E- regulatory network links TCR signaling to effector Treg cell differentiation

Xiaojuan Hana,b, Huarong Huanga,b, Ping Gaoa,b, Qi Zhanga, Xinyuan Liua,b, Baoqian Jiaa, Warren Stroberc, Baidong Houd, Xuyu Zhoua,e, George Fu Gaoa,e,f,g, and Fuping Zhanga,e,1

aChinese Academy of Sciences (CAS) Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, China; bCollege of Life Science, University of Chinese Academy of Sciences, 100049 Beijing, China; cMucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892; dCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, China; eSavaid Medical School, University of Chinese Academy of Sciences, 101408 Beijing, China; fNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206 Beijing, China; and gResearch Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, 100101 Beijing, China

Edited by Ananda W. Goldrath, University of California San Diego, La Jolla, CA, and accepted by Editorial Board Member Philippa Marrack January 17, 2019 (received for review January 11, 2018)

T cell antigen receptor (TCR) signaling is essential for the differenti- erance in the steady state and that restrain immune responses ation and maintenance of effector regulatory T (Treg) cells. However, directed against commensal bacteria, environmental antigens, the contribution of individual TCR-dependent in Treg cells to and pathogens, also require further elucidation. the maintenance of immunotolerance remains largely unknown. Here One important way in which effector Treg cell differentiation we demonstrate that Treg cells lacking E protein undergo further may be regulated is via the activity of E , a family of differentiation into effector cells that exhibit high expression of transcriptional activators or repressors that bind to E-box sites at effector Treg signature genes, including IRF4, ICOS, CD103, KLRG-1, transcriptional sites and thus determine expression (13). γ Previous studies have shown that down-regulation of E-protein and ROR t. E protein-deficient Treg cells displayed increased stability + and enhanced suppressive capacity. Transcriptome and ChIP-seq anal- activity by TCR signaling accompanies the development of Foxp3 yses revealed that E protein directly regulates a large proportion of regulatory T cells (14, 15), whereas increased E-protein activity the genes that are specific to effector Treg cell activation, and impor- accompanies Treg cell dysfunction (16). These observations sug-

tantly, most of the up-regulated genes in E protein-deficient Treg cells gested that recognition of self-antigen in matured Treg cells may INFLAMMATION IMMUNOLOGY AND are also TCR dependent; this indicates that E proteins comprise a further down-regulate E-protein activity, thus driving Treg cells critical gene regulatory network that links TCR signaling to the control to differentiate toward particular specificities that may potentiate of effector Treg cell differentiation and function. their suppressive capacity during immunological challenges. Never- theless, whether E protein regulates matured Treg cell activation E protein | effector Treg cell | TCR signaling | gene regulatory network | or whether E protein directly regulates certain activated Treg differentiation and function signature gene expression has never been demonstrated. To directly address the requirement of E-protein activity in the regulation of Treg cell homeostasis, we studied Treg cells in t is well established that regulatory T cells (Treg cells) play a fl/fl fl/fl E2A HEB Foxp3-Cre-GFP mice with E-protein deletion spe- pivotal role in maintaining immune tolerance and thus in I cific to Treg cells. Notably, we found that deficiency of E protein preventing autoimmunity and chronic inflammation (1). These in differentiated Treg cells results in up-regulated expression of cells can be subdivided into different subsets with somewhat unique characteristics. One type of subset classification is based on site of development and consists of a natural Treg subset that Significance develops in the thymus (called nTreg or tTreg cells) and a pe- ripheral Treg cell subset that develops in the periphery (called Effector Treg cells comprise the subset of the Treg cell pop- iTreg or pTreg cells) (2). Another type of subset classification is ulation that exhibits enhanced regulatory function. Whereas based on function. This classification recognizes a “central” Treg the induction and maintenance of this subset are known to + population that is equivalent to naive CD4 T cells with respect depend on TCR signaling, the underlying molecular mecha- to various markers and circulatory patterns and that differs from nisms downstream of such signaling and the contributions of one or more “effector” Treg cell populations that exhibit en- individual TCR-dependent genes to effector Treg cell genera- hanced regulatory function and migration through nonlymphoid tion are still poorly understood. In the studies described here tissues (3–5). Such effector Treg cells make up a minor fraction differentiated Treg cells in which E-protein (E2A/HEB) expression of Treg cells in the circulation and secondary lymphoid or- has been deleted were utilized to demonstrate that E proteins gans and are referred to as “activated” Treg cells in some are transcriptional suppressors of a large number of genes as- studies because they share phenotypic features with activated sociated with effector Treg cell differentiation, localization, conventional T cells. As such, they are variously defined as function, and proliferation. Thus, this finding indicates that + + + + CD62LlowCCR7lowCD44hiKLRG1 cells, ICOS IRF4 CD103 continuous TCR signals modulating E-protein activity is a major cells, or CD45RAlowCD25hi cells, depending on the study. mechanism underlying Treg cell acquisition of effector functions. Importantly, Treg cells comprising the effector subpopulation defined above are thought to have encountered antigens and Author contributions: F.Z. designed research; X.H., H.H., P.G., Q.Z., X.L., and B.J. have undergone T cell antigen receptor (TCR) stimulation more performed research; B.H., X.Z., and G.F.G. contributed new reagents/analytic tools; X.H., B.H., X.Z., and G.F.G. analyzed data;andX.H.,W.S.,andF.Z.wrotethepaper. recently than central Treg cells (6–9). This coincides with the fact that TCR signaling in differentiated Treg cells is essential to The authors declare no conflict of interest. Treg cell homeostasis, suppressor function, and signature gene This article is a PNAS Direct Submission. A.W.G. is a guest editor invited by the Editorial expression, especially to those signature genes that produce Treg Board. cell effector molecules (10–12). It should be noted, however, that Published under the PNAS license. the underlying molecular mechanisms downstream of TCR sig- 1To whom correspondence should be addressed. Email: [email protected]. naling that account for the above Treg cell characteristics are still This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. poorly understood. In addition, the contributions of individual 1073/pnas.1800494116/-/DCSupplemental. TCR-dependent genes in Treg cells to maintain immunotol-

www.pnas.org/cgi/doi/10.1073/pnas.1800494116 PNAS Latest Articles | 1of10 Downloaded by guest on September 25, 2021 various effector Treg cell markers such as CD103, ICOS, IRF4, Since Helios is a marker of nTreg cells (19), this suggests that E KLRG1, RORγt, as well as enhanced Treg cell stability and protein regulates both nTreg cells as well as iTreg cells. suppressive capacity. Further study demonstrated that E-protein Collectively, these results indicate that the E-protein level in activity was essential for Treg cells to maintain the expression of a mature Treg cells plays a substantial role in regulating Treg cell large proportion of genes found to be expressed almost exclusively homeostasis particularly at nonlymphoid organs exposed to TCR in effector Treg cells. In addition, gene expression profile analysis signaling by ambient antigens. indicated that a substantial amount of effector Treg signature genes regulated by E protein are also regulated by TCR. E proteins reg- E Proteins Regulate Treg Cell Homeostasis in a Cell-Intrinsic Manner. ulate effector Treg cells by directly binding to a large number of To determine if the effects of E protein on Treg cell homeostasis genes that associated with effector Treg cell differentiation, locali- are cell-intrinsic, we generated mixed bone marrow (BM) chi- + zation, function, and proliferation. Thus, our findings demonstrated meric mice by mixing equal numbers of BM cells from CD45.1 + + that downstream of TCR signaling, E-protein activity plays an es- CD45.2 WT Foxp3Cre mice and CD45.1 E2Afl/flHEBfl/fl sential role in the regulation of Treg cell activation. Foxp3Cre mice and then transferred the cell mixtures into lethally + irradiated CD45.2 mice. Analyses of the number of Treg cells Results + + + from WT (CD45.1 CD45.2 ) or E protein-deficient (CD45.1 ) Treg Cell Homeostasis Is Regulated by Specific Deletion of E Proteins cells in lymphoid organs 2 mo after cell transfer showed that in Mature Treg Cells. To explore the specific role of E proteins in E protein-deficient Treg cells were significantly increased com- fl/fl fl/fl + + + + mature Treg cells, we crossed E2A HEB mice with mice that pared with WT Treg cells (Foxp3 CD45.1 /Foxp3 CD45.1 + have a bacterial artificial transgene encoding green CD45.2 ratio: 1.5) (Fig. 1B). In addition, in nonlymphoid or- fluorescent protein (GFP) and humanized Cre recombinase gans of these mice, an even more pronounced increase in + + (hCre) under the control of the Foxp3 promoter (Foxp3-GFP- E protein-deficient Treg cells was observed (Foxp3 CD45.1 / hCre mice) (17, 18). Thus, the resultant E2Afl/flHEBfl/flFoxp3- + + + fl/fl fl/fl Foxp3 CD45.1 CD45.2 ratio: 2.5) (Fig. 1B). To investigate GFP-hCre mice (designated hereafter as E2A HEB mice) E-protein effects on Treg cell homeostasis under inflammatory exhibited specific deletion of E2A and HEB in Treg cells but not + conditions, we immunized mixed bone marrow chimeric mice with in CD4 conventional T cells (SI Appendix, Fig. S1A). We did + + not observe changes in development of CD4 and CD8 T cells myelin oligodendrocyte glycoprotein (MOG) to induce experimental or the proportion of Treg cells in the thymus (SI Appendix, Fig. allergic encephalomyelitis (EAE) or administered dextran sodium S1 B and C). However, we did observe a small, but statistically sulfate (DSS) in drinking water to induce colitis. As expected, significant, increase in the proportion of Treg cells in the spleen, peripheral lymph nodes (pLN), and mesenteric lymph nodes (mLN) (SI Appendix, Fig. S1D), demonstrating a modest, but A SI-LP CLP lung liver SI-LP CLP lung liver integral role of E protein in the regulation of peripheral Treg cell 7.0 8.5 5.9 8.2 15 * 20 ** 20 *** 20 *** + WT homeostasis. The lack of increase in Foxp3 cells in the 10 15 15 15

