Foxp3 Inhibits RORγt-mediated IL-17A mRNA Transcription through Direct Interaction with RORγt*

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg. The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg. T helper (Th) 3The abbreviations used are: Th, T helper; Th17, IL-17-producing helper T cell; TGF-β1, transforming growth factor-β1; CA-TβRI, constitutively activated TGF-β receptor I; Treg, regulatory T cell; iTreg, inducible Treg; IL, interleukin; RORγt, retinoic acid-related orphan receptor γt; RORE, ROR-responsive element; Foxp3, forkhead box protein 3; RT-PCR, reverse transcription-PCR; ChIP, chromatin immunoprecipitation; Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; NFAT, nuclear factor of activated T cells; ZnF, zinc finger; Zip, leucine zipper; FKH, forkhead domain; Luc, luciferase. cells play a pivotal role in adaptive immunity. Upon stimulation by antigens, Th cells undergo distinct developmental pathways, attaining specialized properties and effector functions. CD4+ T cells are traditionally thought to differentiate into Th1 and Th2 cell subsets (1Mosmann T.R. Coffman R.L. Annu. Rev. Immunol. 1989; 7: 145-173Crossref PubMed Scopus (6863) Google Scholar). Recently, a third subset of the polarized T cell subset Th17, characterized by the production of IL-17A, was identified and found to play an important role in autoimmune diseases, elimination of extracellular bacteria, and cancer (2Kikly K. Liu L. Na S. Sedgwick J.D. Curr. Opin. Immunol. 2006; 18: 670-675Crossref PubMed Scopus (267) Google Scholar, 3Aujla S.J. Dubin P.J. Kolls J.K. Semin. Immunol. 2007; 19: 377-382Crossref PubMed Scopus (230) Google Scholar, 4Kottke T. Sanchez-Perez L. Diaz R.M. Thompson J. Chong H. Harrington K. Calderwood S.K. Pulido J. Georgopoulos N. Selby P. Melcher A. Vile R. Cancer Res. 2007; 67: 11970-11979Crossref PubMed Scopus (79) Google Scholar). It has been shown that TGF-β1 plays a pivotal role in the induction of Th17 (5Li M.O. Wan Y.Y. Flavell R.A. Immunity. 2007; 26: 579-591Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Th17 differentiation of naive T cells is initiated by IL-6 and TGF-β1 (6Mangan P.R. Harrington L.E. O'Quinn D.B. Helms W.S. Bullard D.C. Elson C.O. Hatton R.D. Wahl S.M. Schoeb T.R. Weaver C.T. Nature. 2006; 441: 231-234Crossref PubMed Scopus (2611) Google Scholar, 7Bettelli E. Carrier Y. Gao W. Korn T. Strom T.B. Oukka M. Weiner H.L. Kuchroo V.K. Nature. 2006; 441: 235-238Crossref PubMed Scopus (5591) Google Scholar, 8Veldhoen M. Hocking R.J. Atkins C.J. Locksley R.M. Stockinger B. Immunity. 2006; 24: 179-189Abstract Full Text Full Text PDF PubMed Scopus (3020) Google Scholar). In addition, IL-23 as well as IL-21 are thought to be a key cytokine for the maturation and/or maintenance of Th17 cells (9McKenzie B.S. Kastelein R.A. Cua D.J. Trends. Immunol. 2006; 27: 17-23Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, 10Nurieva R. Yang X.O. Martinez G. Zhang Y. Panopoulos A.D. Ma L. Schluns K. Tian Q. Watowich S.S. Jetten A.M. Dong C. Nature. 2007; 448: 480-483Crossref PubMed Scopus (1237) Google Scholar, 11Korn T. Bettelli E. Gao W. Awasthi A. Jager A. Strom T.B. Oukka M. Kuchroo V.K. Nature. 2007; 448: 484-487Crossref PubMed Scopus (1514) Google Scholar). However, TGF-β1 is a suppressor of Th1 and Th2 cell differentiation and drives the conversion of naive T cells to Foxp3-positive Th cells with a regulatory phenotype, so-called inducible regulatory T cells (iTreg) (12Chen W. Jin W. Hardegen N. Lei K.J. Li L. Marinos N. McGrady G. Wahl S.M. J. Exp. Med. 2003; 198: 1875-1886Crossref PubMed Scopus (3821) Google Scholar). These observations suggest that the presence or absence of proinflammatory cytokines might lead to opposing immune consequences induced by TGF-β1. RORγt has been shown to direct Th17 cell differentiation by inducing the IL-23 receptor (13Ivanov I.I. McKenzie B.S. Zhou L. Tadokoro C.E. Lepelley A. Lafaille J.J. Cua D.J. Littman D.R. Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3988) Google Scholar). However, it has not been clarified whether RORγt is directly involved in IL-17A transcription. RORγt could be a STAT3 target since Th17 was not induced in STAT3-deficient T cells (14Yang X.O. Panopoulos A.D. Nurieva R. Chang S.H. Wang D. Watowich S.S. Dong C. J. Biol. Chem. 2007; 282: 9358-9363Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar). STAT3 binding to the RORγt promoter may enhance its expression. However, it has been reported that RORγt can be induced in CD4+ T cells in response to TGF-β1 (15Zhou L. Ivanov I.I. Spolski R. Min R. Shenderov K. Egawa T. Levy D.E. Leonard W.J. Littman D.R. Nat. Immun. 2007; 8: 967-974Crossref Scopus (1685) Google Scholar). Despite the existence of RORγt, naive T cells activated by TGF-β1 alone differentiate into iTreg but not Th17. The mechanism in which Th17 development is suppressed by TGF-β1 alone has not been clarified. CD4+CD25+Foxp3+ Tregs are crucial for the maintenance of immunological tolerance (16Schwartz R.H. Nat. Immun. 2005; 6: 327-330Crossref Scopus (194) Google Scholar, 17Hori S. Nomura T. Sakaguchi S. Science. 2003; 299: 1057-1061Crossref PubMed Scopus (51) Google Scholar). Treg produced in the thymus (naturally occurring Treg) constitutes 3–6% of CD4+ T cells (18Nelson B.H. J. Immunol. 2004; 172: 3983-3988Crossref PubMed Scopus (460) Google Scholar). More recent studies have shown that Foxp3 is induced in CD4+Foxp3– T cells after stimulation with TGF-β1 (19Zheng S.G. Wang J.H. Gray J.D. Soucier H. Horwitz D.A. J. Immunol. 2004; 172: 5213-5221Crossref PubMed Scopus (557) Google Scholar, 20Fu S. Zhang N. Yopp A.C. Chen D. Mao M. Chen D. Zhang H. Ding Y. Bromberg J.S. Am. J. Transplant. 2004; 4: 1614-1627Crossref PubMed Scopus (438) Google Scholar). Foxp3 functions as a transcriptional repressor for various transcription factors such as nuclear factor of activated T cells (NFAT), NF-κB, and Runx1 (21Bettelli E. Dastrange M. Oukka M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5138-5143Crossref PubMed Scopus (450) Google Scholar, 22Wu Y. Borde M. Heissmeyer V. Feuerer M. Lapan A.D. Stroud J.C. Bates D.L. Guo L. Han A. Ziegler S.F. Mathis D. Benoist C. Chen L. Rao A. Cell. 2006; 126: 375-387Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar, 23Ono M. Yaguchi H. Ohkura N. Kitabayashi I. Nagamura Y. Nomura T. Miyachi Y. Tsukada T. Sakaguchi S. Nature. 2007; 446: 685-689Crossref PubMed Scopus (512) Google Scholar), which could be a part of the mechanisms for Foxp3-medaited suppression of immune responses. In this study, using promoter analysis, we found that RORγt potently up-regulates IL-17A reporter activity without any other factors such as NFAT. We identified the two conserved ROR-responsive elements (ROREs) on the IL-17A promoter, which are essential for RORγt-mediated IL-17A promoter activation. Interestingly, we found that IL-6 is not necessary for the induction of RORγt; TGF-β1 alone can up-regulate RORγtin primary naive T cells as well as the EL4 T cell line. IL-6 rather suppressed Foxp3 expression in TGF-β1-treated naive T cells. Thus, we hypothesized that Foxp3 negatively regulates IL-17A transcription. We found that overexpression of Foxp3 reduced IL-17A expression in the EL4 cells and inhibited RORγt-mediated IL-17A promoter activity in the HEK 293T cells. This suppression was partly dependent on the physical interaction between RORγt and Foxp3. Our study provides a novel mechanism for the regulation of Th17 development by Foxp3. Mice—C57BL/6 mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Six- to twelve-week-old mice were used as experimental animals. All experiments were approved by the Animal Ethics Committee of Kyusyu University. Plasmid Constructions—PCR was done to generate the IL-17A promoter plasmid by using mouse genomic DNA as a template. In the case of IL-17A promoter, an ∼6-kb fragment corresponding to nucleotides from –6021 to +37 relative to the determined transcriptional staring site of IL-17A gene was subcloned into pGV-basic 2 vector (TOYOINKI). Series of deletion mutants were generated by restriction enzyme digestion (–1812, –613, –301) or by PCR (–239, –153, –115, –94). The deletion mutants, –1812, –613, and –301, were constructed by the use of the restriction sites Bpu1102I, SpeI, and PstI, respectively. The point mutations (TGCAAT or GTTGTA) in a putative RORE-1 (TGACCT) and RORE-2 (GTGTCA) were introduced by PCR. The point mutants were generated by using –239 plasmid as a template. The mouse RORγt cDNA was amplified by reverse transcription (RT)-PCR and was subcloned into pCMVT7 vector for T7 tag. The Foxp3 mutants were subcloned into pCMV14 vector (Sigma) for FLAG tag. A constitutively activated TGF-β receptor I (CA-TβRI) was provided from Dr. Joan Massagué and described previously (24Takaki H. Minoda Y. Koga K. Takaesu G. Yoshimura A. Kobayashi T. Genes Cells. 