由于浸润率低、扩增能力差、肿瘤内T细胞耗竭等多种因素，嵌合抗原受体（CAR）-T细胞对实体瘤的抗肿瘤作用有限。 NR4A 转录因子已被证明在小鼠 T 细胞耗竭中发挥重要作用。然而，每个NR4a因子对人类T细胞分化的精确贡献仍有待阐明。在本研究中，我们在识别人类表皮生长因子受体类型的人类CAR-T细胞中删除了NR4A家族因子NR4A1、NR4A2和NR4A3 2 (HER2) 通过使用 CRISPR/Cas9 系统。我们通过将 CAR-T 细胞与 Her2 A549 肺腺癌细胞反复共培养，在体外诱导这些细胞的 T 细胞耗竭，并评估细胞表面标志物，如记忆和耗竭表型、增殖能力、细胞因子产生和代谢活性。我们通过将 CAR-T 细胞转移到 A549 荷瘤免疫缺陷小鼠体内，验证了 NR4A1/2/3 三重敲除 (TKO) CAR-T 细胞的体内抗肿瘤毒性。人 NR4A-TKO CAR-T 细胞对体外重复抗原刺激，与对照 CAR-T 细胞相比，在体外和体内均保持更高的肿瘤杀伤活性。对 NR4A 单击、双击和 TKO 效果的比较表明，三重 KO 在避免力竭方面最有效。此外，在包括老年人在内的各种供体的 T 细胞中也观察到 NR4A TKO 的抗肿瘤作用显着增强。从机制上讲，NR4A TKO CAR-T细胞表现出增强的线粒体氧化磷酸化，因此可以在肿瘤内持续更长时间。NR4A因子通过线粒体基因表达调节CAR-T细胞的持久性和干细胞性，因此NR4A是产生CAR-T细胞的非常有前途的靶点。针对实体瘤的优质 CAR-T 细胞。© 作者（或其雇主）2024。根据 CC BY-NC 允许重复使用。禁止商业再利用。请参阅权利和权限。英国医学杂志出版。

Antitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells within the tumor. NR4A transcription factors have been shown to play important roles in T-cell exhaustion in mice. However, the precise contribution of each NR4a factor to human T-cell differentiation remains to be clarified.In this study, we deleted NR4A family factors, NR4A1, NR4A2, and NR4A3, in human CAR-T cells recognizing human epidermal growth factor receptor type 2 (HER2) by using the CRISPR/Cas9 system. We induced T-cell exhaustion in these cells in vitro through repeated co-culturing of CAR-T cells with Her2+A549 lung adenocarcinoma cells and evaluated cell surface markers such as memory and exhaustion phenotypes, proliferative capacity, cytokine production and metabolic activity. We validated the antitumor toxicity of NR4A1/2/3 triple knockout (TKO) CAR-T cells in vivo by transferring CAR-T cells into A549 tumor-bearing immunodeficient mice.Human NR4A-TKO CAR-T cells were resistant against exhaustion induced by repeated antigen stimulation in vitro, and maintained higher tumor-killing activity both in vitro and in vivo compared with control CAR-T cells. A comparison of the effectiveness of NR4A single, double, and TKOs demonstrated that triple KO was the most effective in avoiding exhaustion. Furthermore, a strong enhancement of antitumor effects by NR4A TKO was also observed in T cells from various donors including aged persons. Mechanistically, NR4A TKO CAR-T cells showed enhanced mitochondrial oxidative phosphorylation, therefore could persist for longer periods within the tumors.NR4A factors regulate CAR-T cell persistence and stemness through mitochondrial gene expression, therefore NR4A is a highly promising target for the generation of superior CAR-T cells against solid tumors.© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.