Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 May 1;16(9):2282–2293. doi: 10.1093/emboj/16.9.2282

T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection.

L K Clayton 1, Y Ghendler 1, E Mizoguchi 1, R J Patch 1, T D Ocain 1, K Orth 1, A K Bhan 1, V M Dixit 1, E L Reinherz 1
PMCID: PMC1169830  PMID: 9171343

Abstract

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.

Full Text

The Full Text of this article is available as a PDF (632.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alnemri E. S., Livingston D. J., Nicholson D. W., Salvesen G., Thornberry N. A., Wong W. W., Yuan J. Human ICE/CED-3 protease nomenclature. Cell. 1996 Oct 18;87(2):171–171. doi: 10.1016/s0092-8674(00)81334-3. [DOI] [PubMed] [Google Scholar]
  2. Ashton-Rickardt P. G., Bandeira A., Delaney J. R., Van Kaer L., Pircher H. P., Zinkernagel R. M., Tonegawa S. Evidence for a differential avidity model of T cell selection in the thymus. Cell. 1994 Feb 25;76(4):651–663. doi: 10.1016/0092-8674(94)90505-3. [DOI] [PubMed] [Google Scholar]
  3. Becker M. L., Near R., Mudgett-Hunter M., Margolies M. N., Kubo R. T., Kaye J., Hedrick S. M. Expression of a hybrid immunoglobulin-T cell receptor protein in transgenic mice. Cell. 1989 Sep 8;58(5):911–921. doi: 10.1016/0092-8674(89)90943-4. [DOI] [PubMed] [Google Scholar]
  4. Bluestone J. A. New perspectives of CD28-B7-mediated T cell costimulation. Immunity. 1995 Jun;2(6):555–559. doi: 10.1016/1074-7613(95)90000-4. [DOI] [PubMed] [Google Scholar]
  5. Caputo A., James M. N., Powers J. C., Hudig D., Bleackley R. C. Conversion of the substrate specificity of mouse proteinase granzyme B. Nat Struct Biol. 1994 Jun;1(6):364–367. doi: 10.1038/nsb0694-364. [DOI] [PubMed] [Google Scholar]
  6. Chang H. C., Bao Z., Yao Y., Tse A. G., Goyarts E. C., Madsen M., Kawasaki E., Brauer P. P., Sacchettini J. C., Nathenson S. G. A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11408–11412. doi: 10.1073/pnas.91.24.11408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chinnaiyan A. M., O'Rourke K., Yu G. L., Lyons R. H., Garg M., Duan D. R., Xing L., Gentz R., Ni J., Dixit V. M. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science. 1996 Nov 8;274(5289):990–992. doi: 10.1126/science.274.5289.990. [DOI] [PubMed] [Google Scholar]
  8. Chinnaiyan A. M., Orth K., O'Rourke K., Duan H., Poirier G. G., Dixit V. M. Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases. J Biol Chem. 1996 Mar 1;271(9):4573–4576. doi: 10.1074/jbc.271.9.4573. [DOI] [PubMed] [Google Scholar]
  9. Chinnaiyan A. M., Tepper C. G., Seldin M. F., O'Rourke K., Kischkel F. C., Hellbardt S., Krammer P. H., Peter M. E., Dixit V. M. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J Biol Chem. 1996 Mar 1;271(9):4961–4965. doi: 10.1074/jbc.271.9.4961. [DOI] [PubMed] [Google Scholar]
  10. Crispe I. N. Fatal interactions: Fas-induced apoptosis of mature T cells. Immunity. 1994 Aug;1(5):347–349. doi: 10.1016/1074-7613(94)90064-7. [DOI] [PubMed] [Google Scholar]
  11. Darmon A. J., Nicholson D. W., Bleackley R. C. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature. 1995 Oct 5;377(6548):446–448. doi: 10.1038/377446a0. [DOI] [PubMed] [Google Scholar]
  12. Degermann S., Surh C. D., Glimcher L. H., Sprent J., Lo D. B7 expression on thymic medullary epithelium correlates with epithelium-mediated deletion of V beta 5+ thymocytes. J Immunol. 1994 Apr 1;152(7):3254–3263. [PubMed] [Google Scholar]
  13. Dolle R. E., Hoyer D., Prasad C. V., Schmidt S. J., Helaszek C. T., Miller R. E., Ator M. A. P1 aspartate-based peptide alpha-((2,6-dichlorobenzoyl)oxy)methyl ketones as potent time-dependent inhibitors of interleukin-1 beta-converting enzyme. J Med Chem. 1994 Mar 4;37(5):563–564. doi: 10.1021/jm00031a003. [DOI] [PubMed] [Google Scholar]