Cells (%) 10 10 Cells (%)

Cells (%)

Cells (%) 10

+

+

+ f/f f/f + 5 5 thymus of E2A HEB mice was likely due to the fact that 10 5 5 5 4 12.6 16.3 10.8 14.1 + E2Afl/fl 10 3 0 Foxp3 0 Foxp3 0 Foxp3 Foxp3 0 Foxp3 cells in the thymus are newly developed Tregs in which a 10 fl/fl fl/fl fl/fl fl/fl fl/fl HEB WT 0 WT WT WT Foxp3 HEB HEB TCR-driven decline in E-protein levels determines the number 3 4 5 fl/fl fl/fl HEB fl/fl HEB fl/fl 0 10 10 10 of Tregs rather than Foxp3(Cre)-dependent E-protein deletion. CD4 E2A E2A E2A E2A To investigate this possibility, we took advantage of Rosa26-loxP- Gated Foxp3+ Cells spleen SI-LP CLP YFP B 80 *** 80 *** 80 ***

Cellls

Cellls spleen Cellls SI-LP CLP +

+ Stop-loxP-YFP (R26 ) mice, in which YFP is expressed only after 60 60 + 60

5 fl/fl fl/fl Cre 10 40 KO KO KO 40 40 Cre is expressed. Accordingly, we crossed E2A HEB Foxp3 4 10 20 20 20 YFP WT 70 WT 72 WT 3 62 10 0 0 0 with R26 mice, in which Foxp3-expressing cells that are 28 fl/fl fl/fl fl/fl 0 38 30

% of Foxp3

% of Foxp3 + 3 WT WT % of Foxp3 WT CD45.1 -10 HEB 3 3 4 5 HEB fl/fl HEB fl/fl E-protein gene deleted are labeled with YFP, so that YFP cells are -10 0 10 10 10 fl/fl CD45.2 E-protein knockout (KO) Tregs. As expected, we found that most E2A E2A E2A fl/fl fl/fl Cre YFP Gated Foxp3+ Cells spleen dLN spinal cord Gated CLP CLP of the Tregs in thymus of E2A HEB Foxp3 R26 and WT CD100 *** 80 80 *** Foxp3+ Cells 80 *** 80 ***

Cellls spleen dLN spinal cord Cellls 60 60 Cellls

+ 60

Cellls Cre YFP + 5 + 60 + 10 5 KO Foxp3 R26 mice were YFP-negative (SI Appendix,Fig.S1E), 10 40 40 40 KO KO 4 WT KO 40 10 4 10 WT 20 20 61 WT WT 20 3 20 58 64 10 39 3 55 indicating that at this site TCR-driven decreases in E protein are 10 0 0 0 0 0 0 45 42 36 fl/fl 3 fl/fl fl/fl fl/fl -10

CD45.1 3 3 3 4 5 -10 WT WT WT -10 0 10 10 10 WT

% of Foxp3

CD45.1 3 3 4 5 % of Foxp3

% of Foxp3 largely responsible for Treg development rather than Foxp3 (Cre)- % of Foxp3 -10 0 10 10 10 HEB fl/fl HEB fl/fl HEB fl/fl HEB fl/fl CD45.2 CD45.2 dependent E-protein gene deletion. In contrast, most of the Treg E2A E2A E2A E2A cells in the periphery are YFP-positive, indicating that the cells have Fig. 1. E protein regulates Treg cell homeostasis in a cell-intrinsic manner. undergone E-protein deletion as a result of Foxp3 (Cre) expression. f/f f/f (A) Representative flow cytometric analysis (Left) and the percentages Thus, the lack of increase of Tregs in the thymus of E2A HEB (Right) of Foxp3+ cells within CD4+ cells in indicated organs of WTFoxp3Cre mice is probably due to the fact that the Tregs in thymus are subject and E2Afl/flHEBfl/flFoxp3Cre mice. (B–D) Mixed BM cells from WTFoxp3Cre mice + + + to a lesser degree of Foxp3-Cre mediated E-protein deletion than (CD45.1 CD45.2 cells) and E2Afl/flHEBfl/flFoxp3Cre mice (CD45.1 cells) were + cells in the periphery. transferred into lethally irradiated recipient (CD45.2 ) mice and were analyzed In related studies we found that the GI lamina propria, a 2 mo after reconstitution. (B)DataontheLeft show representative flow mucosal lymphoid site that is exposed to an abundance of foreign cytometry analysis of WT and KO Treg cells in chimeric mice after gating on Foxp3+ cells and data on the Right show frequencies of WT (CD45.1+CD45.2+) antigens and that requires Treg cells for maintenance of intestine + + + homeostasis, exhibited a dramatic increase in Treg cell frequency and KO (CD45.1 ) cells among the reconstituted Tregs (CD4 Foxp3 ) in indicated f/f f/f organs. (C) Two months after reconstitution, EAE was induced in the chimeric in E2A HEB mice and this increase was substantially greater + than the increase in spleen and lymph nodes (Fig. 1A). Similarly, mice; 24 d after induction, Foxp3 cells were analyzed by flow cytometry; data on the Left show representative flow cytometry analysis of WT and KO Treg cells we found that Treg cell increases were also greater in liver and + in chimeric mice after gating on Foxp3 cells and data on the Right show the lung than in lymphoid organs (Fig. 1A). Recognizing that Treg + + + β frequencies of WT (CD45.1 CD45.2 ) and KO (CD45.1 ) cells among the recon- cell development in the lamina propria is highly TGF- de- stituted Tregs (CD4+Foxp3+) in indicated organs. (D) DSS-colitis was induced in pendent, we investigated the role of E protein on iTreg cell the chimeric mice; 10 d after induction Foxp3+ cells were analyzed by flow differentiation in vitro and found that upon stimulation with cytometry; representative flow cytometry analysis (Left) and frequencies (Right) TCR and TGF-β, cells lacking E protein gave rise to an increased + + + + of WT (CD45.1 CD45.2 )andKO(CD45.1) cells in reconstituted Tregs proportion of Foxp3 cells (SI Appendix, Fig. S1F). Finally, we + + – + + (CD4 Foxp3 ) in the CLP. Data in A D, Left are representative of at least observed an increase in the percentages of both Helios Foxp3 three independent experiments. Data in A–D, Right are pooled from at least − + fl/fl fl/fl cells and Helios Foxp3 cells in lamina propria of E2A HEB three independent experiments. Each symbol represents data from one mice compared with that of WT mice (SI Appendix, Fig. S1G). mouse. Graph shows mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1800494116 Han et al. Downloaded by guest on September 25, 2021 24 d after induction, we observed a markedly increased number of A B IL-2 IL-2+IL-12 IL-2+IFN-γ IL-2+IL-4 SI-LP CLP E protein-deficient Treg cells vs. WT Treg cells infiltrating the IL-2+IL-6 * * 30 20 spinal cord as well as in spleen and draining lymph nodes (dLN) WT 3.6 11.2 7.3 15.1 35.8 15 YFP 20

Cells (%) R26 Cells (%)

+ (Fig. 1C) of mice with EAE. A similar observation was obtained + 10 10 in colon lamina propria (CLP) of chimeric mice 10 d after DSS 250K 5 200K

in YFP in fl/fl YFP in

- E2A 3.3 6.3 3.9 6.8 25.4 - 0 0 150K administration (Fig. 1D). It should be noted that the frequency fl/fl fl/fl fl/fl HEB 100K WT WT YFP R26 50K fl/fl HEB HEB

Foxp3 fl/fl of Treg cells in KO mice without inflammation was comparable Foxp3 0

FSC 3 3 4 5 -10 0 10 10 10 E2A E2A to that of KO mice with EAE or colitis (compare Fig. 1B with Foxp3

Fig. 1 C and D). These studies thus indicated that the increased YFP + + spleen pLN mLN WT R26 spleen fl/fl fl/fl YFP percentage of Foxp3 cells among CD4 cells due to E-protein C 63.4 D Id2 Id3 R26 80 *** WT WT ** deletion is maintained in a cell-intrinsic manner under both R26YFP 11.3 10.1 9.3 50 60 R26YFP *** Cells (%) 1.1 40 *** + 0.9 1.0 steady-state and inflammatory conditions. Cells (%)

40 + 100 30 5 fl/fl 10 E2A 80 34.7 20 fl/fl 4 20 fl/fl 60 Id2 10

in YFP in HEB - 0 fl/fl

in YFP in

E-Protein Deletion Stabilizes Foxp3 Expression in Matured Treg Cells. 40 fl/fl Id3 3 - 10 YFP 10 4.0 3.3 3.9 R26 WT YFP 20 R26 0 GFP HEB While Treg cells are generally stable, under lymphopenic or in- cells 0 fl/fl Foxp3 0 0.8 1.1 0.9 3 3 4 5 -10 0 10 10 10 3 3 4 5 -10 0 10 10 10 Foxp3 pLN mLN flammatory conditions a fraction of these cells lose Foxp3 expres- GFP E2A YFP spleen

sion and undergo other phenotypic changes, such as acquisition IL-2 IL-2+IL-12 IL-2+IFN-γ IL-2+IL-4 IL-2+IL-6 of ability to secrete cytokines that mediate diverse effector func- E tions (18). To elucidate the effect of E proteins on Treg cell sta- WT 9.9 16.5 11.0 12.8 54.0 bility, we used cells from E2Afl/flHEBfl/flFoxp3CreR26YFP mice 250K

