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. 1994 Mar 1;179(3):785–796. doi: 10.1084/jem.179.3.785

Thy-1 triggers mouse thymocyte apoptosis through a bcl-2-resistant mechanism

PMCID: PMC2191406  PMID: 7906706

Abstract

Programmed cell death plays an important role during thymocyte development, since a vast majority (97%) of mouse cortical thymocytes die in thymus, whereas only 3% of these cells are rescued from cell death and positively selected. Although it seems well established that thymocyte fate depends upon appropriate surface-expressed T cell receptor, little is known about the molecular mechanism(s) responsible for the massive thymocyte elimination that occurs in the thymus. We report here that Thy-1 is capable of triggering mouse thymocyte death in vitro through a bcl-2-resistant mechanism. We have previously shown that Thy-1 is involved in mouse thymocyte adhesion to thymic stroma through interaction with an epithelial cell ligand. To examine the Thy- 1 signaling function in thymocytes, we have mimicked its interaction with stromal cells by culturing mouse thymocytes onto tissue culture plates coated with monoclonal antibodies (mAb) directed at distinct Thy- 1 epitope regions. mAb recognizing determinants in a defined Thy-1 structural domain, but not others, were found to induce marked thymocyte apoptosis as evidenced by morphological and biochemical data. Use of a quantitative DNA dot blot assay indicated that Thy-1-mediated thymocyte apoptosis was not blocked by RNA or protein synthesis inhibitors, EGTA, or by cyclosporin A, and differed, therefore, from "activation-driven cell death". Moreover, Thy-1(+)-transfected, but not wild-type AKR1 (Thy-1-d) thymoma cells underwent apoptosis after ligation with apoptosis-inducing, Thy-1-specific mAb. In contrast to thymocytes, the latter event was inhibitable by RNA and protein synthesis inhibitors, an indication that thymocytes, but not thymoma cells, contain the molecular components necessary for Thy-1-driven apoptosis. We further showed that Thy-1-triggered thymocyte death is a developmentally regulated process operative in fetal thymocytes from day 17 of gestation, but not in peripheral T cells. Indeed, the target of apoptosis by anti-Thy-1 was found to reside mainly within the CD4+8+3- and CD4+8+3lo double positive immature thymocyte subsets. Finally, it is of major interest that Thy-1-mediated apoptosis, which was found to be readily detectable in thymocytes from bcl-2-transgenic mice, represents a thus far unique experimental system for studying bcl- 2-resistant thymocyte death mechanism(s).

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Selected References

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  1. Aldrich C. J., Hammer R. E., Jones-Youngblood S., Koszinowski U., Hood L., Stroynowski I., Forman J. Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the alpha 3 domain of MHC I molecules. Nature. 1991 Aug 22;352(6337):718–721. doi: 10.1038/352718a0. [DOI] [PubMed] [Google Scholar]
  2. Aoki J., Umeda M., Takio K., Titani K., Utsumi H., Sasaki M., Inoue K. Neural cell adhesion molecule mediates contact-dependent inhibition of growth of near-diploid mouse fibroblast cell line m5S/1M. J Cell Biol. 1991 Dec;115(6):1751–1761. doi: 10.1083/jcb.115.6.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bill J., Palmer E. Positive selection of CD4+ T cells mediated by MHC class II-bearing stromal cell in the thymic cortex. Nature. 1989 Oct 19;341(6243):649–651. doi: 10.1038/341649a0. [DOI] [PubMed] [Google Scholar]
  5. Blackman M. A., Burgert H. G., Gerhard-Burgert H., Woodland D. L., Palmer E., Kappler J. W., Marrack P. A role for clonal inactivation in T cell tolerance to Mls-1a. Nature. 1990 Jun 7;345(6275):540–542. doi: 10.1038/345540a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  8. Brown D. The tyrosine kinase connection: how GPI-anchored proteins activate T cells. Curr Opin Immunol. 1993 Jun;5(3):349–354. doi: 10.1016/0952-7915(93)90052-t. [DOI] [PubMed] [Google Scholar]
  9. Carlow D. A., van Oers N. S., Teh S. J., Teh H. S. Deletion of antigen-specific immature thymocytes by dendritic cells requires LFA-1/ICAM interactions. J Immunol. 1992 Mar 15;148(6):1595–1603. [PubMed] [Google Scholar]
  10. He H. T., Naquet P., Caillol D., Pierres M. Thy-1 supports adhesion of mouse thymocytes to thymic epithelial cells through a Ca2(+)-independent mechanism. J Exp Med. 1991 Feb 1;173(2):515–518. doi: 10.1084/jem.173.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hockenbery D. M. The bcl-2 oncogene and apoptosis. Semin Immunol. 1992 Dec;4(6):413–420. [PubMed] [Google Scholar]
  12. Hueber A. O., Pierres M., He H. T. Sulfated glycans directly interact with mouse Thy-1 and negatively regulate Thy-1-mediated adhesion of thymocytes to thymic epithelial cells. J Immunol. 1992 Jun 15;148(12):3692–3699. [PubMed] [Google Scholar]
  13. Hugo P., Boyd R. L., Waanders G. A., Scollay R. CD4+CD8+CD3high thymocytes appear transiently during ontogeny: evidence from phenotypic and functional studies. Eur J Immunol. 1991 Nov;21(11):2655–2660. doi: 10.1002/eji.1830211103. [DOI] [PubMed] [Google Scholar]
  14. Ingold A. L., Landel C., Knall C., Evans G. A., Potter T. A. Co-engagement of CD8 with the T cell receptor is required for negative selection. Nature. 1991 Aug 22;352(6337):721–723. doi: 10.1038/352721a0. [DOI] [PubMed] [Google Scholar]
  15. Jacobson M. D., Burne J. F., King M. P., Miyashita T., Reed J. C., Raff M. C. Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature. 1993 Jan 28;361(6410):365–369. doi: 10.1038/361365a0. [DOI] [PubMed] [Google Scholar]
  16. Kroczek R. A., Gunter K. C., Germain R. N., Shevach E. M. Thy-1 functions as a signal transduction molecule in T lymphocytes and transfected B lymphocytes. Nature. 1986 Jul 10;322(6075):181–184. doi: 10.1038/322181a0. [DOI] [PubMed] [Google Scholar]
  17. Lehuen A., Monteiro R. C., Kearney J. F. Identification of a surface protein (p100) associated with two glycosyl-phosphatidylinositol-linked molecules (Thy-1 and ThB) by natural anti-lymphocyte autoantibodies. Eur J Immunol. 1992 Sep;22(9):2373–2380. doi: 10.1002/eji.1830220929. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Matzinger P. The JAM test. A simple assay for DNA fragmentation and cell death. J Immunol Methods. 1991 Dec 15;145(1-2):185–192. doi: 10.1016/0022-1759(91)90325-a. [DOI] [PubMed] [Google Scholar]
  20. Merćep M., Noguchi P. D., Ashwell J. D. The cell cycle block and lysis of an activated T cell hybridoma are distinct processes with different Ca2+ requirements and sensitivity to cyclosporine A. J Immunol. 1989 Jun 1;142(11):4085–4092. [PubMed] [Google Scholar]
  21. Millrain M. M., Hederer R., Griffiths S., Fryer P., Tomonari K., Robinson P. J. Novel cell junctions induced by activating Thy-1-specific antibodies. Int Immunol. 1992 Oct;4(10):1137–1143. doi: 10.1093/intimm/4.10.1137. [DOI] [PubMed] [Google Scholar]
  22. Morris R. Thy-1, the enigmatic extrovert on the neuronal surface. Bioessays. 1992 Oct;14(10):715–722. doi: 10.1002/bies.950141014. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Nakashima I., Zhang Y. H., Rahman S. M., Yoshida T., Isobe K., Ding L. N., Iwamoto T., Hamaguchi M., Ikezawa H., Taguchi R. Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death. J Immunol. 1991 Aug 15;147(4):1153–1162. [PubMed] [Google Scholar]
  25. Nakayama K., Nakayama K., Negishi I., Kuida K., Shinkai Y., Louie M. C., Fields L. E., Lucas P. J., Stewart V., Alt F. W. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science. 1993 Sep 17;261(5128):1584–1588. doi: 10.1126/science.8372353. [DOI] [PubMed] [Google Scholar]
  26. Naquet P., Barbet J., Pont S., Marchetto S., Barad M., Devaux C., Rougon G., Pierres M. Characterization of Thy-1 with monoclonal antibodies and evidence of Thy-3. Immunol Ser. 1989;45:99–117. [PubMed] [Google Scholar]
  27. Naquet P., MacDonald H. R., Brekelmans P., Barbet J., Marchetto S., Van Ewijk W., Pierres M. A novel T cell-activating molecule (THAM) highly expressed on CD4-CD8- murine thymocytes. J Immunol. 1988 Dec 15;141(12):4101–4109. [PubMed] [Google Scholar]
  28. Nickas G., Meyers J., Hebshi L. D., Ashwell J. D., Gold D. P., Sydora B., Ucker D. S. Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex. Mol Cell Biol. 1992 Jan;12(1):379–385. doi: 10.1128/mcb.12.1.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pierres A., Naquet P., Van Agthoven A., Bekkhoucha F., Denizot F., Mishal Z., Schmitt-Verhulst A. M., Pierres M. A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones. J Immunol. 1984 Jun;132(6):2775–2782. [PubMed] [Google Scholar]
  30. Pierres M., Goridis C., Golstein P. Inhibition of murine T cell-mediated cytolysis and T cell proliferation by a rat monoclonal antibody immunoprecipitating two lymphoid cell surface polypeptides of 94 000 and 180 000 molecular weight. Eur J Immunol. 1982 Jan;12(1):60–69. doi: 10.1002/eji.1830120112. [DOI] [PubMed] [Google Scholar]
  31. Pont S., Regnier-Vigouroux A., Naquet P., Blanc D., Pierres A., Marchetto S., Pierres M. Analysis of the Thy-1 pathway of T cell hybridoma activation using 17 rat monoclonal antibodies reactive with distinct Thy-1 epitopes. Eur J Immunol. 1985 Dec;15(12):1222–1228. doi: 10.1002/eji.1830151215. [DOI] [PubMed] [Google Scholar]
  32. Reif A. E. The discovery of Thy-1 and its development. Immunol Ser. 1989;45:3–45. [PubMed] [Google Scholar]
  33. Rougon G., Alterman L. A., Dennis K., Guo X. J., Kinnon C. The murine heat-stable antigen: a differentiation antigen expressed in both the hematolymphoid and neural cell lineages. Eur J Immunol. 1991 Jun;21(6):1397–1402. doi: 10.1002/eji.1830210611. [DOI] [PubMed] [Google Scholar]
  34. Rouvier E., Luciani M. F., Golstein P. Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity. J Exp Med. 1993 Jan 1;177(1):195–200. doi: 10.1084/jem.177.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Scollay R., Shortman K. Thymocyte subpopulations: an experimental review, including flow cytometric cross-correlations between the major murine thymocyte markers. Thymus. 1983 Sep;5(5-6):245–295. [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. Simon M. I. Summary: the cell surface regulates information flow, material transport, and cell identity. Cold Spring Harb Symp Quant Biol. 1992;57:673–688. doi: 10.1101/sqb.1992.057.01.073. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Strasser A., Harris A. W., Cory S. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell. 1991 Nov 29;67(5):889–899. doi: 10.1016/0092-8674(91)90362-3. [DOI] [PubMed] [Google Scholar]
  42. Swat W., Ignatowicz L., von Boehmer H., Kisielow P. Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells. Nature. 1991 May 9;351(6322):150–153. doi: 10.1038/351150a0. [DOI] [PubMed] [Google Scholar]
  43. Tentori L., Pardoll D. M., Zuñiga J. C., Hu-Li J., Paul W. E., Bluestone J. A., Kruisbeek A. M. Proliferation and production of IL-2 and B cell stimulatory factor 1/IL-4 in early fetal thymocytes by activation through Thy-1 and CD3. J Immunol. 1988 Feb 15;140(4):1089–1094. [PubMed] [Google Scholar]
  44. Tomonari K. A rat antibody against a structure functionally related to the mouse T-cell receptor/T3 complex. Immunogenetics. 1988;28(6):455–458. doi: 10.1007/BF00355379. [DOI] [PubMed] [Google Scholar]
  45. Uchida N., Weissman I. L. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med. 1992 Jan 1;175(1):175–184. doi: 10.1084/jem.175.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ucker D. S., Ashwell J. D., Nickas G. Activation-driven T cell death. I. Requirements for de novo transcription and translation and association with genome fragmentation. J Immunol. 1989 Dec 1;143(11):3461–3469. [PubMed] [Google Scholar]
  47. Van Ewijk W., Jenkinson E. J., Owen J. J. Detection of Thy-1, T-200, Lyt-1 and Lyt-2-bearing cells in the developing lymphoid organs of the mouse embryo in vivo and in vitro. Eur J Immunol. 1982 Apr;12(4):262–271. doi: 10.1002/eji.1830120403. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Volarević S., Burns C. M., Sussman J. J., Ashwell J. D. Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7085–7089. doi: 10.1073/pnas.87.18.7085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Williams A. F., Barclay A. N. The immunoglobulin superfamily--domains for cell surface recognition. Annu Rev Immunol. 1988;6:381–405. doi: 10.1146/annurev.iy.06.040188.002121. [DOI] [PubMed] [Google Scholar]
  51. Wyllie A. H., Arends M. J., Morris R. G., Walker S. W., Evan G. The apoptosis endonuclease and its regulation. Semin Immunol. 1992 Dec;4(6):389–397. [PubMed] [Google Scholar]
  52. Wyllie A. H., Morris R. G., Smith A. L., Dunlop D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol. 1984 Jan;142(1):67–77. doi: 10.1002/path.1711420112. [DOI] [PubMed] [Google Scholar]
  53. von Boehmer H. Developmental biology of T cells in T cell-receptor transgenic mice. Annu Rev Immunol. 1990;8:531–556. doi: 10.1146/annurev.iy.08.040190.002531. [DOI] [PubMed] [Google Scholar]

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