(Itregs) Controls Colitis and IL-10 Produced by Induced Regulatory T

IL-10 Produced by Induced Regulatory T Cells (iTregs) Controls Colitis and Pathogenic Ex-iTregs during Immunotherapy

This information is current as Erica G. Schmitt, Dipica Haribhai, Jason B. Williams, Praful of September 29, 2021. Aggarwal, Shuang Jia, Louis-Marie Charbonnier, Ke Yan, Rachel Lorier, Amy Turner, Jennifer Ziegelbauer, Peter Georgiev, Pippa Simpson, Nita H. Salzman, Martin J. Hessner, Ulrich Broeckel, Talal A. Chatila and Calvin B. Williams Downloaded from J Immunol 2012; 189:5638-5648; Prepublished online 2 November 2012; doi: 10.4049/jimmunol.1200936 http://www.jimmunol.org/content/189/12/5638 http://www.jimmunol.org/

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-10 Produced by Induced Regulatory T Cells (iTregs) Controls Colitis and Pathogenic Ex-iTregs during Immunotherapy

Erica G. Schmitt,* Dipica Haribhai,* Jason B. Williams,*,1 Praful Aggarwal,† Shuang Jia,‡ Louis-Marie Charbonnier,x Ke Yan,{ Rachel Lorier,† Amy Turner,† Jennifer Ziegelbauer,* Peter Georgiev,x Pippa Simpson,{ Nita H. Salzman,‖ Martin J. Hessner,‡ Ulrich Broeckel,† Talal A. Chatila,x and Calvin B. Williams*

“Natural” regulatory T cells (nTregs) that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” regulatory T cells (iTregs) are nonredundant and essential for tolerance at mucosal surfaces, Downloaded from yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg-produced IL-10 and iTreg fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag12/2 mice by the adoptive transfer of naive CD4+ T cells carrying a nonfunctional Foxp3 allele. At the onset of weight loss, mice were treated with both iTregs and nTregs where one marked subset was selectively IL-10 deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTregs were present but IL-10 deficient, iTreg-produced IL-10 was necessary and sufficient for the treatment of disease, and vice http://www.jimmunol.org/ versa. Invariably, ∼85% of the transferred iTregs lost Foxp3 expression (ex-iTregs) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTregs and ex-iTregs, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10–dependent functional reciprocity between regulatory T cell subsets that maintains mucosal tolerance. The niche supporting stable iTregs is limited and readily saturated, which promotes a large population of ex-iTregs with pathogenic potential during immunotherapy. The Journal of Immunology, 2012, 189: 5638–5648.

D4+ CD25+ Foxp3+ regulatory T cells (Tregs) are essen- which develop in the thymus, and induced regulatory T cells tial to the balance between pro-inflammatory and anti- (iTregs), which arise from conventional T (Tconv) cells in the by guest on September 29, 2021 C inflammatory responses at mucosal surfaces (1). There periphery (2–6). Immunologic tolerance requires both Treg sub- are two subsets of Tregs: natural regulatory T cells (nTregs), sets, which act synergistically (6, 7). Additionally, iTregs can be generated in vitro by T cell activation in the presence of TGF-b1 *Section of Rheumatology, Department of Pediatrics, Medical College of Wisconsin, and IL-2, which makes them an attractive alternative for the Milwaukee, WI 53226; †Human and Molecular Genetics Center, Medical College of treatment of human autoimmune disorders unresponsive to cur- ‡ Wisconsin, Milwaukee, WI 53226; Max McGee National Research Center for Juvenile rent approaches (8–11). In vitro–derived iTregs also suppress Diabetes, Medical College of Wisconsin, Milwaukee, WI 53226; xDivision of Immu- nology, Boston Children’s Hospital, Department of Pediatrics, Harvard Medical School, inflammation in animal models of inflammatory bowel disease, Boston, MA 02115; {Section of Quantitative Health Sciences, Department of Pediatrics, ‖ diabetes, and autoimmune gastritis (12–14). Importantly, in vitro– Medical College of Wisconsin, Milwaukee, WI 53226; and Section of Gastroenterology, derived iTregs contribute to tolerance in disease models where Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226 in vivo–derived iTregs are not present (6, 7). The pathways used 1Current address: University of Chicago, Chicago, IL. by iTregs to support disease resolution in these models remain Received for publication March 28, 2012. Accepted for publication October 10, 2012. unknown, and it is unclear to what extent the method of iTreg derivation influences the acquisition of the full complement of This work was supported by National Institutes of Health Grants RO1 AI073731 and RO1 AI085090 (to C.B.W. and T.A.C.) and RO1 AI078713 (to M.J.H.), a Treg suppressive mechanisms. senior research award from the Crohn’s and Colitis Foundation of America (to C.B.W.), Although much work has been done to uncover the molecular the D.B. and Marjorie A. Reinhart Family Foundation (to C.B.W.), and the Children’s Hospital of Wisconsin (to C.B.W.). mechanisms of Treg suppressive activity, the “division of labor” between nTregs and iTregs remains largely unresolved (2). Mole- The microarray data presented in this article have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/gds) under accession cules such as CTLA-4, granzyme B, IL-10, and TGF-b have been number GSE35543. proposed as mechanisms of nTreg-mediated suppression (15–18). Address correspondence and reprint requests to Dr. Calvin B. Williams, Medical Of these mechanisms, the overall importance of IL-10 to immune College of Wisconsin, 8701 Watertown Plank Road, MACC Fund Research Center, homeostasis is exemplified by the chronic colitis that develops Room 5052, Milwaukee, WI 53226. E-mail address: [email protected] with complete or APC-specific IL-10 deficiency (19, 20). IL-10 is The online version of this article contains supplemental material. particularly important for Tregs at environmental interfaces, as a Abbreviations used in this article: CNS2, conserved noncoding sequence 2; EGFP, enhanced GFP; ex-iTreg, iTreg that lost Foxp3 expression; IEL, intraepithelial lymphocyte; Treg-specific inactivation of IL-10 results in spontaneous colitis (16). hi iTreg, induced regulatory T cell; MHI, Morisita–Horn Index; MLN, mesenteric lymph In the CD45RB transfer model of colitis, IL-10 present in the node; nTreg, natural regulatory T cell; Tconv, conventional T; Treg, regulatory T cell; CD45RBlow population, which contains Tregs, was found to be nec- TSDR, Treg-specific demethylation region; WT, wild-type. essary for disease prevention and treatment (21, 22). Furthermore, Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Treg-derived IL-10 was shown to control Th17 and Th1+Th17 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200936 The Journal of Immunology 5639 cells (23). To date, studies have been limited to the role of IL-10 in tinuous Percoll gradient (67%, 44%). Washed intestinal sections were nTreg function, leaving unresolved the role of IL-10 as an iTreg digested with collagenase D (1 mg/ml; Roche) in the presence of DNase I suppressive mechanism. (Invitrogen). A discontinuous Percoll gradient (67%, 44%) was used to isolate washed lamina propria lymphocytes. In this study, we used cells from mice that harbor a cassette encoding a mutant Foxp3-enhanced green fluorescent fusion protein Abs and flow cytometry ΔEGFP within the Foxp3 locus (Foxp3 ) to create colitis in an envi- Cells were collected from the spleen, MLN, colon and small intestine and ronment free of iTregs (6, 24). After immunotherapy with nTregs stained as indicated. The anti-mouse Abs used were Pacific blue–conjugated and iTregs that were selectively deficient in IL-10, we investigated anti-CD4 (RM4-5; Invitrogen), PerCP-conjugated anti-CD90.1 (OX-7; BD how the source of IL-10 impacted disease progression. We also Biosciences), Alexa Fluor 700–conjugated anti-CD44 (IM7; BioLegend), PE-conjugated anti-CD62L (MEL-14; BD Biosciences), Alexa Fluor 647– examined the clonal and transcriptional relationships between conjugated anti-CD103 (2E7; BioLegend), PE–Texas red–conjugated anti- those iTregs that maintained Foxp3 expression or lost Foxp3 ex- CD25 (PC61 5.3; Invitrogen), PE–Cy7–conjugated Klrg1 (2F1; eBio- pression (ex-iTregs). Our results identify in vivo selection of science), allophycocyanin eFluor 780–conjugated anti-TCRb (H57-597; a potent iTreg pool that is clonally distinct from pathogenic ex- eBioscience), and allophycocyanin-conjugated anti-GITR (DTA-1; eBio- science). A four-laser custom LSRII was used to collect the data, and iTregs and IL-10–dependent reciprocal compensation between FlowJo software was used for analysis. Treg subsets as critical for the maintenance of mucosal tolerance. Intracellular staining and cytokine analysis Materials and Methods Intracellular cytokine staining was performed after a 5-h restimulation Mice with PMA (5 ng/ml; Sigma-Aldrich) and ionomycin (0.5 mM; Sigma-Aldrich)

