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Active Demethylation of the Foxp3 Locus Leads to the Generation of Stable Regulatory T Cells within the

This information is current as Aras Toker, Dirk Engelbert, Garima Garg, Julia K. of September 25, 2021. Polansky, Stefan Floess, Takahisa Miyao, Udo Baron, Sandra Düber, Robert Geffers, Pascal Giehr, Sonja Schallenberg, Karsten Kretschmer, Sven Olek, Jörn Walter, Siegfried Weiss, Shohei Hori, Alf Hamann and Jochen Huehn Downloaded from J Immunol published online 18 February 2013 http://www.jimmunol.org/content/early/2013/02/17/jimmun ol.1203473 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/02/19/jimmunol.120347 Material 3.DC1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 18, 2013, doi:10.4049/jimmunol.1203473 The Journal of Immunology

Active Demethylation of the Foxp3 Locus Leads to the Generation of Stable Regulatory T Cells within the Thymus

Aras Toker,* Dirk Engelbert,† Garima Garg,* Julia K. Polansky,* Stefan Floess,* Takahisa Miyao,‡ Udo Baron,x Sandra Du¨ber,{ Robert Geffers,‖ Pascal Giehr,# Sonja Schallenberg,** Karsten Kretschmer,** Sven Olek,x Jo¨rn Walter,# Siegfried Weiss,{ Shohei Hori,‡ Alf Hamann,† and Jochen Huehn*

Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we

show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of Downloaded from lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guaran-

tees full functionality and long-term lineage stability of Tregs. The Journal of Immunology, 2013, 190: 000–000. http://www.jimmunol.org/

2 D4+ regulatory T cells (Tregs) play an essential role in (CD4SP; i.e., CD4+CD8 ) stage (9, 10). Thymic Treg develop- maintaining immune and preventing autoim- ment requires that CD4SP encounter cognate Ag C mune reactivity of potentially self-reactive presented by thymic APCs in the context of MHC class II (11–14), that have escaped mechanisms (1). For proper and CD4SP thymocytes appear to be predisposed to upregulate development and function, Tregs crucially depend on the forkhead Foxp3 expression (15). According to the two-step model of thymic box Foxp3, and loss of Foxp3 function in hu- Treg development, this TCR signaling event induces upregulation mans and rodents results in devastating (2–7). of IL-2R a-chain (CD25), rendering these thymocytes receptive to by guest on September 25, 2021 The vast majority of Foxp3+ Tregs is generated during subsequent signals that foster their development into development in the thymus (8). Development of thymic Tregs fully functional Foxp3+ Tregs (16, 17). IL-2 plays a predominant mainly takes place in medullary regions at the CD4 single-positive role in this second step of thymic Treg development. However, its loss can be compensated by other that signal through receptors containing the common g subunit, such as IL-7 and IL- *Experimental Immunology, Helmholtz Centre for Infection Research, Braunsch- weig 38124, Germany; †Experimental Rheumatology, Medical Clinic–Rheumatology 15 (16, 18, 19). and Clinical Immunology, Charite´ University Medicine, Berlin 10117, Germany; Stability of Foxp3 expression correlates to DNA demethylation ‡Research Unit for Immune Homeostasis, RIKEN Research Centre for and Immunology, Yokohama 230-0045, Japan; xEpiontis GmbH, Berlin 12489, at a conserved intronic CpG-rich region within the Foxp3 gene Germany; {Molecular Immunology, Helmholtz Centre for Infection Research, locus, designated Treg-specific demethylated region (TSDR) (20). ‖ Braunschweig 38124, Germany; Genome Analytics, Helmholtz Centre for Infection Demethylation at the TSDR (also known as CNS2) is not required Research, Braunschweig 38124, Germany; #Department of Genetics/, Saarland University, Saarbru¨cken 66123, Germany; and **Deutsche Forschungsge- for initiation of Foxp3 expression, but for its long-term mainte- meinschaft–Center for Regenerative Therapies Dresden 01307, Dresden, Germany nance (21–23). Accordingly, stable Tregs display a fully demeth- + Received for publication December 20, 2012. Accepted for publication January 24, ylated TSDR, whereas the TSDR of conventional CD4 T cells 2013. and in vitro–induced Tregs is heavily methylated (22). In addition, This work was supported by the German Research Foundation (SFB738 to J.H. and forced TSDR demethylation confers stability to in vitro–induced SFB650 and Ha1505-10/1 to A.H.), the German Ministry for Education and Research (Deutsches Epigenom Programm to J.W. and A.H.), and by Grants-in-Aid for Sci- Tregs (21). Hence, understanding the mechanisms that cause TSDR entific Research from the Ministry of Education, Culture, Sports, Science and Tech- demethylation in developing Tregs could open up ways to manip- nology of Japan (19059014 and 20689012 to S.H.). ulate TSDR methylation and allow safe application of in vitro– The sequences presented in this article have been submitted to the Gene Expression generated Tregs for therapeutic approaches (24). Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE42021. In this study, we provide direct evidence that commitment to Address correspondence and reprint requests to Prof. Jochen Huehn, Helmholtz a stable Foxp3-expressing lineage is initiated already during early Centre for Infection Research, Inhoffenstrasse 7, Braunschweig 38124, Germany. E-mail address: [email protected] stages of thymic Treg development and is completed by TSDR + The online version of this article contains supplemental material. demethylation in mature thymic Foxp3 Tregs, ensuring full Abbreviations used in this article: FTOC, fetal thymic organ culture; 5hmC, 5- functionality and long-term lineage stability. In developing Tregs, hydroxymethylcytosine; 5mC, 5-methylcytosine; RTE, recent thymic emigrant; SP, CpGs of the TSDR are demethylated through an active mechanism single-positive; Tet, ten-eleven-translocation; Treg, ; TSDR, regula- that involves the recently discovered intermediate of active DNA tory T cell–specific demethylated region. demethylation pathways, 5-hydroxymethylcytosine (5hmC), and Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 enzymes of the ten-eleven-translocation (Tet) family.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203473 2 STABLE Treg INDUCTION IN THE THYMUS

