Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC.

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this "affinity profiling" approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

ß-Actin G342D as a Cause of NK Cell Deficiency Impairing Lytic Synapse Termination.

NK cell deficiency (NKD) occurs when an individual's major clinical immunodeficiency derives from abnormal NK cells and is associated with several genetic etiologies. Three categories of ß-actin-related diseases with over 60 ACTB (ß-actin) variants have previously been identified, none with a distinct NK cell phenotype. An individual with mild developmental delay, macrothrombocytopenia, and susceptibility to infections, molluscum contagiosum virus, and EBV-associated lymphoma had functional NKD for over a decade. A de novo ACTB variant encoding G342D ß-actin was identified and was consistent with the individual's developmental and platelet phenotype. This novel variant also was found to have direct impact in NK cells because its expression in the human NK cell line YTS (YTS-NKD) caused increased cell spreading in lytic immune synapses created on activating surfaces. YTS-NKD cells were able to degranulate and perform cytotoxicity, but they demonstrated defective serial killing because of prolonged conjugation to the killed target cell and thus were effectively unable to terminate lytic synapses. G342D ß-actin results in a novel, to our knowledge, mechanism of functional NKD via increased synaptic spreading and defective lytic synapse termination with resulting impaired serial killing, leading to overall reductions in NK cell cytotoxicity.

A Noncanonical CD56dimCD16dim/- NK Cell Subset Indicative of Prior Cytotoxic Activity Is Elevated in Patients with Autoantibody-Mediated Neurologic Diseases.

Neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein Ab disease, and autoimmune myasthenia gravis (MG) are autoantibody-mediated neurologic conditions where autoantibodies can induce Ab-dependent cellular cytotoxicity (ADCC), a NK cell-mediated effector function. However, whether ADCC is a pathogenic mechanism in patients with these conditions has not been confirmed. We sought to characterize circulatory NK cells using functional assays, phenotyping, and transcriptomics to elucidate their role in pathology. NK cells from NMOSD patients and MG patients with elevated disease burden exhibited reduced ADCC and CD56dimCD16hi NK cells, along with an elevated frequency of CD56dimCD16dim/- NK cells. We determined that ADCC induces a similar phenotypic shift in vitro. Bulk RNA sequencing distinguished the CD56dimCD16dim/- population from the canonical CD56dimCD16hi cytotoxic and CD56hiCD16- immunomodulatory subsets, as well as CD56hiCD16+ NK cells. Multiparameter immunophenotyping of NK cell markers, functional proteins, and receptors similarly showed that the CD56dimCD16dim/- subset exhibits a unique profile while still maintaining expression of characteristic NK markers CD56, CD94, and NKp44. Notably, expression of perforin and granzyme is reduced in comparison with CD56dimCD16hi NK cells. Moreover, they exhibit elevated trogocytosis capability, HLA-DR expression, and many chemokine receptors, including CCR7. In contrast with NMOSD and MG, myelin oligodendrocyte glycoprotein Ab disease NK cells did not exhibit functional, phenotypic, or transcriptomic perturbations. In summary, CD56dimCD16dim/- NK cells are a distinct peripheral blood immune cell population in humans elevated upon prior cytotoxic activity by the CD56dimCD16hi NK cell subset. The elevation of this subset in NMOSD and MG patients suggests prior ADCC activity.

Human PLCG2 haploinsufficiency results in a novel natural killer cell immunodeficiency.

BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet, despite its ubiquitous expression on NK cells, CD56 remains a poorly understand protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function, and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2, accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

cellPLATO: an unsupervised method for identifying cell behaviour in heterogeneous cell trajectory data.

Advances in imaging, cell segmentation, and cell tracking now routinely produce microscopy datasets of a size and complexity comparable to transcriptomics or proteomics. New tools are required to process this 'phenomics' type data. Cell PLasticity Analysis TOol (cellPLATO) is a Python-based analysis software designed for measurement and classification of diverse cell behaviours based on clustering of parameters of cell morphology and motility. cellPLATO is used after segmentation and tracking of cells from live cell microscopy data. The tool extracts morphological and motility metrics from each cell per timepoint, before being using them to segregate cells into behavioural subtypes with dimensionality reduction. Resultant cell tracks have a 'behavioural ID' for each cell per timepoint corresponding to their changing behaviour over time in a sequence. Similarity analysis allows the grouping of behavioural sequences into discrete trajectories with assigned IDs. Trajectories and underlying behaviours generate a phenotypic fingerprint for each experimental condition, and representative cells are mathematically identified and graphically displayed for human understanding of each subtype. Here, we use cellPLATO to investigate the role of IL-15 in modulating NK cell migration on ICAM-1 or VCAM-1. We find 8 behavioural subsets of NK cells based on their shape and migration dynamics, and 4 trajectories of behaviour. Therefore, using cellPLATO we show that IL-15 increases plasticity between cell migration behaviours and that different integrin ligands induce different forms of NK cell migration.

iPSC-based modeling of helicase deficiency reveals impaired cell proliferation and increased apoptosis after NK cell lineage commitment.

