PRDM1 knockout maintains an early memory phenotype in CD19-directed CAR-T cells. (A) CD19-targeting CAR-T cells were electroporated with a Cas9/sgRNA ribonucleoprotein (RNP) complex against PRDM1 or electroporated without RNP (no target). The CAR-T cells were then stimulated thrice by K562-CD19 or NALM6. (B) Immunoblotting of Blimp-1 in control or PRDM1-knockout CAR-T cells generated from 3 different donors. (C) Fold expansion of T cells during the 3 stimulations (n = 11, paired 2-tailed Student t test). (D-F) Memory markers were analyzed in the CD8⁺ CAR-T cell population after the third stimulation. The data shown are representative flow cytometry plots of primary CD8⁺ T cells or CAR-T cells stimulated by K562-CD19 (D) and the frequency of CD45RA^+/−CD62L⁺CCR7⁺CD27⁺CD28⁺ cells (E) or CD45RA^+/−CD62L⁺CCR7⁺CD27⁺CD28⁺IL7R⁺ cells (F) in the CD8⁺ CAR-T cell population (n = 12 or 11, paired 2-tailed Student t test for each). (G) Knockout efficiency was evaluated by Inference of CRISPR Edits (ICE) analysis using the genome extracted before and after repeated stimulation (n = 11, repeated measures one-way ANOVA with multiple comparisons test). The data were obtained from different donor samples. Horizontal lines denote the mean values. NS, not significant.