Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins.

B cell antigen receptor (BCR) signaling induces actin cytoskeleton remodeling by stimulating actin severing, actin polymerization, and the nucleation of branched actin networks via the Arp2/3 complex. This enables B cells to spread on antigen-bearing surfaces in order to increase antigen encounters and to form an immune synapse (IS) when interacting with antigen-presenting cells (APCs). Although the WASp, N-WASp, and WAVE nucleation-promoting factors activate the Arp2/3 complex, the role of WAVE2 in B cells has not been directly assessed. We now show that both WAVE2 and the Arp2/3 complex localize to the peripheral ring of branched F-actin when B cells spread on immobilized anti-Ig antibodies. The siRNA-mediated depletion of WAVE2 reduced and delayed B cell spreading on immobilized anti-Ig, and this was associated with a thinner peripheral F-actin ring and reduced actin retrograde flow compared to control cells. Depleting WAVE2 also impaired integrin-mediated B cell spreading on fibronectin and the LFA-1-induced formation of actomyosin arcs. Actin retrograde flow amplifies BCR signaling at the IS, and we found that depleting WAVE2 reduced microcluster-based BCR signaling and signal amplification at the IS, as well as B cell activation in response to antigen-bearing cells. Hence, WAVE2 contributes to multiple actin-dependent processes in B lymphocytes.

TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation.

TIGIT is an inhibitory receptor expressed on lymphocytes and can inhibit T cells by preventing CD226 co-stimulation through interactions in cis or through competition of shared ligands. Whether TIGIT directly delivers cell-intrinsic inhibitory signals in T cells remains unclear. Here we show, by analysing lymphocytes from matched human tumour and peripheral blood samples, that TIGIT and CD226 co-expression is rare on tumour-infiltrating lymphocytes. Using super-resolution microscopy and other techniques, we demonstrate that ligation with CD155 causes TIGIT to reorganise into dense nanoclusters, which coalesce with T cell receptor (TCR)-rich clusters at immune synapses. Functionally, this reduces cytokine secretion in a manner dependent on TIGIT's intracellular ITT-like signalling motif. Thus, we provide evidence that TIGIT directly inhibits lymphocyte activation, acting independently of CD226, requiring intracellular signalling that is proximal to the TCR. Within the subset of tumours where TIGIT-expressing cells do not commonly co-express CD226, this will likely be the dominant mechanism of action.

"Africans, we know how to adapt indeed": Adaptations to family planning and reproductive health services in humanitarian settings in Nigeria during the COVID-19 pandemic.

On March 30, 2020, the Government of Nigeria implemented its first COVID-19 related lockdown. We worked with two humanitarian projects in Nigeria, the Integrated Humanitarian Assistance to Northeast Nigeria (IHANN II) in Borno State and the United Nations High Commissioner for Refugees South-South Health and Nutrition Intervention (UNHCR-SS-HNIR) for Cameroon Refugees and vulnerable populations in Cross River State, to document the programmatic adaptations to Family Planning/Reproductive Health (FP/RH) services in response to COVID-19 and identify successes and challenges of those adaptations. A mixed methods approach including quantitative analysis of data from routine programmatic activities, qualitative data from in-depth interviews (IDIs) with project staff and process documentation of programmatic activities and modifications was used to 1) identify modifications in FP/RH services due to COVID-19, 2) understand staff perception of their utility and impact, and 3) gauge trends in key FP/RH in-service delivery indicators to assess changes prior to and after the March 2020 lockdown. Monitoring data shows notable declines in service utilization after lockdowns in antenatal care, postnatal care, and outreach campaigns, followed by a return to pre-lockdown levels by July 2020. Results show projects introduced numerous COVID-19 precaution strategies including: community sensitization; triage stations and modification of service flow in facilities; and appointment scheduling for essential services. Findings from IDIs speak to a well-coordinated and implemented COVID-19 response with project staff noting improvements in their time management and interpersonal communication skills. Lessons learned included the need to better sensitize and educate communities, maintain FP commodities and increase support provided to health workers. Deliberate adaptations in IHANN II and UNHCR-SS-HNIR projects turned challenges to opportunities, ensuring continuity of services to the most vulnerable populations.

