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Thymus-derived Foxp3+ regulatory T cells upregulate RORγt expression under inflammatory conditions

Journal of Molecular Medicine volume 96, pages 1387–1394 (2018)Cite this article

Abstract

Foxp3+ regulatory T cells (Tregs) co-expressing the Th17-lineage specification factor RORγt represent a unique Treg subpopulation that has been reported to be induced upon response to gut microbiota within the intestinal immune system. Hence, RORγt+ Tregs are considered to solely consist of peripherally induced Foxp3+ Tregs (pTregs), and the possibility that also thymus-derived Treg (tTregs) can upregulate RORγt expression and contribute to the pool of RORγt+ Tregs was largely ignored. Here, we expand our knowledge on the origin of RORγt+ Tregs by demonstrating that also tTregs can attain RORγt expression. In transgenic Foxp3∆CNS1-Cre reporter mice, a substantial fraction of CNS1-independent Tregs, predominantly consisting of tTregs, was found to co-express RORγt. In addition, genuine tTregs isolated from thymi of Foxp3hCD2RAGGFP reporter mice initiated RORγt expression both in vitro and in vivo, particularly under inflammatory conditions. In conclusion, our data demonstrate that tTregs can upregulate RORγt expression under inflammatory conditions and that hence RORγt+ Tregs can be regarded as a heterogeneous population consisting of both pTregs and tTregs.

Key messages

  • RORγt cannot be considered as a marker for pTregs.

  • CNS1-independent tTregs within the colon display RORγt expression.

  • RORγt can be induced in genuine tTregs, particularly under inflammatory conditions.

  • RORγt+ Tregs are a heterogeneous population consisting of both pTregs and tTregs.

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Abbreviations

BAC:

Bacterial artificial chromosome

mLN:

Mesenteric lymph node

Treg:

Regulatory T cell

pTreg:

Peripherally induced Treg

tTreg:

Thymus-derived Treg

References

  1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133:775–787

    CAS  Article  Google Scholar 

  2. Hsieh CS, Lee HM, Lio CW (2012) Selection of regulatory T cells in the thymus. Nat Rev Immunol 12:157–167

    CAS  Article  Google Scholar 

  3. Tanoue T, Atarashi K, Honda K (2016) Development and maintenance of intestinal regulatory T cells. Nat Rev Immunol 16:295–309

    CAS  Article  Google Scholar 

  4. Huehn J, Beyer M (2015) Epigenetic and transcriptional control of Foxp3 regulatory T cells. Semin Immunol 27:10–18

    CAS  Article  Google Scholar 

  5. Lochner M, Peduto L, Cherrier M, Sawa S, Langa F, Varona R, Riethmacher D, Si-Tahar M, Di Santo JP, Eberl G (2008) In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORgamma t+ T cells. J Exp Med 205:1381–1393

    CAS  Article  Google Scholar 

  6. Zhou L, Lopes JE, Chong MM, Ivanov II, Min R, Victora GD, Shen Y, Du J, Rubtsov YP, Rudensky AY et al (2008) TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 453:236–240

    CAS  Article  Google Scholar 

  7. Sefik E, Geva-Zatorsky N, Oh S, Konnikova L, Zemmour D, McGuire AM, Burzyn D, Ortiz-Lopez A, Lobera M, Yang J et al (2015) MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce a distinct population of RORgamma+ regulatory T cells. Science 349:993–997

    CAS  Article  Google Scholar 

  8. Ohnmacht C, Park JH, Cording S, Wing JB, Atarashi K, Obata Y, Gaboriau-Routhiau V, Marques R, Dulauroy S, Fedoseeva M et al (2015) MUCOSAL IMMUNOLOGY. The microbiota regulates type 2 immunity through RORgammat+ T cells. Science 349:989–993

