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A mechanism for expansion of regulatory T cell repertoire and its role in self tolerance

SUMMARY T cell receptor (TCR) signaling plays a key role in T cell fate determination. Precursor cells expressing TCRs within a certain low affinity range for self peptide-MHC complexes undergo positive selection and differentiate into naïve T cells expressing a highly diverse self-MHC restricted TCR repertoire. In contrast, precursors displaying TCRs with a high affinity for “self” are either eliminated through TCR agonist induced apoptosis (negative selection)1 or restrained by regulatory CD4+ T (Treg) cells, whose differentiation and function are controlled by the X-chromosome encoded transcription factor Foxp3 (review2). Foxp3 is expressed in a fraction of self-reactive T cells that escape negative selection in response to agonist driven TCR signals combined with interleukin-2 (IL-2) receptor signaling. In addition to Treg cells, TCR agonist-driven selection results in the generation of several other specialized T cell lineages like NKT and MAIT cells3. Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs4-6, Here we explored whether a specialized mechanism enables agonist driven selection of Treg cells with a diverse TCR repertoire and its significance for self-tolerance. We found that intronic Foxp3 enhancer CNS3 acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to effectively control self-reactive T cells, especially when thymic negative selection was genetically impaired. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.

driven selection results in the generation of several other specialized T cell lineages like NKT and MAIT cells 3 . Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs 4 -6 , Here we explored whether a specialized mechanism enables agonist driven selection of Treg cells with a diverse TCR repertoire and its significance for self-tolerance. We found that intronic Foxp3 enhancer CNS3 acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to effectively control self-reactive T cells, especially when thymic negative selection was genetically impaired. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.
TCR signaling plays an essential role in Treg cell differentiation and function 7 -10 . Recent studies have shown that a broad range of self-reactivity can promote Treg cell differentiation in the thymus, consistent with the highly diverse TCR repertoire of these cells 4 -6 . We reasoned that a dedicated mechanism, linked to the regulation of Foxp3 gene expression, might enable selection of Treg cells with a diverse TCR repertoire. Previously, we showed that an intronic element of the Foxp3 gene, CNS3, increases the efficiency of Treg cell generation, raising the possibility that it might affect the composition of the Treg TCR repertoire. To account for the potential effects of a mixed 129/B6 genetic background in our previous study, we backcrossed the Foxp3 ΔCNS3-gfp allele onto a B6 genetic background and generated male Foxp3 ΔCNS3-gfp and Foxp3 gfp littermates carrying identical N-terminal eGFP reporters 11 , 12 . Consistent with our previous observation 11 , we found a ~40% reduction in Foxp3 + CD4 + thymocytes in CNS3-deficient mice, compared to CNS3-sufficient littermate controls (2.05 ± 0.38% and 3.38 ± 0.70% of CD4 single-positive thymocytes respectively). The size of other thymocyte subsets was unaffected (Extended Data Fig. 1a, b). In contrast, peripheral Treg cells were present at comparable frequencies, most likely due to homeostatic expansion 7 , 13 -15 (Extended Data Fig. 1a). Interestingly, CNS3 deficiency had no effect on Foxp3 expression in differentiated Treg cells (Extended Data Fig. 1c). Our previous study suggested that CNS3 is epigenetically marked in precursor cells, raising the question at which stage of T cell differentiation CNS3 acts to facilitate Treg cell development. We found that ablation of a conditional CNS3 allele in double positive (DP) or double-negative (DN) thymocytes using Cd4 Cre or Lck Cre drivers, respectively, resulted in similarly defective thymic Treg cell generation (Extended Data Fig. 1d, e). To assess the requirement for CNS3 immediately preceding Foxp3 induction, we acutely ablated CNS3 using tamoxifeninducible Cre and observed decreased Foxp3 induction upon activation of naive CD4 + T cells in the presence of TGF-β and IL-2 (Extended Data Fig. 1f). Interestingly, in mature Treg cells CNS3 was fully dispensable for the maintenance of Foxp3 expression during cell division in the presence of pro-inflammatory cytokines (Extended Data Fig. 1g, h), and for their suppressor function in vivo (Extended Data Fig. 2).
