PRDM1-knockout CAR-T cells maintain polyfunctional cytokine production after repeated stimulation. (A-B) The expression of immunoinhibitory molecules was analyzed after the third restimulation. Representative flow cytometry plots of primary CD8⁺ T cells or CAR-T cells repeatedly stimulated by K562-CD19 (A) and mean fluorescence intensity of PD1, LAG3, and TIM3 in the CD8⁺ CAR-T cell population (B) (n = 6, paired 2-tailed Student t test). (C) Expression of TOX in repeatedly stimulated CAR-T cells with or without PRDM1 knockout was analyzed by immunoblotting. (D-F) Cytokine production after the third stimulation was analyzed by flow cytometry (IL2, IFNγ, and TNFα). Representative flow cytometry plots of CAR-T cells restimulated by K562-CD19 (D), pie charts of the percentages of CAR-T cells producing single, double, and triple cytokines (E), and the frequency of IL2⁺IFNγ⁺TNFα⁺ cells in the CD8⁺ CAR-T cell population are shown (F) (n = 11, paired 2-tailed Student t test). The data presented in B and F are derived from different donor samples. Horizontal lines indicate the mean values. NS, not significant.