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Supplementary Data from Identification and Targeting of the Developmental Blockade in Extranodal Natural Killer/T-cell Lymphoma

Supplementary Figure from Identification and Targeting of the Developmental Blockade in Extranodal Natural Killer/T-cell Lymphoma

Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with poor clinical outcomes. Here we used phenotypic and molecular profiling, including epigenetic analyses, to investigate how ENKTL ontogeny relates to normal NK-cell development. We demonstrate that neoplastic NK cells are stably, but reversibly, arrested at earlier stages of NK-cell maturation. Genes downregulated in the most epigenetic immature tumors were associated with polycomb silencing along with genomic gain and overexpression of EZH2. ENKTL cells exhibited genome-wide DNA hypermethylation. Tumor-specific DNA methylation gains were associated with polycomb-marked regions, involving extensive gene silencing and loss of transcription factor binding. To investigate therapeutic targeting, we treated novel patient-derived xenograft (PDX) models of ENKTL with the DNA hypomethylating agent, 5-azacytidine. Treatment led to reexpression of NK-cell developmental genes, phenotypic NK-cell differentiation, and prolongation of survival. These studies lay the foundation for epigenetic-directed therapy in ENKTL.Significance:Through epigenetic and transcriptomic analyses of ENKTL, a rare, aggressive malignancy, along with normal NK-cell developmental intermediates, we identified that extreme DNA hypermethylation targets genes required for NK-cell development. Disrupting this epigenetic blockade in novel PDX models led to ENKTL differentiation and improved survival.This article is highlighted in the In This Issue feature, p. 85

Background Pediatric nonmalignant lymphoproliferative disorders (PLPDs) are clinically and genetically heterogeneous. Long-standing immune dysregulation and lymphoproliferation in children may be life-threatening, and a paucity of data exists to guide evaluation and treatment of children with PLPD. Objective The primary objective of this study was to ascertain the spectrum of genomic immunologic defects in PLPD. Secondary objectives included characterization of clinical outcomes and associations between genetic diagnoses and those outcomes. Methods PLPD was defined by persistent lymphadenopathy, lymph organ involvement, or lymphocytic infiltration for more than 3 months, with or without chronic or significant Epstein-Barr virus (EBV) infection. Fifty-one subjects from 47 different families with PLPD were analyzed using whole exome sequencing. Results Whole exome sequencing identified likely genetic errors of immunity in 51% to 62% of families (53% to 65% of affected children). Presence of a genetic etiology was associated with younger age and hemophagocytic lymphohistiocytosis. Ten-year survival for the cohort was 72.4%, and patients with viable genetic diagnoses had a higher survival rate (82%) compared to children without a genetic explanation (48%, P = .03). Survival outcomes for individuals with EBV-associated disease and no genetic explanation were particularly worse than outcomes for subjects with EBV-associated disease and a genetic explanation (17% vs 90%; P = .002). Ascertainment of a molecular diagnosis provided targetable treatment options for up to 18 individuals and led to active management changes for 12 patients. Conclusions PLPD defines children at high risk for mortality, and whole exome sequencing informs clinical risks and therapeutic opportunities for this diagnosis.

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.

Recurrent respiratory papillomatosis (RRP), a rare chronic disease caused primarily by human papillomavirus (HPV) 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 non-classical 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 PGE

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.

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.

International audience; Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. This coupling often depends on the function of multidomain scaffolds that orchestrate transient interactions among multiple signaling intermediates and regulatory proteins on organelles. The number of possible scaffold interaction partners and the ability for these interactions to occur at different timescales makes investigations of scaffold functions challenging. This work employs live cell imaging to probe how the multidomain scaffold IQ motif containing GTPase activating protein 1 (IQGAP1) coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling. Using endosomes that are confined by a local actin network as a model system, we demonstrate that IQGAP1 can transition between different actin and endosomal membrane tethered states. Fast scaffold binding/disassociation transitions are shown to be driven by interactions between C-terminal scaffold domains and Rho GTPases at the membrane. Fluctuations in these binding modes are linked to negative regulation of actin polymerization. Although this control governs core elements of IQGAP1 dynamics, actin binding by the N-terminal calponin homology domain of the scaffold is shown to help the scaffold track the temporal development of endosome membrane markers, implying actin associations bolster membrane and actin coordination. Importantly, these effects are not easily distilled purely through standard (static) co-localization analyses or traditional pathway perturbations methods and were resolved by performing dynamic correlation and multiple regression analyses of IQGAP1 scaffold mutants. Using these capabilities with pharmacological inhibition, we provide evidence that membrane tethering is dependent on the activities of the lipid kinase phosphoinositide 3-kinase in addition to the Rho GTPases Rac1 and Cdc42. Overall, these methods and results point to a scaffold tethering mechanism that allows IQGAP1 to help control the amplitude of phosphoinositide lipid messenger signaling by coordinating signaling intermediate activities with the development and disassembly of local actin cytoskeletal networks.

