T-CELL RECEPTOR-INDUCED FAS LIGAND EXPRESSION IN CYTOTOXIC T-LYMPHOCYTE CLONES IS BLOCKED BY PROTEIN-TYROSINE KINASE INHIBITORS AND CYCLOSPORINE-A

Fas/APO-1 is a member of the tumor necrosis factor receptor family of proteins, that induces apoptosis when cross-linked with monoclonal ant ibody (mAb) or with its physiological ligand. Recently, both a perfori n-based and a Fas-based mechanism have been proposed to account for T cell-mediated cytotoxicity. In the present study we used a murine CD8( +) cytotoxic T lymphocyte (CTL) clone (KB5.C20) specific for H-2K(b) a nd a T cell receptor (TcR)-negative variant of the same clone (2005(-) D4) to test (i) whether the same cell can exert both cytotoxic effecto r mechanisms and (ii) the role of TcR engagement in the induction of F as-based cytotoxicity. We demonstrate that both the TcR(+) and TcR(-) clones were able to express the Fas ligand after stimulation with phor bol 12-myristate 13-acetate (PMA)/ionomycin, and that TcR engagement o f the KB5.C20 clone by means of antigen-bearing cells or of its anticl onotypic mAb (Desire-1), which leads to Ca2+-dependent, presumably per forin-based, cytotoxicity, was also able to induce Fas-based cytotoxic ity. In addition, using inhibitors we investigated the signal transduc tion pathway(s) involved in the induction of Fas-based cytotoxicity an d expression of the Fas ligand mRNA in the CTL clones. The involvement of src-like protein tyrosine kinases (PTK) in Fas ligand induction th rough TcR engagement, was strongly suggested by inhibition with the sr c-like PTK inhibitor herbimycin A. Inhibition of Fas ligand induction by genistein, a more general TPK inhibitor, even upon stimulation by P MA plus ionomycin, suggested the possible involvement of PTK activitie s downstream of protein kinase C (PKC) in Fas ligand induction in CTL. Finally, the implication of the Ca2+/calmodulin-dependent protein pho sphatase calcineurin in Fas ligand induction was demonstrated by the p artial inhibition of Fas ligand induction with cyclosporin A. Thus, in CTL clones, Fas ligand expression is inducible by TcR engagement thro ugh a pathway similar to that involved in expression of some lymphokin e genes.