Expression Of Natural Killer Cell Receptors Research Articles

NK-cell receptor modulation in viral infections.

Natural killer (NK) cells play a crucial role in controlling viral infections. The ability to kill infected cells without prior immunization, yet being tolerant to self, healthy cells, depends on the balance of germ-line encoded surface receptors. NK-cell receptors are divided into either activating, leading to activation of NK cell and its cytotoxic and pro-inflammatory activity, or inhibitory, providing tolerance for a target cell. The signals from inhibitory receptors dominate and NK-cell activation requires stimulation of activating receptors. In viral infections, NK-cell interaction with infected cells can result in activation, memory-like NK-cell differentiation, or NK-cell exhaustion, which constitutes one of the viral immune evasion mechanisms. All of these states are associated with the modulation of NK-cell receptor expression. In this review, we summarize the current knowledge of NK-cell receptors and their role in viral infection control, as well as the alterations of their expression observed in acute or chronic infections. We present recently discovered SARS-CoV-2-mediated modulation of NK-cell receptor expression and compare them with other human viral infections. Finally, since modulation of NK-cell receptor activation gives a promising addition to currently used antiviral therapies, we briefly discuss the clinical significance and future perspective of the application of agonists or antagonists of activating and inhibitory receptors, respectively. In sum, our review shows that although much is known about NK-cell receptor biology, a deeper understanding of NK-cell receptors role in viral infections is still needed.

Establishing NK-Cell Receptor Restriction by Flow Cytometry and Detecting Potential NK-Cell Clones of Uncertain Significance

Natural killer (NK) cells develop a complex inhibitory and/or activating NK-cell receptor system, including killer cell immunoglobulin-like receptors (KIRs or CD158) and CD94/NKG2 dimers, which are variably combined to generate the individual’s NK-cell receptor repertoire. Establishing NK-cell receptor restriction by flow cytometric immunophenotyping is an important step in diagnosing NK-cell neoplasms, but reference interval (RI) data for interpreting these studies are lacking. Specimens from 145 donors and 63 patients with NK-cell neoplasms were used to identify discriminatory rules based on 95% and 99% nonparametric RIs for CD158a+, CD158b+, CD158e+, KIR-negative, and NKG2A+ NK-cell populations to establish NK-cell receptor restriction. These 99% upper RI limits (NKG2a >88% or CD158a >53% or CD158b >72% or CD158e >54% or KIR-negative >72%) provided optimal discrimination between NK-cell neoplasm cases and healthy donor controls with an accuracy of 100% compared with the clinicopathologic diagnosis. The selected rules were applied to 62 consecutive samples received in our flow cytometry laboratory that were reflexed to an NK-cell panel due to an expanded NK-cell percentage (exceeding 40% of total lymphocytes). Twenty-two (35%) of 62 samples were found to harbor a very small NK-cell population with restricted NK-cell receptor expression based on the rule combination, suggestive of NK-cell clonality. A thorough clinicopathologic evaluation for the 62 patients did not reveal diagnostic features of NK-cell neoplasms; therefore, these potential clonal populations of NK cells were designated as NK-cell clones of uncertain significance (NK-CUS). In this study, we established decision rules for NK-cell receptor restriction from the largest published cohorts of healthy donors and NK-cell neoplasms. The presence of small NK-cell populations with restricted NK-cell receptors does not appear to be an uncommon finding, and its significance requires further exploration.

Immune profile of primary and recurrent epithelial ovarian cancer cases indicates immune suppression, a major cause of progression and relapse of ovarian cancer

