Hhex negatively regulates Treg cells by inhibiting Foxp3 expression and function

Sung Woong Janga,1, Soo Seok Hwanga,b,1, Hyeong Su Kima, Min Kyung Kima, Woo Ho Leea, Soh Un Hwanga, Jinu Gwakb, Si Kyoung Yewb, Richard A. Flavellb,c,2,3, and Gap Ryol Leea,2,3

aDepartment of Life Science, Sogang University, 04107 Seoul, Korea; bDepartment of Immunobiology, Yale University, New Haven, CT 06520; and cHoward Hughes Medical Institute, Yale University, New Haven, CT 06520

Contributed by Richard A. Flavell, October 29, 2019 (sent for review May 3, 2019; reviewed by Hongbo Chi, Sankar Ghosh, and Ming O. Li) Regulatory T (Treg) cells play an essential role in maintaining which allows other transcription factors to bind and regulate their immune homeostasis, but the suppressive function of Treg cells expression. can be an obstacle in the treatment of cancer and chronic infectious Transforming growth factor beta (TGF-β), which is critical for diseases. Here, we identified the homeobox protein Hhex as a maintaining pTreg cells (16), can also induce Foxp3 in naïve CD4 negative regulator of Treg cells. The expression of Hhex was lower in T cells and promote their differentiation into induced Treg cells Treg cells than in conventional T (Tconv) cells. Hhex expression was (iTreg cells) with suppressive function (17). TGF-β phosphorylates repressedinTregcellsbyTGF-β/Smad3 signaling. Retroviral overex- Smad3, resulting in the formation of Smad3/Smad4 heterodimers, pression of Hhex inhibited the differentiation of induced Treg (iTreg) which can translocate to the nucleus and bind to the Foxp3 en- cells and the stability of thymic Treg (tTreg) cells by significantly hancer (conserved noncoding sequence 1 [CNS1]), inducing Foxp3 reducing Foxp3 expression. Moreover, Hhex-overexpressing Treg expression (18, 19). cells lost their immunosuppressive activity and failed to prevent co- Many transcription factors have been shown to transactivate litis in a mouse model of inflammatory bowel disease (IBD). Hhex the regulatory elements of Foxp3, which include a promoter and expression was increased; however, Foxp3 expression was decreased 3 intronic enhancers (CNS1, CNS2, and CNS3). Signal transducer in Treg cells in a delayed-type hypersensitivity (DTH) reaction, a type I immune reaction. Hhex directly bound to the promoters of Foxp3 and activator of transcription 5 (STAT5; downstream of IL-2), and other Treg signature , including Il2ra and Ctla4,andre- c-Rel, nuclear factor of activated T cells (NFAT), Forkhead box pressed their transactivation. The homeodomain and N-terminal re- O (Foxo), activator protein-1 (AP-1), and more have been shown IMMUNOLOGY AND INFLAMMATION pression domain of Hhex were critical for inhibiting Foxp3 and other to directly bind and activate Foxp3 regulatory elements (4, 20–23). Treg signature genes. Thus, Hhex plays an essential role in inhibiting By contrast, only a few negative regulators of Foxp3 have been Treg cell differentiation and function via inhibition of Foxp3. reported. GATA-binding protein 3 (GATA3), the master regula- tor of Th2 cells, and RAR-related orphan γt(RORγt), regulatory | Hhex | Foxp3 the master regulator of Th17 cells, bind to the Foxp3 promoter to

egulatory T (Treg) cells are a distinct subset of CD4 T cells Significance Rthat regulate immune responses and maintain immune ho- meostasis. Treg cells are generated in the (tTreg cells) or Regulatory T (Treg) cells play an essential role in maintaining differentiate in the periphery (pTreg cells), then circulate immune homeostasis. Studying factors that control Treg dif- through lymphoid organs and reside in tissues to control a variety ferentiation and function are critically important to understand of immune responses (1–4). However, under inflammatory or immune homeostasis. In this manuscript, we discovered that pathogenic conditions, Treg cells become unstable, lose their Hhex exerts an inhibitory effect on Treg immunosuppressive properties, and become more like effector cell differentiation and function. Hhex-overexpressing Treg T cells, resulting in their failure to regulate autoimmune or in- cells lose their Foxp3 expression and fail to suppress immune Foxp3 flammatory diseases (5). On the other hand, in the tumor mi- responses. Hhex directly binds to Foxp3 protein and the croenvironment, accumulation of Treg cells can inhibit antitumor locus and inhibits expression of Foxp3 and its target genes. Thus, Hhex plays an essential role in inhibiting Treg cell dif- immunity. Tumor-infiltrating Treg cells are a major cause of poor ferentiation and function via inhibition of Foxp3. This study clinical outcomes (6). Thus, with increasing numbers of clinical will benefit clinical research in developing a therapeutic strat- trials of various immune therapies, studying Treg cell regulators egy for Treg cell-related diseases. is becoming even more important. Forkhead box P3 (Foxp3) serves as a lineage specification Author contributions: S.W.J., S.S.H., R.A.F., and G.R.L. designed research; S.W.J., S.S.H., transcription factor for Treg cells. Foxp3 controls both the identity H.S.K., M.K.K., W.H.L., S.U.H., J.G., and S.K.Y. performed research; S.W.J., S.S.H., R.A.F., and function of Treg cells. Ectopic expression of Foxp3 in con- and G.R.L. analyzed data; and S.W.J. wrote the paper. ventional CD4 T (Tconv) cells induces a regulatory phenotype and Reviewers: H.C., St. Jude Children’s Research Hospital; S.G., Columbia University Medical function, whereas deletion of the Foxp3 in Treg cells results Center; and M.O.L., Memorial Sloan Kettering Cancer Center. in the loss of their suppressive function (7–10). Foxp3 also induces The authors declare no competing interest. the expression of Treg signature genes including Il2ra (encodes Published under the PNAS license. CD25), Ctla4, Tnfrsf18 (encodes GITR), and Icos (8, 11, 12). Data deposition: The data reported in this paper have been deposited in the Gene Ex- pression Omnibus (GEO) database, http://www.ncbi.nlm.nih.gov/geo (accession no. CD25 (interleukin-2 [IL-2] receptor alpha-chain) is required for GSE139297). Treg cell survival and IL-2 consumption as part of Treg-mediated 1S.W.J. and S.S.H. contributed equally to this work. suppression (13). CTLA4 mediates Treg-dependent down-regulation 2R.A.F. and G.R.L. contributed equally to this work. of CD80 and CD86 on antigen-presenting cells (14). Along with 3To whom correspondence may be addressed. Email: [email protected] or grlee@ Foxp3, CD25 and CTLA4 are commonly accepted as markers sogang.ac.kr. of Treg cells. Recently, Treg-specific superenhancers in genes such This article contains supporting information online at https://www.pnas.org/lookup/suppl/ as Foxp3, Il2ra,andCtla4 have been reported (15). These sites are doi:10.1073/pnas.1907224116/-/DCSupplemental. in a poised state at the early stages of tTreg cell differentiation,

