Single-cell ATAC-seq maps the comprehensive and dynamic chromatin accessibility landscape of CAR-T cell dysfunction.

Chimeric antigen receptor T cells (CAR-T) therapy has achieved remarkable therapeutic success in treating a variety of hematopoietic malignancies. However, the high relapse rate and poor in vivo persistence, partially caused by CAR-T cell exhaustion, are still important barriers against CAR-T therapy. It remains largely elusive on the mechanisms of CAR-T exhaustion and how to attenuate exhaustion to achieve better therapeutic efficacy. In this study, we initially observed that CAR-T cells showed rapid differentiation and increased exhaustion after co-culture with tumor cells in vitro, and then performed single-cell ATAC-seq to depict the comprehensive and dynamic landscape of chromatin accessibility of CAR-T cells during tumor cell stimulation. Analyses of differential chromatin accessible regions and motif accessibility revealed that TFs were distinct in each cell type and reconstituted a coordinated regulatory network to drive CAR-T exhaustion. Furthermore, we performed scATAC-seq in patient-derived CAR-T cells and identified BATF and IRF4 as pivotal regulators in CAR-T cell exhaustion. Finally, knockdown of BATF or IRF4 enhanced the killing ability, inhibited exhaustion, and prolonged the persistence of CAR-T cells in vivo. Together, our study unraveled the epigenetic regulatory mechanisms of CAR-T exhaustion and provided new insights into CAR-T engineering to achieve better clinical treatment benefits.