BackgroundOvarian cancer is the third most prevalent cancer in Indian women. Relative frequency of High grade serous epithelial ovarian cancer (HGSOC) and its associated deaths are highest in India which suggests the importance of understanding their immune profiles for better treatment modality. Hence, the present study investigated the NK cell receptor expression, their cognate ligands, serum cytokines, and soluble ligands in primary and recurrent HGSOC patients. We have used multicolor flow cytometry for immunophenotyping of tumor infiltrated and circulatory lymphocytes. Procartaplex, and ELISA were used to measure soluble ligands and cytokines of HGSOC patients.ResultsAmong the enrolled 51 EOC patients, 33 were primary high grade serous epithelial ovarian cancer (pEOC) and 18 were recurrent epithelial ovarian cancer (rEOC) patients. Blood samples from 46 age matched healthy controls (HC) were used for comparative analysis. Results revealed, frequency of circulatory CD56Bright NK, CD56Dim NK, NKT-like, and T cells was reduced with activating receptors while alterations in immune subsets with inhibitory receptors were observed in both groups. Study also highlights differential immune profile of primary and recurrent ovarian cancer patients. We have found increased soluble MICA which might have acted as “decoy” molecule and could be a reason of decrease in NKG2D positive subsets in both groups of patients. Furthermore, elevated level of serum cytokines IL-2, IL-5, IL-6, IL-10, and TNF-α in ovarian cancer patients, might be associated with ovarian cancer progression. Profiling of tumor infiltrated immune cells revealed the reduced level of DNAM-1 positive NK and T cells in both groups than their circulatory counterpart, which might have led to decrease in NK cell’s ability of synapse formation.ConclusionsThe study brings out differential receptor expression profile on CD56BrightNK, CD56DimNK, NKT-like, and T cells, cytokines levels and soluble ligands which may be exploited to develop alternate therapeutic approaches for HGSOC patients. Further, few differences in the circulatory immune profiles between pEOC and rEOC cases, indicates the immune signature of pEOC undergoes some changes in circulation that might facilitated the disease relapse. They also maintains some common immune signatures such as reduced expression of NKG2D, high level of MICA as well as IL-6, IL10 and TNF-α, which indicates irreversible immune suppression of ovarian cancer patients. It is also emphasized that a restoration of cytokines level, NKG2D and DNAM-1on tumor infiltrated immune cells may be targeted to develop specific therapeutic approaches for high-grade serous epithelial ovarian cancer.

SUMOylation and related post-translational modifications in natural killer cell anti-cancer responses.

SUMOylation is a reversible modification that involves the covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins, leading to changes in their localization, function, stability, and interactor profile. SUMOylation and additional related post-translational modifications have emerged as important modulators of various biological processes, including regulation of genomic stability and immune responses. Natural killer (NK) cells are innate immune cells that play a critical role in host defense against viral infections and tumors. NK cells can recognize and kill infected or transformed cells without prior sensitization, and their activity is tightly regulated by a balance of activating and inhibitory receptors. Expression of NK cell receptors as well as of their specific ligands on target cells is finely regulated during malignant transformation through the integration of different mechanisms including ubiquitin- and ubiquitin-like post-translational modifications. Our review summarizes the role of SUMOylation and other related pathways in the biology of NK cells with a special emphasis on the regulation of their response against cancer. The development of novel selective inhibitors as useful tools to potentiate NK-cell mediated killing of tumor cells is also briefly discussed.

High dimensional analysis reveals distinct NK cell subsets but conserved response to stimulation in umbilical cord blood and adult peripheral blood.

Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20-color flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56neg as well as mature NKp46neg and CD56+ CD16+ NK cell populations in UCB whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL-12, IL-15, and IL-18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16- NK cell populations, TIGIT+ NK cells produced more IFN-γ than their TIGIT- counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion.

Epigenetic modulation and prostate cancer: Paving the way for NK cell anti-tumor immunity.

Immunoepigenetics is a growing field, as there is mounting evidence on the key role played by epigenetic mechanisms in the regulation of tumor immune cell recognition and control of immune cell anti-tumor responses. Moreover, it is increasingly acknowledgeable a tie between epigenetic regulation and prostate cancer (PCa) development and progression. PCa is intrinsically a cold tumor, with scarce immune cell infiltration and low inflammatory tumor microenvironment. However, Natural Killer (NK) cells, main anti-tumor effector immune cells, have been frequently linked to improved PCa prognosis. The role that epigenetic-related mechanisms might have in regulating both NK cell recognition of PCa tumor cells and NK cell functions in PCa is still mainly unknown. Epigenetic modulating drugs have been showing boundless therapeutic potential as anti-tumor agents, however their role in immune cell regulation and recognition is scarce. In this review, we focused on studies addressing modulation of epigenetic mechanisms involved in NK cell-mediated responses, including both the epigenetic modulation of tumor cell NK ligand expression and NK cell receptor expression and function in different tumor models, highlighting studies in PCa. The integrated knowledge from diverse epigenetic modulation mechanisms promoting NK cell-mediated immunity in various tumor models might open doors for the development of novel epigenetic-based therapeutic options for PCa management.