Cre YFP 200K and WT Foxp3 R26 mice as described above to compare fl/fl Id2 150K 26.2 26.0 26.4 26.2 72.3 + − Id3fl/fl 100K the effect of E-protein deletion on the generation of YFP Foxp3 50K − FSC 0 3 3 4 5 “ ” -10 0 10 10 10 (GFP ) exTreg cells. In initial studies, we compared the impact Foxp3 of inflammatory cytokines on the stability of highly purified E + protein-deficient vs. WT spleen YFP cells after culture of the Fig. 2. E-protein deletion stabilizes Foxp3 expression in matured Treg + Cre YFP cells in the presence of IL-2 plus various proinflammatory cyto- cells. (A) Highly purified YFP Treg cells from WT Foxp3 R26 and E2Afl/flHEBfl/flFoxp3CreR26YFP mice were activated and cultured in the pres- kines (IL-12, IFN-γ, IL-4, or IL-6). We found that even in the + ence of IL-2 plus indicated cytokine for 4 d; Foxp3 expression (among YFP presence of optimal amounts of IL-2, the stability of WT Treg cells −

cells) was then analyzed by flow cytometry. (B) The frequency of Foxp3 INFLAMMATION + was severely compromised compared with that of E protein-deficient in YFP cells (Right) in indicated organs of WT Foxp3CreR26YFP and IMMUNOLOGY AND Treg cells as indicated by the greater percentage of exTreg cells that E2Afl/flHEBfl/flFoxp3CreR26YFP mice. (C) Highly purified Treg (YFP+) cells from appeared in the WT cells (Fig. 2A). Intriguingly, the exTreg cells from WT Foxp3CreR26YFPFoxp3GFP or E2Afl/flHEBfl/flFoxp3CreR26YFPFoxp3GFPmicewere − − theWTmiceproducedmoreIFN-γ (SI Appendix,Fig.S2A)andIL- sorted by FACS and were transferred into Rag2 / -recipient mice. GFP expression + among YFP cells was analyzed by flow cytometry 4 wk after cell transfer (Left) 17A (SI Appendix,Fig.S2B) than exTreg cells from E protein- − − + deficient mice; this suggests that E proteins may promote exTreg and the frequency of GFP (Foxp3 )amongYFP cells is shown (Right). (D) Mixed BM chimeric mice were generated by transferring BM cells from cells to become pathogenic T helper cells during inflammations such Cre YFP fl/fl fl/fl Cre YFP + − WT Foxp3 R26 and Id2 Id3 Foxp3 R26 mice into lethally irradiated as EAE. In addition, we observed a lower percentage of YFP Foxp3 + + recipient mice. Flow cytometry analysis of Foxp3 and YFP expression in CD4 − + (exTreg) cells among YFP cells in the small intestine lamina cells (Left) and the frequency of Foxp3 cells among the YFP cells (Right)of fl/fl fl/fl Cre YFP propria and CLP of E2A HEB Foxp3 R26 mice than in WT Foxp3CreR26YFP and Id2fl/flId3fl/flFoxp3CreR26YFP donors in indicated organs in + WT Foxp3CreR26YFP mice (Fig. 2B and SI Appendix, Fig. S2E). the chimeric mice are shown. (E) Highly purified CD25hi CD4 Treg cells from However, in this case the difference, while significant, was less WTFoxp3Cre and Id2fl/flId3fl/flFoxp3Cre mice were activated and cultured in the than that observed in cultured cells. In contrast, there were no dif- presence of IL-2 plus indicated cytokine for 4 d; Foxp3 expression was analyzed by flow cytometry. (A, E,andB–D, Left) Data are representative of at least three ferences in the percentage of exTregs in the spleen and pLN cells – f/f f/f YFP independent experiments. (B D, Right) Data are pooled from at least three in- of E2A HEB R26 mice and WT mice perhaps because cells in dependent experiments. Each symbol represents data from one mouse. Graph these tissues are less subject to antigen stimulation than those in the shows mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001). mucosal tissues (SI Appendix,Fig.S2F). To further investigate the role of E protein on the stability of + − + Treg cells, we transferred highly purified YFP Treg cells from (Foxp3 ) cells among YFP cells was greatly increased in spleen, spleen and lymph nodes of E2Afl/flHEBfl/flFoxp3CreR26YFP pLN, and mLN cells derived from Id protein-deficient mice Cre YFP −/− or WTFoxp3 R26 mice into Rag2 recipients and assessed compared with cells derived from WT mice (Fig. 2D). In com- + + + + Foxp3 (GFP ) cell stability under lymphopenic conditions 4 wk panion studies we cultured highly purified CD25 CD4 Treg after cell transfer. Consistent with the findings above, fewer E cells from WTFoxp3Cre and Id2fl/flId3fl/flFoxp3Cre mice in the protein-deficient Treg cells lose Foxp3 expression than WT Treg presence of IL-2 and proinflammatory cytokines as described cells, indicating that the E protein- deficient Treg cells are more above, and found that the inflammatory cytokines compromised stable under lymphopenic conditions (Fig. 2C). Foxp3 expression in Id protein-deficient Treg cells to a greater To confirm the above findings concerning Treg cell stability, extent than in WT Tregs (Fig. 2E); in addition, these exTreg cells we determined the role of E-protein activity in maintaining Treg fl/fl fl/fl Cre fl/fl fl/fl Cre YFP from Id2 Id3 Foxp3 mice produced increased amounts cell stability in cells from Id2 Id3 Foxp3 R26 mice, i.e., of cytokines (IFN-γ, IL-4) compared with that of WT exTreg cells (SI mice in which E-protein effects are increased because they are Appendix,Fig.S2C and D). Collectively, these studies provide strong not blocked by Id protein. Recognizing that mice with deletion of evidence that lack of E-protein expression in E2Afl/flHEBfl/flFoxp3Cre Id2/Id3 in matured Treg cells exhibit spontaneous inflammation fl/fl fl/fl Cre that might have a secondary effect on Treg cell stability (16), we mice and increased E-protein activity in Id2 Id3 Foxp3 mice affects Treg stability in reciprocal ways, most likely via un- examined the effect of Id proteins on Treg cell stability under the + noninflammatory conditions in mixed bone marrow chimeric derlying effects on survival or proliferation of Foxp3 cells. mice. Bone marrow cells from WT Foxp3CreR26YFP and Id2fl/fl Id3fl/flFoxp3CreR26YFP mice were mixed at 1:1 ratio and trans- E Protein-Deficient Treg Cells Exhibit Enhanced Immunosuppressive ferred into lethally irradiated recipient mice; subsequently (i.e., Function both in Vitro and in Vivo. Previous studies have demon- at 8 wk after cell transfer when the recipient mice were robust strated that Treg cells have a prominent capacity to limit in- and thus apparently free of inflammation), we analyzed the flammation during experimental autoimmune diseases such as fraction of exTreg cells. We found that the percentage of exTreg EAE or cell-transfer colitis. Based on the above findings, we

