Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development

Abstract

The neonatal thymus generates Foxp3+ regulatory T (tTreg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tTreg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tTreg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IAb-Padi4 with moderate dwell times within a conventional docking orientation are exported as tTreg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4+ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tTreg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tTreg selection window.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: TCRs expressed on neonate-derived tTreg cells can recognize steady-state antigens as well as inflammation- and age-dependent self-antigens.
Fig. 2: Identification of self-ligands recognized by neonatal tTreg TCRs using an immunopeptidome library screen.
Fig. 3: The development of Padi492–105-specific tTreg cells is restricted to the neonatal thymus.
Fig. 4: Padi4-specific tTreg cells seed the peripheral repertoire during the neonatal window and respond to inflammation.
Fig. 5: Padi4-specific thymocytes are subject to temporally regulated, stage-specific changes in negative selection.
Fig. 6: Limiting negative selection restores the development of Padi4-specific tTreg cells in the adult thymus.
Fig. 7: Padi4-specific neonatal tTreg cells express TCRs with modest dwell times.
Fig. 8: TCRs that promote neonatal negative selection, tTreg cell differentiation or CD4 Tconv cell development use conventional docking orientations on IAb-Padi4.

Similar content being viewed by others

Data availability

TCR sequence data have been deposited in the NCBI Sequence Read Archive with accession code PRJNA534321. Coordinates and structure factors for the complexes 6235 TCR:IAb-Padi4, 4699 TCR:IAb-Padi4, 6256 TCR:IAb-Padi4, 5287 TCR:IAb-Padi4, 4378 TCR:IAb-Padi4 and 6236 TCR:IAb-Padi4 are available from the Protein Data Bank under accession codes 6MNO, 6MKD, 6MNM, 6MKR, 6MNG and 6MNN, respectively. All additional data that support the findings of this study are available from the corresponding author upon request.

References

  1. Hogquist, K. A. & Jameson, S. C. The self-obsession of T cells: how TCR signaling thresholds affect fate ‘decisions’ and effector function. Nat. Immunol. 15, 815–823 (2014).

    Article  CAS  Google Scholar 

  2. Klein, L., Robey, E. A. & Hsieh, C. S. Central CD4+ T cell tolerance: deletion versus regulatory T cell differentiation. Nat. Rev. Immunol. 19, 7–18 (2019).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Yang, S., Fujikado, N., Kolodin, D., Benoist, C. & Mathis, D. Immune tolerance. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance. Science 348, 589–594 (2015).

    Article  CAS  Google Scholar 

  5. Li, M. O. & Rudensky, A. Y. T cell receptor signalling in the control of regulatory T cell differentiation and function. Nat. Rev. Immunol. 16, 220–233 (2016).

    Article  CAS  Google Scholar 

  6. Mathis, D. & Benoist, C. Aire. Annu. Rev. Immunol. 27, 287–312 (2009).

    Article  CAS  Google Scholar 

  7. Takaba, H. et al. Fezf2 orchestrates a thymic program of self-antigen expression for immune tolerance. Cell 163, 975–987 (2015).

    Article  CAS  Google Scholar 

  8. Takahama, Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat. Rev. Immunol. 6, 127–135 (2006).

    Article  CAS  Google Scholar 

  9. Daley, S. R., Hu, D. Y. & Goodnow, C. C. Helios marks strongly autoreactive CD4+ T cells in two major waves of thymic deletion distinguished by induction of PD-1 or NF-κB. J. Exp. Med. 210, 269–285 (2013).

    Article  CAS  Google Scholar 

  10. Cowan, J. E. et al. The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development. J. Exp. Med. 210, 675–681 (2013).

    Article  CAS  Google Scholar 

  11. Kishimoto, H. & Sprent, J. Negative selection in the thymus includes semimature T cells. J. Exp. Med. 185, 263–271 (1997).

