Clonal deletion and the fate of autoreactive thymocytes that survive negative selection

Journal name:
Nature Immunology
Volume:
13,
Pages:
569–578
Year published:
DOI:
doi:10.1038/ni.2292
Received
Accepted
Published online

Abstract

Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4+CD8lo intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4CD8 double-negative thymocytes positive for the T cell antigen receptor αβ subtype (TCRαβ) that 'preferentially' migrated to the intestine, where they re-expressed CD8α and were sequestered as CD8αα+ intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8αα+ IELs as the developmental fate of autoreactive thymocytes that survive negative selection.

At a glance

Figures

  1. CD28 costimulation-deficient mice have more TCR[alpha][beta]+ DN thymocytes.
    Figure 1: CD28 costimulation–deficient mice have more TCRαβ+ DN thymocytes.

    (a) Staining of CD4 and CD8 (top) in thymocytes from wild-type (WT), Cd28−/− and B7-deficient (B7-DKO) mice on the C57BL/6 background (left) or BALB/c background (right), and surface TCRβ expression on gated DN thymocytes (bottom). Numbers at top of plots (above) indicate total number of thymocytes; numbers adjacent to outlined areas (above) indicate percent DN cells; numbers in parentheses (below) indicate percent DN cells that are TCRβ+ (bracketed lines). (b) Quantification (frequency and number) of TCRαβ+ DN thymocytes in wild-type, Cd28−/− and B7-deficient mice. *P < 0.01, **P < 0.001 and ***P < 0.0001, compared with wild-type (Student's two-tailed t-test). (c) Staining of TCRαβ+ DN thymocytes from wild-type and B7-deficient C57BL/6 mice with a tetramer of CD1d and the α-galactosylceramide analog PBS57 (CD1d-PBS57). Data are representative of five independent experiments (a,b; mean and s.e.m.) or two independent experiments (c).

  2. Effect of transgenes encoding Bcl-2 and Mcl-1 on the appearance of TCR[alpha][beta]+ DN thymocytes.
    Figure 2: Effect of transgenes encoding Bcl-2 and Mcl-1 on the appearance of TCRαβ+ DN thymocytes.

    (a) Staining of CD4 and CD8 in thymocytes from wild-type mice, Bcl-2-transgenic (Bcl-2-TG) mice and Mcl-1-transgenic (Mcl-1-TG) mice (left), and surface expression of TCRβ (middle) and CD5 and (right) on gated DN thymocytes (numbers as in Fig. 1a). (b) Quantification of TCRαβ+ DN thymocytes in wild-type and transgenic mice. *P < 0.05 and **P < 0.001, compared with wild-type (Student's two-tailed t-test). Data represent four independent experiments (mean and s.e.m.).

  3. Expression of Mtv-reactive V[beta]+ TCRs in pre- and post-selection thymocyte subsets.
    Figure 3: Expression of Mtv-reactive Vβ+ TCRs in pre- and post-selection thymocyte subsets.

    (a) Expression of Mtv-9-reactive Vβ5+ and Mtv-8- and Mtv-9-reactive Vβ11+ TCRs and control Vβ8+ TCRs on various thymocyte subsets from wild-type, Cd28−/− and B7-deficient mice on the BALB/c background. Horizontal dashed lines indicate the frequency of preselection DP thymocytes in wild-type mice expressing TCRs of each Vβ specificity. (b) Quantification (frequency and number) of specific Mtv-6-reactive Vβ3+, Mtv-9-reactive Vβ5+ and Mtv-8- and Mtv-9-reactive Vβ11+ TCRs and control Vβ8+ TCRs expressed by TCRαβ+ DN thymocytes from the progeny of BALB/c mice crossed with C57BL/6 mice (CB6) that were wild-type, Bcl-2-transgenic or Mcl-1-transgenic. *P < 0.01, **P < 0.001 and ***P < 0.0001, compared with wild-type (Student's two-tailed t-test). Data represent at least three independent experiments with at least five mice per group (mean and s.e.m.).

