CD8 T Cell Cytotoxicity Assay

CD8 cytotoxicity assays were performed as previously described.^{91 (link)} Briefly, CD8 T cells from TRP1^high Mice^{57 (link)} were isolated with EasySep Mouse CD8+ T cell Isolation Kit (StemCell), resuspended in complete RPMI +100U/mL hIL2 (Peprotech #200–02) and stimulated with CD3/CD28 beads Dynabeads (Thermofisher #11456D) for 48 h. Target B16^Nectin1 or ex vivo cultures were plated in complete DMEM with 50 ng/mL IFNγ (Peprotech #315–05) on the day before co-culture. On day seven following T cell isolation, CD8 T cells were plated at various E:T ratios with or without the presence of tumor cells. 24 h following co-incubation of T cells with tumor cells, media was carefully removed and cell viability was assessed using CellTiterGlo (Promega). Relative luminescence was normalized to wells containing the same number of T cells without tumor cells present. For T cell cytotoxicity comparing wildtype B16^Necti1 to β2m^−/− B16^Nectin1, tumor cells were stained with 5μM CFSE prior to plating with 50 ng/mL IFNγ; T cell cytotoxicity was measured as fluorescent confluence loss comparing T cell containing wells to wells with tumor cells only using a Celigo image cytometer (Nexcelom 200-BFFL-5c).