The plasma membrane is composed of a lipid bilayer characterized by the asymmetrical distribution of phospholipids. Specifically, the acidic phospholipids phosphatidylserine (PS) and phosphatidylethanolamine form a negatively charged inner layer of the plasma membrane. This inner layer interacts with important intracellular signalling molecules, including Src family protein tyrosine kinases, which phosphorylate ITAM domains found on NK cell activation receptors and induce NK cell effector functions.

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Previous research has shown that during cell death or other physiological processes such as platelet activation, there is a loss of lipid asymmetry resulting in the exposure of the PS at the cell surface. Interestingly, NK cell activation has also been shown to result in externalization of PS at the cell surface. However, it is not known whether this event occurs due to NK cell death after activation or to directly regulate NK cell activity.

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Using a combination of flow cytometry and microscopy techniques, Wu and co-authors characterize how lipid scrambling of the cell membrane negatively regulates NK cell function independently of cell death.

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The authors first demonstrated that PS exposure as a result of lipid scrambling at the cell membrane positively correlates with NK cell activation. Incubation with target cells expressing ligands for the NK cell activating receptors CD244 (2B4) and CD226 (DNAM-1) increased PS exposure, while target cells with inhibitory receptor SLAMF7 reduced PS exposure. Further, the authors found no enhancement in PI staining, which indicates cell membrane permeability, demonstrating that lipid scrambling in NK cells is not associated with cell death. 

To investigate how inducing lipid scrambling would affect NK cell activation, the authors overexpressed either the wild type lipid scramblase TMEM16F or the calcium-hypersensitive D408G TMEM16F mutant in YT-S NK cells. As expected, overexpression of TMEM16F increased PS in presence of calcium or when activated by tumor cells expressing CD48, the ligand for 2B4, and this effect was higher when using the D408G TMEM16F mutant. These results demonstrate that enhanced lipid scramblase TMEM16F expression increases PS exposure in a calcium-dependent manner.

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Next, Wu et al. characterized the influence of lipid scrambling on NK cell cytotoxicity. Interestingly, increased lipid scrambling in NK cells by D408G TMEM16F expression actually reduced NK cell-mediated cytotoxicity against tumor target cells. Further, Wu et al. found that lipid scrambling also reduced protein tyrosine phosphorylation, calcium flux, and Erk activation when stimulated with anti-2B4 antibodies. Altogether this suggests that lipid scrambling negatively regulates NK cell-mediated cytotoxicity. 

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Since lipid scrambling regulated NK cell cytotoxicity, the authors hypothesized that this mechanism involves reduced activation receptor signaling. Analysis by flow cytometry demonstrated that lipid scrambling reduced expression of the 2B4 cell surface receptor by 50-70%. In addition, they found that NK cells expressing D408G TMEM16F also had lower levels of Src kinases Fyn and Lck localizing at the plasma membrane. 

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Prior to this paper, increased PS exposure in activated NK cells following a loss of lipid asymmetry had been previously described. However, these studies also observed increased PI staining or activated caspases, suggesting that PS exposure is indicative of eminent NK cell death. Here, Wu et al. show that PS exposure is not only associated with cell death, but it also appears to be an indicator of increased NK cell activation and negatively regulates NK cell function. As PS exposure has been observed during activation of T cells and B cells, the findings of this paper suggest that lipid scrambling may also negatively regulate function of other immune cells. 

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Understanding the mechanisms by which lipid scrambling regulates NK cell signalling and function could uncover novel pathways to prevent NK cell dysregulation in various disease states.

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