TCRalpha/beta and TCRgamma/delta CD4^-/CD8^- HLA-DR alloreactive CTL clones do not use FAS/FAS ligand pathway to lyse their specific target cells

The expression of Fas Ligand (FasL) on human TCRalpha/beta and TCRgamma/delta CD4^-/CD8^- MHC class II-alloreactive clones and Fas/FasL- mediated cytotoxicity were investigated. These clones mediated a HLA-DR2- restricted cytotoxicity toward E418 B cell line (Fas¹). Northern blot analysis demonstrated that all the clones expressed FasL mRNA upon stimulation with E418 specific target. FasL surface expression was detected by immunofluorescence analysis using Fas-Fc soluble protein as well as anti- FasL polyclonal antibodies. Cytotoxicity experiments performed in the presence of anti-Fas, anti-FasL and Fas-Fc molecule indicated that these reagents were unable to inhibit T cell clone mediated lysis toward E418. In addition, when emetine, known to inhibit the induction of Fas-mediated killing, was added during the cytolysis effector phase, no inhibition was observed. These data strongly suggest that Fas/FasL pathway is not involved in this particular T-cell clone-mediated lysis. This cytotoxicity is extracellular Ca²⁺-dependent and is abolished in the presence of EGTA suggesting that it is mainly perforin/granzyme-based.