Fig. 3: Specific gene transcriptional signatures are used to assess CAR T cell functionality.

a Principal component analysis (PCA) of pseudo-bulked scRNA-seq data from the top 40 most represented CAR variants studied in Fig. 2. Also included are cells expressing 28z (CD28-CD3Z) and BBz (4-1BB-CD3Z) colored in red and CAR negative T cells (TCR-) colored in blue. To avoid batch effect variation, only data from Donor 3 is used. Overlayed over each data point, pie charts represent the enrichment of cells in CICs from Fig. 2f. b Expression levels of a set of 42 T cell marker genes across CD8+ pseudo-bulked scRNA-seq samples of a small panel of 10 different CAR T cell variants, 28z and BBz benchmark CAR T cells, TCR- T cells and unstimulated 28z CAR T cells. CAR variants were selected based on their distinct enrichment in CD8 CICs. Marker genes describe phenotypes such as memory, activation, cytotoxicity and exhaustion. Color indicates normalized gene expression deviation from average. Genes and samples are ordered by hierarchical clustering. On the top, a bar plot indicates the percentage of cells identified to be part of the two main CD8+ CICs. c Heatmap showing the difference in gene-set scores (score based on the simultaneous expression of different gene sets computed for each single cell) between the 40 most represented CAR variants, 28z and BBz CARs, unstimulated 28z CAR and CAR negative T cells, for 18 different gene sets. The mean score per variant is given as a fold change measurement when compared to 28z WT CAR.