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Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance

Nature Immunology volume 16, pages 197–206 (2015)Cite this article

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Abstract

Regulatory T cells (Treg cells) can express the transcription factors T-bet and GATA-3, but the function of this expression and whether such cells represent stable subsets is still unknown. By using various reporter tools, we found that the expression of T-bet and GATA-3 in Treg cells was dynamically influenced by the cytokine environment. Treg cell–specific deletion of the gene encoding either T-bet (Tbx21) or GATA-3 (Gata3) alone did not result in loss of Treg cell function; however, mice with combined deficiency in both genes in Treg cells developed severe autoimmune-like diseases. Loss of Treg cell function correlated with upregulation of expression of the transcription factor RORγt and reduced expression of the transcription factor Foxp3. Thus, in the steady state, activated Treg cells transiently upregulated either T-bet or GATA-3 to maintain T cell homeostasis.

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Figure 1: The expression of T-bet and GATA-3 in Treg cells can be induced by cytokines.
Figure 2: Dynamic expression of T-bet or GATA-3 in Treg cells in vitro and in vivo.
Figure 3: Confirmation of dynamic expression of T-bet in Treg cells by the T-bet fate-mapping tool.
Figure 4: Treg cells singly devoid of either T-bet or GATA-3 are functional in protecting against T cell–mediated colitis.
Figure 5: Ablation of both T-bet and GATA-3 in Treg cells results in autoimmune lymphoproliferative disease.
Figure 6: Treg cells doubly deficient in both T-bet and GATA-3 do not suppress colitis.
Figure 7: The dysfunction of Tbx21fl/flGata3fl/flFoxp3-Cre Treg cells is cell intrinsic.

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References

  1. Zhu, J., Yamane, H. & Paul, W.E. Differentiation of effector CD4 T cell populations. Annu. Rev. Immunol. 28, 445–489 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lazarevic, V., Glimcher, L.H. & Lord, G.M. T-bet: a bridge between innate and adaptive immunity. Nat. Rev. Immunol. 13, 777–789 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yagi, R., Zhu, J. & Paul, W.E. An updated view on transcription factor GATA3-mediated regulation of Th1 and Th2 cell differentiation. Int. Immunol. 23, 415–420 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ivanov, I.I. et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Hwang, E.S, Szabo, S.J., Schwartzberg, P.L. & Glimcher, L.H. T helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science 307, 430–433 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Zhu, J. et al. The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses. Immunity 37, 660–673 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Szabo, S.J., et al. Distinct effects of T-bet in TH1 lineage commitment and IFN-γ production in CD4 and CD8 T cells. Science 295, 338–342 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Zhang, F., Meng, G. & Strober, W. Interactions among the transcription factors Runx1, RORγt and Foxp3 regulate the differentiation of interleukin 17-producing T cells. Nat. Immunol. 9, 1297–1306 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lazarevic, V. et al. T-bet represses TH17 differentiation by preventing Runx1-mediated activation of the gene encoding RORγt. Nat. Immunol. 12, 96–104 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Josefowicz, S.Z., Lu, L.F. & Rudensky, A.Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Abbas, A.K. et al. Regulatory T cells: recommendations to simplify the nomenclature. Nat. Immunol. 14, 307–308 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Shevach, E.M. Mechanisms of Foxp3+ T regulatory cell-mediated suppression. Immunity 30, 636–645 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Sakaguchi, S., Miyara, M., Costantino, C.M. & Hafler, D.A. FOXP3+ regulatory T cells in the human immune system. Nat. Rev. Immunol. 10, 490–500 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Chen, W. et al. Conversion of peripheral CD4+CD25-naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Korn, T., Bettelli, E., Oukka, M. & Kuchroo, V.K. IL-17 and Th17 cells. Annu. Rev. Immunol. 27, 485–517 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Zhou, L. et al. TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORγt function. Nature 453, 236–240 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Komatsu, N. et al. Pathogenic conversion of Foxp3+ T cells into TH17 cells in autoimmune arthritis. Nat. Med. 20, 62–68 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Koch, M.A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat. Immunol. 10, 595–602 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wohlfert, E.A. et al. GATA3 controls Foxp3+ regulatory T cell fate during inflammation in mice. J. Clin. Invest. 121, 4503–4515 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rudra, D. et al. Transcription factor Foxp3 and its protein partners form a complex regulatory network. Nat. Immunol. 13, 1010–1019 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang, Y., Su, M.A. & Wan, Y.Y. An essential role of the transcription factor GATA-3 for the function of regulatory T cells. Immunity 35, 337–348 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wan, Y.Y. & Flavell, R.A. Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc. Natl. Acad. Sci. USA 102, 5126–5131 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hosoya, T. et al. GATA-3 is required for early T lineage progenitor development. J. Exp. Med. 206, 2987–3000 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Duarte, J.H., Zelenay, S., Bergman, M.L., Martins, A.C. & Demengeot, J. Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions. Eur. J. Immunol. 39, 948–955 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Koch, M.A. et al. T-bet+ Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor β2. Immunity 37, 501–510 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hall, A.O. et al. The cytokines interleukin 27 and interferon-gamma promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 37, 511–523 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Intlekofer, A.M. et al. Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science 321, 408–411 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yagi, R. et al. The transcription factor GATA3 actively represses RUNX3 protein-regulated production of interferon-γ. Immunity 32, 507–517 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rubtsov, Y.P. et al. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28, 546–558 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Izcue, A., Coombes, J.L. & Powrie, F. Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunol. Rev. 212, 256–271 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Delgoffe, G.M. et al. Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis. Nature 501, 252–256 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Liston, A. & Gray, D.H. Homeostatic control of regulatory T cell diversity. Nat. Rev. Immunol. 14, 154–165 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Zheng, Y. et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control TH2 responses. Nature 458, 351–356 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Cretney, E. et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat. Immunol. 12, 304–311 (2011).

