Cytotoxic T Lymphocyte (CTL) assay

CTL assay was done in two set of experiments: 1) CD8⁺ T cells from OTI/6PGD^−/− and pmel/6PGD^−/− mice were cultured with soluble αCD3 (1μg/ml), soluble αCD28 (1μg/ml) and mIL-2 (20 IU/ml) for 4 days. Corresponding OTI/6PGD^fl/fl and pmel/6PGD^fl/fl controls also were cultured in the same manner. 2) CD8⁺ T cells from OTI/6PGD^fl/fl or pmel/6PGD^fl/fl mice were isolated and stimulated with αCD3 (1μg/ml), αCD28 (1μg/ml) and mIL-2 (20 IU/ml) for 4 days in the presence of 6-AN or vehicle control (DMSO). After 4 days of T cells culture, 1×10⁵ cells were cultured with EG7 (for OTI) or B16-F10 (for pmel) in T: tumor cell ratio of 1:1 for 18 hr. T cells alone and tumor cells alone were considered as controls. After incubation, toxicity percentage of total was determined by Lactate Dehydrogenase (LDH) Assay Kit (Abcam) following manufacturer’s protocol.