  4. TCR[alpha][beta]+ DN thymocytes are the progeny of DP thymocytes.
    Figure 4: TCRαβ+ DN thymocytes are the progeny of DP thymocytes.

    (a) Methylation status of the Cd8b1 promoter in Bcl-2-transgenic thymocytes, determined by bisulfite conversion and sequencing of genomic DNA from sorted thymocyte subpopulations from three individual mice and presented as the frequency of methylated or unmethylated Cd8b1 promoter sequences (Supplementary Fig. 2). *P < 0.0001 (Student's two-tailed t-test). (b) Staining of CD4 and CD8 (above) in thymocytes from mice of various genotypes (above plots) on the C57BL/6 background, and surface TCRβ expression on gated DN thymocytes (below; numbers as in Fig. 1a). Data are from two independent experiments.

  5. Effect of thymic selection on the expression of Mtv-reactive V[beta]+ TCRs.
    Figure 5: Effect of thymic selection on the expression of Mtv-reactive Vβ+ TCRs.

    (a) Frequency of cells expressing Mtv-9-reactive Vβ5+ and Mtv-8- and Mtv-9-reactive Vβ11+ TCRs and control Vβ8+ TCRs in various TCRαβ+ thymocyte subsets from wild-type, Cd28−/− and B7-deficient BALB/c mice, presented as relative to that among preselection DP thymocytes, set as 100%. (b) Surface expression of CD28 on intermediate and TCRαβ+ DN thymocyte subsets from B7-deficient and Bcl-2-transgenic mice, presented as mean fluorescence intensity relative to that of preselection DP thymocytes, set as 100%. (c) TCR-induced calcium mobilization in B7-deficient thymocyte populations depleted of CD8+ cells; downward arrow indicates crosslinkage of biotinylated monoclonal anti-TCR (5 μg/ml) by avidin. (d) Proliferation of purified TCRαβ+ DN and SP8 B7-deficient thymocytes (2 × 104 cells per well) stimulated with plate-bound anti-TCR (5 μg) plus anti-CD28 (10 μg) in the presence (+ IL-2) or absence (Med) of exogenous IL-2 (200 U/ml), assessed as incorporation of [3H]thymidine. Data are from five independent experiments (a; mean ± s.e.m.), represent three independent experiments (b,c; mean ± s.e.m. in b) or are representative of two independent experiments (d; mean and s.e.m. of triplicate cultures).

  6. Developmentally diverted TCR[alpha][beta]+ DN thymocytes migrate to the intestine, where they become CD8[alpha][alpha]+ IELs.
    Figure 6: Developmentally diverted TCRαβ+ DN thymocytes migrate to the intestine, where they become CD8αα+ IELs.

    (a) Homing and fate of developmentally diverted TCRαβ+ DN thymocytes, assessed in the small intestine (IEL) and lymph nodes (LN) of recombination-activating gene 2–deficient (RAG-KO) recipient mice (right) 5 weeks after adoptive transfer of 0.8 × 106 sorted CD5+ DN thymocytes (top) or CD5+CD8+ T cells (bottom) from the lymph nodes of B7-deficient donor mice (left). (b) Flow cytometry of developmentally diverted TCRαβ+ DN thymocytes (right) generated by culture of sorted B7-deficient or Bcl-2 transgenic CD5+ DN thymocytes (2 × 104 cells per well; left) for 4 d in medium alone (Med) or with plate-bound monoclonal anti-TCRβ and soluble IL-15 (100 ng/ml; Anti-TCR + IL-15). (c) Expression of CD8β versus CD8α on TCRβ+ cells from the small intestines of mice of various strains (above plots). Numbers adjacent to or in outlined areas indicate percent cells in each gate throughout. Data are representative of two independent experiments.

  7. Developmentally diverted TCR[alpha][beta]+ DN thymocytes become CD8[alpha][alpha]+ IELs.
    Figure 7: Developmentally diverted TCRαβ+ DN thymocytes become CD8αα+ IELs.

