Abstract
Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule-cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell-mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid "shields" against self-destruction.
Highlights
We demonstrate that perforin binding to natural killer (NK) cell membranes is inhibited by them having high lipid ordering caused by the coalescence of high order lipid membrane domains [18,19,20]
NK cells form immunological synapses with target cells to initiate a series of tightly regulated downstream events that eventually release perforin and other cytolytic molecules into synaptic clefts [21]
Cytotoxic lymphocytes including NK cells and cytotoxic T lymphocyte (CTL) are fundamental to immune defense in large part through their ability to mediate the killing of target cells identified as being dangerous
Summary
NK cells are generally not susceptible to the released cytotoxic contents of their lytic granules Even though both the NK and the target cell membranes are exposed to perforin within the confined space of synaptic cleft, less than 5% of NK cells undergo autolysis during target engagement [9]. Several hypotheses have been proposed to explain this relative and potentially augmented resistance of NK cells during target engagement These include lytic granule– derived cell surface cathepsin B providing self-protection for cytotoxic lymphocytes [13]. We find that an aggressive breast cancer cell line utilizes the same strategy to evade NK cell–mediated killing by assembling a densely packed postsynaptic membrane to block perforin attack Disrupting these protective membrane packing mechanisms reverses NK cell protection against autolysis and, in the case of the breast cancer cell line, effectively switches it to an NK-susceptible state
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