The development and ongoing function of a non-autoreactive and self-limited immune repertoire is dependent on the persisting ability to stringently induce the elimination of functionally inappropriate lymphocytes. Deletion of inappropriate T-cells occurs selectively via the process of activation-driven cell death in response to T-cell receptor-mediated stimulation. Activating stimulation that can trigger T-cell proliferative responses also can induce cell death. In the periphery, an initial activating stimulus induces cell cycle transit and the expression of activation markers including CD95; only upon subsequent stimulation is death induced. Thus, an obligate, temporally ordered pattern regulates the acquisition of susceptibility to activation-driven cell death. This appears to be the case in the thymus as well, although activation-driven negative selection there occurs by a distinct, CD95-independent process. An ordered sequence of events also is central to the process of physiological cell death. The principal investigator has found a thematically conserved, sequential pathway of death in different cell types induced by distinct suicidal stimuli. Cytoplasmic Ced-3-like Asp-specific cysteine protease activity acts upstream of step(s) generally inhibitable by Bcl-2 to modulate the death response, and nuclear cyclin-dependent kinase (cdk) activity functions downstream as an apparent effector of cell death. A similarly ordered sequence of events appears to pertain in all cases of physiological death, although particular elements of the pathway induced by specific suicidal stimuli may be distinct. In the activation-driven cell death response, the induction of cdk activity is dependent on Ced-3-like protease activation but is not inhibited by Bcl-2. The experiments in this application focus on defining, at the molecular level, the ordered events involved in activation-driven cell death. Studies are designed to identify molecularly Ced-3-like cysteine protease, cyclin-dependent kinase, and other trans-dominant activities involved in the death response in mature T lymphocytes and in immature thymocytes. Studies will also refine the discrimination of modulatory and effector aspects of this ordered process. The proposed experiments will also contrast the elements of the activation-driven negative selection pathway to cell death resulting from a failure of positive selection. In addition to providing insight into the control of lymphocyte ontogeny and selection against autoimmune reactivity, a detailed understanding of the mechanism of activation-driven dell death may be revealing of potential mechanisms of in vivo tumor control.