    Article  CAS  PubMed  Google Scholar 

  35. Chaudhry, A. et al. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science 326, 986–991 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yamaguchi, T., Wing, J.B. & Sakaguchi, S. Two modes of immune suppression by Foxp3+ regulatory T cells under inflammatory or non-inflammatory conditions. Semin. Immunol. 23, 424–430 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Oldenhove, G. et al. Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity 31, 772–786 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhou, X. et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 10, 1000–1007 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bailey-Bucktrout, S.L. et al. Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response. Immunity 39, 949–962 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Thornton, A.M. & Shevach, E.M. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287–296 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Levine, A.G., Arvey, A., Jin, W. & Rudensky, A.Y. Continuous requirement for the TCR in regulatory T cell function. Nat. Immunol. 15, 1070–1078 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Durbin, J.E., Hackenmiller, R., Simon, M.C. & Levy, D.E. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84, 443–450 (1996).

    Article  CAS  PubMed  Google Scholar 

  43. Szymczak, A.L. et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat. Biotechnol. 22, 589–594 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Indra, A.K. et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases. Nucleic Acids Res. 27, 4324–4327 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Sun, C.M. et al. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 Treg cells via retinoic acid. J. Exp. Med. 204, 1775–1785 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J.D. Engel (University of Michigan) for GATA-3–GFP reporter mice on the C57BL/6 background; S. Reiner (University of Columbia) for Tbx21fl/fl mice; A. Rudensky (Memorial Sloan Kettering Cancer Center) for Foxp3-IRES-YFP-Cre mice; D. Jankovic (National Institute of Allergy and Infectious Diseases (NIAID)) for Stat1−/− mice; E. Shevach, Y. Belkaid and W. Paul for critical reading of the manuscript and discussions; A. Thornton for discussions; L. Guo for the help with enzyme-linked immunosorbent assay experiments; N. Cereb and S.Y. Yang (Histogenetics) and S. Reiner (Columbia University) for constructing and providing Tbx21fl/fl mice; and the flow cytometry core facility of the NIAID for cell sorting. Supported by the Division of Intramural Research of the NIAID (US National Institutes of Health).

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Authors and Affiliations

  1. Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Fang Yu, Suveena Sharma, Julie Edwards & Jinfang Zhu

  2. Laboratory Animal Sciences Program, National Cancer Institute, Frederick, Maryland, USA

    Lionel Feigenbaum

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Contributions

F.Y. performed all experiments; S.S. and J.E. helped in some experiments and made suggestions on the manuscript; L.F. helped make the transgenic mouse strains; and F.Y. and J.Z. designed the experiments, analyzed the data and wrote the paper.

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Correspondence to Jinfang Zhu.

