The in vitro restimulation assay for measuring the persistence of CAR-T cells under chronic tumor exposure. (A) Schematic of experiment for modeling T cell persistence in vitro. (B) CAR-T cell expansion curve upon chronic tumor exposure. CAR-T cells (5 × 10⁵) were mixed with U87 cells (5 × 10⁵) in 24 wells. After eradication of tumor cells in 2 days, CAR-T cell numbers were counted and were added to fresh tumor cells for another round of tumor challenge (n = 3 biological replicates). (C) The exhaustion marker CD39 levels were measured by flow cytometry after each stimulation (n = 3 biological replicates). (D) The functional markers of CAR-T cells were measured after the sixth round of tumor challenge (n = 3 biological replicates). Data represent the mean ± SD from three independent experiments. Statistics by two-way ANOVA (B and C) or one-way ANOVA with Tukey’s multiple comparison test (D). *, P < 0.05; **, P < 0.01; N.S., no significance.