    CAS  Article  Google Scholar 

  9. Yang BH, Hagemann S, Mamareli P, Lauer U, Hoffmann U, Beckstette M, Fohse L, Prinz I, Pezoldt J, Suerbaum S et al (2016) Foxp3+ T cells expressing RORgammat represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation. Mucosal Immunol 9:444–457

    CAS  Article  Google Scholar 

  10. Tartar DM, Van Morlan AM, Wan X, Guloglu FB, Jain R, Haymaker CL, Ellis JS, Hoeman CM, Cascio JA, Dhakal M et al (2010) FoxP3+RORgammat+ T helper intermediates display suppressive function against autoimmune diabetes. J Immunol 184:3377–3385

    CAS  Article  Google Scholar 

  11. Kluger MA, Luig M, Wegscheid C, Goerke B, Paust HJ, Brix SR, Yan I, Mittrucker HW, Hagl B, Renner ED et al (2014) Stat3 programs Th17-specific regulatory T cells to control GN. J Am Soc Nephrol 25:1291–1302

    CAS  Article  Google Scholar 

  12. Kluger MA, Meyer MC, Nosko A, Goerke B, Luig M, Wegscheid C, Tiegs G, Stahl RA, Panzer U, Steinmetz OM (2016) RORgammat+Foxp3+ cells are an independent bifunctional regulatory T cell lineage and mediate crescentic GN. J Am Soc Nephrol 27:454–465

    CAS  Article  Google Scholar 

  13. Chellappa S, Hugenschmidt H, Hagness M, Line PD, Labori KJ, Wiedswang G, Tasken K, Aandahl EM (2016) Regulatory T cells that co-express RORgammat and FOXP3 are pro-inflammatory and immunosuppressive and expand in human pancreatic cancer. Oncoimmunology 5: e1102828. DOI

    Article  Google Scholar 

  14. Kim B-S, Lu H, Ichiyama K, Chen X, Zhang Y-B, Mistry NA, Tanaka K, Lee Y-h, Nurieva R, Zhang L et al (2017) Generation of RORγt+ antigen-specific T regulatory 17 cells from Foxp3+ precursors in autoimmunity. Cell reports 21:195–207

    CAS  Article  Google Scholar 

  15. Solomon BD, Hsieh CS (2016) Antigen-specific development of mucosal Foxp3+RORgammat+ T cells from regulatory T Cell precursors. J Immunol 197:3512–3519

    CAS  Article  Google Scholar 

  16. Tone Y, Furuuchi K, Kojima Y, Tykocinski ML, Greene MI, Tone M (2008) Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer. Nat Immunol 9:194–202

    CAS  Article  Google Scholar 

  17. Huehn J, Polansky JK, Hamann A (2009) Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage? Nat Rev Immunol 9:83–89

    CAS  Article  Google Scholar 

  18. Schlenner SM, Weigmann B, Ruan Q, Chen Y, von Boehmer H (2012) Smad3 binding to the foxp3 enhancer is dispensable for the development of regulatory T cells with the exception of the gut. J Exp Med 209:1529–1535

    CAS  Article  Google Scholar 

  19. Zheng Y, Josefowicz S, Chaudhry A, Peng XP, Forbush K, Rudensky AY (2010) Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463:808–812

    CAS  Article  Google Scholar 

  20. Kitagawa Y, Ohkura N, Kidani Y, Vandenbon A, Hirota K, Kawakami R, Yasuda K, Motooka D, Nakamura S, Kondo M et al (2017) Guidance of regulatory T cell development by Satb1-dependent super-enhancer establishment. Nat Immunol 18:173–183

    CAS  Article  Google Scholar 

  21. Josefowicz SZ, Niec RE, Kim HY, Treuting P, Chinen T, Zheng Y, Umetsu DT, Rudensky AY (2012) Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature 482:395–399

    CAS  Article  Google Scholar 

  22. Zhang Z, Zhang W, Guo J, Gu Q, Zhu X, Zhou X (2017) Activation and functional specialization of regulatory T cells lead to the generation of Foxp3 instability. J Immunol 198:2612–2625

