Letter | Published:

A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance

Nature volume 528, pages 132136 (03 December 2015) | Download Citation


T-cell receptor (TCR) signalling has a key role in determining T-cell fate. Precursor cells expressing TCRs within a certain low-affinity range for complexes of self-peptide and major histocompatibility complex (MHC) undergo positive selection and differentiate into naive T cells expressing a highly diverse self-MHC-restricted TCR repertoire. In contrast, precursors displaying TCRs with a high affinity for ‘self’ are either eliminated through TCR-agonist-induced apoptosis (negative selection)1 or restrained by regulatory T (Treg) cells, whose differentiation and function are controlled by the X-chromosome-encoded transcription factor Foxp3 (reviewed in ref. 2). Foxp3 is expressed in a fraction of self-reactive T cells that escape negative selection in response to agonist-driven TCR signals combined with interleukin 2 (IL-2) receptor signalling. In addition to Treg cells, TCR-agonist-driven selection results in the generation of several other specialized T-cell lineages such as natural killer T cells and innate mucosal-associated invariant T cells3. Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs4,5,6. Here we explore in mice whether a specialized mechanism enables agonist-driven selection of Treg cells with a diverse TCR repertoire, and the importance this holds for self-tolerance. We show that the intronic Foxp3 enhancer conserved noncoding sequence 3 (CNS3) acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to control self-reactive T cells effectively, especially when thymic negative selection is genetically impaired. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.

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Primary accessions

Gene Expression Omnibus

Data deposits

All RNA and TCR sequencing data have been deposited in the Gene Expression Omnibus under accession numbers GSE71309 and GSE71162, respectively.


  1. 1.

    , , & Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see). Nature Rev. Immunol. 14, 377–391 (2014)

  2. 2.

    , & Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012)

  3. 3.

    & Activation and function of iNKT and MAIT cells. Adv. Immunol. 127, 145–201 (2015)

  4. 4.

    , , , & An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nature Immunol. 7, 401–410 (2006)

  5. 5.

    et al. Recognition of the peripheral self by naturally arising CD25+CD4+ T cell receptors. Immunity 21, 267–277 (2004)

  6. 6.

    , , , & A broad range of self-reactivity drives thymic regulatory T cell selection to limit responses to self. Immunity 37, 475–486 (2012)

  7. 7.

    , , & Continuous requirement for the TCR in regulatory T cell function. Nature Immunol. 15, 1070–1078 (2014)

  8. 8.

    et al. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nature Immunol. 2, 301–306 (2001)

  9. 9.

    , , & High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice. Cell 78, 399–408 (1994)

  10. 10.

    , , & Origin of regulatory T cells with known specificity for antigen. Nature Immunol. 3, 756–763 (2002)

  11. 11.

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

  12. 12.

    et al. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity 22, 329–341 (2005)

  13. 13.

    , , & Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J. Exp. Med. 201, 723–735 (2005)

  14. 14.

    , , & A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nature Immunol. 6, 1142–1151 (2005)

  15. 15.

    , , & Long-term survival but impaired homeostatic proliferation of naive T cells in the absence of p56lck. Science 290, 127–131 (2000)

  16. 16.

    et al. Suppression of inflammation by a synthetic histone mimic. Nature 468, 1119–1123 (2010)

  17. 17.

    et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 504, 451–455 (2013)

  18. 18.

    Inhibition of histone deacetylase activity by butyrate. J. Nutr. 133, 2485S–2493S (2003)

  19. 19.

    et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504, 446–450 (2013)

  20. 20.

    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)

  21. 21.

    , & Endogenous antigen tunes the responsiveness of naive B cells but not T cells. Nature 489, 160–164 (2012)

  22. 22.

    et al. Towards error-free profiling of immune repertoires. Nature Methods 11, 653–655 (2014)

  23. 23.

    et al. VDJtools: unifying post-analysis of T cell receptor repertoires. PLOS Comput. Biol. (in the press)

  24. 24.

    , , , & How the thymus designs antigen-specific and self-tolerant T cell receptor sequences. Proc. Natl Acad. Sci. USA 105, 16671–16676 (2008)

  25. 25.

    , , , & Immune tolerance. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance. Science 348, 589–594 (2015)

  26. 26.

    , , & Antigen presentation in the thymus for positive selection and central tolerance induction. Nature Rev. Immunol. 9, 833–844 (2009)

  27. 27.