    Article  CAS  Google Scholar 

  12. Weissler, K. A. & Caton, A. J. The role of T-cell receptor recognition of peptide:MHC complexes in the formation and activity of Foxp3+ regulatory T cells. Immunol. Rev. 259, 11–22 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Perry, J. S. A. et al. Distinct contributions of Aire and antigen-presenting-cell subsets to the generation of self-tolerance in the thymus. Immunity 41, 414–426 (2014).

    Article  CAS  Google Scholar 

  15. Guerau-de-Arellano, M., Martinic, M., Benoist, C. & Mathis, D. Neonatal tolerance revisited: a perinatal window for Aire control of autoimmunity. J. Exp. Med. 206, 1245–1252 (2009).

    Article  CAS  Google Scholar 

  16. Gratz, I. K. & Campbell, D. J. Organ-specific and memory Treg cells: specificity, development, function, and maintenance. Front. Immunol. 5, 333 (2014).

    Article  Google Scholar 

  17. Leonard, J. D. et al. Identification of natural regulatory T cell epitopes reveals convergence on a dominant autoantigen. Immunity 47, 107–117e8 (2017).

    Article  CAS  Google Scholar 

  18. Spence, A. et al. Revealing the specificity of regulatory T cells in murine autoimmune diabetes. Proc. Natl Acad. Sci. USA 115, 5265–5270 (2018).

    Article  CAS  Google Scholar 

  19. Liu, X. et al. T cell receptor CDR3 sequence but not recognition characteristics distinguish autoreactive effector and Foxp3+ regulatory T cells. Immunity 31, 909–920 (2009).

    Article  CAS  Google Scholar 

  20. Kieback, E. et al. Thymus-derived regulatory T cells are positively selected on natural self-antigen through cognate interactions of high functional avidity. Immunity 44, 1114–1126 (2016).

    Article  CAS  Google Scholar 

  21. Malhotra, D. et al. Tolerance is established in polyclonal CD4+ T cells by distinct mechanisms, according to self-peptide expression patterns. Nat. Immunol. 17, 187–195 (2016).

    Article  CAS  Google Scholar 

  22. Leventhal, D. S. et al. Dendritic cells coordinate the development and homeostasis of organ-specific regulatory T cells. Immunity 44, 847–859 (2016).

    Article  CAS  Google Scholar 

  23. Kalekar, L. A. et al. CD4+ T cell anergy prevents autoimmunity and generates regulatory T cell precursors. Nat. Immunol. 17, 304–314 (2016).

    Article  CAS  Google Scholar 

  24. Xing, Y., Wang, X., Jameson, S. C. & Hogquist, K. A. Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling. Nat. Immunol. 17, 565–573 (2016).

    Article  CAS  Google Scholar 

  25. Fontenot, J. D., Dooley, J. L., Farr, A. G. & Rudensky, A. Y. Developmental regulation of Foxp3 expression during ontogeny. J. Exp. Med. 202, 901–906 (2005).

    Article  CAS  Google Scholar 

  26. Li, J., Park, J., Foss, D. & Goldschneider, I. Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus. J. Exp. Med. 206, 607–622 (2009).

    Article  CAS  Google Scholar 

  27. Govern, C. C., Paczosa, M. K., Chakraborty, A. K. & Huseby, E. S. Fast on-rates allow short dwell time ligands to activate T cells. Proc. Natl Acad. Sci. USA 107, 8724–8729 (2010).

    Article  CAS  Google Scholar 

  28. Aleksic, M. et al. Dependence of T cell antigen recognition on T cell receptor-peptide MHC confinement time. Immunity 32, 163–174 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Apostolou, I., Sarukhan, A., Klein, L. & von Boehmer, H. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3, 756–763 (2002).

    Article  CAS  Google Scholar 

  31. Aschenbrenner, K. et al. Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells. Nat. Immunol. 8, 351–358 (2007).

    Article  CAS  Google Scholar 

  32. Legoux, F. P. et al. CD4+ T cell tolerance to tissue-restricted self antigens is mediated by antigen-specific regulatory T cells rather than deletion. Immunity 43, 896–908 (2015).

