Figure 5.

MC38‐Sia^null cells induce CD8⁺ T cell apoptosis. (a) and (b) MC38‐MOCK and MC38‐Sia^null cells were pulsed with OVA_257‐264 and labeled with CFSE or Cellvue Claret respectively (a). After 5 h of incubation with activated OT‐I CD8⁺ T cells, viability of MC38 cells was analyzed by flow cytometry and calculated by dividing the number of viable (7‐AAD⁻Annexin V⁻) MC38 cells present in the CD8⁺ T cell co‐culture by the viable MC38 cells cultured without T cells and multiplied by 100% (b). (c–f), Viable CD8⁺ T cells were analyzed with flow cytometry and gated as CD8b⁺7‐AAD⁻Annexin V⁻. (c) C, C57Bl/6 splenocytes were co‐cultured with MC38‐MOCK or MC38‐Sia^null cells in presence of medium or PHA‐L for 48 h. (d) Activated OT‐I CD8⁺ T cells were co‐cultured with MC38‐MOCK or MC38‐Sia^null cells for 24 h. (e) Activated OT‐I CD8⁺ T cells were co‐cultured with MC38‐MOCK in presence of medium or MC38‐Sia^null supernatant for 24 h. (f) and (g) Activated OT‐I CD8⁺ T cells were cultured with control (f) or heat inactivated (g) medium, MC38‐MOCK or MC38‐Sia^null supernatants for 24 h. Data are representative of one (g), two (c, e and f) or three (b and d) independent experiments. Bar, mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. [Color figure can be viewed at wileyonlinelibrary.com]