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Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells

Nature Immunology volume 15, pages 473–481 (2014)Cite this article

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Abstract

Regulatory T cells (Treg cells) express members of the tumor-necrosis factor (TNF) receptor superfamily (TNFRSF), but the role of those receptors in the thymic development of Treg cells is undefined. We found here that Treg cell progenitors had high expression of the TNFRSF members GITR, OX40 and TNFR2. Expression of those receptors correlated directly with the signal strength of the T cell antigen receptor (TCR) and required the coreceptor CD28 and the kinase TAK1. The neutralization of ligands that are members of the TNF superfamily (TNFSF) diminished the development of Treg cells. Conversely, TNFRSF agonists enhanced the differentiation of Treg cell progenitors by augmenting responsiveness of the interleukin 2 receptor (IL-2R) and transcription factor STAT5. Costimulation with the ligand of GITR elicited dose-dependent enrichment for cells of lower TCR affinity in the Treg cell repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated the development of Treg cells. Thus, expression of members of the TNFRSF on Treg cell progenitors translated strong TCR signals into molecular parameters that specifically promoted the development of Treg cells and shaped the Treg cell repertoire.

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Figure 1: Expression of GITR, OX40 and TNFR2 on thymic Treg cell progenitors and of GITRL, OX40L and TNF on APCs in the thymic medulla.
Figure 2: Treg cell progenitors express select members of the TNFRSF in direct proportion to TCR signal strength.
Figure 3: TNFRSF agonists enhance the conversion of Treg cell progenitors into Foxp3+ Treg cells.
Figure 4: Deficiency in GITR or OX40 imposes a modest cell-intrinsic block on the thymic development of Treg cells.
Figure 5: Neutralization of members of the TNFSF by antibodies in thymic organ cultures inhibits the development of Treg cells and the acquisition of maturation markers.
Figure 6: GITR, OX40 and TNFR2 redundantly drive the development of Treg cells in vivo.
Figure 7: An excess of ligands of the TNFSF broadens the Treg cell repertoire to include a greater fraction of cells expressing TCRs with lower affinity for self.

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References

  1. Klein, L., Hinterberger, M., Wirnsberger, G. & Kyewski, B. Antigen presentation in the thymus for positive selection and central tolerance induction. Nat. Rev. Immunol. 9, 833–844 (2009).

    CAS  PubMed  Google Scholar 

  2. Stritesky, G.L., Jameson, S.C. & Hogquist, K.A. Selection of self-reactive T cells in the thymus. Annu. Rev. Immunol. 30, 95–114 (2012).

    CAS  PubMed  Google Scholar 

  3. Brunkow, M.E. et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27, 68–73 (2001).

    CAS  PubMed  Google Scholar 

  4. Wildin, R.S. et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27, 18–20 (2001).

    CAS  PubMed  Google Scholar 

  5. Yamanouchi, J. et al. Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat. Genet. 39, 329–337 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Lowe, C.E. et al. Large-scale genetic fine mapping and genotype-phenotype associations implicate polymorphism in the IL2RA region in type 1 diabetes. Nat. Genet. 39, 1074–1082 (2007).

    CAS  PubMed  Google Scholar 

  7. Garg, G. et al. Type 1 diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function. J. Immunol. 188, 4644–4653 (2013).

    Google Scholar 

  8. Lio, C.W. & Hsieh, C.S. A two-step process for thymic regulatory T cell development. Immunity 28, 100–111 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Burchill, M.A. et al. Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire. Immunity 28, 112–121 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Isomura, I. et al. c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells. J. Exp. Med. 206, 3001–3014 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Ruan, Q. et al. Development of Foxp3+ regulatory T cells is driven by the c-Rel enhanceosome. Immunity 31, 932–940 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Long, M., Park, S.G., Strickland, I., Hayden, M.S. & Ghosh, S. Nuclear factor-κB modulates regulatory T cell development by directly regulating expression of Foxp3 transcription factor. Immunity 31, 921–931 (2009).