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.

Migration is an important function for natural killer cells. Cell motility has implications in development, tissue infiltration, and cytotoxicity, and measuring the properties of natural killer (NK) cell migration using in vitro assays can be highly informative. Many researchers have an interest in studying properties of NK cell migration in the context of genetic mutation, disease, or in specific tissues and microenvironments. Motility assays can also provide information on the localization of proteins during different phases of cell migration. These assays can be performed on different surfaces for migration or coupled with chemoattractants and/or target cells to test functional outcomes or characterize cell migration speeds and phenotypes. NK cells undergo migration during differentiation in tissue, and these conditions can be modeled by culturing NK cells on a confluent bed of stromal cells on glass and imaging cell migration. Alternatively, fibronectin- or ICAM-1-coated surfaces promote NK cell migration and can be used as substrates. Here, we will describe techniques for the experimental setup and analysis of NK cell motility assays by confocal microscopy or in-incubator imaging using commercially available systems. Finally, we describe open-source software for analyzing cell migration using manual tracking or automated approaches and discuss considerations for the implementation of each of these methods.

The cell autonomous balance of immune-inhibitory and -stimulatory signals is a critical yet poorly understood process in cancer immune evasion. Using patient-derived co-culture models and humanized mouse models, we show that an intact CD58:CD2 interaction is necessary for anti-tumor immunity. Defects in this axis lead to multi-faceted immune evasion through impaired CD2-dependent T cell polyfunctionality, T cell exclusion, impaired intra-tumoral proliferation, and concurrent protein stabilization of PD-L1. We performed genome-scale CRISPR-Cas9 and CD58 coimmunoprecipitation mass spectrometry screens identifying CMTM6 as a key stabilizer of CD58, and show that CMTM6 is required for concurrent upregulation of PD-L1 in CD58 loss. Single-cell RNA-seq analysis of patient melanoma samples demonstrates that most TILs lack expression of primary costimulatory signals required for response to PD-1 blockade (e.g. CD28), but maintain strong CD2 expression, thus providing an opportunity to mobilize a so far therapeutically untapped pool of TILs for anti-tumor immunity. We identify two potential therapeutic avenues, including rescued activation of human CD2-expressing TILs using recombinant CD58 protein, and targeted disruption of PD-L1/CMTM6 interactions. Our work identifies an underappreciated yet critical axis at the nexus of cancer immunity and evasion, uncovers a fundamental mechanism of co-inhibitory and -stimulatory signal balancing, and provides new approaches to improving cancer immunotherapies.

SummaryPD-1/PD-L1-blockade immunotherapies have limited efficacy in the treatment of muscle-invasive bladder cancer (MIBC) and metastatic urothelial carcinoma. Here, we show thatKLRC1(NKG2A) expression associates with improved survival and responsiveness to PD-L1 blockade immunotherapy inCD8Ahighbladder tumors. The loss of antigen presentation is a common mechanism for tumor escape in bladder cancer. NKG2A+CD8 T cells are able to circumvent HLA-ABC loss through TCR-independent cytotoxicity, which is partly mediated by DNAM-1. In bladder tumors, NKG2A is acquired on a subset of PD-1+CD8 T cells, alongside stronger tissue-residency memory features, TCR-independent cytotoxicity and evidence of recent proliferation. HLA-E is low but variably expressed on bladder tumors. When expressed, NKG2A+CD8 T cell anti-tumor responses to HLA-ABC-deficient tumors are inhibited and partly restored upon NKG2A blockade. Overall, our study identifies an alternative path for CD8 T cell exhaustion, that is mediated by NKG2A upregulation and TCR-independent cytotoxicity.

Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with poor clinical outcomes. Here we used phenotypic and molecular profiling, including epigenetic analyses, to investigate how ENKTL ontogeny relates to normal NK-cell development. We demonstrate that neoplastic NK cells are stably, but reversibly, arrested at earlier stages of NK-cell maturation. Genes downregulated in the most epigenetic immature tumors were associated with polycomb silencing along with genomic gain and overexpression of EZH2. ENKTL cells exhibited genome-wide DNA hypermethylation. Tumor-specific DNA methylation gains were associated with polycomb-marked regions, involving extensive gene silencing and loss of transcription factor binding. To investigate therapeutic targeting, we treated novel patient-derived xenograft (PDX) models of ENKTL with the DNA hypomethylating agent, 5-azacytidine. Treatment led to reexpression of NK-cell developmental genes, phenotypic NK-cell differentiation, and prolongation of survival. These studies lay the foundation for epigenetic-directed therapy in ENKTL. Significance: Through epigenetic and transcriptomic analyses of ENKTL, a rare, aggressive malignancy, along with normal NK-cell developmental intermediates, we identified that extreme DNA hypermethylation targets genes required for NK-cell development. Disrupting this epigenetic blockade in novel PDX models led to ENKTL differentiation and improved survival. This article is highlighted in the In This Issue feature, p. 85

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

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

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.

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.

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.

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. MCM10 mutant cells demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in MCM10-mutant cells was predominantly comprised of replication initiation defects. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel modulator of DNA replication timing.

Human papillomavirus (HPV) infections underlie a wide spectrum of both benign and malignant epithelial diseases. In this report, we describe the case of a young man who had encephalitis caused by herpes simplex virus during adolescence and currently presented with multiple recurrent skin and mucosal lesions caused by HPV. The patient was found to have a pathogenic germline mutation in the X-linked interleukin-2 receptor subunit gamma gene (IL2RG), which was somatically reverted in T cells but not in natural killer (NK) cells. Allogeneic hematopoietic-cell transplantation led to restoration of NK cytotoxicity, with normalization of the skin microbiome and persistent remission of all HPV-related diseases. NK cytotoxicity appears to play a role in containing HPV colonization and the ensuing HPV-related hyperplastic or dysplastic lesions. (Funded by the National Institutes of Health and the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources.).

A baby girl born at 38 weeks of gestation with no perinatal complications was brought to our attention for possible severe combined immunodeficiency (SCID) because of low T cell receptor excision circles (TRECs) (11/mcL, reference range > 18) on newborn screen. Blood was collected for confirmatory flow cytometry and the absolute number of T cells was borderline low (2116/mcL, reference range 2500–5500) but sufficient to rule out SCID. However, her NK cell enumeration showed absent (< 1%) NK cells (CD3(−) CD56(+) CD16(+) by BD Biosciences 6-color TBNK, San Jose, CA). Subsequent testing at a local laboratory identified normal numbers of NK cells (26%), but at this laboratory, NK cells were defined as (CD3(−) CD56(+) by locally derived reagents). Complete laboratory values can be found in Supplementary Table E1. We therefore pursued further testing to determine if CD16 was absent from the patient’s cells or if there was a lack of targeting by the specific anti-CD16 monoclonal antibody used.

We report the natural history, clinical manifestations, genetics, and immunohematological findings in 14 patients from 11 families with ARPC1B deficiency, delineating the spectrum of the disease that appears progressive and challenging to manage clinically.

Germline mutations in GATA2 are associated with a complex immunodeficiency and cancer predisposition syndrome. Somatic GATA2mut in myeloid malignancies may impart a similar phenotype. We reviewed adult patients with a diagnosis of GATA2mut hematological malignancy who were referred to our HHMC for genetic testing, and identified to have somatic GATA2mut. Nine patients with a median age of 63 years were included. Six patients (66.7%) were males. Atypical CML and acute myeloid leukemia were the most common initial presentation. The median overall VAF was 47.14%. Monocytopenia was pronounced when the GATA2mut involved the C-terminal ZFD. GATA2 N-terminal ZFD mutations tend to be co-mutated with biCEBPAmut. Unlike germline GATA2 mutations, monocytopenia associated with somatic GATA2 mutations often resolved at remission. We concluded that similar to germline GATA2 mutations, a subset of somatic GATA2 mutations can impart a germline phenotype.

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.