Cell proliferation is a ubiquitous process required for organismal development and homeostasis. However, individuals with partial loss-of-function variants in DNA replicative helicase components often present with immunodeficiency due to specific loss of natural killer (NK) cells. Such lineage-specific disease phenotypes raise questions on how the proliferation is regulated in cell type-specific manner. We aimed to understand NK cell-specific proliferative dynamics and vulnerability to impaired helicase function using iPSCs from individuals with NK cell deficiency (NKD) due to hereditary compound heterozygous GINS4 variants. We observed and characterized heterogeneous cell populations that arise during the iPSC differentiation along with NK cells. While overall cell proliferation decreased with differentiation, early NK cell precursors showed a short burst of cell proliferation. GINS4 deficiency induced replication stress in these early NK cell precursors, which are poised for apoptosis, and ultimately recapitulate the NKD phenotype.

Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.

Mapping human natural killer cell development in pediatric tonsil by imaging mass cytometry and high-resolution microscopy.

Natural killer (NK) cells develop from CD34+ progenitors in a stage-specific manner defined by changes in cell surface receptor expression and function. Secondary lymphoid tissues, including tonsil, are sites of human NK cell development. Here we present new insights into human NK cell development in pediatric tonsil using cyclic immunofluorescence and imaging mass cytometry. We show that NK cell subset localization and interactions are dependent on NK cell developmental stage and tissue residency. NK cell progenitors are found in the interfollicular domain in proximity to cytokine-expressing stromal cells that promote proliferation and maturation. Mature NK cells are primarily found in the T-cell rich parafollicular domain engaging in cell-cell interactions that differ depending on their stage and tissue residency. The presence of local inflammation results in changes in NK cell interactions, abundance, and localization. This study provides the first comprehensive atlas of human NK cell development in secondary lymphoid tissue.

An inference model gives insights into innate immune adaptation and repertoire diversity.

The innate immune system is the body's first line of defense against infection. Natural killer (NK) cells, a vital part of the innate immune system, help to control infection and eliminate cancer. Studies have identified a vast array of receptors that NK cells use to discriminate between healthy and unhealthy cells. However, at present, it is difficult to explain how NK cells will respond to novel stimuli in different environments. In addition, the expression of different receptors on individual NK cells is highly stochastic, but the reason for these variegated expression patterns is unclear. Here, we studied the recognition of unhealthy target cells as an inference problem, where NK cells must distinguish between healthy targets with normal variability in ligand expression and ones that are clear "outliers." Our mathematical model fits well with experimental data, including NK cells' adaptation to changing environments and responses to different target cells. Furthermore, we find that stochastic, "sparse" receptor expression profiles are best able to detect a variety of possible threats, in agreement with experimental studies of the NK cell repertoire. While our study was specifically motivated by NK cells, our model is general and could also apply more broadly to explain principles of target recognition for other immune cell types.

The CD58-CD2 axis is co-regulated with PD-L1 via CMTM6 and shapes anti-tumor immunity.

The cell-autonomous balance of immune-inhibitory and -stimulatory signals is a critical process in cancer immune evasion. Using patient-derived co-cultures, humanized mouse models, and single-cell RNA-sequencing of patient melanomas biopsied before and on immune checkpoint blockade, we find that intact cancer cell-intrinsic expression of CD58 and ligation to CD2 is required for anti-tumor immunity and is predictive of treatment response. Defects in this axis promote immune evasion through diminished T cell activation, impaired intratumoral T cell infiltration and proliferation, and concurrently increased PD-L1 protein stabilization. Through CRISPR-Cas9 and proteomics screens, we identify and validate CMTM6 as critical for CD58 stability and upregulation of PD-L1 upon CD58 loss. Competition between CD58 and PD-L1 for CMTM6 binding determines their rate of endosomal recycling over lysosomal degradation. Overall, we describe an underappreciated yet critical axis of cancer immunity and provide a molecular basis for how cancer cells balance immune inhibitory and stimulatory cues.

Deciphering the localization and trajectory of human natural killer cell development.

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.