www.pnas.org/cgi/doi/10.1073/pnas.1907224116 PNAS Latest Articles | 1of10 Downloaded by guest on September 24, 2021 repress Foxp3 expression during Th2 or Th17 differentiation, re- Results spectively (24, 25). In addition, STAT3, which lies downstream of Expression of Hhex Is Low in Treg Cells. To identify regulators of IL-6, competes with STAT5 to down-regulate Foxp3 (23). Our Treg cells, the transcriptomes of Th2, Th9, and Treg cells were group also identified yin yang 1 (YY1) as an inhibitor of Foxp3 compared by microarray analysis. Naïve CD4 T cells were isolated expression and activity (26), but negative regulators of Foxp3 and from mouse spleens and cultured under each differentiation con- Treg cell activity and function need to be further studied. dition. All conditions included anti-CD3/anti-CD28 stimulation Hematopoietically expressed homeobox (Hhex) is a highly and IL-2, with addition of IL-4 for Th2 cells, IL-4 and TGF-β conserved transcription factor belonging to the homeobox protein for Th9 cells, and TGF-β for Treg cells. To identify candidates family. The human and murine Hhex are 94% homolo- for direct suppressors of Treg differentiation or Foxp3, cell gous, with only a single amino acid difference in the homeodomain differentiation-related ( Consortium) transcription (27, 28). Hhex was first identified in hematopoietic cells (29, 30). factors (gene cards) that were expressed at lower levels in Treg Hhex is expressed in early hematopoietic progenitors and is down- cells than in Th2 and Th9 cells were selected (SI Appendix,Fig.S1; regulated during differentiation (31, 32). Hhex has been reported to play an essential role in lineages, but is not well studied Omnibus [GEO] accession no. GSE139297). The in T cells because of its low expression level (32, 33). Hhex is a homeobox gene Hhex was one of the genes with the largest dif- ference in expression. To confirm the expression of Hhex in CD4 homooligomer-forming transcription factor that regulates target + − + + genes directly by binding to DNA through homeodomains or in- T cells, CD4 CD25 Tconv cells and CD4 CD25 Treg cells directly by modulating other transcription factors through protein– were isolated from mouse spleens and mesenteric lymph nodes protein interactions (27, 34). Hhex can both enhance and repress (mLNs) and Hhex mRNA was evaluated by quantitative reverse target genes, but it has been better characterized as a transcrip- transcription PCR (qRT-PCR) (Fig. 1A). Expression of Hhex was tional repressor (27). significantly lower in Treg cells. Naïve CD4 T cells were also dif- In this study, we examined the role of Hhex in Treg cells. Hhex ferentiated in vitro, and the expression of Hhex mRNA (Fig. 1B) expression was lower in Treg cells than in Tconv cells, and was and protein (Fig. 1C) was measured in each subset. The expression down-regulated by TGF-β/Smad3 signaling. Ectopic expression of Hhex was notably low in Th17 and Treg cells. of Hhex impaired the identity and function of Treg cells. Hhex directly bound to the Foxp3 locus and to the promoters of Treg Hhex Expression Is Inhibited by the TGF-β/Smad3 Signaling Pathway. signature genes such as Il2ra and Ctla4. Furthermore, Hhex- The low expression of Hhex in Th17 and Treg cells, which require overexpressing Treg cells showed lower expression of Foxp3 and TGF-β for differentiation, prompted us to investigate whether Treg signature genes and could not prevent mouse inflammatory TGF-β regulates Hhex expression. Naïve CD4 T cells were cul- bowel disease (IBD). These results strongly suggest that Hhex is an tured under anti-CD3/anti-CD28 stimulation with IL-2 (1 ng/mL) important negative regulator of the Treg lineage. and the indicated concentrations of TGF-β (Fig. 1D), and Hhex

