The Regulation of CD95 Ligand Expression and Function in CTL

Abstract

Previous studies with CTL lines and CTL hybridomas have suggested that functional CD95 (APO-1/Fas)-ligand (CD95L) expression on effector CTLs is a consequence of specific CTL-target recognition and TCR triggering of newly transcribed CD95L. Such a control on the expression of CD95L could provide a double safeguard for killing only cognate target cells. Here the regulation of CD95L expression and function was tested in in vivo primed, alloreactive peritoneal exudate CTL (PEL) from perforin-deficient (P0) mice. CD95L-based, PEL-mediated cytotoxicity was blocked by brefeldin A, an inhibitor of intracellular protein transport, but not by the protein synthesis inhibitor emetine, the immunosuppressive drug cyclosporin A, or the DNA transcription inhibitor actinomycin D. CD95L mRNA transcripts in freshly isolated PEL were shown by RT-PCR; CD95L surface expression was evident by staining with Fas-Fc as well as CD95L Abs. Undiminished CD95L expression and cytocidal activity were found in PEL incubated for 48 h in culture, without adding Ag, mitogen, or cytokines. PEL expressed functional CD95L, yet exhibited target cell-specific killing, except when encountering high CD95-expressing cells. The results indicate that PEL use CD95L probably expressed in the Golgi and/or on the cell surface and do not require newly transcribed CD95L upon target cell conjugation. Hence the TCR-triggered recruitment of preformed CD95L, rather than its biosynthesis, controls CD95L-based specific lysis induced by CTL.