These findings raised the question of how, mechanistically, CNS3 could selectively facilitate the initiation, but not the maintenance of Foxp3 expression. To begin addressing this problem, we sought to identify the stage of thymocyte differentiation at which the CNS3 region first acquires features characteristic of a poised enhancer. We previously found that CNS3 is marked by lysine 4 monomethylation of histone H3 (H4K4me1) in DP thymocytes 11 . Unexpectedly, we found increased H3K4me1 at CNS3 at DN1 stage and in hematopoietic stem cells (HSC), comparable to the levels observed in DP, CD4 SP thymocytes, and naïve CD4 + and CD8 + T cells (Fig. 1a-c and data not shown). In contrast, CNS3 chromatin was not enriched for H3K4me1 in embryonic stem cells (ESC), macrophages (Mϕ), or dendritic cells (DC) (Fig. 1b, c). These results indicate that the poised state of CNS3 is established at a very early stage of hematopoiesis, but is lost in "non-T" cell lineages. Because CNS3 appeared to be the earliest epigenetically modified region in the Foxp3 locus, it might exert its function by facilitating chromatin remodeling at the Foxp3 promoter.
While deposition of the "active" histone modifications H3K4me3 and H3K27Ac at the Foxp3 promoter occurred exclusively in Treg cells (Extended Data Fig. 3a, b), we found an enrichment of H3K4me1 in mature CD4 SP thymocytes and naïve CD4 + T cells (Fig 1d). In the absence of CNS3, both mature CD4 SP thymocytes and naïve CD4 + T cells showed impaired H3K4me1 accumulation at the Foxp3 promoter (Fig. 1e,  To address whether the CNS3-dependent poised state of the Foxp3 promoter assists deposition of additional permissive marks and further chromatin remodeling that facilitates the initiation of Foxp3 expression, we cultured naive CD4 + T cells from male Foxp3 gfp and Foxp3 ΔCNS3-gfp littermates under Treg cell differentiation conditions and isolated Foxp3 − cells that had been exposed to Foxp3-inducing conditions, but did not yet acquire Foxp3 expression. We observed a CNS3-dependent increase in H3K27Ac at the Foxp3 promoter preceding Foxp3 expression (Extended Data Fig. 3f), consistent with the defect in Treg cell differentiation in the absence of CNS3 (Extended Data Fig 1f). As a proof, blocking the recruitment of bromodomain-containing histone acetylation readers using iBET sharply reduced Treg cell induction efficiency in a dose-dependent manner (Extended Data Fig.  3g) 16 . Conversely, blocking histone deacetylase (HDAC) activity using butyrate increased H3K27Ac at the Foxp3 promoter in agreement with recent reports 17 -19 (Fig. 1g).
Importantly, provision of butyrate rescued impaired in vitro Treg cell differentiation associated with CNS3 deficiency (Fig. 1h). These observations suggest that a CNS3dependent poised state at the Foxp3 promoter, likely via looping, in precursor cells may enhance their sensitivity to Foxp3-inducing signals (Extended Data Fig. 3h).
While CNS3-dependent poising of the Foxp3 promoter could facilitate Foxp3 induction in a probabilistic manner, it might also enable lower strength TCR signals to promote Treg cell differentiation.
To address this possibility we tested whether impaired Foxp3 induction in CNS3-deficient naïve CD4 + T cells could be rescued by increasing amounts of CD3 antibody under in vitro Treg cell differentiation conditions. We found that the relative difference in the efficiency of Foxp3 induction between CNS3-sufficient and -deficient CD4 + T cells was markedly decreased in the presence of higher amounts of CD3 antibody ( Fig. 2a and Extended Data Fig. 4a), suggesting that increased TCR signal strength can partially compensate for the lack of Foxp3 promoter poising in the absence of CNS3, and imply that differentiation of Treg precursors receiving lower TCR stimulation might be disproportionally impeded by CNS3 deficiency. A corollary of this notion is that the mature Treg cells differentiated from CNS3-deficient precursors are enriched for TCRs at the higher end of the self-reactivity spectrum, and depleted of those with lower self-reactivity.
