Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1995 Mar 1;181(3):927–941. doi: 10.1084/jem.181.3.927

T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection

PMCID: PMC2191909  PMID: 7532685

Abstract

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin- dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H- 2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

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

  1. Acha-Orbea H., Zinkernagel R. M., Hengartner H. Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells. Eur J Immunol. 1985 Jan;15(1):31–36. doi: 10.1002/eji.1830150107. [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. Bendelac A., Matzinger P., Seder R. A., Paul W. E., Schwartz R. H. Activation events during thymic selection. J Exp Med. 1992 Mar 1;175(3):731–742. doi: 10.1084/jem.175.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benoist C., Mathis D. Positive selection of the T cell repertoire: where and when does it occur? Cell. 1989 Sep 22;58(6):1027–1033. doi: 10.1016/0092-8674(89)90501-1. [DOI] [PubMed] [Google Scholar]
  5. Berg L. J., Pullen A. M., Fazekas de St Groth B., Mathis D., Benoist C., Davis M. M. Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand. Cell. 1989 Sep 22;58(6):1035–1046. doi: 10.1016/0092-8674(89)90502-3. [DOI] [PubMed] [Google Scholar]
  6. Bill J., Kanagawa O., Woodland D. L., Palmer E. The MHC molecule I-E is necessary but not sufficient for the clonal deletion of V beta 11-bearing T cells. J Exp Med. 1989 Apr 1;169(4):1405–1419. doi: 10.1084/jem.169.4.1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blackman M. A., Marrack P., Kappler J. Influence of the major histocompatibility complex on positive thymic selection of V beta 17a+ T cells. Science. 1989 Apr 14;244(4901):214–217. doi: 10.1126/science.2784868. [DOI] [PubMed] [Google Scholar]
  8. Blackman M., Kappler J., Marrack P. The role of the T cell receptor in positive and negative selection of developing T cells. Science. 1990 Jun 15;248(4961):1335–1341. doi: 10.1126/science.1972592. [DOI] [PubMed] [Google Scholar]
  9. Bryson J. S., Caywood B. E., Kaplan A. M. Relationship of cyclosporine A-mediated inhibition of clonal deletion and development of syngeneic graft-versus-host disease. J Immunol. 1991 Jul 15;147(2):391–397. [PubMed] [Google Scholar]
  10. Chan S. H., Cosgrove D., Waltzinger C., Benoist C., Mathis D. Another view of the selective model of thymocyte selection. Cell. 1993 Apr 23;73(2):225–236. doi: 10.1016/0092-8674(93)90225-f. [DOI] [PubMed] [Google Scholar]
  11. Cohen J. J., Duke R. C., Fadok V. A., Sellins K. S. Apoptosis and programmed cell death in immunity. Annu Rev Immunol. 1992;10:267–293. doi: 10.1146/annurev.iy.10.040192.001411. [DOI] [PubMed] [Google Scholar]
  12. Crompton T., Lees R. K., Pircher H., MacDonald H. R. Precommitment of CD4+CD8+ thymocytes to either CD4 or CD8 lineages. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8982–8986. doi: 10.1073/pnas.90.19.8982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crump A. L., Grusby M. J., Glimcher L. H., Cantor H. Thymocyte development in major histocompatibility complex-deficient mice: evidence for stochastic commitment to the CD4 and CD8 lineages. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10739–10743. doi: 10.1073/pnas.90.22.10739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Davis C. B., Killeen N., Crooks M. E., Raulet D., Littman D. R. Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes. Cell. 1993 Apr 23;73(2):237–247. doi: 10.1016/0092-8674(93)90226-g. [DOI] [PubMed] [Google Scholar]
