Fig. 5: Knockout of FASLG in 1928ζ CAR-T cells does not impair antitumor cytolytic activity across multiple B cell cancers.
From: CAR-engineered lymphocyte persistence is governed by a FAS ligand–FAS autoregulatory circuit
a, Schematic for the CRISPR/Cas9-mediated KO of FASLG in human CD8+ T cells expressing a 1928ζ CAR. b, Scatter-plot of FAS RNA-seq values from n = 85 B cell cancer lines featured in the CCLE. Horizontal line represents the median and vertical bars represent the interquartile range. TPM, transcript per million. c, Correlation of FAS transcript counts to total FAS protein levels from n = 15 B cell malignancy lines featured in the CCLE with matched RNA-seq and quantitative proteomic data. All cell lines express WT FAS except for KARPAS-422, which contains a FAS (W176G) mutation located in the protein’s transmembrane domain. Line represents linear regression. d, Measurement of FAS expression on Nalm6 B-ALL cells using FACS and cytolytic activity of FASLG KO versus AAVS1 KO T cells transduced with tEGFR or tEGFR-1928ζ against Nalm6/NLS–mCherry cells. e, Same as d but using Raji B-NHL and Raji/NLS–mCherry B-NHL cells. f, Same as in d but using JVM2 B cell prolymphocytic leukemia (B-PLL) and JVM2/NLS–mCherry B-PLL cells. FAS mean fluorescence intensity (MFI) values compared to an isotype control using an unpaired Student’s t-test using n = 3 biologically independent samples (d–f). Cytolytic activity was measured at indicated E:T ratios using Incucyte with data shown as mean ± s.d. using n = 3 biologically independent samples (d–f). Statistical comparisons were performed using a one-way ANOVA. NS, not significant (P > 0.05). The measured FASLG insertion/deletion frequency following FASLG KO was 93.0 ± 1.0% and 95.7 ± 1.5% in T cells transduced with tEGFR alone and tEGFR-1928ζ, respectively.