To test this possibility we analyzed Foxp3 − and Foxp3 + CD4 SP thymocytes and naïve CD4 + T cells for the expression of orphan nuclear receptor Nr4a1 (Nur77), a prominent TCR target gene in Treg and conventional T cells that accurately reports the strength of TCR signaling 20 , 21 . In agreement with the in vitro Foxp3 induction studies, we found that both thymic and peripheral CNS3-deficient Treg, but not Foxp3 − CD4 + T cells were markedly enriched for cells expressing higher levels of Nur77 in comparison to their CNS3-sufficient counterparts. This trend was observed in non-competitive settings of male  Fig. 5a, c, e and data not shown). These results further supported the notion that CNS3 deficiency resulted in an enrichment for thymic Treg cells with a heightened TCR signal strength.
To assess the autoreactivity of CNS3-deficient vs. -sufficient Treg cells in vivo, we examined their capacity for MHC class II (MHC-II) dependent homeostatic expansion under lymphopenic conditions, known to be proportional to TCR affinity for "self" 5 . CNS3deficient Treg cells expanded markedly more than CNS3-sufficient counterparts, upon cotransfer with congenically labeled effector T cells into lymphopenic hosts (Tcrb −/− Tcrd −/− ), which was likely driven by the recognition of self-antigens presented by MHC-II molecules, because antibody mediated blockage of MHC-II prevented expansion Treg cells and erased the advantage of CNS3-deficient Treg cells over their CNS3-sufficient counterparts (Fig. 2e, f, Extended Data Fig. 5f). Accordingly, the frequency of CNS3-deficient Treg cells was noticeably increased in the periphery of Foxp3 ΔCNS3-gfp/+ compared to Foxp3 gfp/+ heterozygous females (Extended Data Fig. 5g). Thus, Treg cells developed from precursors lacking CNS3 resulted in a skewed TCR repertoire.
We next examined the TCR repertoires of Treg cells, naïve and activated CD4 + T cells in Foxp3 ΔCNS3-gfp or Foxp3 gfp Tcra −/+ mice expressing the DO11.10 TCRβ chain transgene where TCR diversity is limited to a single functional TCRα chain locus 5 . Barcoded TCRα libraries were generated using an optimized protocol and high throughput sequencing data were analyzed using MIGEC software package 22 . Cluster analysis using VDJtools 23 showed that TCRα repertoires of thymic and peripheral CNS3-deficient and -sufficient Treg, but not naïve or activated effector CD4 + T cells were distinct (Fig. 2g). Further analysis showed a significantly reduced TCRα diversity of CNS3-deficient Treg, but not naïve or effector CD4 + T cells in comparison to their CNS3-sufficient counterparts (Fig. 2h). Since the TCR Complementarity-Determining Region 3 (CDR3) largely determines TCR specificity for peptide-MHC complexes (pMHC), we assessed the frequencies of strongly interacting amino acid residues in TCRα CDR3 by leveraging a mathematical model linking the features of amino acid residues in the CDR3 to TCR affinity for pMHC 24 . Interestingly, the TCRα CDR3s were significantly enriched for strongly interacting amino acid residues (Extended Data Fig. 5h, i), and for more randomly added nucleotides (Supplementary Table  1) in CNS3-deficient vs. -sufficient Treg, but not naïve or effector CD4 + T cells. These results implied higher affinities of CNS3-deficient Treg TCRs for self-antigens and further supported the notion that CNS3 shapes Treg TCR repertoire by increasing its diversity, likely, by enabling Treg differentiation in response to a broad range of self-reactivity.