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+ − + + Fig. 1. Expression of Hhex is low in Treg cells and is repressed by TGF-β/Smad3 signaling. (A) CD4 CD25 (Tconv) and CD4 CD25 (Treg) cells were sorted from mouse spleens and mLNs. Hhex mRNA expression was measured by qRT-PCR. (B and C) Naïve CD4 T cells were isolated from splenocytes and differ- entiated into various CD4 T cell subsets (Th1, Th2, Th17, and Treg) for 3 d. Relative Hhex mRNA expression was determined by qRT-PCR (B), and Hhex protein expression was detected by immunoblot analysis (C). (D) Naïve CD4 T cells were cultured for 3 d under Th0 conditions in the presence of the indicated concentrations of TGF-β. Relative expression of Foxp3 and Hhex mRNA was measured by qRT-PCR. (E) Transactivation of the Hhex promoter by Smad proteins was measured by transient reporter assay. EL4 cells were transfected with the Hhex promoter–luciferase (LUC) reporter construct together with an empty vector (ctrl) or expression vectors for Smad2d and Smad4 or Smad3d and Smad4. Promoter activities are shown as the fold change (FC) relative to the ctrl vector. All error bars indicate the SD, and P values were calculated using Student’s t tests. *P < 0.05, **P < 0.01.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1907224116 Jang et al. Downloaded by guest on September 24, 2021 and Foxp3 mRNA levels were examined. The expression of Foxp3 in Hhex-transduced Th17 cells (SI Appendix,Fig.S3B and C). To was increased by TGF-β,asreported(17),butHhex expression was examine whether altered IFN-γ expression affects Hhex-mediated significantly decreased by TGF-β in a concentration-dependent suppression of Foxp3, we blocked IFN-γ activity with a neutraliz- manner (Fig. 1D). ing during iTreg differentiation (SI Appendix,Fig.S4). In Treg cells TGF-β signaling is transmitted through the Anti–IFN-γ antibody only slightly increased Foxp3 expression in phosphorylation of Smad3, but not Smad2, and its binding with a both control and Hhex-overexpressing iTreg cells (SI Appendix, common partner, Smad4 (19, 35). We made a reporter construct Fig. S4A). The inhibition of Foxp3 by Hhex was maintained, re- containing the Hhex promoter (nucleotides −309/+22, as pre- gardless of IFN-γ presence (SI Appendix,Fig.S4B), and the extent viously reported) (36) and measured the transactivation of the of the Hhex effect also did not differ much (SI Appendix,Fig. Hhex promoter after ectopic expression of various Smad proteins S4C). Together, Hhex increases the expression of IFN-γ in dif- (Fig. 1E). Expression of Smad3 along with Smad4 significantly ferentiating CD4 T cells, while IFN-γ is not critical for suppression repressed the activity of Hhex promoter, but expression of Smad2 of Foxp3 expression. along with Smad4 did not affect the promoter activity. Taken together, these results demonstrate that TGF-β inhibits expres- Hhex Inhibits the Immunosuppressive Function of Treg Cells. To ex- sion of Hhex in CD4 T cells through Smad3. amine whether Hhex affects the function of Treg cells, in vitro immunosuppression assays were performed. iTreg and tTreg cells Hhex Negatively Regulates the Differentiation and Stability of Treg were transduced with either the empty vector or the Hhex ex- + Cells. To characterize the role of Hhex in Treg differentiation, pression vector, and GFP , successfully transduced cells, were naïve CD4 T cells were transduced with a retroviral vector con- sorted. Murine naïve CD4 T cells were labeled with PKH26, a taining GFP and the Hhex gene during their in vitro differentiation lipophilic red fluorescent dye, and mixed with the transduced Treg into Treg cells. Foxp3 expression was dramatically decreased in cells in various ratios. The cocultures were incubated for 3 d in the Hhex-transduced Treg cells compared with empty vector-transduced presence of anti-CD3/anti-CD28 beads, and the proliferation of (control) Treg cells (Fig. 2 A–C). However, the expression of the naïve CD4 T cells was measured. Both Hhex-overexpressing Foxp3 in GFP (nontransduced) cells was not different between iTreg and tTreg cells failed to effectively suppress the proliferation Hhex-transduced and control cells, suggesting that the effect of of naïve CD4 T cells, even at a low naïve T cell:iTreg cell ratio Hhex on Treg cells is cell intrinsic. In Hhex-transduced cells, the (Fig. 3 A and B), suggesting that their suppressive function higher the GFP expression, the lower was the expression of Foxp3 was impaired. (Fig. 2A). Moreover, Treg signature genes, including Il2ra and Next, the function of Hhex-overexpressing Treg cells was ex-