    Article  CAS  Google Scholar 

  33. Barthlott, T., Kassiotis, G. & Stockinger, B. T cell regulation as a side effect of homeostasis and competition. J. Exp. Med. 197, 451–460 (2003).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. Stadinski, B. D. et al. Hydrophobic CDR3 residues promote the development of self-reactive T cells. Nat. Immunol. 17, 946–955 (2016).

    Article  CAS  Google Scholar 

  36. Van Santen, H. M., Benoist, C. & Mathis, D. Number of T reg cells that differentiate does not increase upon encounter of agonist ligand on thymic epithelial cells. J. Exp. Med. 200, 1221–1230 (2004).

    Article  CAS  Google Scholar 

  37. Pacholczyk, R. et al. Nonself-antigens are the cognate specificities of Foxp3+ regulatory T cells. Immunity 27, 493–504 (2007).

    Article  CAS  Google Scholar 

  38. Hsieh, C. S., Zheng, Y., Liang, Y., Fontenot, J. D. & Rudensky, A. Y. An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat. Immunol. 7, 401–410 (2006).

    Article  CAS  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

  40. Andreotti, A. H., Joseph, R. E., Conley, J. M., Iwasa, J. & Berg, L. J. Multidomain control over TEC kinase activation state tunes the T cell response. Annu. Rev. Immunol. 36, 549–578 (2018).

    Article  CAS  Google Scholar 

  41. Hahn, M., Nicholson, M. J., Pyrdol, J. & Wucherpfennig, K. W. Unconventional topology of self peptide-major histocompatibility complex binding by a human autoimmune T cell receptor. Nat. Immunol. 6, 490–496 (2005).

    Article  CAS  Google Scholar 

  42. Adams, J. J. et al. T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex. Immunity 35, 681–693 (2011).

    Article  CAS  Google Scholar 

  43. Schubert, D. A. et al. Self-reactive human CD4 T cell clones form unusual immunological synapses. J. Exp. Med. 209, 335–352 (2012).

    Article  CAS  Google Scholar 

  44. Beringer, D. X. et al. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex. Nat. Immunol. 16, 1153–1161 (2015).

    Article  CAS  Google Scholar 

  45. Altan-Bonnet, G. & Germain, R. N. Modeling T cell antigen discrimination based on feedback control of digital ERK responses. PLoS Biol. 3, e356 (2005).

    Article  Google Scholar 

  46. Bautista, J. L. et al. Intraclonal competition limits the fate determination of regulatory T cells in the thymus. Nat. Immunol. 10, 610–617 (2009).

    Article  CAS  Google Scholar 

  47. Thiault, N. et al. Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors. Nat. Immunol. 16, 628–634 (2015).

    Article  CAS  Google Scholar 

  48. Weist, B. M., Kurd, N., Boussier, J., Chan, S. W. & Robey, E. A. Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition. Nat. Immunol. 16, 635–641 (2015).

    Article  CAS  Google Scholar 

  49. Huseby, E. S., Sather, B., Huseby, P. G. & Goverman, J. Age-dependent T cell tolerance and autoimmunity to myelin basic protein. Immunity 14, 471–481 (2001).

    Article  CAS  Google Scholar 

  50. Dong, M. et al. Alterations in the thymic selection threshold skew the self-reactivity of the TCR repertoire in neonates. J. Immunol. 199, 965–973 (2017).

    Article  CAS  Google Scholar 

  51. Gilligan, D. M. et al. Targeted disruption of the β adducin gene (Add2) causes red blood cell spherocytosis in mice. Proc. Natl Acad. Sci. USA 96, 10717–10722 (1999).

    Article  CAS  Google Scholar 

  52. Hemmers, S., Teijaro, J. R., Arandjelovic, S. & Mowen, K. A. PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection. PLoS One 6, e22043 (2011).

