Assessment of tumor recognition and cytolytic assays

An enzyme-linked immunospot (ELISpot) IFN-γ release assay (Cellular Technology Limited, Shaker Heights, OH, USA) [8] was used to assess specific T cell avidity and reactivity. Cultured T cells were washed and cocultured, either alone or with autologous tumor cell lines, as established above. In the ELISpot assays, effector cells (E) (1×10⁵) were added to target cell lines (T) (1×10⁴) at an E: T ratio of 10: 1 per well in a 96-well plate and incubated for 48 h, according to the manufacturer’s instructions (Cellular Technology Limited, Shaker Heights, OH, USA). The raw data were analyzed and plotted using Immunospot software (Cellular Technology Limited, Shaker Heights, OH, USA). More than 40 spots and twice background was defined as positive T cell reactivity.

The cell counting kit-8 (CCK-8) assay was used to detect cytolysis. Target cells were plated per well with effector cells at varying effector to target ratios (1.8: 1, 5.5: 1, 16.6: 1, and 50: 1) in 96-well plates for 24 hours in an incubator. The supernatants were obtained for absorbance measurement at OD 450 nm. The percentage of lysed cells were calculated according to the formula: control absorbance–experimental absorbance)/control absorbance - control blank × 100%.