Fig. 7: Targeting A_2AR by CRISPR/Cas9 enhances the effector function of human anti-Lewis Y CAR T cells.

Human anti-Lewis Y CAR T cells were generated and then treated with CRISPR/Cas9 and a sgRNA targeting either human A_2AR or a nontargeting control. CAR T cells were then cultured for 6–9 days prior to downstream assays. A INDEL frequency as determined by ICE analysis. Data represent the mean ± SEM of four different donors. B cAMP accumulation following stimulation with indicated doses of NECA. Where indicated, SCH58261 (SCH, 1 µM) was added as a control. Data represent the mean ± SD of triplicate samples. Data from a representative experiment of n = 2. C Proportion of CD8⁺ CAR T cells expressing a T_SCM CD45RA⁺CD62L⁺CD27⁺ phenotype. Left—representative experiment, Right—data represented as the mean ± SEM of four individual donors. Statistics determined using a paired t test. D, E. 2 × 10⁵ anti-Lewis Y CAR T cells were cocultured with MDA-MB-435 (Lewis Y negative, LeY⁻), OVCAR-3, or MCF7 cells in the presence or absence of NECA (10 µM) or SCH58261 (10 µM). After 8 h, supernatants were harvested and production of IFNγ, TNF, or MIP1α determined. Data shown are the mean ± SD of triplicate/quadruplicate cultures from n = 6 experiments, ****p < 0.0001, ***p < 0.001, **p < 0.01, two-way ANOVA. F Percentage suppression of IFNγ and TNF production mediated by NECA following anti-Lewis Y CAR T-cell coculture with OVCAR-3 tumor cells. ***p < 0.001, *p < 0.05 paired t test, n = 6 (TNF) or 7 (IFNγ) independent donors. G, H Anti-Lewis Y CAR T cells were stimulated with an anti-idiotype antibody (anti-LeY CAR) for 8 h in the presence or absence of 10 µM NECA and RNA analyzed by 3′RNA-Seq. G Principal component analysis based on top 100 most variable genes. H Gene set enrichment analyses for indicated pathways comparing anti-CAR-activated CAR T cells in the presence or absence of NECA (10 µM) for both nontargeting sgRNA and A_2AR sgRNA-edited CAR T cells. G, H Statistical tests were performed with indicated R packages as outlined in the “Methods”. I Heatmap of gene expression for pathways shown in (H). NT sgRNA—nontargeting sgRNA. Source data are provided as a Source Data file.