in the presence of brefeldin A (1 ml/ml; BD Biosciences). Surface staining Downloaded from Foxp3EGFP and Foxp3DEGFP mice on the BALB/c background were gen- of cells was performed using a modified FACS buffer containing 10 mg/ml erated and screened as previously described (24). Thy1.2+ Foxp3DEGFP brefeldin A. Cells were stained on ice for 30 min with the primary anti- newborn mice were rescued by i.p. transfer of 60 3 106 unfractionated mouse Abs PE–Cy7–conjugated anti-CD4 (RM4-5; BD Biosciences), Thy1.1+ BALB/c splenocytes to generate naive Thy1.2+ CD4+ CD45RBhi PerCP-conjugated anti-CD90.1 (OX-7; BD Biosciences), and allophyco- T cells with the nonfunctional Foxp3DEGFP allele. Rag12/2 and IL102/2 cyanin eFluor 780–conjugated anti-TCRb (H57-597; eBioscience) then mice were obtained from The Jackson Laboratory. The Animal Resource washed with the modified FACS buffer and fixed in 1% paraformal- Committee at the Medical College of Wisconsin approved all animal dehyde overnight at 4˚C. After this incubation, cells were washed with experiments. 1 ml PBS and then permeabilized with 1 ml 0.1% Triton-X. Intracellular http://www.jimmunol.org/ staining was performed for 30 min at room temperature with allophy- Cell purification and adoptive transfer cocyanin-conjugated anti–IFN-g (XMG1.2; BD Biosciences), Alexa Fluor 700–conjugated anti–TNF-a (MP6-XT22; BD Biosciences), Pacific blue– Pooled splenocytes and lymph node cells (axillary, brachial, inguinal, and conjugated anti–IL-17A (TC11-18H10.1; BioLegend) or with Pacific blue– mesenteric) were stained with either anti-CD4–allophycocyanin (RM4-5; conjugated anti-Helios (22F6; BioLegend), and allophycocyanin-conjugated BD Biosciences) or anti-CD4–Pacific blue (RM4-5; Invitrogen), plus anti- anti–CTLA-4 (UC10-4B9; BioLegend). A four-laser custom LSRII was CD90.1–PerCP (OX-7; BD Biosciences) and anti-CD45RB–allophycocya- used to collect the data, and FlowJo software was used for analysis. Serum nin (C363.16A; eBioscience) as appropriate and sorted on the basis of Ab cytokines were measured using the eBioscience FlowCytomix kit follow- and enhanced GFP (EGFP) fluorescence. All sorting was done on a FACS- ing the manufacturer’s recommendations. Aria (BD Biosciences). The average purity and viability of the sorted CD4+ populations was 98.96 6 0.14 and 84.31 6 0.68 (n = 120), respectively. Histology by guest on September 29, 2021 Colitis was induced in 6- to 8-wk-old Rag12/2 BALB/c mice by i.p. injection of 4 3 105 CD4+ CD90.12 EGFP2 CD45RBhi cells. Mice were Complete colons were fixed in formalin, processed, and stained with H&E weighed twice weekly. In some experiments, when mice lost 2.5% (65.7%) using a histology core facility. Blinded sections from the entire colon were of their initial body weight or began to exhibit symptoms of colitis (di- examined by a pathologist (N.H.S.), and large intestine colitis scores were arrhea, hunched posture), they were treated by i.p. injection of nTregs determined for inflammatory changes on a 4-point semiquantitative scale (Thy1.2+) plus iTregs (Thy1.1+)(53 105 each) purified by cell sorting. with 0 representing no change (26). The following features were consid- The nTregs were isolated on the basis of EGFP expression from the ered: severity, depth and chronic nature of the inflammatory infiltrate, crypt spleen and lymph nodes of Foxp3EGFP BALB/c mice or IL102/2 Foxp3EGFP abscess formation, granulomatous inflammation, epithelial cell hyperpla- BALB/c mice. The iTregs were generated in culture (as described below) sia, mucin depletion, ulceration, and crypt loss. and isolated on the basis of EGFP expression. RNA and cDNA isolation For serial adoptive transfer experiments, 1 3 105 CD4+ Thy1.1+ EGFP– ex-iTregs were isolated by sorting cells from the mesenteric lymph node Spleen and MLN cells of mice that were successfully treated with 0.5 3 (MLN) and spleen of mice that were successfully treated with Thy1.1+ wild- 106 Thy1.1+ WT iTregs plus 0.5 3 106 Thy1.2+ WT nTregs were stained 2 2 type (WT) iTregs plus Thy1.2+ WT nTregs and injected i.p. into Rag1 / with Pacific blue–conjugated anti-CD4 (RM4-5; Invitrogen) and PerCP- hosts. In some experiments, the mice were treated with 0.5 3 106 Thy1.2+ conjugated anti-CD90.1 (OX-7; BD Biosciences) and were sorted on the nTregs plus 0.5 3 106 Thy1.2+ iTregs and were coinjected with 15,000 basis of Ab and EGFP fluorescence. Ex-iTregs (CD4+ Thy1.1+ EGFP–), Thy1.1+ EGFP– T cells (based on a 3% sort impurity, three times the cal- iTregs (CD4+ Thy1.1+ EGFP+), and nTregs (CD4+ Thy1.1– EGFP+) were culated impurity). The Thy1.1+ EGFP– T cells were isolated from iTregs that isolated by flow cytometry sorting on a FACSAria. Total RNA was had lost Foxp3 expression in culture. extracted with the RNeasy Micro Kit for ,100,000 cells or the RNeasy Mini Kit for .100,000 cells (Qiagen) according to the manufacturer’s TGF-b1–mediated in vitro conversion protocol. cDNA was synthesized with the Superscript III First Strand 2 2 2 Synthesis System and oligonucleotide (dT) primers (Invitrogen) according Sorted CD4+ EGFP cells from Foxp3 EGFP or IL10 / Foxp3 EGFP mice to the manufacturer’s protocol. Isolated cDNA was used for quantitative (1 3 106/ml) were cultured with anti-CD3 mAb (clone 14-2C11 at 2.5 mg/ml) PCR, spectratype analysis, or CDR3 sequencing. coated dishes in the presence of soluble anti-CD28 mAb (1 mg/ml; clone 37.51), TGF-b1 (5 ng/ml; R&D Systems), and 100 U/ml IL-2. After 72 h, Quantitative PCR cells were resorted based on EGFP fluorescence and used for adoptive transfer or maintained in culture with IL-2. Quantitative PCR was performed in a StepOnePlus PCR System (Applied Biosystems) using the TaqMan Fast Universal PCR Master Mix (Applied Intraepithelial lymphocyte and lamina propria lymphocyte Biosystems) and predeveloped specific TaqMan primers for Il10 isolation (Mm00439616_m1). GAPDH was used (Mm99999915_g1) as a house- keeping gene (Applied Biosystems). The standard curve for Il10 was de- The entire colon and the distal 15 cm of the small intestine were used as veloped by isolating bone marrow cells from C57BL/6J and culturing the the source of intraepithelial lymphocytes (IELs) and lamina propria lym- cells at 4 3 104 cells/ml in DMEM/F12-10 medium (DMEM/F12 [Life phocytes (25). IELs were removed by gentle shaking of 0.5-cm intestinal Technologies], 10% [v/v] FBS [Atlantic Biologics], 10 mM L-glutamine sections for 30 min in buffer containing 5% (v/v) FCS, 1 mM DTT (Sigma- [Life Technologies], 100 IU penicillin/ml [Life Technologies], 100 mg/ml Aldrich), and 5 mM EDTA. IELs were washed and isolated on a discon- streptomycin [Life Technologies]) at 4 3 106 cells/ml in the presence of 10 5640 iTregs PROVIDE IL-10 BUT ARE UNSTABLE IN VIVO