Materials and Methods lowed: low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Mice Tris [pH 8.1], and 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X- 100, 2 mM EDTA, 20 mM Tris [pH 8.1,] and 500 mM NaCl), LiCl buffer BALB/c and C57BL/6J mice were purchased from Janvier. BALB/c 3 (25 mM LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid, 1 mM EDTA, and Thy1.1 and Foxp3GFP reporter mice (kindly provided by Alexander 10 mM Tris [pH 8]), and TE buffer (10 mM Tris and 1 mM EDTA). After- Rudensky, Memorial Sloan-Kettering Center, New York, NY) were wards, Ab-bound fragments were collected with hot elution buffer (1% SDS bred at the Helmholtz Centre for Infection Research (Braunschweig, and 100 mM NaHCO3) and purified using the Nucleospin Extract II kit Germany). Indu-Rag1fl/fl 3 Rosa26-CreERT2 mice were obtained by (Macherey-Nagel) according to the procedure recommended for the purifi- crossing Indu-Rag1fl/fl mice (25) with Rosa26-CreERT2 mice (kindly cation of samples containing SDS. The amount of immunoprecipitated DNA provided by Prof. Anton Berns, The Netherlands Cancer Institute, and input DNA was quantified by real-time PCR using SYBR Green Amsterdam, The Netherlands) and bred at the Helmholtz Centre for In- (Invitrogen) with the following primer pair: TSDR forward: 59-AACC- fection Research. development was induced in Indu-Rag1fl/fl TTGGGCCCCTCTGGCA-39; and reverse: 59-GGCCGGATGCATTGGG- mice by oral administration of 400 ml tamoxifen (20 mg/ml; Ratiopharm) CTTCA-39. Immunoprecipitated DNA was set in relation to input DNA hCD2 GFP in ClinOleic (Baxter). Foxp3 3 RAG1 mice were obtained by using the following expression: enrichment = 2^ 2 [CtDIP 2 CtInput]. For crossing Foxp3hCD2 mice (26) with RAG1GFP mice (27) (kindly provided normalization, mean enrichment factor for the samples within one experiment by Nobuo Sakaguchi, Kumamoto, Japan) and bred at the RIKEN Research was set in relation to the mean enrichment factor for all three experiments. Centre for Allergy and Immunology (Yokohama, Japan). RAG2GFP mice (28) were bred at the Centre for Regenerative Therapies (Dresden, Ger- Quantitative PCR many). Mice were kept under specific pathogen-free conditions in accor- RNA was isolated using the DNA/RNA AllPrep Kit (Qiagen). cDNA was dance with institutional, state, and federal guidelines. produced from equivalent amounts of RNA using Superscript II reverse Cell sorting transcriptase (Invitrogen). For quantification, samples were amplified by real-time PCR using SYBR Green (Invitrogen). Relative mRNA expression Downloaded from Peripheral CD4+ T cells were enriched from pooled LN and spleen cells was calculated by comparative threshold cycle method using hypoxanthine using anti-CD4 microbeads and magnetic separation on an autoMACS phosphoribosyltransferase for normalization. Primer pairs that were pub- separator (Miltenyi Biotec). For sorting, total thymocytes were lished previously were used for Tet1 and Tet3 (30) or Tet2 (31). depleted of CD8+ cells using APC-conjugated CD8 Abs and anti-APC microbeads. Depleted cells were stained, and subsets were sorted on an DNA microarray hybridization and analysis FACSAria II (BD Biosciences). RNA was isolated using the DNA/RNA AllPrep Kit (Qiagen), and quality and integrity of total RNA was controlled on Agilent Technologies 2100 Flow cytometry http://www.jimmunol.org/ Bioanalyzer (Agilent Technologies). A total of 500 ng total RNA was used Single-cell suspensions were prepared from lymphoid organs and stained for biotin labeling according to the 39 IVT Express Kit (Affymetrix). with fluorochrome-conjugated anti-mouse CD4 (clone RM4-5), CD8 (53- A total of 7.5 mg biotinylated cRNA was fragmented and placed in hy- 6.7), CD24 (M1/69), CD25 (PC61), Thy1.1 (HIS51), and anti-human CD2 bridization mixture containing four biotinylated hybridization controls (RPA-2.10). Intracellular staining was performed with Foxp3 (FJK-16s) and (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Ki-67 (B56) Abs. Flow cytometric analyses were performed on LSR II (BD Samples were hybridized to an identical lot of Affymetrix GeneChip MOE Biosciences), and data were analyzed with FlowJo software (Tree Star). 430 2.0 (Affymetrix) for 16 h at 45˚C. Steps for washing and SA-PE staining were processed on the fluidics station 450 using the recom- BrdU incorporation mended FS450 protocol (Affymetrix). Image Analysis was performed on the GCS3000 Scanner and GCOS1.2 Software Suite (Affymetrix). Anal-

Mice received two i.p. injections of 1 mg BrdU in PBS 9 and 3 h before ysis of microarray data were performed using GeneSpring 11.5.1 (Agilent by guest on September 25, 2021 analysis. Thymocytes were stained for cell-surface molecules, followed by Technologies). Signal intensities (raw data) were log2 transformed and fixation, permeabilization, and intracellular staining for Foxp3 and BrdU normalized using RMA. Gene expression values per condition are given as using the BrdU flow kit (BD Biosciences). relative expression to mean expression value calculated from intensities of all conditions (mean centralization, normalized data). The entire micro- Adoptive transfers array data set is available under Gene Expression Omnibus accession Total CD4+ T cells were isolated from pooled LN and spleen cells of number GSE42021 (http://www.ncbi.nlm.nih.gov/geo/). BALB/c 3 Thy1.1 mice. Isolated cells were i.v. injected into BALB/c recipients in 100 ml sterile PBS. Transferred cells (2 3 106 per recipient) Methylation analysis . + were 95% CD4 . DNA methylation analysis was performed by bisulphite sequencing as de- scribed previously (20). Only cells from male mice were used for methyl- Fetal thymic organ cultures ation analyses. Thymic lobes from embryonic day 18.5 fetuses of Foxp3GFP mice were placed on a polyethersulfonate transwell membrane with a pore size of 0.4 mm (Corning), and the transwell insert was placed in a well containing Results 400 ml serum-free AIM V medium (Life Technologies) in a 24-well plate. Partial TSDR demethylation in thymic Tregs Lobes were incubated for up to 14 d, and medium was replaced every 5 d. Gender of fetuses was determined by PCR as described previously (29). The TSDR contains 14 CpG motifs (Fig. 1A), which are fully demethylated in Tregs isolated from secondary lymphoid organs Cell culture (20), whereas CD4SP Foxp3+ thymocytes show only partial de- Thymic Treg subsets were stimulated with anti-CD3/anti-CD28–coated methylation at the TSDR (32). We first confirmed this phenotype GFP+ microbeads (Life Technologies) and 50 ng/ml recombinant mouse IL-2 using FACS-purified CD4SP Foxp3 thymocytes (Fig. 1B). GFP+ (R&D Systems) for 5 d. In some experiments, 125–250 mM L-mimosine The partially methylated TSDR of bulk CD4SP Foxp3 thy- (Sigma-Aldrich) was added. mocytes prompted us to identify the developmental stages at DNA immunoprecipitation which TSDR demethylation occurred. Thereto, we sorted three subpopulations of thymic Foxp3+ Tregs based on CD24 expres- Genomic DNA was prepared using the DNA/RNA AllPrep Kit (Qiagen) sion, which is known to correlate inversely to maturity of CD4SP according to the manufacturer’s recommendations. DNA was sonicated to produce random fragments ranging in size from 200–1000 bp, and 1 mg and CD8SP thymocytes (33). TSDR demethylation was most pro- lo GFP+ sheared DNA was used for immunoprecipitation by overnight incubation with nounced in mature CD24 CD4SP Foxp3 thymocytes (Fig. 1C). Abs against 5-hydroxymethylcytosine (39769; Active Motif) in a final volume The immature CD24hi subset displayed a largely methylated TSDR, of 250 ml IP buffer (10 mM Na-phosphate [pH 7], 140 mM NaCl, and 0.05% and the CD24int subset showed intermediate methylation. These Triton X-100) at 4˚C. An appropriate aliquot of sheared DNA was kept as input control. Enrichment of Ab-bound DNA was performed using protein A/ findings suggest that TSDR demethylation is a continuous process G-microbeads (Miltenyi Biotec), and magnetic separation was done on cor- that begins at an early CD4SP stage subsequent to Foxp3 protein responding separation columns by several washing steps, the order as fol- expression and proceeds during further thymocyte maturation. The Journal of Immunology 3 Downloaded from

+ FIGURE 1. TSDR demethylation in thymic Foxp3 Tregs. (A) Schematic of the Foxp3 gene locus (modified from Ref. 20). The TSDR is located http://www.jimmunol.org/ upstream of exon 21 and contains 14 CpG motifs. (B) TSDR methylation was analyzed by bisulphite sequencing on genomic DNA of FACS-purified CD4SP GFP+ and GFP2 thymocytes from Foxp3GFP mice. Percentage of methylation is color-coded according to the scale; each horizontal bar represents an individual CpG motif. Percentages indicate average demethylation within the TSDR. One representative experiment of five is shown. (C) TSDR methylation analysis on genomic DNA of FACS-purified thymic CD24hi, CD24int, and CD24lo GFP+ CD4SP Treg subsets from Foxp3GFP mice. One representative experiment out of three is shown.

