Fig. 2

Generation and characterization of A20 CAR4 T and A20 CAR5 T cells. A Schematic representations of lentiviral constructs encoding the A20 CAR4 and A20 CAR5 sequences. The original A20 CAR4 construct (upper panel) was modified by adding a cDNA sequence for an anti-PD-L1 scFv at the 3’ end, linked to an HA tag, resulting in the A20 CAR5 construct (lower panel). B Histogram plots showing CAR surface expression in primary human T cells detected using an anti-c-Myc antibody. C Summary of CAR surface expression data from five independent experiments. D Immunoblot analysis of anti-PD-L1 scFv in the supernatants of NT, A20 CAR4 T, and A20 CAR5 T cells cultured for 5 days was detected using an anti-HA tag antibody. E Anti-PD-L1 scFv secretion from A20 CAR5 T cells over time: supernatants were collected at 0, 24, 48, and 72 h. F Schematic representation of A20 CAR5 T cells secreting anti-PD-L1 scFv, which targets PD-L1 + CCA cells (KKU-213A). G PD-L1 expression on effector cells was evaluated after culture. H Anti-HA antibody staining was performed to demonstrate the binding of the secreted anti-PD-L1 scFv from A20 CAR5 T cells to PD-L1 on effector cells. I, J Flow cytometric histograms showing the binding of HA-tagged anti-PD-L1 scFv to KKU-213A cells. The results represent the mean ± standard error of the mean (SEM) from at least three independent experiments using blood samples from different healthy donors. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)