Preventing Surgery-Induced NK Cell Dysfunction Using Anti-TGF-β Immunotherapeutics.

Natural Killer (NK) cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed postoperatively. This dysfunction is associated with increased morbidity and cancer recurrence. NK activity depends on the integration of activating and inhibitory signals, which may be modulated by transforming growth factor-beta (TGF-β). We hypothesized that impaired postoperative NK cell IFNγ production is due to altered signaling pathways caused by postoperative TGF-β. NK cell receptor expression, downstream phosphorylated targets, and IFNγ production were assessed using peripheral blood mononuclear cells (PBMCs) from patients undergoing cancer surgery. Healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma and in the presence/absence of a TGF-β-blocking monoclonal antibody (mAb) or the small molecule inhibitor (smi) SB525334. Single-cell RNA sequencing (scRNA-seq) was performed on PBMCs from six patients with colorectal cancer having surgery at baseline/on POD1. Intracellular IFNγ, activating receptors (CD132, CD212, NKG2D, DNAM-1), and downstream target (STAT5, STAT4, p38 MAPK, S6) phosphorylation were significantly reduced on POD1. Furthermore, this dysfunction was phenocopied in healthy NK cells through incubation with rTGF-β1 or POD1 plasma and was prevented by the addition of anti-TGF-β immunotherapeutics (anti-TGF-β mAb or TGF-βR smi). Targeted gene analysis revealed significant decreases in S6 and FKBP12, an increase in Shp-2, and a reduction in NK metabolism-associated transcripts on POD1. pSmad2/3 was increased and pS6 was reduced in response to rTGF-β1 on POD1, changes that were prevented by anti-TGF-β immunotherapeutics. Together, these results suggest that both canonical and mTOR pathways downstream of TGF-β mediate phenotypic changes that result in postoperative NK cell dysfunction.

IL-2 Combined with IL-15 Enhanced the Expression of NKG2D Receptor on Patient Autologous NK Cells to Inhibit Wilms' Tumor via MAPK Signaling Pathway.

Objective The dysfunction of immune surveillance, a hot spot in cancer research, could lead to the occurrence and development in multicancers. However, the potential mechanisms of immunity in Wilms' tumor (WT) remain unclear on Wilms' tumor (WT). In this study, we aim to investigate the immune cell in WT and explore the underlying treatment strategy. Method We quantified stromal and immune scores by using ESTIMATE algorithm based on gene expression matrix of WT patients in TCGA and GEO databases. Different expression genes (DEGs) and functional enrichments were analyzed by R studio and DAVID tools. Flow cytometry, immunofluorescence staining, ELISA assay, and qRT-PCR were used for detecting the NK cells, cytotoxic cytokines (INF-γ, PRF, and GZMB), and NK cell receptor expression, respectively. WT patient autologous NK cells were stimulated by IL-2 and IL-15, and the cytotoxicity of NK cells against WT cell lines was detected by LDH assay. Western blot experiment was used for measuring the MAPK signaling pathway protein maker in NK cells. Results ESTIMATE indicated that WT tissue had a lower immune score than adjacent kidney tissue. Meanwhile, the low immune score group was associated with poorly outcomes. DEG functional enrichment analysis showed that NK cell-mediated cytotoxicity was significantly different in low and high immune score groups. Although few of proportion of NK cells in WT patients were increased, most of that were significantly lower than normal children. Moreover, the proportion of NK cells and the expression level of INF-γ, PRF, and GZMB in WT tissue were lower than adjacent kidney tissue. Importantly, the NKG2D expression level of NK cells was significantly lower in WT tissue. Furthermore, in vitro, compared with uncultured NK cells, IL-2 and IL-15 could effectively enhance the cytotoxicity of NK cells on killing the WT cell lines. The FACS and WB results showed that the NKG2D and p-PI3K ratio PI3K, MEK1/2, and p-ERK1/2 ratio ERK1/2 were significantly increased in IL-2 and IL15 group compared with uncultured groups. Conclusion The abnormal NK cell-mediated cytotoxicity may cause the occurrence of WT. Costimulation of WT patients autologous NK cells could effectively enhance the antitumor reaction which involved in activation of NKG2D-mediated MAPK signaling pathway.

