mech- and TNF-a, respectively. This suggests an important role for anism commonly observed for ROR family members. RORC2 in the inhibition of Treg differentiation under inflammatory ROR family transcription factors induce repression of target conditions. Reduced levels of IL-17A are presumably a direct genes by recruiting corepressors, which induce chromatin deace- consequence of RORC2 knockdown, which is known to regulate tylation and therefore gene repression (39–41). Several corepressors IL-17A expression on a transcriptional level (49). In contrast, the have been described to interact with ROR family members, and it decrease in IFN-g is a novel observation and might be caused in- remains to be determined which of them mediates the repression of directly by the increased FOXP3 expression, as FOXP3 has been the foxp3 gene observed in this study. Retinoic acid has been re- shown to repress IFN-g production (50). Tregs with reduced ported to be a key factor in regulating the balance of Treg versus RORC2 levels tended to be more suppressive, although the results Th17 cells (42–45). Although retinoic acid seems to mediate these did not reach statistical significance. The small increase in sup- effects by its cognate receptor retinoic acid receptor a (46, 47), the pressive activity might be explained by the fact that the siRNA- mechanism possibly also involves RORC2, as retinoic acid has mediated RORC2 knockdown is maximal after 48 h and might be been found to bind to RORC2 and inhibit its transcriptional activity less pronounced at the time point of suppression. Furthermore, it (48). Therefore, retinoic acid might favor Treg cell differentiation has been shown that RORC2 expression does not completely ex- by binding and inhibiting RORC2, leading to an increased FOXP3 clude suppressive activity (51, 52). The extent to which RORC2 expression. However, addition of retinoic acid did not change NF reduces suppressive activity is likely to depend on the relative levels binding to the ROREs on the FOXP3 promoter (data not shown). of FOXP3 and RORC2. 6168 RORC2 INHIBITS Tregs THROUGH INTERACTION WITH FOXP3 PROMOTER

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