T-cell Activation and IFN-γ Production

T‐cell clones M170.48 and 10C10 were stimulated for 5 h in the presence of brefeldin A (10 µg·mL⁻¹, Sigma) with the tumor cell lines M113 and M6 (pretreated or not with thapsigargin for 24 h and washed extensively over another 24 h period) at an E : T ratio of 1 : 2. Cells were first stained with a PE‐conjugated anti‐CD8 mAb (BioLegend, clone RPA‐T8), fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with saponin 0.1%, stained with an APC‐conjugated anti‐ IFNγ mAb (BioLegend, clone B27) as previously described [11 (link)], and analyzed by flow cytometry. The expression of HLA‐A*0201 was assessed by flow cytometry using a PE‐conjugated anti‐HLA‐A*0201‐specific antibody (clone BB7.2, BD, Le pont de Claix, France).