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Innate immune cells represent the first line of cellular immunity, comprised of both circulating and tissue-resident natural killer cells and innate lymphoid cells. These innate lymphocytes arise from a common CD34+ progenitor that differentiates into mature natural killer cells and innate lymphoid cells. The successive stages in natural killer cell maturation are characterized by increased lineage restriction and changes to phenotype and function. Mechanisms of human natural killer cell development have not been fully elucidated, especially the role of signals that drive the spatial localization and maturation of natural killer cells. Cytokines, extracellular matrix components, and chemokines provide maturation signals and influence the trafficking of natural killer cell progenitors to peripheral sites of differentiation. Here we present the latest advances in our understanding of natural killer and innate lymphoid cell development in peripheral sites, including secondary lymphoid tissues (i.e. tonsil). Recent work in the field has provided a model for the spatial distribution of natural killer cell and innate lymphoid cell developmental intermediates in tissue and generated further insights into the developmental niche. In support of this model, future studies using multifaceted approaches seek to fully map the developmental trajectory of human natural killer cells and innate lymphoid cells in secondary lymphoid tissues.

NK cells are innate immune cells that reside within tissue and circulate in peripheral blood. They interact with a variety of microenvironments, yet how NK cells engage with these varied microenvironments is not well documented. The adhesome represents a molecular network of defined and predicted integrin-mediated signaling interactions. In this study, we define the integrin adhesome expression profile of NK cells from human tonsil, peripheral blood, and those derived from human hematopoietic precursors through stromal cell coculture systems. We report that the site of cell isolation and NK cell developmental stage dictate differences in expression of adhesome associated genes and proteins. Furthermore, we define differences in cortical actin content associated with differential expression of actin regulating proteins, suggesting that differences in adhesome expression are associated with differences in cortical actin homeostasis. These data provide understanding of the diversity of human NK cell populations and how they engage with their microenvironment.

Lymphocyte development is a complex and coordinated pathway originating from pluripotent stem cells during embryogenesis and continuing even as matured lymphocytes are primed and educated in adult tissue. Hematopoietic stem cells develop in a specialized niche that includes extracellular matrix and supporting stromal and endothelial cells that both maintain stem cell pluripotency and enable the generation of differentiated cells. Cues for lymphocyte development include changes in integrin-dependent cell motility and adhesion which ultimately help to determine cell fate. The capacity of lymphocytes to adhere and migrate is important for modulating these developmental signals both by regulating the cues that the cell receives from the local microenvironment as well as facilitating the localization of precursors to tissue niches throughout the body. Here we consider how changing migratory and adhesive phenotypes contribute to human natural killer (NK)- and T-cell development as they undergo development from precursors to mature, circulating cells and how our understanding of this process is informed by in vitro models of T- and NK cell generation.

CD56 is the main identifying cell surface molecule of NK cells and has been recently identified as a regulator of cytotoxic function in NK cell lines. Despite its newly defined role in lytic granule polarization and exocytosis, biological questions remain involving the localization and function of CD56 at the immunological synapse. Here we use confocal and structured illumination microscopy to demonstrate recruitment of CD56 to the peripheral supramolecular activating cluster (pSMAC) of the immunological synapse of lytic effector cells. We provide additional data demonstrating that cell lines that are less dependent on CD56 for function are not utilizing alternative pathways of cytotoxicity, and that those that are dependent on CD56 have normal expression of activating and adhesion receptors. Finally, we use actin reporter (LifeAct) expressing NK92 cell lines and live cell confocal microscopy to visualize live cell killing events with WT and CD56-KO cells. This work further characterizes the novel role for CD56 in cytotoxic function of NK cells and provides deeper insight into the role of CD56 at the NK cell immunological synapse.

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients., Minichromosome maintenance protein 10 (MCM10) is critical for eukaryotic DNA replication. Here, by modelling MCM10 variants in human cell lines, the authors reveal a mechanism of MCM10-associated disease, finding that loss of MCM10 function constrains telomerase activity.

Integrins are transmembrane receptors associated with adhesion and migration and are often highly differentially expressed receptors amongst natural killer cell subsets in microenvironments. Tissue resident natural killer cells are frequently defined by their differential integrin expression compared to other NK cell subsets, and integrins can further localize tissue resident NK cells to tissue microenvironments. As such, integrins play important roles in both the phenotypic and functional identity of NK cell subsets. Here we review the expression of integrin subtypes on NK cells and NK cell subsets with the goal of better understanding how integrin selection can dictate tissue residency and mediate function from the nanoscale to the tissue environment.

Natural killer (NK) cells play critical roles in physiologic and pathologic conditions such as pregnancy, infection, autoimmune disease and cancer. In cancer, numerous strategies have been designed to exploit the cytolytic properties of NK cells, with variable success. A major hurdle to NK-cell focused therapies is NK cell recruitment to and infiltration into tumors. While the chemotaxis pathways regulating NK recruitment to different tissues are well delineated, the mechanisms human NK cells employ to physically migrate are ill-defined. We show for the first time that human NK cells express fibroblast activation protein (FAP), a cell surface protease previously thought to be primarily expressed by activated fibroblasts. FAP degrades the extracellular matrix to facilitate cell migration and tissue remodeling. We used novel in vivo zebrafish and in vitro 3D culture models to demonstrate that FAP regulates NK cell migration, extravasation, and infiltration into tumors, ultimately affecting tumor cell lysis. These findings demonstrate the necessity of proteolytic migration in NK cell function, suggest novel mechanisms of action of FAP targeting drugs, and provide an entirely new way to regulate NK cell activity.Graphical Abstract