TSDR demethylation is initiated during thymic Treg were repopulated with T cells (Supplemental Fig. 2B). At day 14, + development when Foxp3 Tregs were readily detectable in the thymus but by guest on September 25, 2021 + Activated peripheral T cells are to a low extent capable of recir- hardly any Foxp3 Tregs were present in peripheral lymphoid organs (Supplemental Fig. 2B, 2C), we purified CD4SP CD25hi culating to the thymus (34, 35); hence the possibility was given fl/fl that TSDR methylation analysis in thymic Foxp3+ Treg subsets cells from thymi of Indu-Rag1 mice (Fig. 2B). Foxp3 protein was blurred by recirculating peripheral Foxp3+ Tregs. In adoptive expression in 70–90% of sorted cells was confirmed by intracel- + lular staining (Supplemental Fig. 2D). TSDR demethylation was transfer studies, we identified essentially no donor-derived Foxp3 hi fl/fl Tregs within thymi of lymphocompetent recipients (data not clearly evident in CD4SP CD25 cells of Indu-Rag1 mice, + although not as pronounced as in steady-state wild-type thymi shown), indicating that recirculation of peripheral Foxp3 Tregs is 2 fl/fl very limited. Nevertheless, to formally exclude that recirculating (Fig. 2B). In contrast, CD4SP CD25 cells from both Indu-Rag1 Tregs of peripheral origin were responsible for the partial TSDR and wild-type mice displayed a fully methylated TSDR. demethylation in thymi of untreated mice, we investigated the We next analyzed the TSDR methylation status in recent thymic emigrants (RTEs) by using RAG1GFP mice (27), in which RTEs TSDR methylation status of developing Tregs in fetal thymic or- lo gan cultures (FTOCs), where first Foxp3+ cells were observed on can be identified in peripheral lymphoid organs as GFP cells, due to delayed degradation of GFP protein after cessation of Rag1 day 4 of culture (Supplemental Fig. 1A), corresponding to post- GFP hCD2 natal days 1 to 2 (36). We used FTOCs from Foxp3GFP fetuses to gene expression. RAG1 mice were crossed to Foxp3 re- porter mice (26). TSDR methylation analysis of peripheral CD4+ purify Tregs on day 10 of culture and found that these FTOC- + + lo GFP+ hCD2 CD25 GFP RTEs revealed complete demethylation (Fig. derived Foxp3 Tregs had a partially demethylated TSDR, akin + + + 2 GFP2 2C), similar to CD4 hCD2 CD25 GFP mature peripheral Tregs. to their in vivo counterparts, whereas Foxp3 cells isolated GFP from FTOCs displayed a fully methylated TSDR (Fig. 2A, Sup- In the RAG2 mouse line that also allows tracking of RTEs (28), we observed only slight (,25%) methylation of the TSDR in plemental Fig. 1B). These results showed that the thymic micro- + hi lo environment is sufficient to trigger TSDR demethylation in CD4 CD25 GFP RTEs (data not shown), further supporting the developing Tregs. In a second approach we used an in vivo model notion that Tregs bear epigenetic marks that confer lineage iden- fl/fl tity at the time of thymic emigration. In summary, these results of inducible T cell development, Indu-Rag1 mice, in which + inversion of the floxed coding sequence of the Rag1 gene causes demonstrated that TSDR demethylation in Foxp3 Tregs is initi- lymphopenia (25). In a background that ubiquitously expresses ated during their development in the thymus, and progressive a tamoxifen-inducible Cre recombinase, T cell development can demethylation occurs during maturation of these thymocytes. be induced at a given time by tamoxifen administration (Supple- mental Fig. 2A). This model allows the interrogation of TSDR Commitment to a stable Treg lineage during early stages of methylation in developing thymic Foxp3+ Tregs without running Treg development the risk of including peripheral contaminants to the analysis. We next investigated at which developmental stage commitment to Following tamoxifen administration, thymi of Indu-Rag1fl/fl mice a stable Foxp3-expressing phenotype takes place by assessing 4 STABLE Treg INDUCTION IN THE THYMUS Downloaded from

FIGURE 2. TSDR demethylation is initiated in the thymus. (A) Fetal thymi were collected from embryonic day 18.5 Foxp3GFP fetuses, cultured for 10 d, and TSDR methylation analysis was performed on genomic DNA from FACS-purified Foxp3GFP+ or Foxp3GFP2 cells. Percentage of methylation is color- coded according to the scale; each horizontal bar represents an individual CpG motif. Percentages indicate average methylation within the TSDR. Results http://www.jimmunol.org/ shown are representative for four independent experiments. (B) CD4SP CD25hi and CD252 thymocytes were FACS-sorted from C57BL/6 (wild-type [WT]) or Indu-Rag1fl/fl mice 14 d after tamoxifen administration. TSDR methylation analysis was performed on genomic DNA from sorted cells. Results shown are representative for three independent experiments. (C) CD4+hCD2+CD25+GFP+ and CD4+hCD2+CD25+GFP2 cells were purified from spleens and lymph nodes of Foxp3hCD2 3 RAG1GFP mice. TSDR methylation analysis was performed on genomic DNA from sorted cells. Results shown are rep- resentative for two independent experiments. stability of Foxp3 expression in thymic Foxp3+ Treg subsets of 5-d-culture (Fig. 4B). The two-step model of thymic Treg devel- different maturity. Interestingly, even the most immature CD24hi opment suggests that Foxp32CD25+ Treg precursors only need subset among thymic Tregs fully maintained Foxp3 expression cytokine signaling by IL-2 to mature into Foxp3+ Tregs (16, 17). by guest on September 25, 2021 upon in vitro stimulation, despite a substantially methylated TSDR We therefore tested whether IL-2 signaling alone would also (Fig. 3A), and stability of Foxp3 protein expression was accom- suffice to induce TSDR demethylation in Treg precursors. When panied by complete TSDR demethylation during the 5-d culture. cultured with IL-2 only, 30–40% of Treg precursors became In fact, all thymic Treg subsets maintained Foxp3 expression (Fig. Foxp3GFP+, and mild TSDR demethylation was observed (Fig. 3B), and near-to-complete demethylation was observed in all 4C). Concomitantly with Foxp3 upregulation, Treg precursors subsets after in vitro stimulation (Fig. 3C). These findings suggest responded to IL-2 by substantial proliferation. However, the per- that commitment to a stable Treg lineage is established during an centage of Foxp3GFP+ cells and the extent of TSDR demethylation early stage of thymic Treg development and consists of differen- were similar when Treg precursors were cultured with IL-15, tiation programs that initiate Foxp3 expression and subsequently despite a drastically reduced cell-division rate (Fig. 4C). induce TSDR demethylation. We compared gene expression Foxp3GFP2CD25+ Treg precursors also displayed a set of differ- profiles of Foxp3+ Treg subsets of different maturity and could entially expressed genes compared with conventional Foxp3GFP2 identify a set of genes that were specifically up- or downregulated CD4SP thymocytes (Fig. 4D, Supplemental Table II), and this in Foxp3+ Tregs, but not in Foxp32 conventional T cells, in differential expression pattern persisted in later Treg differentia- a maturation-dependent manner (Fig. 3D). Genes that showed the tion stages (Fig. 4E), indicating that Foxp3-independent compo- most pronounced differential regulation during maturation were nents of the Treg differentiation program are already triggered in Axl, Bcl2l15, Cxcr5, Il1r2, and Prr5l. Well-known Treg-associated precursors. Collectively, our results suggest that thymic Treg genes such as Socs2, Ebi3, and Ccr6 were also upregulated by precursors are already primed by the thymic microenvironment to thymic Tregs during maturation (Fig. 3D, Supplemental Table I). differentiate into stable Foxp3+ Tregs with a demethylated TSDR This finding indicates that Tregs consolidate their unique pheno- along with acquisition of Treg-specific expression programs; and type in the thymus after Foxp3 expression is initiated by estab- demethylation can be accomplished, albeit less efficiently, in the lishment of their specific gene expression profile and functional absence of additional TCR signals. maturation. Our results suggested that TSDR demethylation is part of this overall consolidation program that is initiated during early The TSDR is demethylated in an active fashion in thymic Tregs stages of Treg development. DNA demethylation mostly occurs in a passive manner by impaired We next asked whether thymic Foxp32CD25+ Treg precursors transfer of methyl groups to newly synthesized, unmethylated DNA (16, 17) already have the potential to become stable Foxp3+ Tregs. strands, and thus depends on cell division. Increasing evidence is Thymic CD4SP Foxp3GFP2CD25+ cells displayed a fully meth- now available showing that active (i.e., cell-division–independent) ylated TSDR (Fig. 4A). However, when stimulated in vitro via the DNA demethylation, initially described in plants, also occurs in TCR in presence of IL-2, part of these cells became Foxp3GFP+, mammalian cells (37). It was therefore of interest whether cell and partial TSDR demethylation was observed at the end of the division was necessary for TSDR demethylation. To this end, we The Journal of Immunology 5 Downloaded from

