Cytotoxicity assay

Effector cytotoxic CD8⁺ T cells were prepared from naïve CD8⁺ T cells isolated from spleen of OT‐I mice, and activated with Listeria‐OVA BacB cells, or with OVAp‐I‐loaded splenocytes as positive control, during 7 days.

EL‐4 cells were incubated alone or with 0.5 μM OVAp‐I for 1 h. After washing with PBS, OVAp‐I‐loaded EL‐4 cells were stained with 5 μM CellTrace Violet and unloaded EL‐4 cells with 0.5 μM CellTrace Violet. After washing with complete medium (with FBS), the populations were mixed and incubated with CTL at various ratios (5:1, 2:1, 1:1, 0.5:1 EL‐4:CTL; for 4 h). Next, they were analyzed by flow cytometry. Specific cytotoxicity was calculated as 1 − (% EL‐4 CellViolet^high/% EL‐4 CellViolet^low) × 100 (Lang et al, ²⁰⁰⁵). Relative cytotoxicity was calculated by subtracting the specific cytotoxicity of the negative control (EL‐4 cells incubated without CTL).