New Insights and Implications of Natural Killer Cells in Parkinson's Disease.

Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by the loss of dopaminergic neurons in the substantia nigra and the abnormal aggregation and accumulation of the alpha-synuclein (α-syn) protein into Lewy bodies. It is established that there is an association between inflammation and PD; however, the time course of the inflammatory process as well as the immune cells involved are still debated. Natural killer (NK) cells are innate lymphocytes with numerous functions including targeting and killing infected or malignant cells, antimicrobial defense, and resolving inflammation. NK cell subsets differ in their effector function capacities which are modulated by activating and inhibitory receptors expressed at the cell surface. Alterations in NK cell numbers and receptor expression have been reported in PD patients. Recently, NK cell numbers and frequency were shown to be altered in the periphery and in the central nervous system in a preclinical mouse model of PD. Moreover, NK cells have recently been shown to internalize and degrade α-syn aggregates and systemic NK cell depletion exacerbated synuclein pathology in a preclinical mouse model of PD, indicating a potential protective role of NK cells. Here, we review the inflammatory process in PD with a particular focus on alterations in NK cell numbers, phenotypes, and functions.

Highly active selenium nanotherapeutics combined with metformin to achieve synergistic sensitizing effect on NK cells for osteosarcoma therapy

Abstract NK cells-based cancer therapy combined with chemotherapeutic drugs for the treatment of tumors can enhance the immunosensitivity of NK cells, increase the expression of NK cell receptors, and eventually boost the killing effect of NK cells on cancer cells. Selenium (Se) with different chemical structures can be metabolized into selenoproteins to regulate tumor and immune cells’ fate and functions. Herein, we found that, functionalized Se nanoparticles (SeNPs) combining with metformin (met) could amply the immunotherapeutic effects of NK92 cells against osteosarcoma cancer. The results revealed that TW80-SeNPs combined with met had the optimum performance on NK92 cells for HepG2 cells, owing to the increased ROS in HepG2 cells and the augmented expression of cell surface receptor proteins ULBP-3/4, PD-L1, MICA, and NK92 cell surface receptor proteins PD-1 and FasL. Additionally, TW80-SeNPs were gradually metabolized into selenoproteins (Gpx4 and TR1) into human osteosarcoma MG63 cells to reinforce the anticancer effect of NK92 cells by regulating the redox balance in the tumor microenvironment. This study provides a therapeutic approach in treating cancer itself or diabetes coupled with cancer. Moreover, it provides a multidrug strategy to improve immune cell function in practical applications, especially for synergistic immunotherapy of osteosarcoma.

