Bioluminescence-based cytotoxicity assay

Luciferase-expressing EBV-LCL tumor cells were counted and resuspended at a concentration of 3 × 10⁵ cells/mL. Cells were given D-Luciferin (75 µg/ml; Perkin Elmer, Waltham, MS, USA) and were placed in 96-well white flat-bottomed plates at 100 µl cells/well in triplicates. The EBV-LCLs were then loaded with different 10 µM KRAS mutated peptides as indicated. Effector T cells were added at a 20:1 E:T ratio. In order to determine spontaneous and maximal killing, wells with target cells only or with target cells in 1% Triton™ X-100 (Sigma-Aldrich, St Louis, MO, USA) were seeded. Cells were left at 37°C and the bioluminescence (BLI) was measured with a luminometer (VICTOR Multilabel Plate Reader, Perkin Elmer) as relative light units (RLU) at indicated time points. Target cells incubated without any effector cells were used to determine baseline spontaneous death RLU at each time point. Triplicate wells were averaged and lysis percentage was calculated using the following equation: % specific lysis = 100x(spontaneous cell death RLU – sample RLU)/(spontaneous death RLU – maximal killing RLU). Metrics were then computed using Igor Pro 8.1 (Wavemetrics, Portland, OR, USA). Sigmoid curves (no Hill equation) were fitted for every set of points (using Igor Pro 8.1) for visualization, with standard deviation as weighting factor.