Uncovering a New Cytotoxic Mechanism of Natural Killer Cells

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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.