2006; 11: 871-882Crossref PubMed Scopus (46) Google Scholar). Transfection into HEK 293T cells and EL4 cells and luciferase assay were described previously (15Zhou L. Ivanov I.I. Spolski R. Min R. Shenderov K. Egawa T. Levy D.E. Leonard W.J. Littman D.R. Nat. Immun. 2007; 8: 967-974Crossref Scopus (1685) Google Scholar, 17Hori S. Nomura T. Sakaguchi S. Science. 2003; 299: 1057-1061Crossref PubMed Scopus (51) Google Scholar, 18Nelson B.H. J. Immunol. 2004; 172: 3983-3988Crossref PubMed Scopus (460) Google Scholar). Naive T Cell Preparation and Differentiation—CD4+CD25– T cells were isolated from spleens and lymph nodes by negative selection using magnetic beads (Milteny Biotech) (typically >95% purity). For Th17 differentiation, 1 × 106 CD4+CD25– T cells were cultured with 1 μg/ml plate-bound anti-CD3 antibody (Ab) (clone 145-2C11) and 0.5 μg/ml soluble anti-CD28 Ab (eBioscience) in the presence of 0.2 ng/ml recombinant murine and μg/ml Ab, μg/ml Ab in the presence of 2 ng/ml recombinant TGF-β1 and ng/ml recombinant IL-6 IL-6 was for iTreg differentiation Cell and cytokine were as described Y. Kobayashi T. K. R. M. T. K. T. T. T. K. Yoshimura A. J. Immunol. 2007; PubMed Scopus Google Scholar). Cell was by cell as described I. H. S. S. Yoshimura T. Koga K. H. K. Takaesu G. Kobayashi T. Yoshimura A. J. Exp. Med. 2006; PubMed Scopus Google Scholar). and cells were in for was with a The expression of was as an The were as GAPDH, and TGF-β1, and Foxp3, and RORγt, and IL-17A, and PCR was on cDNA using the The relative is as is the between the of of the and of the assay was using to the The cell were with Ab or for Ab and protein magnetic beads for The were for for reverse and with K. DNA were to PCR. The PCR for the IL-17A promoter region to and was using the and The PCR were using an was of were All shown in the in this are RORγt and Foxp3 by Naive T reported Foxp3 was induced in naive T cells in response to TGF-β1 IL-17A was induced and Foxp3 was reduced in response to TGF-β1 IL-6 However, the in which IL-17A production is has not been clarified. It has been that TGF-β1 IL-6 RORγt, which in IL-17A transcription. However, the that TGF-β1 alone also the expression of Foxp3 and RORγtin CD4+ T cells (15Zhou L. Ivanov I.I. Spolski R. Min R. Shenderov K. Egawa T. Levy D.E. Leonard W.J. Littman D.R. Nat. Immun. 2007; 8: 967-974Crossref Scopus (1685) Google Scholar). Foxp3 and RORγt are in a cell in the of CD4+ T cell the between Foxp3 and RORγt expression and IL-17A we the expression the iTreg or Th17 using In with studies (15Zhou L. Ivanov I.I. Spolski R. Min R. Shenderov K. Egawa T. Levy D.E. Leonard W.J. Littman D.R. Nat. Immun. 2007; 8: 967-974Crossref Scopus (1685) Google Scholar), we found that TGF-β1 induced the expression of RORγt and Foxp3 in CD4+CD25– T cells IL-17A was induced Th17 and iTreg the Th17 induced of IL-17A the iTreg RORγt was induced of stimulation regardless of the presence or absence of IL-6. Foxp3 was after stimulation and a IL-6 reduced the expression of Foxp3 This is a direct of IL-6 on the Foxp3 promoter IL-6 suppressed Foxp3 promoter activity in primary T cells not These suggest that IL-17A were not determined by RORγt Foxp3 and IL-17A expression to opposing we the that Foxp3 negatively regulates RORγt-mediated IL-17A expression. RORγt the IL-17A the regulatory mechanism of IL-17A we a reporter with the use of the HEK 293T cell line. we an ∼6-kb DNA fragment of the of the IL-17A gene using the mouse genomic this IL-17A promoter region was subcloned of a luciferase reporter gene This IL-17A promoter was activated by an anti-CD3 Ab in the EL4 cells EL4 is a cell line but properties and RORγt constitutively IL-17A promoter activity was with or without an RORγt expression vector in the HEK 293T cells in which RORγt was not In the presence of RORγt, the promoter activity was the IL-17A expression. the expression of various in EL4 cell line was determined by were