Tolerance is established in polyclonal CD4+ T cells by distinct mechanisms, according to self-peptide expression patterns

Abstract

Studies of repertoires of mouse monoclonal CD4+ T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4+ T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4+ T cells was maintained by distinct mechanisms, according to self-peptide expression patterns.

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Figure 1: Three main patterns of tolerance to self antigens.
Figure 2: Ins1eGFP mice 'ignore' the eGFPp–I-Ab epitope.
Figure 3: eGFPp–I-Ab–specific CD4+ T cells undergo limited clonal deletion in the thymus.
Figure 4: High-affinity T cells are deleted in Ins2eGFP mice.
Figure 5: Aire-mediated thymic expression of eGFP promotes clonal deletion and Treg induction in Ins2eGFP mice.
Figure 6: Expression of a self epitope by thymic antigen-presenting cells induces intrathymic clonal deletion.
Figure 7: Frequency of self antigen–positive thymic antigen-presenting cells correlates with the responsiveness of the corresponding CD4+ T cell population.
Figure 8: Tolerance mechanisms identified in eGFP-expressing mouse strains govern tolerance to true self antigens.

References

  1. 1

    Xing, Y. & Hogquist, K.A. T-cell tolerance: central and peripheral. Cold Spring Harb. Perspect. Biol. 4, a006957 (2012).

    PubMed  PubMed Central  Google Scholar 

  2. 2

    Wing, K. & Sakaguchi, S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat. Immunol. 11, 7–13 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Semana, G., Gausling, R., Jackson, R.A. & Hafler, D.A. T cell autoreactivity to proinsulin epitopes in diabetic patients and healthy subjects. J. Autoimmun. 12, 259–267 (1999).

    CAS  PubMed  Google Scholar 

  4. 4

    Moon, J.J. et al. Quantitative impact of thymic selection on Foxp3+ and Foxp3 subsets of self-peptide/MHC class II-specific CD4+ T cells. Proc. Natl. Acad. Sci. USA 108, 14602–14607 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Yu, W. et al. Clonal deletion prunes but does not eliminate self-specific αβ CD8+ T lymphocytes. Immunity 42, 929–941 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Maeda, Y. et al. Detection of self-reactive CD8+ T cells with an anergic phenotype in healthy individuals. Science 346, 1536–1540 (2014).

    CAS  PubMed  Google Scholar 

  7. 7

    Ohashi, P.S. et al. Ablation of “tolerance” and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 65, 305–317 (1991).

    CAS  Google Scholar 

  8. 8

    Oldstone, M.B., Nerenberg, M., Southern, P., Price, J. & Lewicki, H. Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response. Cell 65, 319–331 (1991).

    CAS  Google Scholar 

  9. 9

    Jenkins, M.K. & Schwartz, R.H. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J. Exp. Med. 165, 302–319 (1987).

    CAS  PubMed  Google Scholar 

  10. 10

    Akkaraju, S. et al. A range of CD4 T cell tolerance: partial inactivation to organ-specific antigen allows nondestructive thyroiditis or insulitis. Immunity 7, 255–271 (1997).

    CAS  PubMed  Google Scholar 

  11. 11

    Zehn, D. & Bevan, M.J. T cells with low avidity for a tissue-restricted antigen routinely evade central and peripheral tolerance and cause autoimmunity. Immunity 25, 261–270 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Hataye, J., Moon, J.J., Khoruts, A., Reilly, C. & Jenkins, M.K. Naive and memory CD4+ T cell survival controlled by clonal abundance. Science 312, 114–116 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Marzo, A.L. et al. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6, 793–799 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

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

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Bouneaud, C., Kourilsky, P. & Bousso, P. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity 13, 829–840 (2000).

    CAS  Google Scholar 

  16. 16

    Nelson, R.W. et al. T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity. Immunity 42, 95–107 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Reich, M. et al. GenePattern 2.0. Nat. Genet. 38, 500–501 (2006).

    CAS  Google Scholar 

  18. 18

    Hara, M. et al. Transgenic mice with green fluorescent protein-labeled pancreatic beta-cells. Am. J. Physiol. Endocrinol. Metab. 284, E177–E183 (2003).

    CAS  PubMed  Google Scholar 

  19. 19

    Anderson, M.S. et al. Projection of an immunological self shadow within the thymus by the aire protein. Science 298, 1395–1401 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Carreres, M.I. et al. Transcription factor Foxd1 is required for the specification of the temporal retina in mammals. J. Neurosci. 31, 5673–5681 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Kobayashi, A. et al. Identification of a multipotent self-renewing stromal progenitor population during mammalian kidney organogenesis. Stem Cell Rep. 3, 650–662 (2014).

