Fig. 8. Targeting A_2AR by CRISPR/Cas9 enhances the in vivo efficacy of human anti-Lewis Y CAR T cells.

NSG mice were injected subcutaneously with 5 × 10⁶ OVCAR-3 tumor cells. Once tumors were established (15–20 mm²), mice were irradiated (1 Gy) and treated with 9 × 10⁶ anti-Lewis Y CAR T cells generated as per Fig. 6 on two subsequent days. Mice were then treated with 50,000U of IL-2 on days 0–4 post CAR T cell treatment. A Tumor growth, data shown are the mean ± SEM of 6–7 mice per group, ****p < 0.0001 per group, two-way ANOVA. B Survival determined as when tumors exceeded 80 mm². ****p < 0.0001 Log-Rank test. C, D Mice were bled at day 8 post treatment. C The number of human T cells per µl of blood was determined. Data shown are the mean ± SEM, **p < 0.01, ****p < 0.0001 paired t test. A–C n = 6 (nontreated) or 7 (mock sgRNA or A_2AR sgRNA) mice per group. D The phenotype of CD8⁺ CAR T cells in terms of CD62L and PD-1 expression. Data shown are concatenated from n = 7 per group. E, F. At day 14 post treatment, tumors were excised and the proportion of (E) CD8⁺ and (F) CD4⁺ CAR T cells expressing IFNγ, TNF, and Ki-67 was determined. Data shown are the mean ± SEM of 9 mice per group. *p < 0.05 paired t test. G Serum was collected at day 40 post treatment and the concentration of defined serum factors determined. Data shown as the mean ± SEM of n = 6 (nontreated, mock sgRNA) or 7 (A_2AR sgRNA) mice per group. H At the experimental endpoint, lungs, liver, and spleens were collected and sections analyzed by hematoxylin and eosin staining. One representative mouse of three per group is shown. A scale bar indicating a size of 200 µm is shown. Source data are provided as a Source Data file.