Ctla4, were markedly reduced in Hhex-overexpressing Treg cells amined in vivo using a transfer-induced colitis model, a mouse IMMUNOLOGY AND INFLAMMATION + + (Fig. 2D). These genes are directly involved in both the develop- model of IBD. CD4 CD62L CD45RBhi naïve CD4 T cells were ment and function of Treg cells (13, 14). The expression of Il17a,a transferred into RAG1-deficient recipient mice alone or in com- + + Th17 cytokine, was also decreased by Hhex, but the expression of bination with CD4 CD25 tTreg cells (control or Hhex over- Ifng, a Th1 cytokine, was increased (Fig. 2D). Cell proliferation and expressing). Transfer of control tTreg cells along with naïve CD4 apoptosis of Hhex-transduced Treg cells, examined by Ki-67 and T cells efficiently blocked the signs of colitis, including weight loss Annexin V staining, were not different from those of controls (Fig. (Fig. 3C), colon swelling, splenomegaly (Fig. 3D), and infiltration 2 E and F), The proliferation rates examined by CellTrace Violet of the lamina propria by inflammatory cells (Fig. 3E). However, (CTV) in control and Hhex-overexpressing Treg cells stimulated Hhex-expressing tTreg cells failed to suppress these inflammatory with anti-CD3/CD28 beads were comparable (Fig. 2G). These phenotypes (Fig. 3 B–D). Adoptively transferred naïve CD4 T cells + results suggest that the decreased Foxp3 expression in Hhex- differentiated into effector CD4 T cells, including IFN-γ Th1 and + transduced Treg cells was not due to cell proliferation or death. IL-17a Th17 cells, when they were injected alone (Fig. 3 F and Next, the effect of Hhex overexpression was examined in tTreg G). Cotransfer of control tTreg cells successfully suppressed the + + cells, which had already developed in vivo. CD25 CD4 tTreg differentiation of naïve CD4 T cells, but transfer of Hhex- cells were isolated from mouse spleens and transduced with the overexpressing tTreg cells did not (Fig. 3 F and G), suggesting Hhex expression vector. Hhex also reduced Foxp3 expression that Hhex impairs the suppressive function of tTreg cells in vivo. significantly in tTreg cells, although the reduction was not as great Together, these results suggest that Hhex inhibits the immuno- as in iTreg cells (SI Appendix,Fig.S2A and B). This was an in- suppressive function of Treg cells both in vitro and in vivo. teresting finding, because tTreg cells are known to be very stable (7). Treg signature genes, including Il2ra, Ctla4,andIcos, were also Hhex Expression Is Increased under Inflammatory Conditions. Treg decreasedbyHhex(SI Appendix,Fig.S2C). ICOS is involved in cells can lose Foxp3 expression and suppressive activity under Treg cell function and is also used as a marker of highly sup- inflammatory conditions (5, 39, 40). Our data above suggest that pressive T cells (12, 37, 38). By contrast, Ifng expression was in- Hhex may play a role in type I immune responses. To examine creased in response to Hhex expression (SI Appendix,Fig.S2C). the role of Hhex in Treg cells under physiologically relevant Collectively, these data indicate that Hhex interferes with the conditions, we performed delayed-type hypersensitivity (DTH) differentiation of iTreg cells and disrupts the stability of tTreg cells reaction, a type I immune response. Sensitization (day 0) and by reducing the expression of Foxp3 and Treg signature genes. challenge (day 7) with topical application of oxazolone on mice back skin induced epidermal thickening and increased cell in- Hhex Promotes a Th1-Like Phenotype in CD4 T Cells. In both iTreg filtration in the skin at day 9 (Fig. 4A). The total number of CD4 + cells and tTreg cells, ectopic expression of Hhex induced Ifng T cells and the proportion of IFN-γ cells among the CD4 T cells while reducing Foxp3 (Fig. 2D and SI Appendix,Fig.S2C). were increased in the draining lymph nodes, indicating that type Therefore, we examined the effect of Hhex on effector CD4 T cell I inflammatory responses were developed in the mice (Fig. 4 B differentiation by overexpressing Hhex in Th0 cells, obtained by and C). Treg cells were isolated from the draining lymph nodes culturing with only anti-CD3/anti-CD28 stimulation and IL-2 of control and DTH-induced mice, and gene expression of Hhex, (1 ng/mL). Only Ifng was increased, while Il4, Il17a,andFoxp3 Foxp3, and Ifng was measured by qRT–PCR. Expression of Hhex were all decreased (SI Appendix,Fig.S3A). Notably, Tbx21 was increased in Treg cells under the inflammatory environment (encoding T-bet) expression was not altered (SI Appendix,Fig. (Fig. 4D). In addition, Foxp3 expression was decreased (Fig. 4E) S3A). Hhex was also introduced into Th1 and Th17 cells. Con- and Ifng expression was increased (Fig. 4F). These results show sistent with the above results, expression of IFN-γ was increased in that Hhex in Treg cells may play a role under physiological Hhex-transduced Th1 cells, while IL-17a expression was decreased inflammatory conditions.

Jang et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 24, 2021 A lrtc xehH B 100 ctrl Hhex 73.8% 74.3% 65.9% 12.7% **** /GFP- /GFP+ /GFP- /GFP+ 80 Foxp3

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Fig. 2. Hhex overexpression inhibits the expression of Foxp3 and Treg signature genes. (A–F) Naïve CD4 T cells were stimulated for 24 h and transduced with a retroviral vector expressing GFP with or without (ctrl) Hhex. Cells were cultured for an additional 2 d under iTreg differentiation conditions. Foxp3 ex- + + + pression was detected by flow cytometry, and the ratio of Foxp3 cells among GFP (transduced) cells is shown (A). GFP cells were sorted, and the relative amount of Foxp3 protein was detected by immunoblot analysis (B). The expression of Hhex, Foxp3, and other Treg signature genes was measured by qRT-PCR + (C and D). Proliferation of the GFP cells was determined by Ki-67 staining (E). Apoptosis was measured by Annexin V staining (F). (G) Transduced iTreg cells were stained with CTV and cultured for an additional 4 d in the presence of anti-CD3/CD28 beads and IL-2. All error bars represent the SD, and P values were calculated using Student’s t tests. *P < 0.05, ***P < 0.001, ****P < 0.0001. n.d., not detected.

