Fig. 5. Single-cell sequencing reveals specific distribution of CAR^High T cells.

Annotation and further analysis of CAR^High T in the single-cell data were performed by applying the gene signatures, developed in this work, associated to both CD4⁺ and CD8⁺ CAR^High T cells that showed higher correlation with the CAR protein level than that yielded by the CAR gene expression. (A) UMAP plot showing CAR^High T cell distribution across analyzed cells. (B) Quantification of the percentage of CAR^High T cell along the different clusters. In accordance with phenotypic results, CAR^High T cells are mainly localized within activated CD4⁺ cells (clusters 6 and 17) and pre-exhausted CD8⁺ T cells (cluster 9). (C) UMAP plots overlaid with the score of activation and tonic signaling signatures, showing their distribution across cells. CAR^High T cells were enriched in the score for both signatures. (D) Quantification of the signature score in CAR^High T cells from clusters 6 (CD4⁺ activated), 8 (CD8⁺ cytotoxic), and 9 (CD8⁺ pre-exhausted), in comparison with the rest of the cells, for both activation and tonic signaling signatures. CAR^High T cells presented a significant increase for both signatures in almost all three clusters analyzed. Wilcoxon test (D). ***P < 0.001.