Circulating Natural Killer Cells Research Articles

An altered natural killer cell immunophenotype characterizes clinically severe pediatric RSV infection.

Respiratory syncytial virus (RSV) infects nearly all children by 2 years of age and is a leading cause of pediatric hospitalizations. A subset of children with RSV infection (RSV+ children) develop respiratory failure requiring intensive care, but immune mechanisms distinguishing severe pediatric RSV infection are not fully elucidated. Natural killer (NK) cells are key innate immune effectors of viral host defense. In this study of 47 critically ill RSV+ children, we coupled NK cell immunophenotype and cytotoxic function with clinical parameters to identify an NK cell immune signature of severe pediatric RSV disease. Airway NK cells were increased in intubated RSV+ children with severe hypoxemia and prolonged duration of mechanical ventilation and were correlated with clinical severity scores. Peripheral blood NK cells were decreased in RSV+ patients and had altered activating receptor expression, with increased expression of CD69 and decreased expression of NKG2D. Ex vivo, circulating NK cells from RSV+ patients exhibited functional impairment characterized by decreased cytotoxicity as well as aberrant immune synapse assembly and lytic granule trafficking. NK cell frequency and phenotype correlated with clinical measures that defined disease severity. These findings implicate a role for NK cells in mediating RSV immunopathology and suggest that an altered NK cell immunophenotype is associated with severe RSV disease in young children.

Progressive accumulation of hyperinflammatory NKG2Dlow NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic comorbidities remain ill-defined. Here, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aeroallergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed coincident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine tumor necrosis factor-α. These observations provide important insights into a potential mechanism underlying the development of allergic comorbidity in early life in children with AD, which involves altered NK cell functional responses, and define an endotype of severe AD.

Decreased Peripheral Blood Natural Killer Cell Count in Untreated Juvenile Dermatomyositis Is Associated with Muscle Weakness.

Juvenile Dermatomyositis (JDM) is the most common inflammatory myopathy in pediatrics. This study evaluates the role of Natural Killer (NK) cells in Juvenile Dermatomyositis (JDM) pathophysiology. The study included 133 untreated JDM children with an NK cell count evaluation before treatment. NK cell subsets (CD56low/dim vs. CD 56bright) were examined in 9 untreated children. CD56 and perforin were evaluated in situ in six untreated JDM and three orthopedic, pediatric controls. 56% of treatment-naive JDM had reduced circulating NK cell counts, designated "low NK cell". This low NK group had more active muscle disease compared to the normal NK cell group. The percentage of circulating CD56low/dim NK cells was significantly lower in the NK low group than in controls (0.55% vs. 4.6% p < 0.001). Examination of the untreated JDM diagnostic muscle biopsy documented an increased infiltration of CD56 and perforin-positive cells (p = 0.023, p = 0.038, respectively). Treatment-naive JDM with reduced circulating NK cell counts exhibited more muscle weakness and higher levels of serum muscle enzymes. Muscle biopsies from treatment-naive JDM displayed increased NK cell infiltration, with increased CD56 and perforin-positive cells.

Fasting reshapes tissue-specific niches to improve NK cell-mediated anti-tumor immunity

Fasting is associated with improved outcomes in cancer. Here, we investigated the impact of fasting on natural killer (NK) cell anti-tumor immunity. Cyclic fasting improved immunity against solid and metastatic tumors in an NK cell-dependent manner. During fasting, NK cells underwent redistribution from peripheral tissues to the bone marrow (BM). In humans, fasting also reduced circulating NK cell numbers. NK cells in the spleen of fasted mice were metabolically rewired by elevated concentrations of fatty acids and glucocorticoids, augmenting fatty acid metabolism via increased expression of the enzyme CPT1A, and Cpt1a deletion impaired NK cell survival and function in this setting. In parallel, redistribution of NK cells to the BM during fasting required the trafficking mediators S1PR5 and CXCR4. These cells were primed by an increased pool of interleukin (IL)-12-expressing BM myeloid cells, which improved IFN-γ production. Our findings identify a link between dietary restriction and optimized innate immune responses, with the potential to enhance immunotherapy strategies.

