针对 T 细胞肿瘤的嵌合抗原受体 T (CAR-T) 细胞疗法仍然面临许多挑战，其中之一就是由于 CAR-T 细胞上表达的靶基因而造成的自相残杀。尽管如此，这些 CAR-T 细胞可以通过延长培养时间并有效消除恶性 T 细胞来进行体外扩增。然而，CAR-T 细胞在细胞亚群中存活的机制、所涉及的分子及其调控仍然未知。我们进行了单细胞转录组分析，以研究针对 T 细胞的自相残杀 CAR-T 产品（CD26 CAR-T 和 CD44v6 CAR-T），并以来自同一供体的针对 B 细胞的 CD19 CAR-T 作为对照。与 CD19 CAR-T 相比，自相残杀的 CAR-T 细胞没有表现出独特的细胞亚群，但具有更多的耗尽 T 细胞、更少的细胞毒性 T 细胞和更多的 T 细胞受体 (TCR) 克隆扩增。此外，我们观察到自相残杀的 CAR-T 细胞存活伴随着靶基因表达。基因表达结果表明，自相残杀的 CAR-T 细胞可能会下调其人类白细胞抗原 (HLA) 分子以逃避 T 细胞识别。单细胞调控网络分析和抑制实验表明，关键调控因素介导的耗竭可能有助于自相残杀的 CAR-T 细胞存活。这些数据共同为自相残杀的 CAR-T 细胞的生存提供了宝贵的首次见解。© 2024 作者。

Chimeric antigen receptor T (CAR-T) cell therapy targeting T cell tumors still faces many challenges, one of which is its fratricide due to the target gene expressed on CAR-T cells. Despite this, these CAR-T cells can be expanded in vitro by extending the culture time and effectively eliminating malignant T cells. However, the mechanisms underlying CAR-T cell survival in cell subpopulations, the molecules involved, and their regulation are still unknown. We performed single-cell transcriptome profiling to investigate the fratricidal CAR-T products (CD26 CAR-Ts and CD44v6 CAR-Ts) targeting T cells, taking CD19 CAR-Ts targeting B cells from the same donor as a control. Compared with CD19 CAR-Ts, fratricidal CAR-T cells exhibit no unique cell subpopulation, but have more exhausted T cells, fewer cytotoxic T cells, and more T cell receptor (TCR) clonal amplification. Furthermore, we observed that fratricidal CAR-T cell survival was accompanied by target gene expression. Gene expression results suggest that fratricidal CAR-T cells may downregulate their human leukocyte antigen (HLA) molecules to evade T cell recognition. Single-cell regulatory network analysis and suppression experiments revealed that exhaustion mediated by critical regulatory factors may contribute to fratricidal CAR-T cell survival. Together, these data provide valuable and first-time insights into the survival of fratricidal CAR-T cells.© 2024 The Authors.