FIGURE 4

gzmB⁺CTL-induced activation of caspase-3 is independent of Bid or Bak and Bax and is crucial for PS exposure but not for mitochondrial perturbation. (a) Gp33-pulsed MEF.wt, MEF.Bid^-/- and MEF.Bak Bax^-/- were incubated with ex vivo virus-immune gzmB⁺CTL (1 and 4 h as indicated, 10:1 effector/target ratio). Activation of caspase-3 was monitored with an FITC-labeled mAb against the active form of the enzyme. Histograms show a representative experiment. Numbers correspond to the percentage of cells as indicated by the horizontal bars. (b) gp33-pulsed MEF.wt was incubated with ex vivo virus-immune gzmB⁺CTL (4 h, 10:1 effector/target ratio) in the presence or absence of the general caspase inhibitor Ac-ZVAD-fmk (80 M). Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake (b) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and Methods. A representative experiment is shown in the upper panels. Numbers correspond to the percentage of cells in each quadrant. Data in the graph (PS/PI) are represented as meanS.E.M. of three independent experiments. Ctr: only target cells; ZVAD: target cells+ZVAD-fmk