Analyzing Single Cell Secretions by "Shadow Imaging".

Here, we describe a method, which we term "shadow imaging," to analyze the secretions of individual cells at immune synapses or other cell contacts. Following immune synapse formation and cellular activation on ligand-rich slides, the position of each cell is recorded using a pulsed immunofluorescence stain against the proteins on the ligand-rich slide surface. The pulsed stain does not penetrate the synaptic cleft, resulting in an unlabeled region or "shadow" beneath cells that is retained following cellular detachment. The secreted components, such as perforin, exosomes, or other types of extracellular vesicles, are retained on the slide and can be analyzed on a single-cell basis using immunofluorescence. The ability to identify single cells secreting different combinations of particles, proteins, and vesicles enables us to better understand the heterogeneity in immune cell secretions and can be used as a novel approach for phenotyping cell populations.

Four reasons for adopting a life course approach to health in the COVID-19 era and beyond.

The life course approach effectively responds to pressing health needs and fills critical gaps to improve health outcomes in the era of COVID-19 and beyond. This article outlines four main reasons to adopt and implement the life course approach in public health at national and local levels: (i) the approach effectively responds to new health trends and evidence, (ii) it fills longstanding gaps in care, (iii) it best addresses health inequities, and (iv) it can help achieve more with less.

El enfoque del curso de vida da respuestas eficaces a las urgentes necesidades de salud y salva brechas críticas para mejorar los resultados de salud en la era de la COVID-19 y los años posteriores. En este artículo se describen las cuatro razones principales para adoptar y poner en práctica el enfoque del curso de vida en la salud pública a nivel local y nacional: este enfoque (i) da respuestas eficaces a las tendencias y la evidencia nuevas en el ámbito de la salud, (ii) salva brechas de larga data en la atención, (iii) aborda de la mejor manera posible las inequidades de salud, y (iv) puede contribuir a lograr más con menos recursos.

A abordagem de curso de vida responde de forma efetiva a necessidades urgentes de saúde e preenche lacunas críticas para melhorar os resultados de saúde na era da COVID-19 e mais além. Este artigo descreve quatro motivos principais para adotar e implementar a abordagem de curso de vida na saúde pública em nível nacional e local: (i) a abordagem responde de forma efetiva a novas evidências e tendências em saúde, (ii) preenche lacunas antigas nos cuidados de saúde, (iii) lida melhor com iniquidades em saúde e (iv) pode ajudar a conquistar mais com menos recursos.

Four reasons for adopting a life course approach to health in the COVID-19 era and beyond

[ABSTRACT]. The life course approach effectively responds to pressing health needs and fills critical gaps to improve health outcomes in the era of COVID-19 and beyond. This article outlines four main reasons to adopt and implement the life course approach in public health at national and local levels: (i) the approach effectively responds to new health trends and evidence, (ii) it fills longstanding gaps in care, (iii) it best addresses health inequities, and (iv) it can help achieve more with less.

[RESUMEN]. El enfoque del curso de vida da respuestas eficaces a las urgentes necesidades de salud y salva brechas críticas para mejorar los resultados de salud en la era de la COVID-19 y los años posteriores. En este artículo se describen las cuatro razones principales para adoptar y poner en práctica el enfoque del curso de vida en la salud pública a nivel local y nacional: este enfoque (i) da respuestas eficaces a las tendencias y la evi- dencia nuevas en el ámbito de la salud, (ii) salva brechas de larga data en la atención, (iii) aborda de la mejor manera posible las inequidades de salud, y (iv) puede contribuir a lograr más con menos recursos.

[RESUMO]. A abordagem de curso de vida responde de forma efetiva a necessidades urgentes de saúde e preenche lacunas críticas para melhorar os resultados de saúde na era da COVID-19 e mais além. Este artigo descreve quatro motivos principais para adotar e implementar a abordagem de curso de vida na saúde pública em nível nacional e local: (i) a abordagem responde de forma efetiva a novas evidências e tendências em saúde, (ii) preenche lacunas antigas nos cuidados de saúde, (iii) lida melhor com iniquidades em saúde e (iv) pode ajudar a conquistar mais com menos recursos.

Impact of transitioning to virtual delivery of a cardiovascular health improvement program for Latinos during the COVID-19 pandemic.

BACKGROUND: Community Heart Health Actions for Latinos at Risk (CHARLAR) is a promotora-led cardiovascular disease (CVD) risk-reduction program for socio-demographically disadvantaged Latinos and consists of 11 skill-building sessions. The COVID-19 pandemic has led to worsening health status in U.S. adults and necessitated transition to virtual implementation of the CHARLAR program. METHODS: A mixed-methods approach was used to evaluate virtual delivery of CHARLAR. Changes in health behaviors were assessed through a pre/post program survey. Results from virtual and historical (in-person delivery) were compared. Key informant interviews were conducted with promotoras and randomly selected participants and then coded and analyzed using a thematic approach. RESULTS: An increase in days of exercise per week (+ 1.52), daily servings of fruit (+ 0.60) and vegetables (+ 0.56), and self-reported general health (+ 0.38), were observed in the virtual cohort [all p < 0.05]. A numeric decrease in PHQ-8 (-1.07 p = 0.067) was also noted. The historical cohort showed similar improvements from baseline in days of exercise per week (+ 0.91), daily servings of fruit (+ 0.244) and vegetables (+ 0.282), and PHQ-8 (-1.89) [all p < 0.05]. Qualitative interviews revealed that the online format provided valuable tools supporting positive behavior change. Despite initial discomfort and technical challenges, promotoras and participants adapted and deepened valued relationships through additional virtual support. CONCLUSION: Improved health behaviors and CVD risk factors were successfully maintained through virtual delivery of the CHARLAR program. Optimization of virtual health programs like CHARLAR has the potential to increase reach and improve CVD risk among Latinos.

Escaping Death: How Cancer Cells and Infected Cells Resist Cell-Mediated Cytotoxicity.

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.

Heterogeneity in extracellular vesicle secretion by single human macrophages revealed by super-resolution microscopy.

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 M2-like 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.

Does hsa-miR-223-3p from platelet-derived extracellular vesicles regulate tissue factor expression in monocytic cells?

Extracellular vesicles (EVs) released from activated platelets contain microRNAs, the most abundant of which is hsa-miR-223-3p. Endogenous hsa-miR-223-3p suppresses the expression of tissue factor (TF), the initiator of the extrinsic coagulation pathway, in endothelial cells. Monocytes can be induced to express TF to enhance coagulation, but the role of hsa-miR-223-3p in regulating monocyte TF remains unknown. This study examined whether hsa-miR-223-3p from platelet-derived EVs (pdEVs) affects TF expression in monocytes. THP-1 cells, differentiated into a monocyte-like phenotype with 1α,25-dihydroxyvitaminD3, were transfected with hsa-miR-223-3p mimic or control microRNA. Alternatively, THP-1 cells were incubated with pdEVs from PAR1-agonist peptide activated-platelets, as platelet releasate, or pdEVs isolated by ultracentrifugation. Transfection with hsa-miR-223-3p mimic resulted in significant reductions in TF protein, determined by western blotting and flow cytometry and reduced procoagulant activity, measured by a TF-specific factor Xa generation assay, compared to cells transfected with control microRNA. This reduction was reversed by co-transfection with hsa-miR-223-3p inhibitor, AntagomiR-223. Incubation of THP-1 cells with pdEVs also decreased TF expression; however, this was not reversed by AntagomiR-223. Taken together, monocyte TF expression is downregulated by hsa-miR-223-3p, but when transferred via pdEVs the effect was not reversed with Antagomir-223, suggesting other pdEV components may contribute to TF regulation.Abbreviations: Tissue factor (TF), Factor VII (FVII), activated Factor VII (FVIIa), Factor X (FX), activated Factor X (FXa), extracellular vesicles (EVs), microvesicles (MVs), platelet-derived extracellular vesicles (pdEVs), protease-activated receptor 1 agonist peptide (PAR1-AP), lipopolysaccharide (LPS), P-selectin glycoprotein ligand-1 (PSGL-1), Tris-Buffered Saline Tween (TBST), room temperature (RT)[Figure: see text].

Radiotherapy transiently reduces the sensitivity of cancer cells to lymphocyte cytotoxicity.

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.

Four reasons for adopting a life course approach to health in the COVID-19 era and beyond / Cuatro razones para adoptar el enfoque del curso de vida en el ámbito de la salud en la era de la COVID-19 y los años posteriores / Quatro motivos para adotar uma abordagem de curso de vida à saúde na era da COVID-19 e mais além

ABSTRACT The life course approach effectively responds to pressing health needs and fills critical gaps to improve health outcomes in the era of COVID-19 and beyond. This article outlines four main reasons to adopt and implement the life course approach in public health at national and local levels: (i) the approach effectively responds to new health trends and evidence, (ii) it fills longstanding gaps in care, (iii) it best addresses health inequities, and (iv) it can help achieve more with less.

RESUMEN El enfoque del curso de vida da respuestas eficaces a las urgentes necesidades de salud y salva brechas críticas para mejorar los resultados de salud en la era de la COVID-19 y los años posteriores. En este artículo se describen las cuatro razones principales para adoptar y poner en práctica el enfoque del curso de vida en la salud pública a nivel local y nacional: este enfoque (i) da respuestas eficaces a las tendencias y la evidencia nuevas en el ámbito de la salud, (ii) salva brechas de larga data en la atención, (iii) aborda de la mejor manera posible las inequidades de salud, y (iv) puede contribuir a lograr más con menos recursos.

RESUMO A abordagem de curso de vida responde de forma efetiva a necessidades urgentes de saúde e preenche lacunas críticas para melhorar os resultados de saúde na era da COVID-19 e mais além. Este artigo descreve quatro motivos principais para adotar e implementar a abordagem de curso de vida na saúde pública em nível nacional e local: (i) a abordagem responde de forma efetiva a novas evidências e tendências em saúde, (ii) preenche lacunas antigas nos cuidados de saúde, (iii) lida melhor com iniquidades em saúde e (iv) pode ajudar a conquistar mais com menos recursos.

Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1ß Secretion in Human Macrophages.

Interleukin 1ß (IL-1ß) 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-1ß 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-1ß 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-1ß release in human macrophages.

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells.

One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.

Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages.

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.

Improving maternal and newborn care: cost-effectiveness of an innovation to rebrand traditional birth attendants in Sierra Leone.

OBJECTIVES: This paper evaluates the cost-effectiveness of rebranding former traditional birth attendants (TBAs) to conduct health promotion activities and refer women to health facilities. METHODS: The project used 200 former TBAs, 100 of whom were also enrolled in a small income generating business. The evaluation had a three-arm, quasiexperimental design with baseline and endline household surveys. The three arms were: (a) Health promotion (HP) only; (b) Health promotion plus business (HP+); and (c) the comparison group. The Lives Saved Tool is used to estimate the number of lives saved. RESULTS: The HP+ intervention had a statistically significant impact on health facility delivery and four or more antenatal care (ANC) visits during pregnancy. The cost-effectiveness ratio was estimated at US$4130 per life year saved in the HP only arm, and US$1539 in the HP+ arm. Therefore, only the HP+ intervention is considered to be cost-effective. CONCLUSIONS: It is critical to prioritize cost-effective interventions such as, in the case of rural Sierra Leone, community-based strategies involving rebranding TBAs as health promoters and enrolling them in health-related income generating activities.

Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles.

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.

Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells.

Natural Killer (NK) cells can engage multiple virally infected or tumor cells sequentially and deliver perforin for cytolytic killing of these targets. Using microscopy to visualize degranulation from individual NK cells, we found that repeated activation via the Fc receptor CD16 decreased the amount of perforin secreted. However, perforin secretion was restored upon subsequent activation via a different activating receptor, NKG2D. Repeated stimulation via NKG2D also decreased perforin secretion, but this was not rescued by stimulation via CD16. These different outcomes of sequential stimulation could be accounted for by shedding of CD16 being triggered by cellular activation. The use of pharmacological inhibitors and NK cells transfected to express a noncleavable form of CD16 revealed that CD16 shedding also increased NK cell motility and facilitated detachment of NK cells from target cells. Disassembly of the immune synapse caused by CD16 shedding aided NK cell survival and boosted serial engagement of target cells. Thus, counterintuitively, shedding of CD16 may positively impact immune responses.