Single-cell transcriptomic sequencing identifies the genes related to polyfunctionality of persistent CAR-T cells targeting B7-H3 (CD276) in mice bearing human neuroblastoma xenografts

To understand the molecular determinants of B12(VHH)-CAR-T polyfunctionality, we simultaneously measured the protein and RNA expression levels of 95 single CD4+ B12(VHH)-CAR-T cells isolated from IMR5 tumor-bearing mouse and re-stimulated with IMR5 tumor cells in vitro. We identified two CAR-T clusters, low polyfunctionality and high polyfunctionality subsets, in a 2D t-SNE plot. Moreover, our single-cell mRNA expression profiling revealed 32 genes that displayed statistically significant, concordant differences between the two cell subsets. Overall design: IMR5 tumor-bearing mice persistent B12(VHH)-CAR-T CAR T cells were stimulated with IMR5 tumor cells in vitro, then grouped into high-polyfunctionality and low-polyfunctionality subsets based on the cytokine secretion profile. Then scRNA-seq was used to analyze upregulated genes in each subset.