U/ml MCSF (14-8983-80; eBioscience). The cells were incubated at 37˚C was performed using the Ion Torrent 314 or 316 chip and respective for 7 d (medium was changed on day 3). Macrophages were isolated using reagents. Sequence analysis and base calling was performed using the Cellstripper (Cellgro), washed, and resuspended in DMEM/F12-10 at built-in sequence software (v.1.9). 2 3 105 cells/ml. The cells were plated for 8 h at 37˚C in a 24-well plate containing 50 ng/ml LPS (Sigma), 1 mg/ml OVA (323–339 peptide), and CDR3 repertoire analysis by subcloning m 15 l/ml anti-OVA (0220-1682; AbD Serotec). cDNA was isolated as The purified PCR products were TA cloned into the pCR4-TOPO vector Il10 described, and the gene from the exon 2/3 boundary (forward primer: (Invitrogen) to create cDNA libraries. Individual colonies were subcloned, 9 9 5 -AATGCAGGACTTTAAGGGTTACTTGGG-3 ) to the exon 4/5 boundary and plasmids containing inserts were grown for 16 h in Luria–Bertani 9 9 (reverse primer: 5 -CTTGTAGACACCTTGGTCTTGGAG-3 ) was cloned medium, frozen at 280˚C in 50% glycerol, and sent to Beckman Coulter into the pCR4-TOPO vector (Invitrogen) and transformed into DH5a cells. for sequencing. The CDR3 regions were identified as the sequence be- The plasmid was isolated with a mini prep (Promega) and then purified tween the second conserved cysteine encoded by the 59 Vb gene segment by agarose gel extraction. The concentration was determined using log- and the conserved phenylalanine encoded by the 39 Jb segment (IMGT). dilutions and measured with a NanoDrop (Invitrogen). The standard curve was created with 10-fold dilutions based on copy number. Statistics Gene expression analysis The comparisons between groups for overall survival functions were done Total RNA for the iTreg, nTreg, and ex-iTreg sets was isolated with using the log-rank test. The random coefficient model was used to generate TRIzol (Invitrogen), according to the manufacturer’s protocol, from sorted a quadratic fit of the weight change over time. For the colitis scores, cell cells pooled from 14 mice treated with 0.5 3 106 WT nTregs plus 0.5 3 frequencies and numbers, and serum cytokine levels, a non-parametric 106 WT iTregs. Labeled target was prepared and hybridized to Affymetrix Kruskal–Wallis test was used to compare the measurements between the 430 2.0 GeneChips in accordance with the manufacturer’s protocol. Two groups. For the pairwise comparisons, a Mann–Whitney U test was per- technical replicates were performed, and the results were averaged. Probe formed. The TCR repertoire data were analyzed using the Morisita–Horn Index, which was calculated using EstimateS software. Downloaded from sets that revealed a 2-fold difference (|log2 ratio| .1.0) relative to Tconv cells were identified and used in subsequent analyses. The data were normalized with the robust multi-array analysis algorithm derived by the Bioconductor group (http://www.bioconductor.org) (27). The mean fold Results change was calculated from two independent arrays for each cell type Selective Treg IL-10 deficiency in the treatment of colitis , , and was scored p 0.05 with a false discovery rate 10% by the non- To establish the relevance of iTreg-produced IL-10 in a setting parametric rank product test (28). A false negative result is recovered for 2/2 + containing the nTreg subset, we induced colitis in BALB/c Rag1 Foxp3 Tregs because the gene array probes for Foxp3 lie distal to the http://www.jimmunol.org/ 5 + – hi poly-A initiation site in the Foxp3 EGFP allele. The microarray data are mice by transferring 4 3 10 Thy 1.2 EGFP CD45RB cells available in the Gene Expression Omnibus database (http://www.ncbi.nlm. isolated by cell sorting from BALB/c Thy1.2+ Foxp3ΔEGFP mice. nih.gov/gds) under the accession number GSE35543. Tconv cell data were The transferred CD45RBhi cells have a nonfunctional Foxp3 allele, taken from Gene Expression Omnibus microarray data set GSE6875. and colitis develops with accelerated kinetics in the absence of Methylation analysis in vivo–derived iTregs (6). When mice lost ∼2.5% of their initial body weight, we treated them with different combinations of The methylation status of the Treg-specific demethylation region (TSDR) 5 5 of Foxp3 in donor male nTregs, iTregs, and ex-iTregs purified from mice 5 3 10 iTregs plus 5 3 10 nTregs, where one or both of the treated with WT nTregs plus WT iTregs by cell sorting was assessed by Treg subsets lacked the capacity to produce IL-10 (Fig. 1). For + – bisulfite sequence analysis (29). Briefly, genomic DNAwas treated by bisulfite, all experiments, the iTregs were derived in vitro from CD4 EGFP by guest on September 29, 2021 to convert unmethylated cytosines into uracil leaving methylated cytosines cells isolated from the spleens and lymph nodes of Foxp3 EGFP unchanged. Bisulfite conversion of DNA was performed using EZ DNA 2/2 EGFP Methylation-Direct Kit (Zymo Research) according to the manufacturer’s or Il10 Foxp3 mice and cultured with TGF-b1andTCR instructions. The TSDR of converted DNA was amplified by methylation- cross-linking to upregulate Foxp3 expression. The nTregs were specific primer sequences 59-TATTTTTTTGGGTTTTGGGATATTA-39 isolated by cell sorting from the spleen and lymph nodes of these (forward) and 59-AACCAACCAACTTCCTACACTATCTAT-39 (reverse). same mice. The PCR product was purified and inserted into a TOPO TA cloning vector (Invitrogen). The ligation product was used to transform competent bacteria In the absence of any therapeutic intervention, mice rapidly lost (10-b competent Escherichia coli, New England Biolabs), and clones were weight, became moribund, and were sacrificed, with a mean sur- selected on kanamycin. Plasmid DNA was extracted by Qiagen miniprep vival of 45 d (Fig. 1A, 1B). In treated mice, where the transferred kit, and clones that present a 300-bp fragment after EcoRI digestion were iTreg compartment supplied the only source of Treg-derived IL- selected. Sequencing was done with M13R primer. Blast analysis was done 10, weight gain and survival were similar to those of control mice by comparing the M13R sequence and converted Foxp3 gene sequence. that were treated with WT iTregs plus WT nTregs (Fig. 1A). Spectratype analysis Reversing the experiment, where the nTreg compartment supplied cDNA was amplified by PCR with a Cb primer (59-CTCAAACAAGGA- the IL-10, showed similar patterns of weight gain and survival. In GACCTTGGGTGG-39) and a Vb primer from one of 22 Vbs (30). An contrast, complete absence of all IL-10 produced by Tregs, while ABI 3100 Genetic Analyzer was used to analyze the length distribution of better than no therapy, still resulted in weight loss, and only 25% amplified cDNA products, and Xplorer v2.4.2 (http://www.dnatools.com/ of the mice survived to 125 d (Fig. 1A, 1B). When Treg-derived download.html) was used to create histograms. IL-10 was limited to iTregs, there was also less inflammatory Ion torrent sequencing infiltrate in the colons of treated mice (Fig. 1C, 1D). Serum levels cDNA was amplified by PCR with the Cb primer and a Vb8.2 primer (59- of IFN-g were reduced when IL-10 was produced by at least one GCTACCCCCTCTCAGACATCAGTG-39). PCR products were purified Treg compartment, and TNF-a was reduced in all treatment using the QIAquick PCR purification kit (Qiagen) according to the man- groups irrespective of the capacity of Tregs to produce IL-10 (Fig. ufacturer’s protocol and concentrated using ethanol precipitation. The 1E). Serum levels of IL-17A were not reduced with Treg treat- DNA sample was purified using Agencourt AMpure beads. The purified ment. samples were used to generate libraries for Next Generation Sequencing using the Ion Torrent Personal Genome Machine (Life Technologies, An increase in iTreg frequency and/or number was seen in the 2/2 Carlsbad, CA) following the Ion Fragment Library Kit protocol. During MLNs, colon, and spleen of mice treated with WT iTregs plus Il10 preparation of the libraries, samples were size selected using the Sage nTregs compared with control mice treated with WT iTregs plus Science Pippen Prep instrument and 2% agarose cassettes. Completed li- WT nTregs (Fig. 2A, 2B, Supplemental Fig. 1A, 1B, and data not braries were analyzed and quantified on the Agilent 2100 BioAnalyzer. The obtained libraries were processed for sequencing by dilution following shown). However, the number of nTregs recovered from these the recommendations for the Ion Xpress Template Kit protocol that used tissues was similar between all treatment groups, demonstrating an emulsion PCR, breaking, and enrichment of each sample. Sequencing that there was no effect of Treg-derived IL-10 on nTreg recovery The Journal of Immunology 5641 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Treatment of experimental colitis with iTregs plus nTregs in the presence of selective IL-10 deficiency. (A) Quadratic regression analysis of the weight change over time after the induction of experimental colitis with cells isolated from Foxp3DEGFP mice (black; n = 21). Mice were treated with Il102/2 iTregs plus Il102/2 nTregs (red, n = 17), WT iTregs plus Il102/2 nTregs (blue, n = 18), Il102/2 iTregs plus WT nTregs (orange, n =22),orWTiTregsplus WT nTregs (green, n = 40). Control Rag12/2 mice (gray; n = 6) did not receive cells. Individual mice are represented by dashed lines, and solid lines indicate a quadratic curve fit to the data. (B) Kaplan–Meier survival curves for the mice in (A). (C) Representative H&E-stained histological sections of the colons from the mice in (A), shown at original magnification 310 and 340. (D) Colitis scores from mice where histology was obtained. (E) Serum levels of IFN-g, TNF-a, and IL-17A from the indicated mice. For all treatment groups, seven or more experiments were performed. For these and all subsequent scatterplots, each colored dot represents an individual mouse, and the horizontal bars represent mean values. *p , 0.05, **p , 0.005, ***p , 0.0005.