Hhex Directly Binds to and Represses Foxp3 and Other Treg Signature defined. Some studies suggested that repeats of core 5′-ATTA-3′ Genes. Hhex is a DNA-binding transcription factor that has been or 5′-TAAT-3′ sequences are important for the binding specificity characterized mostly as a repressor (27). Hhex binds to DNA with of Hhex (31, 34). We found multiple repeats of these sequences at its homeodomain, but the target sequence has yet to be accurately the Foxp3 locus, so we investigated whether Hhex regulates Foxp3

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1907224116 Jang et al. Downloaded by guest on September 24, 2021 ABC100 100 120 * ** ** 80 110 80 60 100

40 90 60 ctrl Treg naive T Hhex Treg 20 80 naive T + ctrl tTreg proliferated cells (%) proliferated cells (%)

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Fig. 3. Hhex impairs the immunosuppressive function of Treg cells in vitro and in vivo. (A and B) The in vitro suppressive activity of Treg cells was measured by analyzing the PKH26+ proliferating cells among naïve CD4 T cells cocultured with the indicated ratios of iTreg cells (A) or a 1:1 ratio of tTreg cells (B). Empty vector (ctrl)- or Hhex-transduced (GFP+) Treg cells were sorted for the assay. (C–G) In vivo suppressive activity was assessed in an inflammatory bowel disease + + model. CD4 CD25 tTreg cells were sorted from mouse splenocytes and retrovirally transduced with an empty vector (ctrl) or a Hhex expression vector. Naïve + − + − − CD4 T (CD4 CD25 CD62L CD45RBhi) cells were administered to RAG1 / mice alone or together with ctrl-transduced or Hhex-expressing Treg cells. Mice were killed at day 22. (C) Weight loss of mice. (D) Morphology of colons, spleens, and mLNs. (E) Hematoxylin and eosin (H&E) staining of colon sections. (Scale bar, 200 μm.) (F and G) Populations of cytokine-expressing effector T cells in the spleen and mLNs. All error bars represent the SD, and P values were calculated using Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

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Fig. 4. Hhex expression is increased under type I inflammatory conditions in vivo. DTH reaction was induced by double topical applications of oxazolone (day 0, sensitization and day 7, challenge) on the mice back skin. The mice were killed at 2 d after challenge and analyzed for disease phenotypes. (A) H&E staining of back skin section. (B) Total CD4 T cell numbers in draining lymph nodes (dLNs) of untreated control and oxazolone-treated DTH groups were counted. (C) + + Populations of cytokine-expressing CD4 T cells in dLNs. (D–F) CD4 CD25 Treg cells were isolated from dLNs of control and DTH mice. Total RNA was extracted and expression of Hhex, Foxp3, and Ifng was measured by qRT-PCR. All error bars represent the SD, and P values were calculated using Student’s t tests. *P < 0.05, **P < 0.01.

gene expression through direct binding, using chromatin immu- expression of Foxp3, IL2ra, Ctla4,andIcos by directly binding noprecipitation (ChIP) assays. A mutant form of Hhex lacking the to their gene loci. homeodomain (ΔHD), the DNA-binding domain, was used as a negative control. Both the WT and ΔHD Hhex genes were FLAG Each of the Hhex Domains Has a Specific Role in Inhibiting Treg tagged at the C terminus. An anti-FLAG antibody was used to Differentiation. Hhex consists of 3 major domains, an N-terminal precipitate WT or ΔHD Hhex from transduced iTreg cells. WT proline-rich (NT) domain, which represses transcriptional activ- Hhex, but not ΔHD Hhex, bound directly to the Foxp3 promoter ity, the homeodomain (HD), which binds to DNA, and an acidic at CNS1 and CNS2, but not at CNS3 (Fig. 5A). Hhex also bound C-terminal (CT) domain, which induces transcriptional activity to the promoters of some Treg signature genes, including Il2ra, (27, 41, 42). The first 50 amino acids of the NT domain also have a Ctla4,andIcos (Fig. 5B). These results suggest that Foxp3 and role in the dimerization of Hhex (43). Therefore, Hhex was di- some Treg signature genes are direct targets of the transcription vided into 4 domains (NT1, NT2, HD, and CT) (Fig. 6A), and 4 factor Hhex. mutants were constructed with each domain deleted (ΔNT1, Next, transient reporter assays were used to examine whether ΔNT2, ΔHD, and ΔCT). To characterize the function of each binding of Hhex regulates the activity of target gene promoters. domain in Treg differentiation, each mutant Hhex construct was EL4 T cells were transfected with reporter constructs containing transduced into naïve CD4 T cells, and the cells were differenti- the promoter of each gene together with an empty vector or a ated into iTreg cells. In contrast to the WT protein, the 4 mutant Hhex expression vector. Stimulation with phorbol 12-myristate proteins could not inhibit Foxp3 expression in iTreg cells (Fig. 6 B– 13-acetate (PMA) and ionomycin was used to mimic activation E). The NT and HD, but not the CT, domains were essential for by TCR signaling and costimulation. With or without CNS1, the the inhibition of the Treg signature genes Il2ra and Ctla4 (Fig. 6E). promoter activity of Foxp3 was significantly decreased by Hhex Hhex can interact with many cellular proteins, mainly through overexpression (Fig. 5C). Other binding targets, such as the its NT domain (27). We therefore investigated whether Hhex promoters of IL2ra, Ctla4, and Icos, were also inhibited by Hhex physically interacts with Foxp3 and regulates its activity. Coim- (Fig. 5D). The Tnfrsf18 promoter, where Hhex binding was not munoprecipitation (co-IP) assays revealed a binding interaction detected, was not affected by Hhex transfection (Fig. 5 B and D). between Hhex and Foxp3 (Fig. 6F). To examine which domain Taken together, these results suggest that Hhex regulates the of Hhex is important for binding to Foxp3, co-IPs were performed