Changes in subset distribution and impaired function of circulating natural killer cells in patients with colorectal cancer

Natural killer (NK) cells are closely associated with malignant tumor progression and metastasis. However, studies on their relevance in colorectal cancer (CRC) are limited. We aimed to comprehensively analyze the absolute counts, phenotypes, and function of circulating NK cells in patients with CRC using multiparametric flow cytometry. The distribution of NK cell subsets in the peripheral circulation of patients with CRC was significantly altered relative to the control group. This is shown by the decreased frequency and absolute count of CD56dimCD16+ NK cells with antitumor effects, contrary to the increased frequency of CD56bright NK and CD56dimCD16- NK cells with poor or ineffective antitumor effects. NK cells in patients with CRC were functionally impaired, with decreased intracellular interferon (IFN)-γ secretion and a significantly lower percentage of cell surface granzyme B and perforin expression. In addition, IFN-γ expression decreased significantly with the tumor stage progression. Based on a comprehensive analysis of the absolute counts, phenotypes, and functional markers of NK cells, we found an altered subset distribution and impaired function of circulating NK cells in patients with CRC.

Abstract PO4-03-09: Profile of circulating natural killer cells in stage III triple negative breast cancer before and after neoadjuvant chemotherapy

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. The treatment for locally advanced disease is based on neoadjuvant chemotherapy and immunotherapy, with complete pathological response (pCR) serving as a predictor of disease-free survival. Although the presence of immune cell infiltration in the tumor has a favorable prognostic impact, there is a lack of predictive biomarkers for complete pathological response to neoadjuvant chemotherapy (NAC). Objective: to analyze the profile of circulating Natural Killer (NK) cells and NK subpopulations (NK FAS+ and NK PD1+) in association with the occurrence of complete pathological response after neoadjuvant chemotherapy in women with clinical stage III TNBC. Methods: This was a longitudinal prospective and translational cohort study, including women aged 18 to 60 years with stage III TNBC diagnosed between March 2015 and July 2017, with a follow-up of 24 months. A comparison control group consisted of 30 healthy women aged 18 to 60 years without prior or current cancer diagnosis and no family history of breast cancer. Peripheral blood samples were collected before NAC and before surgery. Flow cytometry was used to analyze the percentage levels of NK cells and NK subpopulations (NK FAS+ and NK PD1+). The Shapiro-Wilk test was initially applied to assess the normality of quantitative variables. The non-parametric Mann-Whitney test was used for comparison between two groups. The statistical significance level was set at p&amp;lt; 0.05. Overall survival (OS) was estimated using the Kaplan-Meier method. Statistical analysis was performed using GraphPad Prism v9.5.1. Results: The median age was 45 years. Stage IIIA was identified in 28% of the patients, while IIIB was observed in 72%. Regarding pathological response, 14 patients (48%) achieved pCR, while 15 patients (52%) had a non-complete pathological response (non-pCR). The 2-year OS was 86%, with no median reached. Higher percentage levels of NK cells, NK FAS+, and NK PD1+ were observed in the patient group compared to the control group, with significant differences. There was a significant increase in the percentage levels of NK cells after NAC compared to pre-NAC levels, and a reduction in NK PD1+ levels. NK cell levels were significantly higher in patients with stage IIIA compared to stage IIIB, while NK FAS+ levels were lower. NK cell and NK FAS+ subpopulation levels were significantly higher in patients who achieved pCR compared to non-pCR patients, with no difference in NK PD1+ cell levels. Conclusions: Elevated levels of circulating NK cells and NK FAS+ subpopulation collected from peripheral blood before NAC may serve as potential biomarkers for pCR in patients with TNBC. Citation Format: Luiz Jose de Barros Batista, Jurema Telles, Candice Santos, Ariani Souza, Marcelo Salgado, Leuridan Torres. Profile of circulating natural killer cells in stage III triple negative breast cancer before and after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-09.