Han et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 25, 2021 reasoned that E-protein deletion specific to Treg cells would The RNA-seq study showed that 801 genes were up- or down- lead to enhanced Treg cell function. To investigate this possi- regulated at least twofold in E protein-deficient Treg cells rela- bility, we induced EAE in WT and E2Afl/flHEBfl/flFoxp3Cre mice tive to the genes in WT Treg cells (Fig. 4A, Top and Fig. 4B, and found that E2Afl/flHEBfl/flFoxp3Cre mice were more resistant Venn A). These genes were termed “E protein-dependent genes,” to EAE, as evidenced by lower disease scores, not only during whereas 1,611 genes changed expression at least twofold in E the initial progressive phase of disease, but also during the protein-deficient Treg cells subjected to TCR stimulation relative remitting-relapsing phase of disease occurring after peak disease to genes in WT Treg cells also subjected to TCR stimulation (Fig. 3A). This finding could be attributed to differences in Treg (Fig. 4A, Bottom and Fig. 4B, Venn B). Meanwhile, 6,606 gene + cell migration since significantly more Foxp3 cells were pre- expressions were changed in WT Treg cells after TCR stimula- sent in the spinal cords of E2Afl/flHEBfl/flFoxp3Cre mice than tion (WT vs. WT-48 h), which were termed “TCR-dependent those of WT Foxp3Cre mice (Fig. 3B). Of interest, there were more genes” (Fig. 4B, Venn C). In further analysis, we found that a MOG-specific Treg cells in the E2Afl/flHEBfl/flFoxp3Cre mice than similar proportion of genes regulated by E protein [WT vs. KO that of WT Foxp3Cre mice (Fig. 3C); accordingly, MOG-specific cells (Fig. 4B, Venn A) and WT-TCR 48-h vs. KO-TCR 48-h + CD4 T cells were greatly decreased in E2Af/fHEBf/f mice com- cells (Fig. 4B, Venn B)] were also TCR dependent (Fig. 4B, Right pared with that of WT mice (Fig. 3C), suggesting that E protein- Top, Venn A overlap C, 433/801 genes = 54% and Fig. 4B, Right deficient Treg cells are more responsive to antigen stimulation, and Bottom, Venn B overlap C, 887/1,611 genes = 55%, respectively). antigen-specific E protein-deficient Treg cells exhibit enhanced This similarity in the proportion of genes regulated by E protein suppressive capacity. E2Afl/flHEBfl/flFoxp3Cre mice spinal cords alone and by E protein plus TCR stimulation suggests that the harbored significantly fewer IL-17– and IFN-γ–producing cells effects of E protein on gene regulation are parallel to or slightly (Fig. 3 D and E), which also suggests that E2Afl/flHEBfl/flFoxp3Cre amplified by TCR stimulation. In addition, comparison of E protein mice were more resistant to EAE. and TCR-dependent genes showed that most of the genes up- As E2Afl/flHEBfl/flFoxp3Cre mice have an increased percentage regulated in E protein-deficient Treg cells were also TCR- of Treg cells in the spinal cords, the increased suppressive dependent genes (Fig. 4C). This suggests that a large proportion function of Treg cells in E2Afl/flHEBfl/flFoxp3Cre mice with EAE of Treg cell-specific gene expression downstream of TCR signaling, may simply be due to the presence of an increased Treg cell pool is dependent on E protein and therefore, the E-protein regulatory rather than because the Treg cells are more suppressive. To network ties TCR signaling to matured Treg cell homeostasis. clarify this issue, we performed in vitro suppressive assays in Examination of the specific genes up-regulated in KO vs. WT which titrated Treg cells from WT or E2Afl/flHEBfl/flFoxp3Cre (TCR 48-h) disclosed several genes encoding transcription fac- were cocultured with an equal number of carboxyfluorescein tors shown previously to be involved in the differentiation and + diacetate succinimidyl ester (CFSE)-labeled CD4 T cells and function of effector Treg cells such as Nfat5, Ikzf3, and Irf4,as + the CD4 T cell proliferation was assessed. We found that E well as genes encoding factors with a similar potential func- protein-deficient Treg cells had an increased capacity to suppress tion, Itgae, Icos, Cd44, and Lag3 (Fig. 4D). In addition, genes + conventional CD4 T cell proliferation compared with WT Treg encoding secreted factors associated with Treg cell suppressive function, including Granzyme B, Il10, and Fgl2 were also signif- cells. Thus, Treg cells with E-protein deletion do exhibit in- icantly increased in KO vs. WT (TCR 48-h) as were genes in- creased intrinsic suppressive activity (Fig. 3F). volved in the TGF-β signaling pathways, such as Tgfbr3 and To further investigate the suppressive function of E protein- Smad3 (Fig. 4D). In a related vein, the expression of RORγt was deficient Treg cells in vivo, we turned to the well-characterized increased in E protein-deficient Treg cells following TCR stim- cell transfer-induced colitis model. Here, we compared colitis −/− hi ulation. This is significant because an effector Treg cell lineage developing in Rag2 mice following transfer of naïve CD45RB + + + subset that expresses both Foxp3 and RORγt has been shown CD4 T cells together with Treg cells from WT Foxp3CreR26YFP fl/fl fl/fl Cre YFP to have enhanced suppressive capacity and increased ability to or E2A HEB Foxp3 R26 mice. Recipient mice cotransferred regulate Th17-mediated immune responses (20–22). Finally, E protein- deficient Treg cells exhibited less colitis than mice several chemokine and chemokine receptor or -encoding cotransferred WT Treg cells as indicated by their increased genes were up-regulated in the E protein-deficient cells sub- weight gain (Fig. 3G), decreased cell infiltration in the colon jected to TCR stimulation, suggesting that such Treg cells have (SI Appendix,Fig.S2G), and by the dramatic decrease of both + + an increased ability to migrate to inflammatory sites (Fig. 4D). total CD4 T cells and total IL-17– and IFN-γ–producing CD4 – The up-regulation of specific genes in E protein-deficient cells Tcells(Fig.3H J and SI Appendix,Fig.S2H). Importantly, subjected to TCR stimulation noted above was accompanied by although the Treg cell percentages were increased in spleen, mLN, the up-regulation of a large number of genes involved in more and colon of mice that were transferred E protein-deficient Tregs + general cell functions. This includes genes encoding ribosome (Fig. 3 H and K) due to decreased total CD4 T cell numbers (Fig. RNA-processing genes, indicating that protein translation was 3H) compared with mice transferred WT Treg cells, the absolute greatly up-regulated by E-protein deletion in these cells; in ad- numbers of Treg cells in spleen, mLN, and colon were comparable dition, consistent with (GO) analysis, genes corre- in the two groups (Fig. 3K). Thus, these data further support the lating with cell proliferation, cell cycling, and cell apoptosis, such view that decreased E-protein levels in Treg cells result in Treg as cdk6, cdkn1a,andBcl2 (Fig. 4D), were also greatly up-regulated cells with increased regulatory function. in E protein-deficient Treg cells. Thus, loss of E protein in dif- ferentiated Treg cells affect the expression of a large proportion of E Protein Globally Regulates Effector Treg Cell Signature Gene genes underlying the multifaceted function of effector Treg cells. Expression. The above findings showing that E protein-deficient Interestingly, one set of genes not up-regulated by TCR stim- Treg cells exhibit increased homeostasis, enhanced suppressive ulation in E protein-deleted cells are genes associated with function and increased stability prompted us to hypothesize that IL-2 and IL-7 signaling, i.e., genes that have been shown to be E protein might control a distinct, lineage-specific, transcrip- critically involved in initial Treg cell induction and maintenance tional program in Treg cells. To investigate this possibility, we (23). In particular, neither CD25, CD122 (IL-2Rα and IL-2Rβ used high-throughput RNA-sequencing (RNA-seq) analysis to chains, respectively), CD127 (IL-7Rα chain) expression was compare the global gene-expression patterns of E protein- significantly altered upon E-protein depletion in matured Treg deficient and -sufficient Treg cells. Recognizing that Treg cells cells (SI Appendix, Fig. S4A) nor was IL-2 signaling as evidenced are constantly being stimulated by self-antigens and that E pro- by phosphorylation of STAT5 (SI Appendix, Fig. S3A). In addi- teins are regulated by TCR stimulation, we compared E protein- tion, c-Rel, a pioneering transcription factor that initiates Treg deficient and -sufficient Treg cells without stimulation [KO vs. cell development was also not modulated by E-protein depletion WT (no TCR hereafter)] or stimulated with anti-CD3/CD28 for (SI Appendix, Fig. S3B). It is thus apparent that while decreased 48 h [KO vs. WT (TCR 48 h hereafter)] in the studies. E-protein activity is a key feature of the cell signaling pathways

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1800494116 Han et al. Downloaded by guest on September 25, 2021 WT Gated spleen A BCspinal cord Foxp3+ Cells E2Afl/flHEBfl/fl * 4 * 50 Cells 6 *

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spinal cord 0 IFN-γ fl/fl fl/fl +

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IFNγ E2A E2A E2A Treg: Tconv 1:1 1:2 1:4 1:8 1:16 1:32 0:1 F 100 ** *** 80 WT 39.6 58.4 70.1 80.2 84.9 85.1 88.9 *** 60 *** no Treg 40 WT Treg KO Treg 150 % Divided 20 fl/fl 25.0 40.3 59.7 70.1 81.0 83.4 E2A 100 fl/fl 0 HEB 50 1:1 1:2 1:4 1:8 0:1 cells 0 3 3 4 5 1:161:32 -10 0 10 10 10 CFSE Treg: Tconv ratio INFLAMMATION

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Fig. 3. Treg cells deficient in E protein exhibit enhanced immunosuppressive function in vivo. (A–E) EAE was induced in WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre mice and the extent of disease assessed at day 24. (A) The disease score of WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre mice after EAE induction (WTFoxp3Cre n = 4, + + E2Afl/flHEBfl/flFoxp3Cre n = 4). (B) Representative flow cytometric analysis (Left) and the frequency (Right)ofFoxp3 cells among CD4 cells infiltrating the spinal cord of WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre mice. (C) Flow cytometric analysis of MOG-specific Treg cells in spleens of WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre + + + + mice (Left), frequency of MOG35–55 among Foxp3 cells (Middle), and the frequency of MOG35–55 among CD4 cells (Right). (D and E) Cells from spinal cord of WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre mice were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin. (D) Cytokine production of the cells analyzed by flow cytometry. (E) The frequency of IFN-γ+, IL-17A+, and IFN-γ+IL-17A+ cells. (B–E) Data are pooled from two independent experiments; + − each symbol represents data from one mouse, mean ± SD are shown (*P < 0.05, **P < 0.01). (F) CFSE-labeled Tconv cells (CD4 CD25 CD45RBhi) from WT mice + were cultured with or without titrated numbers of YFP Treg cells from WT Foxp3CreR26YFP or E2Afl/flHEBfl/flFoxp3CreR26YFP mice for 3 d after which cell division was analyzed by flow cytometry. Shown are representative cell proliferation dye dilution profiles of Tconv cells of four independent experiments (Left) and percentages of these cells, divided at different Tregs. Tconv cell ratios (Right); n = 4, mean ± SD are shown (**P < 0.01, ***P < 0.001). (G–K) Six- to 8- wk old RAG2-deficient mice were adoptively transferred with WT naive CD45RBhi CD4+ T cells together with either PBS (CD45RBhi+PBS) or sorted Treg cells derived from WT Foxp3CreR26YFP mice (CD45RBhi+WT Treg) or from E2Afl/flHEBfl/flFoxp3CreR26YFP mice (CD45RBhi+KO Treg) (n = 6 in each group). (G) Body weight curve of mice in the various groups. One-way ANOVA with the Dunnett’s multiple comparisons test were used to compare the weight loss curve. *P < + + + + 0.05. (H) The absolute number of CD4 cells (Top), the frequency of Foxp3 cells in CD4 cells (Middle), and the absolute number of Foxp3 cells (Bottom)in + + + + + + + indicated organs. (I) Flow cytometry of IFN-γ and IL-17A cells in CD4 cells in the spleen. (J) The absolute number of IFN-γ , IL-17A , and IFN-γ IL-17A cells. + (K) Flow cytometry analysis of Foxp3 cells in indicated organs. (I and K) Data are representative of two independent experiments. (H and J) Data are pooled from two independent experiments; each symbol represents data from one mouse, mean ± SD are shown. (One-way ANOVA with the Dunnett’s multiple comparisons test were used. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.)

