Extended Data Fig. 8: Disabling FAS signaling restrains terminal differentiation of CAR-T cells following serial antigen encounter.
From: CAR-engineered lymphocyte persistence is governed by a FAS ligand–FAS autoregulatory circuit
(a) Experimental design for testing the impact of ΔFAS coexpression on the distribution of memory T cell subsets following serial engagement of 1928ζ CAR-T cells with Nalm6 B-ALL cells. T cells transduced either with tLNGFR-1928ζ or tEGFR-1928ζ-ΔFAS were individually co-cultured at a 1:2 ratio with Nalm6 cells. Beginning nine days after the first exposure to tumor cells, Nalm6 cells were re-added to the co-culture every five to six days for a total of three rounds of stimulation. Following each round, an aliquot of cells was harvested and analyzed by FACS. (b) Summary of the distribution of TCM (CD45RA−CD45RO+CCR7+) and TEMRA (CD45RA+CD45RO+CCR7−) phenotype CAR-T cells following one, two, or three rounds of stimulation with Nalm6 cells. (c) Experimental design for testing the impact of T cell-derived FASLG on the distribution of memory T cell subsets following serial engagement of tEGFR-1928ζ-ΔFAS CAR-T cells with Nalm6 B-ALL cells. FASLG was knocked out (KO) of transduced T cells using CRISPR/Cas9 or left untreated as controls. Time points were identical to those shown in (a). (d) Summary of the distribution of TCM and TEMRA phenotype CAR-T cells following one, two, or three rounds of stimulation with Nalm6 cells. Data in panels (b) and (d) is displayed as the mean ± s.e.m. of the indicated memory subset (n = 3 biologically independent samples after gating on transduced T cells identified by EGFR or LNGFR expression). Groups were compared using a paired two-tailed t-test. ns = not significant.