FIGURE 3. Stability of Foxp3 expression is imprinted during thymic Treg development. (A) Thymic CD4SP GFP+CD24hi immature Tregs were purified from Foxp3GFP mice and stimulated with anti-CD3/anti-CD28–coated beads. After 5 d, stability of Foxp3 expression was analyzed by flow cytometry (top panels). TSDR methylation analyses were performed on genomic DNA of ex vivo–isolated as well as stimulated cells (bottom panels). Percentage of

methylation is color-coded according to the scale; each horizontal bar represents an individual CpG motif. Percentages indicate average methylation within http://www.jimmunol.org/ the TSDR. (B) CD4SP Foxp3GFP+ Tregs were sorted into CD24hi, CD24int, and CD24lo subsets. Sorted cells were stimulated as in (A), and stability of Foxp3 expression was analyzed after 5 d of culture. Pooled data from five independent experiments (n = 14) are shown. (C) TSDR methylation analysis on genomic DNA of ex vivo isolated CD24hi, CD24int, and CD24lo thymic Foxp3GFP+ Treg subsets or after a 5-d in vitro stimulation of the subsets as described in (A). Pooled data from three independent experiments (n = 5) are shown. (D) Gene expression profiling was performed on FACS-purified GFP+ and GFP2 CD24hi, CD24int, and CD24lo CD4SP thymocyte subsets from Foxp3GFP mice. Genes that are up- or downregulated in Foxp3GFP+ thymocytes during maturation, but are exempt from such regulation in Foxp3GFP2 thymocytes, are displayed in the scatter plot. Only genes showing consistent regulation in three independent RNA microarrays are shown. The outer green lines depict a corridor of 2-fold change. Known Treg signature genes and genes showing highest differential expression are annotated. by guest on September 25, 2021 cultured thymic CD4SP Foxp3GFP+ Tregs in the presence of DNA from thymic Treg subsets by an Ab that specifically rec- L-mimosine, an inhibitor of late G1 phase of the cell cycle (38). ognizes 5hmC to examine the relative abundance of 5hmC within GFP+ TSDR demethylation in thymic Tregs cultured in presence of L- the TSDR in developing thymic Tregs. All three thymic Foxp3 mimosine was identical to the dividing PBS control (Fig. 5A, 5B). Treg subsets showed substantial enrichment of 5hmC within the CD4SP Foxp3GFP2 cells remained heavily methylated at the TSDR compared with conventional Foxp3GFP2CD252CD4SP as TSDR when stimulated in vitro, both in presence and absence of well as DP thymocytes (Fig. 6A). Notably, thymic Treg precursors GFP2 + GFP+ hi L-mimosine, excluding nonspecific effects of the inhibitor on DNA (Foxp3 CD25 ) and mature Foxp3 CD25 Tregs from methylation (data not shown). Thus, TSDR demethylation occurs peripheral lymphoid organs did not show enrichment of 5hmC in an active fashion without the need for cell division in vitro. within the TSDR over conventional T cells (Fig. 6A), further To assess the proliferative state of thymocytes during in vivo supporting the notion that TSDR demethylation is initiated and Treg differentiation, we made use of BrdU incorporation. Thy- to a great extent finalized during thymic Treg development. mic Foxp3+ Treg subsets only poorly incorporated i.p.-injected Expression of all three Tet dioxygenases was detectable in BrdU, suggesting a low degree of proliferation (Fig. 5C, 5E). In thymic Foxp3+ Treg subsets, and expression of Tet2 and Tet3 was contrast, double-positive (i.e., CD4+CD8+) thymocytes, which are markedly elevated compared with conventional CD4SP thymo- known to proliferate extensively, incorporated substantially more cytes (Fig. 6B). Tet3 was expressed at much higher levels than BrdU. The vast majority of CD24hi and CD24int thymic Foxp3+ Tet1 and Tet2 in all thymic cell populations analyzed, and highest Tregs were also negative for Ki-67 expression (Fig. 5D, 5F), levels of Tet3 expression were detected in CD24hi and CD24int showing that they were not actively cycling, further supporting Treg subsets. Hence, 5mC is likely converted to 5hmC by Tet a mechanism of TSDR demethylation that did not depend on dioxygenases in developing thymic Tregs. Taken together, these progression through the cell cycle. results strongly support an active mechanism of TSDR demethy- lation in developing thymic Tregs, which ensures long-term lin- Conversion of methylated cytosine to 5hmC facilitates DNA eage stability and suppressive potential after thymic egress. demethylation at the TSDR The family of Tet dioxygenases has recently been shown to catalyze Discussion oxidation of 5-methylcytosine (5mC) to 5hmC (30, 39). Enzymes Stable Foxp3 expression depends on selective DNA demethylation of the base excision repair pathway can then excise 5hmC from at the TSDR within the Foxp3 locus. The experiments presented in the DNA and replace it by an unmethylated cytosine; hence 5hmC this study deliver a precise temporal delineation of TSDR deme- acts as an intermediate state during active DNA demethylation. thylation in developing thymic Tregs: the demethylation machinery Standard bisulphite sequencing cannot discriminate 5mC from is triggered in Foxp32CD25+ Treg precursors, but demethylation 5hmC (40). Thus, we performed immunoprecipitation of genomic only starts after Foxp3 protein is detectable. It then continues as 6 STABLE Treg INDUCTION IN THE THYMUS Downloaded from http://www.jimmunol.org/

