Figure 6.

Role of caspases and Bcl-x_L in the generation of immunity against EL4 antigens. (A) EL4 cells were incubated with ex vivo gp33-specific CD8⁺ T cells from virus-immunized C57BL/6 mice in the presence of the gp33 Ag for 20 hours at an effector:target ratio 3:1 (EL4.gp33^TcLCMV Group) or at an effector:target ratio 7:1 in the presence of the pan-caspase inhibitor Q-VD-OPh (30 µM; EL4.gp33^TcLCMV + Q VD group). After this time, cell cultures were collected and used to immunize C57B/L6 mice via intraperitoneal. At day 0 and day 7, as control, mice were immunized with PBS. On day 14, the different groups were inoculated with 2×10⁵ EL4 cells in the right flank. Tumor development was monitored over 25 days as described in the Methods section. The data correspond to 12 mice from three independent experiments, where ***p<0.001. Two-way analysis of variance (ANOVA) with Bonferroni post-test and log-rank test (Mantel-Cox). (B) The same experiment as in (A) was performed but using EL4 cells and the mutants thereof overexpressing Bcl-X_L or DNC3. The data correspond to 12 mice from three independent experiments, where **p<0.01; ***p<0.001. Two-way ANOVA with Bonferroni post test and log-rank test (Mantel-Cox). (C) wt mice were immunized with the indicated gp33-pulsed EL4 dead cells killed as indicated in (A), with PBS (CTR) or with gp33-specific Tc cells (Tc^LCMV). On day 10, splenocytes from these mice were isolated and incubated at an effector:taget ratio 100:1 with EL4 cells in the absence of the viral peptide gp33. After 18 hours, PS exposure on plasma membrane was measured by three-color flow cytometry using Annexin-V. Data are represented as the mean±SD of three independent experiments, using six mice in total, where *p<0.05, analyzed by unpaired t-test.