Han et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 25, 2021 A Up: 338 B C D WT vs KO ( no TCR) Down: 463 20 A B 150 100 10 E-protein dependent

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-dependent genes

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2 among E-protein

dependent genes

-dependent genes

%TCR & E-protein 12.5 AB C significant: 647 150

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2 10 100 7.5 KO-48h vs WT-48h upregulated 5.0 50

2.5 Genes (%) 55% TCR dependent 0

WT-48h vs WT WT-48h 1.0 -5 -1 1 5 10 -10 A: WT-48h vs WT Expression abs log Expression log2 (fold) KO vs WT (TCR 48h) B: KO-48h vs WT-48h (log2fold>1) D no TCR TCR 48h no TCR TCR 48h no TCR TCR 48h no TCR TCR 48h no TCR TCR 48h no TCR TCR 48h no TCR TCR 48h KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT KO/WT Transcription factors Cell surface proteins Function associated Chemkine & receptor integring RNA-processing proliferation&apoptosis Ahr Lag3 Il10 Ccl20 Itga3 Rrp15 Cdkn1a Irf4 Ccr5 Tgfb Ccl3 Itgb3 Rrp7a Cdk17 Cnbd2 Batf3 Il18r1 Furin Ccl28 Itgae Rrp8 Maf Tgfbr3 Gzmb Itgav Dis3 Bcl11b Ccl4 Ki67 Nfat5 Il1r1 Lta Ccr5 Itgb5 Lars2 Smad3 Bcl2 Cd44 Fgl2 Cxcr6 Rp1l2 Cdk6 Rorc Icos Ccr2 Khnyn Ikzf3 Tnfsf9 Rrp1b Expression (g(log2fold)) Tbx21 Il12rb2 Exosc5 Irf8 -2 0 2

Fig. 4. E protein globally regulates effector Treg cell signature gene expression. (A) Genes expressed differently in Treg cells from E2Afl/flHEBfl/flFoxp3Cre mice (KO) vs. from WTFoxp3Cre mice (WT) (Top) or after anti-CD3/CD28 stimulated for 48 h (WT-48 h, KO-48 h, respectively) (Bottom) were plotted; numbers in plots indicate genes up-regulated (red) or down-regulated (green) by twofold or more (P < 0.05). (B, Left) Venn diagram. (Right) Genes differently expressed twofold or more in KO Treg cells vs. WT Treg cells (Venn A) that also changed expression upon TCR stimulation (Venn C) (Top), and genes differently expressed twofold or more in KO-48 h Treg cells vs. WT-48 h Treg cells (Venn B) that were also TCR dependent (Venn C) (Bottom). (C, Left) Genes up-regulated in E-protein KO-48 h cells vs. WT-48 h plotted against those differently expressed in WT-48 h vs. WT cells; (C, Middle) Venn diagram of genes differently expressed twofold or more in WT-48 h vs. WT Treg cells (TCR-dependent genes) (Venn A) relative to those up-regulated twofold or more in KO-48 h vs. WT-48 h Treg cells (up-regulated genes in E-protein KO Tregs (Venn B); (C, Right) gene% = TCR-dependent genes among those up-regulated twofold or more in E-protein KO Treg cells. (D) Heat map of E protein-regulated genes.

+ necessary for initial development of Foxp3 Treg cells, its further more remarkably in nonlymphoid tissues than in the spleen or decreased expression in matured Treg cells does not regulate lymph nodes, and that many E protein-dependent genes were these signaling pathways. associated with effector Treg cell signatures as indicated in In a final set of studies relating to the above RNA-seq studies, RNA-seq analysis, suggested that E protein-deficient Treg cells we validated the various findings with quantitative RT-PCR + are prone to differentiate toward effector Treg cells. To confirm analyses. Accordingly, YFP Treg cells sorted from spleens of the impact of E-protein deletion on effector Treg cell differ- fl/fl fl/fl Cre YFP Cre YFP E2A HEB Foxp3 R26 or WT Foxp3 R26 mice were entiation, we compared E2Afl/flHEBfl/flFoxp3Cre mice and WT stimulated with anti-CD3/28 for 48 h after which mRNA extracted Foxp3Cre control mice with respect to expression of molecules from cultured cells was subjected to quantitative RT-PCR. Con- previously known to associate with Treg cell activation and/or sistent with the RNA-seq data, a host of molecules essential for effector function. This analysis revealed that surface molecules effector Treg cell differentiation and function were significantly indicative of activated or effector Treg cells, such as CD103, increased in TCR-stimulated E protein-deficient Treg cells vs. WT Treg cells (SI Appendix, Fig. S3C). In confirmation, similar studies ICOS, and KLRG1, were significantly increased in E protein- conducted with Treg cells sorted from Id2fl/flId3fl/flFoxp3CreR26YFP deficient Treg cells (SI Appendix, Fig. S4A). As expected, these mice vs. cells from WT Foxp3CreR26YFP mice showed that the changes in expression were more pronounced in the CLP and other nonlymphoid organs such as lung and liver, as indicated by expression of most of these genes was decreased in the Id2/Id3- + depleted Treg cells (SI Appendix,Fig.S3D). the fact that the proportion of Foxp3 cells that express CD103 and KLRG1 were significantly increased at these tissue E-Protein Activity Regulates Effector Treg Cell Differentiation, sites (Fig. 5 A and B). Similarly, the expression of ICOS and Trafficking, Proliferation, and Survival. The observation that the IRF4, the latter a transcription factor important for effector + ratio of Foxp3 cells of E2Afl/flHEBfl/flFoxp3Cre mice increased Treg differentiation and function (24), was also greatly increased

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1800494116 Han et al. Downloaded by guest on September 25, 2021 + in Foxp3 cells from E2Afl/flHEBfl/flFoxp3Cre mice compared with hanced suppressive capacity during intestinal inflamma- WT mice (Fig. 5C). tion. Similarly, GATA3 and T-bet are expressed in activated Treg cells with effector characteristics display an increased Treg cells and are required for Treg cell suppressive function ability to accumulate in inflammatory sites to suppress in- under specific physiological circumstances for the maintenance flammation (25). In a functional study of the effect of E-protein of Treg cell homeostasis (26–30). Given that RORγt as well as deletion on Treg cell effector characteristics, we determined the T-bet expression was greatly increased upon TCR stimulation in effect of deletion of E protein on the ability of Treg cells to E protein-deficient Treg cells in the RNA-seq analysis, we inves- accumulate in the spinal cords of mice with EAE, i.e., a site of tigated whether E protein specifically regulated certain effector + + + + inflammation where they can exert suppressive function as Treg cell lineage differentiation. Foxp3 RORγt , Foxp3 T-bet , + + demonstrated in Fig. 3. We found that in parallel with cells in the and Foxp3 GATA3 effector Treg cell subsets were detected in spleen (SI Appendix, Fig. S4B) and draining lymph nodes (SI spleen, lung, liver, and CLP of WT Foxp3Cre and E2Afl/flHEBfl/fl + + Appendix, Fig. S4C)ofE2Afl/flHEBfl/flFoxp3Cre mice undergoing Foxp3Cre mice. However, whereas the Foxp3 RORγt subset was EAE, the spinal cords of E2Afl/flHEBfl/flFoxp3Cre mice contained significantly increased in E2Afl/flHEBfl/flFoxp3Cre mice relative to + + + a significantly increased proportion of Treg cells expressing WT Foxp3Cre mice (Fig. 6 A and B), Foxp3 T-bet and Foxp3 + CD103 and KLRG1 (SI Appendix,Fig.S4D)aswellasin- GATA3 Treg cell subsets were not comparably increased (SI creased amounts of ICOS and IRF4 (SI Appendix,Fig.S4E). Appendix, Fig. S5 A and B). In a series of further studies, we In additional studies to determine whether the increased ex- investigated different Treg subset cells under inflammatory pression of activation/effector markers in Treg cells above were conditions, including EAE, DSS-induced colitis, and Listeria cell intrinsic, we conducted studies of effector Treg cells de- monocytogenes infection; we reproducibly observed that only + + veloping in mixed BM chimeric mice generated as described Foxp3 RORγt Treg cells were dramatically increased in all of + + above and analyzed 8 wk after reconstitution. As expected, we these disease conditions (Fig. 6 C–E), whereas the Foxp3 T-bet + + found a higher percentage of CD103 , KLRG-1 Treg cells (SI subset did not change significantly even in the Th1-type Listeria Appendix, Fig. S4F) and up-regulated expression of ICOS and infection (Fig. 6F). Thus, our findings indicated that E-protein ac- + + IRF4 in Treg cells (SI Appendix, Fig. S4G) derived from the tivity specifically regulates Foxp3 RORγt Treg cell differentiation. fl/fl fl/fl Cre Given that E-protein activity was down-regulated by TCR stimu- E2A HEB Foxp3 mice donors. In parallel studies, cells + lation, this finding suggests that RORγt effector Treg cell differ- from chimeric mice in which EAE had been induced were ex- + + + entiation requires relatively stronger TCR stimulation than T-bet amined. Here again, the percentage of CD103 Foxp3 cells or + + + KLRG-1 Foxp3 cells infiltrating in spinal cord (Fig. 5D)or and GATA3 Treg cell differentiation.

