Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies

Abstract

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 via extending culture time and effectively eliminating malignant T cells. However, the mechanisms underlying CAR-T cell survival from cell subpopulations, molecules and regulation are still unknown. We performed single-cell transcriptome profiling to investigate the fratricidal CAR-T products (CD26 CAR-T and CD44v6 CAR-T) targeting T cells, taking CD19 CAR-T targeting B cells from the same donor as a control. Compared with CD19 CAR-T, fratricidal CAR-T cells exhibit no unique cell subpopulation, but have more exhausted T cells, fewer cytotoxic T cells and more TCR clonal amplification. Furthermore, we observed that fratricidal CAR-T cell survival was accompanied by target gene expression. Gene expression results suggests that fratricidal CAR-T cells may downregulate their 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.