Fig. 7: CAR-NK survival is regulated by a FAS/FAS-ligand autoregulatory circuit.

a,b, Representative FACS (a) and summary scatter-plot (b) quantifying FAS expression by NK cells at rest and 5 d following activation. Data are shown as mean ± s.e.m. for n = 3 biologically independent samples. Statistical analysis was performed by two-sided Student’s t-test. c, Transduction efficiencies of T cells or NK cells displayed as median ± interquartile range from n = 26 T cell and n = 14 NK cell experiments. P values were calculated using an unpaired two-tailed t-test with Welch’s correction. d, Representative FACS plots quantifying lzFAS-L induced apoptosis in nontransduced NK cells or NK cells transduced with indicated vectors. Numbers indicate mean ± s.e.m. of activated caspase 3/7⁺/annexin V⁺ cells (n = 3 biologically independent samples). e, Western blot for FAS-L protein in lysates from FASLG KO or control (Ctrl) NK cells transduced with tEGFR-1928ζ-ΔFAS (ECF) or tLNGFR-1928ζ (LC). Frequency of frameshift Indels in FASLG are displayed beneath each lane. Representative results from two independent experiments are shown. f, Relative antigen-driven in vitro expansion of control and FASLG KO 1928ζ CAR-NK cells ± ΔFAS. CAR-NK cells were combined ~1:1 and serially restimulated at indicated time points with K562-CD19 FASLG KO cells or left unstimulated as controls. Data are displayed as the mean ratio of tEGFR⁺/tLNGFR⁺ cells ± s.e.m. (n = 3 biologically independent samples). Groups compared using a paired two-tailed Student’s t-test for accumulated differences between each time point. NS, not significant (P > 0.05). g, Cytolysis of Raji/mCherry cells co-cultured at indicated E:T ratios with FASLG KO versus FASLG-WT tEGFR-1928ζ CAR-NK cells. Data are shown as mean ± s.e.m. (n = 3 biologically independent samples). Statistical comparisons were performed using a one-way ANOVA. NS, not significant (P > 0.05). NA, not applicable. h, Experimental design to test the in vivo persistence of NK cells that express a 1928ζ CAR ± ΔFAS in Raji B-NHL-bearing mice. i, Scatter-plot comparing the ratio of tEGFR⁺/tLNGFR⁺ CAR-NK cells before infusion and following adoptive transfer in the bone marrow. Symbols represent individually evaluated mice, n = 3 for baseline measurement and n = 10 per time point. P values compare infusion product to each time point using an unpaired, two-sided, Welch’s t-test.

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