    (a) Expression of Mtv-reactive Vβ5+, Vβ11+ and Vβ12+ TCRs and control Vβ4+ TCRs in various thymocyte subsets and IELs from B7-deficient BALB/c mice. (b) Total TCRαβ+ CD8αβ+ or CD8αα+ IELs in wild-type and B7-deficient BALB/c mice. *P < 0.002 (Student's two-tailed t-test). (c) Expression of Mtv-reactive Vβ5+, Vβ11+ and Vβ12+ TCRs and control Vβ4+ TCRs in various T cell subsets from wild-type BALB/c mice. *P < 0.05 and **P < 0.01 (Student's two-tailed t-test). (d) Proliferation of sorted TCRαβ+CD8αα+ IELs and SP4 lymph node T cells (LNT; 2.5 × 104 cells per well) stimulated with soluble monoclonal anti-CD3 (5 μg) and irradiated syngenic antigen-presenting cells in the presence of medium alone or recombinant IL-2 (200 U/ml) or IL-15 (100 ng/ml), assessed as incorporation of [3H]thymidine. Data represent three independent experiments (mean and s.e.m.).

  8. Runx3 is required for the differentiation of TCR[alpha][beta]+ DN thymocytes into developmentally diverted CD8[alpha][alpha]+ IELs.
    Figure 8: Runx3 is required for the differentiation of TCRαβ+ DN thymocytes into developmentally diverted CD8αα+ IELs.

    (a) Expression of the Runx3-YFP reporter in thymocyte subsets and IELs from Bcl-2-transgenic mice (left) and mean fluorescent intensity (MFI) of Runx3-YFP (right). (b) Expression of Runx3-YFP (middle) and CD8β and CD8α (right) on developmentally diverted TCRαβ+ DN thymocytes generated by culture of sorted Runx3-sufficient (Runx3+/YFP) or Runx3-deficient (Runx3YFP/YFP) Bcl-2-transgenic CD5+ DN thymocytes (2.5 × 104 cells per well; left) for 4 d in medium alone or with plate-bound monoclonal anti-TCRβ and soluble IL-15 (100 ng/ml). Numbers adjacent to or in outlined areas indicate percent cells in that gate; numbers in plots (middle) indicate MFI of Runx3-YFP. (c) Total TCRαβ+ DN thymocytes and TCRαβ+CD8αα+ IELs in Runx3-sufficient and Runx3-deficient Bcl-2-transgenic mice. *P < 0.01 (Student's two-tailed t-test). Data are representative of three independent experiments (a,c; mean and s.e.m.) or two experiments (b).

