Fig. 3

Assessment of the re-challenge killing potential and long-term antitumor efficacy of CAR T cells against KKU-213A cells. A Schematic diagram of the re-challenge killing experiment. A20 CAR T cells were co-cultured with KKU-213A target cells at a 5:1 effector-to-target (E:T) ratio. Antigenic stimulation was performed every three days for a total of three cycles over a 9-day period. B Cytotoxicity percentages of A20 CAR4 T and A20 CAR5 T cells against target cells were assessed through a flow cytometry-based assay. C Timeline and schematic of the long-term antigen challenge, showing CAR T cell proliferation following co-culture with KKU-213A cells at an E:T ratio of 2:1 for 6 days. D Cytotoxicity in the long-term antigen challenge assay, assessed via crystal violet staining. E Percentage of cytotoxicity by A20 CAR4 T and A20 CAR5 T cells against target cells after prolonged antigen challenge on days 3 and 6. Data from three individual healthy donors are presented as the mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)