嵌合抗原受体（CAR）-T 细胞疗法的进展显着改善了复发或难治性血液恶性肿瘤患者的临床结果。然而，由于仅一小部分患者可以获得临床益处，因此进展仍然受到阻碍。对CAR-T细胞在单细胞水平上的体内行为缺乏了解阻碍了其在临床实践中更广泛的应用。越来越多的证据表明，单细胞测序技术可以帮助完善受体设计，指导基于基因的T细胞修饰，并优化CAR-T制造条件，所有这些对于长期免疫监视和更有利的临床结果至关重要。采用这些方法产生的信息也可能有助于我们理解决定治疗效果和毒性的众多复杂因素。在这篇综述中，我们讨论了 CAR-T 免疫疗法在临床实践中失败的原因，以及自单细胞测序技术里程碑以来该领域所学到的知识。我们进一步概述了单细胞分析在 CAR-T 免疫疗法中应用的最新进展。具体来说，我们概述了单细胞研究，重点关注靶抗原、CAR-转基因整合以及临床前研究和临床应用，然后讨论它将如何影响 CAR-T 细胞疗法的未来。© 2023。作者（ s）。

Advances in chimeric antigen receptor (CAR)-T cell therapy have significantly improved clinical outcomes of patients with relapsed or refractory hematologic malignancies. However, progress is still hindered as clinical benefit is only available for a fraction of patients. A lack of understanding of CAR-T cell behaviors in vivo at the single-cell level impedes their more extensive application in clinical practice. Mounting evidence suggests that single-cell sequencing techniques can help perfect the receptor design, guide gene-based T cell modification, and optimize the CAR-T manufacturing conditions, and all of them are essential for long-term immunosurveillance and more favorable clinical outcomes. The information generated by employing these methods also potentially informs our understanding of the numerous complex factors that dictate therapeutic efficacy and toxicities. In this review, we discuss the reasons why CAR-T immunotherapy fails in clinical practice and what this field has learned since the milestone of single-cell sequencing technologies. We further outline recent advances in the application of single-cell analyses in CAR-T immunotherapy. Specifically, we provide an overview of single-cell studies focusing on target antigens, CAR-transgene integration, and preclinical research and clinical applications, and then discuss how it will affect the future of CAR-T cell therapy.© 2023. The Author(s).