    CAS  PubMed  Google Scholar 

  13. Vang, K.B. et al. Cutting edge: CD28 and c-Rel-dependent pathways initiate regulatory T cell development. J. Immunol. 184, 4074–4077 (2010).

    CAS  PubMed  Google Scholar 

  14. Deenick, E.K. et al. c-Rel but not NF-κB1 is important for T regulatory cell development. Eur. J. Immunol. 40, 677–681 (2010).

    CAS  PubMed  Google Scholar 

  15. Zheng, Y. et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Burchill, M.A., Yang, J., Vogtenhuber, C., Blazar, B.R. & Farrar, M.A. IL-2 receptor β-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. J. Immunol. 178, 280–290 (2007).

    CAS  PubMed  Google Scholar 

  17. Klinger, M. et al. Thymic OX40 expression discriminates cells undergoing strong responses to selection ligands. J. Immunol. 182, 4581–4589 (2009).

    CAS  PubMed  Google Scholar 

  18. Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y. & Sakaguchi, S. Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3, 135–142 (2002).

    CAS  PubMed  Google Scholar 

  19. Ronchetti, S. et al. GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur. J. Immunol. 34, 613–622 (2004).

    CAS  PubMed  Google Scholar 

  20. Valzasina, B. et al. Triggering of OX40 (CD134) on CD4+CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR. Blood 105, 2845–2851 (2005).

    CAS  PubMed  Google Scholar 

  21. Croft, M. The role of TNF superfamily members in T-cell function and diseases. Nat. Rev. Immunol. 9, 271–285 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Richter, C. et al. The tumor necrosis factor receptor stalk regions define responsiveness to soluble versus membrane-bound ligand. Mol. Cell. Biol. 32, 2515–2529 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Grell, M. et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 83, 793–802 (1995).

    CAS  PubMed  Google Scholar 

  24. Moran, A.E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Coquet, J.M. et al. Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27–CD70 pathway. J. Exp. Med. 210, 715–728 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wan, Y.Y., Chi, H., Xie, M., Schneider, M.D. & Flavell, R.A. The kinase TAK1 integrates antigen and cytokine receptor signaling for T cell development, survival and function. Nat. Immunol. 7, 851–858 (2006).

    CAS  PubMed  Google Scholar 

  27. Sato, S. et al. TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells. Int. Immunol. 18, 1405–1411 (2006).

    CAS  PubMed  Google Scholar 

  28. Salomon, B. et al. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12, 431–440 (2000).

    CAS  PubMed  Google Scholar 

  29. Lio, C.W., Dodson, L.F., Deppong, C.M., Hsieh, C.S. & Green, J.M. CD28 facilitates the generation of Foxp3− cytokine responsive regulatory T cell precursors. J. Immunol. 184, 6007–6013 (2010).

    CAS  PubMed  Google Scholar 

  30. Tai, X., Cowan, M., Feigenbaum, L. & Singer, A. CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T cell differentiation independently of interleukin 2. Nat. Immunol. 6, 152–162 (2005).

    CAS  PubMed  Google Scholar 

  31. Hogquist, K.A. Assays of thymic selection. Fetal thymus organ culture and in vitro thymocyte dulling assay. Methods Mol. Biol. 156, 219–232 (2001).

    CAS  PubMed  Google Scholar 

  32. Cheng, G. et al. IL-2 receptor signaling is essential for the development of Klrg1+ terminally differentiated T regulatory cells. J. Immunol. 189, 1780–1791 (2012).

    CAS  PubMed  Google Scholar 

  33. Yamaguchi, T. et al. Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity 27, 145–159 (2007).

    CAS  PubMed  Google Scholar 

  34. Tai, X. et al. Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals. Immunity 38, 1116–1128 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Pacholczyk, R., Ignatowicz, H., Kraj, P. & Ignatowicz, L. Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells. Immunity 25, 249–259 (2006).

