Fig. 5.

Demethylation-mediated reversal of cGAS silencing enhances antigenicity and T cell recognition of melanoma. Schematic of the experimental procedure for the IFN-γ release assay. cGAS^absent human melanoma cell lines were pretreated with 5AZADC and cocultured with their HLA-matched human melanoma TIL in the presence or absence of dsDNA for 24 h (A). IFN-γ (Top) and IFN-β (Bottom) concentrations in supernatants were measured using ELISA (B). In some experimental conditions, tumor cells were preincubated with an MHC class I blocking Ab (W6/32) to determine CD8⁺ TIL reactivity. Representative histograms (C) and mean fluorescence intensity (MFI) of HLA-A.B.C expression on indicated melanoma cell lines (D). Schematic of the experimental procedure for the ⁵¹Cr release cytotoxicity assay using MART-1–pulsed WM39 cells (±5AZADC and/or ±dsDNA pretreatment) as target cells and MART-1–reactive TIL as effector cells (E). Frequency of MART-1 tetramer⁺ T cells in human melanoma TIL 123 (F). Specific lysis of MART-1–pulsed WM39 (±5AZADC and/or ±dsDNA) targets by TIL 123 at the indicated effector/target (E/T) ratios (G). All data are mean ± SD for two or three biological replicates. P values were calculated by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).