Evaluating the Functional Capability of Circulating Natural Killer Cells in Human Studies

Abstract Introduction: Interleukin-15 (IL-15) appears to be involved in T cell and natural killer (NK) cell homeostasis and immune-mediated diseases. In a first-in-human study of a monoclonal antibody against IL-15 (TEV-53408), a reduction of circulating NK cells was observed in healthy volunteers. In order to determine if there was also a qualitative effect on NK cells, in-vitro functional assessments were performed on the remaining NK cells. Methods: PBMCs were obtained from 81 healthy participants(administered TEV-53408 or placebo) in a single-dose (SD) and a multiple dose (MD) study. Samples were stimulated with phorbol myristate acetate (PMA)/ionomycin to induce degranulation, stained with an NK cell-extracellular panel and CD107a. After fixation, permeabilization, and intracellular staining for perforin, cells were run through a flow-cytometry assay. Results: The collected data demonstrated a standard CD56 bright:dim ratio at all time points. Perforin load (MFI) and % of CD107a expression with or without stimulation suggest that TEV-53408 had no effect on the degranulation ability of NK cells. Discussion: These assessments provide an effective method to assess the phenotype and functionality of remaining NK cells following anti-IL-15 treatment. The results suggest that treatment with TEV-53408 does not affect the functionality of the remaining NK cells during the studied timeframe, despite the reduction of circulating NK cells.

Lingering Effects of Early Institutional Rearing and Cytomegalovirus Infection on the Natural Killer Cell Repertoire of Adopted Adolescents.

Adversity during infancy can affect neurobehavioral development and perturb the maturation of physiological systems. Dysregulated immune and inflammatory responses contribute to many of the later effects on health. Whether normalization can occur following a transition to more nurturing, benevolent conditions is unclear. To assess the potential for recovery, blood samples were obtained from 45 adolescents adopted by supportive families after impoverished infancies in institutional settings (post-institutionalized, PI). Their immune profiles were compared to 39 age-matched controls raised by their biological parents (non-adopted, NA). Leukocytes were immunophenotyped, and this analysis focuses on natural killer (NK) cell populations in circulation. Cytomegalovirus (CMV) seropositivity was evaluated to determine if early infection contributed to the impact of an atypical rearing. Associations with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), two cytokines released by activated NK cells, were examined. Compared to the NA controls, PI adolescents had a lower percent of CD56bright NK cells in circulation, higher TNF-α levels, and were more likely to be infected with CMV. PI adolescents who were latent carriers of CMV expressed NKG2C and CD57 surface markers on more NK cells, including CD56dim lineages. The NK cell repertoire revealed lingering immune effects of early rearing while still maintaining an overall integrity and resilience.

FcαRI (CD89) is upregulated on subsets of mucosal and circulating NK cells and regulates IgA-class specific signaling and functions

Immunoglobulin A (IgA) is the predominant mucosal antibody class with both anti- and pro-inflammatory roles [1–3]. However, the specific role of the IgA receptor CD89, expressed by a subset of natural killer (NK) cells, is poorly explored. We found that CD89 protein expression on circulating NK cells is infrequent in humans and rhesus macaques, but transcriptomic analysis showed ubiquitous CD89 expression, suggesting an inducible phenotype. Interestingly, CD89+ NK cells were more frequent in cord blood and mucosae, indicating a putative IgA-mediated NK cell function in the mucosae and infant immune system. CD89+ NK cells signaled through upregulated CD3ζ, SYK, ZAP70, and SLAMF1, but also showed high expression of inhibitory receptors such as KLRG1 and reduced activating NKp46 and NKp30. CD89-based activation or ADCC with monomeric IgA1 reduced NK cell functions, while ADCC with combinations of IgG and IgA2 was enhanced compared to IgG alone. These data suggest that functional CD89+ NK cells survey mucosal sites, but CD89 likely serves as regulatory receptor which can be further modulated depending on IgA and IgG subclass. Although the full functional niche of CD89+ NK cells remains unexplored, these intriguing data suggest the CD89 axis could represent a novel immunotherapeutic target in the mucosae or early life.

The proportion of CD161 on CD56+ NK cells in peripheral circulation associates with clinical features and disease activity of primary Sjögren's syndrome.

