Co-culture of cancer cells and T cells was performed as previously described (66 (link)). Briefly, B16F10 cells were maintained in complete DMEM media (10% FBS and 50U/ml of Penicillin-Streptomycin). CD8 T cells isolated from spleen and lymph nodes from Pmel-1 or OT-I mice were stimulated with anti-CD3/CD28 beads (ThermoFisher, 11452D) and then cultured in complete RPMI 1640 media (10%FBS, 20mM HEPES, 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, 2mM L-glutamine, and 50U/ml streptomycin and penicillin, 20ng/ml recombinant mouse IL-2).
To test the sensitivity of cancer cells to T cell-driven cytotoxicity (OT-I or Pmel-1 model), we plated B16F10 cells (sgRosa26, sgTraf3, pEF1a-Empty, or pEF1a-Traf3) at equal density in all wells, and added T cells at ratios to cancer cells. With the OT-I model, we first incubated the B16F10 cells with 1nM SIINFEKL peptide for 2 hours prior to co-culture with T cells. With the Pmel-1 model, B16F10 cells were either pre-treated with 1ng/ml IFNγ overnight or untreated prior to the co-culture. There are 2–4 cell-culture replicates for each condition. After a one-day or three-day co-culture with T cells, we counted the remaining cancer cells by FACS using the precision count beads (BioLegend, 424902). T cells present in these cultures were gated out based on antibodies specific for CD45 (Biolegend, clone 30-F11) or CD8 (BioLegend, clone 53-6.7).
To test the sensitivity of cancer cells to T cell-driven cytotoxicity (OT-I or Pmel-1 model), we plated B16F10 cells (sgRosa26, sgTraf3, pEF1a-Empty, or pEF1a-Traf3) at equal density in all wells, and added T cells at ratios to cancer cells. With the OT-I model, we first incubated the B16F10 cells with 1nM SIINFEKL peptide for 2 hours prior to co-culture with T cells. With the Pmel-1 model, B16F10 cells were either pre-treated with 1ng/ml IFNγ overnight or untreated prior to the co-culture. There are 2–4 cell-culture replicates for each condition. After a one-day or three-day co-culture with T cells, we counted the remaining cancer cells by FACS using the precision count beads (BioLegend, 424902). T cells present in these cultures were gated out based on antibodies specific for CD45 (Biolegend, clone 30-F11) or CD8 (BioLegend, clone 53-6.7).