    CAS  Article  Google Scholar 

  23. Thiault N, Darrigues J, Adoue V, Gros M, Binet B, Perals C, Leobon B, Fazilleau N, Joffre OP, Robey EA et al (2015) Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors. Nat Immunol 16:628–634

    CAS  Article  Google Scholar 

  24. Ohkura N, Hamaguchi M, Morikawa H, Sugimura K, Tanaka A, Ito Y, Osaki M, Tanaka Y, Yamashita R, Nakano N et al (2012) T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity 37:785–799

    CAS  Article  Google Scholar 

  25. Toker A, Engelbert D, Garg G, Polansky JK, Floess S, Miyao T, Baron U, Duber S, Geffers R, Giehr P et al (2013) Active demethylation of the Foxp3 locus leads to the generation of stable regulatory T cells within the thymus. J Immunol 190:3180–3188

    CAS  Article  Google Scholar 

  26. Wheaton JD, Yeh CH, Ciofani M (2017) Cutting edge: c-Maf Is required for regulatory T Cells to adopt RORgammat+ and follicular phenotypes. J Immunol. https://doi.org/10.4049/jimmunol.1701134

    CAS  Article  Google Scholar 

  27. Samanta A, Li B, Song X, Bembas K, Zhang G, Katsumata M, Saouaf SJ, Wang Q, Hancock WW, Shen Y et al (2008) TGF-beta and IL-6 signals modulate chromatin binding and promoter occupancy by acetylated FOXP3. Proc Natl Acad Sci U S A 105:14023–14027

    CAS  Article  Google Scholar 

  28. Lal G, Zhang N, van der Touw W, Ding Y, Ju W, Bottinger EP, Reid SP, Levy DE, Bromberg JS (2009) Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J Immunol 182:259–273

    CAS  Article  Google Scholar 

  29. O’Connor RA, Floess S, Huehn J, Jones SA, Anderton SM (2012) Foxp3+ Treg cells in the inflamed CNS are insensitive to IL-6-driven IL-17 production. Eur J Immunol 42:1174–1179

    Article  Google Scholar 

  30. Gao Y, Tang J, Chen W, Li Q, Nie J, Lin F, Wu Q, Chen Z, Gao Z, Fan H et al (2015) Inflammation negatively regulates FOXP3 and regulatory T-cell function via DBC1. Proc Natl Acad Sci U S A 112:E3246–E3254

    CAS  Article  Google Scholar 

  31. Haribhai D, Lin W, Edwards B, Ziegelbauer J, Salzman NH, Carlson MR, Li SH, Simpson PM, Chatila TA, Williams CB (2009) A central role for induced regulatory T cells in tolerance induction in experimental colitis. J Immunol 182:3461–3468

    CAS  Article  Google Scholar 

  32. Huang YJ, Haist V, Baumgartner W, Fohse L, Prinz I, Suerbaum S, Floess S, Huehn J (2014) Induced and thymus-derived Foxp3+ regulatory T cells share a common niche. Eur J Immunol 44:460–468

    CAS  Article  Google Scholar 

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Acknowledgments

We thank all members of the animal facility, the flow cytometry facility, and the Department Experimental Immunology of the Helmholtz Centre for Infection Research (HZI), especially Dr. Lothar Gröbe, Yassin Elfaki, and Ahmed Elfiky for the technical assistance, and Dr. Shohei Hori (University of Tokyo, Japan) for providing Foxp3hCD2RAGGFP mice.

Funding

This work was supported by the German Research Foundation (SFB738-C7) and the Alexander von Humboldt Foundation.

