Blimp-1 KO CAR T cells successfully eliminate tumor cells in multiple rounds of cocultures. (A) Schematic of cell culture protocol for repeated challenges of Mock and Blimp-1 KO CAR T cells with GFP-expressing ARP-1 (ARP-1-GFP) cells. (B) Survival of ARP-1-GFP cells at the end of the consecutive cocultures, assessed by flow cytometry to detect GFP⁺ cells and displayed as fluorescence-activated cell sorting (FACS) plots. Control cells are ARP-1-GFP cells cultured without CAR T cells. (C) CAR T-cell expansion from before the first challenge (PRE) and after each of the 4 successive challenges (C1-C4), based on cell counts and displayed as the cumulative number of population doublings (n = 10). (D) Ratio of CD4⁺ and CD8⁺ cells at the end of the fourth challenge. (E) Efficiency of Blimp-1 deletion in KO CAR T cells before (pre) and after (post) repeated challenges measured by Inference of CRISPR Edits analysis to calculate percentage of cells containing indels in the Prdm1 gene. (F-G) Forward scatter (F) and side scatter (G) analysis of CD4⁺ and CD8⁺ CAR⁺ Mock and Blimp-1 KO CAR T cells. Shown are representative histograms (n = 8). (H) Total protein content of repeatedly challenged Mock and Blimp-1 KO CAR T cells, measured by Bradford assay. (I) De novo protein synthesis in CD4⁺ and CD8⁺ CAR⁺ Mock and Blimp-1 KO CAR T cells, as measured by the incorporation of O-propargyl-puromycin (OPP). Negative control is cells treated with cycloheximide (CHX). Shown are representative histograms (n = 2). Graphs show means ± SD. GFP, green fluorescent protein.