    , , , & Aire regulates negative selection of organ-specific T cells. Nature Immunol. 4, 350–354 (2003)

  28. 28.

    et al. Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells. Proc. Natl Acad. Sci. USA 109, 535–540 (2012)

  29. 29.

    et al. Aire-dependent thymic development of tumor-associated regulatory T cells. Science 339, 1219–1224 (2013)

  30. 30.

    Theories and quantification of thymic selection. Front. Immunol. 5, 13 (2014)

  31. 31.

    et al. Control of the inheritance of regulatory T cell identity by a cis element in the Foxp3 locus. Cell 158, 749–763 (2014)

  32. 32.

    et al. Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells: possible role of T cellbound Ia antigens as targets of immunoregulatory T cells. J. Immunol. 132, 662–667 (1984)

  33. 33.

    , & Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes. Nature 420, 429–434 (2002)

  34. 34.

    et al. Identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays. J. Clin. Invest. 115, 3428–3439 (2005)

  35. 35.

    et al. Quantitative profiling of immune repertoires for minor lymphocyte counts using unique molecular identifiers. J. Immunol. 194, 6155–6163 (2015)

  36. 36.

    et al. Age-related decrease in TCR repertoire diversity measured with deep and normalized sequence profiling. J. Immunol. 192, 2689–2698 (2014)

  37. 37.

    , & Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014)

  38. 38.

    et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  39. 39.

    & Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010)

  40. 40.

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

  41. 41.

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

  42. 42.

    & Active induction of experimental allergic encephalomyelitis. Nature Protocols 1, 1810–1819 (2006)

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We thank P. Bos, A. Arvey, C. Konopacki, G. Gasteiger, S. Lee, T. Chinen and K. Wu for technical assistance, CKP IBCH for equipment, and R. Prinjha for providing iBET. Y.F. was supported by a Postdoctoral Fellowship of the Cancer Research Institute. This study was supported by NIH grants R37 AI034206 and U01 HG007893, Cancer Center Support Grant P30 CA008748, and the Howard Hughes Medical Institute (A.Y.R.). M.S., E.V.P. and D.M.C. were supported by MCB program RAS and RFBR grants 14-04-01247 and 15-34-21052.

Author information

Author notes

    • Yongqiang Feng
    •  & Joris van der Veeken

    These authors contributed equally to this work.


  1. Howard Hughes Medical Institute and Immunology Program, Ludwig Center at Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Yongqiang Feng
    • , Joris van der Veeken
    • , Stanislav Dikiy
    • , Beatrice E. Hoyos
    • , Bruno Moltedo
    • , Saskia Hemmers
    •  & Alexander Y. Rudensky
  2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, Moscow 117997, Russia

    • Mikhail Shugay
    • , Ekaterina V. Putintseva
    •  & Dmitriy M. Chudakov
  3. Pirogov Russian National Research Medical University, Ostrovityanova 1, Moscow 117997, Russia

    • Mikhail Shugay
    •  & Dmitriy M. Chudakov
  4. Central European Institute of Technology, Masaryk University, Kamenice 753/5, Brno 62500, Czech Republic

    • Mikhail Shugay
    • , Ekaterina V. Putintseva
    •  & Dmitriy M. Chudakov
  5. Computational Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Hatice U. Osmanbeyoglu
    •  & Christina S. Leslie
  6. Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, Washington 98195, USA

    • Piper Treuting


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Y.F. and A.R. conceived and designed the study. Y.F. performed animal and in vitro studies, flow cytometric, TCR sequencing and gene expression analyses. J.v.d.V. analysed the epigenetic modifications of CNS3 and how they affect Foxp3 transcriptional regulation. M.S., E.V.P. and D.M.C. analysed TCR sequencing data. H.U.O. and C.S.L. analysed RNA sequencing data. B.E.H. performed serum Ig isotype analysis. S.D. and S.H. participated in phenotypic analysis of mice. S.H. generated the Cre retroviral construct. B.M. and S.D. participated in optimizing TCR sequencing protocol. P.T. analysed histopathology. Y.F. and A.Y.R. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alexander Y. Rudensky.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Table 1

    The file shows the numbers of added nucleotides in TCRα CDR3 .

  2. 2.

    Supplementary Data 1

    This file shows up‐regulated genes in activated vs resting Treg cells.

  3. 3.

    Supplementary Data 2

    This files shows down‐regulated genes in activated vs resting Treg cells.

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