Fig. 2: CAR-T derived FASLG auto-regulates cellular persistence in vivo.

(a) Schematic overview of the experimental design to test the in vivo persistence of human T cells that express a 1928ζ CAR ± a FAS dominant negative receptor (ΔFAS) in tumor-bearing mice. T cells were co-transferred at a ~1:1 ratio into NSG mice bearing established Nalm6 B-cell acute lymphoblastic leukemia (B-ALL) and tracked based on expression of tLNGFR or tEGFR. (b) Percentage of CD8⁺ and CD4⁺ T cells with a given memory phenotype at the time of adoptive transfer following transduction with tLNGFR-1928ζ or tEGFR-1928ζ- ΔFAS. Bar graphs displayed as mean ± s.e.m. using n=3 biological replicates. (c) Representative FACS and (d) summary scatter plots measuring the ratio of tEGFR⁺ to tLNGFR⁺ T cells at the time of infusion and four weeks following adoptive transfer. 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 expression in lysates from control or FASLG-KO 1928ζ CAR-transduced T cells. Cells were analyzed at rest and 48h after anti-CD3/CD28 restimulation. The frequency of frameshift Indels in FASLG for each cell type are shown beneath each lane. (f) Relative antigen-driven in vitro expansion of control and FASLG-KO 1928ζ CAR-T cells with or without ΔFAS co-expression. CAR-T cells were combined in ~1:1 ratio on day 0 and serially restimulated at indicated time points (▲) with K562-CD19 FASLG-KO leukemia cells (left panel) or left unstimulated as controls (right panel). Data is displayed as the mean ratio of tEGFR/tLNGFR T cells ± s.e.m. using n=3 biological replicates. Groups were compared using a paired two-tailed Student’s T test for accumulated differences between each time point. (g) Schematic overview of the 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 at a ~1:1 ratio with control or FASLG-KO tEGFR-1928ζ- ΔFAS CAR-T cells into Nalm6 B-ALL bearing NSG mice. (h) Representative FACS and (i) summary scatter plot comparing the ratio of tEGFR to tLNGFR cells at the time of infusion and four weeks following adoptive transfer. Symbols displayed as mean ± s.e.m. Groups compared using a two-sided Student’s T test. ns, not significant (P>0.05).