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IMMUNOTHERAPY

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

Abstract

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.

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Fig. 1: The dynamic changes of CAR-T cell proliferation, differentiation, activation, and exhaustion after tumor cell stimulation.
Fig. 2: Chromatin accessibility landscape of CAR-T cells after tumor cells stimulation in vitro.
Fig. 3: The dynamic changes of chromatin accessibility and TF activities during CAR-T cells differentiation.
Fig. 4: The coordinated regulatory TFs network during CAR-T cell exhaustion.
Fig. 5: Chromatin accessibility landscape of CAR-T cells in multiple myeloma patients.
Fig. 6: CAR-T cells with BATF or IRF4 knockdown showed increased effector functions, persistence, and enhanced antitumor potency.

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Acknowledgements

This work was supported by grants from the National Key R&D Program of China, Stem Cell and Translation Research (2018YFA0109300), the Zhejiang Province Science Foundation for Distinguished Young Scholars (LR19H080001), the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2020R01006), and the National Natural Science Foundation of China (81870080, 91949115, 82000187, 81900176). Thanks for the technical support by the Core Facilities, Zhejiang University School of Medicine.

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PJ and PQ conceived the project and designed the experiments. PJ analyzed data and wrote the manuscript. ZZ conducted CAR-T cells with BATF and IRF4 knockdown and performed assays in vivo. ZL performed assays in vitro. YXH and XL were involved in patient sample acquisition. YLH, XZ, HZ, MZ, JD, TG provided cell culture and molecular biology assistance. HH and PQ supervised the overall project and co-wrote the manuscript. All authors contributed to reading and editing the manuscript.

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Correspondence to He Huang or Pengxu Qian.

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Jiang, P., Zhang, Z., Hu, Y. et al. Single-cell ATAC-seq maps the comprehensive and dynamic chromatin accessibility landscape of CAR-T cell dysfunction. Leukemia (2022). https://doi.org/10.1038/s41375-022-01676-0

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