Extended Data Fig. 9: Experimental design, functional assays, and melanoma co-culture experiments with NY-ESO-1 TCR T cells engineered with variants identified in base editing screens.
From: Mapping variant effects on anti-tumor hallmarks of primary human T cells with base-editing screens
a, Schematic for engineering and expanding NY-ESO-1 specific T cells. b, Representative flow cytometry dotplot of NY-ESO-1 specific T cells prior to sorting. c, Viable A375-dsRed cells relative to time t0 after culture with NY-ESO-1 specific T cells for 48 hours at varying effector to target ratios. aMHCI = MHC class I-blocking antibody (n = 3 independent biological replicates). d, Representative flow cytometry histograms of ABE-mediated knockout of B2M or CD2 in NY-ESO-1 specific T cells. e, Representative contour plots of single or multiplexed base editing of B2M and CD2. f, AKT phosphorylation (pS473), in NY-ESO-1 specific T cells after either 15 minutes of co-culture with A375 cells (+) or media alone (-) (n = 3 independent biological replicates). g, MFI of TNFα and h, GrzB in NY-ESO-1 specific T cells with indicated base edits after 8-hour co-culture with A375-dsRed cells at a 1:1 effector to target ratio (n = 3 independent biological replicates). i, Frequency of NY-ESO-1 specific T cells with indicated genotypes co-expressing TNFα, IL2, and GrzB after 8 hours of co-culture with A375 cells at a 1:1 effector to target ratio (n = 3 independent biological replicates.). NT = non-targeting control sgRNA j, Viable wild-type (WT) or CD58-KO A375 cells relative to time t0 after 48 hours of co-culture with NY-ESO-1 specific T cells at a 1:1 effector to target ratio (n = 3 independent biological replicates.) k, Viable B2M-KO A375 cells relative to time t0 after 48 hours of co-culture with NY-ESO-1 specific T cells with indicated genotypes. Dotted lines in (f-i) represent mean of the control. Dotted lines in (j-k) represent relative viable cell count at time t0. One-way ANOVA with Tukey’s test for multiple comparisons (panel c). One-way ANOVA with Dunnett’s test for multiple comparisons (panels f-i, k). Student’s t test (panel j). Error bars represent mean +/− SD (panels c, f-k).
