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

研究目的：比较细胞培养第10天时，CD19-28z与GD2-28z人嵌合抗原受体T细胞（chimeric antigen receptor T cell, CAR T）的细胞状态。
研究方法：将人T细胞活化后，采用CD19-28z或GD2-28z CAR构建体进行慢病毒转导，继续体外培养10天，随后送至斯坦福功能基因组学实验室，依托10X Genomics平台开展3'端单细胞RNA测序。
研究结果：对表达CD19-28z与GD2-28z CAR的CD8+ T细胞进行单细胞RNA测序分析并比较转录因子谱，结果证实，在功能耗竭的GD2-28z CAR T细胞中，bZIP家族成员JUN、JUNB、JUND及ATF4为差异表达最显著且互作网络覆盖最广泛的基因之一。
研究结论：本研究揭示了细胞状态层面的特征，可解释培养第10天时，高功能CD19-28z CAR T细胞与易耗竭型GD2-28z CAR T细胞之间的内在差异。
整体实验设计：本次研究共对1530个CAR T细胞完成测序。其中804个CD19-28z CAR T细胞的平均测序深度为350587条reads/细胞，单细胞捕获的唯一分子标识符（unique molecular identifier, UMI）中位数为12675，可比对到的独特基因中位数为2990个。726个GD2-28z CAR T细胞的平均测序深度为405585条reads/细胞，单细胞捕获的UMI中位数为15708，可比对到的独特基因中位数为3446个。