Abstract 6169: Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions

Abstract Pancreatic stellate cells (PSCs) are the main sources of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma (PDAC), which account for at least 50% of PDAC’s total tumor volume and its dense desmoplastic stroma. In recent years, immune-stromal cell interactions have been the subject of research in a variety of diseases. Interestingly, natural killer (NK) cells are relatively understudied in PDAC and may be exploited to identify new treatment opportunities. We have explored how NK cell-PSC interactions are mediated by known NK cell ligands. Human PSC (hPSC, CRC-811) or PDAC (MIA-PaCa-2, PANC-1) cell lines were co-cultured with DiO stained NK cells (NK-92, donor NK cells) for 4 hours, then target cells were assessed for apoptosis by flow cytometry using AnnexinV and Sytox stains. We found that although NK cells lyse both PSCs and PDAC cells, there was more NK cell-mediated lysis against PSCs. Using flow cytometry, we found that PSCs express the well-known MICA/B ligand, which binds the activating NKG2D receptor on NK cells to initiate its cytotoxic activity. In co-culture, neutralizing MICA/B in vitro did not completely inhibit NK cell-mediated lysis of PSCs, suggesting other interactions need to be explored. To identify other molecular determinants, NK cell ligand and receptor expression was evaluated by quantitative PCR in human PSC, PDAC, NK, and other leukocytic (K562) cell lines. Cell surface validation of highly expressed NK cell ligands and receptors were confirmed by flow cytometry and/or Western blot. NK cell ligands that were both highly expressed and localized to the cell surface were: NKG2D ligands—MICA and ULBP2 (both activating), KIR2DS4 (CD158I) ligand—HLA-A (activating), and NKp44 ligands—NKp44L and PCNA (activating and inhibitory, respectively). Their corresponding receptors were also expressed and localized to the cell surface by NK cells. These results highlight novel NK cell-PSC interactions that may mediate immune suppression in PDAC. These findings warrant further investigation. Citation Format: Zoe X. Malchiodi, Allison Fitzgerald, Elena Galvano, Louis M. Weiner. Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6169.

Inhibition of O-GlcNAcylation Decreases the Cytotoxic Function of Natural Killer Cells.

Natural killer (NK) cells mediate killing of malignant and virus-infected cells, a property that is explored as a cell therapy approach in the clinic. Various cell intrinsic and extrinsic factors affect NK cell cytotoxic function, and an improved understanding of the mechanism regulating NK cell function is necessary to accomplish better success with NK cell therapeutics. Here, we explored the role of O-GlcNAcylation, a previously unexplored molecular mechanism regulating NK cell function. O-GlcNAcylation is a post-translational modification mediated by O-GlcNAc transferase (OGT) that adds the monosaccharide N-acetylglucosamine to serine and threonine residues on intracellular proteins and O-GlcNAcase (OGA) that removes the sugar. We found that stimulation of NK cells with the cytokines interleukin-2 (IL-2) and IL-15 results in enhanced O-GlcNAcylation of several cellular proteins. Chemical inhibition of O-GlcNAcylation using OSMI-1 was associated with a decreased expression of NK cell receptors (NKG2D, NKG2A, NKp44), cytokines [tumor necrosis factor (TNF)-α, interferon (IFN-γ)], granulysin, soluble Fas ligand, perforin, and granzyme B in NK cells. Importantly, inhibition of O-GlcNAcylation inhibited NK cell cytotoxicity against cancer cells. However, increases in O-GlcNAcylation following OGA inhibition using an OGA inhibitor or shRNA-mediated suppression did not alter NK cell cytotoxicity. Finally, we found that NK cells pretreated with OSMI-1 to inhibit O-GlcNAcylation showed compromised cytotoxic activity against tumor cells in vivo in a lymphoma xenograft mouse model. Overall, this study provides the seminal insight into the role of O-GlcNAcylation in regulating NK cell cytotoxic function.

Combinatorial Expression of NK Cell Receptors Governs Cell Subset Reactivity and Effector Functions but Not Tumor Specificity.

NK cell receptors allow NK cells to recognize targets such as tumor cells. Many of them are expressed on a subset of NK cells, independently of each other, which creates a vast diversity of receptor combinations. Whether these combinations influence NK cell antitumor responses is not well understood. We addressed this question in the C57BL/6 mouse model and analyzed the individual effector response of 444 mouse NK cell subsets, defined by combinations of 12 receptors, against tumor cell lines originating from different tissues and mouse strains. We found a wide range of reactivity among NK subsets, but the same hierarchy of responses was observed for the different tumor types, showing that the repertoire of NK cell receptors does not encode for different tumor specificities but for different intrinsic reactivities. The coexpression of CD27, NKG2A, and DNAM-1 identified subsets with relative cytotoxic specialization, whereas reciprocally, CD11b and KLRG1 defined the best IFN-γ producers. The expression of educating receptors Ly49C, Ly49I, and NKG2A was also strongly correlated with IFN-γ production, but this effect was suppressed by unengaged receptors Ly49A, Ly49F, and Ly49G2. Finally, IL-15 coordinated NK cell effector functions, but education and unbound inhibitory receptors retained some influence on their response. Collectively, these data refine our understanding of the mechanisms governing NK cell reactivity, which could help design new NK cell therapy protocols.