To understand the functional significance of CNS3-dependent regulation of the Treg cell repertoire, we first assessed the immune status of male Foxp3 ΔCNS3-gfp and their wild type Foxp3 gfp littermates. We observed no effect of CNS3 deficiency on the numbers of activated or memory CD4 + or CD8 + T cells and on cytokine production by T cells in the secondary lymphoid organs in 8-12 week old mice (Fig. 3a-c and data not shown). Although CNS3deficient Treg cells were capable of preventing systemic autoimmunity ( Fig. 3a-c, Extended Data Fig. 1a), it remained possible that the skewed Treg TCR repertoire might have "holes". Thus, we reasoned that select few non-lymphoid organs may exhibit focused immune activation in CNS3-deficient vs. CNS3-sufficient mice, whereas others might be similarly or even more protected against autoimmunity by the overrepresented highly autoreactive Treg cells. Indeed, we found increased numbers of activated effector T cells and elevated IL-13 and IFNγ production by T cells in the lungs of Foxp3 ΔCNS3-gfp mice ( Fig. 3a-c). We also observed markedly elevated titers of circulating autoantibodies against a number of selfantigens in the sera of CNS3-deficient mice vs. their wild-type Foxp3 gfp littermates, whereas the absolute amounts of Ig isotypes were comparable (Fig. 3d, Extended Data Fig. 6a and data not shown). This notion was further supported by the observed modest, but consistent, decrease in the severity of experimental autoimmune encephalomyelitis (EAE) in Foxp3 ΔCNS3-gfp vs. Foxp3 gfp littermates (Extended Data Fig. 6b-f). To directly compare the suppressive capacity of Treg cells developed in CNS3-deficient and -sufficient mice, we transferred these Ly5.2 + Treg cells together with Ly5.1 + Foxp3 null (ΔFoxp3) effector T cells into T cell-deficient recipients (Fig. 3e). Despite comparable expansion of CNS3-sufficient and -deficient Treg cells in the recipients, we observed more pronounced weight loss and increased pro-inflammatory cytokine production by effector ΔFoxp3 T cells in the presence of CNS3-deficient Treg cells in comparison to the control ( Fig. 3f-h and Extended Data Fig.  6g, h). These results indicate that Treg cells developed from CNS3-deficient precursors were selectively impaired in their capacity to suppress self-reactive effector T cells.
Both negative selection and Treg cell generation are driven by self-antigen recognition in the thymus and likely play complementary roles in self-tolerance 1 , 2 , 25 , 26 . We reasoned that the relatively mild impairment in suppressive capacity of Treg cells from CNS3-deficient mice on a B6 genetic background, resistant to autoimmunity, may not fully reveal the biological significance of CNS3-dependent broadening of the Treg cell repertoire because of efficient negative selection. Therefore, we assessed the consequences of combined deficiency in CNS3 and Aire (Autoimmune Regulator), a nuclear factor required for thymic negative selection and optimal Treg cell generation. Aire deficiency leads to diminished expression of a subset of tissue-restricted antigens in the thymus and, consequently, an enlarged selfreactive effector T cell pool and diminished Treg repertoire 25 , 27 -29 . In contrast to a late-onset and rather mild autoimmunity observed in Aire deficient mice on a B6 genetic background, double deficiency in CNS3 and Aire resulted in fatal early-onset aggressive autoimmune lesions in multiple tissues as early as 3-4 weeks of age, when detectable autoimmune inflammation was lacking in littermates with a single deficiency in Aire or CNS3 ( Fig. 4a and Extended Data Fig. 7a). We noticed a 100% (n>35) penetrance with a stochastic gender independent variation in manifestations expected from perturbations in randomly generated repertoires of self-reactive T cells as well as the probabilistic nature of negative selection 30 (Extended Data Fig. 7a and data not shown). This was accompanied by significant increases in CD4 + T cell activation, IFNγ production ( Our studies suggest that CNS3, an intronic Foxp3 regulatory element, establishes a poised state of the Foxp3 promoter in precursor cells and increases the probability of Foxp3 induction in response to TCR stimulation, particularly, within a lower range of signal strength (Extended Data Fig. 8). Similar mechanisms of promoter poising may operate in other cell types and enable them to respond to a wider spectrum of growth factor or morphogen concentrations through receptor-triggered analogue signaling. CNS3 mediated Foxp3 promoter poising expands the TCR repertoire of Treg cells, which is essential for keeping pathogenic self-reactive T cells that escape negative selection in check.

