Figure 3.

BATF3 and TLR4 are required for the generation of cancer immunity after immunization with dead EL4 cells. (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. After this time, cell cultures were collected and used to immunize wild-type (wt), TLR4^KO and BATF3^KO mice at day 0 and day 7. As control, mice were immunized with PBS. On day 14, mice 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 10 mice from two independent experiments, where ***p<0001. Two-way analysis of variance (ANOVA), with Bonferroni post-test and log-rank test (Mantel-Cox) in the survival graph’s. WT, TLR4^KO and BATF3^KO mice were immunized with gp33-pulsed EL4 dead cells as indicated in (A). On day 10, splenocytes from these mice were isolated and incubated at an effector:target ratio 100:1 with EL4 cells in the presence or 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<0,1; analyzed by unpaired t-test.