PDF(Pubmed)
Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：由于各种因素，嵌合抗原受体（CAR）-T细胞对实体瘤的抗肿瘤作用有限，如低渗透率，扩展能力差，以及肿瘤内T细胞的耗尽。NR4A转录因子已被证明在小鼠T细胞耗竭中起重要作用。然而,每个NR4a因子对人T细胞分化的确切贡献仍有待阐明.
方法：在本研究中，我们删除了NR4A家族因子，NR4A1，NR4A2和NR4A3在人CAR-T细胞中通过使用CRISPR/Cas9系统识别人表皮生长因子受体2型（HER2）。我们通过反复共培养CAR-T细胞与Her2+A549肺腺癌细胞在体外诱导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细胞的抗肿瘤毒性。
结果：人NR4A-TKOCAR-T细胞对体外反复抗原刺激引起的耗竭具有抗性，与对照CAR-T细胞相比，在体外和体内均保持较高的肿瘤杀伤活性。NR4A单的有效性比较，双,TKO证明了三重KO在避免疲惫方面最有效。此外，在包括老年人在内的各种供体的T细胞中也观察到NR4ATKO的抗肿瘤作用的强烈增强。机械上,NR4ATKOCAR-T细胞显示线粒体氧化磷酸化增强，因此可以在肿瘤内持续更长的时间。
结论：NR4A因子通过线粒体基因表达调节CAR-T细胞的持久性和干性，因此NR4A是产生针对实体瘤的优异CAR-T细胞的非常有希望的靶标。
方法：在本研究中，我们删除了NR4A家族因子，NR4A1，NR4A2和NR4A3在人CAR-T细胞中通过使用CRISPR/Cas9系统识别人表皮生长因子受体2型（HER2）。我们通过反复共培养CAR-T细胞与Her2+A549肺腺癌细胞在体外诱导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细胞的抗肿瘤毒性。
结果：人NR4A-TKOCAR-T细胞对体外反复抗原刺激引起的耗竭具有抗性，与对照CAR-T细胞相比，在体外和体内均保持较高的肿瘤杀伤活性。NR4A单的有效性比较，双,TKO证明了三重KO在避免疲惫方面最有效。此外，在包括老年人在内的各种供体的T细胞中也观察到NR4ATKO的抗肿瘤作用的强烈增强。机械上,NR4ATKOCAR-T细胞显示线粒体氧化磷酸化增强，因此可以在肿瘤内持续更长的时间。
结论：NR4A因子通过线粒体基因表达调节CAR-T细胞的持久性和干性，因此NR4A是产生针对实体瘤的优异CAR-T细胞的非常有希望的靶标。