Natural killer (NK) cells are effector cells of the innate immune system involved in defense against virus-infected and transformed cells. The effector function of NK cells is linked to their ability to migrate to sites of inflammation or damage. Therefore, understanding the factors regulating NK cell migration is of substantial interest. Here, we show that in the absence of aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, NK cells have reduced capacity to migrate and infiltrate tumors in vivo. Analysis of differentially expressed genes revealed that ankyrin repeat and SOCS Box containing 2 (Asb2) expression was dramatically decreased in Ahr–/– NK cells and that AhR ligands modulated its expression. Further, AhR directly regulated the promoter region of the Asb2 gene. Similar to what was observed with murine Ahr–/– NK cells, ASB2 knockdown inhibited the migration of human NK cells. Activation of AHR by its agonist FICZ induced ASB2-dependent filamin A degradation in NK cells; conversely, knockdown of endogenous ASB2 inhibited filamin A degradation. Reduction of filamin A increased the migration of primary NK cells and restored the invasion capacity of AHR-deficient NK cells. Our study introduces AHR as a new regulator of NK cell migration, through an AHR-ASB2-filamin A axis and provides insight into a potential therapeutic target for NK cell-based immunotherapies.

Natural killer (NK) cells are innate immune cells that reside within tissue and circulate in peripheral blood. As such, they interact with a variety of complex microenvironments, yet how NK cells engage with these varied microenvironments is not well documented. The integrin adhesome represents a molecular network of defined and predicted integrin-mediated signaling interactions. Here, we define the integrin adhesome expression profile of NK cells from tonsil, peripheral blood and those derived from hematopoietic precursors through stromal cell coculture systems. We report that the site of cell isolation and NK cell developmental stage dictate differences in expression of adhesome associated genes and proteins. Furthermore, we define differences in cortical actin content associated with differential expression of actin regulating proteins, suggesting that differences in adhesome expression are associated with differences in cortical actin homeostasis. Together, these data provide new understanding into the diversity of human NK cell populations and how they engage with their microenvironment.

An inherited disorder makes WAVEs The WAVE regulatory complex (WRC) is a multiunit complex that regulates actin cytoskeleton formation. Although other actin-regulatory proteins modulate human immune responses, the precise role for the WRC has not yet been established. Cook et al. studied five patients from four unrelated families who harbor missense variants of the gene encoding the WRC component HEM1. These patients presented with recurrent infections and poor antibody responses, along with enhanced allergic and autoimmune disorders. HEM1 was found to be required for the regulation of cortical actin and granule release in T cells and also interacted with a key metabolic signaling complex contributing to the disease phenotype. By linking these interactions to immune function, this work suggests potential targets for future immunotherapies. Science , this issue p. 202

Human NK cells develop in tonsils through discrete NK cell developmental intermediates (NKDIs), yet the mechanistic regulation of this process is unclear. We demonstrate that Notch activation in human tonsil-derived stage 3 (CD34−CD117+CD94−NKp80−) and 4A (CD34−CD117+/−CD94+NKp80−) NKDIs promoted non–NK innate lymphoid cell differentiation at the expense of NK cell differentiation. In contrast, stage 4B (CD34−CD117+/−CD94+NKp80+) NKDIs were NK cell lineage committed despite Notch activation. Interestingly, whereas NK cell functional maturation from stage 3 and 4A NKDIs was independent of Notch activation, the latter was required for high NKp80 expression and a stage 4B–like phenotype by the NKDI-derived NK cells. The Notch-dependent effects required simultaneous engagement with OP9 stromal cells and were also stage-specific, with NOTCH1 and NOTCH2 receptors regulating stage 3 NKDIs and NOTCH1 primarily regulating stage 4A NKDIs. These data establish stage-specific and stromal-dependent roles for Notch in regulating human NK cell developmental plasticity and maturation.

Adaptive immune responses are acknowledged to evolve from innate immunity. However, limited information exists regarding whether encounters between innate cells direct the generation of specialized T-cell subsets. We aim to understand how natural killer (NK) cells modulate cell-mediated immunity in humans. We found that human CD14+CD16− monocytes that differentiate into inflammatory dendritic cells (DCs) are shaped at the early stages of differentiation by cell-to-cell interactions with NK cells. Although a fraction of monocytes is eliminated by NK-cell–mediated cytotoxicity, the polarization of interferon-γ (IFN-γ) at the NKp30-stabilized synapses triggers a stable IFN-γ signature in surviving monocytes that persists after their differentiation into DCs. Notably, NK-cell–instructed DCs drive the priming of type 17 CD8+ T cells (Tc17) with the capacity to produce IFN-γ and interleukin-17A. Compared with healthy donors, this cellular network is impaired in patients with classical NK-cell deficiency driven by mutations in the GATA2 gene. Our findings reveal a previously unrecognized connection by which Tc17-mediated immunity might be regulated by NK-cell–mediated tuning of antigen-presenting cells.

Human natural killer (NK) cells are defined as CD56+CD3−. Despite its ubiquitous expression on human NK cells the role of CD56 (NCAM) in human NK cell cytotoxic function has not been defined. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis through interactions with focal adhesion kinase (FAK). Here we demonstrate that deletion of CD56 on the NK92 cell line leads to impaired cytotoxic function. CD56-knockout (KO) cells fail to polarize during immunological synapse (IS) formation and have severely impaired exocytosis of lytic granules. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 is decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 are rescued by the reintroduction of CD56. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells., eLife digest The immune system deploys different cell types to take out cancer cells. True to their name, one type of immune cell known as natural killer cells kills tumor target cells by releasing toxic proteins that kill the harmful cells. In humans, these immune cells are defined, among other things, by the presence of a protein called CD56 on their cell surface. This protein (which is also known as NCAM) is thought to help cells to stick to their surroundings and control their movements. However, it was not clear whether CD56 also plays a role in the destructive abilities of natural killer cells. Gunesch et al. have now looked to see what would happen if natural killer cells lacked CD56 on their surface. The experiments included deleting the gene for CD56 from two kinds of human natural killer cell that are commonly grown in the laboratory (called NK92 and YTS). In both cases, the cells lacking CD56 killed fewer cancer cells than the unedited natural killer cells. The NK92 cells were much more affected by the loss of CD56 than the YTS cells, and after Gunesch et al. compared the two kinds of cell they identified another protein called Pyk2 as the potential reason behind the difference. The Pyk2 protein is known to help a natural killer cell latch onto target cancer cells and release its toxic proteins. To do this, Pyk2 must first be activated with phosphate groups via a process known as phosphorylation. Gunesch et al. showed that Pyk2 protein in unedited NK92 cells was more highly phosphorylated than those of the YTS cells, and that Pyk2 activation by phosphorylation was greatly decreased in NK92 cells when the gene for CD56 was deleted. Together these and other results suggest that CD56 on natural killer cells helps to promote Pyk2 to activate the cells’ cancer-killing abilities through Pyk2 phosphorylation, especially in NK92 cells. These findings open up new lines of investigation into the relationship between sticky surface proteins and the activation of immune cells. They may also have important implications for the use of the immune system to treat cancer via immunotherapy.

