Fig. 5. Targeting A_2AR by CRISPR/Cas9 enhances the effector function of both CD8⁺ and CD4⁺ CAR T cells.

Anti-Her2 CAR T cells were generated as per Fig. 4 following CRISPR/Cas9-mediated editing of CAR T cells with either an A_2AR sgRNA or a nontargeting sgRNA control. A–E 1.2 × 10⁶ CAR T cells were stimulated with anti-myc Tag (anti-CAR) for 5 h in the presence or absence of NECA (1 µM) and cells then isolated via FACS sorting into CD4⁺ and CD8⁺ subsets. Significantly modulated genes were defined based on a threshold of log₂FC (fold change) of ≥0.75 or ≤−0.75 and FDR < 0.05 > log² 0.75-fold change and FDR < 0.05. Statistical tests were performed with indicated R packages as outlined in the “Methods”. A CPMs for Adora2a gene in CD8⁺ and CD4⁺ CAR T cells before (control) and after (anti-CAR) activation. Data shown as mean ± SD of technical duplicates. B Principal component analysis based on top 100 most variable genes. C MA plot for genes increased or decreased by NECA in the indicated subsets. Significant genes are highlighted in green (increased by NECA) or blue (decreased by NECA). D Venn diagram showing the number of genes significantly modulated by NECA in each subset. E Heat maps for genes significantly suppressed by NECA in CD8⁺, CD4⁺, or both CD8⁺ and CD4⁺ CAR T cells. F, G. Anti-Her2 CAR T cells were generated as per Fig. 4G and CD8⁺ enrichment performed where indicated. Mice received one dose of 2 × 10⁷ anti-Her2 CAR T cells. F Flow cytometry analysis of CD8 and CD4 expression within the CAR T-cell population before and after CD8⁺ enrichment. G Tumor growth represented as the mean ± SEM of six (nontreated, A_2AR sgRNA CD8⁺-enriched CAR T cells), seven (non-targeting sgRNA, bulk CAR T cells, and non-targeting sgRNA, CD8⁺-enriched CAR T cells), or eight (A_2AR sgRNA, bulk CAR T cells) mice per group, n = 1 experiment. ***p < 0.001. Two-way ANOVA. Source data are provided as a Source Data file.