Uncovering a New Cytotoxic Mechanism of Natural Killer Cells

揭示自然杀伤细胞的新细胞毒性机制

• 批准号: RGPIN-2019-05042
• 负责人: Haddad, Elie
• 金额: $ 2.33万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684477
• 关键词: Uncovering; New; Cytotoxic; Mechanism; Natural

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. **

自然杀伤(NK)细胞以其杀死受感染或恶性细胞的能力而闻名。多年来，根据传统检测方法获得的数据，几种恶性细胞被认为对NK细胞介导的杀伤具有抵抗力。以急性淋巴细胞白血病(ALL)细胞系为模型，我们证明了这种明显的耐药性相当于延迟杀伤，依赖于一种未被描述的细胞毒途径，该途径不涉及颗粒胞吐或死亡受体细胞毒途径。有趣的是，我们发现gp91Phox，经典地参与NADPH氧化酶复合体，是这种杀死所必需的，不同于NADPH氧化酶的其他成分。事实上，我们观察到穿孔素缺陷的NKAES和脱颗粒缺陷的NKAES可以有效地杀死所有细胞。此外，所有稳定转导FADD显性阴性的细胞仍被NKAES细胞杀死，FADD显性阴性阻止死亡受体凋亡信号。另一方面，gp91Phox缺陷的NKAES细胞并不能杀死所有细胞。虽然从未报道过NADPH氧化酶在NK细胞中表达，但我们的结果表明，尽管没有黄色素b558，gp91实际上在NK细胞中表达，这表明它可能参与了另一种复合体。此外，我们观察到NK细胞在所有杀伤过程中发生的代谢修饰与在经典细胞毒途径中观察到的非常不同。我们建议通过以下方法进一步破译这一新的细胞毒途径：*1.鉴定NK细胞代谢修饰并研究gp91在这一新的细胞毒途径中的作用。对NKAES细胞的转录和海马数据分析表明，新陈代谢是这一新的细胞毒途径的根源。为了了解代谢修饰在这一新的细胞毒途径中的意义，我们将研究面对ALL的NKAES细胞的OXPHOS和糖酵解。此外，我们的数据表明gp91在NK细胞中表达，但不在经典的NADPH氧化酶复合体中表达。我们将表征gp91及其伙伴的细胞定位。*2.鉴定NK细胞在靶细胞中诱导的死亡过程。我们的数据表明，NK细胞通过我们新的细胞毒途径介导的死亡是通过一种不需要caspase激活或线粒体去极化的机制发生的。我们将比较NKAES敏感和耐药靶点的转录组，并使用靶细胞系上的CRISPR文库来确定对这一新的细胞毒途径敏感的途径。*对这一新的细胞毒途径的了解将代表着NK细胞生物学的真正突破。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。**