Regnase-1 deletion improves human CAR-T-cell expansion and function. (A) Editing efficacy of candidate Regnase-1 targeting sgRNAs in human CD19 CAR-T cells as determined by targeted deep sequencing. (B) Western blot analysis of Regnase-1 in hCD19 CAR-T cells 24 hours after ribonucleoprotein (RNP) transfection. (C-F) Human WT and Regnase-1 KO CAR-T cells were cocultured with irradiated Raji cells at a 1:2 E:T ratio. CAR-T cells were harvested and counted 7 days later, followed by restimulation with irradiated Raji cells. Phenotypic analysis was performed 24 hours after stimulation. Data are shown as mean plus or minus SEM and represent 3 independent experiments with CAR-T cells from 3 donors targeted with sgRNA-1. Similar results were obtained with sgRNA-3. (C) Number of CD8⁺ and CD4⁺ WT and Regnase-1 KO CAR-T cells or untransduced donor T cells after each round of stimulation. (D) Proportion of CD25⁺CD8⁺ and CD4⁺ CAR-T cells after each stimulation. (E) Proportion of TNFα⁺, IL-2⁺, IFNγ⁺, and granzyme B⁺CD8⁺ and CD4⁺ CAR-T cells after 3 rounds of stimulation. (F) Proportion of CCR7⁺CD8⁺ and CD4⁺ CAR-T cells after 3 rounds of stimulation. (G-J) NSG mice bearing Raji tumors were treated with untransduced human T cells, or with activated CD8⁺ and CD4⁺ (1:1) Regnase-1 KO or WT CAR-T cells. (G) Tumor growth monitored by bioluminescence imaging. Solid lines represent means and dotted lines represent individual mice. Data were pooled from 2 independent experiments. (H) CD8⁺ and CD4⁺ CAR-T-cell number in spleen and bone marrow 7 and 35 days after transfer. (I) Percentage of CD8⁺ and CD4⁺ CAR-T cells in blood at indicated time points. (J) Percentage of TCF-1⁺ CD8⁺ and CD4⁺ CAR-T cells in spleen and bone marrow 35 days after transfer. Significance was determined by multiple Student t test with Holm-Sidak correction for multiple comparisons (C-E), unpaired Student t test (F), or Mann-Whitney test (H-J). Data are shown as mean plus or minus SEM and represent 2 independent experiments with 4 to 5 mice per group (H-J). *P < .05; **P < .01; ***P < .001; ****P < .0001.