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. 2022 Jun 16;30(10):3155–3175. doi: 10.1016/j.ymthe.2022.06.006

Figure 8.

Figure 8

Effects of single CAR signaling and concomitant CAR/endogenous TCR co-signaling on molecular signature of SB-CD19-28z.CAR/TCR KO and SB-CD19-28zCAR/scrambled T cells

(A) Schematic representative of experimental design for analysis of single CAR-induced molecular signaling profile. A total of 3 × 106 SB-CD19-28z.CAR/TCR KO or SB-CD19-28z.CAR/scrambled T cells was stimulated with irradiated NALM6 cell (effector-to-target [E:T] = 1:1) for inducing CAR signaling for 24–48 h. As a negative control of resting state, CD19 CAR T cells were cultured in the absence of stimulus. (B) Quantitative analysis of ZAP70, PLCγ1, and p38 protein phosphorylation in CAR T cells after 24 h of stimulation. (C) Quantitative analysis of RNA expression profiling of selected genes associated with the PI3K signaling pathway, apoptosis, metabolism, and cell proliferation in SB-CD19-28z.CAR/TCR KO and SB-CD19-28z.CAR/scrambled T cells at 48 h post-stimulation. (D) Schematic representative of experimental design for analysis of simultaneous CAR/endogenous TCR-induced molecular signaling profile. A total of 3 × 106 SB-CD19-28z.CAR/TCR KO or SB-CD19-28z.CAR/scrambled T cells was stimulated with irradiated NALM6 cell (E:T = 1:1) and anti-CD3 antibody (bead:cell = 1:1) for inducing CAR and endogenous TCR co-signaling for 24–48 h. As a negative control of resting state, CD19 CAR T cells were cultured in the absence of stimulus. (E) Quantitative analysis of ZAP70, PLCγ1, and p38 protein phosphorylation in CAR T cells after 24 h of stimulation. (F) Quantitative analysis of RNA expression profiling of selected genes associated with the PI3K signaling pathway, apoptosis, metabolism, and cell proliferation in SB-CD19-28z.CAR/TCR KO and SB-CD19-28z.CAR/scrambled T cells at 48 h post-stimulation. Differentially expressed genes (DEGs) are defined as the genes showing p < 0.05 for differential expression. Data are shown as means ± SEM (n = 3 biologically independent donors); Student’s t test; n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.