PGE2 expression by HPV6/11-induced respiratory papillomas blocks NK cell activation in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP), a rare chronic disease caused primarily by human papillomavirus types 6 and 11, consists of repeated growth of premalignant papillomas in the airway. RRP is characterized by multiple abnormalities in innate and adaptive immunity. Natural killer (NK) cells play important roles in immune surveillance and are part of the innate immune responses that help prevent tumor growth. We identified that papillomas lack classical class I MHC and retain nonclassical class I MHC expression. Moreover, in this study, we have identified and characterized the mechanism that blocks NK cell targeting of papilloma cells. Here, we show for the first time that the PGE2 secreted by papilloma cells directly inhibits NK cells activation/degranulation principally through the PGE2 receptor EP2, and to a lesser extent through EP4 signaling. Thus, papilloma cells have a potent mechanism to block NK cell function that likely supports papilloma cell growth.

Bearing a grudge-How human natural killer cells remember past insults.

Natural killer (NK) cell pathogen-specific memory is selected and maintained in the absence of antigen receptor rearrangement.

Human natural killer cells: Form, function, and development.

Human natural killer (NK) cells are innate lymphoid cells that mediate important effector functions in the control of viral infection and malignancy. Their ability to distinguish "self" from "nonself" and lyse virally infected and tumorigenic cells through germline-encoded receptors makes them important players in maintaining human health and a powerful tool for immunotherapeutic applications and fighting disease. This review introduces our current understanding of NK cell biology, including key facets of NK cell differentiation and the acquisition and execution of NK cell effector function. Further, it addresses the clinical relevance of NK cells in both primary immunodeficiency and immunotherapy. It is intended to provide an up-to-date and comprehensive overview of this important and interesting innate immune effector cell subset.

An ELF4 hypomorphic variant results in NK cell deficiency.

NK cell deficiencies (NKD) are a type of primary immune deficiency in which the major immunologic abnormality affects NK cell number, maturity, or function. Since NK cells contribute to immune defense against virally infected cells, patients with NKD experience higher susceptibility to chronic, recurrent, and fatal viral infections. An individual with recurrent viral infections and mild hypogammaglobulinemia was identified to have an X-linked damaging variant in the transcription factor gene ELF4. The variant does not decrease expression but disrupts ELF4 protein interactions and DNA binding, reducing transcriptional activation of target genes and selectively impairing ELF4 function. Corroborating previous murine models of ELF4 deficiency (Elf4-/-) and using a knockdown human NK cell line, we determined that ELF4 is necessary for normal NK cell development, terminal maturation, and function. Through characterization of the NK cells of the proband, expression of the proband's variant in Elf4-/- mouse hematopoietic precursor cells, and a human in vitro NK cell maturation model, we established this ELF4 variant as a potentially novel cause of NKD.

Partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia.

Human NK cell deficiency (NKD) is a primary immunodeficiency in which the main clinically relevant immunological defect involves missing or dysfunctional NK cells. Here, we describe a familial NKD case in which 2 siblings had a substantive NKD and neutropenia in the absence of other immune system abnormalities. Exome sequencing identified compound heterozygous variants in Go-Ichi-Ni-San (GINS) complex subunit 4 (GINS4, also known as SLD5), an essential component of the human replicative helicase, which we demonstrate to have a damaging impact upon the expression and assembly of the GINS complex. Cells derived from affected individuals and a GINS4-knockdown cell line demonstrate delayed cell cycle progression, without signs of improper DNA synthesis or increased replication stress. By modeling partial GINS4 depletion in differentiating NK cells in vitro, we demonstrate the causal relationship between the genotype and the NK cell phenotype, as well as a cell-intrinsic defect in NK cell development. Thus, biallelic partial loss-of-function mutations in GINS4 define a potentially novel disease-causing gene underlying NKD with neutropenia. Together with the previously described mutations in other helicase genes causing NKD, and with the mild defects observed in other human cells, these variants underscore the importance of this pathway in NK cell biology.

NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer.

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8+ T cells. In bladder tumors, NKG2A is acquired on CD8+ T cells later than PD-1 as well as other well-established immune checkpoints. NKG2A+ PD-1+ CD8+ T cells diverge from classically defined exhausted T cells through their ability to react to human leukocyte antigen (HLA) class I-deficient tumors using T cell receptor (TCR)-independent innate-like mechanisms. HLA-ABC expression by bladder tumors is progressively diminished as disease progresses, framing the importance of targeting TCR-independent anti-tumor functions. Notably, NKG2A+ CD8+ T cells are inhibited when HLA-E is expressed by tumors and partly restored upon NKG2A blockade in an HLA-E-dependent manner. Overall, our study provides a framework for subsequent clinical trials combining NKG2A blockade with other T cell-targeted immunotherapies, where tumors express higher levels of HLA-E.