Author information

Affiliations

  1. Department Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany

    Juhao Yang, Mangge Zou, Joern Pezoldt & Jochen Huehn

  2. Key Laboratory of Pathogenic Microbiology and Immunology, University of the Chinese Academy of Sciences, Beijing, 100080, China

    Mangge Zou & Xuyu Zhou

Authors
  1. Juhao Yang
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  2. Mangge Zou
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  3. Joern Pezoldt
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  4. Xuyu Zhou
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  5. Jochen Huehn
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Corresponding author

Correspondence to Jochen Huehn.

Ethics declarations

All animal experiments were approved by the Lower Saxony Committee on the Ethics of Animal Experiments as well as the responsible state office (Lower Saxony State Office of Consumer Protection and Food Safety) under the permit numbers 33.9-42502-04-13-1170 and 33.9-42502-04-12/1012. Foxp3∆CNS1-CreRosa26YFP mice (C57BL/6 background) were bred in-house (Institute of Microbiology, Chinese Academy of Sciences). All experiments were conducted according to the guidelines of the Institute of Microbiology, Chinese Academy of Sciences Institutional Animal Care and Use Committee (permit no. PZIMCAS2011005).

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Supplementary Figure 1.

Genuine tTregs virtually lack RORγt expression and can be identified upon adoptive transfer into immunocompetent hosts. Genuine tTregs (CD25+Foxp3hCD2+RAGGFP+ CD4SP thymocytes) isolated from thymi of Foxp3hCD2RAGGFP reporter mice (CD45.1) were adoptively transferred into three-week-old immunocompetent recipient mice (CD45.2). PBS-injected mice served as controls. (a) Scatterplot summarizes RORγt expression among sorted CD25+Foxp3hCD2+RAGGFP+ CD4SP thymocytes before transfer. Data pooled from nine independent experiments are shown as mean ± SD. (b) Representative dotplot confirms lack of false-positive CD45.1+ cells among CD4+ cells from mLN of PBS-injected control mice four weeks post transfer. Number indicates frequency of CD45.1+ cells. (c) Scatterplot summarizes frequencies of CD45.1+ cells among CD4+ cells in mLN of recipient mice four weeks after adoptive transfer of genuine tTregs. (d) Scatterplot summarizes absolute number of CD45.1+CD4+ cells recovered from mLN of recipient mice four weeks after adoptive transfer of genuine tTregs. Each dot represents one mouse. Data pooled from three independent experiments are shown as mean ± SD (n = 6-9). (EPS 671 kb)

Supplementary Figure 2.

Impact of IL-6 on phenotype, survival and proliferation of in vitro cultured tTregs. Genuine tTregs (CD25+Foxp3hCD2+RAGGFP+ CD4SP thymocytes) isolated from thymi of Foxp3hCD2RAGGFP reporter mice were cultured for four days with or without IL-6, followed by flow cytometric analysis. (a) Scatterplots summarize mean fluorescence intensity (MFI) of RORγt (left) and Foxp3 expression (right) in indicated groups normalized to medium control. Each dot represents data from one independent experiment (n=5). (b) Representative dotplots determine LIVE/DEAD (LD) fixable Blue expression (left), and scatterplot (right) summarizes frequencies of LD+ cells among RORγt+ or RORγt- tTregs at the end of the culture. Each dot represents one independent experiment. Data are shown as mean ± SD. (c) Representative histograms depicts CellTrace Violet (CTV) signal of RORγt+ or RORγt- tTregs at the end of the culture (left), and scatterplot summarizes MFI of CTV signal among RORγt+ or RORγt- tTregs at the end of the culture. Unstimulated naïve T cells served as control. Each dot represents one independent experiment. Data are shown as mean ± SD. (EPS 735 kb)

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Yang, J., Zou, M., Pezoldt, J. et al. Thymus-derived Foxp3+ regulatory T cells upregulate RORγt expression under inflammatory conditions. J Mol Med 96, 1387–1394 (2018). https://doi.org/10.1007/s00109-018-1706-x

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  • DOI: https://doi.org/10.1007/s00109-018-1706-x

Keywords

  • Regulatory T cells
  • Thymus-derived
  • Peripherally induced