The purpose of this study was to examine the proportion of CD161 on CD56+ natural killer (NK) cells in peripheral blood of primary Sjögren's syndrome (pSS) and investigate its clinical relevance of pSS. The proportion of CD56+ NK cells and CD161 on CD56+ NK cells was detected by flow cytometry in 31 pSS patients and 29 healthy controls (HCs). The correlations between the proportion of CD161+CD56+ NK cells and clinical features and disease activity of pSS were further analyzed. Meanwhile, we drew the receiver operating characteristic curve to evaluate the diagnostic value of CD161+CD56+ NK cells in pSS. In addition, we evaluated the differences in the effects of CD161+ cells and CD161- cells in peripheral blood on the function of CD56+ NK cells in 5 pSS patients. The proportion of CD56+ NK cells and CD161+CD56+ NK cells decreased markedly in pSS patients compared to HCs. The correlation analysis showed that the proportion of CD161+CD56+ NK cells negatively correlated with white blood cells, Immunoglobulin A (IgA), IgM, IgG, European League Against Rheumatism Sjogren's Syndrome Patient Reported Index andEuropean League Against Rheumatism Sjogren's Syndrome Disease Activity Index, and positively correlated with complement C4. The proportion of CD161+CD56+ NK cells in pSS patients with decayed tooth, fatigue, arthralgia, skin involvement, primary biliary cirrhosis, interstitial lung disease, anti-SSA/Ro60 positive, anti-SSB positive and high IgG was lower than that in negative patients. Furthermore, compared with inactive patients, the proportion of CD161+CD56+ NK cells decreased obviously in active patients. The area under the curve was 0.7375 (p = .0016), the results indicated that CD161+CD56+ NK cells had certain diagnostic values for pSS. In addition, the proportion of CD86, HLA-DR, Ki67, FasL, TNF-α, and IFN-γ on CD161+CD56+ NK cells was lower than that on CD161-CD56+ NK cells in the peripheral blood of pSS patients. This study suggested that the proportion of CD56+ NK cells and CD161+CD56+ NK cells decreased significantly in pSS patients, and the proportion of CD161+CD56+ NK cells negatively associated with the clinical features and disease activity of pSS patients. CD161 expression inhibited the function of CD56+ NK cells in peripheral blood of pSS patients. The CD161+CD56+ NK cells may present as a potential target for therapy and a biomarker of disease activity in pSS.

Abstract 5786: Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC

Abstract Introduction: The prevailing histologic subtype of nasopharyngeal carcinoma (NPC) in the endemic regions is non-keratinizing carcinoma which shows almost consistent association with latent infection by Epstein-Barr virus (EBV). Previously our group developed a series of Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes, which can disrupt the EBV latency maintained by EBNA1, and such lytic induction could alert the immune cells. Methods: Natural killer (NK) cells of the innate immunity play a significant role in controlling EBV infection. In this study, we investigated whether our EBNA1 probes (ZRL5P4 &amp; L2P4) could activate NK cells to eliminate NPC. Results: Studies of clinical specimens indicated that tumor infiltration of the cytotoxic NK cell subset is a favorable prognostic marker for NPC patients. Furthermore, the cytotoxicity of circulating NK cells derived from NPC patients is equally well the cells from healthy individuals. If those patients’ NK cells can be attracted to infiltrate into the tumors, the tumor burden should be relieved. Indeed, the NPC xenograft with the ZRL5P4 treatment showed heavy mouse NK cell infiltration and tumor size reduction. The essential role of those mouse NK cells was demonstrated. Next, we showed that ZRL5P4 could induce the production of a soluble factor by NPC cells to stimulate the NK cell cytotoxicity. Such NK cell activity was verified in a metastasis NPC model. Mechanistically, the EBNA1 probe treatment could induce the expression of EBV early lytic gene Rta and EBNA1 itself. Both Rta and EBNA1 are known transcription factors to drive the expression of LMP1, the NF-κB signaling was then activated, which turned on the production of the well-known NK cell activation cytokine IFN-β. Conclusion: Taken together, our results demonstrated that our EBNA1 probes can mobilize NK cells to the tumor sites and activate their anti-tumor activity via the EBV reactivation and stimulation of IFN-β production. Citation Format: Luo Chen, Xiao Han Liu, Hei Tung Yuen, William C. Cho, Ka-Leung Wong, Hong Lok Lung. Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5786.

Soluble MICA in endometriosis pathophysiology: Impairs NK cell degranulation and effector functions.

Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.

Integrated Analysis of Single-Cell and Bulk RNA Sequencing Data Reveals Memory-like NK Cell Subset Associated with Mycobacterium tuberculosis Latency.