References

  1. Singer, A., Adoro, S. & Park, J.H. Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat. Rev. Immunol. 8, 788801 (2008).
  2. Starr, T.K., Jameson, S.C. & Hogquist, K.A. Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139176 (2003).
  3. Gascoigne, N.R. & Palmer, E. Signaling in thymic selection. Curr. Opin. Immunol. 23, 207212 (2011).
  4. McCaughtry, T.M. & Hogquist, K.A. Central tolerance: what have we learned from mice? Semin. Immunopathol. 30, 399409 (2008).
  5. Kappler, J.W., Roehm, N. & Marrack, P. T cell tolerance by clonal elimination in the thymus. Cell 49, 273280 (1987).
  6. Bendelac, A., Savage, P.B. & Teyton, L. The biology of NKT cells. Annu. Rev. Immunol. 25, 297336 (2007).
  7. Jordan, M.S. et al. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2, 301306 (2001).
  8. Baldwin, T.A., Hogquist, K.A. & Jameson, S.C. The fourth way? Harnessing aggressive tendencies in the thymus. J. Immunol. 173, 65156520 (2004).
  9. Laufer, T.M., DeKoning, J., Markowitz, J.S., Lo, D. & Glimcher, L.H. Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex. Nature 383, 8185 (1996).
  10. Kishimoto, H. & Sprent, J. Negative selection in the thymus includes semimature T cells. J. Exp. Med. 185, 263271 (1997).
  11. Kishimoto, H. & Sprent, J. Several different cell surface molecules control negative selection of medullary thymocytes. J. Exp. Med. 190, 6573 (1999).
  12. Punt, J.A., Havran, W., Abe, R., Sarin, A. & Singer, A. T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus. J. Exp. Med. 186, 19111922 (1997).
  13. Punt, J.A., Osborne, B.A., Takahama, Y., Sharrow, S.O. & Singer, A. Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28. J. Exp. Med. 179, 709713 (1994).
  14. McKean, D.J. et al. Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation. J. Immunol. 166, 34683475 (2001).
  15. Ramsdell, F., Lantz, T. & Fowlkes, B.J. A nondeletional mechanism of thymic self tolerance. Science 246, 10381041 (1989).
  16. Roberts, J.L., Sharrow, S.O. & Singer, A. Clonal deletion and clonal anergy in the thymus induced by cellular elements with different radiation sensitivities. J. Exp. Med. 171, 935940 (1990).
  17. Takahama, Y. Medullary interplay for central tolerance. Blood 118, 23802381 (2011).
  18. Dautigny, N., Le Campion, A. & Lucas, B. Timing and casting for actors of thymic negative selection. J. Immunol. 162, 12941302 (1999).
  19. Jones, L.A., Izon, D.J., Nieland, J.D., Linsley, P.S. & Kruisbeek, A.M. CD28–B7 interactions are not required for intrathymic clonal deletion. Int. Immunol. 5, 503512 (1993).
  20. Sentman, C.L., Shutter, J.R., Hockenbery, D., Kanagawa, O. & Korsmeyer, S.J. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 67, 879888 (1991).
  21. Tan, R., Teh, S.J., Ledbetter, J.A., Linsley, P.S. & Teh, H.S. B7 costimulates proliferation of CD48+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes. J. Immunol. 149, 32173224 (1992).
  22. Walunas, T.L., Sperling, A.I., Khattri, R., Thompson, C.B. & Bluestone, J.A. CD28 expression is not essential for positive and negative selection of thymocytes or peripheral T cell tolerance. J. Immunol. 156, 10061013 (1996).
  23. Simpson, E. T cell repertoire selection by mouse mammary tumour viruses. Eur. J. Immunogenet. 20, 137149 (1993).
  24. Buhlmann, J.E., Elkin, S.K. & Sharpe, A.H. A role for the B7–1/B7–2:CD28/CTLA-4 pathway during negative selection. J. Immunol. 170, 54215428 (2003).
  25. Carbone, A.M., Marrack, P. & Kappler, J.W. Demethylated CD8 gene in CD4+ T cells suggests that CD4+ cells develop from CD8+ precursors. Science 242, 11741176 (1988).
  26. Negishi, I. et al. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 376, 435438 (1995).
  27. Yu, X., Fournier, S., Allison, J.P., Sharpe, A.H. & Hodes, R.J. The role of B7 costimulation in CD4/CD8 T cell homeostasis. J. Immunol. 164, 35433553 (2000).
  28. Riley, J.L. PD-1 signaling in primary T cells. Immunol. Rev. 229, 114125 (2009).
  29. Gangadharan, D. et al. Identification of pre- and postselection TCRαβ+ intraepithelial lymphocyte precursors in the thymus. Immunity 25, 631641 (2006).
  30. Sato, T. et al. Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes. Immunity 22, 317328 (2005).
