Fig. 3: CAR T cells engineered to express A1R under the control of the NR4A2 promoter exhibit enhanced CAR T cell function without loss of memory precursors. | Nature Communications

Fig. 3: CAR T cells engineered to express A1R under the control of the NR4A2 promoter exhibit enhanced CAR T cell function without loss of memory precursors.

From: Tumor site-directed A1R expression enhances CAR T cell function and improves efficacy against solid tumors

Fig. 3

A Cytokine secretion of anti-Lewis Y CAR T cells that were pre-conditioned in culture with DPCPX (100 nM) prior to co-culture with OVCAR-3 tumors, representative with mean ± SD of triplicate cultures. B CRISPR/HDR template with homology arms for the NR4A2 gene, to knock-in payload transgene under the control of the NR4A2 promoter. C RNA-seq counts per million (CPM) of ADORA1 gene after serial coculture with OVCAR-3 tumors, represented as the mean ± SD (D) Cytokine secretion and E intracellular cytokine staining in CAR T cells after serial coculture with OVCAR-3 tumors, represented as the mean ± SD of triplicate cultures. F CD69 and LAG3 expression in CD8+/CD4+ CAR T cells without tumor stimulation. G Quantification of %TSCM (CD45RO-CD45RA+CD27+CD62L+) in CD4+/CD8+ human anti-Lewis Y CAR T cells shown as pooled data of means ± SEM from 3 donors. H Expression of markers following coculture with OVCAR-3 tumor cells, represented as the mean ± SD of triplicate cultures. I Cytokine secretion by anti-HER2 CAR T cells against MCF-7 breast cancer after serial coculture, represented as the mean ± SD of triplicate cultures. J–K Differential accessibility peaks from ATAC-sequencing in J unstimulated or (K) OVCAR-3 tumor stimulated human CAR T cells. L HOMER motif analyses of enriched motifs in NR4A2/hA1R CAR T cells. M PCA plot for each round of coculture. N Quantificaiton of DEGs with each round of stimulation. O Magnitude of up or downregulated DEGs from the previously identified A1R signature in NR4A2/hA1R CAR T cells. Box-plot defined by box (1st and 3rd interquartile range), median-line, whiskers extending to points within 1.5x interquartile range. P–Q GSEA of gene-sets from mSigDB Hallmarks, KEGG and Sade-feldman gene signatures (GSE120575) for P hA1R vs Control or (Q) hA1R vs NR4A2KO CAR T cell groups after first stimulation with tumor cells. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. A, F, G, K, O One-way ANOVA or (D, E, H, I, L, M, N) two-way ANOVA. C, J–Q Human bulk ATAC-seq and RNA-seq was performed in independent experiments, 3 technical replicates each.

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