T Cell Cytotoxicity Assay Protocol

T cell killing assay was performed as previously described (34 (link)). Human primary T cells (70024, STEMCELL Technologies) were maintained in RPMI-1640 with 10% FBS and 10 ng/mL of IL-2 (589102, BioLegend). For T cell expansion and activation, T cells were cultured in ImmunoCult-XF T Cell Expansion Medium (10981, STEMCELL Technologies) with 25 μL/mL of ImmunoCult Human CD3/CD28 T Cell Activator (10971, STEMCELL Technologies) and 10 ng/mL of IL-2 for 7 days. To analyze the killing effect of T cells, 3 × 10⁵ of tumor cells were cocultured with 3 × 10⁶ of activated T cells in DMEM with 10% FBS, 25 μL/mL of T cell activator and 10 ng/mL of IL-2 for 48 hours. Then LDH release assay was performed.