Figure 3.

FAP CAR activation-dependent stringent regulation of ML CAR expression and activity in multiplex engineered FAPCAR_ΔPD1_{ML CAR} UT cells

(A) Flow cytometry plots depicting FAP CAR and ML CAR expression in indicated UT cell groups, with or without human FAP (FAP) protein-mediated FAP CAR activation for 48 h. (B) Graph representing quantitation of percentage of ML CAR-positive viable T cells, as determined in (A). Bars show the means ± SD, n = 3 donors; p values determined by Student t test (two-tailed, unpaired). ns, not significant, ∗p ≤ 0.05. (C) Graph depicting kinetics of FAP CAR and ML CAR expression following FAP protein-mediated activation of FAP CAR on FAPCAR_ΔPD1_{ML CAR} UT cells at 0 h. Each data point represents mean ± SD, n = 3 donors. (D) Graph representing time course of FAP CAR and MLCAR expression upon FAP CAR stimulation and withdrawal of stimulus (FAP protein) from FAPCAR_ΔPD1_{ML CAR} UT cells. Each data point represents mean ± SD, n = 3 donors. (E) Schematic for assessing ML CAR cytotoxicity of indicated UT cells against ML⁺FAP⁻ NCI-H226-LUC tumor cells upon FAP CAR stimulation with FAP for 3 days, and subsequent withdrawal of stimulus (FAP protein) for 4 days. (F) Bar graph representing percentage ML⁺FAP⁻ NCI-H226-LUC tumor cell killing at different time points defined in (E). Cytotoxicity was measured 24 h post incubation of UT cells taken from indicated time points in (E) with target cells at Effector:Target ratio = 1:1. Bars show the means ± SD, n = 2 independent experiments; p values determined by Student t test (two-tailed, unpaired). ns, not significant, ∗p ≤ 0.05.