TCR-mediated up-regulation of c-FLIPshort correlates with resistance toward CD95-mediated apoptosis by blocking death-inducing signaling complex activity

To investigate apoptosis resistance upon restimulation in human peripheral blood T lymphocytes, we used the following in vitro model. This model represents the main features of T cell reactivity: freshly isolated PKA-activated T cells cultured in IL-2 for a prolonged period of time develop a CD95 (APO-1/Fas) apoptosis-sensitive phenotype, These T cells represent activation-induced cell death-sensitive T cells during the down phase of an immune response, A fraction of apoptosis-sensitive activated T cells becomes apoptosis resistant upon TCR/CD3 restimulation, CD95 apoptosis sensitivity requires formation of a functional receptor associated death-inducing signaling complex (DISC), i.e., a protein complex of CD95 receptors, the adaptor Pas-associated death domain protein (FADD)/MORT1 and caspase-8 (PADD-like IL-1 beta -converting enzyme (FLICE), MACH, Mch5), We identified activation of procaspase-8 at the DISC as the main target for the protective activity of TCR/CD3 restimulation, We found that procaspase-8 cleavage Is reduced in T cells after TCR/CD3 restimulation, In addition, we detected up-regulation of c-FLIPS (the short splice variant of the cellular FLICE inhibitory protein) and strongly enhanced recruitment of c-FLIPS into the DISC, These data suggest that the recruitment of c-FLIPS into the DISC results in reduced DISC and caspase-8 activity.