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1907224116 Jang et al. Downloaded by guest on September 24, 2021 A 0.3 ** B 0.3 ** ctrl * Hhex ** ** ** Hhex dHD 0.2 ** 0.2 *** *** ** *** *** ns % input ns % input 0.1 0.1 * *

0.0 0.0 promoter CNS1 CNS2 CNS3 CD25 P Ctla4 P Gitr P Icos P sucol3pxoF seneggerT C D Foxp3 P Foxp3 P + CNS1 CD25 P Ctla4 P Gitr P Icos P 3 3 ** 4 5 4 4 nontreated *** stim. 4 3 ** 3 3 2 * 2 3 *** 2 2 2 2 1 1 1 1 1 1

0 0 0 0 0 0 relative luciferase activity (FC) relative luciferase activity (FC) ctrl Hhex ctrl Hhex ctrl Hhex ctrl Hhex ctrl Hhex ctrl Hhex IMMUNOLOGY AND INFLAMMATION

Fig. 5. Hhex binds to and suppresses the Foxp3 locus and promoters of Treg signature genes. (A and B) Binding of Hhex to the Foxp3 locus and promoters of Treg signature genes was measured by ChIP. iTreg cells transduced with empty (ctrl), Hhex-FLAG, or HhexΔHD-FLAG (without the homeodomain) expression vectors were used. Enrichment of the target DNA was quantified by qPCR following immunoprecipitation with a FLAG antibody. (C and D) Transactivation of the promoters of Foxp3 and Treg signature genes by Hhex was measured by transient reporter assay. EL4 cells were transfected with Foxp3P-, Foxp3P+CNS1-, CD25P-, Ctla4P-, GitrP-, or IcosP–LUC reporter constructs together with an empty vector (ctrl) or a Hhex expression vector. After 16 h, cells were left untreated (nontreated) or were stimulated with PMA and ionomycin (stim.). Promoter activity is shown as the fold change (FC) relative to the nontreated ctrl. All error bars indicate the SD, and P values were calculated using Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

with full-length Hhex and the 4 mutants. Deletion of the NT2 Hhex Deficiency Does Not Alter the Development and Function of domain of Hhex caused loss of the interaction with Foxp3 (Fig. Treg Cells. To investigate how Hhex deficiency affects Treg cells, 6G), suggesting that Hhex directly binds to Foxp3 through its NT2 T cell phenotypes were analyzed in Hhexfl/fl Foxp3-Cre conditional domain. To further investigate whether Hhex binding impacts the knockout (Hhex cKO) mice. Development of CD4, CD8, and Treg stability of Foxp3 protein, we coexpressed WT or ΔNT2 Hhex with cells in the thymus and spleen of Hhex cKO mice was not different Foxp3 and measured the stability of Foxp3 by immunoblot assay from that of Hhexfl/fl littermate control mice (SI Appendix,Fig.S6 after treating with a translation inhibitor, cycloheximide (SI Ap- A–G). B, NK, and NKT cell populations in the spleen were also pendix,Fig.S5). The stability of Foxp3 protein was not altered by not affected (SI Appendix,Fig.S6F). In addition, Hhex deficiency Hhex binding. did not affect Foxp3 expression or cell proliferation during in vitro Since Foxp3 has been reported to induce CD25 and CTLA4 differentiation of Treg cells (SI Appendix,Fig.S6H and I). The expression in Treg cells through direct binding to their genes immunosuppressive function of iTreg cells was also not altered by (11), we examined whether Hhex and its physical interaction Hhex deficiency (SI Appendix,Fig.S6J). The finding that Hhex with Foxp3 affects Foxp3-dependent transactivation of the deficiency did not affect the differentiation and function of Treg Il2ra and Ctla4 genes. The full-length Hhex or 4 domain- cells may be due to its low expression in Treg cells. deleted mutants of Hhex were transfected into EL4 T cells together with Foxp3 and reporter constructs containing the Discussion promoters of Il2ra and Ctla4. Full-length Hhex significantly Since Foxp3 is the most specific marker for characterizing Treg repressed the activity of the Il2ra and Ctla4 promoters even in cells, the terms that indicate the functional status of Treg cells are the presence of Foxp3 (Fig. 6 H and I). However, the mutant also based on Foxp3 expression. Treg cell “differentiation” is de- + Hhex proteins were only able to partially repress these target termined by whether non-Treg cells become Foxp3 Treg cells; + genes compared with the full-length Hhex. All 4 domains of Treg cell “stability” is determined by whether Foxp3 Treg cells − Hhexcontributedtotheregulation of Foxp3 target genes. become Foxp3 ex-Treg cells; and Treg cell “plasticity” is de- These results suggest that all 4 domains of Hhex (the HD, as a termined by whether Treg cells undergo functional changes while DNA-binding domain, the NT, as a repression domain, and the maintaining Foxp3 expression (7). In this study, we show that CT, limited role in repressing Foxp3 but critical for binding to Hhex, as a critical inhibitor of Foxp3, regulates the differentiation, the promoters of Foxp3 target genes) are critical for inhibiting stability, and plasticity of Treg cells. Retroviral overexpression Foxp3 and Treg signature genes, and NT2 serves as an in- of Hhex inhibited Foxp3 induction during iTreg differentiation. + terface for protein–protein interactions with Foxp3. In tTreg cells, Hhex reduced Foxp3 cell numbers. Finally,