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Integrated supplementary information

Supplementary Figure 1 AmCyan is a faithful reporter for T-bet expression.

(a) Splenocytes from the T-bet-AmCyan BAC transgenic mice were sorted for AmCyan+ and AmCyan- populations and then intracellular staining for T-bet expression in these two populations was carried out. (b) Naïve CD4+ (CD4+CD25-CD45RBhiAmCyan-) T cells were isolated by cell sorting from the T-bet-AmCyan BAC transgenic mice and then cultured under TH1 or TH2 polarizing conditions for 4 days. AmCyan was then assessed by flow cytometry. Each number above the gate indicates the percentage of AmCyan positive cells. Data are representative of two independent experiments.

Supplementary Figure 2 Analysis of Treg cell subsets before and after cell sorting.

(a) Splenocytes from the T-bet-AmCyan:GATA3-GFP:Foxp3-mRFP tri-color reporter mice were stained with anti-CD4 and anti-CXCR3. Plot on the left was gated on CD4+Foxp3-RFP+ population; histograms on the right show CXCR3 expression on each Treg subset. (b, c) Treg subsets were separated from the T-bet-AmCyan:GATA3-GFP:Foxp3-RFP tri-color reporter mice by cell sorting. The purity of each sorted population was shown (b). Plots were gated on CD4+Foxp3-RFP+ population. Some sorted cells were first stained with anti-CXCR3 and then fixed. They were then permeabilized and stained intracellularly with anti-T-bet overnight (c). Plots show T-bet and CXCR3 expression by each Treg subset. Data are representative of three (a), more than five (b) and two (c) independent experiments.

Supplementary Figure 3 T-bet expression in Treg cells depends on STAT1-activating cytokines.

Cells from the spleens and lymph nodes of wild type, Stat1-/-, Stat4-/- and IFN-gr1-/- T-bet-ZsGreen mice were fixed and stained for CD4, CD25 and Foxp3 and then analyzed by flow cytometry. Dot plots were gated on CD4+CD25+Foxp3+ population. Data are representative of two independent experiments.

Supplementary Figure 4 Combined deficiency of T-bet and GATA-3 in Treg cells does not affect the development of Treg cells.

(a) Flow cytometric analysis of percentage of CD4+ and CD8+ T cells in the thymi of 8-week old Foxp3-Cre and Tbx21fl/flGata3fl/fl-Foxp3-Cre (DKO) mice. (b) Graphical presentation of percentage of Foxp3+ Treg cells among CD4 single positive thymocytes of 8-week old Foxp3-Cre and DKO mice. Each dot represents an individual mouse. (c) Graphical presentation of percentage of Foxp3+ Treg cells among CD4+ T cells in the spleen and lymph nodes of 8-week old Foxp3-Cre and DKO mice. Data are representative of two independent experiments. Error bars represent standard deviation of the mean (n=6). Statistical significance was determined by an ordinary one-way ANOVA (b and c). ns, not significant, *p<0.05, ****p<0.0001.

Supplementary Figure 5 Combined deficiency in T-bet and GATA-3 in Treg cells does not affect the proliferation or survival of Treg cells.

The splenocytes from 8-week old Foxp3-Cre and Tbx21fl/flGata3fl/fl-Foxp3-Cre (DKO) mice were stained with anti-CD4, anti-Foxp3, anti-CD25 and Ki-67 (a) or Bcl-2 (b). Dot plots were gated on CD4+Foxp3+ live cells. Numbers indicate the percentages in each quadrant. Data are representative of two independent experiments, each experiment with 2-3 mice in each group.

Supplementary Figure 6 Tbx21fl/flGata3fl/flFoxp3-Cre (DKO) Treg cells have normal expression of CD25 and CTLA-4 but lower expression of GITR, CD103 and Nrp-1.

The splenocytes from 8-week old Foxp3-Cre and DKO mice were stained with anti-CD4, anti-Foxp3 and antibodies against various “Treg-specific” cell surface markers. All the stainings were performed without cell permeablization except for CTLA-4 staining. Histograms were gated on CD4+Foxp3+ live cells. Data are representative of two independent experiments, each experiment with 2-3 mice in each group.

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Yu, F., Sharma, S., Edwards, J. et al. Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance. Nat Immunol 16, 197–206 (2015). https://doi.org/10.1038/ni.3053

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