Fig. 4

Identification of high-confident hits and validation of their knockdown effect in enhancing CAR T cell-mediated tumor killing. A-C Comparing screening results in U87 MG cells that were co-cultured with B7-H3-targeting CAR T cells (A, this study) and with EGFR-targeting CAR T cells (B, Larson et al. [41]) revealed common hits showing CAR T enhancing phenotypes (C). D Knockdown of PI4KA, ATP6V1A, NDUFV1, UBA1 and ARPC4 in CRISPRi-U87 cells. The relative mRNA level of each targeted gene was calculated as the ratio of its expression in cells expressing a targeting sgRNA as compared to a non-targeting control sgRNA measured by qPCR. Error bars denote SEM. E Strategies for further characterization of the selected high-confident hits. F-J Tumor killing effect of B7-H3 CAR T cells against CRISPRi-U87 cells with PI4KA (F), ATP6V1A (G), NDUFV1 (H), ARPC4 (I) and UBA1 (J) knockdown after 2-day co-culture at E: T ratios of 1:1,1:2, and 1:4 (n = 3). Error bars denote SEM. K Tumor killing effect of B7-H3 CAR T cells against U87 MG cells treated with vehicle (Ctrl) or 5 nM PI4KA inhibitor GSK-A1 at E: T ratios of 1:1, 1:2 and 1:4. Data shown are the percentages of residual tumor cells after 2-day co-culture (n = 3). Error bars denote SEM. L Cell surface levels of B7-H3 in CRISPRi-U87 cells expressing the indicated sgRNAs stained with the B7-H3-PE antibody as assessed by flow cytometry. M Intracellular staining of granzyme B in CAR T cells after 24 h co-culture with the CRISPRi-U87 cells expressing the indicated sgRNAs as assessed by flow cytometry