FIGURE 4. Foxp32CD25+ Treg precursors are primed for TSDR demethylation. (A) TSDR methylation was analyzed by bisulphite sequencing of genomic DNA isolated from ex vivo–sorted CD4SP GFP2CD25+ or GFP2CD252 thymocytes from Foxp3GFP mice. Percentage of methylation is color- coded according to the scale; each horizontal bar represents an individual CpG motif. Percentages indicate average methylation within the TSDR. (B) Thymocyte populations were sorted as in (A) and stimulated with anti-CD3/anti-CD28–coated microbeads in the presence of IL-2 for 5 d. TSDR meth- ylation analysis was performed on total cells at the end of culture. One representative experiment out of two is shown. (C) CD4SP GFP2CD25+ thymocytes sorted from Foxp3GFP mice were labeled with Cell Trace Violet and cultured for 5 d with IL-2 or IL-15 in the absence of TCR stimulation. Foxp3 ex- pression and TSDR methylation of total cells was assessed at the end of the culture. One representative experiment out of two is shown. (D) Gene ex- by guest on September 25, 2021 pression profiles of CD4SP Foxp3GFP2CD25+ Treg precursors and Foxp3GFP2CD252 thymocytes were analyzed. Scatter plot shows genes that are differentially expressed in Treg precursors compared with CD252 thymocytes consistently in three independent RNA microarrays. Selected Treg-associated genes are annotated. The outer green lines depict a corridor of 2-fold change. (E) Expression of genes shown in (D) was compared between Foxp3GFP2 CD25+ Treg precursors and total Foxp3GFP+ thymic Tregs. The same genes as in (D) are highlighted. part of the maturation program through which Tregs pass in the has been considered as passive demethylation. Intriguingly, our thymic medulla and is to a great extent completed at time of egress data show that TSDR demethylation in the thymus occurs inde- to the periphery. To draw this conclusion safely, we had to exclude pendently of proliferation, as CD4SP Foxp3+ Tregs hardly divide the possibility that recirculating Tregs of peripheral origin con- during maturation, and inhibitors of mitosis do not prevent deme- tribute to the overall methylation levels we observed in thymocytes. thylation during in vitro cultures, implying an active mechanism of By using several complementary approaches, we could provide TSDR demethylation. clear evidence that recirculating peripheral cells do not significantly It has recently been discovered that 5mC can be modified to contribute to the methylation pattern of thymic Tregs, but rather 5hmC by Tet family enzymes (30, 39). 5hmC can either iteratively thymic maturation drives progressive TSDR demethylation. be further oxidized to 5-formylcytosine and 5-carboxylcytosine by Somewhat surprisingly, immature CD24hiFoxp3+ thymic Tregs, Tet enzymes or deaminated to 5-hydroxymethyluracil by AID/ which similar to in vitro TGF-b–induced Foxp3+ Tregs (21) showed APOBEC deaminases (43). 5-carboxylcytosine and 5-hydrox- a largely methylated TSDR, almost fully maintained Foxp3 ex- ymethyluracil, in turn, are recognized and excised by DNA gly- pression when stimulated in vitro, in stark contrast to TGF-b–in- cosylases like TDG and SMUG1 (44–47), creating an abasic site duced Tregs. Hence, signals that confer stability to Tregs appear to that is filled with an unmethylated cytosine by further components be received at early stages of thymic Treg differentiation, possibly of the base excision repair pathway. Alternatively, 5-carbox- even as early as the Foxp32 precursor stage. TSDR demethylation ylcytosine may be excised by a hitherto unknown putative decar- is subsequently accomplished during the consolidation phase in an boxylase to directly yield unmethylated cytosine (43). Hence, autopilot state and serves to imprint stable and long-term Foxp3 conversion of 5mC to 5hmC by Tet enzymes is a key event that expression. The early signals are yet to be discovered, though it is can initiate several mechanisms of DNA demethylation. Recent tempting to speculate that signals that induce Foxp3 expression at studies have shown that self-renewal of hematopoietic stem cells the same time initiate Foxp3-independent pathways of Treg dif- as well as their differentiation into myeloid cells is perturbed in ferentiation, including TSDR demethylation (23, 32). mice lacking Tet2, concomitant with altered genomic levels of Regulation of DNA demethylation in immune cells mostly has 5hmC (48, 49). We now demonstrate for the first time, to our been found to be associated with proliferation [except for a few knowledge, the presence of 5hmC at a specific gene locus during reports, such as demethylation of the Il-2 gene (41, 42)] and hence a defined stage of T cell differentiation. Whether one of the three The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. TSDR demethylation occurs in absence of cell division. (A) Thymic CD4SP GFP+ Tregs were sorted from Foxp3GFP mice. Left panels show postsort purity and ex vivo TSDR methylation analysis. Sorted cells were stimulated with anti-CD3/anti-CD28–coated microbeads in the presence or absence of the late G1-phase inhibitor L-mimosine. After 5 d, TSDR methylation status was analyzed by bisulphite sequencing. Percentage of methylation is color-coded according to the scale; each horizontal bar represents an individual CpG motif. Numbers indicate average demethylation within the TSDR.(B) Summary TSDR methylation analyses performed as in (A) from three independent experiments (n = 5). (C and E) BALB/c mice received two i.p. injections by guest on September 25, 2021 of 1 mg BrdU. Nine hours after the first and 3 h after the second injection, BrdU incorporation of thymocyte subsets was analyzed by flow cytometry. Histograms are gated on indicated subsets, and numbers indicate the percentage of BrdU+ cells. (E) Summary of three mice. (D and F) Ki-67 staining of thymocyte subsets. Histograms are gated on indicated subsets and numbers indicate percentage of Ki-67+ cells. Shaded histograms represent controls. (F) Summary of five mice. Results are representative for two independent experiments. DP, Double-positive.

Tet enzymes plays a predominant role for conversion of 5mC to Signals that initiate functional differentiation of thymic Tregs are 5hmC in thymic Foxp3+ Tregs or whether they act in a redundant better known. Upregulation of CD25 upon TCR ligation is the first manner remains to be clarified. At present, very little is known step of Treg differentiation in CD4SP thymocytes (16). Because about how expression of Tet enzymes is regulated. It is therefore first signs of TSDR demethylation are already observed in Foxp32 a highly challenging question, which signals lead to induction of CD25+ Treg precursors upon exposure to IL-2, one might consider Tet dioxygenases and their specific recruitment to the Foxp3 locus IL-2, or other cytokines that signal through common g subunit– in developing thymic Tregs. containing receptors, as being one of the signals involved in

