Fig. 2: Constitutive A₁R expression drives terminal differentiation of CAR T cells.

C57BL6-HER2 transgenic mice bearing orthotopic E0771-HER2 tumors were pre-treated with 4 Gy of total body irradiation prior to adoptive transfer of 2 doses of 10e⁶ anti-HER2 CAR T cells. 5 doses of 50,000 IU of IL-2 were injected i.p every 24 h. A Tumor growth curve, representative of 2 independent experiments with n = 4-6 mice per group. B CAR T cell counts in spleen and tumors from n = 5-6 mice except for non-treated group (n = 2 mice). Data represented as the mean ± SEM (A, B). C Quantification of %T_SCM (CD45RO^-CD45RA⁺CD27⁺CD62L⁺) in CD4⁺ and CD8⁺ NGFR⁺ human anti-Lewis Y CAR T cells shown as pooled data of means ± SEM from n = 4 independent healthy donors. D CD69 and LAG3 expression in human CAR T cells cultured with or without A₁R antagonist DPCPX (1 μM) in CD8⁺/CD4⁺ CAR T cells prior to tumor stimulation, representative of n = 4 donors. E Volcano plot of DEGs for unstimulated CAR T cells. F GSEA of DEGs enriched in A₁R CAR T cells using the KEGG and Sade-Feldman single-cell RNA-seq datasets (GSE120575). G Correlation of significant DEGs between tumor stimulated and unstimulated hA1R vs Ctrl CAR T cells. H CD8⁺ T cell meta-clusters from integrated atlas of single cell RNA-seq experiments across 21 tumor types and cells from 316 donors⁵⁶. Derived A₁R signature genes up or down-regulated are overlayed onto meta-clusters, I GSEA of up or down-regulated genes from each meta-cluster for unstimulated hA₁R CAR T cells vs control. J Quantification of gene expression in each meta-cluster GSEA for select core-enriched genes from the A₁R signature as defined by ref. ⁵⁶. K Heatmap of significant DEGs from the A₁R signature upregulated or downregulated with a logFC≥1 or logFC≤1 respectively between tumor stimulated and unstimulated hA₁R vs Ctrl CAR T cells. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. C paired two-sided t-test (B) One-way ANOVA or (A) two-way ANOVA. Human bulk RNA-seq was performed on a single donor with 3 technical replicates. F, I GSEA P-value estimation is based on an adaptive multi-level split Monte-Carlo scheme based off the fgsea package.