(Fig. 2B, Supplemental Fig. 1B). These data indicate that a com- Next, we examined the impact of iTreg-produced IL-10 on ef- pensatory expansion of WT iTregs occurs when nTregs lack the fector T cells in the target tissues. Overall, the number of CD4+ capacity to produce IL-10 and that differences in clinical outcome T cells was reduced in the MLN and spleen of treated mice between the treatment groups are not due to an overall reduction compared with untreated controls, irrespective of the IL-10 status in Treg numbers. of the Tregs used to treat the mice (Fig. 2C and data not shown). 5642 iTregs PROVIDE IL-10 BUT ARE UNSTABLE IN VIVO Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Analysis of Treg and effector T cells isolated from the MLN of treated mice. (A) Representative ﬂow cytometry (top panel: n = 16, 17, 19, 13, left to right) of the mice of Fig. 1 showing in vitro–derived iTregs (Thy1.1+ EGFP+) and nTregs (Thy1.1– EGFP+). Scatterplots (bottom panels) show the frequency of iTregs and nTregs contained within the TCRb+ CD4+ gate. (B) The number of iTregs (left) and nTregs (right) recovered from mice in each treatment group. (C) The mean number of TCRb+ CD4+ T cells recovered from the indicated treatment groups (n = 16, 17, 19, 13, left to right). Error bars depict the SEM. (D) Representative intracellular cytokine staining of cells stimulated ex vivo. Cells from the indicated treatment groups were stained for IFN-g and IL-17A, and the analysis shows the TCRb+ CD4+ gate (n = 15, 15, 18, 14, left to right). (E) The number of MLN TCRb+ CD4+ T cells that are IFN-g+, IL-17A+, and IFN-g+ IL-17A+. For these and all subsequent representative FACS plots, numbers indicate the mean percent of cells in the quadrant. *p , 0.05, **p , 0.005.

CD4+ T cells were further reduced in the MLN of mice treated experimental design. Additional mechanisms of Treg-mediated with WT iTregs plus Il102/2 nTregs, suggesting a modest effect of suppression may be operative, as a modest Treg-associated ef- iTreg-produced IL-10 in this location. Consistent with the reduc- fect on weight change and survival was observed in the complete tion in CD4+ T cells, the number of IFN-g+, IL-17A+, or IFN-g+ absence of Treg-derived IL-10. IL-17A+ effector CD4+ T cells in the MLN of treated mice was also reduced when the iTreg compartment selectively produced Stability and fate of iTregs IL-10 (Fig. 2D, 2E) and closely matched that seen in mice treated In all treatment groups, many Thy1.1+ Foxp3+ iTregs lost Foxp3 with Il102/2 iTregs plus WT nTregs. In the colon, the frequency expression (ex-iTregs) and were maintained in the MLN, colon, of IL-17A+ and IFN-g+ IL-17A+ T cells was reduced when Treg spleen, and small intestine of treated mice (Fig. 2A, Supplemental IL-10 production was limited to iTregs (Supplemental Fig. 1C). Fig. 1A, and data not shown). Contaminating EGFP– cells from These data demonstrate that iTregs are a potent source of IL-10 the sorted iTreg cultures contributed little (12%) to the ex-iTreg and that iTreg suppression of lymphoproliferation and Th1 and population (Supplemental Fig. 1D, 1E). When comparing groups, Th17 cell differentiation is closely associated with IL-10 pro- the frequency of ex-iTregs was highest in the MLN and spleen duction. The data also confirm a similar outcome with a reverse in of mice treated with IL-10–sufficient iTregs (Fig. 3A, 3B). This The Journal of Immunology 5643 pattern was reversed in the colon and small intestine (Supple- regulated relative to naive CD4+ T cells (Supplemental Table I). mental Fig. 1F). The ex-iTregs isolated from mice treated with This analysis captured 319 of the 603 probe sets found in the WT nTregs plus WT iTregs did not continue to produce IL-10 as canonical Treg transcriptional signature (31). Notably, 1437 (44%) demonstrated by RT-PCR of sorted cells (Supplemental Fig. 1G). probe sets were common to all three groups and included ∼40% However, in vitro stimulation of ex-iTregs isolated from multiple (132 of 319) of the Treg probe sets that we recovered (Fig. 4C). tissues resulted in the production of IFN-g and IL-17A (Fig. 3C These included directionally concordant expression of the Treg and Supplemental Fig. 1H). Importantly, the frequency of ex- signature genes Itgae, Lef1, Dusp4, Ctla4, Ahr, Gzmb, Ccr6, and iTregs in the MLN that were IL-17A+ or IFN-g+ IL-17A+ was Rora. In most cases, however, the ex-iTregs had the smallest fold reduced when iTregs produced IL-10 (Fig. 3C). The frequency of changes in level of expression (Fig. 4D). Most of the remaining ex-iTregs producing IL-17A was increased (18–27%) in the small Treg signature genes were suppressed or not differentially regu- intestines of mice from all treatment groups relative to the other lated in the ex-iTreg expression profile, including Ikzf4, Gpr83, tissues (data not shown). These data demonstrate that ex-iTregs Nrp1, Ikzf2, Pde3b, Il2ra, Ebi3, and Klrg1. The genetic signatures can produce proinflammatory cytokines and that the capacity of of in vitro–derived iTregs that were maintained in vivo and nTregs ex-iTregs to produce IL-17A and the localization of ex-iTregs may were remarkably similar (64% overlap, Fig. 4C), consistent with be influenced by iTreg-produced IL-10. our previous data examining the relationship between in vivo– derived iTregs and nTregs (7). Phenotypic and transcriptional analysis of iTregs and ex-iTregs Ex-iTregs also differentially regulated a number of genes not We investigated the phenotypic and molecular relationship between contained within the canonical Treg signature (Fig. 4E). Ex-iTregs nTregs, iTregs, and ex-iTregs by examining their cell surface showed induction of Il17a (30-fold versus Tconv cells) and Ifng Downloaded from phenotype, gene expression profile, and their methylation status at (11-fold versus Tconv cells), consistent with the intracellular cy- the TSDR of conserved noncoding sequence 2 (CNS2) within the tokine staining. In contrast, Il10 expression was largely confined Foxp3 promoter. Flow cytometric analysis of cells from the MLN to nTregs and iTregs (52-fold and 27-fold increases versus Tconv of mice treated with WT iTregs plus WT nTregs revealed that cells, respectively). The ex-iTregs expressed Il2, Il22, Ccl5, and iTregs and nTregs had similar expression of several cell surface Gzmk. Several other genes not associated with the Treg signature

markers including CD25, CD62L, CD44, CD103, KLRG1, GITR, were expressed in ex-iTregs as well as both Treg subsets, such as http://www.jimmunol.org/ and CTLA-4 (Fig. 4A). Helios was expressed in 20.6% (65.8%) Lyz1, Gp49a, S100a8, S100a9, and Klf4. In all three cell types, of the iTregs in the spleen, but was generally not found in iTregs Ikzf1 (Ikaros) and Sox4 were repressed. in the MLN or in ex-iTregs (Fig. 4A, 4B, and data not shown). To investigate further the molecular relationship between the Several differences were noted between ex-iTregs and iTregs nTreg, iTreg, and ex-iTreg subsets, we analyzed the methylation found in the MLN. Compared to iTregs, the ex-iTregs had lower status of the TSDR within CNS2 of the Foxp3 promoter of mice levels of CD25, CD103, KLRG1, and CTLA-4, although they had rescued with WT nTregs plus WT iTregs. As expected, the CpG similar expression of CD62L, CD44, and GITR (Fig. 4B). islands in the TSDR of WT nTregs were demethylated (Fig. 4F, Next, we sorted nTregs, iTregs, and ex-iTregs from the spleens top panel) (7, 29). Consistent with their lack of Foxp3 expression, and MLNs of mice treated with WT iTregs plus WT nTregs and the CpG islands in ex-iTregs showed extensive methylation at the by guest on September 29, 2021 examined their gene expression proﬁles. From the three groups, examined CpG motifs. In aggregate, CpG islands in the TSDR of we identiﬁed a total of 3250 probe sets that were differentially stable in vitro–derived iTregs isolated from successfully treated