Abstract MP05: The Impact Of Neighborhood Social Environment On Natural Killer Cells In A Community-based Cohort Of African American Women

Introduction: Chronic exposure to environmental stressors is associated with racial and ethnic disparities in cardiovascular disease (CVD) risk factors. African American (AA) women are at highest risk for obesity and subsequent CVD development. Obesity as a CVD risk factor and psychosocial stressors have been associated with altered Natural Killer (NK) cell distribution and function. Less is known about how perceived and objective neighborhood social environment relates to NK cell distribution and function. Hypothesis: More favorable neighborhood social environment would associate with a greater number and more functional NK cell population. Methods: We recruited 40 AA women from a Washington, DC community-based cohort and determined the NK cell profile using flow cytometry according to their CD3/CD56 expression on the cell surface. Neighborhood social environment was measured by neighborhood environment (NE) perceptions and objective neighborhood deprivation index (NDI). NE perceptions were measured using validated questionnaires to calculate overall NE perceptions, perceived neighborhood violence, physical/social environment, social cohesion, and safety (higher score=more favorable perceptions). NDI was measured using U.S. Census data on census-tract level features (i.e., housing characteristics, education, income, poverty) where higher NDI=lower NE socioeconomic level. Multivariable regression models were used to determine associations between neighborhood social environment and NK cell number. Additionally, regression models were adjusted for Atherosclerotic Cardiovascular Disease (ASCVD) risk and BMI, unless otherwise specified. Results: Among this community-based cohort of AA women (N=40 mean age 58.8 ±12.0; BMI 34.2 ±6.9), overall NE perceptions were associated with overall NK cell numbers before and after adjustments for ASCVD and BMI (β=0.48, p=0.007, β=0.50, p=0.004). Perceived lack of violence was associated with increased NK cell number (β=0.38, p=0.04) even after the adjustment for ASCVD and BMI (β=0.39, p=0.03). Similar trends were observed with social cohesion and NK cells (β=0.32, p=0.06) and with perceived favorable physical/social environment and NK cells (β=0.34, p=0.07). In contrast, we found that safety did not show any significant association with NK cell numbers (β=0.28, p=0.13). Finally, NDI as an objective measure of neighborhood SES did not associate with NK cell numbers (β=-0.05, p=0.74). Conclusion: Together, our data suggest that perceived neighborhood social environment associates with NK cell numbers, while objective measures do not. These findings suggest that changes in NK cell populations may be driven by perceived social environment factors and could alter CV risk. Future studies are needed to determine the potential impact of social environment stressors on NK cell subtypes, receptor expression, and function.

Impact of NK Cell Activating Receptor Gene Variants on Receptor Expression and Outcome of Immunotherapy in Acute Myeloid Leukemia.

Natural killer cells are important effector cells in the immune response against myeloid malignancies. Previous studies show that the expression of activating NK cell receptors is pivotal for efficient recognition of blasts from patients with acute myeloid leukemia (AML) and that high expression levels impact favorably on patient survival. This study investigated the potential impact of activating receptor gene variants on NK cell receptor expression and survival in a cohort of AML patients receiving relapse-preventive immunotherapy with histamine dihydrochloride and low-dose IL-2 (HDC/IL-2). Patients harboring the G allele of rs1049174 in the KLRK1 gene encoding NKG2D showed high expression of NKG2D by CD56bright NK cells and a favorable clinical outcome in terms of overall survival. For DNAM-1, high therapy-induced receptor expression entailed improved survival, while patients with high DNAM-1 expression before immunotherapy associated with unfavorable clinical outcome. The previously reported SNPs in NCR3 encoding NKp30, which purportedly influence mRNA splicing into isoforms with discrete functions, did not affect outcome in this study. Our results imply that variations in genes encoding activating NK cell receptors determine receptor expression and clinical outcome in AML immunotherapy.