Human NK cells are effectors of the innate immune system that originate from hematopoietic precursors in the bone marrow. While stromal cell lines that support NK cell development from hematopoietic precursors are often used to generate mature NK cells from lymphoid precursors in vitro, the nature of contributing factors of these stromal cells to the generation of functionally mature NK cells has been poorly described. Previous studies have shown that developing NK cells adhere to, and migrate on, developmentally supportive stroma. Here, we describe the generation of cell-derived matrices (CDMs) from a commonly used murine fetal liver stromal cell line. These CDMs are derived directly from the same EL08.1D2 stromal cell line known to support NK cell differentiation and contain ECM structural components fibronectin and collagen. We demonstrate that CDMs support NK cell adhesion and migration with similar properties as intact cells. Further, we show that CDMs support NK cell maturation from lymphoid precursors in vitro, albeit with reduced cell survival compared to intact cell-based differentiation. Together, these results describe a cell-free system that supports NK cell development and that can serve as a useful model for studying the nature of the biochemical interactions between NK cell developmental intermediates and developmentally supportive substrates.

Human NK cells are innate immune effectors that play a critical roles in the control of viral infection and malignancy. The importance of their homeostasis and function can be demonstrated by the study of patients with primary immunodeficiencies (PIDs), which are part of the family of diseases known as inborn defects of immunity. While NK cells are affected in many PIDs in ways that may contribute to a patient's clinical phenotype, a small number of PIDs have an NK cell abnormality as their major immunological defect. These PIDs can be collectively referred to as NK cell deficiency (NKD) disorders and include effects upon NK cell numbers, subsets, and/or functions. The clinical impact of NKD can be severe including fatal viral infection, with particular susceptibility to herpesviral infections, such as cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. While NKD is rare, studies of these diseases are important for defining specific requirements for human NK cell development and homeostasis. New themes in NK cell biology are emerging through the study of both known and novel NKD, particularly those affecting cell cycle and DNA damage repair, as well as broader PIDs having substantive impact upon NK cells. In addition, the discovery of NKD that affects other innate lymphoid cell (ILC) subsets opens new doors for better understanding the relationship between conventional NK cells and other ILC subsets. Here, we describe the biology underlying human NKD, particularly in the context of new insights into innate immune cell function, including a discussion of recently described NKD with accompanying effects on ILC subsets. Given the impact of these disorders upon human immunity with a common focus upon NK cells, the unifying message of a critical role for NK cells in human host defense singularly emerges.

The lytic immunological synapse (IS) is a discrete structural entity formed after the ligation of specific activating receptors that leads to the destruction of a cancerous cell. The formation of an effector cell IS in cytotoxic T lymphocytes or natural killer cells is a hierarchical and stepwise rearrangement of structural and signaling components and targeted release of the contents of lytic granules. While recent advances in the generation and testing of cytotoxic lymphocytes expressing chimeric antigen receptors (CARs) has demonstrated their efficacy in the targeted lysis of tumor targets, the contribution and dynamics of IS components have not yet been extensively investigated in the context of engineered CAR cells. Understanding the biology of the CAR IS will be a powerful approach to efficiently guide the engineering of new CARs and help identify mechanistic problems in existing CARs. Here, we review the formation of the lytic IS and describe quantitative imaging-based measurements using multiple microscopy techniques at a single cell level that can be used in conjunction with established population-based assays to provide insight into the important cytotoxic function of CAR cells. The inclusion of this approach in the pipeline of CAR product design could be a novel and valuable innovation for the field.

IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks., Summary: Super-resolution imaging and multiplexed live-cell analyses indicate the multi-domain scaffold IQGAP1 contributes to a complex regulatory network that coordinates the actin encapsulation and membrane processing of a novel endosomal compartment.

Minichromosome maintenance protein 10 (Mcm10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 mutations in patients with distinctive but overlapping clinical phenotypes – natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of Mcm10-associated disease, we modeled these mutations in human cell lines. Mcm10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of Mcm10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in Mcm10-deficient cells require endonucleolytic processing by Mus81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic mutations in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes in both NKD and CM patients.

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here we report a novel cause of NKD resulting from compound heterozygous mutations in MCM10 that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. By modeling MCM10 deficiency in human NK cell lines and primary NK cell precursors, we demonstrate that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function.

Natural killer cell deficiencies (NKDs) are an emerging phenotypic subtype of primary immune deficiency. NK cells provide a defense against virally infected cells using a variety of cytotoxic mechanisms, and patients who have defective NK cell development or function can present with atypical, recurrent, or severe herpesviral infections. The current pipeline for investigating NKDs involves the acquisition and clinical assessment of patients with a suspected NKD followed by subsequent in silico, in vitro, and in vivo laboratory research. Evaluation involves initially quantifying NK cells and measuring NK cell cytotoxicity and expression of certain NK cell receptors involved in NK cell development and function. Subsequent studies using genomic methods to identify the potential causative variant are conducted along with variant impact testing to make genotype-phenotype connections. Identification of novel genes contributing to the NKD phenotype can also be facilitated by applying the expanding knowledge of NK cell biology. In this review, we discuss how NKDs that affect NK cell cytotoxicity can be approached in the clinic and laboratory for the discovery of novel gene variants.