  31. Egawa, T. & Littman, D.R. ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage. Nat. Immunol. 9, 11311139 (2008).
  32. Cheroutre, H., Lambolez, F. & Mucida, D. The light and dark sides of intestinal intraepithelial lymphocytes. Nat. Rev. Immunol. 11, 445456 (2011).
  33. 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, 152162 (2005).
  34. Taniuchi, I. et al. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell 111, 621633 (2002).
  35. Brugnera, E. et al. Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells. Immunity 13, 5971 (2000).
  36. Bouillet, P. et al. BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes. Nature 415, 922926 (2002).
  37. Hu, Q., Sader, A., Parkman, J.C. & Baldwin, T.A. Bim-mediated apoptosis is not necessary for thymic negative selection to ubiquitous self-antigens. J. Immunol. 183, 77617767 (2009).
  38. Kovalovsky, D., Pezzano, M., Ortiz, B.D. & Sant'Angelo, D.B. A novel TCR transgenic model reveals that negative selection involves an immediate, Bim-dependent pathway and a delayed, Bim-independent pathway. PLoS ONE 5, e8675 (2010).
  39. Suen, A.Y. & Baldwin, T.A. Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens. Proc. Natl. Acad. Sci. USA 109, 893898 (2012).
  40. Erlacher, M. et al. Puma cooperates with Bim, the rate-limiting BH3-only protein in cell death during lymphocyte development, in apoptosis induction. J. Exp. Med. 203, 29392951 (2006).
  41. McCaughtry, T.M., Baldwin, T.A., Wilken, M.S. & Hogquist, K.A. Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla. J. Exp. Med. 205, 25752584 (2008).
  42. Gao, J.X. et al. Perinatal blockade of b7–1 and b7–2 inhibits clonal deletion of highly pathogenic autoreactive T cells. J. Exp. Med. 195, 959971 (2002).
  43. Wang, R., Wang-Zhu, Y. & Grey, H. Interactions between double positive thymocytes and high affinity ligands presented by cortical epithelial cells generate double negative thymocytes with T cell regulatory activity. Proc. Natl. Acad. Sci. USA 99, 21812186 (2002).
  44. Lambolez, F., Kronenberg, M. & Cheroutre, H. Thymic differentiation of TCRαβ+ CD8αα+ IELs. Immunol. Rev. 215, 178188 (2007).
  45. Leishman, A.J. et al. Precursors of functional MHC class I- or class II-restricted CD8αα+ T cells are positively selected in the thymus by agonist self-peptides. Immunity 16, 355364 (2002).
  46. Levelt, C.N. et al. High- and low-affinity single-peptide/MHC ligands have distinct effects on the development of mucosal CD8αα and CD8αβ T lymphocytes. Proc. Natl. Acad. Sci. USA 96, 56285633 (1999).
  47. Baldwin, T.A., Sandau, M.M., Jameson, S.C. & Hogquist, K.A. The timing of TCR α expression critically influences T cell development and selection. J. Exp. Med. 202, 111121 (2005).
  48. Takahama, Y., Shores, E.W. & Singer, A. Negative selection of precursor thymocytes before their differentiation into CD4+CD8+ cells. Science 258, 653656 (1992).
  49. Cruz, D. et al. An opposite pattern of selection of a single T cell antigen receptor in the thymus and among intraepithelial lymphocytes. J. Exp. Med. 188, 255265 (1998).
  50. Liu, B., Tahk, S., Yee, K.M., Fan, G. & Shuai, K. The ligase PIAS1 restricts natural regulatory T cell differentiation by epigenetic repression. Science 330, 521525 (2010).

Download references

Author information

Affiliations

  1. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

    • Leonid A Pobezinsky,
    • Xuguang Tai,
    • Susanna Jeurling,
    • François Van Laethem,
    • Jung-Hyun Park &
    • Alfred Singer
  2. Ludwig Center for Cancer Research, University of Lausanne, Epalinges, Switzerland.

    • Georgi S Angelov
  3. Science Applications International Corporation-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, USA.

    • Lionel Feigenbaum

Contributions

L.A.P. designed the study, did experiments, analyzed data and contributed to the writing of the manuscript; G.S.A., X.T., S.J. and F.V.L. did experiments and analyzed data; J.-H.P. and L.F. generated transgenic mice, and A.S. designed the study, analyzed data and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (455 KB)

    Supplementary Figures 1–7

Additional data