Single Cell Transcriptomic Characterization of CAR T-Cell Products Reveals Subpopulations, Stimulation and Exhaustion Signatures

Chimeric antigen receptor (CAR) T-cell adoptive therapy is set to transform the treatment of a rapidly expanding range of malignancies. Although the activation process of normal T cells is well characterized, comparatively little is known about the activation of cells via the CAR. Here we have used flow cytometry together with single cell transcriptome profiling to characterize the starting material (peripheral blood mononuclear cells –PBMCs-) and CAR therapeutic products of 3 healthy donors in the presence and absence of antigen specific stimulation. Bioinformatic analysis of the 57,676 single cell transcriptomes showed the CAR products to contain several subpopulations of cells, with the cellular composition reproducible from donor to donor, and all major cellular subsets compatible with CAR expression. Only 50% of CAR-expressing cells displayed transcriptional changes upon CAR-specific antigen exposure. The resulting molecular signature for CAR T-cell activation provides a rich resource for future dissection of the underlying mechanisms. Targeted data interrogation also revealed that a small proportion of antigen-responding CAR-expressing cells displayed an exhaustion signature, with both known markers and genes not previously associated with T-cell exhaustion. Comprehensive single cell transcriptomic analysis thus represents a powerful way to guide the assessment and optimization of clinical-grade CAR-T-cells, and inform future research into the underlying molecular processes. To characterize CAR-T cell products

嵌合抗原受体（Chimeric antigen receptor, CAR）T细胞过继免疫疗法即将革新日益增多的恶性肿瘤的治疗格局。尽管正常T细胞的激活过程已得到充分阐明，但学界对CAR介导的T细胞激活机制却知之甚少。本研究采用流式细胞术（flow cytometry）联合单细胞转录组谱分析（single cell transcriptome profiling），对3名健康志愿者在抗原特异性刺激存在与缺失状态下的起始样本（外周血单个核细胞，peripheral blood mononuclear cells, PBMCs）以及CAR治疗产品进行表征分析。对57676个单细胞转录组的生物信息学分析（bioinformatic analysis）显示，CAR治疗产品包含多个细胞亚群，其细胞组成在不同供体间具有良好可重复性，且所有主要细胞亚群均符合CAR表达的特征。仅50%的CAR阳性细胞在受到CAR特异性抗原刺激后出现转录组层面的改变。本研究获得的CAR T细胞激活分子特征谱，为后续解析其潜在作用机制提供了丰富的研究资源。靶向数据挖掘还发现，一小部分对抗原产生应答的CAR阳性细胞呈现出T细胞耗竭（T-cell exhaustion）特征谱，其中既包含已知的耗竭标志物，也涵盖了此前未被报道与T细胞耗竭相关的基因。因此，全面的单细胞转录组分析可作为一种有力工具，用于指导临床级CAR-T细胞产品的评估与优化，并为后续探索其潜在分子过程提供研究思路。本研究旨在表征CAR-T细胞产品。