Fig. 3: CAR-T derived FASLG auto-regulates cellular persistence in vivo.
From: CAR-engineered lymphocyte persistence is governed by a FAS ligand–FAS autoregulatory circuit
a, Experimental design to test persistence of T cells expressing a 1928ζ CAR ± ΔFAS in tumor-bearing mice. T cells were co-transferred into NSG mice bearing Nalm6 B-ALL and tracked based on tLNGFR or tEGFR expression. i.v., intravenous. b, Distribution of memory T cell subsets before transfer. Bar graphs displayed as mean ± s.e.m. using n = 3 biologically independent samples. TEM, effector memory T cell; TEMRA, terminal effector memory T cell. c,d, Representative FACS (c) and summary scatter-plots (d) measuring the ratio of tEGFR+/tLNGFR+ T cells at the time of infusion (n = 3 biologically independent samples) and following adoptive transfer. Symbols represent individually evaluated mice (n = 10) and are displayed as mean ± s.e.m. P values calculated based on comparison to the infusion product using a two-sided Student’s t-test. e, Western blot for FAS-L protein from control or FASLG KO 1928ζ CAR-transduced T cells at rest or 48 h after anti-CD3/CD28 restimulation. The 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-T cells ± ΔFAS coexpression. CAR-T cells were combined ~1:1 and serially restimulated at indicated time points with K562-CD19 FASLG KO leukemia cells (left) or left unstimulated (right). Data are displayed as the mean ratio of tEGFR/tLNGFR T cells ± s.e.m. using n = 3 biologically independent samples. Groups compared using a paired two-tailed Student’s t-test for accumulated differences between each time point. g, Experimental design to test the influence of CAR-T-derived FASLG on in vivo persistence in mice bearing established Nalm6 B-ALL. Control or FASLG KO tLNGFR-1928ζ CAR-T cells were co-transferred ~1:1 with control or FASLG KO tEGFR-1928ζ-ΔFAS CAR-T cells into Nalm6 B-ALL-bearing NSG mice. h,i, Representative FACS (h) and summary scatter-plot (i) comparing the ratio of tEGFR to tLNGFR cells at the time of infusion (n = 2 biologically independent samples) and 4 weeks following transfer. Symbols represent values from individually evaluated mice (FASLG wild-type (WT), n = 5; FASLG KO, n = 7) and are displayed as mean ± s.e.m. Groups were compared using a two-sided Student’s t-test. NS, not significant (P > 0.05).
