Uncovering a New Cytotoxic Mechanism of Natural Killer Cells

自然杀伤（NK）细胞以其杀死感染或恶性细胞的能力而闻名。多年来，基于使用传统测定法获得的数据，认为几种恶性细胞对NK细胞介导的杀伤具有抗性。使用急性淋巴细胞白血病（ALL）细胞系作为模型，我们证明了这种明显的耐药性是相当延迟的杀伤依赖于一个未描述的细胞毒性途径，不涉及颗粒胞吐或死亡受体细胞毒性途径。有趣的是，我们发现，gp91phox，经典参与NADPH氧化酶复合物，是必要的这种杀伤，不像其他组件的NADPH氧化酶。事实上，我们观察到穿孔素缺陷型NKAES和脱颗粒缺陷型NKAES可以有效地杀死ALL细胞。此外，用FADD显性阴性稳定转导的ALL细胞（其阻止死亡受体凋亡信号传导）仍然被NKAES细胞杀死。另一方面，gp91phox缺陷型NKAES细胞不能杀死ALL细胞。虽然NADPH氧化酶从未被报道在NK细胞中表达，但我们的结果表明，尽管没有黄细胞色素b558，但gp91实际上在NK细胞中表达，这表明它可能参与替代复合物。此外，我们观察到在ALL杀伤过程中NK细胞中发生的代谢修饰与经典细胞毒性途径中观察到的代谢修饰非常不同。我们建议通过以下方式进一步解释这种新的细胞毒性途径：*1。鉴定NK细胞代谢修饰并研究gp91在这种新的细胞毒性途径中的作用。对NKAES细胞的转录组学和SeaHorse数据分析表明，代谢是这种新的细胞毒性途径的根源。为了了解这种新的细胞毒性途径中代谢修饰的含义，我们将研究NKAES细胞面对ALL时的氧化磷酸化和糖酵解。此外，我们的数据表明，gp91在NK细胞中表达，但不是在经典的NADPH氧化酶复合物。我们将描述gp91及其伙伴的细胞定位。2. NK细胞诱导的靶细胞死亡过程的鉴定。我们的数据表明，NK细胞介导的死亡通过我们的新的细胞毒性途径发生的机制，不需要半胱天冬酶激活或线粒体去极化。我们将比较NKAES敏感和耐药靶标的转录组，并将在靶细胞系上使用CRISPR文库来鉴定对这种新的细胞毒性途径敏感的途径。* 对这种新的细胞毒性途径的理解将代表NK细胞生物学的真实的突破。我们的初步数据和我们在NK细胞生物学方面的专业知识使我们的团队处于实现目标的战略地位。**

Natural Killer (NK) cells are known for their ability to kill infected or malignant cells. For many years, based on data obtained using traditional assays, several malignant cells were considered to be resistant to NK-cell mediated killing. Using acute lymphoblastic leukemia (ALL) cell lines as a model, we demonstrated that this apparent resistance was rather delayed killing relying on an undescribed cytotoxic pathway that does not involve granule exocytosis or the death receptors cytotoxic pathways. Interestingly, we found that gp91phox, classically involved in the NADPH oxidase complex, is necessary for this killing, unlike other components of the NADPH oxidase. Indeed, we observed that perforin-deficient NKAES and degranulation-deficient NKAES could kill efficiently ALL cells. Also, ALL cells stably transduced with FADD-dominant negative, which prevents death receptor apoptotic signaling, were still killed by NKAES cells. On the other hand, gp91phox-deficient NKAES cells could not kill ALL cells. Although NADPH oxidase has never been reported to be expressed in NK cells, our results indicate that gp91 is actually expressed in NK cells despite the absence of the flavocytochrome b558, suggesting that it could be involved in an alternative complex. In addition, we observed that metabolic modifications occurring in NK cells during ALL killing were very different from the ones observed during classical cytotoxic pathways. We propose to further decipher this new cytotoxic pathway by: ***1. Identifying NK cell metabolism modifications and studying the role of gp91 during this new cytotoxic pathway. Transcriptomic and SeaHorse data analyses on NKAES cells suggest that metabolism is at the root of this new cytotoxic pathway. To understand the implication of metabolic modifications in this new cytotoxic pathway, we will study the oxphos and glycolysis of NKAES cells facing ALL. Moreover, our data suggest that gp91 is expressed in NK cells but not in the classical NADPH oxidase complex. We will characterize the cell localization of gp91 and its partners.***2. Identification of the NK-cell induced death processes in target cells. Our data suggest that death mediated by NK cells via our new cytotoxic pathway occurs through a mechanism that does not require caspase activation or mitochondrial depolarization. We will compare the transcriptome of NKAES-sensitive and resistant targets, and we will use CRISPR library on target cell lines to identify the pathways responsible for sensitivity to this new cytotoxic pathway. ***The understanding of this new cytotoxic pathway would represent a real breakthrough in NK cell biology. Our preliminary data and our expertise in NK cell biology position our team in a strategic situation to achieve our goals. **