Fig. 6: FAS-L/FAS signaling is dispensable for CAR-T antitumor efficacy.
From: CAR-engineered lymphocyte persistence is governed by a FAS ligand–FAS autoregulatory circuit
a, Growth kinetics of Nalm6 B-ALL, Raji B-NHL or activated T cells in the presence or absence of lzFAS-L. Each cell type was transduced with mCherry. Data are shown as mean ± s.e.m. using n = 3 biologically independent samples. FC, fold change. b,c, Experimental design (b) and Kaplan–Meier survival curve (c) comparing the in vivo antitumor efficacy of human CD8+ T cells that express a 1928ζ CAR with CRISPR/Cas9-mediated KO of FASLG or AAVS1 against established Nalm6 B-ALL. tEGFR alone, n = 8; tEGFR-1928ζ AAVS1 KO, n = 15; tEGFR-1928ζ FASLG KO, n = 15. Statistical comparisons were made using a log-rank test. d, Scatter-plots displaying the enrichment or depletion of sgRNAs targeting indicated genes in the death receptor pathway by Cas9-expressing Nalm6 B-ALL cells. Tumor cells were placed under selection by T cells transduced with a 1928ζ CAR (left), a 41BBζ CAR (right) or left nontransduced as specificity controls (Ctrl). Data were reanalyzed from two published genome-scale CRISPR/Cas9 screens and are shown as mean log2FC ± s.e.m. of sgRNAs targeting indicated genes. NTC, nontargeted control sgRNAs. n = 6 unique sgRNAs per gene for 1928ζ CAR experiment and n = 8 unique sgRNAs per gene for the 19BBζ experiment. Gene level significance was determined using a one-way ANOVA corrected for multiple comparisons by Dunnett’s test. e, Schematic for the CRISPR/Cas9-mediated KO of FAS using an individual sgRNA in Nalm6 B-ALL. f, Time-dependent cytolytic activity of 1928ζ CAR-T cells against FAS KO versus FAS-WT Nalm6/mCherry cells at a high (left) or low (right) E:T ratio. Data are shown as mean ± s.e.m. using n = 3 biologically independent samples. Statistical comparisons were performed using a one-way ANOVA. NS, not significant (P > 0.05).