Jang et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 24, 2021 A N-ter Homeodomain C-ter C **

NT1 NT2 HD CT 80 ****

0 50 137 197 271 60 B ctrl Hhex ΔNT1 40 74.3% 12.7% 75.6% /GFP+ /GFP+ /GFP+ 20

Foxp3+ / GFP+ cells (%) 0 T ex T1 D ctrl h dC H dD dDT2 dH

D ΔNT2 ΔHD ΔCT 68.6% 74.6% 55.6% ctrl Hhex ΔNT1 ΔNT2 ΔHD ΔCT

/GFP+ /GFP+ /GFP+ 3pxoF Foxp3

β-Actin

GFP

E Foxp3 Il2ra Ctla4 Tnfrsf18 F normal Input αFoxp3 5 0.3 20 400 IgG

4

) 15 300 HHEX 0.2 3

gapdh 10 200 αFLAG normal 2 Input 0.1 (HHEX) IgG (gene/ 5 100 1 relative expression Foxp3 0 0.0 0 0 T x 2 T rl 1 2 T ex T1 T2 D T1 D t ex T D trl ex D ctrl dC ctrl N dC c h dCT c dC Hh dN dN dH Hhe d dNT dH H dN dNT dH Hh dNT1 dNT2 dH

GHIFoxp3+ +++++ Hhex WT ೚NT1 ೚NT2 ೚HD ೚CT - CD25 promoter activity Ctla4 promoter activity IP(αFLAG) Foxp3 + ctrl Foxp3 + ctrl αFoxp3 Foxp3 + Hhex **** Foxp3 + Hhex *** Foxp3 + dNT1 ** Foxp3 + dNT1 ns Input Foxp3 + dNT2 **** Foxp3 + dNT2 *

αFLAG Foxp3 + dHD * Foxp3 + dHD ns (Hhex) Foxp3 + dCT ** Foxp3 + dCT ** 0.0 0.2 0.4 0.6 0.8 1.0 0.00.20.40.60.81.0