  14. Dolle R. E., Singh J., Rinker J., Hoyer D., Prasad C. V., Graybill T. L., Salvino J. M., Helaszek C. T., Miller R. E., Ator M. A. Aspartyl alpha-((1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)methyl ketones as interleukin-1 beta converting enzyme inhibitors. Significance of the P1 and P3 amido nitrogens for enzyme-peptide inhibitor binding. J Med Chem. 1994 Nov 11;37(23):3863–3866. doi: 10.1021/jm00049a001. [DOI] [PubMed] [Google Scholar]
  15. Duan H., Chinnaiyan A. M., Hudson P. L., Wing J. P., He W. W., Dixit V. M. ICE-LAP3, a novel mammalian homologue of the Caenorhabditis elegans cell death protein Ced-3 is activated during Fas- and tumor necrosis factor-induced apoptosis. J Biol Chem. 1996 Jan 19;271(3):1621–1625. doi: 10.1074/jbc.271.3.1621. [DOI] [PubMed] [Google Scholar]
  16. Duan H., Orth K., Chinnaiyan A. M., Poirier G. G., Froelich C. J., He W. W., Dixit V. M. ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B. J Biol Chem. 1996 Jul 12;271(28):16720–16724. doi: 10.1074/jbc.271.28.16720. [DOI] [PubMed] [Google Scholar]
  17. Fearnhead H. O., Dinsdale D., Cohen G. M. An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes. FEBS Lett. 1995 Nov 20;375(3):283–288. doi: 10.1016/0014-5793(95)01228-7. [DOI] [PubMed] [Google Scholar]
  18. Fernandes-Alnemri T., Armstrong R. C., Krebs J., Srinivasula S. M., Wang L., Bullrich F., Fritz L. C., Trapani J. A., Tomaselli K. J., Litwack G. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7464–7469. doi: 10.1073/pnas.93.15.7464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fowlkes B. J., Schwartz R. H., Pardoll D. M. Deletion of self-reactive thymocytes occurs at a CD4+8+ precursor stage. Nature. 1988 Aug 18;334(6183):620–623. doi: 10.1038/334620a0. [DOI] [PubMed] [Google Scholar]
  20. Hanson R. D., Sclar G. M., Kanagawa O., Ley T. J. The 5'-flanking region of the human CGL-1/granzyme B gene targets expression of a reporter gene to activated T-lymphocytes in transgenic mice. J Biol Chem. 1991 Dec 25;266(36):24433–24438. [PubMed] [Google Scholar]
  21. Heusel J. W., Hanson R. D., Silverman G. A., Ley T. J. Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2. J Biol Chem. 1991 Apr 5;266(10):6152–6158. [PubMed] [Google Scholar]
  22. Hogquist K. A., Jameson S. C., Heath W. R., Howard J. L., Bevan M. J., Carbone F. R. T cell receptor antagonist peptides induce positive selection. Cell. 1994 Jan 14;76(1):17–27. doi: 10.1016/0092-8674(94)90169-4. [DOI] [PubMed] [Google Scholar]
  23. Kisielow P., Blüthmann H., Staerz U. D., Steinmetz M., von Boehmer H. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature. 1988 Jun 23;333(6175):742–746. doi: 10.1038/333742a0. [DOI] [PubMed] [Google Scholar]
  24. Krantz A., Copp L. J., Coles P. J., Smith R. A., Heard S. B. Peptidyl (acyloxy)methyl ketones and the quiescent affinity label concept: the departing group as a variable structural element in the design of inactivators of cysteine proteinases. Biochemistry. 1991 May 14;30(19):4678–4687. doi: 10.1021/bi00233a007. [DOI] [PubMed] [Google Scholar]
  25. Kuida K., Lippke J. A., Ku G., Harding M. W., Livingston D. J., Su M. S., Flavell R. A. Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science. 1995 Mar 31;267(5206):2000–2003. doi: 10.1126/science.7535475. [DOI] [PubMed] [Google Scholar]
  26. Kuida K., Zheng T. S., Na S., Kuan C., Yang D., Karasuyama H., Rakic P., Flavell R. A. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature. 1996 Nov 28;384(6607):368–372. doi: 10.1038/384368a0. [DOI] [PubMed] [Google Scholar]
  27. Li P., Allen H., Banerjee S., Franklin S., Herzog L., Johnston C., McDowell J., Paskind M., Rodman L., Salfeld J. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell. 1995 Feb 10;80(3):401–411. doi: 10.1016/0092-8674(95)90490-5. [DOI] [PubMed] [Google Scholar]
