Fas-specific apoptosis in thymic subsets. (A) After 18 hours of incubation with agonistic anti-Fas antibody (clone CH-11, 2 μg/mL) or IgM antibody (2 μg/mL) in the presence of 10 μg/mL cycloheximide, double-staining of CD4/CD8 subsets was performed in thymocytes before labeling with annexin-V. The proportion of (□) CD4⁺, (▧) CD4⁺CD8⁺ (DP), and (▪) CD8⁺cells that underwent Fas-specific apoptosis was calculated. Data are the means ± SEM of 3 independent experiments. The proportion of apoptotic CD4⁺ cells was three times higher in cytokine-activated thymocytes than in anti-CD3–activated thymocytes. (B) Fas staining was performed before annexin-V labeling, and the proportion of cells undergoing Fas-specific apoptosis was examined in Fas^+/− and Fas^hi cells. A representative experiment is shown. Annexin-V stainings in the presence of anti-Fas CH-11 are presented and 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. Fas^hi cells were always enriched in apoptotic cells, but cytokine-activated Fas^hi cells contained more apoptotic cells than did anti-CD3–activated cells.