Figure 3.

Caspase inhibitors completely block PS exposure induced by gzmA-deficient CTL. (A–F) EL4.F15 cells were incubated with ex vivo virus-specific CD8⁺ cells (MACS selected, ≥95% CD8⁺ cells) from B6, gzmA^−/−, or gzmB^−/− mice for 2 h at a 10:1 effector/target ratio in the presence or absence of the LCMV peptide gp33. (A–D) CML cultures were incubated in the presence or absence of 100 μM of the pan caspase inhibitor Z-VAD-fmk or the caspase 3 inhibitor Z-DEVD-fmk. Subsequently, PS exposure on plasma membrane (annexin-V-FITC; A) and PI uptake (B) and, in parallel, ΔΨ_m loss (DiOC₆(3) staining; C) and ROS generation (2-HE; D) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). Results are the mean of three independent experiments and are given as the difference of percentages in the presence or absence of gp33 ± SD. (E and F) Inhibition of caspase 3 or 9 activity reduces, respectively, caspase 9 or 3 activation induced by B6 and gzmA^−/− CTL. Caspase 3 activity was analyzed using an FITC-labeled mAb against the active form of the enzyme (E), whereas caspase 9 activity was analyzed by using the specific fluorescent substrate FAM-LEHD-fmk (F) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). CMLs were also developed in the presence of the caspase 3 (100 μM Z-DEVD-fmk; F, bottom, gray) or caspase 9 (100 μM Z-LEHD-fmk; E, bottom, orange; and F, middle, orange) inhibitors. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars shown in the top panels.