  28. Murphy K. M., Heimberger A. B., Loh D. Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science. 1990 Dec 21;250(4988):1720–1723. doi: 10.1126/science.2125367. [DOI] [PubMed] [Google Scholar]
  29. Nalin C. M. Apoptosis research enters the ICE age. Structure. 1995 Feb 15;3(2):143–145. doi: 10.1016/s0969-2126(01)00144-7. [DOI] [PubMed] [Google Scholar]
  30. Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995 Jul 6;376(6535):37–43. doi: 10.1038/376037a0. [DOI] [PubMed] [Google Scholar]
  31. Orth K., O'Rourke K., Salvesen G. S., Dixit V. M. Molecular ordering of apoptotic mammalian CED-3/ICE-like proteases. J Biol Chem. 1996 Aug 30;271(35):20977–20980. doi: 10.1074/jbc.271.35.20977. [DOI] [PubMed] [Google Scholar]
  32. Patten P. A., Rock E. P., Sonoda T., Fazekas de St Groth B., Jorgensen J. L., Davis M. M. Transfer of putative complementarity-determining region loops of T cell receptor V domains confers toxin reactivity but not peptide/MHC specificity. J Immunol. 1993 Mar 15;150(6):2281–2294. [PubMed] [Google Scholar]
  33. Quan L. T., Tewari M., O'Rourke K., Dixit V., Snipas S. J., Poirier G. G., Ray C., Pickup D. J., Salvesen G. S. Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1972–1976. doi: 10.1073/pnas.93.5.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rotonda J., Nicholson D. W., Fazil K. M., Gallant M., Gareau Y., Labelle M., Peterson E. P., Rasper D. M., Ruel R., Vaillancourt J. P. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat Struct Biol. 1996 Jul;3(7):619–625. doi: 10.1038/nsb0796-619. [DOI] [PubMed] [Google Scholar]
  35. Rozzo S. J., Eisenberg R. A., Cohen P. L., Kotzin B. L. Development of the T cell receptor repertoire in lpr mice. Semin Immunol. 1994 Feb;6(1):19–26. doi: 10.1006/smim.1994.1004. [DOI] [PubMed] [Google Scholar]
  36. Sarin A., Wu M. L., Henkart P. A. Different interleukin-1 beta converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli. J Exp Med. 1996 Dec 1;184(6):2445–2450. doi: 10.1084/jem.184.6.2445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schlegel J., Peters I., Orrenius S., Miller D. K., Thornberry N. A., Yamin T. T., Nicholson D. W. CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem. 1996 Jan 26;271(4):1841–1844. doi: 10.1074/jbc.271.4.1841. [DOI] [PubMed] [Google Scholar]
  38. Sebzda E., Wallace V. A., Mayer J., Yeung R. S., Mak T. W., Ohashi P. S. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science. 1994 Mar 18;263(5153):1615–1618. doi: 10.1126/science.8128249. [DOI] [PubMed] [Google Scholar]
  39. 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. 1991 Nov 29;67(5):879–888. doi: 10.1016/0092-8674(91)90361-2. [DOI] [PubMed] [Google Scholar]
  40. Shinkai Y., Rathbun G., Lam K. P., Oltz E. M., Stewart V., Mendelsohn M., Charron J., Datta M., Young F., Stall A. M. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 1992 Mar 6;68(5):855–867. doi: 10.1016/0092-8674(92)90029-c. [DOI] [PubMed] [Google Scholar]
  41. Smith C. A., Williams G. T., Kingston R., Jenkinson E. J., Owen J. J. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature. 1989 Jan 12;337(6203):181–184. doi: 10.1038/337181a0. [DOI] [PubMed] [Google Scholar]
  42. Smith K. G., Strasser A., Vaux D. L. CrmA expression in T lymphocytes of transgenic mice inhibits CD95 (Fas/APO-1)-transduced apoptosis, but does not cause lymphadenopathy or autoimmune disease. EMBO J. 1996 Oct 1;15(19):5167–5176. [PMC free article] [PubMed] [Google Scholar]
  43. Steller H. Mechanisms and genes of cellular suicide. Science. 1995 Mar 10;267(5203):1445–1449. doi: 10.1126/science.7878463. [DOI] [PubMed] [Google Scholar]
  44. Surh C. D., Sprent J. T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature. 1994 Nov 3;372(6501):100–103. doi: 10.1038/372100a0. [DOI] [PubMed] [Google Scholar]
