CFSE-based Cytotoxicity Assay

In vitro cytotoxiocity assay was performed using CFSE-based labeling6 (link). SK37 or peptide-pulsed SK29 were labeled with 0.5 μM CFSE, whereas peptide-unpulsed SK29 was labeled with 5 μM CFSE. In some experiments, cancer cells were treated with 20 μg/ml Fas-ligand antibodies (eBiosciences) for 1 hour before addition to T cells. Fas-ligand antibody was also present during co-culture. 2 U/ml of recombinant IL-2, combination of 10 μg/ml anti-CD25 (IL-2Rα) antibody (BD Bioscience) and anti-IL-2 antibody (eBioscience), 0.1 nM concanamycin A (Sigma-Aldrich) or 100 μM Z-AAD-CMK (Enzo Biochem, Inc.) were added at the initiation of T cell culture. Cells were incubated in 5 mL round-bottom tubes for 14–16 hours. Cancer cells were harvested by treatment with trypsin/EDTA and analyzed for CFSE staining levels by FACSCalibur flow cytometer (BD Biosciences) to enumerate the percentages of SK37 and SK29. Acquisition data were analyzed by FCS Express Version 3 software (De Novo Software) or FlowJo software. Cytotoxicity was calculated using the following formula: % cytotoxicity = 100 × {1−(%SK29/%SK37)_{without T cells}/(%SK29/%SK37)_{with T cells}}.