FIGURE 6. 5hmC is enriched in thymic Treg subsets. (A) Indicated cell populations were purified from thymi or pooled spleens and lymph nodes (LN) of Foxp3GFP mice. Genomic DNA was extracted from sorted cells and subjected to DNA immunoprecipitation with an anti-5hmC Ab. Quantitative PCR was performed on precipitated DNA with TSDR-specific oligonucleotides. Pooled data from three independent experiments (n = 7) are shown. (B) Total RNA was extracted from indicated cell populations and quantitative RT-PCR was performed to assess the expression levels of Tet1, Tet2, and Tet3. Pooled data from three independent experiments (n = 7) are shown. DP, Double-positive; HPRT, hypoxanthine phosphoribosyltransferase. 8 STABLE Treg INDUCTION IN THE THYMUS driving epigenetic fixation of Foxp3 expression. IL-2 signals have regulatory T cells specific for self expressed and presented by Aire+ medullary thymic epithelial cells. Nat. Immunol. 8: 351–358. been recently suggested to contribute to TSDR demethylation in 14. Lee, H. M., J. L. Bautista, J. Scott-Browne, J. F. Mohan, and C. S. Hsieh. 2012. in vitro–generated Tregs after their adoptive transfer (50). Nev- A broad range of self-reactivity drives thymic regulatory T cell selection to limit ertheless, it appears unlikely that IL-2 alone is sufficient to induce responses to self. 37: 475–486. 15. Wirnsberger, G., F. Mair, and L. Klein. 2009. Regulatory T cell differentiation of full TSDR demethylation, as only very mild epigenetic changes thymocytes does not require a dedicated antigen-presenting cell but is under 2 + were observed when Foxp3 CD25 Treg precursors were cultured T cell-intrinsic developmental control. Proc. Natl. Acad. Sci. USA 106: 10278– with IL-2 in the absence of TCR signaling. 10283. 16. Lio, C. W., and C. S. Hsieh. 2008. A two-step process for thymic regulatory In this study, we present evidence that commitment to the Treg T cell development. Immunity 28: 100–111. lineage takes place in the thymus through induction of stable Foxp3 17. Burchill, M. A., J. Yang, K. B. Vang, J. J. Moon, H. H. Chu, C. W. Lio, expression that is consolidated through demethylation at the A. L. Vegoe, C. S. Hsieh, M. K. Jenkins, and M. A. Farrar. 2008. Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell TSDR, allowing the majority of Tregs to retain Foxp3 expression repertoire. Immunity 28: 112–121. and their regulatory capacity under homeostatic and inflammatory 18.Bayer,A.L.,J.Y.Lee,A.delaBarrera,C.D.Surh,andT.R.Malek.2008.A + + + conditions (26, 51–53). In addition, we demonstrate the presence function for IL-7R for CD4 CD25 Foxp3 T regulatory cells. J. Immunol. 181: 225–234. of 5hmC within the Foxp3 locus of developing thymic Tregs to- 19. Vang, K. B., J. Yang, S. A. Mahmud, M. A. Burchill, A. L. Vegoe, and gether with elevated levels of Tet family enzymes, suggesting the M. A. Farrar. 2008. IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development. J. Immunol. involvement of Tet enzymes in active DNA demethylation at this 181: 3285–3290. site. To our knowledge, this is the first report showing a role of 20. Floess, S., J. Freyer, C. Siewert, U. Baron, S. Olek, J. Polansky, K. Schlawe, 5hmC in active DNA demethylation processes at a specific gene H. D. Chang, T. Bopp, E. Schmitt, et al. 2007. Epigenetic control of the locus in regulatory T cells. PLoS Biol. 5: e38. locus that occur during development and differentiation of 21. Polansky, J. K., K. Kretschmer, J. Freyer, S. Floess, A. Garbe, U. Baron, S. Olek, Downloaded from lymphocytes. It is conceivable that 5hmC-mediated active DNA A. Hamann, H. von Boehmer, and J. Huehn. 2008. DNA methylation controls demethylation is more broadly involved in the specification and Foxp3 gene expression. Eur. J. Immunol. 38: 1654–1663. 22. Huehn, J., J. K. Polansky, and A. Hamann. 2009. Epigenetic control of FOXP3 ex- epigenetic imprinting of lineage identity in cells of the immune pression: the key to a stable regulatory T-cell lineage? Nat. Rev. Immunol. 9: 83–89. system or other somatic tissues. The clarification of signals that 23. Zheng, Y., S. Josefowicz, A. Chaudhry, X. P. Peng, K. Forbush, and induce expression of Tet enzymes and their recruitment to the A. Y. Rudensky. 2010. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463: 808–812.

Foxp3 locus to catalyze hydroxylation of 5mC as well as the 24. Toker, A., and J. Huehn. 2011. To be or not to be a Treg cell: lineage decisions http://www.jimmunol.org/ factors that further process 5hmC to yield demethylated CpG controlled by epigenetic mechanisms. Sci. Signal. 4: pe4. 25. Du¨ber, S., M. Hafner, M. Krey, S. Lienenklaus, B. Roy, E. Hobeika, M. Reth, motifs warrant intensified research. It can be expected that iden- T. Buch, A. Waisman, K. Kretschmer, and S. Weiss. 2009. Induction of B-cell tification of these factors will pave the way for improved strategies development in adult mice reveals the ability of to produce B-1a to generate and expand Tregs for therapeutic applications, for cells. 114: 4960–4967. 26. Miyao, T., S. Floess, R. Setoguchi, H. Luche, H. J. Fehling, H. Waldmann, which the lack of stability is still a major concern. J. Huehn, and S. Hori. 2012. Plasticity of Foxp3(+) T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells. Immunity 36: 262–275. Acknowledgments 27. Kuwata, N., H. Igarashi, T. Ohmura, S. Aizawa, and N. Sakaguchi. 1999. Cutting edge: absence of expression of RAG1 in peritoneal B-1 cells detected by knocking

We thank Lothar Gro¨be for cell sorting. by guest on September 25, 2021 into RAG1 locus with green fluorescent protein gene. J. Immunol. 163: 6355–6359. 28. Yu, W., H. Nagaoka, M. Jankovic, Z. Misulovin, H. Suh, A. Rolink, F. Melchers, E. Meffre, and M. C. Nussenzweig. 1999. Continued RAG expression in late Disclosures stages of development and no apparent re-induction after immunization. The authors have no financial conflicts of interest. Nature 400: 682–687. 29. Clapcote, S. J., and J. C. Roder. 2005. Simplex PCR assay for sex determination in mice. Biotechniques 38: 702–, 704, 706. 30. Ito, S., A. C. D’Alessio, O. V. Taranova, K. Hong, L. C. Sowers, and Y. Zhang. References 2010. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and 1. Klein, L., and K. Jovanovic. 2011. Regulatory T cell lineage commitment in the inner cell mass specification. Nature 466: 1129–1133. thymus. Semin. Immunol. 23: 401–409. 31. Ko, M., Y. Huang, A. M. Jankowska, U. J. Pape, M. Tahiliani, H. S. Bandukwala, 2. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell de- J. An, E. D. Lamperti, K. P. Koh, R. Ganetzky, et al. 2010. Impaired hydrox- velopment by the transcription factor Foxp3. Science 299: 1057–1061. ylation of 5-methylcytosine in myeloid with mutant TET2. Nature 468: 3. Khattri, R., T. Cox, S. A. Yasayko, and F. Ramsdell. 2003. An essential role for 839–843. Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4: 337–342. 32. Ohkura, N., M. Hamaguchi, H. Morikawa, K. Sugimura, A. Tanaka, Y. Ito, 4. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the M. Osaki, Y. Tanaka, R. Yamashita, N. Nakano, et al. 2012. T cell receptor development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: stimulation-induced epigenetic changes and Foxp3 expression are independent and 330–336. complementary events required for Treg cell development. Immunity 37: 785–799. 5. Lahl, K., C. Loddenkemper, C. Drouin, J. Freyer, J. Arnason, G. Eberl, A. Hamann, 33. Marodon, G., and B. Rocha. 1994. Generation of mature T cell populations in the H. Wagner, J. Huehn, and T. Sparwasser. 2007. Selective depletion of Foxp3+ thymus: CD4 or CD8 down-regulation occurs at different stages of thymocyte regulatory T cells induces a scurfy-like disease. J. Exp. Med. 204: 57–63. differentiation. Eur. J. Immunol. 24: 196–204. 6. Kim, J. M., J. P. Rasmussen, and A. Y. Rudensky. 2007. Regulatory T cells 34. Kirberg, J., N. Bosco, J. C. Deloulme, R. Ceredig, and F. Agene`s. 2008. Pe- prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. ripheral T lymphocytes recirculating back into the thymus can mediate thymo- Immunol. 8: 191–197. cyte positive selection. J. Immunol. 181: 1207–1214. 7. Gambineri, E., T. R. Torgerson, and H. D. Ochs. 2003. , 35. McCaughtry, T. M., M. S. Wilken, and K. A. Hogquist. 2007. Thymic emigration polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome revisited. J. Exp. Med. 204: 2513–2520. of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of 36. Fontenot,J.D.,J.L.Dooley,A.G.Farr,andA.Y.Rudensky.2005.Developmental T-cell homeostasis. Curr. Opin. Rheumatol. 15: 430–435. regulation of Foxp3 expression during ontogeny. J. Exp. Med. 202: 901–906. 8. Hsieh, C. S., H. M. Lee, and C. W. Lio. 2012. Selection of regulatory T cells in 37. Bhutani, N., D. M. Burns, and H. M. Blau. 2011. DNA demethylation dynamics. the thymus. Nat. Rev. Immunol. 12: 157–167. Cell 146: 866–872. 9. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, and 38. Lalande, M. 1990. A reversible arrest point in the late G1 phase of the mam- A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead malian cell cycle. Exp. Cell Res. 186: 332–339. transcription factor foxp3. Immunity 22: 329–341. 39. Tahiliani, M., K. P. Koh, Y. Shen, W. A. Pastor, H. Bandukwala, Y. Brudno, 10. Lee, H. M., and C. S. Hsieh. 2009. Rare development of Foxp3+ thymocytes in S. Agarwal, L. M. Iyer, D. R. Liu, L. Aravind, and A. Rao. 2009. Conversion of the CD4+CD8+ subset. J. Immunol. 183: 2261–2266. 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL 11. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, partner TET1. Science 324: 930–935. M. A. Lerman, A. Naji, and A. J. Caton. 2001. Thymic selection of CD4+CD25+ 40. Huang, Y., W. A. Pastor, Y. Shen, M. Tahiliani, D. R. Liu, and A. Rao. 2010. The regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2: 301–306. behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5: 12. Apostolou, I., A. Sarukhan, L. Klein, and H. von Boehmer. 2002. Origin of e8888. regulatory T cells with known specificity for antigen. Nat. Immunol. 3: 756–763. 41. Bruniquel, D., and R. H. Schwartz. 2003. Selective, stable demethylation of the 13. Aschenbrenner, K., L. M. D’Cruz, E. H. Vollmann, M. Hinterberger, -2 gene enhances transcription by an active process. Nat. Immunol. 4: J. Emmerich, L. K. Swee, A. Rolink, and L. Klein. 2007. Selection of Foxp3+ 235–240. The Journal of Immunology 9