    CAS  PubMed  Google Scholar 

  36. Lee, H.M., Bautista, J.L., Scott-Browne, J., Mohan, J.F. & Hsieh, C.S. A broad range of self-reactivity drives thymic regulatory T cell selection to limit responses to self. Immunity 37, 475–486 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Sekiya, T. et al. Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis. Nat. Immunol. 14, 230–237 (2013).

    CAS  PubMed  Google Scholar 

  38. Stritesky, G.L. et al. Murine thymic selection quantified using a unique method to capture deleted T cells. Proc. Natl. Acad. Sci. USA 110, 4679–4684 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Lu, L.F. et al. Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity 30, 80–91 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Song, J., So, T., Cheng, M., Tang, X. & Croft, M. Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion. Immunity 22, 621–631 (2005).

    CAS  PubMed  Google Scholar 

  41. Song, J., So, T. & Croft, M. Activation of NF-κB1 by OX40 contributes to antigen-driven T cell expansion and survival. J. Immunol. 180, 7240–7248 (2008).

    CAS  PubMed  Google Scholar 

  42. Chen, X., Baumel, M., Mannel, D.N., Howard, O.M. & Oppenheim, J.J. Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. J. Immunol. 179, 154–161 (2007).

    CAS  PubMed  Google Scholar 

  43. Chen, X. et al. TNFR2 is critical for the stabilization of the CD4+Foxp3+ regulatory T. cell phenotype in the inflammatory environment. J. Immunol. 190, 1076–1084 (2013).

    CAS  PubMed  Google Scholar 

  44. Domen, J. & Weissman, I.L. Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2. Exp. Hematol. 31, 631–639 (2003).

    CAS  PubMed  Google Scholar 

  45. Siegel, R.M. et al. Inhibition of thymocyte apoptosis and negative antigenic selection in bcl-2 transgenic mice. Proc. Natl. Acad. Sci. USA 89, 7003–7007 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Shahinian, A. et al. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261, 609–612 (1993).

    CAS  PubMed  Google Scholar 

  47. Sakanishi, T. & Yagita, H. Anti-tumor effects of depleting and non-depleting anti-CD27 monoclonal antibodies in immune-competent mice. Biochem. Biophys. Res. Commun. 393, 829–835 (2010).

    CAS  PubMed  Google Scholar 

  48. Sheehan, K.C. et al. Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75. J. Exp. Med. 181, 607–617 (1995).

    CAS  PubMed  Google Scholar 

  49. Pear, W.S. et al. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 92, 3780–3792 (1998).

    CAS  PubMed  Google Scholar 

  50. Soneoka, Y. et al. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23, 628–633 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Riccardi (Universita Di Perugia) and E. Shevach (US National Institutes of Health) for Gitr−/− mice; R. Schreiber (Washington University) for neutralizing antibody to TNFR2; G. Hubbard, A. Kne and C. Reis for assistance with animal husbandry; T. Martin, J. Motl and P. Champoux for cell sorting and maintenance of the Flow Cytometry Core Facility at the University of Minnesota (5P01AI035296); and C. Katerndahl, A. Moran, A. Pagán, G. Stritesky, K. Pauken, L. Heltemes-Harris and K. Berquam-Vrieze for comments and for reviewing the manuscript. Supported by the Immunology Training Program of the University of Minnesota (2T32AI007313 to S.A.M. and J.M.S.), the University of Minnesota Medical Scientist Training Program (5T32GM008244 to S.A.M. and J.M.S.), the US National Institutes of Health (F30DK096844 and F30DK100159 to S.A.M. and J.M.S.; HL062683 to J.S.B. and J.M.G.; AI101407 and NS64599 to H.C. and Y.W.; AI088209 to K.A.H. and Y.X.; and CA154998, CA151845 and AI061165 to M.A.F.), the US National Cancer Institute (1F31CA183226 to L.S.M.), the University of Minnesota Undergraduate Research Opportunities Program (H.M.S.) and the Leukemia and Lymphoma Society (M.A.F.).

