Extended Data Fig. 10: Design and results of leukemia co-culture with CD19 CAR-T cells equipped 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, Representative histograms of GFP expression, indicating transduction efficiency of first- and second-generation CD19-CAR constructs (CD19-CD3z or CD19-BBz, respectively) in primary human T cells. Blue histograms represent untransduced control T cells. b, Expression of CTLA4 on CD19-CAR T cells, edited with a control non-targeting sgRNA (NT) or CTLA4-KO sgRNA, following 48-hour co-culture with Nalm6 leukemia cells at an 0.5:1 effector to target ratio. c, Relative cell numbers of Nalm6 cells 48 hours after co-culture with CD19-BBz CAR T variants at several E:T ratios, compared to time t0. d, Relative number of CD19-KO Nalm6 cells 48 hours after co-culture with CD19-BBz CAR T cells at an 0.25:1 effector to target ratio, compared to time t0. e, Quantification of CD19-CD3z CAR T cell AKT phosphorylation (pS473) by flow cytometry after 15 minutes of culture with either Nalm6 leukemia (+) or media alone (−) with representative flow histograms. f, Quantification of CD19-CD3z CAR T cell intracellular expression of TNFα and g, IL2 after 8-hour culture with Nalm6 leukemia (+) or in media only (-). h, Relative cell numbers of wild-type and i, CD19-KO Nalm6 cells 48 hours after co-culture with CD19-CD3z CAR T cells at an 0.5:1 effector to target ratio, compared to time t0. Dotted lines in (e-g) represent mean of the control population. Dotted lines in (panel c, d, h, i) represent relative viable cell count at time t0. One-way ANOVA with Tukey’s test for multiple comparisons (panel b), one-way ANOVA with Dunnett’s test for multiple comparisons (panels d-i). Error bars represent mean +/- SD (panels b-i).