NK Cell Subpopulations and Receptor Expression in Recovering SARS-CoV-2 Infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic of coronavirus disease (COVID-19). Whereas in most cases COVID-19 is asymptomatic or pauci-symptomatic, extremely severe clinical forms are observed. In this case, complex immune dysregulations and an excessive inflammatory response are reported and are the main cause of morbidity and mortality. Natural killer cells are key players in the control of viral infection, and their activity is regulated by a tight balance between activating and inhibitory receptors; an alteration of NK activity was suggested to be associated with the development of severe forms of COVID-19. In this study, we analyzed peripheral NK cell subpopulations and the expression of activating and inhibitory receptors in 30 patients suffering from neurological conditions who recovered from mild, moderate, or severe SARS-CoV-2 infection, comparing the results to those of 10 SARS-CoV-2-uninfected patients. Results showed that an expansion of NK subset with lower cytolytic activity and an augmented expression of the 2DL1 inhibitory receptor, particularly when in association with the C2 ligand (KIR2DL1-C2), characterized the immunological scenario of severe COVID-19 infection. An increase of NK expressing the ILT2 inhibitory receptor was instead seen in patients recovering from mild or moderate infection compared to controls. Results herein suggest that the KIR2DL1-C2 NK inhibitory complex is a risk factor toward the development of severe form of COVID-19. Our results confirm that a complex alteration of NK activity is present in COVID-19 infection and offer a molecular explanation for this observation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-021-02517-4.

Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients.

Despite rapid progress in the treatment of many cancers, glioblastoma remains a devastating disease with dismal prognosis. The aim of this study was to identify chaperone- and immune-related biomarkers to improve prediction of outcome in glioblastoma. Depending on its intra- or extracellular localization the major stress-inducible heat shock protein 70 (Hsp70) fulfills different tasks. In the cytosol Hsp70 interferes with pro-apoptotic signaling pathways and thereby protects tumor cells from programmed cell death. Extracellular Hsp70 together with pro-inflammatory cytokines are reported to stimulate the expression of activatory NK cell receptors, recognizing highly aggressive human tumor cells that present Hsp70 on their cell surface. Therefore, intra-, extracellular and membrane-bound Hsp70 levels were assessed in gliomas together with activatory NK cell receptors. All gliomas were found to be membrane Hsp70-positive and high grade gliomas more frequently show an overexpression of Hsp70 in the nucleus and cytosol. Significantly elevated extracellular Hsp70 levels are detected in glioblastomas with large necrotic areas. Overall survival (OS) is more favorable in patients with low Hsp70 serum levels indicating that a high Hsp70 expression is associated with an unfavorable prognosis. The data provide a first hint that elevated frequencies of activated NK cells at diagnosis might be associated with a better clinical outcome.

Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.

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

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

Role of NKG2a/c+CD8+ T cells in pathogenic versus non-pathogenic SIV infections

SummarySome viruses have established an equilibrium with their host. African green monkeys (AGM) display persistent high viral replication in the blood and intestine during Simian immunodeficiency virus (SIV) infection but resolve systemic inflammation after acute infection and lack intestinal immune or tissue damage during chronic infection. We show that NKG2a/c+CD8+ T cells increase in the blood and intestine of AGM in response to SIVagm infection in contrast to SIVmac infection in macaques, the latter modeling HIV infection. NKG2a/c+CD8+ T cells were not expanded in lymph nodes, and CXCR5+NKG2a/c+CD8+ T cell frequencies further decreased after SIV infection. Genome-wide transcriptome analysis of NKG2a/c+CD8+ T cells from AGM revealed the expression of NK cell receptors, and of molecules with cytotoxic effector, gut homing, and immunoregulatory and gut barrier function, including CD73. NKG2a/c+CD8+ T cells correlated negatively with IL-23 in the intestine during SIVmac infection. The data suggest a potential regulatory role of NKG2a/c+CD8+ T cells in intestinal inflammation during SIV/HIV infections.