42. Murayama, A., K. Sakura, M. Nakama, K. Yasuzawa-Tanaka, E. Fujita, Translocation 2 (TET2) negatively regulates homeostasis and differentiation Y. Tateishi, Y. Wang, T. Ushijima, T. Baba, K. Shibuya, et al. 2006. A specific of hematopoietic stem cells in mice. Proc. Natl. Acad. Sci. USA 108: 14566– CpG site demethylation in the human gene promoter is an epige- 14571. netic memory. EMBO J. 25: 1081–1092. 49. Li, Z., X. Cai, C. L. Cai, J. Wang, W. Zhang, B. E. Petersen, F. C. Yang, and 43. Branco, M. R., G. Ficz, and W. Reik. 2012. Uncovering the role of 5-hydrox- M. Xu. 2011. Deletion of Tet2 in mice leads to dysregulated hematopoietic stem ymethylcytosine in the epigenome. Nat. Rev. Genet. 13: 7–13. cells and subsequent development of myeloid malignancies. Blood 118: 4509– 44. Nilsen, H., K. A. Haushalter, P. Robins, D. E. Barnes, G. L. Verdine, and 4518. T. Lindahl. 2001. Excision of deaminated cytosine from the vertebrate genome: 50. Chen, Q., Y. C. Kim, A. Laurence, G. A. Punkosdy, and E. M. Shevach. 2011. role of the SMUG1 uracil-DNA glycosylase. EMBO J. 20: 4278–4286. IL-2 controls the stability of Foxp3 expression in TGF-beta-induced Foxp3+ 45. He, Y. F., B. Z. Li, Z. Li, P. Liu, Y. Wang, Q. Tang, J. Ding, Y. Jia, Z. Chen, L. Li, T cells in vivo. J. Immunol. 186: 6329–6337. et al. 2011. Tet-mediated formation of 5-carboxylcytosine and its excision by 51. Komatsu, N., M. E. Mariotti-Ferrandiz, Y. Wang, B. Malissen, H. Waldmann, TDG in mammalian DNA. Science 333: 1303–1307. and S. Hori. 2009. Heterogeneity of natural Foxp3+ T cells: a committed regu- 46. Guo, J. U., Y. Su, C. Zhong, G. L. Ming, and H. Song. 2011. Hydroxylation of 5- latory T-cell lineage and an uncommitted minor population retaining plasticity. methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Proc. Natl. Acad. Sci. USA 106: 1903–1908. Cell 145: 423–434. 52. Zhou, X., S. L. Bailey-Bucktrout, L. T. Jeker, C. Penaranda, M. Martı´nez- 47. Cortellino, S., J. Xu, M. Sannai, R. Moore, E. Caretti, A. Cigliano, M. Le Coz, Llordella, M. Ashby, M. Nakayama, W. Rosenthal, and J. A. Bluestone. 2009. K. Devarajan, A. Wessels, D. Soprano, et al. 2011. Thymine DNA glycosylase is Instability of the transcription factor Foxp3 leads to the generation of pathogenic essential for active DNA demethylation by linked deamination-base excision memory T cells in vivo. Nat. Immunol. 10: 1000–1007. repair. Cell 146: 67–79. 53. Rubtsov, Y. P., R. E. Niec, S. Josefowicz, L. Li, J. Darce, D. Mathis, C. Benoist, 48. Ko, M., H. S. Bandukwala, J. An, E. D. Lamperti, E. C. Thompson, R. Hastie, and A. Y. Rudensky. 2010. Stability of the regulatory T cell lineage in vivo. A. Tsangaratou, K. Rajewsky, S. B. Koralov, and A. Rao. 2011. Ten-Eleven- Science 329: 1667–1671. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

Supplementary Table I. List of genes that are up or downregulated in CD4SP Foxp3GFP+CD24lo mature compared to CD4SP Foxp3GFP+CD24hi immature thymic Tregs but are exempt from such regulation in CD4SP Foxp3GFP-CD24lo compared to CD4SP Foxp3GFP- CD24hi conventional thymocytes.

Gene Symbol fold change (CD24lo vs. CD24hi) Axl 6.239677 Il1r2 5.881265 Cxcr5 5.3293266 Bcl2l15 5.014403 Nrn1 4.7928724 Wisp1 4.526591 Gpr15 4.506774 Zdhhc2 4.435472 Zdhhc2 3.7784417 Gucy1a3 3.5954132 Ebi3 3.5723066 Zdhhc2 3.4620485 D430019H16Rik 3.4522789 Cpe 3.2346325 Ass1 3.1771343 Klrg1 3.0320733 Gprin3 2.9788878 Hdac9 2.9180486 Ng23 2.8958302 Itgb1 2.889645 Casp4 2.8829408 Nucb2 2.813509 Fcrl1 2.8074145 6330509M05Rik 2.8026779 Arrdc4 2.774836 Il10ra 2.7218251 Ifitm2 2.702996 Prdm1 2.6864183 BC021614 2.6478858 Cldnd1 2.60181 Dusp14 2.5901976 Cpd 2.5807667 Ccr6 2.4915957 Vegfc 2.480992 Cyp4f18 2.4670775 Scin 2.4508944 Rapgef5 2.441831 Tmem136 2.3995364 Igh-6 2.395534 Myo3b 2.3934593 4930524L23Rik 2.380861 Rapgef5 2.38002 Penk 2.374319 Tmem154 2.3663764 Ccdc69 2.3524907 Cd86 2.3490262 Vsig10 2.3280559 Gpr160 2.3272724 Aldoc 2.3221562 Socs2 2.3181946 Zdhhc2 2.3090017 Syt11 2.268028 Ptger2 2.252341 Osbpl3 2.247804 Slc7a10 2.2378561 Gpr18 2.227269 Syt11 2.212123 Egln3 2.19657 Frzb 2.1861818 Pnkd 2.1792536 Il6ra 2.167455 Procr 2.1660428 Jdp2 2.1603873 Ms4a4d 2.1551373 Bag3 2.1470113 Nfil3 2.1439946 Zbtb32 2.1403935 Lclat1 2.1385684 Pros1 2.1328092 Pck2 2.1326435 Slc12a8 2.1299155 Arl6 2.1245008 Atxn1 2.1132507 4930420K17Rik 2.1050997 Itgb1 2.098189 Hist2h3c2-ps 2.0956345 Syt11 2.09395 Syt11 2.0914862 Cd86 2.08756 Arhgef12 2.0778224 Spock2 2.0723658 Tiam1 2.0499682 Kbtbd11 2.040483 Tox2 2.035614 Kbtbd11 2.0280893 Wisp1 2.0270083 Cldnd1 2.025516 Hif1a 2.0206156 Cd55 2.0206022 Thy1 2.0197856 Neb 2.0153816 Cpd 2.011974 Nt5e 2.009614 Gzma -2.0056956 Lmo4 -2.014986 Stmn1 -2.025018 Nr4a2 -2.0461369 Cybrd1 -2.0523472 Trpm1 -2.1062672 Pole -2.1217935 C1ql3 -2.1252172 Tbc1d4 -2.1750808 Chek1 -2.1849325 Cd160 -2.2008986 Chek1 -2.2123108 Pkdcc -2.2199476 Car8 -2.227121 Tm6sf1 -2.254276 Slc16a6 -2.2836988 Crispld2 -2.3833144 Kctd12 -2.3914354 Napb -2.420042 Ltbp1 -2.4380653 Phldb2 -2.4407494 Hc -2.5214062 Dscc1 -2.7055852 Prr5l -2.731818 Slc35d3 -2.8253717 Ccnj -2.912255 C1ql3 -3.1602077 Fibcd1 -3.2313013 Scel -3.305623 Nrp2 -3.3336837 Xcl1 -3.3754945 Prr5l -3.5428612 Camk2n1 -3.5887823 Lox -3.6450756 Ptger3 -3.9861462 Lox -4.052528 Mcf2l -4.088174 Nrp2 -4.1465755 Serpine2 -4.1764064 Slc45a3 -4.3319244 Trpm1 -4.775267 Prr5l -6.489393