Natural killer (NK) cells are innate-like lymphocytes that belong to the family of type-1 innate lymphoid cells and rapidly respond to virus-infected and tumor cells. In this study, we have combined scRNA-seq data and bulk RNA-seq data to define the phenotypic and molecular characteristics of peripheral blood NK cells. While the role of NK cells in immune surveillance against virus infections and tumors has been well established, their contribution to protective responses to other intracellular microorganisms, such as Mycobacterium tuberculosis (Mtb), is still poorly understood. In this study, we have combined scRNA-seq data and bulk RNA-seq data to illuminate the molecular characteristics of circulating NK cells in patients with active tuberculosis (TB) disease and subjects with latent Mtb infection (LTBI) and compared these characteristics with those of healthy donors (HDs) and patients with non-TB other pulmonary infectious diseases (ODs). We show here that the NK cell cluster was significantly increased in LTBI subjects, as compared to patients with active TB or other non-TB pulmonary diseases and HD, and this was mostly attributable to the expansion of an NK cell population expressing KLRC2, CD52, CCL5 and HLA-DRB1, which most likely corresponds to memory-like NK2.1 cells. These data were validated by flow cytometry analysis in a small cohort of samples, showing that LTBI subjects have a significant expansion of NK cells characterized by the prevalence of memory-like CD52+ NKG2C+ NK cells. Altogether, our results provide some new information on the role of NK cells in protective immune responses to Mtb.

Isolation and Phenotypic Characterization of Tumor-Infiltrating NK Cells in Skin Carcinoma.

In oncological research, the function of tumor-infiltrating natural killer (NK) cells in skin carcinoma presents a viable avenue for novel therapeutic methods. NK cells are essential to the body's defense against malignancies, including skin cancer, and are especially important in more sophisticated cancer immunotherapies such as vaccinations containing dendritic cells. The deadliest type of skin cancer, malignant melanoma, still has a poor prognosis even with advancements in early-stage therapies, which emphasizes the need for novel therapeutic strategies. NK cells from human melanoma metastases were subjected to single-cell RNA-seq analysis, which demonstrated notable variations in the transcriptional programs of tumor-infiltrating and circulating NK cells. Different transcriptional states are displayed by NK cells that have invaded tumors, indicating that they are functionally specialized in areas like chemokine production and cytotoxicity. These results emphasize the functions of NK cells in recruiting other significant immune cell types, such as cross-presenting dendritic cells, and in direct cytotoxicity against malignant cells. Investigating NK cells that infiltrate tumors in skin carcinomas presents a viable approach to comprehending and may be modifying the immune environment surrounding these cancers. It is essential to comprehend the distinct characteristics and roles of NK cells inside the tumor microenvironment in order to create more potent immunotherapeutic approaches to treat skin cancer. In order to perhaps open the door for new directions in cancer immunotherapy, the project intends to establish a thorough technique for the isolation and thorough phenotypic characterization of tumor-infiltrating NK cells in skin carcinoma.

Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

Interleukin-10 gene intervention ameliorates liver fibrosis by enhancing the immune function of natural killer cells in liver tissue

Background and aimsInterleukin 10 (IL-10) and natural killer (NK) cells have the potential to combat liver fibrosis. However, whether NK cells play an important role in the anti-fibrotic effects of IL-10 is not sufficiently elucidated. In this study, we investigated the regulatory effects of IL-10 on NK cells during liver fibrosis. MethodsFibrotic mice induced with carbon tetrachloride were treated with or without IL-10 in the presence or absence of NK cells. Liver damage and fibrosis were assessed using hematoxylin and eosin and Sirius Red staining and serum transaminase and liver hydroxyproline assays, respectively. NK cell distribution, quantity, activation, cytotoxicity, development, and origin were analyzed using immunohistochemistry, immunofluorescence, and flow cytometry. Enzyme-linked immunosorbent assay was used to determine chemokine levels. ResultsIn the presence of NK cells, IL-10 gene intervention improved liver fibrosis and enhanced NK cell accumulation and function in the liver, as evidenced by increased NKG2D, interferon-γ, and CD107a expression. Furthermore, IL-10 promoted the migration of circulating NK cells to the fibrotic liver and elevated C-C motif ligand 5 levels. However, depletion of NK cells exacerbated liver fibrosis and impaired the anti-fibrotic effect of IL-10. ConclusionsThe anti-fibrotic effect of IL-10 relies on the enhancement of NK cell immune function, including activation, cytotoxicity, development, and migration. These results provide valuable insights into the mechanisms through which IL-10 regulates NK cells to limit the progression of liver fibrosis.

A Pan-Cancer Single-Cell Transcriptomic Atlas of Natural Killer (NK) Cells Reveals Intrinsic and Extrinsic Mediators of NK Cell Anti-Tumor Functions

Background: Natural killer (NK) cells are critical mediators of anti-tumor immunity, engaging in direct cytotoxic responses and promoting pro-inflammatory immune microenvironments. The molecular mechanisms underlying these functions, however, have yet to be fully elucidated. This is due in part to a lack of understanding of the phenotypic and functional diversity of NK cells across cancer types. Here, we present a single-cell transcriptomic atlas of &amp;gt;250,000 NK cells spanning 340 patients across 17 cancer types to advance our understanding of tumor-infiltrating NK cells. Methods: We re-analyzed publicly available single-cell RNA-sequencing (scRNA-seq) datasets of primary, therapy-naïve tumors, as well as matching peripheral blood samples and healthy donor blood samples. We employed systematic computational analyses to deconvolute the phenotypic and functional diversity of tumor-infiltrating NK cells and define key regulators of NK cell functions in both a cancer type-specific and pan-cancer manner. Feature expression module scoring was used to infer phenotypic and functional characteristics of NK cell clusters in each cancer type. Transcription factor regulon and cell-cell communication prediction algorithms were applied to understand the mechanistic drivers of NK cell populations within tumors. Results: Tumor-infiltrating NK cells display distinct patterns of phenotypic and functional features across cancer types. Nonetheless, we identify consensus NK cell populations across cancers that demonstrate the utility and performance of our gene module-based phenotypic and functional annotations. Interestingly, we observe the enrichment of adaptive-like and CD56 bright NK cell populations in tumors that exhibit features of tissue residency, including the upregulation of ITGA1, ITGAE, and CD69 alongside the downregulation of FCG3RA. Notably, these tissue-resident populations display enhanced chemokine and cytokine activity compared to their recruited counterparts, suggesting the significance of tissue-resident NK cell populations in immune recruitment to tumors. We find the phenotypic composition of intra-tumoral NK cells is vastly different than that of circulating NK cells which harbor predominantly CD56 dim populations, as well as a significant proportion of adaptive-like NK cells in several cancer types. Leveraging the paired nature of several tumor and peripheral blood samples, we performed temporal comparative analyses that revealed the robust expression of KLF2 and S1PR5 in circulation which is then lost in paired tumor tissues. While KLF2 and S1PR5 have been previously implicated in the homing and migration of immune cells, their roles in the trafficking of NK cells to tumors have yet to be defined. In modeling transitions from canonical to stressed NK cell states, we not only identify well-established NK cell checkpoints ( TIM-3, LAG3, and CISH) but also identify novel putative checkpoints yet to be investigated in NK cells, such as the AP-1 family of transcription factors. Additionally, we examine extrinsic factors contributing to intra-tumoral NK cell phenotypes, detecting significant crosstalk between NK cells and immunomodulatory macrophages and CD4+ T cells mediated by galectin-9 and TGFβ interactions. Conclusions: We define the phenotypic and functional pan-cancer landscape of NK cells and uncover both intrinsic and extrinsic factors influencing their anti-tumor functions. We envision our NK cell atlas will facilitate the development of strategies to enhance NK cell-based immunotherapies against cancer.

NK cells from COVID-19 positive patients exhibit enhanced cytotoxic activity upon NKG2A and KIR2DL1 blockade.

