The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus This information is current as of September 26, 2021. Kazue Tsuji-Takayama, Motoyuki Suzuki, Mayuko Yamamoto, Akira Harashima, Ayumi Okochi, Takeshi Otani, Toshiya Inoue, Akira Sugimoto, Terumasa Toraya, Makoto Takeuchi, Fumiyuki Yamasaki, Shuji Nakamura and Masayoshi Kibata Downloaded from J Immunol 2008; 181:3897-3905; ; doi: 10.4049/jimmunol.181.6.3897 http://www.jimmunol.org/content/181/6/3897 http://www.jimmunol.org/ References This article cites 51 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/181/6/3897.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! 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The Journal of Immunology The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus Kazue Tsuji-Takayama,* Motoyuki Suzuki,* Mayuko Yamamoto,* Akira Harashima,* Ayumi Okochi,* Takeshi Otani,* Toshiya Inoue,* Akira Sugimoto,* Terumasa Toraya,* Makoto Takeuchi,* Fumiyuki Yamasaki,† Shuji Nakamura,1* and Masayoshi Kibata* STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pa- thology due to a reduced number of CD4؉CD25؉ naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production Downloaded from through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA http://www.jimmunol.org/ methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element. The Journal of Immunology, 2008, 181: 3897–3905. he STAT molecules play key roles in intracellular signal revealed the contribution of STAT5 molecules to nTreg develop- transduction after cellular stimulation by cytokines, ment because a subset of these mice exhibited autoimmune pa- T growth factors, and hormones. Among seven mammalian thology very similar to IL-2 or IL-2R knockout mice (7). A human by guest on September 26, 2021 family members, two highly related STAT5 gene products, patient with a STAT5b mutation displayed immune dysregulation STAT5a and STAT5b, have been of particular interest because associated with decreased numbers and impaired suppressive abil- their activation can be induced by a wide spectrum of cytokines. ity of CD4ϩCD25high cells, indicating both developmental and Recently, especially in regulatory T (Treg)2 biology, STAT5 pro- functional defects of nTreg cells (8). An essential role of STAT5 teins have been recognized as particularly important due to their molecules in the IL-2/IL-2R signaling pathway was confirmed by essential downstream roles in the IL-2/IL-2R signaling pathway experiments using mice with an IL-2R␤ mutation, which exclu- (1–4). Naturally occurring Treg (nTreg) cells are generated and sively activates STAT5 (4). Therefore, the IL-2-STAT5 signaling maintained in an IL-2-dependent manner. This was demonstrated axis contributes to some of the essential properties of nTreg cells. in studies of mice deficient for IL-2, IL-2R␣, and IL-2R␤, in which Furthermore, one result of the IL-2-STAT5 signaling axis is the autoimmune diseases developed due to lower numbers of nTregs enhancement of nTreg-specific gene expression, the transcription (5, 6). Also, studies of STAT5a and STAT5b double-knockout mice factor FOXP3 (4). It has been shown that STAT5 molecules di- rectly control FOXP3 gene expression, and STAT consensus se- quences were identified within promoter or intron regions (4, 9). *Cell Biology Institute, Research Center, Hayashibara Biochemical Laboratories, Fu- jisaki, Okayama, Japan; and †Kurashiki Medical Center, Bakuro-cho, Kurashiki, Although roles of IL-2/IL-2R signaling were firmly established in Japan nTreg cells, its roles in other types of Treg cells are poorly Received for publication April 15, 2008. Accepted for publication July 10, 2008. understood. The costs of publication of this article were defrayed in part by the payment of page IL-10 is an inhibitory cytokine mediating suppression by regu- charges. This article must therefore be hereby marked advertisement in accordance latory or suppressor T cells (10). Despite its essential roles in im- with 18 U.S.C. Section 1734 solely to indicate this fact. mune responses, the mechanisms regulating IL-10 production are 1 Address correspondence and reprint requests to Dr. Shuji Nakamura, Cell Biology not well understood. We previously reported that IL-2 was effec- Institute, Research Center, Hayashibara Biochemical Laboratories, 675-1 Fujisaki, Okayama 702-8006, Japan. E-mail address: [email protected] tive for enhancement of IL-10 production in certain types of Treg 2 Abbreviations used in this paper: Treg, regulatory T; ARRE, Ag receptor-responsive cells (11). Such cells are categorized as IL-10-producing Treg elements; ChIP, chromatin immunoprecipitation; CNS, conserved noncoding se- cells, including Tr1-like cells, IL-10-Treg, and a newly character- quences; conT, conventional T; DC, dendritic cell; HS, hypersensitivity; I-SRE-4, ized Treg cell line (designated HOZOT) with a phenotype of STAT-responsive element within intron 4; nTreg, naturally occurring regulatory T; ϩ ϩ P-SRE, STAT-responsive element within proximal region; pAb, polyclonal Ab; CD4 CD8 . In that study, we analyzed the mechanisms of high rVISTA, Regulatory Visualization Tools for Alignment; SRE, STAT-responsive el- IL-10 production by these Treg cells in comparison with other T ement; TSS, transcription start site; UCB, umbilical cord blood. cells, especially Th2 cells, which are typical high producers of Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 IL-10 among non-Treg cells. For Th2 cells, both transcriptional www.jimmunol.org 3898 Treg PRODUCED IL-10 THROUGH AN INTRONIC ENHANCER and epigenetic mechanisms have been reported in the regulation of Systems) and CD28 mAb (37407.111; R&D Systems) (CD3/CD28) in the IL-10 production. At the transcriptional level, transcriptional acti- presence of 10 ng/ml IL-2. The IL-10-producing Treg (IL-10-Treg) cell line was also obtained from vators, such as c-jun, jun B (12), and NF-AT (13), have been ϩ Ϫ CD4 CD25 mononuclear cells, as previously described (27). The cells documented as essential molecules for IL-10 production. At an were cultured on immature DC derived from UCB in the presence of 1 nM epigenetic level, a Th2-specific transcription factor, GATA-3, has vitamin D3 (Sigma-Aldrich) and 50 nM dexamethasone (Sigma-Aldrich). been described as a chromatin remodeling molecule. GATA3 can The cells were expanded for another 2 wk in the presence of vitamin D3/dexamethasone and IL-2. bind to DNase I hypersensitivity (HS) sites residing on both the ϩ Ϫ Ј The Th2 cell line was obtained from CD4 CD25 mononuclear cells, 5 -proximal region and intron 4, and then induce remodeling of as described previously (28). The cells were cultured with plate-bound chromatin structure by increasing histone acetylation (14). In this CD3/CD28 in the presence of 10 ng/ml human rIL-4 (PeproTech) and 4 situation, GATA-3 acts as a stabilizing factor, and not a transcrip- ␮g/ml anti-human IFN-␥ mAb (Hayashibara Biochemical Laboratories) tional activator, keeping an open chromatin configuration for the for 48 h. Then, the cells were expanded by addition of 10 ng/ml IL-2. After ␥ IL-10 gene. Our previous study indicated that, in contrast to Th2 6 days, the production of IL-10, IFN- , and IL-4 was assessed in the su- pernatants from the CD3/CD28-treated cells. cells, GATA-3 plays a less important role in IL-10-producing Treg Naive T cells were prepared as CD4ϩCD25Ϫ mononuclear cells. The cells (IL-10-Treg and HOZOT) because GATA-3 expression in percentage of CD45RAϩ cells was Ͼ90%.