    CAS  Google Scholar 

  22. 22

    Ben-Yehudah, A. et al. Specific dynamic and noninvasive labeling of pancreatic beta cells in reporter mice. Genesis 43, 166–174 (2005).

    CAS  PubMed  Google Scholar 

  23. 23

    Kaplan, D.H. et al. Autocrine/paracrine TGFβ1 is required for the development of epidermal Langerhans cells. J. Exp. Med. 204, 2545–2552 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Heng, T.S., Painter, M.W. & Immunological Genome Project Consortium. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008).

    CAS  Google Scholar 

  25. 25

    Gong, S. et al. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917–925 (2003).

    CAS  PubMed  Google Scholar 

  26. 26

    Crawford, F., Kozono, H., White, J., Marrack, P. & Kappler, J. Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. Immunity 8, 675–682 (1998).

    CAS  PubMed  Google Scholar 

  27. 27

    Warren, H.S., Vogel, F.R. & Chedid, L.A. Current status of immunological adjuvants. Annu. Rev. Immunol. 4, 369–388 (1986).

    CAS  PubMed  Google Scholar 

  28. 28

    Portnoy, D.A., Auerbuch, V. & Glomski, I.J. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J. Cell Biol. 158, 409–414 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Cordier, A.C. & Haumont, S.M. Development of thymus, parathyroids, and ultimo-branchial bodies in NMRI and nude mice. Am. J. Anat. 157, 227–263 (1980).

    CAS  PubMed  Google Scholar 

  30. 30

    Schaefer, B.C., Schaefer, M.L., Kappler, J.W., Marrack, P. & Kedl, R.M. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell. Immunol. 214, 110–122 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Lindquist, R.L. et al. Visualizing dendritic cell networks in vivo. Nat. Immunol. 5, 1243–1250 (2004).

    CAS  PubMed  Google Scholar 

  33. 33

    Panneck, A.R. et al. Cholinergic epithelial cell with chemosensory traits in murine thymic medulla. Cell Tissue Res. 358, 737–748 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Tallini, Y.N. et al. BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons. Physiol. Genomics 27, 391–397 (2006).

    CAS  PubMed  Google Scholar 

  35. 35

    Snippert, H.J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    CAS  PubMed  Google Scholar 

  36. 36

    Gardner, J.M. et al. Deletional tolerance mediated by extrathymic Aire-expressing cells. Science 321, 843–847 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Pepper, M., Pagan, A.J., Igyarto, B.Z., Taylor, J.J. & Jenkins, M.K. Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells. Immunity 35, 583–595 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Geginat, G., Schenk, S., Skoberne, M., Goebel, W. & Hof, H. A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes. J. Immunol. 166, 1877–1884 (2001).

    CAS  PubMed  Google Scholar 

  39. 39

    Rees, W. et al. An inverse relationship between T cell receptor affinity and antigen dose during CD4+ T cell responses in vivo and in vitro. Proc. Natl. Acad. Sci. USA 96, 9781–9786 (1999).

    CAS  PubMed  Google Scholar 

  40. 40

    Takeshima, H., Komazaki, S., Nishi, M., Iino, M. & Kangawa, K. Junctophilins: a novel family of junctional membrane complex proteins. Mol. Cell 6, 11–22 (2000).

    CAS  PubMed  Google Scholar 

  41. 41

    Fu, X.M., Dai, X., Ding, J. & Zhu, B.T. Pancreas-specific protein disulfide isomerase has a cell type-specific expression in various mouse tissues and is absent in human pancreatic adenocarcinoma cells: implications for its functions. J. Mol. Histol. 40, 189–199 (2009).

    CAS  PubMed  Google Scholar 

  42. 42

    Rosette, C. et al. The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model. Immunity 15, 59–70 (2001).

    CAS  PubMed  Google Scholar 

  43. 43

    Au-Yeung, B.B. et al. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation. Proc. Natl. Acad. Sci. USA 111, E3679–E3688 (2014).

    CAS  PubMed  Google Scholar 

  44. 44

    Klein, L., Kyewski, B., Allen, P.M. & Hogquist, K.A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see). Nat. Rev. Immunol. 14, 377–391 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Tubo, N.J. et al. Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection. Cell 153, 785–796 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    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).

    CAS  PubMed  Google Scholar 

  47. 47

    Tanchot, C., Barber, D.L., Chiodetti, L. & Schwartz, R.H. Adaptive tolerance of CD4+ T cells in vivo: multiple thresholds in response to a constant level of antigen presentation. J. Immunol. 167, 2030–2039 (2001).