Immunodeficiency often coincides with immune hyperresponsiveness such as autoimmunity, lymphoproliferation, or atopy, but the molecular basis of this paradox is typically unknown. We describe four families with immunodeficiency coupled with atopy, lymphoproliferation, cytokine overproduction, hemophagocytic lymphohistocytosis, and autoimmunity. We discovered loss-of-function variants in the gene NCKAP1L, encoding the hematopoietic-specific Hem1 protein. Three mutations cause Hem1 protein and WAVE regulatory complex (WRC) loss, thereby disrupting actin polymerization, synapse formation, and immune cell migration. Another mutant, M371V encodes a stable Hem1 protein but abrogates binding of the Arf1 GTPase and identifies Arf1 as a critical Hem1 regulator. All mutations reduce the cortical actin barrier to cytokine release explaining immune hyperresponsiveness. Finally, Hem1 loss blocked mTORC2-dependent AKT phosphorylation, T cell proliferation, and effector cytokine production during T cell activation. Thus, our data show that Hem1 independently governs two key regulatory complexes, the WRC and mTORC2, and how Hem1 loss causes a combined immunodeficiency and immune hyperresponsiveness disease.One sentence summaryHem1 loss of function mutations cause a congenital immunodysregulatory disease and reveal its role regulating WAVE2 and mTORC2 signaling.

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific signaling and regulatory processes. While central to many aspects of cell physiology, this coupling often depends on the function of multi-domain scaffolds that orchestrate transient interactions and dynamic feedback among a spectrum of signaling intermediates and regulatory proteins on organelles. Understanding scaffold protein functions has remained challenging given this complexity. This work employs live-cell imaging and statistical analyses to deconvolve (demultiplex) how the multi-domain scaffold IQGAP1 coordinates phosphoinositide signaling with organelle-specific actin regulation and membrane processing events. Using actin-ensconced endosomes that localize to the basal cortex of polarized epithelial cells as a model system, we demonstrate abilities to dissect how IQGAP1 transitions between different actin and endosomal-membrane tethered states. We provide evidence IQGAP1 functions as a transient inhibitor of actin growth around the endosomes in at least one of these states. While not easily distilled via standard (static) colocalization analyses or traditional pathway perturbations methods, this negative regulation was revealed via a series of dynamic correlation and multiple regression analyses. These methods also uncovered that the negative actin regulation is linked to GTPase-dependent tethering to the endosomal membrane. Moreover, the scaffold transitions underlying this control are shown to depend on the production of PIP3 lipid messengers by the lipid kinase PI3K. Overall, these methods and results provide new insights in to how IQGAP1 act as a signaling hub by orchestrating time-dependent membrane and cytoskeletal protein interactions and provide new routes to dissect scaffold-mediated pathway regulation in a variety of settings.

Many therapeutic monoclonal antibodies require binding to Fc γ receptors (FcγRs) for full effect and increasing the binding affinity increases efficacy. Preeminent among the five activating human FcγRs is FcγRIIIa/CD16a expressed by natural killer (NK) cells. CD16a is heavily processed, and recent reports indicate that the composition of the five CD16a asparagine(N)-linked carbohydrates (glycans) impacts affinity. These observations indicate that specific manipulation of CD16a N-glycan composition in CD16a-expressing effector cells including NK cells may improve treatment efficacy. However, it is unclear if modifying the expression of select genes that encode processing enzymes in CD16a-expressing effector cells is sufficient to affect N-glycan composition. We identified substantial processing differences using a glycoproteomics approach by comparing CD16a isolated from two NK cell lines, NK92 and YTS, with CD16a expressed by HEK293F cells and previous reports of CD16a from primary NK cells. Gene expression profiling by RNA-Seq and qRT-PCR revealed expression levels for glycan-modifying genes that correlated with CD16a glycan composition. These results identified a high degree of variability between the processing of the same human protein by different human cell types. N-glycan processing correlated with the expression of glycan-modifying genes and thus explained the substantial differences in CD16a processing by NK cells of different origins.

Lytic granule convergence to the MTOC prepares NK cells for cytotoxic effector function. Hsu et al. demonstrate that convergence increases specificity in directed secretion, thereby preventing bystander killing., Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells.

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.

While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells, which we term the developmental synapse. Finally, we identify a role for CD56 in developmental synapse structure, NK cell motility and NK cell development. Thus, we define the developmental synapse leading to human NK cell functional maturation., CD56, a splicing variant of NCAM, marks human NK cell differentiation stages. Here the authors show that developing human NK cells form CD56-enriched synapses with stromal cells, and CD56 is critical to promote motility of NK cells that increases with their maturation.