    Article  CAS  Google Scholar 

  53. Stadinski, B. D. et al. A role for differential variable gene pairing in creating T cell receptors specific for unique major histocompatibility ligands. Immunity 35, 694–704 (2011).

    Article  CAS  Google Scholar 

  54. Moon, J. J. et al. Tracking epitope-specific T cells. Nat. Protoc. 4, 565–581 (2009).

    Article  CAS  Google Scholar 

  55. Bogunovic, B., Srinivasan, P., Ueda, Y., Tomita, Y. & Maric, M. Comparative quantitative mass spectrometry analysis of MHC class II-associated peptides reveals a role of GILT in formation of self-peptide repertoire. PLoS One 5, e10599 (2010).

    Article  Google Scholar 

  56. Bozzacco, L. et al. Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells. J. Proteome Res. 10, 5016–5030 (2011).

    Article  CAS  Google Scholar 

  57. Dongre, A. R. et al. In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses. Eur. J. Immunol. 31, 1485–1494 (2001).

    Article  CAS  Google Scholar 

  58. Sofron, A., Ritz, D., Neri, D. & Fugmann, T. High-resolution analysis of the murine MHC class II immunopeptidome. Eur. J. Immunol. 46, 319–328 (2016).

    Article  CAS  Google Scholar 

  59. Fugmann, T., Sofron, A., Ritz, D., Bootz, F. & Neri, D. The MHC class II immunopeptidome of lymph nodes in health and in chemically induced colitis. J. Immunol. 198, 1357–1364 (2017).

    Article  CAS  Google Scholar 

  60. Nanaware, P. P., Jurewicz, M. M., Leszyk, J., Shaffer, S. A. & Stern, L. J. HLA-DO modulates the diversity of the MHC-II self-peptidome. Mol. Cell. Proteomics 18, 490–503 (2018).

    Article  Google Scholar 

  61. Rudensky, A., Preston-Hurlburt, P., al-Ramadi, B. K., Rothbard, J. & Janeway, C. A. Jr Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs. Nature 359, 429–431 (1992).

    Article  CAS  Google Scholar 

  62. Yang, X. et al. TCRklass: a new K-string-based algorithm for human and mouse TCR repertoire characterization. J. Immunol. 194, 446–454 (2015).

    Article  CAS  Google Scholar 

  63. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  64. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  65. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  66. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  67. Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

    Article  CAS  Google Scholar 

  68. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  Google Scholar 

  69. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  Google Scholar 

  70. Rudolph, M. G., Stanfield, R. L. & Wilson, I. A. How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24, 419–466 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institutes of Health (DK095077, AR071269 and AI109858 to E.S.H.). X-ray diffraction data were collected at the LRL-CAT (31-ID) beamline at APS at the Argonne National Laboratory for PDB IDs 6MKD and 6MKR, and the FMX (17-ID-2) beamline at NSLS II at the Brookhaven National Laboratory for PDB IDs 6MNM, 6MNO, 6MNN and 6MNG.

Author information

Authors and Affiliations

Authors

Contributions

B.D.S., S.J.B. and E.S.H. conceived and designed the project and interpreted the experiments. B.D.S. performed the TCR cloning and sequencing, flow cytometry and T cell activation experiments. B.D.S. and S.J.B. performed the structural, biophysical and statistical analyses. N.A.S., B.R.D. and P.G.H. performed the experiments. L.J.S. aided the mass spectrometry analyses. B.D.S., S.J.B. and E.S.H. wrote the manuscript.

Corresponding author

Correspondence to Eric S. Huseby.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Supplementary Tables 1–3.

Reporting Summary

Supplementary Dataset 1

IAb peptide library.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Stadinski, B.D., Blevins, S.J., Spidale, N.A. et al. A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development. Nat Immunol 20, 1046–1058 (2019). https://doi.org/10.1038/s41590-019-0414-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-019-0414-1

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing