A long-standing unknown is how an immune synapse disassembles. In this study, Srpan et al. show that shedding of CD16 promotes the detachment of NK cells from target cells to aid serial engagement of multiple targets and to sustain NK cell viability.

No abstract

Natural killer (NK) cells form immune synapses to ascertain the state of health of cells they encounter. If a target cell triggers NK cell cytotoxicity, lytic granules containing proteins including perforin and granzyme B, are secreted into the synaptic cleft inducing target cell death. Secretion of these proteins also occurs from activated cytotoxic T lymphocytes (CTLs) where they have recently been reported to complex with thrombospondin-1 (TSP-1) in specialized structures termed supramolecular attack particles (SMAPs). Here, using an imaging method to define the position of each NK cell after removal, secretions from individual cells were assessed. NK cell synaptic secretion, triggered by ligation of NKp30 or NKG2D, included vesicles and SMAPs which contained TSP-1, perforin, and granzyme B. Individual NK cells secreted SMAPs, CD63+ vesicles, or both. A similar number of SMAPs were secreted per cell for both NK cells and CTLs, but NK cell SMAPs were larger. These data establish an unexpected diversity in NK cell synaptic secretions.

Supplementary information Supplementary methodsPreparation of pooled plasma for CAT assays; Citrated blood tubes were simultaneously collected from 20 healthy donors. Corn trypsin inhibitor (CTI; Cambridge Biosciences) was immediately added to tubes at a final concentration of 18.3 µg/mL. The blood tubes were then centrifuged at 1800g for 30 minutes at RT, plasma was collected, equal volumes of plasma from each donor were pooled, the plasma was further centrifuged at 20000g for 30 minutes to pellet large pdEV and the resulting plasma was stored in 1ml single use aliquots at -80 o C.

Cytotoxic lymphocytes are critical in our immune defence against cancer and infection. Cytotoxic T lymphocytes and Natural Killer cells can directly lyse malignant or infected cells in at least two ways: granule-mediated cytotoxicity, involving perforin and granzyme B, or death receptor-mediated cytotoxicity, involving the death receptor ligands, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL). In either case, a multi-step pathway is triggered to facilitate lysis, relying on active pro-death processes and signalling within the target cell. Because of this reliance on an active response from the target cell, each mechanism of cell-mediated killing can be manipulated by malignant and infected cells to evade cytolytic death. Here, we review the mechanisms of cell-mediated cytotoxicity and examine how cells may evade these cytolytic processes. This includes resistance to perforin through degradation or reduced pore formation, resistance to granzyme B through inhibition or autophagy, and resistance to death receptors through inhibition of downstream signalling or changes in protein expression. We also consider the importance of tumour necrosis factor (TNF)-induced cytotoxicity and resistance mechanisms against this pathway. Altogether, it is clear that target cells are not passive bystanders to cell-mediated cytotoxicity and resistance mechanisms can significantly constrain immune cell-mediated killing. Understanding these processes of immune evasion may lead to novel ideas for medical intervention.

Interleukin 1β (IL-1β) plays a major role in inflammation and is secreted by immune cells, such as macrophages, upon recognition of danger signals. Its secretion is regulated by the inflammasome, the assembly of which results in caspase 1 activation leading to gasdermin D (GSDMD) pore formation and IL-1β release. During inflammation, danger signals also activate the complement cascade, resulting in the formation of the membrane attack complex (MAC). Here, we report that stimulation of LPS-primed human macrophages with sub-lytic levels of MAC results in activation of the NOD-like receptor 3 (NLRP3) inflammasome and GSDMD-mediated IL-1β release. The MAC is first internalized into endosomes and then colocalizes with inflammasome components; adapter protein apoptosis associated speck-like protein containing a CARD (ASC) and NLRP3. Pharmacological inhibitors established that MAC-triggered activation of the NLRP3 inflammasome was dependent on MAC endocytosis. Internalization of the MAC also caused dispersion of the trans-Golgi network. Thus, these data uncover a role for the MAC in activating the inflammasome and triggering IL-1β release in human macrophages.

The diverse origins, nanometre-scale and invasive isolation procedures associated with extracellular vesicles (EVs) mean they are usually studied in bulk and disconnected from their parental cell. Here, we used super-resolution microscopy to directly compare EVs secreted by individual human monocyte-derived macrophages (MDMs). MDMs were differentiated to be M0-, M1-or M2-like, with all three secreting EVs at similar densities following activation. However, M0-like cells secreted larger EVs than M1-and M2-like macrophages. Proteomic analysis revealed variations in the contents of differently sized EVs as well as between EVs secreted by different MDM phenotypes. Super resolution microscopy of single-cell secretions identified that the class II MHC protein, HLA-DR, was expressed on ∼40% of EVs secreted from M1-like MDMs, which was double the frequency observed for M0-like and M2like EVs. Strikingly, human macrophages, isolated from the resected lungs of cancer patients, secreted EVs that expressed HLA-DR at double the frequency and with greater intensity than M1-like EVs. Quantitative analysis of single-cell EV profiles from all four macrophage phenotypes revealed distinct secretion types, five of which were consistent across multiple sample cohorts. A sub-population of M1-like MDMs secreted EVs similar to lung macrophages, suggesting an expansion or recruitment of cells with a specific EV secretion profile within the lungs of cancer patients. Thus, quantitative analysis of EV heterogeneity can be used for single cell profiling and to reveal novel macrophage biology.

The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell–mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy–immunotherapy protocols.