OBJECTIVES/GOALS: Natural killer (NK) cells are a potential cancer therapeutic but expanding NK cells efficiently in vitro is difficult. Natural killer cell deficiency (NKD), a primary immune deficiency affecting only NK cells, is caused by defects in several DNA replication proteins. By studying NKD we will achieve better NK cell in vitro differentiation. METHODS/STUDY POPULATION: One patient with NKD has a compound heterozygous mutation in the essential DNA replication protein MCM10. We hypothesize that in individuals with NKD, dramatic telomere erosion from abnormal DNA replication leads to premature senescence and the loss of NK cells. To test our hypothesis, we will knockout one allele of MCM10 or over express MCM10 in NK cells isolated from blood. We will then monitor telomere length, expansion and cytotoxic activity of these NK cells. To understand the role of MCM10 in early stages of NK cell development we will deplete MCM10 in induced pluripotent stem cells and differentiate these cells into NK cells. During this differentiation we will monitor progression through NK cell developmental stages as well as telomere length and senescence markers. RESULTS/ANTICIPATED RESULTS: Telomeres insulate chromosomes and induce permanent growth arrest (senescence) when they are critically short. We have demonstrated that depletion of a DNA replication protein causes telomere erosion and increases senescence markers. NK cells have shorter telomeres and lower telomerase expression than other immune cells. We predict, this relatively poor telomere maintenance sensitizes NK cells to telomere loss upon depletion of replication proteins. During in vitro differentiation, we expect NK cell precursors to undergo premature senescence secondary to telomere shortening. Furthermore, we expect supplementation of DNA replication proteins will enhance NK cell expansion and maturation. DISCUSSION/SIGNIFICANCE OF IMPACT: NKD patients have provided the scientific community with clues as to what proteins NK cells rely on for their development. This project aims not only to understand why these proteins are critical, but to harness that information for cellular anti-cancer therapeutics.

Background Patients with signal transducer and activator of transcription 5b (STAT5b) deficiency have impairment in T-cell homeostasis and natural killer (NK) cells which leads to autoimmunity, recurrent infections, and combined immune deficiency. Objective In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b−/− mice. Methods We used multiparametric flow cytometry, functional NK cell assays, microscopy, and a Stat5b−/− mouse model to elucidate the effect of impaired and/or absent STAT5b on NK cell development and function. Results This alteration generated a nonfunctional CD56bright NK cell subset characterized by low cytokine production. The CD56dim NK cell subset had decreased expression of perforin and CD16 and a greater frequency of cells expressing markers of immature NK cells. We observed low NK cell numbers and impaired NK cell maturation, suggesting that STAT5b is involved in terminal NK cell maturation in Stat5b−/− mice. Furthermore, human STAT5b-deficient NK cells had low cytolytic capacity, and fixed-cell microscopy showed poor convergence of lytic granules. This was accompanied by decreased expression of costimulatory and activating receptors. Interestingly, granule convergence and cytolytic function were restored after IL-2 stimulation. Conclusions Our results show that in addition to the impaired terminal maturation of NK cells, human STAT5b mutation leads to impairments in early activation events in NK cell lytic synapse formation. Our data provide further insight into NK cell defects caused by STAT5b deficiency.

The HLH-2004 criteria are used to diagnose hemophagocytic lymphohistiocytosis (HLH), yet concern exists for their misapplication, resulting in suboptimal treatment of some patients. We sought to define the genomic spectrum and associated outcomes of a diverse cohort of children who met the HLH-2004 criteria. Genetic testing was performed clinically or through research-based whole-exome sequencing. Clinical metrics were analyzed with respect to genomic results. Of 122 subjects enrolled over the course of 17 years, 101 subjects received genetic testing. Biallelic familial HLH (fHLH) gene defects were identified in only 19 (19%) and correlated with presentation at younger than 1 year of age (P < .0001). Digenic fHLH variants were observed but lacked statistical support for disease association. In 28 (58%) of 48 subjects, research whole-exome sequencing analyses successfully identified likely molecular explanations, including underlying primary immunodeficiency diseases, dysregulated immune activation and proliferation disorders, and potentially novel genetic conditions. Two-thirds of patients identified by the HLH-2004 criteria had underlying etiologies for HLH, including genetic defects, autoimmunity, and malignancy. Overall survival was 45%, and increased mortality correlated with HLH triggered by infection or malignancy (P < .05). Differences in survival did not correlate with genetic profile or extent of therapy. HLH should be conceptualized as a phenotype of critical illness characterized by toxic activation of immune cells from different underlying mechanisms. In most patients with HLH, targeted sequencing of fHLH genes remains insufficient for identifying pathogenic mechanisms. Whole-exome sequencing, however, may identify specific therapeutic opportunities and affect hematopoietic stem cell transplantation options for these patients.

Human natural killer (NK) cell precursors undergoing in vitro differentiation from hematopoietic stem cell precursors are tracked by long-term live-cell imaging. As differentiation progresses, NK cells acquire increasingly motile and complex modes of migration., Human natural killer (NK) cells are generated from CD34+ precursors and can be differentiated in vitro by coculture with developmentally supportive stromal cells. We have previously described the acquisition of cell migration as a feature of NK cell terminal maturation in this system. Here we perform continuous long-term imaging and tracking of NK cell progenitors undergoing in vitro differentiation. We demonstrate that NK cell precursors can be tracked over long time periods on the order of weeks by utilizing phase-contrast microscopy and show that these cells acquire increasing motility as they mature. Additionally, we observe that NK cells display a more heterogeneous range of migratory behaviors at later stages of development, with the acquisition of complex modes of migration that are associated with terminal maturation. Together these data demonstrate previously unknown migratory behaviors of innate lymphocytes undergoing lineage differentiation revealed by long-term imaging and analysis workflows.

Natural killer (NK) cells are critical in immune defense against infected, stressed or transformed cells. Their function is regulated by the heterogeneous expression of a wide array of surface receptors that shape its phenotypic diversity. Although NK cells develop in the bone marrow and secondary lymphoid tissues, substantive differentiation is apparent in the peripheral blood including known age-related variation. In order to gain greater insight into phenotypic and functional variation within peripheral blood NK cells across age groups, we used multi-parametric, polyfunctional flow cytometry to interrogate the NK cell variability in 20 healthy adults and 15 5-10, 11-15 and 16-20 year-old children. We found that the normative ranges in both adults and children displayed great inter-individual variation for most markers. While the expression of several receptors did not differ, among those that did, the majority of the differences existed between adults and the three pediatric groups, rather than among children of different ages. Interestingly, we also identified variation in the individual expression of some markers by sex and ethnicity. Combinatorial analysis of NK cell receptors revealed intermediate subsets between the CD56bright and CD56dim NK cells. Furthermore, on examining the NK cell diversity by age, adults were discovered to have the lowest developmental diversity. Thus, our findings identify previously unappreciated NK cell subsets potentially distinguishing children from adults and suggest functional correlates that may have relevance in age-specific host defense.