  15. Gao E. K., Lo D., Cheney R., Kanagawa O., Sprent J. Abnormal differentiation of thymocytes in mice treated with cyclosporin A. Nature. 1988 Nov 10;336(6195):176–179. doi: 10.1038/336176a0. [DOI] [PubMed] [Google Scholar]
  16. Jenkins M. K., Schwartz R. H., Pardoll D. M. Effects of cyclosporine A on T cell development and clonal deletion. Science. 1988 Sep 23;241(4873):1655–1658. doi: 10.1126/science.241.4873.1655. [DOI] [PubMed] [Google Scholar]
  17. Kappler J. W., Staerz U., White J., Marrack P. C. Self-tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex. Nature. 1988 Mar 3;332(6159):35–40. doi: 10.1038/332035a0. [DOI] [PubMed] [Google Scholar]
  18. Kappler J., White J., Wegmann D., Mustain E., Marrack P. Antigen presentation by Ia+ B cell hybridomas to H-2-restricted T cell hybridomas. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3604–3607. doi: 10.1073/pnas.79.11.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Kosugi A., Sharrow S. O., Shearer G. M. Effect of cyclosporin A on lymphopoiesis. I. Absence of mature T cells in thymus and periphery of bone marrow transplanted mice treated with cyclosporin A. J Immunol. 1989 May 1;142(9):3026–3032. [PubMed] [Google Scholar]
  21. Kubo M., Kincaid R. L., Webb D. R., Ransom J. T. The Ca2+/calmodulin-activated, phosphoprotein phosphatase calcineurin is sufficient for positive transcriptional regulation of the mouse IL-4 gene. Int Immunol. 1994 Feb;6(2):179–188. doi: 10.1093/intimm/6.2.179. [DOI] [PubMed] [Google Scholar]
  22. Kubo R. T., Born W., Kappler J. W., Marrack P., Pigeon M. Characterization of a monoclonal antibody which detects all murine alpha beta T cell receptors. J Immunol. 1989 Apr 15;142(8):2736–2742. [PubMed] [Google Scholar]
  23. Kubo S., Nakayama T., Furutani-Seiki M., Kishimoto H., Hashimoto K., Bae M. J., Yokochi T., Takeda N., Aizawa S., Asano Y. A novel form of self tolerance dictated in the thymus of transgenic mice with autoreactive TCR alpha and beta chain genes. Int Immunol. 1994 Apr;6(4):593–602. doi: 10.1093/intimm/6.4.593. [DOI] [PubMed] [Google Scholar]
  24. Ledbetter J. A., Herzenberg L. A. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol Rev. 1979;47:63–90. doi: 10.1111/j.1600-065x.1979.tb00289.x. [DOI] [PubMed] [Google Scholar]
  25. Leo O., Foo M., Sachs D. H., Samelson L. E., Bluestone J. A. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1374–1378. doi: 10.1073/pnas.84.5.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leo O., Foo M., Segal D. M., Shevach E., Bluestone J. A. Activation of murine T lymphocytes with monoclonal antibodies: detection on Lyt-2+ cells of an antigen not associated with the T cell receptor complex but involved in T cell activation. J Immunol. 1987 Aug 15;139(4):1214–1222. [PubMed] [Google Scholar]
  27. Lo D., Ron Y., Sprent J. Induction of MHC-restricted specificity and tolerance in the thymus. Immunol Res. 1986;5(3):221–232. doi: 10.1007/BF02919203. [DOI] [PubMed] [Google Scholar]
  28. Lyons A. B., Samuel K., Sanderson A., Maddy A. H. Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry. Cytometry. 1992;13(8):809–821. doi: 10.1002/cyto.990130803. [DOI] [PubMed] [Google Scholar]
  29. MacDonald H. R., Lees R. K., Schneider R., Zinkernagel R. M., Hengartner H. Positive selection of CD4+ thymocytes controlled by MHC class II gene products. Nature. 1988 Dec 1;336(6198):471–473. doi: 10.1038/336471a0. [DOI] [PubMed] [Google Scholar]