FIGURE 3. Stability and fate of iTregs. (A)The frequency (left) and number (right)ofTCRb+ CD4+ Thy1.1+ EGFP– ex-iTregs found in the MLN of the miceshowninFig.1(n = 16, 17, 10, 13, left to right). (B)Thefrequency(left)andnumber(right)of ex-iTregs found in the spleen of the mice shown in Fig. 1 (n = 16, 17, 10, 13, left to right). (C) Repre- sentative ﬂow cytometry (top panels: n = 15, 15, 9, 13, left to right) showing intracellular cytokine staining of ex-iTregs from the MLN of treated mice after ex vivo stimulation. Staining for IFN-g and IL-17A is shown for cells within the TCRb+ CD4+ Thy1.1+ EGFP– gate. Scatterplots (bottom panels) showing the percentage of MLN TCRb+ CD4+ Thy1.1+ EGFP– T cells that are IFN-g+, IL-17A+, and IFN-g+ IL-17A+.*p , 0.05, **p , 0.005. 5644 iTregs PROVIDE IL-10 BUT ARE UNSTABLE IN VIVO Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Phenotypic and transcriptional profile of iTregs and ex-iTregs. (A and B) Representative flow cytometric staining of iTregs (TCRb+ CD4+ Thy1.1+ EGFP+) and nTregs (TCRb+ CD4+ Thy1.1– EGFP+)(A) or iTregs and ex-iTregs (TCRb+ CD4+ Thy1.1+ EGFP–)(B) from the MLN of mice treated with WT iTregs plus WT nTregs (from Fig. 1), stained as indicated. Data are representative of at least five mice and three experiments. (C) Commonly and uniquely regulated probe sets found in stable in vitro–derived iTregs, ex-iTregs, and nTregs. Probe sets that revealed a 2-fold difference (|log2 ratio| .1.0) and rank product false discovery rate ,10% relative to Tconv cells were identified and used in subsequent analyses. (D) Heat map showing the fold change in expression of select genes found within the canonical Treg transcriptome. Genes are organized into coregulated gene clusters (1 to 7) with cluster 1 containing genes tightly correlated with Foxp3 expression and cluster 4 and 5 containing genes influenced by TCR and IL-2 signaling. For further information on the clustering, please see Hill et al. (31). (E) Heat map showing the fold change in expression of select differentially regulated probe sets not found within the Treg transcriptome. For (C) and (D), the scale (–3.0-fold to +3-fold) represents the fold change relative to the (Figure legend continues) The Journal of Immunology 5645 mice showed partial demethylation, although there was consider- dominated by a single CDR3 length were occasionally observed able variability in the methylation patterns found in individual and in general did not correlate between cell populations and mice. Notably, iTregs isolated from at least two mice were largely mice. demethylated (Fig. 4F, bottom panel). The extensive iTreg We next examined the clonal relationship between iTregs and ex- demethylation seen in these two mice did not correlate with Treg iTregs by comparing TCR sequences from mouse 4 and mouse 6. or ex-iTreg recovery, rate of weight loss after colitis induction, or Vb8.2 cDNA fragments were selected for TCR CDR3 sequencing the weight gained after treatment (data not shown). based on the broad distribution of CDR3 lengths with both Taken together, these gene and protein expression data indicate Gaussian and skewed distributions seen in all analyzed mice (Fig. that ex-iTregs retain a component of the Treg canonical signature. 6A). CDR3 regions from each population were amplified by However, loss of Foxp3 expression is associated with changes in PCR, linked to beads, and examined using non-optical integrated the expression of several genes important for Treg suppressive semiconductor sequencing. We obtained 416,207 in-frame reads function and in the acquisition of genes related to the inflammatory encoding 5,162 iTreg and 3,738 ex-iTreg CDR3 polypeptides response and T cell effector capability (32–36). The general failure (Supplemental Table II). Repertoire comparisons based on amino of most in vitro–derived iTregs to demethylate CpG islands in the acid sequences revealed minimal overlap (∼2%) between unique TSDR likely contributed to the large number of ex-iTregs present iTreg and ex-iTreg CDR3 regions (Fig. 6B). Consistent with this in the treated mice, as demethylation of these residues has been observation, the Morisita–Horn Index (MHI) was 0.11 and 0.12 shown to be important to iTreg stability (37). Fully demethylated for the two comparisons, indicating little similarity. Ln-rank fre- iTreg TSDRs were occasionally observed, suggesting that iTregs quency versus ln-rank plots revealed a two-component distribution per se are not excluded from stable Foxp3 expression. consisting of clones that follow a power law relationship and Downloaded from multiple high-rank clonotypes. To determine if the same high-rank Pathogenicity of ex-iTregs clonotypes expanded after transfer, we compared the two iTreg The ex-iTregs expressed genes associated with regulation but also and the two ex-iTreg repertoires that developed in treated mice expressed genes associated with inflammation. Because of the dual that received aliquots from a common Treg pool (Fig. 6C). There nature exhibited by ex-iTregs, we tested their pathogenic potential was little overlap between the iTreg populations (∼1%, MHI = 5 2/2 by the adoptive transfer of 1 3 10 sorted ex-iTregs into Rag1 0.092). The ex-iTreg populations also showed a small 1.4% http://www.jimmunol.org/ hosts. Recipients rapidly lost weight, became moribund, and were overlap, although the MHI for this comparison was higher (MHI = sacrificed after ∼40 d (Fig. 5A, 5B). Histological analysis of the 0.488). The higher MHI here indicates that a few of the high-rank colon revealed lymphocytic infiltration and severe colitis (Fig. clonotypes were recovered from both mice. For control experi- 5C). Notably, an average of 2% of the transferred cells found in ments, we compared the iTreg clonotypes recovered from the the MLN regained Foxp3 expression in vivo. This frequency of same samples run on two different semiconductor chips or by iTregs matched that seen after the transfer of 1 3 105 CD4+ direct cloning and sequencing of all four populations (Fig. 6D, EGFP– CD45RBhi cells into Rag12/2 mice (Fig. 5D). Thus, in the Supplemental Fig. 2B, Supplemental Table II). As expected, the absence of Tregs, ex-iTregs cause severe disease. The capacity of TCR repertoires recovered by the different methods were highly ex-iTregs to upregulate Foxp3 is retained and is similar to that of similar (average MHI ∼0.8) and in some instances nearly identical by guest on September 29, 2021 naive CD4+ T cells. (MHI = 0.971). These data validate non-optical integrated semiconductor sequencing in this application and demonstrate that Clonal relationship between iTregs and ex-iTregs we can find similarity between repertoires where it exists. Col- The capacity of ex-iTregs to reacquire Foxp3 expression after lectively, these data indicate that each mouse expanded unique transfer into Rag12/2 hosts (Fig. 5D) suggested that the ex-iTregs populations of iTregs and ex-iTregs, and the ex-iTreg populations and iTregs could be interconverting and thus clonally related. To also shared a few high-rank clonotypes between the two mice. test this hypothesis, we isolated iTregs and ex-iTregs from six Within an individual mouse, the iTreg and ex-iTreg populations mice treated with WT iTregs plus WT nTregs and characterized were clonally unrelated. the TCR repertoires. Notably, mouse 1 and mouse 2 received equivalent aliquots drawn from the same population of pooled Discussion Tregs. In a separate experiment, we also treated mouse 4 and In this study, we modified a T cell transfer model of colitis to create mouse 6 by splitting a pooled Treg sample. Mouse 3 and mouse 5 chimeric mice where nTregs and iTregs were selectively deficient each received an unrelated population of cells. This experimental in IL-10. Although iTregs composed a fraction (∼20%) of all Tregs design allowed us to compare the iTreg and ex-iTreg populations recovered from treated mice, the IL-10 supplied by iTregs could that were maintained within a single individual and between two replace nTreg-derived IL-10 in the cure of disease. In the reverse individuals that received equal fractions of the same inoculum. experiment, nTreg-derived IL-10 was equally effective. These Fragment analysis of 12 Vb CDR3 regions from six treated mice results demonstrate the principle of reciprocal compensation be- showed both skewed and Gaussian distributions with limited tween Treg subsets, which is an essential tolerogenic mechanism. similarity between the iTreg and ex-iTreg populations, both within Importantly, we identified iTregs as a particularly potent source individuals and between those individuals that received equivalent of IL-10 in the gastrointestinal tract that can be used to augment Treg populations (Supplemental Fig. 2A). For example, only the regulatory function in situations where nTregs are defective or Vb1 and Vb2 analyses showed a dominant peak at the same CDR3 depleted. Reductions in Treg numbers and/or function have been length distribution in more than half of the samples. Spectratypes implicated in many human autoimmune diseases (38). This ther-

mean normalized intensity value (log2 ratio) across all four groups (nTreg, iTreg, ex-iTreg, Tconv cells). (F) Methylation status of individual CpG motifs within the TSDR of CNS2 in Foxp3. Individual CpG motifs are numbered with reference to the transcription initiation site of Foxp3. The average percent methylation is shown in the bar graph for nTregs (n = 3), iTregs (n = 11), and ex-iTregs (n = 10) isolated from mice treated with WT nTregs plus WT iTregs (top panel). Methylation patterns at each of the examined TSDR motifs of Tregs and ex-iTregs from the individual mice, numbered 1–11, are shown in the heat map (bottom panel). The color code ranges from yellow (no methylation) to blue (100% methylation). 5646 iTregs PROVIDE IL-10 BUT ARE UNSTABLE IN VIVO

FIGURE 5. Pathogenicity of ex-iTregs. Weight change over time (A) and Kaplan–Meier survival curves (B) after the adoptive transfer of 1 3 105 ex- iTregs sorted from mice successfully treated with WT iTregs plus WT nTregs and transferred into Rag12/2 hosts. Each line represents an individual mouse (n = 5, three experiments). (C) Representa- tive H&E-stained histological section (left) from the colons and colitis scores (right) for the mice in (A). (D) Representative flow cytometry from the MLN of mice in (A)(left) and control mice that received 1 3 105 CD4+ EGFP– CD45RBhi cells (right) showing staining for CD4 and EGFP fluorescence. Downloaded from apeutic approach may therefore have broad application, as long- in treated mice may not be equivalent to in vivo–derived iTregs term stable tolerance can be achieved through iTreg transfers under the same conditions. Further studies are needed to compare when nTregs are also present. the function and stability of iTregs and nTregs derived in vivo. Prior work has shown that the iTreg transcriptional signature 72 h This caveat notwithstanding, we have identified important char- http://www.jimmunol.org/ after in vitro induction of Foxp3 with TGF-b was largely inde- acteristics of stable in vitro–derived iTregs relevant to their use in pendent of Foxp3 expression and distinct from that of nTregs (6, immunotherapy. 39). In this study, we extended these studies by determining the Notably, ∼85% of the surviving Thy1.1+ population no longer transcriptional profile of in vitro–derived iTregs that were main- expressed Foxp3 three months after transfer. Although these ex- tained in treated mice for approximately 3 mo. We now demon- iTregs retained some elements of the canonical Treg transcrip- strate that nTregs and in vitro–derived iTregs that are stable in vivo tional signature, the TCRb CDR3 repertoires of iTregs and ex- share similar transcriptional profiles, including the expression of iTregs were essentially non-overlapping. Thus, the generation of many genes associated with Treg suppressive function in addition a large population of ex-iTregs in vivo may be the unintended

to Il10. These data provide further support for the hypothesis that consequence of in vitro conversion based on nonspeciﬁc TCR by guest on September 29, 2021 the two Treg subsets use similar suppressive mechanisms. This cross-linking. In the absence of Treg control, the prior history of result also illustrates that the in vitro–derived iTreg and nTreg Foxp3 expression was not sufﬁcient to control the pathogenic transcriptional signatures converge as the iTregs are selected and potential of ex-iTregs upon retransfer into Rag12/2 hosts. However, maintained in vivo. One caveat to these studies is that we have iTreg-derived IL-10 limited the frequency of ex-iTregs adopting compared in vivo–derived nTregs to in vitro–derived iTregs and a Th1, Th17, or Th1+Th17 cell phenotype. This is consistent with not to in vivo–derived iTregs. In vitro–derived iTregs that persist previous studies showing that IL-10 provided exogenously or by