draining lymph nodes (SI Appendix, Fig. S4H) as well as the INFLAMMATION expression of ICOS and IRF4 on Treg cells in spinal cord (Fig. E Protein Regulates Effector Treg Cell Signature Gene Expression by IMMUNOLOGY AND Directly Binding to the Regulatory Element of These Genes. To fur- 5E) or draining lymph nodes (SI Appendix, Fig. S4I) was greatly ther investigate the mechanism by which E protein influences E2Afl/flHEBfl/flFoxp3Cre increased from the mice donors than gene expression in matured Treg cells, we performed genome- from WT donors. In reciprocal studies to determine if the phe- fl/fl fl/fl Cre wide ChIP-seq assays. These showed that E2A binds to putative notype of Treg cells from Id2 Id3 Foxp3 mice was cell promoter or enhancer regions of a host of genes, including the intrinsic, we conducted studies of Treg cells in mixed bone Icos, Itgae, Klrg1, Irf4, Rorc, GranzymeB, Il10, and Cdk6, Bcl2 marrow chimeras reconstituted with cells from WT Foxp3Cre and fl/fl fl/fl Cre genes (Fig. 7A and SI Appendix, Fig. S5C). These data were then Id2 Id3 Foxp3 mice. We found that CD103, ICOS, KLRG1, supported by ChIP-qPCR analysis that showed that E47 binding fl/fl fl/fl Cre and IRF4 expression in Treg cells from Id2 Id3 Foxp3 to genomic fragments containing promoter or conserved en- donor mice were significantly decreased compared with cells from hancer sequences specific for Itgae, Irf4, Icos, Klrg1, and Rorc WT Foxp3Cre donor mice (Fig. 5 F and G). These various studies (Fig. 7B). In related studies we determined patterns of histone of chimeras thus provided firm evidence that E protein modulates methylation of E-protein binding sites in various genes modified effector Treg cell differentiation in a cell-intrinsic manner. by E-protein deletion. We found that trimethylation of histone The RNA-seq analysis also indicated that E protein-deficient Treg H3 at Lys4, a histone change consistent with active transcription, cells were also marked by increased expression of genes associated was increased in the Itgae, Irf4, Icos, Klrg1, and Rorc genes of + with “apoptosis” and “cell cycle.” On the basis of these findings, we effector Treg cells compared with conventional CD4 T cells, reasoned that the increased Treg cell number in peripheral tissue of E and the trimethylation of this region was significantly increased protein-deficient mice (Fig. 1) was a consequence of increased Treg in corresponding genes of E protein-deficient Treg cells (Fig. cell proliferation or survival in response to continuous autoantigen 7C). Taken together, these data indicate that E protein directly + stimulation. To investigate this possibility, YFP Treg cells were sorted regulates the expression of genes that are specific for effector from E2Afl/flHEBfl/flFoxp3CreR26YFP and WT Foxp3CreR26YFP mice, Treg cells by directly binding to the regulatory element of these labeled with CFSE and cultured in the presence of anti- genes and by remodeling of the chromatin at regulatory elements CD3/CD28 and IL-2 for various lengths of time. Treg cells from of key genes affecting effector Treg cell differentiation so as to E2Afl/flHEBfl/flFoxp3Cre mice exhibited a greater number of cell di- render these sites less transcriptionally active; in contrast, re- + visions in each time period than WT Foxp3 cells (Fig. 5H) as well duced E-protein activity causes these sites to be more tran- + as increased numbers of Ki67 cells (Fig. 5I); in addition, they scriptionally active and gives rise to differentiation of effector + exhibited decreased numbers of AnnexinV (apoptotic cells) Treg cells with enhanced suppressor function. over 24 h (SI Appendix, Fig. S4J). Accordingly, this was accom- panied by an increased accumulation of Treg cells over the 4-d Discussion period of study (Fig. 5J). Despite major progress in our understanding of TCR engage- Collectively, these data provided strong evidence that E protein ment in the differentiation of effector Treg cells, the downstream exerts broad regulation of the gene expression required for effector molecular mechanisms underlying TCR regulation of Treg cell Treg cell differentiation, function, survival, and proliferation. homeostasis and suppressive capacity is still poorly defined. Here we demonstrate that specific deletion of E protein in matured + + + + + + Foxp3 RORγt but Not Foxp3 Tbet or Foxp3 GATA3 Effector Treg Treg cells (in E2Afl/flHEBfl/flFoxp3-GFP-hCre mice) resulted in Cell Subsets Are Regulated by E Protein. Previous reports indicate Treg cells with increased stability/survival and enhanced sup- + that Foxp3 Treg cells can co-opt the expression of specific pressive function. We also showed that a large proportion of transcription factors that are associated with the differentiation genes regulated by E protein was also TCR dependent, strongly + and function of effector CD4 T cell lineages. For example, it suggesting that the effects of E protein on gene regulation are + has been demonstrated that Foxp3 T cells expressing RORγt parallel to or slightly amplified by TCR stimulation. Finally, we represent a stable effector lineage with an en- showed with ChiP-seq analysis that E protein binds to genes

Han et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 25, 2021 A B C lung liver CLP lung liver CLP lung liver CLP

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40 + + 40 40 3 400 20 20 20 2 95.2 Ki67 80.1 90.2 Ki67 300 Ki67 E2Afl/fl 0 0 0 1 200 fl/fl fl/fl fl/fl HEBfl/fl WT WT WT 100 HEB HEB HEB Cell number (×10 0 fl/fl fl/fl fl/fl 0 2 31 4

cells 0 3 3 4 5 -10 0 10 10 10 E2A E2A E2A Time (days) Ki67

Fig. 5. E-protein activity regulates effector Treg cell differentiation, trafficking, proliferation, and survival. (A) Cells of indicated organs from WTFoxp3Cre + + and E2Afl/flHEBfl/flFoxp3Cre mice were stained directly with CD4, Foxp3, CD103, and KLRG-1. Dot plots were gated on CD4 cells. (B) Frequencies of CD103 + + (Top) and KLRG-1 (Bottom) cells in Foxp3 cells in indicated organs from WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre mice. (C) Mean fluorescence of ICOS (Top) + and IRF4 (Bottom) on Foxp3 cells in indicated organs of WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre mice. (D and E) Mixed BM chimeric mice were generated by transferring BM cells from CD45.1+CD45.2+ WT Foxp3Cre and CD45.1+ E2Afl/flHEBfl/flFoxp3Cre mice into lethally irradiated recipient mice. (D) Flow cytometric analysis of CD103, KLRG-1, and Foxp3 expression among CD4+ cells (Left) and the frequencies of CD103+ (Right Top) and KLRG-1+ (Right Bottom) cells among CD45.1+CD45.2+ WT Foxp3Cre and CD45.1+ E2Afl/flHEBfl/flFoxp3Cre Foxp3+ cells in the spinal cord of BM chimeric mice 24 d after EAE induction. (E) Flow + + cytometric analysis of ICOS and IRF4 expression (Left) and the mean fluorescence of ICOS (Right Top) and IRF4 (Right Bottom) among CD45.1 CD45.2 WT + Foxp3Cre and CD45.1 E2Afl/flHEBfl/flFoxp3Cre Treg cells in the spinal cord of BM chimeric mice 24 d after EAE induction. (F and G) Mixed BM chimeric mice were generated by transferring BM cells from WT Foxp3Cre and Id2fl/flId3fl/flFoxp3Cre mice into lethally irradiated recipient mice. (F) Flow cytometric analysis of + + + CD103, KLRG-1, and Foxp3 expression among CD4 cells (Left) and the frequencies of CD103 (Right Top) and KLRG-1 (Right Bottom) cells among WT Foxp3Cre and Id2fl/flId3fl/flFoxp3Cre Foxp3+ cells in the spleen of the mixed BM chimeric mice. (G) Flow cytometric analysis of ICOS and IRF4 expression of WT Foxp3Cre and Id2fl/flId3fl/flFoxp3Cre Foxp3+ cells in the spleen of the mixed BM chimeric mice. (H–J) Highly purified Treg cells from WT Foxp3CreR26YFP and E2Afl/flHEBfl/flFoxp3CreR26YFP mice were (H) labeled with CFSE and stimulated with anti-CD3 plus anti-CD28 in the presence of human IL-2 for the indicated time. CFSE dilution in the Treg cells at the indicated time points were analyzed by flow cytometry (Left) and the frequencies of CFSE-diluted cells + + + + (Right); or (I)Ki67 in Foxp3 cells at the indicated time (Left) and the frequencies of Ki67 in Foxp3 cells (Right). (J) Total cell numbers at the indicated time points were counted separately. (A, G and D, E, F, H,andI, Left) Data are representative of at least three independent experiments. (B, C, J and D, E, F, H,andI, Right) Data are pooled from at least three independent experiments, each symbol represents data from one mouse, mean ± SD are shown (*P < 0.05, **P < 0.01, ***P < 0.001).

