Extended Data Fig. 3: Quantification of FASLG expression by 1928ζ CAR-expressing T cells following in vitro co-culture with CD19+ leukemia cells using RNA in situ hybridization.
From: CAR-engineered lymphocyte persistence is governed by a FAS ligand–FAS autoregulatory circuit
(a) Representative immunofluorescent confocal images and (b) summary violin plots quantifying FASLG mRNA expression by nontransduced or 1928ζ CAR-expressing T cells at rest or 24 h following in vitro co-culture with CD19+ Nalm6 B-ALL. Samples were co-hybridized with DAPI (blue) and multiplexed RNA-FISH probes specific for the mRNA sequence of the CAR’s single-chain variable fragment (scFv) (green), CD3E mRNA (white), and FASLG mRNA (red). Data shown is representative of results from n = 12, n = 13, and n = 15 gated regions from nontransduced T cells, CAR-T cells alone, and CAR-T cells co-cultured with Nalm6 cells, respectively. T cells were derived from n = 2 healthy donors. Violin distributions are centered around the median (red horizontal line) with quartiles ranges displayed above and below (dashed horizontal lines). The maxima and minima are represented by the top and bottom of each plot. Each dot represents mean FASLG mRNA expression within a particular cell type from a region of interest. P-values calculated using a one-way ANOVA with a Šídák’s multiple comparisons test. a.u. = arbitrary fluorescence units.