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Authors and Affiliations

  1. and the Department of Laboratory Medicine and Pathology, Center for Immunology, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

    Shawn A Mahmud, Luke S Manlove, Heather M Schmitz, Yan Xing, David L Owen, Kristin A Hogquist & Michael A Farrar

  2. Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Yanyan Wang & Hongbo Chi

  3. Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, USA

    Jason M Schenkel

  4. Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA

    Jonathan S Boomer & Jonathan M Green

  5. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

    Jonathan M Green

  6. Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

    Hideo Yagita

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Contributions

S.A.M. designed and did experiments and wrote the manuscript; L.S.M., H.M.S., Y.X., Y.W., D.L.O., J.M.S. and J.S.B. did some experiments and contributed intellectually to the work; J.M.G., H.Y., H.C. and K.A.H. provided reagents and/or animals and intellectual contributions; M.A.F. designed experiments, supervised research and assisted in the preparation of the manuscript; and all authors read the manuscript and helped with final revisions.

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Correspondence to Michael A Farrar.

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Integrated supplementary information

Supplementary Figure 1 TNFRSF expression during thymocyte development.

Thymocytes from Foxp3-GFP reporter mice were harvested and evaluated by flow cytometry for expression of GITR, OX40, TNFR2, and CD27. Gates used to identify the indicated populations were as follows; DN thymocytes: CD4−CD8− (grad shaded histograms), DP thymocytes: CD4+CD8+ (black lines), conventional CD4SP: CD4+CD8−CD25−Foxp3− (red lines), Treg cell progenitors were CD25+Foxp3− (green lines), and mature Treg cells were CD25+Foxp3+ (blue lines).

Supplementary Figure 2 GITR and OX40 are diminished on Treg cell progenitors from Cd28-/- mice on the C57BL/6 background.

Histograms plotted in the top panels showing GITR and OX40 expression are derived by gating on Treg cell progenitors from Cd28-/- mice (red histograms) and their wild type littermates (B6 background; blue histograms). In the lower panels, cumulative data are shown for the expression of GITR and OX40 on Treg cell progenitors from CD28-deficient mice in comparison to wild type littermates (mean ± SEM, n=3, p-values generated by student's T-test).

Supplementary Figure 3 Frequency of CD25+Foxp3− Treg cell progenitors in dominant-negative mixed−bone marrow chimeras.

Cells in gates drawn in Fig. 6b were evaluated for CD25 and Foxp3 expression to determine the frequencies of CD25+Foxp3− Treg cell progenitors. The percentage of Treg cell progenitors within CD4SP in each group is plotted as a scatter plot (mean ± SEM, n ≥ 3).

Supplementary Figure 4 CD25−FOXP3lo Treg cell progenitors express TNFRSF in proportion to TCR signal strength and are responsive to TNFRSF costimulation

(a,b) CD25+Foxp3− Treg cell progenitors, and the alternately described population of Treg cell progenitors which are CD25−Foxp3lo are gated in red and blue, respectively, and are compared to conventional CD4SP (CD25−Foxp3−; gray shaded histogram) for expression of GITR and OX40. Raw values for GITR and Nur77-GFP from (c) CD25+Foxp3− and (d) CD25−Foxp3lo Treg cell progenitors were plotted and used to calculate Pearson correlation coefficients. P-values assess whether the degree of correlation was statistically significant. (e) CD25−Foxp3lo Treg cell progenitors were sorted from Foxp3-RFP x Nur77-GFP reporter mice and incubated with 1 U/mL IL-2 and increasing concentrations of GITRL-Fc. The percentage of cells which upregulated CD25 and converted into mature CD25+Foxp3+ Treg cells after 72h are shown in the scatter plot with a regression line applied. (f) The MFI of Nur77-GFP in newly formed CD25+Foxp3+ Treg cells is shown after sorting CD25−Foxp3lo Treg cell progenitors from Foxp3-RFP x Nur77-GFP reporter mice and stimulating for 72h with 1 U/mL IL-2 and increasing concentrations of GITRL-Fc.

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Mahmud, S., Manlove, L., Schmitz, H. et al. Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells. Nat Immunol 15, 473–481 (2014). https://doi.org/10.1038/ni.2849

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