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1800494116 Han et al. Downloaded by guest on September 25, 2021 E-protein regulation of Treg cells is pertinent to the genera- A B + + spleen lung liver CLP tion of RORγt Foxp3 cells, a functionally distinct Treg cell spleen lung liver CLP 9.1 0.6 4.2 0.5 4.8 1.1 5.1 6.4 Cells

Cells

** Cells Cells * * + *

+

+ + 20 30 30 80 subset that has been shown to suppress Th17 responses in EAE WT γ + + 15 20 20 60 (22). In the present study we showed that ROR t Foxp3 Treg 88.0 2.3 92.3 3.0 90.8 3.1 79.5 9.0 10 40 cells were significantly increased in mice with E protein-deficient 5 10 10 10 % in Foxp3

% in Foxp3

% in Foxp3 1.5 8.9 % in Foxp3 5 20 12.9 1.2 7.4 2.9 6.4 9.1 + +

+

+

4 t fl/fl 10 E2A 0 γt 0 0 Treg cells, both in the steady state and in inflammatory states. In 3 0 fl/fl 10 fl/fl fl/fl fl/fl fl/fl HEB WT 2 WT WT WT 10

ROR addition, we found that the gene expression patterns of E

RORγ

0 RORγt 2 3.4 7.7 RORγt fl/fl HEB fl/fl HEB fl/fl HEB fl/fl HEB Foxp3 -10 82.5 83.4 82.9 5.3 75.3 9.2 + 3 3 4 5 -10 0 10 10 10 γ E2A E2A E2A E2A protein-deficient Treg cells are very similar to that of ROR t RORγt + dLN spleen CLP Foxp3 cells found in the intestine and in immunization-induced spleen CLP + + CDdLN 11.9 0.9 7.5 6.5 γ

Cells

Cells ROR t Foxp3 cells (22) since in all cases one sees up-regulation

Cells * * ** +

+

3.0 + 20 80 WT 20 WT 15 15 60 of the Icos, Itgae, Rorc, Il1r1, Il23r, Havrc2,andIl10 genes. Finally, 82.4 4.8 77.9 8.1 10 10 40 E protein-deficient Treg cells exhibited enhanced suppressive ac- 5 4 10 10 14.6 8.9 5 20 3 2.1 5.5 % in Foxp3 5 10 % in Foxp3 4 % in Foxp3

fl/fl 10 + tivity and stability, also in line with characteristics of previously fl/fl +

6.5 + t E2A t E2A 2 0 0 + 10 fl/fl 3 0 10 fl/fl fl/fl fl/fl γ HEB fl/fl HEB 1 0 10 WT WT described ROR t Treg cells. These findings suggest that E pro- 3 WT Foxp3 0 + -10 10 76.8 4.5 76.6 9.0 HEB Foxp3 HEB 3 4 5 0 1 2 3 4 fl/fl fl/fl HEB RORγ 0 10 10 10 fl/fl RORγ γ 10 10 10 10 10 RORγt tein is a key regulator of the ROR t Treg cell subset and that this RORγt RORγt E2A E2A E2A subset arises when Tregs are subjected to a sufficient and possibly lung liver lung liver lung liver lung liver higher level of TCR signaling and E-protein deletion that allows EF4.2 0.4 4.4 0.8 5.7 1.0 6.1 2.2 ns ns Cells 20 50 ** * Cells

Cells

+

Cells

+

+ WT 40 30 + transcription of the RORγt gene. 15 40 + 30 20 30 92.4 3.0 91.7 3.1 20 74.5 18.8 57.134.6 10 20 Recent studies of mice with Id2/Id3 deficiency in Foxp3 Treg 4 10 4 10 10 5 3 % in Foxp3 10

8.9 2.9 18.5 4.6 % in Foxp3 10 7.4 1.5 % in Foxp3 3 13.2 10 1.5 % in Foxp3 cells provide data in some ways parallel to those obtained in this + 10

+

+ fl/fl + 0 E2A 2 0 10 2 0 10 fl/fl fl/fl fl/fl 0 study in that these mice also manifest E-protein abnormalities HEB fl/fl 1 fl/fl 1 WT WT

10 10 T-bet WT WT T-bet HEB fl/fl HEB fl/fl 0 7.7 RORγt 0 Foxp3 5.3 10 83.4 84.2 RORγt 10 82.3 13.0 49.5.28.4

HEB HEB Foxp3 3 4 5 fl/fl fl/fl 0 1 2 3 4 (16). However, in this case increased E-protein function results 01010 10 10 10 10 10 10 E2A E2A RORγt E2A E2A Tbet in decreased Treg cell stability and in vivo suppressive function, Fig. 6. Foxp3+RORγt+ but not Foxp3+Tbet+ or Foxp3+GATA3+ effector Treg the latter manifesting as spontaneous inflammation of the lungs, cell subset is regulated by E protein. (A and B) Flow cytometric analysis of eyelids, skin, and esophagus (16). These changes at first glance RORγt and Foxp3 expression in cells from indicated organs from WTFoxp3Cre suggest that Id2/Id3 deletion provides a mirror image of + and E2Afl/flHEBfl/flFoxp3Cre mice (A) and the frequencies of RORγt cells E-protein deletions and thereby predicts the effects of E-protein + within Treg cells (B). (C) Flow cytometric analysis of RORγtexpressioninFoxp3 deletion. However, this is probably not the case for several cells in the draining lymph node from WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre reasons: first, Id2/Id3 deletion impacts mainly on regulation of B + INFLAMMATION mice 21 d after EAE induction (Left) and the frequency of RORγt cells cell/Th2 responses and results in allergic manifestations; in IMMUNOLOGY AND within these Treg cells (Right). (D) DSS-colitis was induced in WTFoxp3Cre contrast, E-protein deletion has a more general effect on Treg and E2Afl/flHEBfl/flFoxp3Cre mice; flow cytometric analysis of RORγtand cell suppressive function and leads to enhanced suppression of + Foxp3 expression in indicated organs (Left) and the frequencies of RORγt Th1/Th17-mediated inflammation in EAE and transfer colitis; cells in Treg cells 10 d after colitis induction (Right). (E and F)WTFoxp3Cre and E2Afl/flHEBfl/flFoxp3Cre mice were infected by L. monocytogenes;(E)flowcyto- + metric analysis of RORγt and Foxp3 expression and the frequencies of RORγt cells in isolated lung and liver Treg cells (Right). (F) Flow cytometric analysis of + A T-bet and Foxp3 expression (Left) and the frequencies of T-bet cells among Treg cells (Right). (A and C–F, Left) Data are representative of at least three in- dependent experiments. (B and C–F, Right) Data are pooled from at least three independent experiments; each symbol represents data from one mouse, mean ± SD were shown (ns, not significant, *P < 0.05, **P < 0.01).

involved in Treg cell effector function and that decreased bind- ing (resulting from down-regulation) leads to enhanced expression of these genes. Thus, TCR signaling regulates Treg cell function B by releasing these genes from the negative effects of E protein. Regarding the specific effects of E proteins, the above- mentioned ChIP-seq studies showed that before down-regulation by TCR signaling, E proteins bind to a large number of the genes that are associated with effector Treg cell differentiation (Irf4, Rorc), localization (Itgae and Ccr5, Ccr2,etc.),function(Icos, C Lag3, Il10,andGranzymeB), survival (Bcl2), and proliferation (Cdk6); in addition, E proteins bind to the gene encoding Klrg1,a marker of mature or terminally differentiated cells, including Treg cells. In studies probing the effects of E-protein binding we found that histone changes indicative of active gene transcription (tri- methylation at Lys4 of H3) were present in many of the above Fig. 7. E protein regulates effector Treg cell signature gene expression by genes following E-protein down-regulation. Thus, the picture that directly binding to the regulatory element of these genes. (A) Thymocytes emerges is that E proteins have a potent capacity to suppress were isolated from WT mice; E47 ChIP was performed and subjected to li- transcription of those genes necessary for effector Treg cell brary preparation and sequencing as described in Methods. Representative function. Of particular interest in this context is that IRF4 is of ChIP sequencing tracks showing composite binding of E2A to Itgae, Icos, dramatically up-regulated during inflammation in E protein- Irf4, Klrg1, and Rorc genes. (B) Chromatin from thymocytes were subjected deficient Treg cells, indicating that E protein negatively regulates to immunoprecipitation with anti-E47. Binding of E proteins to the indicated the expression IRF4. This observation is significant because genes was analyzed by quantitative real-time PCR. Results are representative of at least three independent experiments; mean ± SD are shown. (C)Chro- + IRF4 is a TCR-induced gene that has been shown to be critical matin from purified naive CD4 cells, WT Treg cells, and E protein-deficient for the optimal function of effector Treg cells and to be a mediator Treg cells were subjected to immunoprecipitation with anti-H3K4. Binding of effector Treg cell differentiation; thus, this finding greatly of H3K4 to the indicated genes was analyzed by quantitative real-time PCR. strengthens the link between Treg cell differentiation and TCR- Results are representative of two independent experiments; mean ± SD are regulated E-protein activity. shown (*P < 0.05, **P < 0.01, ***P < 0.001).

