Functional characterization of CD70-CAR T cells after repeated antigen stimulation in vitro. CD70-CAR/NT T cells and AML cells (Molm-13) were cocultured for 5 days, and fresh Molm-13 cells were repeatedly added every 5 days thereafter. The absolute cell count of tumor and T cells at the end of each coculture was determined by flow cytometry with CountBright counting beads. (A) The absolute cell count of Molm-13 cells at the end of each coculture. (B) The absolute cell count of CD70-CAR/NT T cells at the end of each coculture. (C-D) Cytokine production by CD70-CAR and NT T cells after antigen stimulation was determined with the multiplex detection assay. Data show the quantitative determination of TH1/TC1– and TH2/TC2–associated cytokines 24 hours after the first (C) and second (D) antigen stimulation. Data are the mean of results from 4 different donors. P values indicating the statistical significance for secreted cytokines of CD27z-CAR-T cells in comparison with all other constructs and NT T cells were calculated by 2-way ANOVA. (E-F) Expression of LAG-3, TIM-3 and PD-1, markers associated with T-cell exhaustion on CD70-CAR T cells after antigen stimulation. Data are the mean frequency ± standard deviation (SD) of triple-negative (E) and triple-positive (F) T cells from 3 different donors 72 hours after the third antigen stimulation. The error bars indicate the SD. **P < .01; ***P < .001; ****P < .0001; ns, not significant, by unpaired Student t test.