cultured with or without anti-CD3 Ab for and HEK 293T cells were with IL-17A promoter and RORγt or promoter and with the of Foxp3 of of of experiments are assay of RORγt and on IL-17A promoter in HEK 293T cells with a and/or Foxp3 was by with the RORγt binding sites the IL-17A promoter, we generated a of mutants the of the promoter These were for promoter activity in the HEK 293T cells with or without The the to not on the RORγt-mediated IL-17A promoter However, a deletion reduced IL-17A promoter and an deletion the RORγt-mediated IL-17A promoter activity This that the region from to has been shown to of the by a A.M. S. E. Res. Biol. PubMed Google Scholar). shown in are two RORE-1 and this These two and are conserved between and an conserved of this RORγt to of the IL-17A the of two we introduced mutations into two mutations in of the two the RORγt-mediated IL-17A promoter and mutations in the RORγt-mediated promoter activity These that two are essential for RORγt-mediated IL-17A promoter that RORγt to the the IL-17A promoter, we the binding of RORγt to this region by HEK 293T cells were with RORγt and nuclear were with DNA with RORγt was amplified by PCR with the region to shown in RORγt was shown to to the IL-17A promoter In addition, were in the presence of RORγt in the absence of that RORγt up-regulates the IL-17A expression by directly binding to the IL-17A promoter region of Foxp3 the IL-17A in EL4 the of Foxp3 on IL-17A expression, we the of IL-17A in EL4 cells. shown in EL4 cells constitutively RORγt, and IL-17A was induced by anti-CD3 Ab expression of Foxp3 reduced the expression of expression of RORγt expression of TGF-β1 was by Foxp3 Thus, that Foxp3 IL-17A expression. the mechanism of IL-17A expression by Foxp3, we whether Foxp3 expression the RORγt-mediated IL-17A transcription in the HEK 293T cells. Foxp3 suppressed RORγt-mediated IL-17A promoter activity in a However, Foxp3 not transcriptional activity with a reporter STAT3 transcriptional activity was not by Foxp3 not we whether Foxp3 with the of RORγt with in the IL-17A assay that Foxp3 expression reduced RORγt binding as well as of in the IL-17A promoter region These suggest that Foxp3 can RORγt-mediated IL-17A transcription by RORγt binding to the IL-17A Foxp3 RORγt by has been shown that Foxp3 the transcription and a with on DNA Y. Borde M. Heissmeyer V. Feuerer M. Lapan A.D. Stroud J.C. Bates D.L. Guo L. Han A. Ziegler S.F. Mathis D. Benoist C. Chen L. Rao A. Cell. 2006; 126: 375-387Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar). However, we could not any direct binding of Foxp3 to the IL-17A promoter not Foxp3 inhibited the minimum IL-17A promoter not which not any Foxp3 binding we hypothesized that Foxp3 the RORγt by direct physical interaction with this we and reporter using a of Foxp3 deletion mutants We strong interaction between Foxp3 and The protein a zinc a leucine and a (FKH) It has been shown that the and domain are essential for the suppression activity of Foxp3. The has been shown to be necessary for T.R. Ziegler S.F. J. Immunol. 2006; PubMed Scopus Google the role of has not been clarified. Interestingly, an region corresponding to the exon 2 region of Foxp3 was necessary for interaction with However, the other the ZnF, Zip, and were A reporter assay that the exon 2 region as well as the domain was necessary for the suppression of RORγt-mediated IL-17A promoter activation This was in IL-17A induction in EL4 cells. EL4 cells were with Foxp3 or mutants to the IL-17A expression was by and PCR. shown in overexpression of Foxp3, but not or inhibited induction of IL-17A transcription observations suggest that Foxp3 with RORγt in the exon 2 region and that the domain plays a role in the suppression of RORγt-mediated IL-17A transcription. IL-17A is a cytokine of Th17, a subset of helper T cells. It has been that forced expression of RORγt is sufficient for the expression of IL-17A in T cells (13Ivanov I.I. McKenzie B.S. Zhou L. Tadokoro C.E. Lepelley A. Lafaille J.J. Cua D.J. Littman D.R. Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3988) Google Scholar). Recently, has also been shown to IL-17A in T cells X.O. Nurieva R. A. Y. Ma L. B. Panopoulos A.D. Schluns Watowich S.S. Tian Q. Jetten A.M. Dong C. Immunity. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the for the activation of the IL-17A promoter by RORγt and its regulation by cytokines have not been clarified. This is the on of the IL-17A promoter in IL-6 was necessary for the induction of IL-17A expression It has been thought that IL-6 is necessary it RORγt in with TGF-β1. However, we and found that RORγt is induced by TGF-β1 We that reduced expression of Foxp3 was with IL-17A expression, RORγt were not directly with IL-17A expression. we that RORγt is essential for the induction of IL-17A, Foxp3 may negatively IL-17A expression in This was by overexpression of Foxp3 in EL4 cells in which RORγt is constitutively we that Foxp3 overexpression suppressed RORγt-mediated IL-17A promoter activity we propose that of the functions of IL-6 on Th17 induction is the suppression of Foxp3 expression. how IL-6 Foxp3 We have reported that TGF-β can Foxp3 promoter in EL4 cells and that this promoter activation H. K. Koga K. T. Takaesu G. Y. S. Yoshimura A. Kobayashi T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We that directly to the Foxp3 promoter and suppressed the Foxp3 promoter activation. However, we could not any of IL-6 or activated STAT3 on the Foxp3 promoter activation in EL4 cells as well as primary T cells. In addition, IL-6 as well as of overexpression not transcriptional we could not how STAT3 Foxp3 is a that the STAT3 binding site was not in on the promoter region may be induced by We also could not the that induced by STAT3 are involved in STAT3 study is necessary for the mechanism of Foxp3 by IL-6. the mechanism for the suppression of Foxp3 by to be we propose a novel mechanism for the regulation of IL-17A expression by IL-6. IL-6 Foxp3 expression, and RORγt is to the IL-17A In this study, we a reporter that of the regulatory mechanism of IL-17A transcription in the HEK 293T cells. This is the that RORγt functions as a direct transcriptional of HEK 293T cells can be used for this may be for that IL-17A promoter we that Foxp3 suppressed RORγt-mediated IL-17A promoter activation by a physical have shown that Foxp3 interacted with and suppressed the effector functions of Th cells by the activity of Y. Borde M. Heissmeyer V. Feuerer M. Lapan A.D. Stroud J.C. Bates D.L. Guo L. Han A. Ziegler S.F. Mathis D. Benoist C. Chen L. Rao A. Cell. 2006; 126: 375-387Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar). Foxp3 has also been shown to transcriptional activity (21Bettelli E. Dastrange M. Oukka M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5138-5143Crossref PubMed Scopus (450) Google Scholar). A mechanism may be for the suppression of RORγt by Foxp3. Interestingly, the interaction between Foxp3 and is reported to be dependent on the domain Y. Borde M. Heissmeyer V. Feuerer M. Lapan A.D. Stroud J.C. Bates D.L. Guo L. Han A. Ziegler S.F. Mathis D. Benoist C. Chen L. Rao A. Cell. 2006; 126: 375-387Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar), we found that between Foxp3 and RORγt was dependent on the exon 2 However, the domain was for the suppression of RORγt Thus, Foxp3 RORγt by direct binding through the exon 2 region and transcriptional activity of RORγt through the It has been reported that the domain is essential for nuclear and repressor functions as well as DNA binding of Foxp3 T.R. Ziegler S.F. J. Immunol. 2006; PubMed Scopus Google Scholar, E. Zhang Y. Ziegler S.F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, a study that mutations in with immune were the domain and the leucine but were also the of the T.R. Ziegler S.F. J. Immunol. 2006; PubMed Scopus Google identified a novel of the which is for of transcription as well as repressor The region may be important for interaction with transcription factors In we found that deletion of the zinc and leucine not suppression of IL-17A transcription. the mechanism of the transcriptional repressor of Foxp3 may to the transcription factors that with Foxp3. In we propose a novel mechanism for regulation of IL-17A expression by TGF-β1 and IL-6. TGF-β1 is important for the induction of RORγt, IL-6 is necessary to expression of the transcriptional repressor Foxp3. Foxp3 also may also Th1 and Th2 through a We Dr. M. for the EL4 cell line. We also T. and M. for and Y. for