Anti-Fas–induced apoptosis in preactivated thymocytes. (A) Representative analysis of the apoptotic effect of an agonistic anti-Fas antibody on human preactivated thymocytes. After 24 hours of culture with IgG1 antibody, anti-CD3 antibody, or IFN-γ + IL-7, human thymocytes were collected and 105 cells were incubated with agonistic anti-Fas antibody (clone CH-11, 2 μg/mL) or IgM antibody (2 μg/mL), with or without 10 μg/mL cycloheximide. Cells were harvested 18 hours later, washed with PBS, and stained with FITC-conjugated annexin-V to quantify apoptosis. Specific anti-Fas–mediated apoptosis was calculated by subtracting the proportion of annexin-positive cells in the presence of control IgM from the proportion of annexin-positive cells in the presence of anti-Fas CH-11. In the absence of cycloheximide, less than 10% of thymocytes underwent Fas-specific apoptosis. In the presence of cycloheximide, thymocytes activated by IFN-γ + IL-7 were more susceptible than anti-CD3–activated thymocytes to Fas-specific apoptosis. (B) Data are the means ± SEM of 5 independent experiments. (□) IgG1; (▧) anti-CD3; (▪) IL-7 + IFN-γ.
