Role of Fas and granule exocytosis pathways in tumor-infiltrating T lymphocyte-induced apoptosis of autologous human lung-carcinoma cells

We have isolated a cytotoxic T lymphocyte (CTL) clone, Heu161, that reacts specifically with the human autologous lung carcinoma cell line IGR-Heu. We first demonstrated that IGR-Heu lacked Fas-receptor expression and was resistant to CD95-induced apoptosis. To further elucidate the role of Fas in tumor immune surveillance, we have stably transfected IGR-Heu with a Fas-expression vector and isolated CD95-sensitive and -resistant clones. Our data indicated that the resistance of 2 selected Fas-transfected clones to CD95-mediated lysis correlated with down-regulation of caspase-8 or its lack of cleavage and subsequent activation. All Fas transfectants, either sensitive or resistant to anti-Fas agonistic antibody, were as efficiently lysed by the CTL clone as the parental cell line. In addition, neither anti-Fas-blocking antibody nor Fas-Fc molecule inhibited T-cell lysis of Fas-sensitive tumor clone. This cytotoxicity was extracellular Ca²⁺-dependent and abolished in the presence of EGTA, indicating that it was mainly granzyme-mediated. Interestingly, although the caspase inhibitor z-VAD-fmk had no effect on tumor-cell lysis, it efficiently blocked target DNA damage triggered by autologous CTLs via the granule exocytosis pathway, indicating that the latter event was caspase-dependent. The present results suggest that lung carcinoma-specific CTLs use mainly a granule exocytosis-dependent pathway to lyse autologous target cells and that these effectors are able to circumvent alteration of the Fas-triggered intracellular signalling pathway via activation of a caspase-independent cytoplasmic death mechanism.