FOXO1 is a master regulator of CAR T memory programming [scRNA_FOXO1_NGFR]

Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3–7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states. Single cell transcriptomic study of HER2.BBz CAR T cells overexpressing empty vector (NGFR) or FOXO1 which were isolated from Her2+ osteosarcoma tumors in NSG mice. 20,000 cells were collected per mouse with 5 total mice per condition. CAR T cells were generated from one human donor.

嵌合抗原受体T（CAR T）细胞的持久性不足，限制了其在B细胞恶性肿瘤与实体瘤中的治疗应用[1,2]。包括TCF7（蛋白名称TCF1）在内的记忆相关基因的表达，与患者的治疗应答及长期持久性密切相关[3-7]，这提示记忆程序与治疗疗效存在关联。本研究证实，先驱转录因子（pioneer transcription factor）FOXO1可促进人源CAR T细胞的记忆程序，并抑制其耗竭。在人源CAR T细胞中对内源FOXO1进行药物抑制或基因编辑，会降低记忆相关基因的表达，诱导耗竭样表型，并削弱其体外与体内的抗肿瘤活性。FOXO1过表达可诱导与T细胞记忆相符的基因表达程序，并增加FOXO1结合基序处的染色质可及性。在慢性刺激条件下，过表达FOXO1的细胞仍可维持功能、记忆潜能与代谢适配性，并在体内表现出更强的持久性与抗肿瘤活性。与之相反，过表达TCF1无法激活典型的记忆程序，也无法增强CAR T细胞的治疗效能。值得注意的是，内源FOXO1的活性与患者体内CAR T细胞及肿瘤浸润淋巴细胞（Tumor Infiltrating Lymphocyte, TIL）的应答相关，这凸显了其在癌症免疫治疗中的临床价值。本研究结果表明，通过FOXO1进行记忆重编程，可增强人源CAR T细胞的持久性与治疗效能，并证实了先驱因子——这类可结合致密染色质并诱导局部表观遗传重塑的因子——在优化治疗性T细胞状态中的应用潜力。本数据集为单细胞转录组研究，样本取自NSG小鼠体内的HER2.BBz CAR T细胞，这些细胞分别转染空载体（NGFR）或FOXO1过表达载体，并从HER2阳性骨肉瘤肿瘤中分离得到。每组实验共使用5只小鼠，每只小鼠收集20000个细胞；CAR T细胞由单名人源供体制备而成。