Figure 1.

EL4.gp33 cells killed by antigen-specific Tc cells express immunogeneic cell death signals. (A) EL4 cells and mutants thereof (Bcl-x_L and DNC3) 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 the indicated effector:target ratios. Subsequently, phosphatydilserine exposure on plasma membrane and cell membrane integrity were measured by three-color flow cytometry using Annexin-V and 7-AAD as described in the Methods section. Data are represented as mean±SD of four independent experiments using eight mice in total. (B–D) EL4 cells and mutants thereof (Bcl-X_L and DNC3) 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 1:1 effector:target ratio. A higher effector:target ratio (3:1) was used when QVD (30 µM) was included. Non-pulsed EL4 cells incubated with Tc cells and gp33-EL4 cells alone were used as controls. (B) Calreticulin exposure was analyzed by flow cytometry as indicated in the Methods section. (C, D) HMGB1 and interleukin (IL)-1β was measured in cell supernatants by ELISA. Data are represented as the mean±SD of four independent experiments, where *p<0,1; **p<0,01; ***p<0001, analyzed by unpaired t-test.