Supplementary Table II. List of genes that are up or downregulated in CD4SP Foxp3GFP-CD25+ Treg precursors compared to CD4SP Foxp3GFP-CD25- conventional thymocyte subsets.

Gene Symbol fold change Precursor vs. Precursor vs. Precursor vs. Foxp3-CD24hi Foxp3-CD24int Foxp3-CD24lo Cd83 9.684526 11.739084 7.8802943 Mpzl2 2.9369102 3.697425 4.708801 Serpinb1a 4.1892276 3.556902 3.3658016 Epcam 2.1079624 2.7845895 2.0635786 P4ha2 -2.5845234 -5.3066316 -2.3182437 Sdc4 6.27508 6.8147426 4.9103146 Gfi1 3.59952 4.8347707 4.8813963 Ggt5 -2.0423088 -3.5910225 -2.2672777 Cyth3 4.2027965 3.099688 3.6639402 Slc22a2 4.466096 4.163616 4.1254034 Map3k8 2.4604 2.2452245 2.5562115 Ctla4 7.7806253 3.939133 6.576891 Il1r2 2.9414065 4.15854 3.52132 Tnfrsf4 4.0616746 4.5559225 10.370335 Lta 3.738609 3.4366446 3.4745483 Il2ra 6.84083 5.341475 6.7154794 Il2ra 13.327448 14.283621 15.315106 Tgfbr1 2.2736588 2.486104 3.011921 Enc1 -2.130344 -2.87327 -3.21021 Itgb3 -2.231528 -2.2151058 -2.4792917 Ikzf2 8.981856 10.184831 3.8421175 Igfbp4 -2.0929825 -6.94971 -3.8169801 Tnfrsf18 2.516459 3.068827 3.8539877 Actg2 6.1986217 7.844236 7.1638327 Mbnl3 4.0502653 3.4079432 2.660783 Dst 3.7273195 4.067266 4.884569 Igfbp4 -2.9967077 -9.615502 -5.3426175 Igfbp4 -2.22426 -6.235966 -3.9751525 Sectm1a 12.833087 13.439294 11.734966 Fcrl1 2.0849612 2.2135265 2.1127849 Oas2 -2.2582762 -4.877152 -3.0640543 Rgs16 15.662028 22.232311 11.2778635 Cd72 7.696787 7.3752103 4.676744 Plagl1 15.680618 10.295593 11.827811 Alcam 2.4441 2.2587502 2.2512076 Igf1r 2.3997097 2.8369021 4.8664565 Penk 19.58215 12.227497 12.852148 Tnfrsf9 10.803429 18.632519 25.73079 Tmem158 2.4929764 2.779612 2.055974 ENSMUSG00000068790 6.2349544 5.479725 7.5936236 Arhgap21 2.0830944 2.2770977 2.2278926 Bmyc 2.3757226 2.4707348 2.1482158 Lrig1 2.7630851 2.4459434 2.349013 Mboat1 -2.130849 -2.7338533 -2.9585931 Igfbp4 -2.7576547 -7.7922087 -4.85066 Igfbp4 -2.2670822 -6.230056 -3.511694 Ikzf4 3.2224693 2.3193343 5.8219 Fam46c -2.1690776 -3.2725773 -2.7727842 Sema4a -2.2590458 -2.882089 -3.9579453 Wisp1 2.1528876 2.2469444 2.0048218 Cish 4.253945 2.6105556 3.5244184 Sdc4 2.9570446 3.1539218 3.4429805 Ptger2 4.9300623 4.496049 5.249808 Lrig1 3.0369596 2.6255794 2.4099813 Enc1 -2.1667466 -2.5495338 -2.835913 Ryr2 3.3863578 3.1101873 3.1378403 Jdp2 4.451784 3.994916 2.5942218 Ccr6 9.099725 6.941294 7.089714 Dntt 2.2637928 6.98071 8.402938 Hdgfrp3 2.2445529 2.755262 3.9561732 Ahcyl2 2.4363678 2.2324553 2.2381663 ENSMUSG00000068790 6.731911 5.934638 8.429105 Oasl2 -2.0047154 -3.04129 -2.6751425 Khdrbs3 -2.044003 -3.577814 -2.5993783 Ntn1 6.145475 5.4245596 9.173744 Cpd 8.704006 8.542906 4.3925476 Sh3rf1 2.8441536 3.4739535 2.8403585 Tnfrsf9 7.1418743 9.86485 16.036419 Dusp4 13.4831 14.733007 25.416016 Igf1r 2.0111954 2.4666502 4.186206 Lypd6b -2.0062625 -2.9897883 -2.1315477 Acpl2 -2.234049 -3.8277073 -2.6685088 Fam46c -2.0294538 -2.7515247 -2.4923592 Hs3st3b1 -2.6867628 -5.1839304 -5.7279453 Il1rl2 -2.0818183 -2.1147027 -2.7298164 Vav2 2.3146935 2.9209704 4.097157 Ppm1l 2.6231213 2.171955 2.169916 Itgb8 7.315993 7.328177 7.124847 Adamts6 2.688951 2.376573 2.3354862 Ikzf2 7.51798 8.407602 2.3043823 Chd7 -2.1453428 -3.2930288 -3.4416618 Ppm1l 2.2205477 2.1409752 2.259559 Dnahc7b 2.6594055 2.186928 2.394492 LOC100504522 5.009973 4.21897 6.1362653 Ston1 3.2198353 4.7058105 6.2663503 Ntn4 3.1198468 3.5113802 4.0997972 Rgmb -2.6584334 -4.414649 -2.9698827 Stx11 6.479795 7.029024 5.4932165 2810001G20Rik -2.1002629 -3.6255589 -2.6789675 Itgb3 -3.4845088 -3.9519362 -3.8456166 Vipr1 -2.7176423 -6.706168 -4.823279 Camk2n1 2.007832 2.900548 2.6445763 Ikzf2 10.193131 11.742596 3.9649687 Ikzf4 3.985024 2.8921018 7.5200152 Stx11 4.9108276 4.0103216 3.722277 Cybrd1 2.8432918 2.795289 5.5242634