Haploinsufficiency of the hematopoietic transcription factor GATA2 underlies monocytopenia and mycobacterial infections; dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency; familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML); and Emberger syndrome (primary lymphedema with MDS). A comprehensive examination of the clinical features of GATA2 deficiency is currently lacking. We reviewed the medical records of 57 patients with GATA2 deficiency evaluated at the National Institutes of Health from January 1, 1992, to March 1, 2013, and categorized mutations as missense, null, or regulatory to identify genotype-phenotype associations. We identified a broad spectrum of disease: hematologic (MDS 84%, AML 14%, chronic myelomonocytic leukemia 8%), infectious (severe viral 70%, disseminated mycobacterial 53%, and invasive fungal infections 16%), pulmonary (diffusion 79% and ventilatory defects 63%, pulmonary alveolar proteinosis 18%, pulmonary arterial hypertension 9%), dermatologic (warts 53%, panniculitis 30%), neoplastic (human papillomavirus+ tumors 35%, Epstein-Barr virus+ tumors 4%), vascular/lymphatic (venous thrombosis 25%, lymphedema 11%), sensorineural hearing loss 76%, miscarriage 33%, and hypothyroidism 14%. Viral infections and lymphedema were more common in individuals with null mutations (P = .038 and P = .006, respectively). Monocytopenia, B, NK, and CD4 lymphocytopenia correlated with the presence of disease (P < .001). GATA2 deficiency unites susceptibility to MDS/AML, immunodeficiency, pulmonary disease, and vascular/lymphatic dysfunction. Early genetic diagnosis is critical to direct clinical management, preventive care, and family screening.

Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell anti-leukemia potential is unclear. Paradigm-shifting reports have shown that NK cells exhibit "memory-like" properties following hapten exposure, virus infection, or combined cytokine pre-activation. Human cytokine-induced memory-like (ML) NK cells display enhanced re-stimulation responses to numerous activating stimuli, including tumor target cells. This has been translated in clinical trials as cellular therapy for rel/ref AML patients (NCT#), and the dose escalation of a phase 1/2 study has been completed (PMID). Donor memory-like NK cells expanded in patients' blood and bone marrow and retained enhanced functionality ex vivo, with 7 of 11 patients achieving CR/CRi. Since NK cell recognition depend on signals from multiple activating and inhibitory receptors, we developed mass cytometry panels to immunophenotype and track the diversity and effector functions of these human in vivo-differentiated memory-like NK cells. Previous work showed that that in vivo-differentiated memory-like (ML) NK cells were distinct from baseline (BL) NK cells from the same donor, as well as NK cells from normal donor PBMC. Multidimensional analyses revealed a memory-like phenotype: CD56hi CD11blo CD62L+ NKG2Ahi NKp30hi Ki-67+. Furthermore, Citrus analyses revealed that higher NKG2A expression was significantly correlated with treatment failure. NKG2A is a C-type lectin receptor with two immunoreceptor tyrosine-based inhibitory motifs. Signaling through NKG2A is achieved when it engages its ligand, HLA-E. HLA-E is a non-classical major histocompatibility complex class I molecule that is expressed abundantly on many normal tissue types as well as tumors, including AML. Based on these findings that NKG2A is upregulated on memory-like NK cells and the intensity of NKG2A on memory-like NK cells correlated with patient responses, we hypothesized that NKG2A/HLA-E interactions represent a major barrier to memory-like NK cell responses. CRISPR based gene editing of primary human NK cells has been technically challenging. In order to interrogate the role NKG2A may play in limiting ML NK cell responses, we optimized the MaxCyte GT electroporation system to introduce Cas9 and guide RNA into freshely isolated, purified human NK cells. As proof of principle, we introduced Cas9 and gRNA targeting CD56 into NK cells and assessed CD56 expression a week later. We observed a 96.5% ± 0.8% (SD) reduction in median CD56 expression as determined by flow cytometry, with little impact on cell viability (90.3% ± 2.7% live v 87.0% ± 3.1% live ΔCD56) after electroporation. Next we introduced Cas9 and gRNA against NKG2A into freshly isolated, purified human NK cells. After electroporation, cells were briefly incubated with IL-12/IL-15/IL-18, overnight. The cytokines are washed away and the cells incubated for 4 days in low-dose IL-15, which was required for their survival. NKG2A frequency was decreased 64.72% (ΔNKG2A v Control; 42.3-85.7% range, ± 13.18% SD) by 4 days post-electroporation. We compared the ability of these cells to respond to HLA-E+ K562 leukemia targets and observed a significantly enhanced ML NK cell response by ΔNKG2A ML NK cells compared to control ML NK cells (19.04 ± 5.9% IFN-γ+ v 34.9 ± 8.8% ΔNKG2A IFNγ+; Mean ± S.D.). Finally, we infused ΔNKG2A ML NK or control ML NK cells into NSG recipient mice and assessed the spleen at D7 and D14 for persistence and NKG2A expression. We were able to detect ΔNKG2A ML NK and control ML NK cells at both time points and the ΔNKG2A ML NK cells remain NKG2A-negative, post-transfer. Using gene-editing approaches, the data reveal an important inhibitory role for NKG2A on ML NK cell responses against HLA-E+ targets. Primary human NK cells are notoriously difficult to modify by virus or electroporation. Indeed, most reports utilize expanded NK cells or cord-blood differentiated NK cells which were edited in the stem cell stage. This report demonstrates that mature NK cells can be modified with little ex vivo manipulation with high efficiency and viability. This method has broad potential to expand our understanding of human NK cell biology using genetic loss or gain of function techniques, as exemplified by identification of NKG2A as a critical ML NK cell checkpoint. Figure Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

