INHIBITION OF CPP32-LIKE PROTEASES PREVENTS GRANZYME B-BASED AND FAS-BASED, BUT NOT GRANZYME A-BASED CYTOTOXICITY EXERTED BY CTL CLONES

The perforin-facilitated entry of granzymes in target cells is a major mechanism used by CTL to induce cell death. It has been reported that granzyme B can cleave and activate the apoptotic cysteine protease p3 2 (CPP32)/Yama and its homologues in vitro. However, the mechanism for granzyme-based cytolysis exerted by intact CTL remains unclear. In th e present work, we have used anti-CD3 mAb-redirected lysis of Fas-nega tive L1210 cells by CTL clones as a model to study perforin/granzyme-b ased cytotoxicity separately from the contribution of the Fas/Fas liga nd system. N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), a specific inhibitor of CPP32-like proteases, completely prevented the former ty pe of lysis in 3-h assays, but not in long-term (16-h) assays. A combi nation of Ac-DEVD-CHO and the granzyme A inhibitor IGA o)-4-chloro-3-( 2-isothioureidoethoxy)-isocoumarin) inhibited long-term cytolysis. 3,4 -Dichloroisocoumarin, a serine-protease inhibitor that efficiently inh ibits granzyme B and poorly inhibits granzyme A, had similar effects a s Ac-DEVD-CHO on anti-CD3 mAb-redirected lysis of L1210 cells. On the other hand, Fas-based cytolysis exerted by the same CTL clones on Fas- transfected L1210 cells (L1210Fas) was inhibited completely by Ac-DEVD -CHO, irrespective of the incubation time. These results suggest that granzyme B- and Fas-based cytotoxicity exerted by CTL clones converge at the level of CPP32-like protease activation, while granzyme A acts via a different, still undefined, pathway. We also demonstrate that pe rforin/granzyme-based cytolysis occurs without increase in the cellula r ceramide content, ruling out the contribution of the sphingomyelinas e pathway to this mechanism of cell death.