Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively

We show that an influenza hemagglutinin-specific CD4(+) murine T cell hybri doma (IP-12-7) enters the apoptotic suicide program via the Fas ligand (Fas L)/Fas-mediated pathway upon T cell receptor (TCR) stimulation. These cells express Fas and Fast mRNA, cell surface Fas and intracellular Fast, but do not enter apoptosis upon Pas ligation prior to TCR stimulation. TCR stimul ation additionally results in protein synthesis-dependent cell surface expr ession of the preformed Fast. Addition of phorbol dibutyrate (PBu2) alone w as sufficient to induce susceptibility to Pas ligation induced apoptosis, w hile addition of both PBu2 and calcium ionophore A23187 were required to in duce Fast cell surface expression. Addition of cyclosporin A completely inh ibited TOP-mediated death and Fast cell surface up-regulation, but had no e ffect on apoptosis induced directly by Pas ligation following TOR stimulati on. Inhibitors of protein kinase C (PKC) (Gd 6976 and GF 109203X) completel y inhibited TOP-induced susceptibility to Pas ligation, but only partially inhibited TOP-induced cell surface expression of Fast. PKC isoenzymes alpha , beta, delta and zeta were expressed by this cell line and only the alpha and beta l isoforms translocated to the membrane fraction upon TCR stimulat ion. Our data suggest that in activation-induced T cell apoptosis PKC is in volved in pathways that mediate the acquisition of Fas susceptibility, whil e calcineurin is required for cell surface expression of the preformed FasL .