Introduction: Pediatric non-malignant lymphoproliferative disorders (LPDs) are a clinically and genetically heterogeneous. While transient lymphadenopathy is extremely common and rarely dangerous, long-standing immune dysregulation and lymphoproliferation in children may be life-threatening. Data to guide evaluation and treatment of children with benign LPD are lacking. The primary objective of this study was to define the genomic spectrum and clinical characteristics of a cohort of children with nonmalignant LPD. Identification of the underlying pathogenic mechanisms may facilitate timely interventions and potentially guide optimal therapeutic strategies. Method s: Patients at Texas Children's Hospital and collaborating referral centers who met criteria for non-malignant LPD were offered participation in this study, approved by the Baylor College of Medicine Institutional Review Board. LPD was defined as persistent lymphadenopathy, lymph organ involvement, or lymphocytic infiltration for more than 3 months, with or without chronic or significant EBV infection. Chronic or significant EBV infection was defined as recurrent or persistent EBV viremia more than 3 months, invasive EBV disease, or EBV PCR copies >100,000. All subjects and/or family members provided written informed consent to have their clinical and genetic information published. Genetic testing was performed clinically or through research-based whole-exome sequencing (WES). Results : Fifty-one subjects from 47 different families with non-malignant LPD were identified. Median age at disease presentation was 3.3 years (range 3.9 weeks - 21 years) with nearly equal proportions of males (n = 26) and females (n = 25). Almost half of subjects were Hispanic (49%), and 29% were non-Hispanic white. Fifteen subjects (29%) met HLH-2004 diagnostic criteria for HLH. Twenty-one patients had EBV-associated lymphoproliferative disorders (EBV-LPD) and 6 of the 51 ultimately developed malignancy. Clinical genetic testing was performed in 29 patients, and research-based WES was performed in 44 patients. Potential disease-causing genetic defects were identified in 62% of families. Of these pathogenic variants, targeted therapies may be effective for treatment in at least 10 of the conditions (17 subjects, 33%). Furthermore, genetic results supported potential for curative HSCT in 35% of the patients. Mechanistically, all of the LPD-associated genes could be placed into 1 of 3 categories: 1) defective control of lymphocyte activity; 2) impaired lymphocyte activation, cytoskeletal organization, and apoptosis; and 3) dysregulated inflammation. Ten-year survival for the entire cohort was 72.4% with a median 5.6 years of follow-up (range 0.10 - 26.6). Patients without evidence of a genetic explanation had a lower ten-year survival compared to those patients for whom a genetic explanation was identified (48% versus 82%, respectively, p=0.03). When both EBV-LPD and genetic explanation were considered, the ten-year survival estimate for those with EBV-associated disease and no genetic explanation was significantly worse than those with EBV-associated disease and a genetic explanation (17% vs. 90%; p =0.002). Patients without EBV-associated disease were at lower risk of death than those with EBV-LPD. Evaluating outcomes associated with maximum treatment received, ten-year survival was lowest (25% survival) among those who underwent HSCT. Conclusion s: Pediatric non-malignant LPD represents a group of conditions with high risk of death. WES identified actionable mutations in the majority of LPD cases in this cohort. Early identification of these mutations can guide therapy by confirming diagnosis, revealing molecular targets and/or supporting definitive therapy with stem cell transplant. Disclosures Forbes: Takeda: Consultancy. Jolles:CSL Behring: Consultancy, Honoraria, Research Funding, Speakers Bureau; LFB: Consultancy, Honoraria, Research Funding, Speakers Bureau; UCB Pharma: Consultancy, Other: Drug Safety Committee; Shire/Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharming: Consultancy, Honoraria, Research Funding, Speakers Bureau; Octapharma: Consultancy, Honoraria, Research Funding, Speakers Bureau. Heslop:Marker Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Allovir: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cell Medica: Research Funding. Rouce:Novartis: Consultancy, Honoraria; Tessa Therapeutics: Research Funding; Kite, a Gilead Company: Consultancy, Honoraria.

Signaling pathways leading to natural killer (NK)-cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of the immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells and that this is required for initiation of downstream signaling and multiple NK-cell effector functions in vitro and in vivo. Surprisingly, we found that 2 types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family-independent SLP-76-dependent signaling pathway was identified. The LAT family-independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family-dependent and ADAP-dependent pathway contributed to interferon-gamma production and cytotoxicity; however, they were not essential for other SLP-76-dependent events, including phosphorylation of AKT and extracellular signal-related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK-cell function.

Natural killer (NK) cells participate in host defense by surveying for and ultimately killing virally infected or malignant target cells. NK cell cytotoxicity is a tightly regulated process that proceeds stepwise from adhesion and activation to the secretion of preformed lytic granule contents onto a diseased or stressed cell. We previously characterized rapid dynein-dependent lytic granule convergence to the microtubule-organizing center (MTOC) as an early, prerequisite step in NK cell cytotoxicity. Although multiple activating receptors can trigger granule convergence, the specific signal or signals responsible remained unknown. Using live cell confocal microscopy, NK cell lytic granule movement after NK cell activation was captured and measured. Using inhibitors of common early signaling mediators, we show that Src kinases are required for lytic granule convergence, but downstream signals that promote actin rearrangement, MTOC polarization, and calcium mobilization are not. Exposure to interleukin 2 was also sufficient to induce lytic granule convergence, which required noncanonical Src-dependent signaling. Thus, NK cell lytic granule convergence, prompted by specific receptor-mediated and soluble cytokine signals, depends on a directly downstream early Src kinase-dependent signal and emphasizes the importance of this step in readying NK cells for cytotoxicity.

