γδ T细胞是强效的抗癌效应细胞，具有广泛靶向肿瘤的潜力，并且不依赖患者特异性新抗原或人类白细胞抗原背景。γδ T细胞能够感知在转化细胞中普遍存在的保守细胞应激信号，然而目前对有应激靶细胞的靶向机制尚未充分揭示。Vγ9Vδ2 T细胞是人类γδ T细胞中最丰富的亚群，能够识别含有丰富由肿瘤细胞产生的磷酸抗原激活的细胞表面蛋白复合物的蛋白2A1 (BTN2A1)和BTN3A1 (参考文献6-8)。本文中，我们结合基因组范围的CRISPR筛选，在靶癌细胞中鉴定了调控γδ T细胞杀伤作用和BTN3A细胞表面表达的通路。筛选结果显示了BTN3A在细胞表面的丰度受到多层次调控，并通过转录、翻译后修饰和膜转运等途径激活γδ T细胞。此外，对癌细胞进行不同的遗传干扰和破坏代谢通路的抑制剂的研究发现，特别是ATP生成过程的干扰，能够改变BTN3A水平。在代谢危机期间，BTN3A和BTN2A1的诱导依赖于AMP激活蛋白激酶 (AMPK)。最后，我们发现小分子激活AMPK在细胞系模型和患者源性肿瘤器官样体内可增加BTN2A1-BTN3A复合物的表达，并提高Vγ9Vδ2 T细胞受体介导的杀伤作用。这种AMPK依赖的代谢应激诱导的配体上调机制加深了我们对γδ T细胞应激监视的认识，并提出了增强γδ T细胞抗癌活性的新途径。© 2023. 作者（们），独家许可给 Springer Nature Limited。

γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1-5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells4-recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.© 2023. The Author(s), under exclusive licence to Springer Nature Limited.