  30. MacDonald H. R., Schneider R., Lees R. K., Howe R. C., Acha-Orbea H., Festenstein H., Zinkernagel R. M., Hengartner H. T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature. 1988 Mar 3;332(6159):40–45. doi: 10.1038/332040a0. [DOI] [PubMed] [Google Scholar]
  31. Marrack P., Kappler J. The T cell receptor. Science. 1987 Nov 20;238(4830):1073–1079. doi: 10.1126/science.3317824. [DOI] [PubMed] [Google Scholar]
  32. Nakayama T., June C. H., Munitz T. I., Sheard M., McCarthy S. A., Sharrow S. O., Samelson L. E., Singer A. Inhibition of T cell receptor expression and function in immature CD4+CD8+ cells by CD4. Science. 1990 Sep 28;249(4976):1558–1561. doi: 10.1126/science.2120773. [DOI] [PubMed] [Google Scholar]
  33. Nakayama T., Samelson L. E., Nakayama Y., Munitz T. I., Sheard M., June C. H., Singer A. Ligand-stimulated signaling events in immature CD4+CD8+ thymocytes expressing competent T-cell receptor complexes. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9949–9953. doi: 10.1073/pnas.88.22.9949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nakayama T., Singer A., Hsi E. D., Samelson L. E. Intrathymic signalling in immature CD4+CD8+ thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain. Nature. 1989 Oct 19;341(6243):651–654. doi: 10.1038/341651a0. [DOI] [PubMed] [Google Scholar]
  35. Nakayama T., Ueda Y., Yamada H., Shores E. W., Singer A., June C. H. In vivo calcium elevations in thymocytes with T cell receptors that are specific for self ligands. Science. 1992 Jul 3;257(5066):96–99. doi: 10.1126/science.1621102. [DOI] [PubMed] [Google Scholar]
  36. Nikolić-Zugić J., Bevan M. J. Functional and phenotypic delineation of two subsets of CD4 single positive cells in the thymus. Int Immunol. 1990;2(2):135–141. doi: 10.1093/intimm/2.2.135. [DOI] [PubMed] [Google Scholar]
  37. Nossal G. J. Negative selection of lymphocytes. Cell. 1994 Jan 28;76(2):229–239. doi: 10.1016/0092-8674(94)90331-x. [DOI] [PubMed] [Google Scholar]
  38. Page D. M., Kane L. P., Allison J. P., Hedrick S. M. Two signals are required for negative selection of CD4+CD8+ thymocytes. J Immunol. 1993 Aug 15;151(4):1868–1880. [PubMed] [Google Scholar]
  39. Prud'homme G. J., Vanier L. E. Cyclosporine, tolerance, and autoimmunity. Clin Immunol Immunopathol. 1993 Mar;66(3):185–192. doi: 10.1006/clin.1993.1024. [DOI] [PubMed] [Google Scholar]
  40. 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. 1994 Feb 1;179(2):709–713. doi: 10.1084/jem.179.2.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ramsdell F., Jenkins M., Dinh Q., Fowlkes B. J. The majority of CD4+8- thymocytes are functionally immature. J Immunol. 1991 Sep 15;147(6):1779–1785. [PubMed] [Google Scholar]
  42. Robey E., Fowlkes B. J. Selective events in T cell development. Annu Rev Immunol. 1994;12:675–705. doi: 10.1146/annurev.iy.12.040194.003331. [DOI] [PubMed] [Google Scholar]
  43. Ronchese F., Schwartz R. H., Germain R. N. Functionally distinct subsites on a class II major histocompatibility complex molecule. Nature. 1987 Sep 17;329(6136):254–256. doi: 10.1038/329254a0. [DOI] [PubMed] [Google Scholar]
  44. Samelson L. E., Germain R. N., Schwartz R. H. Monoclonal antibodies against the antigen receptor on a cloned T-cell hybrid. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6972–6976. doi: 10.1073/pnas.80.22.6972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schreiber S. L., Crabtree G. R. The mechanism of action of cyclosporin A and FK506. Immunol Today. 1992 Apr;13(4):136–142. doi: 10.1016/0167-5699(92)90111-J. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Sha W. C., Nelson C. A., Newberry R. D., Kranz D. M., Russell J. H., Loh D. Y. Positive and negative selection of an antigen receptor on T cells in transgenic mice. Nature. 1988 Nov 3;336(6194):73–76. doi: 10.1038/336073a0. [DOI] [PubMed] [Google Scholar]
  48. Shi Y. F., Sahai B. M., Green D. R. Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes. Nature. 1989 Jun 22;339(6226):625–626. doi: 10.1038/339625a0. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Shortman K., Vremec D., Egerton M. The kinetics of T cell antigen receptor expression by subgroups of CD4+8+ thymocytes: delineation of CD4+8+3(2+) thymocytes as post-selection intermediates leading to mature T cells. J Exp Med. 1991 Feb 1;173(2):323–332. doi: 10.1084/jem.173.2.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Swat W., Dessing M., von Boehmer H., Kisielow P. CD69 expression during selection and maturation of CD4+8+ thymocytes. Eur J Immunol. 1993 Mar;23(3):739–746. doi: 10.1002/eji.1830230326. [DOI] [PubMed] [Google Scholar]
  53. Teh H. S., Kishi H., Scott B., Von Boehmer H. Deletion of autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal TCR levels and low levels of CD8 molecules. J Exp Med. 1989 Mar 1;169(3):795–806. doi: 10.1084/jem.169.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tsien R. Y., Harootunian A. T. Practical design criteria for a dynamic ratio imaging system. Cell Calcium. 1990 Feb-Mar;11(2-3):93–109. doi: 10.1016/0143-4160(90)90063-z. [DOI] [PubMed] [Google Scholar]
  55. Turka L. A., Schatz D. G., Oettinger M. A., Chun J. J., Gorka C., Lee K., McCormack W. T., Thompson C. B. Thymocyte expression of RAG-1 and RAG-2: termination by T cell receptor cross-linking. Science. 1991 Aug 16;253(5021):778–781. doi: 10.1126/science.1831564. [DOI] [PubMed] [Google Scholar]
  56. Uematsu Y., Ryser S., Dembić Z., Borgulya P., Krimpenfort P., Berns A., von Boehmer H., Steinmetz M. In transgenic mice the introduced functional T cell receptor beta gene prevents expression of endogenous beta genes. Cell. 1988 Mar 25;52(6):831–841. doi: 10.1016/0092-8674(88)90425-4. [DOI] [PubMed] [Google Scholar]
  57. Urdahl K. B., Pardoll D. M., Jenkins M. K. Cyclosporin A inhibits positive selection and delays negative selection in alpha beta TCR transgenic mice. J Immunol. 1994 Mar 15;152(6):2853–2859. [PubMed] [Google Scholar]
  58. Yamashita I., Nagata T., Tada T., Nakayama T. CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection. Int Immunol. 1993 Sep;5(9):1139–1150. doi: 10.1093/intimm/5.9.1139. [DOI] [PubMed] [Google Scholar]
  59. Yokoyama W. M., Koning F., Kehn P. J., Pereira G. M., Stingl G., Coligan J. E., Shevach E. M. Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation. J Immunol. 1988 Jul 15;141(2):369–376. [PubMed] [Google Scholar]
  60. Zacharchuk C. M., Merćep M., Chakraborti P. K., Simons S. S., Jr, Ashwell J. D. Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic. J Immunol. 1990 Dec 15;145(12):4037–4045. [PubMed] [Google Scholar]
  61. Ziegler S. F., Levin S. D., Johnson L., Copeland N. G., Gilbert D. J., Jenkins N. A., Baker E., Sutherland G. R., Feldhaus A. L., Ramsdell F. The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex. J Immunol. 1994 Feb 1;152(3):1228–1236. [PubMed] [Google Scholar]
  62. van Meerwijk J. P., Germain R. N. Development of mature CD8+ thymocytes: selection rather than instruction? Science. 1993 Aug 13;261(5123):911–915. doi: 10.1126/science.8102208. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES