Single-cell gene expression in CD19-specific and GD2-specific CAR T cells on day 10 in culture.

Purpose: To compare cell states between CD19-28z and GD2-28z human CAR T cells on day 10 of cell culture. Methods: Human T cells were activated and lentivirally transduced with CD19-28z or GD2-28z CAR constructs and maintained in culture for 10 days, and then delivered to the Stanford Functional Genomics Facility for 3' single-cell RNA-sequencing on the 10X Genomics platform. Results: Comparison of transcription factor profiles by single cell RNA-seq analysis of CD8+ T cells expressing CD19-28z vs. GD2-28z CAR confirmed that the bZIP family members JUN, JUNB, JUND, and ATF4 were among the most differentially expressed and broadly connected in exhausted GD2-28z CAR T cells. Conclusions: This study provides insights into cell states that could explain the underlying differences between highly functional CD19-28z CAR T cells and exhaustion-prone GD2-28z CAR T cells on day 10 in culture. Overall design: A total of 1,530 CAR T cells were sequenced. 804 CD19-28z CAR T cells were sequenced to an average of 350,587 reads per cell capturing a median of 12,675 unique molecular identifier (UMI) counts per cell mapping to a median of 2,990 unique genes per cell. 726 GD2-28z CAR T cells were sequenced to an average of 405,585 reads per cell capturing a median of 15,708 unique molecular identifier (UMI) counts per cell mapping to a median of 3,446 unique genes per cell.