Fig. 6. Domain-deleted forms of Hhex lose their effects in Treg cells. (A) Schematic diagram of Hhex domains showing the NT1 (N-terminal 1), NT2 (N- terminal 2), HD (homeodomain), and CT (C-terminal) domains. (B–E) Retroviral vectors encoding WT Hhex or deletion mutants of Hhex without each domain + + + (ΔNT1, ΔNT2, ΔHD, or ΔCT) were transduced into iTreg cells. Foxp3 cells were measured by flow cytometry (B), and the ratio of Foxp3 cells among GFP cells + is shown (C). Error bars represent the SD, and P values were calculated using Student’s t tests. **P < 0.01, ****P < 0.0001. GFP cells were sorted, and the relative amount of Foxp3 protein was measured by immunoblot analysis (D). mRNA levels of Foxp3 and other Treg signature genes were measured by qRT- PCR (E). (F) Binding of Hhex and Foxp3 was determined by co-IP. Hhex-FLAG and Foxp3 were overexpressed in 293T cells. Cell lysates were immunopreci- pitated with anti-Foxp3, anti-FLAG (for Hhex), or normal IgG (as a negative control). Then, the proteins were immunoblotted with anti-Hhex or anti-Foxp3 . (G) WT Hhex-FLAG, ΔNT1-FLAG, ΔNT2-FLAG, ΔHD-FLAG, or ΔCT-FLAG was cotransfected along with Foxp3 into 293T cells. Cell lysates were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated proteins were detected with an anti-Foxp3 antibody. Input cell lysates were immunoblotted with anti-Foxp3 and anti-FLAG antibodies. (H and I) Transactivation of the promoters of Il2ra and Ctla4 by Foxp3 and Hhex was measured by transient reporter assay. EL4 cells were transfected with CD25P- or Ctla4P–LUC reporter constructs together with a Foxp3 expression vector and a vector expressing full-length or domain-deleted forms of Hhex (dNT1, dNT2, dHD, or dCT). Promoter activities are shown as the fold change (FC) relative to ctrl vector-transduced cells. Error bars represent the SD, and P values were calculated using Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1907224116 Jang et al. Downloaded by guest on September 24, 2021 Hhex-overexpressing iTreg cells and tTreg cells lost their Here, we show that Hhex expression is inhibited by TGF-β in immunosuppressive functions, gaining a Th1-like phenotype. mouse CD4 T cells. The promoter activity of Hhex (nucleotides Our studies reveal several mechanisms by which Hhex can −309/+22, not containing the BRE) was repressed by the active negatively regulate Treg cells. First, Hhex directly bound to the form of Smad3, but not by active Smad2, suggesting a mecha- Foxp3 locus to regulate its expression. Specifically, the activity of nism of TGF-β–dependent regulation of Hhex. the Foxp3 promoter and CNS1 was almost completely inhibited Homeobox proteins are a large family of transcription factors by Hhex binding, which seems to be the most important mech- with a highly conserved 60-amino acid long DNA-binding do- anism for Hhex regulation of Treg cells. Second, Hhex physically main known as the homeodomain. However, only a few ho- interacted with Foxp3. Previous studies suggest that transcription meobox proteins have been reported to have a role in CD4 factors including GATA3, RORγt, and YY1 inhibit the function T cells, one of which is Hlx. Hlx is up-regulated during Th1 cell of Foxp3 via protein–protein interactions (26, 44, 45). We add differentiation and induces IFN-γ expression synergistically with Hhex in the list of Foxp3-interacting proteins. Third, Hhex di- T-bet (50). Moreover, retroviral overexpression of Hlx alone also rectly binds to the promoters of Treg signature genes through its can induce IFN-γ expression in naïve T cells differentiated under HD domain, such as Il2ra and Ctla4. This suggests that Hhex can Th2-polarizing conditions (51). We obtained similar results when also act as a Foxp3-independent Treg cell regulator. Foxp3, Il2ra, we overexpressed Hhex in CD4 T cells. Hhex induced the ex- and Ctla4 have been reported as Treg-specific superenhancers. pression of IFN-γ in Th0 cells, iTreg cells, and even tTreg cells. They may easily be targets of the same transcription factor. It will The expression of T-bet was not altered. Hlx and Hhex, as ho- be interesting to see if Hhex plays a role in the regulation of the meobox proteins, may play a similar role in CD4 T cells. Further superenhancers by directly binding to them. studies on the role of Hhex in Th1 cells and Hlx in Treg cells will Our Hhex domain mutant study suggests specific functions of be informative. each domain. NT has been shown to exert repressive function by This study reveals a role of Hhex in mature T cells, while other protein–protein interactions (27), and NT2 is shown to be critical studies have examined the effect of Hhex overexpression in for interaction with Foxp3 in our study. HD has been shown to T cell lineages. Transgene-driven expression of Hhex in thymo- directly bind to DNA (27), and our data support that it is re- cytes using the Lck promoter disrupted normal T cell maturation quired for Hhex binding to the Foxp3 locus. CT, which has been (52). Retrovirally transduced Hhex-overexpressing hematopoi- shown to exert activation function (27), has the lowest effect in Hhex function in our study, since the ΔCT mutant only slightly etic precursor cells generated immature T lymphocyte-derived affected Foxp3 expression. Therefore, it seems that each domain lymphomas in bone marrow-recipient mice (17). Thus, we ex- of Hhex plays differential and nonredundant roles in Treg amined the proliferation and apoptosis of Hhex-transduced Treg IMMUNOLOGY AND INFLAMMATION function and it indicates that each mutant has partial effects in cells, both of which were found to be similar to those of controls. Foxp3 expression and its activity. However, summation of these There were also no side effects in mice adoptively transferred effects may explain the full function of Hhex. The reason that the with Hhex-overexpressing tTreg cells. Hhex mutants did not show dominant-negative effect is not clear. In conclusion, we identified Hhex as a negative regulator of One possible explanation is the low expression of Hhex in Treg Treg cells. Hhex directly inhibits Foxp3 and other Treg signature cells. Because of low activity of Hhex in Treg cells, additional genes. This study suggests a molecular mechanism of Treg cell Hhex mutants may not cause any further effect. regulation, which may be relevant for the development of ther- Some of the initial studies on Hhex reported that it is not de- apeutic strategies for Treg cell-related diseases, including cancer. tected in T cell lineages (30, 32). However, here we detected protein and mRNA expression of Hhex in CD4 T cells. Hhex Methods expression was lower in Treg cells. Consistent with its low ex- Details of mice, isolation, and culture of mouse CD4 T cells, RNA isolation, and pression in Treg cells, Foxp3-dependent Hhex-deficient mice did qRT-PCR, immunoblot analysis, transient reporter assay, intracellular flow not show any phenotype. We also determined that TGF-β inhibits cytometry, retroviral transduction, in vitro suppression assay, mouse in- Hhex expression in CD4 T cells via Smad3 activation. In CD4 flammatory bowel disease model, mouse delayed type hypersensitivity β model, ChIP assays, and Co-IP assays are described in SI Appendix. All animal T cells, TGF- is known to induce Treg differentiation but inhibits experiments were approved by the institutional animal care and use com- Th1 differentiation (17, 46). In this context, it is conceivable that mittees of Sogang and Yale universities. TGF-β suppresses Hhex, which inhibits Treg differentiation, leading to Th1 differentiation. Inhibition of Hhex could be a sig- Data Availability. All data needed to evaluate the conclusions in the paper are nificant role of TGF-β in the development of Treg cells. present in the paper, SI Appendix, and a dataset in the Gene Expression There have been studies in several species on the mechanisms Omnibus (GEO) database, http://www.ncbi.nlm.nih.gov/geo (accession no. by which TGF-β and Smad proteins regulate Hhex. In human GSE139297). lung fibroblasts, TGF-β1 causes Hhex expression through miR- 21–3p induction (47). In a study of bone morphogenetic proteins ACKNOWLEDGMENTS. This work was supported by the National Research (BMPs), Smad1, but not Smad2 and Smad3, was found to in- Foundation of Korea (NRF) grants funded by the Korean government (NRF-2017R1A2B3008621 and NRF-2015M3C9A2054020 to G.R.L., and teract with the BMP-responsive element (BRE) in the mouse 2014H1A8A1022457 to S.W.J.). This work was supported by the Howard Hhex gene to induce Hhex expression (48). In Xenopus tropicalis, Hughes Medical Institute (R.A.F.). S.S.H. was supported by a Leslie H. ChIP assays using anti-Smad2/3 pulled down the hhex locus (49). Warner Fellowship from the Yale Cancer Center.

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