Despite a high response rate in chimeric antigen receptor (CAR) T cell therapy for acute lymphocytic leukaemia (ALL)^1,2,3, approximately 50% of patients relapse within the first year^4,5,6, representing an urgent question to address in the next stage of cellular immunotherapy. Here, to investigate the molecular determinants of ultralong CAR T cell persistence, we obtained a single-cell multi-omics atlas from 695,819 pre-infusion CAR T cells at the basal level or after CAR-specific stimulation from 82 paediatric patients with ALL enrolled in the first two CAR T ALL clinical trials and 6 healthy donors. We identified that elevated type 2 functionality in CAR T infusion products is significantly associated with patients maintaining a median B cell aplasia duration of 8.4 years. Analysis of ligand–receptor interactions revealed that type 2 cells regulate a dysfunctional subset to maintain whole-population homeostasis, and the addition of IL-4 during antigen-specific activation alleviates CAR T cell dysfunction while enhancing fitness at both transcriptomic and epigenomic levels. Serial proteomic profiling of sera after treatment revealed a higher level of circulating type 2 cytokines in 5-year or 8-year relapse-free responders. In a leukaemic mouse model, type 2^high CAR T cell products demonstrated superior expansion and antitumour activity, particularly after leukaemia rechallenge. Restoring antitumour efficacy in type 2^low CAR T cells was attainable by enhancing their type 2 functionality, either through incorporating IL-4 into the manufacturing process or by priming manufactured CAR T products with IL-4 before infusion. Our findings provide insights into the mediators of durable CAR T therapy response and suggest potential therapeutic strategies to sustain long-term remission by boosting type 2 functionality in CAR T cells.

尽管嵌合抗原受体 （CAR） T 细胞疗法治疗急性淋巴细胞白血病 （ALL） 的反应率很高^1,2,3，但大约 50% 的患者在第一年内复发^4,5,6，这是细胞免疫治疗下一阶段亟待解决的问题。在这里，为了研究超长 CAR T 细胞持久性的分子决定因素，我们从 695,819 个输注前 CAR T 细胞的基础水平或 CAR 特异性刺激后获得了单细胞多组学图谱，这些细胞来自前两项 CAR T ALL 临床试验中入组的 82 名儿科患者和 6 名健康供体。我们发现 CAR T 输注产品中 2 型功能升高与维持中位 B 细胞再生障碍持续时间为 8.4 年的患者显著相关。配体-受体相互作用的分析表明，2 型细胞调节功能失调的亚群以维持全群稳态，并且在抗原特异性激活过程中添加 IL-4 可缓解 CAR T 细胞功能障碍，同时增强转录组和表观基因组水平的适应性。治疗后血清的连续蛋白质组学分析显示，5 年或 8 年无复发反应者的循环 2 型细胞因子水平较高。在白血病小鼠模型中，2 型^高 CAR T 细胞产物表现出优异的扩增和抗肿瘤活性，尤其是在白血病再次发作后。通过增强 2 型^低 CAR T 细胞的 2 型功能，可以通过将 IL-4 掺入制造过程或在输注前用 IL-4 引发制造的 CAR T 产品来恢复其抗肿瘤功效。 我们的研究结果为持久 CAR T 疗法反应的介质提供了见解，并提出了通过增强 CAR T 细胞中的 2 型功能来维持长期缓解的潜在治疗策略。