Mice
Foxp3 CNS3-fl-gfp mice were generated using ES cell line CY2.4 (C56BL/6) as previously described 11  To induce deletion of CNS3 in vivo, tamoxifen solution (40mg/ml in olive oil) was administered by gavage to UBC Cre-ERT2 R26Y Foxp3 CNS3fl-gfp mice more than 3 days before lymphocyte isolation.
All mice were maintained in the MSKCC animal facility under SPF conditions, and the experiments were approved by the institutional review board (IACUC 08-10-023).

Statistical Analysis
Statistical tests were performed with Prism (GraphPad), Excel (Microsoft), or R statistical environment. Box-and-whisker plots show minimum, maximum, first and third quartiles and median.

In vivo suppression assay
To assess Treg cell suppressor capacity in vivo we conducted adoptive T cell transfers into T cell deficient recipients as previously described 31 . Briefly, 2.5~3.0×10 6 Foxp3 − CD4 + and/or CD8 + T cells isolated from Foxp3 gfpko or Foxp3 ΔCNS3-gfp Aire KO fold naïve CD4 + T cells (CD25 − CD44 lo CD62L hi ) isolated from wild type CD45.2 B6 males. To block TCR stimulation by self peptide-MHC class II complexes, 0.5 mg I-A b specific monoclonal antibody Y3P (IgG2a) or control IgG2a (Bio X Cell) was injected i.v. every other day before and after T cell transfer 32 , 33 . The lymphocyte subsets were analyzed by flow cytometry 9 days later.

Flow cytometric analyses and tissue lymphocyte preparation
Tissue lymphocytes were prepared as previously described 31

Autoantibody profiling using Autoantigen Microarrays
Analysis of autoantibody reactivity against a panel of 95 auto-antigens was conducted using the autoantigen microarrays developed by University of Texas Southwestern Medical Center 34 . Briefly, serum samples pretreated with DNase-I and diluted at 1:50 were incubated with the autoantigen arrays. After a second incubation with Cy3-conjugated anti-mouse IgG the arrays were scanned with a Genepix 4200A scanner (Molecular Device). The fluorescent signals for individual auto-antigens were extracted from the resulting images with Genepix Pro 6.0 (Molecular Devices), followed sequentially by subtraction of local background, average of duplicates, normalization with total IgG, and subtraction of a negative PBS control.  To label the 5'end of TCRα mRNA, a DNA-RNA hybrid oligonucleotide with 12 random nucleotides serving as barcodes to tag individual mRNA molecules was synthesized as reported 35 . High-throughput sequencing-Samples were quantified with Kapa Library Quantification kits (Kapa Biosystems) and sequenced on a MiSeq sequencer (Illumina) using 200 cycles of read 1, 6 cycles of index read and 200 cycles of read 2 with the following customized primers:

TCR sequencing and data analysis
Read 1: CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT.
Data analysis-Barcoded sequencing data were analyzed with MIGEC software 22 .
Briefly, unique molecular identifier (UMI) sequences were extracted from raw sequencing data (Read 1) with MIGEC/Checkout routine. Reads (≥5) bearing the same UMI were grouped and assembled to generate consensus sequences with MIGEC/Assemble. Variable (V) and Joining (J) segment mapping, Complementarity Determining Region 3 (CDR3) extraction, and error correction were performed with MIGEC/CdrBlast as previously described 22 , which eliminates PCR and sequencing errors, as well as normalizes the output data as cDNA counts that represent the TCR clonotypes in a population 36 .
Comparison of TCRα repertoires between CNS3-deficient and -sufficient mice at protein level was evaluated using VDJtools post-analysis framework (https://github.com/mikessh/ vdjtools) 23 . Pearson correlation of clonotype frequencies for the shared TCR clones was used for the generation of the dendrogram. Clonal diversities of TCRα repertoires were evaluated using inverse Simpson index computed separately for individual samples after downsampling the repertoires to the size of the smallest sample from the same organ. Similar downsampling strategy, not weighted by clonotype frequencies, was used to compute the average size of added nucleotides in CDR3. A mathematical model proposed by Kosmrlj et al. 24 was used to assess the strength of CDR3 amino-acid interactions with selfpeptide-MHC complexes. Numbers of strongly interacting amino acid residues (LFIMVWCY) were calculated for the V-segment part of TCRα CDR3 and V-J segment junction. Those numbers were then weighted by the corresponding clonotype frequencies and the resulting sums were used for the comparisons between samples. . The distribution of gene expression changes is shown for transcriptional signature genes and the rest of all expressed genes. One-tailed Kolmogorov-Smirnov test is used to determine the significance between the distributions of signature genes and the rest of expressed genes.

Chromatin Immunoprecipitation
1×10 6 cells were cross-linked with 1% formaldehyde for 5 min at room temperature. Crosslinked cells were lysed and nuclei were resuspended in 250 μl nuclear lysis buffer containing 1% SDS. Chromatin input samples were prepared by sonication of cross-linked nuclear lysates. For histone ChIPs, nuclear lysates were subjected to micrococcal nuclease digestion prior to sonication. Nuclei were resuspended in 100ul MNase (New England Biolabs) at 12,000 units/ml for 1 minute at 37°C. The reaction was stopped by addition of 10 μl 0.5M EDTA. Chromatin input samples were incubated overnight at 4°C with antibodies against H3K4me1 (Abcam), H3K4me3 (Millipore), or H3K27Ac (Abcam) and precipitated for 90 minutes at 4°C using protein A Dynabeads (Life Technologies). After thorough washing, bead-bound chromatin was subjected to proteinase K digestion and decrosslinking overnight at 65°C. DNA fragments were isolated using Qiagen PCR purification kit. Relative abundance of precipitated DNA fragments was analyzed by qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems). Primers used for qPCR are the followings:

