PRDM1 knockout augments maintenance of a memory phenotype and cytokine polyfunctionality in multiple types of antitumor T cells. (A) Immunoblotting analysis of Blimp-1 expression in control or PRDM1-knockout 28z CAR-T cells targeting GD2. Representative blots of 3 experiments. (B-C) Surface expression of PD1, TIM-3, and LAG3 in CD19 or GD2-targeting 28z or BBz CAR-T cells with or without PRDM1 knockout was analyzed on day 10. Representative flow cytometry plots (B) and mean fluorescence intensity of each molecule (C) (n = 4, one-way ANOVA with multiple comparisons test). Experiments were repeated twice. **P < .01 compared with the control FMC63-28z CAR-T cells. (D-E) The frequency of T_SCM/T_CM (D) and IL2⁺IFNγ⁺TNFα⁺ cells (E) was analyzed in the CD8⁺ CAR-T cell population on day 11 (n = 3-4, unpaired 2-tailed Student t test). Representative data of 2 independent experiments. (F-I) NSG mice infused with GD2-expressing NALM6-GL were treated with the 14g2a^E101K CAR-T cells with or without PRDM1 knockout (n = 7 for each group). The frequency of human T cells in the PB was analyzed at the indicated time points (G) (unpaired 2-tailed Student t test). Tumor progression was monitored by in vivo bioluminescence imaging (H) (unpaired 2-tailed Student t test of the log-transformed values). Kaplan-Meier curve for the overall survival of mice (I) (n = 7, log-rank test). (J-L) T cells transduced with anti-mesothelin 28z CAR (clone ss1) or HLA-A2/MART1_27–35-specific TCR (clone DMF5) were ablated with PRDM1 and analyzed for memory markers (J) (n = 4, unpaired 2-tailed Student t test), fold expansion (K) (n = 4, unpaired 2-tailed Student t test), and cytokine production (L) (n = 7 for ss1 CAR-T cells and n = 4 for DMF5 TCR-T cells, unpaired 2-tailed Student t test) after 3 antigenic stimulations. (M) The frequency of PRDM1 knockout was evaluated in the ss1 CAR-T cells before and after stimulation (n = 4 samples). In C-E, G, H, and J-M, horizontal lines depict the mean values. NS, not significant.