Apc conjugated anti ifn γ mab

Tumors were cut into pieces and incubated in RPMI-1640 (Nacalai Tesque) supplemented with 1% FBS, 10 mM HEPES, 0.2% collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 2 KU/mL DNase I (Sigma-Aldrich) for 40 min at 37 °C. All material was passed through a 70 μm cell strainer to obtain single cell suspensions. After staining dead cells using a Zombie Yellow Fixable Viability Kit (BioLegend) and blocking of Fc receptors with anti-CD16/32 mAb (2.4G2, Bio X Cell), the cells were stained with mAbs for cell surface antigens, H-2D^b and H-2K^b dimers. For intracellular cytokine staining, 1 × 10⁵ cells were stimulated with peptides, 1 × 10⁵ YTN2 or 1 × 10⁵ YTN16 cells in the presence of 10 μg/mL brefeldin A (Sigma-Aldrich) for 4 h. After staining dead cells with the Fixable Viability Dye eFluor 450 (Thermo Fisher Scientific) and blocking Fc receptors with anti-CD16/32 mAb, the cells were stained with mAbs for cell surface antigens, followed by fixation, permeabilization, and staining with APC-conjugated anti-IFN-γ mAb (BioLegend) using Intracellular Staining Fixation Buffer and Intracellular Staining Permeabilization Wash Buffer (10X) (BioLegend) according to the manufacturer’s protocols.

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).