FIGURE 6. TCRb CDR3 repertoire analysis of iTregs and ex-iTregs. (A)Vb8.2 spectratype analysis of iTregs and ex-iTregs isolated from six mice treated with WT iTregs plus WT nTregs. Mouse 4 (Mu4) and mouse 6 (Mu6) were treated with a single split inoculum of Tregs. (B) Distribution of unique and overlapping iTreg and ex-iTreg CDR3 amino acid sequences of Vb8.2-containing clones isolated from Mu4 and Mu6 using the Ion 316 chip. Ln-rank frequency versus ln-rank plots for each TCRb CDR3 repertoire are shown below the Venn diagram corresponding to that population. (C) Unique and overlapping iTreg sequences (top panel) and ex-iTreg sequences (bottom panel) obtained from Mu4 and Mu6. (D) A comparison of the iTreg TCR CDR3 unique and overlapping sequences recovered by non-optical integrated semiconductor sequencing of the same sample (Mu4) on two separate chips, Ion 314 and Ion 316. The number of sequences and the MHI are listed for each comparison. The Journal of Immunology 5647

CD4+ Foxp3+ Tregs constrains Th17 and Th1+Th17 cell frequen- Foxp3+ regulatory T cells: more of the same or a division of labor?” cies (23). Additionally, IL-10 signaling in Tregs and Th17 cells (2) is that both shared methods and unique characteristics are limits Th17 cell-mediated inflammation (23, 40). Collectively, needed to enforce tolerance. these data support a specific role for iTreg-produced IL-10 in the control of Th17 responses. 2 2 Acknowledgments After retransfer into Rag1 / hosts, ∼2% of the ex-iTregs re- We thank James Verbsky for kindly providing the Foxp3EGFP IL102/2 mice. acquired Foxp3 expression. Thus ex-iTregs retain the potential to We thank Brandon Edwards and Kyle Upchurch for assistance with cell re-express Foxp3 and irrespective of their pathogenic potential sorting. We also thank Mary Williams for administrative assistance and may serve as an iTreg reservoir in instances when iTreg responses William Drobyski and Jack Gorski for critical review of the manuscript. are exhausted or diminished. Unstable Foxp3 expression in both nTregs and iTregs has been described, although others have con- Disclosures cluded that Foxp3 expression in nTregs is heritable and stable (41– The authors have no financial conflicts of interest. 48). Our results clearly demonstrate that many in vitro–derived iTregs have unstable Foxp3 expression in vivo. It has been shown that iTregs derived in vitro are unstable due to a failure to fully References demethylate the TSDR (29, 37). This incomplete demethylation 1. Izcue, A., J. L. Coombes, and F. Powrie. 2009. Regulatory lymphocytes and could contribute to the high number of ex-iTregs observed in intestinal inflammation. Annu. Rev. Immunol. 27: 313–338. 2. Curotto de Lafaille, M. A., and J. J. Lafaille. 2009. Natural and adaptive foxp3+ treated mice. It is interesting, however, that even mice with iTregs regulatory T cells: more of the same or a division of labor? Immunity 30: 626– that had an extensively demethylated TSDR still had a sizeable 635. Downloaded from fraction of in vitro–derived iTregs that lost Foxp3 expression and 3. Apostolou, I., and H. von Boehmer. 2004. In vivo instruction of suppressor commitment in naive T cells. J. Exp. Med. 199: 1401–1408. became ex-iTregs. Furthermore, stable iTregs that never fully 4. Curotto de Lafaille, M. A., A. C. Lino, N. Kutchukhidze, and J. J. Lafaille. 2004. acquired the nTreg TSDR signature were recovered from suc- CD25- T cells generate CD25+Foxp3+ regulatory T cells by peripheral expansion. J. Immunol. 173: 7259–7268. cessfully treated mice, both in this experimental colitis model and 5. Knoechel, B., J. Lohr, E. Kahn, J. A. Bluestone, and A. K. Abbas. 2005. Se- in a model of Foxp3 deficiency, suggesting that pathways other quential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen. J. Exp. Med. 202: 1375–1386. than promoter demethylation may impact the maintenance of http://www.jimmunol.org/ 6. Haribhai, D., W. Lin, B. Edwards, J. Ziegelbauer, N. H. Salzman, M. R. Carlson, iTregs in vivo (7). The amount of intermouse variability in the S. H. Li, P. M. Simpson, T. A. Chatila, and C. B. Williams. 2009. A central role extent of iTreg TSDR demethylation further supports the notion for induced regulatory T cells in tolerance induction in experimental colitis. J. that factors such as subclinical inflammation and disease status Immunol. 182: 3461–3468. 7. Haribhai, D., J. B. Williams, S. Jia, D. Nickerson, E. G. Schmitt, B. Edwards, may contribute to iTreg stability. Because iTregs can be specific J. Ziegelbauer, M. Yassai, S. H. Li, L. M. Relland, et al. 2011. A requisite role for gut bacterial Ags, strategies to derive polyclonal populations of for induced regulatory T cells in tolerance based on expanding antigen receptor diversity. Immunity 35: 109–122. Ag-specific iTregs prior to transfer may enhance the stability of 8. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, and Foxp3 expression (49). Treatment with inhibitors of DNA meth- S. M. Wahl. 2003. Conversion of peripheral CD4+CD25- naive T cells to CD4 yltransferases and histone deacetylases have been shown to en- +CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med. 198: 1875–1886. by guest on September 29, 2021 hance the stability of Foxp3 expression and could be incorporated 9. Davidson, T. S., R. J. DiPaolo, J. Andersson, and E. M. Shevach. 2007. Cutting into in vitro induction protocols (37, 50, 51). edge: IL-2 is essential for TGF-beta-mediated induction of Foxp3+ T regulatory When a pooled population of nTregs and iTregs was used to treat cells. J. Immunol. 178: 4022–4026. 10. Trzonkowski, P., M. Bieniaszewska, J. Juscinska, A. Dobyszuk, A. Krzystyniak, two mice, iTreg populations emerged that composed a similar N. Marek, J. Mysliwska, and A. Hellmann. 2009. First-in-man clinical results of fraction of the Treg compartment but contained distinct TCR the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells. Clin. Immunol. 133: 22–26. repertoires. This result supports two conclusions. First, a limited 11. Hippen, K. L., S. C. Merkel, D. K. Schirm, C. Nelson, N. C. Tennis, J. L. Riley, and fixed pool of in vitro–derived iTregs contains a surprisingly C. H. June, J. S. Miller, J. E. Wagner, and B. R. Blazar. 2011. Generation and large number of clones with TCRs that can be maintained within large-scale expansion of human inducible regulatory T cells that suppress graft- versus-host disease. Am. J. Transplant. 11: 1148–1157. the iTreg niche. In fact, it has been shown that high TCR diversity 12. Fantini, M. C., C. Becker, I. Tubbe, A. Nikolaev, H. A. Lehr, P. Galle, and is important for the optimal function of Tregs in a model of ex- M. F. Neurath. 2006. Transforming growth factor beta induced FoxP3+ regula- perimental acute graft versus host disease (52). Second, the iTreg tory T cells suppress Th1 mediated experimental colitis. Gut 55: 671–680. 13. Weber, S. E., J. Harbertson, E. Godebu, G. A. Mros, R. C. Padrick, B. D. Carson, niche is restricted, as selection of iTregs is likely to be stochastic, S. F. Ziegler, and L. M. Bradley. 2006. Adaptive islet-specific regulatory CD4 and there was minimal overlap in the iTreg repertoires. This T cells control autoimmune diabetes and mediate the disappearance of pathogenic Th1 cells in vivo. J. Immunol. 176: 4730–4739. constraint is probably not due to the number of available Ags, 14. DiPaolo, R. J., C. Brinster, T. S. Davidson, J. Andersson, D. Glass, and given the complexity of the microbiome. Thus, other factors such E. M. Shevach. 2007. Autoantigen-specific TGFbeta-induced Foxp3+ regulatory as the number of tolerogenic APCs, local concentrations of TGF- T cells prevent autoimmunity by inhibiting dendritic cells from activating au- toreactive T cells. J. Immunol. 179: 4685–4693. b1 and IL-2, and signaling via the programmed death 1–pro- 15. Wing, K., Y. Onishi, P. Prieto-Martin, T. Yamaguchi, M. Miyara, Z. Fehervari, grammed death ligand pathway may determine the size of the T. Nomura, and S. Sakaguchi. 2008. CTLA-4 control over Foxp3+ regulatory iTreg population (53–57). In the absence of nTregs, the iTreg T cell function. Science 322: 271–275. 16. Rubtsov, Y. P., J. P. Rasmussen, E. Y. Chi, J. Fontenot, L. Castelli, X. Ye, compartment expands ∼5-fold indicating that nTregs, either di- P. Treuting, L. Siewe, A. Roers, W. R. Henderson, Jr., et al. 2008. Regulatory rectly or indirectly, also influence the size of the iTreg niche (6). T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28: 546–558. Manipulation of these factors may provide mechanisms to expand 17. Chen, M. L., M. J. Pittet, L. Gorelik, R. A. Flavell, R. Weissleder, H. von the iTreg compartment thereby decreasing the propensity for the Boehmer, and K. Khazaie. 2005. Regulatory T cells suppress tumor-specific CD8 formation of pathogenic ex-iTregs while improving therapeutic T cell cytotoxicity through TGF-beta signals in vivo. Proc. Natl. Acad. Sci. USA 102: 419–424. outcome. 18. Gondek, D. C., L. F. Lu, S. A. Quezada, S. Sakaguchi, and R. J. Noelle. 2005. In conclusion, recent work has established that iTregs and nTregs Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells are functionally nonredundant, based in part on differences in TCR involves a granzyme B-dependent, perforin-independent mechanism. J. Immu- nol. 174: 1783–1786. repertoire and presumably TCR specificity (7, 49, 58). We now 19. Ku¨hn, R., J. Lo¨hler, D. Rennick, K. Rajewsky, and W. Mu¨ller. 1993. Interleukin- identify the capacity for reciprocal compensation between the two 10-deficient mice develop chronic enterocolitis. Cell 75: 263–274. 20. Liu, B., S. L. Tonkonogy, and R. B. Sartor. 2011. Antigen-presenting cell pro- Treg subsets that relies on a mutual suppressive mechanism. Thus, duction of IL-10 inhibits T-helper 1 and 17 cell responses and suppresses colitis the answer to the recently posited question “Natural and adaptive in mice. Gastroenterology 141: 653–662. 5648 iTregs PROVIDE IL-10 BUT ARE UNSTABLE IN VIVO

