Cytotoxicity Assay for T-cell-Mediated Tumor Killing

T-cell-mediated tumor cell killing assay was performed as described previously.^{21 (link)} Tumor cells were pre-treated with MMC (0.4 μM) for 24 h. PBMCs were plated at a density of 1 × 10⁶/well in 6-well plates and then co-cultured with tumors cells at 4:1 ratio for 24 h. Anti-human PD-L1 antibody, atezolizumab (Selleck Chemicals, USA) (50 μg/ml) was added afterwards. The co-culture system was maintained for 5 days and the wells were washed with PBS twice to remove PBMCs. The adherent and surviving cancer cells were fixed and stained with crystal violet solution. Cancer cell viability was then estimated by colorimetric determination.