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.
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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.
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).
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).
Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M. Regulatory T cells and immune tolerance. Cell 133, 775–787 (2008).
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).
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).
Mathis, D. & Benoist, C. Aire. Annu. Rev. Immunol. 27, 287–312 (2009).
Takaba, H. et al. Fezf2 orchestrates a thymic program of self-antigen expression for immune tolerance. Cell 163, 975–987 (2015).
Takahama, Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat. Rev. Immunol. 6, 127–135 (2006).
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).
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).
Kishimoto, H. & Sprent, J. Negative selection in the thymus includes semimature T cells. J. Exp. Med. 185, 263–271 (1997).
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).
Malchow, S. et al. Aire-dependent thymic development of tumor-associated regulatory T cells. Science 339, 1219–1224 (2013).
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).
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).
Gratz, I. K. & Campbell, D. J. Organ-specific and memory Treg cells: specificity, development, function, and maintenance. Front. Immunol. 5, 333 (2014).
Leonard, J. D. et al. Identification of natural regulatory T cell epitopes reveals convergence on a dominant autoantigen. Immunity 47, 107–117e8 (2017).
Spence, A. et al. Revealing the specificity of regulatory T cells in murine autoimmune diabetes. Proc. Natl Acad. Sci. USA 115, 5265–5270 (2018).
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).
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).
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).
Leventhal, D. S. et al. Dendritic cells coordinate the development and homeostasis of organ-specific regulatory T cells. Immunity 44, 847–859 (2016).
Kalekar, L. A. et al. CD4+ T cell anergy prevents autoimmunity and generates regulatory T cell precursors. Nat. Immunol. 17, 304–314 (2016).
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).
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).
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).
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).
Aleksic, M. et al. Dependence of T cell antigen recognition on T cell receptor-peptide MHC confinement time. Immunity 32, 163–174 (2010).
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).
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).
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).
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).
Barthlott, T., Kassiotis, G. & Stockinger, B. T cell regulation as a side effect of homeostasis and competition. J. Exp. Med. 197, 451–460 (2003).
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).
Stadinski, B. D. et al. Hydrophobic CDR3 residues promote the development of self-reactive T cells. Nat. Immunol. 17, 946–955 (2016).
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).
Pacholczyk, R. et al. Nonself-antigens are the cognate specificities of Foxp3+ regulatory T cells. Immunity 27, 493–504 (2007).
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).
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).
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).
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).
Adams, J. J. et al. T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex. Immunity 35, 681–693 (2011).
Schubert, D. A. et al. Self-reactive human CD4 T cell clones form unusual immunological synapses. J. Exp. Med. 209, 335–352 (2012).
Beringer, D. X. et al. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex. Nat. Immunol. 16, 1153–1161 (2015).
Altan-Bonnet, G. & Germain, R. N. Modeling T cell antigen discrimination based on feedback control of digital ERK responses. PLoS Biol. 3, e356 (2005).
Bautista, J. L. et al. Intraclonal competition limits the fate determination of regulatory T cells in the thymus. Nat. Immunol. 10, 610–617 (2009).
Thiault, N. et al. Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors. Nat. Immunol. 16, 628–634 (2015).
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).
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).
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).
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).
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).
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).
Moon, J. J. et al. Tracking epitope-specific T cells. Nat. Protoc. 4, 565–581 (2009).
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).
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).
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).
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).
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).
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).
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).
Yang, X. et al. TCRklass: a new K-string-based algorithm for human and mouse TCR repertoire characterization. J. Immunol. 194, 446–454 (2015).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Rudolph, M. G., Stanfield, R. L. & Wilson, I. A. How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24, 419–466 (2006).
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.
The authors declare no competing interests.
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Nature Immunology (2019)