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SUPPLEMENTAL FIGURE LEGENDS

Figure S1. Analysis of Treg, ex-iTreg and effector T cells isolated from treated mice. (A) Representative flow cytometry analysis (top panel; n=7, 5, 8, 8, from left to right) of colon lamina propria lymphocytes (LPL) from the mice in Figure 1 showing in vitro-derived iTreg cells (Thy1.1+ EGFP+) and nTreg cells (Thy1.1– EGFP+). Scatter plots (bottom panels) showing the frequency of colon LPL iTreg cells (left) and nTreg cells (right) contained within the TCRβ+ CD4+ gate. (B) Scatter plot depicting the number of iTreg cells (left) and nTreg cells (right) recovered from the colon LPL of mice from each treatment group. (C) Representative flow cytometry analysis of the colon LPL (left panels; n= 7, 5, 8, 8, from left to right) from mice treated with the indicated combinations of iTreg plus nTreg cells showing intracellular cytokine staining of cells stimulated ex vivo with PMA and ionomycin. Staining for IFN-γ and IL-17A is shown for cells within the TCRβ+ CD4+ gate. Scatter plots (right panels) showing the percentage of colon LPL TCRβ+ CD4+ T cells that are IFN-γ+, IL-17A+ and IFN-γ+ IL-17A+. (D) Representative flow cytometry from the spleen and MLN of mice with colitis that were treated with Thy1.2+ nTreg cells plus Thy1.2+ iTreg cells and were coinjected with 15,000 Thy1.1+ EGFP– T cells isolated from iTreg cells that had lost Foxp3 expression in culture (n=4, 2 experiments). (E) Scatter plots depicting the frequency of TCRβ+ CD4+ Thy1.1+ EGFP– cells found in the spleen and MLN of mice shown in D compared to the frequency of TCRβ+ CD4+ Thy1.1+ EGFP– ex- iTreg cells found in mice successfully treated with WT iTreg cells plus WT nTreg cells from Figure 1. (F) Scatter plot depicting the frequency of TCRβ+ CD4+ Thy1.1+ EGFP– ex-iTreg cells found in the colon LPL (left) and small intestine (SI) LPL (right) of treated mice from Figure 1. (G) qPCR analysis of IL-10 expression in flow cytometry-sorted ex-iTreg (CD4+ Thy1.1+ EGFP–), iTreg (CD4+ Thy1.1+ EGFP+), and nTreg cells (CD4+ Thy1.1– EGFP+). The data is taken from a total of 6 mice done in 3 separate experiments. Each point is the mean of triplicate wells. IL-10 expression is shown as fold change over GAPDH expression. (H) Representative flow cytometry analysis (left panels; n=7, 5, 8, 8, from left to right) showing intracellular cytokine staining of colon LPL ex-iTreg cells stimulated ex-vivo with PMA and ionomycin and stained for IFN-γ and IL-17A gate. Scatter plot (right panels) showing the percentage of colon LPL ex-iTreg cells that are IFN-γ+, IL-17A+ and IFN-γ+ IL-17A+. For all representative FACS plots, numbers indicate the mean percent of cells in the quadrant. For all scatter plots, each colored dot represents an individual mouse and the horizontal bars represent mean values. *P<0.05; **P<0.005

Figure S2. Comprehensive spectratype analysis and CDR3 sequencing evaluation. (A) TCR Vβ spectratypes of iTreg and ex-iTreg cells using 9 different V region 5’ PCR primers. (B) A comparison of the unique and overlapping iTreg and ex-iTreg cell TCRβ CDR3 sequences recovered by non- optical integrated semiconductor sequencing with the 316 chip and the same samples sequenced using a either the 314 chip or subcloned and sequenced by standard methods. The number of sequences in each category and the MHI are listed.

Table S1. Differentially regulated probesets. Probe sets from Figure 4C (n = 3250) that revealed a twofold difference (|log2 ratio| > 1.0) and rank product false discovery rate (FDR) <10%, in at least one cell type, relative to Tconv cells. Each row contains the probeset ID, gene symbol, Fold Change: ex-iTreg vs Tconv, FDR: ex-iTreg vs Tconv, Fold Change: iTreg vs Tconv, FDR: iTreg vs Tconv, Fold Change: nTreg vs Tconv, and FDR: nTreg vs Tconv. The mean fold change was calculated from 2 independent arrays for each cell type and was scored P<0.05 with an FDR <10% by the non-parametric rank product test. The table is sorted based on the column Fold Change: iTreg vs Tconv (smallest to largest).

Table S2. Distribution and frequency of iTreg and ex-Treg cell Vβ8.2 CDR3 amino acid sequences. Table shows the number of times each unique amino acid sequence was recovered from the iTreg and ex-iTreg cells isolated from Mouse 4 and Mouse 6 (Figure 5). Sequences recovered with a total frequency of 10 or more times using the Ion Torrent 316 Chip, 314 Chip, and subcloning methods are included for each population where applicable. Table is sorted by the total frequency (total number of times each unique sequence was recovered).

2 SUPPLEMENTAL FIGURE 1

3 SUPPLEMENTAL FIGURE 2

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Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0#0$$"% 0#0$0"% 0#"$ 0 %# % 0##%' 0#"% 0"'"% 0$%"$ 0 0" 0"% 0" "% 0#$%$0 % 0##% 0#$# % 0$'"% 0# &"% 0#$# 0#0$#"% 00$" 0# (# % 0#%# % 0 %0 0$0"$ 0$ 0#&#"% 0" 0# % 0$#"% 0#$0% 0"" 0# 0")%# % 0## % 0# 0$%# % 0$#$0"% 00 '%# % 0#&"% 0!"" 0# 0" 0 % 0$0' 0#$#$" 0($(!! 0$0%# % 0" 0"% 0## 0#$"# % 0#$ 0%# " 0%"$ 0#0 # 0"$$" 0#00# 0#"% 0#$ 0%"% 0 0$00 0 $$ % 0$#( 00%# % 0 0# 0#$0"% 0#"% 0" 0#&00"% 0$$0 00%!"% 0"%%0 0" 0#0# 0#$$% 0 $# % 0# %# % 0#% 0 % 0# 0#$%# % 0#0&

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0#'% 0#0" 00 0$#$#"$ 0% 00%# % 0!"0 0!"" ' 0"0"% 0 0$"% 0#0$$$!0"% 0# $# % 0#&0% 0$"% 0#$ "% 00$"% 00# 0#0 0%"% 0")0 % 0#0"% 0##$ 0$%% 0#00" 0#00"% 0#00" 0( 0%# % 0#0"% 00% 0" 0"% 0 (# % 0 0#0$$# 0#0$$!0"% 0#&"% 0"""$ 0#$"% 0" 0#"% 0#%# % 0#$"$ 0%%"$ 0")$ 0# 0#&"$ 0#$"# % 0#$ 0#"0"% 0#0"% 0#$0" 0%%0 0#"% 0#$ "% 0#$#&0"% 00%"% 0""% 00$$$!0"% 0#$ "$ 0#0$$$!0% 0#$#% 0# 0'# % 0 0 0" 0#"% 00" 0$" 0###$""% 0$# % 0$"% 0" % 0#0% 0 0"% 0#00" 0"#$0"% 0#0$0"% 0"0"$ 0 !"$ 0$%0"$ 0 0"%

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0 ' 0"$ 0#$"# % 0#$ 0 0# 0#$' % 0" 0%# % 0"$#&"% 0$# 00 0#0%%# % 0!"" 0 0% 0#$0" 0#"% 0"% 0# " 0#&%0 0(%0"% 0 "% 0## 0#&0"% 0# % 0# 0"% 0#0% 0 #"% 0#0$0"% 0 * 0% # 0#"$ 000#0"% 0& "# % 0#$0# 0 0"% 0#0&0% 0#0"% 0"'"% 0$%0" 00$$# 0%" 0" 0#0$$$ 0"% 0# %# % 0##$%0 0#0#"% 0 $0$#'% 0$%# 0##&"% 0 0# 00 " 0# 0""% 00%%% 0"% 0$0" 0%%# % 0#&0!%# % 0#00"$ 0$ % 0 0" 0#$0%# % 0%0" 0&0%# % 0!$" 0"% 0"( % 0#!"% 0 0# 0#('"% 0#0#"% 0#0%0 0% 0$%# % 0#0$0%# % 0($(!! 0#0"% 0#0 # 0&"% 0 %# % 0 %

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0# # 0'$% 0#$ 0"!%# % 0# 0$$# " 0#'"% 0#0$$$$"% 0$" 0$ " 0$0 % 0%($ 0#& #0"% 0#0$$$!0"$ 0$"# % 0'%"% 0#%0"% 0# #0"% 0#0 $% 0#$#!0"% 0"$$0"% 0#0#$'"% 0#% 0# #%# % 0$ " 0#0&00"% 00'% 00 0$%0 00 0#00" 0#0$ %# % 0#0"% 0 $ " 0# 0#0"% 0!"00 0%0"% 0"%# % 00$!%# % 0#$# % 0#% 0 0%%# % 0#0'&"% 0# 0 00" 0' 00#0"% 0" # 00#&0"% 0$"("% 0"%"% 00# 0 0" 0# $$$!0"% 0#0" 0' " 00% " 0$#&"% 0#' 0##"$ 0 "" 0"% 00 # 0#0"$ 0%"% 0#0$ 0" "% 0#!"% 0!"" 0#%0"% 0#0%0"% 0 0#0& ! 0#00# 0" 0"% 00 "%

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0% 0'%0 00'0"% 0 0#0$$$!0"% 0#$0 0# 0#0$% 0$#$'"%* !"" 0"$ # 0#0')(% 0#"(# % 00 % 0"&0"% 0 0 " 0$0" 0#00# % 0# " 0 '%0 0#$%# % 00$ " 0$#0" 0#0&0"% 0$#( * 0$$#"% 0# 0!"" * 0!"$($*! 0$0$# 0#&"% 0#0$$$ 0$#&0" 0#0$$!0"% 0## 0#0"% 0#"&% 00(# 0"% % 0#$0"% 0 %# % 0$0#%# % 0"0$$0 0##*% 0 0) 0 "% $ 0 (0 0 #!$ 0# (0 0#0"% 0!"$0 0#%$(!! 0#0$$ % 0 "$ 0!"" 0%0%0 00%0 0#0$$&!0"% 0#%# % 0"0" 0#0"% 00$ " 0##$"% 0" 0" 0##$"% 0"#$00"% 0 %# % 0&0"% 0" 0" 0$$# 0##'% 0#0$00" 0 " 0 ! 00 (0 " 0" 0#0$ 0" 0#0$$$"%

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0$#$"$0" 0% " 00( % 0!"0 0$# 0%# 0#$"% 0#$0"% 0#0 0"%0" 0#0 % 0#%' % 0# % 0%%0 00 # % 00 0#$$"% 0"% 0%%# % 0#$$%0 0# % 0##&0"% 0!0"% 0#$ 0# "% 0"%% 0' 0"$00"% 00$)"$ 0#0 % 0#0$$$0"% 0#000 0$#&"$0" 0 0 " 00$#&%# % 000"% 00#$%# % #0$$$!0"% 0%# 0#'%# % 0## " 0 %0 0#$ #"% 0#$# % 0#0$$ 0#0"""% 0" 0 0#$# % 0"%% % 0!%0 0#)#0" 0#0( 0$"% 0#0'%0 0$# % 0 " 0%" 00%(# % 0### 00 % 0"% " 00$ $"% 0## 0#0$$$! "% 0#0$# % 0$#&"% 00 %# % 0 " 00$ 0%0 0%# % 0& " " 0#0( % 0 %# % 0$# " 0#' 0#0# 0#&# 0" " 0#0$$0" 0#$0"

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0 %0 0 ! 0% " 0 0 $ 0%%" 0$#( 00"% 0#"% 0%"$ !"" 0#$0 % 0$#&$%0 0$%0" 0#0&"% 00"% 00%0" 0# ! % 0#$ % 0#% 0)$"$!0"% 0#$$$!% 0$%# % 0$$% 0% 0#&% 0$"% 0#$"% 0#&0" 0%# % 0"%$ 0##% 0#% 0 "% 0 # 0#% 0*"% 0 0" 00% 0$# "$ 0#$ " 0#0% % 0#0 0"'$0 0#!% 0#0% 0#&"% 0#"$ 0# "$ 0 0#00" 000" 00"% 0$"$ 000"% 0"0%$0$ 0#%0#"% 0#0$$$!00 0#& % 0#0"$ 0 "% 0#0&00 " 0## 0"$0 % 0# # 0#%0 0#"#% 0#0 #0"% 0"#( 0$0" 00% "0 00% " 0#0$$%!0"% 0" 00"% 00 0" 00%#0"% 0#$"$ 0#'0 " 0$!""

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 0% " 0$0 " 0# 00#0 " 0#0$"% 0#&0" 0#'"% 0#0$$$!00 0#0$$% 0#&% 0#$" " 0##*%# % 00%# % 0#0$"$!0"% 0#0$0"% 00 # 00 0"% 0 #0"% 0#0#$#% #0$$$!0"% 0#!"% 0$# " 0$%# % 00"% 0#$00% 0 $0" 0#000$# % 0#00 % 00"# % 0 # 0#0"% 0*##% 0#% 0& 0"0") 0%%"$ 0%# 0%%($(# 0!"$ 0" $0"% 00$ % 0#%0 0#0$($!0"% 00"% 0% 00"% 0#% 0#0$ $!0"% 0#'$#$0! 00 " 0 0#0"% 0" 0' 0#0&"% 0 0$# % 0#0$$$!0"% 0#0$$$!"% 0#0%")! 00 % 0#$#% 0$#"%* 00 0$#$"$ 00'%# % 0 " 0% 00% " 0#$ 0%# 0"% 0"%# 0 %0 0#%00 0$00 % 0%0 0"$ # 0 "% 0"& 0### 0#0

Mouse 4 ex- Mouse 6 ex- Mouse 4 iTreg Mouse 4 iTreg Mouse 4 iTreg Mouse 4 ex- iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 iTreg Mouse 6 ex- iTreg Total Amino Acid Sequence 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning 316 Chip 314 Chip Subcloning iTreg 316 Chip Subcloning Frequency 00% 0 0 % 00' 0#$ 0#0$%"% 0#0( " 0#0$ 0$%00"$ 0"0 0#$#$0 0#0""% 0#000" 00"$ 0#0$0"% 00)0 0#0$# 0#0"% 0 '0" 0#0$0" 0#$% 0# 0"% 0$"% 0 0" 0#$ % 0 (0"% 00$% 0"'%# % 0 00"% 0 0" 0#00"% 0"%" 0%($ 0#% % 00## 0$#&0" 0#0"% 0#0$ % 000"% 0#""% 0$# 0##%* 0%# % 0"% 0# "$ 0###% 0"%"$ 0"&$%0 0#$ 0 0) "% 0" & 0$%#0 0$%# 0$"% 000"% 0"&" 0$"$ 00#&0"% 0$#% 0$#'"% 0 % 0!"#" 0"%# % 0#0"% 0# 00% "% 0#$ 0*$0 0#0% 0#$% "$ 0#0 0%0"$ 0"0"% 0# 0#&# " 0$&"$0" 00$ 0#0$ # % 0##&00"% 0%