Han et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 25, 2021 second, because deletion of Id2/Id3 in matured Treg cells leads that the molecular mechanism of TCR regulation of Treg cell dif- to spontaneous autoimmune disease, the apparent effect of Id2/ ferentiation is in fact its ability to control E-protein activity. Thus, E Id3 on Treg cell function derived from deletion studies may in protein plays a stage-dependent role in Treg cells, whereby it is not reality be secondary to inflammatory effects of such deletion; only indispensable for their development in the thymus as demon- third, TCR stimulation of Id protein-deleted cells results in both strated previously (15), but also essential for their further differenti- down-regulation of Id and E-protein expression rather than a ation into effector Treg cells in the periphery. On this basis, the reciprocal change that one might expect in the case of opposing underlying regulatory circuits governing E-protein levels are likely to functions. These various differences between the effects of Id2/ provide insight into how Treg cells maintain their essential identity Id3 and E-protein deletion are likely to arise from the fact that at and at the same time exhibit plasticity in various tissue settings. the molecular level there does not exist a strict point/counter- point relation between these sets of transcriptional regulatory Methods factors. For this reason, it is fair to say that direct deletion of Mice. WT CD45.1 mice were obtained from The Jackson Laboratory. Tcf3 E protein provides a better picture of E-protein effects on Treg (E2A)fl/flTcf12(HEB)fl/fl mice, Id2fl/flId3fl/fl mice, and Foxp3-GFPKI mice were cells than the indirect effects of Id protein deletion. gifts from Y. Zhuang, Duke University, Durham, NC, and from M. Oukka, Prior studies have revealed that E-protein expression regulates Brigham and Women’s Hospital, Boston, MA, respectively. Foxp3Cre/Rosa26YFP cell proliferation and survival (apoptosis) (31). This correlates mice were a gift from Xuyu Zhou, Chinese Academy of Sciences, Beijing, China. with the results of RNA-seq analyses performed in this study that Strain-matched WT Foxp3Cre mice served as controls. E2Afl/flHEBfl/fl mice were showed that genes involved in the “cell proliferation and cycling” crossed with Foxp3-GFP-KI mice and Foxp3Cre/Rosa26YFP mice to generate such as Bcl2, Cdk6, and Cdk14 genes, were up-regulated in E E2Afl/flHEBfl/fl Foxp3-GFP-KI/Foxp3Cre/Rosa26YFP mice. The study was approved protein-deficient Treg cells. In addition, the latter cells exhibited by the Research Ethics Committee of the Institute of Microbiology, Chinese increased proliferative responses in vitro upon TCR stimulation, Academy of Sciences (IMCAS), the permit number was APIMCAS2017015. which most likely contributed to the increased percentage of effector Treg cells noted in E2Afl/flHEBfl/flFoxp3-GFP-hCre mice Flow Cytometry and Cell Sorting. Flow cytometry was performed on a in vivo. These findings correlated with the results of ChIP-seq FACSCalibur or FACS CantoII (BD Biosciences) and the data were analyzed by analysis that revealed that genes involved in cell cycling exhibited FlowJo. Cell sorting was performed using a FACS AriaIII (BD). Fluorochrome- a high level of E-protein binding, including the Bcl2 gene and conjugated Abs are provided in SI Appendix. cycle promoter gene, Cdk6; it is thus likely that the increased proliferation and up-regulated expression of cell cycle genes An extended methods section is provided in SI Appendix, SI Materials were also a major functional consequence of E-protein loss in and Methods. Treg cells. ACKNOWLEDGMENTS. We thank Prof. Y. Zhuang (Duke University Medical In summary, these studies provide strong evidence that Center, Durham, NC) for providing E2Af/fHEBf/f and Id2f/fId3f/f mice. This E protein regulates a large set of key effector Treg cell signature study was supported by Strategic Priority Research Program of the Chinese genes that govern the differentiation, effector function, proliferation, Academy of Sciences (Grant XDB29010000), the National Natural Science andsurvivalofalreadyestablishedTregcells.InasmuchasE-protein Foundation of China (Grant 31670894) and the National Key Research and down-regulation is largely under TCR control, these findings indicate Development Project of China (Grant 2016YFC1200302).

1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune 17. Zhou X, et al. (2008) Selective miRNA disruption in T reg cells leads to uncontrolled tolerance. Cell 133:775–787. autoimmunity. J Exp Med 205:1983–1991. 2. Curotto de Lafaille MA, Lafaille JJ (2009) Natural and adaptive + regulatory 18. Zhou X, et al. (2009) Instability of the transcription factor Foxp3 leads to the gener- T cells: More of the same or a division of labor? Immunity 30:626–635. ation of pathogenic memory T cells in vivo. Nat Immunol 10:1000–1007. 3. Campbell DJ, Koch MA (2011) Phenotypical and functional specialization of FOXP3+ 19. Thornton AM, et al. (2010) Expression of Helios, an Ikaros transcription factor family regulatory T cells. Nat Rev Immunol 11:119–130. member, differentiates thymic-derived from peripherally induced Foxp3+ T regula- 4. Lee JH, Kang SG, Kim CH (2007) FoxP3+ T cells undergo conventional first switch to tory cells. J Immunol 184:3433–3441. lymphoid tissue homing receptors in thymus but accelerated second switch to non- 20. Sefik E, et al. (2015) MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce lymphoid tissue homing receptors in secondary lymphoid tissues. J Immunol 178: a distinct population of RORγ+ regulatory T cells. Science 349:993–997. 301–311. 21. Yang BH, et al. (2016) Foxp3(+) T cells expressing RORγt represent a stable regulatory 5. Panduro M, Benoist C, Mathis D (2016) Tissue Tregs. Annu Rev Immunol 34:609–633. T-cell effector lineage with enhanced suppressive capacity during intestinal inflam- 6. Kleinewietfeld M, et al. (2005) CCR6 expression defines regulatory effector/memory- mation. Mucosal Immunol 9:444–457. + like cells within the CD25(+)CD4+ T-cell subset. Blood 105:2877–2886. 22. Kim BS, et al. (2017) Generation of RORγt antigen-specific T regulatory 17 cells from 7. Huehn J, et al. (2004) Developmental stage, phenotype, and migration distinguish Foxp3+ precursors in autoimmunity. Cell Rep 21:195–207. naive- and effector/memory-like CD4+ regulatory T cells. J Exp Med 199:303–313. 23. Setoguchi R, Hori S, Takahashi T, Sakaguchi S (2005) Homeostatic maintenance of 8. Suffia I, Reckling SK, Salay G, Belkaid Y (2005) A role for CD103 in the retention of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction CD4+CD25+ Treg and control of Leishmania major infection. JImmunol174:5444–5455. of autoimmune disease by IL-2 neutralization. J Exp Med 201:723–735. 9. Beyersdorf N, Ding X, Tietze JK, Hanke T (2007) Characterization of mouse CD4 T cell 24. Zheng Y, et al. (2009) Regulatory T-cell suppressor program co-opts transcription subsets defined by expression of KLRG1. Eur J Immunol 37:3445–3454. factor IRF4 to control T(H)2 responses. Nature 458:351–356. 10. Lee HM, Bautista JL, Scott-Browne J, Mohan JF, Hsieh CS (2012) A broad range of self- 25. Liston A, Gray DH (2014) Homeostatic control of regulatory T cell diversity. Nat Rev reactivity drives thymic regulatory T cell selection to limit responses to self. Immunity Immunol 14:154–165. + 37:475–486. 26. Wohlfert EA, et al. (2011) GATA3 controls Foxp3 regulatory T cell fate during in- 11. Vahl JC, et al. (2014) Continuous T cell receptor signals maintain a functional regu- flammation in mice. J Clin Invest 121:4503–4515. latory T cell pool. Immunity 41:722–736. 27. Wang Y, Su MA, Wan YY (2011) An essential role of the transcription factor GATA- 12. Levine AG, Arvey A, Jin W, Rudensky AY (2014) Continuous requirement for the TCR 3 for the function of regulatory T cells. Immunity 35:337–348. in regulatory T cell function. Nat Immunol 15:1070–1078. 28. Yu F, Sharma S, Edwards J, Feigenbaum L, Zhu J (2015) Dynamic expression of tran- 13. Murre C, McCaw PS, Baltimore D (1989) A new DNA binding and dimerization motif in scription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance. immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56: Nat Immunol 16:197–206. 777–783. 29. Koch MA, et al. (2009) The transcription factor T-bet controls regulatory T cell ho- 14. Maruyama T, et al. (2011) Control of the differentiation of regulatory T cells and meostasis and function during type 1 inflammation. Nat Immunol 10:595–602. T(H)17 cells by the DNA-binding inhibitor Id3. Nat Immunol 12:86–95. 30. Levine AG, et al. (2017) Stability and function of regulatory T cells expressing the 15. Gao P, et al. (2014) Dynamic changes in E-protein activity regulate T reg cell devel- transcription factor T-bet. Nature 546:421–425. opment. J Exp Med 211:2651–2668. 31. Bain G, Quong MW, Soloff RS, Hedrick SM, Murre C (1999) Thymocyte maturation 16. Miyazaki M, et al. (2014) Id2 and Id3 maintain the regulatory T cell pool to suppress is regulated by the activity of the helix-loop-helix protein, E47. JExpMed190: inflammatory disease. Nat Immunol 15:767–776. 1605–1616.

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