Immunoglobulin isotype ELISA and immunofluorescence staining
Quantification of serum Ig isotypes was performed by ELISA as previously described 40 .
Tissue sections from gender matched Rag1 −/− mice were used to detect mouse autoantibodies. Briefly, organs from the Rag1 −/− mice were dissected, fixed with neutral buffered formalin, embedded with paraffin and sectioned. After deparaffinization with EZPrep buffer (Ventana Medical Systems) and antigen retrieval with Cell Conditioning Solution (CC1) (Ventana Medical Systems) the sections were blocked for 30 minutes with Background Buster solution (Innovex), followed by Avidin/Biotin blocking for 8 minutes, mouse serum (1:50 dilution) incubation for 5 hours and biotinylated horse anti-mouse IgG (Vector Labs) incubation for 1 hour. The detection was performed with Streptavidin-HRP (Ventana Medical Systems) followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen). The slides were then counterstained with DAPI (Sigma Aldrich) for 10 minutes, mounted, scanned with a Mirax scanner and visualized with Pannoramic Viewer (3DHISTECH). Scanned images were scored and representative snapshots were processed with Photoshop (Adobe) to switch the green and red channels for presentation purpose.

Generation of mixed bone marrow chimeras
Mixed bone marrow chimeras were generated as previously described 31 . Briefly, recipient mice were irradiated (9.5 Gy) 24h before i.v. injection of 10×10 6 bone marrow cells from CD45.1 Foxp3 gfp and CD45.2 Foxp3 ΔCNS3-gfp mixed at a 1:1 ratio. After bone marrow transfer, the recipient mice were administrated with 2mg/ml neomycin in drinking water for 3 weeks and analyzed 8-10 weeks later.

Histological Analysis
Tissue samples were fixed in 10% neutral buffered formalin and processed for hematoxylin and eosin staining. Stained slides were scored for tissue inflammation as previously described 41 .

EAE induction
EAE was induced by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55, GenScript) in Complete Freund's Adjuvant (CFA, Sigma) and mice were monitored for disease as previously described 42 . c-e, ChIP-qPCR analysis of H3K4me1 (c), H3K4me3 (d) and H3K27Ac (e) in the Foxp3 locus in mature Treg cells isolated from wild type Foxp3 gfp and Foxp3 ΔCNS3-gfp male mice normalized to the background binding to the Gm5069 locus. f, CNS3-dependent deposition of H3K27Ac at the Foxp3 promoter in Foxp3 − CD4 + T cells during in vitro Treg cell induction. Foxp3 gfp or Foxp3 ΔCNS3-gfp naïve CD4 + T cells were cultured under in vitro Treg cell differentiation conditions. After three days of culture, GFP − and GFP + cells were sorted for ChIP-qPCR analysis. Two-tailed unpaired t-test.  Linked circles represent samples from the same mice. Data represent one of two independent experiments. h, i, Numbers of strongly interacting amino acid residues (LFIMVWCY) were calculated for the V-segment part of TCRα CDR3 (binned to germline) and V-J segment junction, and weighted by the corresponding clonotype frequencies. Sums of the weighted scores were used for the comparisons between CNS3-deficient and -sufficient groups (unpaired t-test).

Extended Data
The data represent the analysis of pooled TCR sequences derived from the indicated thymic (h) and peripheral (i) CD4 + naïve (Tn), activated effector (Te) and Treg cell subsets isolated from individual Foxp3 gfp (n=5) and Foxp3 ΔCNS3-gfp mice (n=3). Box-and-whisker plots show minimum, maximum, first and third quartiles and median. g-h, Analysis of the proportion of Treg cells in CD4 + TCRβ + cell population (g) and level of Foxp3 expression (MFI) (h) in in vivo suppressor assay of CNS3-deficient or -sufficient Treg cells (Fig. 3e-h). Two-tailed unpaired Mann Whitney test.