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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714563
• 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.

自然杀伤细胞（NK）以其杀死感染细胞或恶性细胞的能力而闻名。多年来，根据使用传统测定法获得的数据，人们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，与NADPH氧化酶的其他成分不同，gp91phox通常参与NADPH氧化酶复合物，是这种杀伤所必需的。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ALL细胞。此外，以fadd -显性阴性（可阻止死亡受体凋亡信号传导）稳定转导的ALL细胞仍被NKAES细胞杀死。另一方面，gp91phox缺失的NKAES细胞不能杀死ALL细胞。虽然NADPH氧化酶从未报道过在NK细胞中表达，但我们的研究结果表明，尽管没有黄细胞色素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物。此外，我们观察到NK细胞在ALL杀伤过程中发生的代谢改变与在经典细胞毒性途径中观察到的代谢改变非常不同。我们建议通过以下方式进一步解读这一新的细胞毒性途径：