    CAS  PubMed  Google Scholar 

  48. 48

    Kearney, E.R., Pape, K.A., Loh, D.Y. & Jenkins, M.K. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1, 327–339 (1994).

    CAS  PubMed  Google Scholar 

  49. 49

    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 

  50. 50

    PrabhuDas, M. et al. Immune mechanisms at the maternal-fetal interface: perspectives and challenges. Nat. Immunol. 16, 328–334 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Gupta, R.K. et al. Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells. Cell Metab. 15, 230–239 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Xin, H.B., Deng, K.Y., Rishniw, M., Ji, G. & Kotlikoff, M.I. Smooth muscle expression of Cre recombinase and eGFP in transgenic mice. Physiol. Genomics 10, 211–215 (2002).

    CAS  PubMed  Google Scholar 

  53. 53

    Fontenot, J.D. et al. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329–341 (2005).

    CAS  Google Scholar 

  54. 54

    Shen, A. & Higgins, D.E. The 5′ untranslated region-mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity. Mol. Microbiol. 57, 1460–1473 (2005).

    CAS  PubMed  Google Scholar 

  55. 55

    Lauer, P., Chow, M.Y., Loessner, M.J., Portnoy, D.A. & Calendar, R. Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J. Bacteriol. 184, 4177–4186 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Brockstedt, D.G. et al. Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proc. Natl. Acad. Sci. USA 101, 13832–13837 (2004).

    CAS  PubMed  Google Scholar 

  57. 57

    Chu, H.H., Moon, J.J., Kruse, A.C., Pepper, M. & Jenkins, M.K. Negative selection and peptide chemistry determine the size of naive foreign peptide-MHC class II-specific CD4+ T cell populations. J. Immunol. 185, 4705–4713 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Moon, J.J. et al. Naive CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity 27, 203–213 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Malhotra, D. et al. Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks. Nat. Immunol. 13, 499–510 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Berzins, S.P., Boyd, R.L. & Miller, J.F. The role of the thymus and recent thymic migrants in the maintenance of the adult peripheral lymphocyte pool. J. Exp. Med. 187, 1839–1848 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    de Hoon, M.J., Imoto, S., Nolan, J. & Miyano, S. Open source clustering software. Bioinformatics 20, 1453–1454 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    CAS  Google Scholar 

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Acknowledgements

We thank D. Mueller for reviewing the manuscript; J. Walter and O. Rainwater for technical assistance; P. Lauer (and Aduro Biotech) for the Lm-eGFP strain PL1113. Supported by the US National Institutes of Health (P01 AI35296 to M.K.J. and K.A.H.; F32 AI114050 to D.M.; T32 GM008244 and F30 DK093242 to R.W.N.; T32 AI07313 to J.L.L.; K99 AI114884 to Y.J.L.), the Wallin Neuroscience Discovery Fund (to H.T.O. and M.K.J.) and the Juvenile Diabetes Research Foundation (2-2011-662 to B.T.F.).

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D.M. designed the study, did experiments, analyzed data, and wrote the manuscript, J.L.L., T.D., Y.J.L., W.E.P., J.V.L., R.W.N. and M.S.A. did experiments, B.T.F., H.T.O. and K.A.H. provided discussions; and M.K.J. designed the study, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Marc K Jenkins.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gating strategy for thymic epithelial cells and dendritic cells.

(a) Contour plots depicting the initial gating strategy for analysis of eGFP and eYFP expression by EpCAM+ thymic epithelial cells and dendritic cells. Briefly, thymuses were enzymatically digested to produce single cell suspensions, which were enriched for EpCAM+ and CD11c+ cells. Doublets were excluded. Numbers indicate the percent of cells in each gate.(b) Contour plots showing the gating strategy for EpCAM+ thymic epithelial cells, proceeding from singlet gate 2 (a). EpCAM+ CD45.2 stromal cells were gated for further analysis by excluding CD90.2+ T cells, dead cells, CD19+ B cells, macrophages and dendritic cells (CD11b+ and CD11c+). Remaining cells were analyzed further in Figure 7. Numbers indicate the percent of cells in the gate.(c) Contour plots showing the gating strategy for thymic dendritic cells, proceeding from singlet gate 2 (a). CD11c+ cells were gated and dead cells, CD90.2+ T cells, CD11chiCD11bhi eosinophils, CD19+ B cells, and autofluorescent macrophages (shaded, blue gate) were then excluded. Remaining CD11c+CD11blo–int cells were analyzed further in Figure 7. Numbers indicate the percent of cells carried forward for analysis.

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Malhotra, D., Linehan, J., Dileepan, T. 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). https://doi.org/10.1038/ni.3327

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