Besides circadian rhythms, oscillations cycling with a 12 hr period exist. However, the prevalence, origin, regulation, and function of mammalian 12 hr rhythms remain elusive. Utilizing an unbiased mathematical approach identifying all superimposed oscillations, we uncovered prevalent 12 hr gene expression and metabolic rhythms in mouse liver, coupled with a physiological 12 hr unfolded protein response oscillation. The mammalian 12 hr rhythm is cell autonomous, driven by a dedicated 12 hr pacemaker distinct from the circadian clock, and can be entrained in vitro by metabolic and ER stress cues. Mechanistically, we identified XBP1s as a transcriptional regulator of the mammalian 12 hr clock. Downregulation of the 12 hr gene expression strongly correlates with human hepatic steatosis and steatohepatitis, implying its importance in maintaining metabolic homeostasis. The mammalian 12 hr rhythm of gene expression also is conserved in nematodes and crustaceans, indicating an ancient origin of the 12 hr clock. Our work sheds new light on how perturbed biological rhythms contribute to human disease.

Defining immunological mechanisms underlying NK cell biology is crucial for the treatment and prevention of immune deficiency and malignancy. The limited availability of human biological specimens presents a challenge to the study of human immunobiology. The use of high throughput, multi-parametric assays will not only aid in the definition and diagnosis of complex human immune disorders affecting NK cell function but also advance NK cell biology through population-based assessment of molecular signaling. In an effort to garner the most information from limited numbers of human cells, we designed a quantitative method to study NK cell function using imaging flow cytometry (IFC), which combines multiparametric flow cytometry and fluorescence microscopy. Specifically, we developed IFC as a tool to measure polarization and secretion of lytic granules at the immunological synapse formed between an NK cell and a susceptible target. We have further validated our approach through quantitative comparison with high-resolution confocal microscopy. We show that IFC can be used as a quantitative, high throughput measure of NK cell biological function possessing greater dimensionality than standard flow cytometry.

Recent advances in imaging technology have enabled significant advances in the study of NK cell cytotoxic effector function through quantitative analysis of the NK cell immunological synapse. This can include the use of high- and super-resolution microscopy to quantify dynamics of cytoskeletal elements and the role they play in the regulation and execution of NK cell directed secretion. Here we describe a protocol for the recapitulation of the NK cell lytic synapse on glass, the acquisition of microscopy images, and suggested approaches for the processing and analysis of microscopy data.

Introduction. Our objective is to understand how HIV infection increases the risk of progression from latent tuberculosis (TB) to active disease. We understand now that immunity is a balance of competing immune responses by multiple cell types. Since T-lymphocyte production of interferon-gamma (IFN-γ) in response toMycobacterium tuberculosis (Mtb) antigens fails to differentiate disease from latent infection, we applied a comprehensive profiling methodology to define immune biomarkers that reliably predict a patient’s TB risk.Methods. We established a cohort of HIV-infected adults with TB disease from Swaziland. Multiparametric flow cytometry was used to quantify the mycobacterial-specific anti-inflammatory (IL-4 and IL-10) and proinflammatory (IFN-γ) immune response.Results. From 12 HIV-infected Swaziland patients with TB disease, the CD4+, CD8+, Double Negative, and CD56+CD3−lymphocytes increase their IL-4 : IFN-γratio as HIV disease worsens (Spearmanrof −0.59; −0.59; −0.60; and −0.59, resp.;p<0.05). Similarly, HIV severity is associated with an increased IL-10 : IFN-γratio (Spearmanrof −0.76;p=0.01).Conclusion. As HIV disease progresses, both the adaptive and innate branches skew away from an inflammatory and towards anti-inflammatory phenotype.

The formation of a dynamic, actin-rich immunological synapse (IS) and the polarization of cytolytic granules toward target cells are essential to the cytotoxic function of NK cells. Following polarization, lytic granules navigate through the pervasive actin network at the IS to degranulate and secrete their toxic contents onto target cells. We examined lytic granule motility and persistence at the cell cortex of activated human NK cells, using high-resolution total internal reflection microscopy and highly quantitative analysis techniques. We illustrate that lytic granules are dynamic and observe substantial motility at the plane of the cell cortex prior to, but not after, degranulation. We also show that there is no significant change in granule motility in the presence of Latrunculin A (which induces actin depolymerization), when added after granule polarization, but that there is a significant decrease in lytic granule persistence subsequent to degranulation. Thus, we show that lytic granules are highly dynamic at the cytolytic human NK cell IS prior to degranulation and that the persistence of granules at the cortex following exocytosis requires the integrity of the synaptic actin network.

Summary According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34−CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34−CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.

The leukocyte integrin LFA-1 is critical for natural killer (NK) cell cytotoxicity as it mediates NK-cell adhesion to target cells and generates activating signals that lead to polarization of the actin cytoskeleton. However, the LFA-1–mediated signaling pathway is not fully understood. Here, we examined the subcellular localization of actin-associated proteins in wild-type, talin-deficient, and Wiskott-Aldrich Syndrome protein (WASP)–deficient NK cells bound to beads coated with the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). In addition, we carried out coimmunoprecipitation analyses and also used a pharmacologic reagent to reduce the level of phosphatidylinositol-4,5-bisphosphate (PIP2). The results revealed the following signaling pathways. Upon ICAM-1 binding to LFA-1, talin redistributes to the site of LFA-1 ligation and initiates 2 signaling pathways. First, talin recruits the actin nucleating protein complex Arp2/3 via constitutive association of vinculin with talin and Arp2/3. Second, talin also associates with type I phosphatidylinositol 4-phosphate 5-kinase (PIPKI) and binding of LFA-1 to ICAM-1 results in localized increase in PIP2. This increase in PIP2 recruits WASP to the site of LFA-1 ligation where WASP promotes Arp2/3-mediated actin polymerization. These processes are critical for the initiation of NK cell–mediated cytotoxicity.