T cell homeostasis and antitumor function require the Na+-K+-ATPase. T cell homeostasis and antitumor function require the Na+-K+-ATPase

The cancer-killing activity of T cells is often compromised within tumors, allowing disease progression. We previously found that intratumoral elevations in extracellular K+ constrain T cell antitumor function. Despite the relevance of K+abundance for T cell antitumor function and the importance of ion gradients for cellular physiology broadly, our understanding of T cell K+ transporters remains rudimentary7,8. Here, we report that the Na+-K+-ATPase is required for T cell quiescence, memory formation, and antitumor activity. Deletion of Atp1a1, the catalytic alpha subunit of the Na+-K+-ATPase, in CD8+ T cells induced constitutive activity in TCR, Akt-mTOR, and MAPK/ Erk signaling pathways. This state of tonic signal transduction was reflected in the acquisition of co-inhibitory surface receptors and terminal differentiation in T cells following Atp1a1 deletion. Mechanistically, we found that the Na+-K+-ATPase functions to support ROS homeostasis as its disruption produced ROS accumulation and the addition of antioxidants prevented the accelerated differentiation and acquisition of co-inhibitory receptors in T cells lacking Atp1a1. The in vivo behavior of T cells lacking Atp1a1 was also consistent with tonic signal transduction and stimulation-induced terminal differentiation. T cells lacking Atp1a1 could not achieve proliferative burst or form memory following pathogen challenge or perform tumor destruction in a syngeneic model of orthotopic murine melanoma. These results highlight the fundamental but underappreciated importance of monovalent ion transporters in T cell biology and have translational implications for the ongoing development of immune checkpoint blockade and T cell transfer therapies (i.e. CAR, TCR, TIL). Overall design: Comparitive gene expression profiling of RNA-seq data for C57BL/6 murine CD8+ T cells electroporated with CRISPR-Cas9 molecules complexed to sgRNAs encoding Atp1a1 specific or scramble sequences

T细胞的杀癌活性常在肿瘤微环境中受损，进而导致疾病进展。我们此前发现，肿瘤内细胞外钾离子（K+）水平升高会抑制T细胞的抗肿瘤功能。尽管钾离子丰度对T细胞抗肿瘤功能至关重要，且离子梯度对细胞生理整体意义重大，但目前我们对T细胞钾离子转运体的认知仍十分有限7,8。本研究证实，钠钾-ATP酶（Na+-K+-ATPase）对于T细胞的静息状态、记忆形成以及抗肿瘤活性均不可或缺。在CD8+ T细胞中敲除钠钾-ATP酶的催化α亚基Atp1a1后，T细胞受体（TCR）、Akt-mTOR以及MAPK/Erk信号通路会出现组成型激活。这种持续性信号转导状态，体现在Atp1a1敲除后T细胞表面共抑制性表面受体的获得以及终末分化过程中。从机制上看，我们发现钠钾-ATP酶可维持活性氧（ROS）稳态；一旦该酶被破坏，就会导致活性氧积累，而添加抗氧化剂则可阻断Atp1a1缺失T细胞的加速分化以及共抑制受体的获得过程。Atp1a1缺失T细胞的体内行为也与持续性信号转导及刺激诱导的终末分化相符：在病原体攻击实验中，这类T细胞无法实现增殖爆发或形成记忆细胞；在同源原位小鼠黑色素瘤模型中，它们也无法完成肿瘤杀伤任务。本研究结果凸显了单价离子转运体在T细胞生物学中基础却未被充分重视的重要性，其研究成果对当前免疫检查点阻断疗法以及T细胞过继转移疗法（即嵌合抗原受体（CAR）、T细胞受体（TCR）、肿瘤浸润淋巴细胞（TIL）疗法）的开发具有转化应用价值。整体实验设计：对C57BL/6小鼠CD8+ T细胞开展比较基因表达谱分析，这些细胞被电转染了与Atp1a1特异性或乱序单引导RNA（sgRNA）结合的CRISPR-Cas9复合物，随后进行RNA测序（RNA-seq）。