  45. Takahashi T., Tanaka M., Brannan C. I., Jenkins N. A., Copeland N. G., Suda T., Nagata S. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell. 1994 Mar 25;76(6):969–976. doi: 10.1016/0092-8674(94)90375-1. [DOI] [PubMed] [Google Scholar]
  46. Tartaglia L. A., Ayres T. M., Wong G. H., Goeddel D. V. A novel domain within the 55 kd TNF receptor signals cell death. Cell. 1993 Sep 10;74(5):845–853. doi: 10.1016/0092-8674(93)90464-2. [DOI] [PubMed] [Google Scholar]
  47. Tewari M., Quan L. T., O'Rourke K., Desnoyers S., Zeng Z., Beidler D. R., Poirier G. G., Salvesen G. S., Dixit V. M. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995 Jun 2;81(5):801–809. doi: 10.1016/0092-8674(95)90541-3. [DOI] [PubMed] [Google Scholar]
  48. Thornberry N. A., Bull H. G., Calaycay J. R., Chapman K. T., Howard A. D., Kostura M. J., Miller D. K., Molineaux S. M., Weidner J. R., Aunins J. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992 Apr 30;356(6372):768–774. doi: 10.1038/356768a0. [DOI] [PubMed] [Google Scholar]
  49. Thornberry N. A., Peterson E. P., Zhao J. J., Howard A. D., Griffin P. R., Chapman K. T. Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry. 1994 Apr 5;33(13):3934–3940. doi: 10.1021/bi00179a020. [DOI] [PubMed] [Google Scholar]
  50. Trauth B. C., Klas C., Peters A. M., Matzku S., Möller P., Falk W., Debatin K. M., Krammer P. H. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science. 1989 Jul 21;245(4915):301–305. doi: 10.1126/science.2787530. [DOI] [PubMed] [Google Scholar]
  51. Vasquez N. J., Kaye J., Hedrick S. M. In vivo and in vitro clonal deletion of double-positive thymocytes. J Exp Med. 1992 May 1;175(5):1307–1316. doi: 10.1084/jem.175.5.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vaux D. L., Aguila H. L., Weissman I. L. Bcl-2 prevents death of factor-deprived cells but fails to prevent apoptosis in targets of cell mediated killing. Int Immunol. 1992 Jul;4(7):821–824. doi: 10.1093/intimm/4.7.821. [DOI] [PubMed] [Google Scholar]
  53. Vaux D. L., Strasser A. The molecular biology of apoptosis. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2239–2244. doi: 10.1073/pnas.93.6.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vaux D. L. Toward an understanding of the molecular mechanisms of physiological cell death. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):786–789. doi: 10.1073/pnas.90.3.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Walker N. P., Talanian R. V., Brady K. D., Dang L. C., Bump N. J., Ferenz C. R., Franklin S., Ghayur T., Hackett M. C., Hammill L. D. Crystal structure of the cysteine protease interleukin-1 beta-converting enzyme: a (p20/p10)2 homodimer. Cell. 1994 Jul 29;78(2):343–352. doi: 10.1016/0092-8674(94)90303-4. [DOI] [PubMed] [Google Scholar]
  56. Wang S., Miura M., Jung Y. k., Zhu H., Gagliardini V., Shi L., Greenberg A. H., Yuan J. Identification and characterization of Ich-3, a member of the interleukin-1beta converting enzyme (ICE)/Ced-3 family and an upstream regulator of ICE. J Biol Chem. 1996 Aug 23;271(34):20580–20587. doi: 10.1074/jbc.271.34.20580. [DOI] [PubMed] [Google Scholar]
  57. Wilson K. P., Black J. A., Thomson J. A., Kim E. E., Griffith J. P., Navia M. A., Murcko M. A., Chambers S. P., Aldape R. A., Raybuck S. A. Structure and mechanism of interleukin-1 beta converting enzyme. Nature. 1994 Jul 28;370(6487):270–275. doi: 10.1038/370270a0. [DOI] [PubMed] [Google Scholar]
  58. Wyllie A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980 Apr 10;284(5756):555–556. doi: 10.1038/284555a0. [DOI] [PubMed] [Google Scholar]
  59. Yang E., Korsmeyer S. J. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood. 1996 Jul 15;88(2):386–401. [PubMed] [Google Scholar]
  60. Yonehara S., Ishii A., Yonehara M. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med. 1989 May 1;169(5):1747–1756. doi: 10.1084/jem.169.5.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES