Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex

Development of tolerance to self Ag occurs during a negative cell selection process in the thymus. This selection process is thought to involve interactions between Ag-specific thymocyte receptors and self Ag presented by the MHC proteins on accessory cells, resulting in deletion of potentially harmful self-reactive precursors. However, the mechanisms underlying this clonal deletion have not been identified. In confirmation of previous findings (C.A. Smith, G.T. Williams, R. Kingston, E.J. Jenkins, J.J.T. Owen, 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181), we have found that an anti-CD3 antibody stimulated DNA fragmentation, characteristic of a suicide mechanism known as apoptosis or programmed cell death (PCD), in suspensions of human thymocytes. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca²⁺ concentration, most of which was of extracellular origin. Although the magnitude and duration of the Ca²⁺ increase were similar to those observed in response to Con A, the mitogen did not stimulate DNA fragmentation or cell death. Phorbol ester prevented Ca²⁺-dependent DNA fragmentation and cell killing in response to anti-CD3 or other agents that stimulated PCD, suggesting that activation of protein kinse C abrogated cell suicide. Disappearance of CD4⁺CD8⁺ immature thymocytes was generally observed in response to all agents that stimulated PCD, whereas mature PBL were insensitive to stimulation of PCD. Our results suggest that antibody-mediated stimulation of immature thymocytes via the TCR complex results in Ca²⁺-dependent, endonuclease-mediated cell killing, depending on the activation status of protein kinase C.