Fig. 2: A2AR knockdown enhances CAR T-cell effector function.
From: CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy
Anti-Her2 CAR T cells were generated as per Fig. 1 and, where indicated, cotransduced with shRNAs directed toward either A2AR or a nontargeting (scramble) shRNA control. NGFR+ CAR T cells were isolated through MACS isolation prior to downstream assays. A, B cAMP accumulation following stimulation of CAR T cells with indicated doses of NECA or adenosine. Where indicated, SCH58261 (SCH, 1 µM) was added as a control. Representative experiments of n = 2. B Data represented as mean ± SD of triplicate replicates. Statistical tests indicated for scramble shRNA vs. other groups, one-way ANOVA. C, D 1 × 105 CAR T cells were cultured with 1 × 105 E0771-Her2 cells for C 16 h or (D) 4 h and supernatants analyzed by CBA to detect IFNγ and TNF. Data are presented as the mean ± SD of triplicate/quadruplicate cultures from a representative experiment of n = 3. E Anti-Her2 CAR T cells were cocultured with 1 × 104 of indicated 51chromium-labeled tumor cells at the indicated effector:target ratios for 4 hours. Chromium release was detected by an automated gamma counter (Wallac Wizard 1470). Data are shown as the mean ± SD of triplicate cultures. Statistics: two-way ANOVA. A–E ****p < 0.0001, ***p < 0.01, *<p < 0.01 *p < 0.05. F Heatmap for expression of indicated genes determined by NanoString analysis. G CAR T cells were transduced as per Fig. 1, but at day 2 post transduction, maintained in IL-7 and IL-15 (10 ng/ml). 2 × 105 CAR T cells were then cultured with 1 × 105 E0771-Her2 tumor cells for 72 h with 1 × 105 tumor cells replaced every 24 h. CAR T cells were then analyzed for their expression of TIM-3 and Granzyme B. Data shown represent concatenated triplicated samples from a representative experiment of n = 2. Source data are provided as a Source Data file.