SARS CoV-2 has caused a global pandemic leading to significant morbidity and mortality. There is a need to elucidate and further understand the implications of COVID-19 disease on the immune system to develop improved therapeutic strategies. In particular, Natural Killer (NK) cells play an essential role in mediating the innate immune response against viral infections. To better understand the role of innate immunity in COVID-19, we characterized the phenotype of circulating NK cells from 74 COVID-19 patients and 25 controls. Through evaluating the protein expression of activating and inhibitory NK cell surface molecules using dimension reduction analysis and clustering, we identified 4 specific clusters of NK cells specific to disease state (COVID-19 positive or COVID-19 negative) and characterized COVID-19 positive NK cells as: NGK2A+KIR2DL1+NKG2C-. Utilizing blocking antibodies specific for receptors NKG2A and KIR2DL1, we found that both NKG2A and KIR2DL1 blockade markedly enhances the ability of NK cells from COVID-19 positive patients to lyse SARS-Cov-2 infected cells. Overall, this study reveals new insights into NK cell phenotypes during SARS-CoV-2 infection and suggests a therapeutic approach worthy of further investigation to enhance NK cell-mediated responses against the virus.

Circulating natural killer cells and their association with breast cancer and its clinico-pathological characteristics.

Natural killer (NK) cells play a critical role in cancer immunosurveillance and hold promise as both therapies and prognostic markers in advanced disease. We explore factors that may influence NK cell concentration in the peripheral blood of women with breast cancer in Côte d'Ivoire compared to healthy controls and implications for future research in our context. In this cross-sectional case-control study, blood samples were taken from 30 women diagnosed with breast cancer within 6 months of diagnosis and fifteen healthy women at University Teaching Hospital [Centre Hospitalier Universitaire (CHU)] Treichville in Abidjan, Côte d'Ivoire, from March to September 2018. The blood draw could take place at any time following diagnosis and through treatment. Demographic and clinical data were collected. NK cells were isolated, stained, analysed and counted using the flow cytometer at the Department of Immunology at CHU of Cocody. All p-values were two-sided. Mean age among 30 women with breast cancer was 49 years old compared to 45 years old for 15 controls (p = 0.41). Among 30 women with breast cancer, 4 (13.3%) had Stage 2 disease, 14 (46.7 %) at Stage 3, and 12 (40%) at Stage 4. Fourteen (46.7%) had breast cancer that was hormone receptor-positive (HR+) HER2-negative, 10 (33.3%) had triple-negative cancer, three (10.0%) had HR+HER2+ disease, and three (10.0%) HR-HER2+ cancer. NK cell concentration was not associated with cancer diagnosis, age, cancer stage, subtype, or type of treatment patients received (p > 0.05). Although we did not find an association between NK cell concentration, cancer characteristics or treatment, our results be limited by the small sample size and timing of blood draw. Our next steps include a larger study to explore circulating NK cells prior to any treatment and NK cell infiltration within breast cancer tumour and correlating this with response to treatment and prognosis.

Interleukin-21 promotes Type-1 activation and cytotoxicity of CD56dimCD16bright natural killer cells during kidney allograft antibody–mediated rejection showing a new link between adaptive and innate humoral allo-immunity

The role of Natural killer (NK) cells during kidney allograft antibody-mediated rejection (ABMR) is increasingly recognized, but an in-depth characterization of mechanisms that contribute to such immune response is still under investigation. Here, we characterized phenotypic, functional, and transcriptomic profiles of peripheral blood circulating and allograft infiltrating CD56dimCD16bright NK cells during anti-HLA donor-specific antibody (DSA)+ ABMR. Cross-sectional analyses performed in 71 kidney transplant recipients identified a unique phenotypic circulating CD56dimCD16bright NK cell cluster expanded in DSA+ ABMR. This cluster co-expressed high levels of the interleukin-21 Receptor (IL-21R); Type-1 transcription factors T-bet and EOMES, CD160 and natural killer group 2D cytotoxic and activating co-stimulatory receptors. CD160+ IL-21R+ NK cells correlated with elevated plasma IL-21, Ki-67+ ICOS+ (CD278) IL-21-producing circulating T follicular helper cells, enhanced Type-1 pro-inflammatory cytokines, NK cell cytotoxicity, worse microvascular inflammation and graft loss. Single-cell transcriptomic analysis of circulating NK cells delineated an expanded cluster in DSA+ ABMR characterized by elevated pro-inflammatory/cytotoxic pathways, IL-21/STAT3 signaling, and leukocyte trans-endothelial migration pathways. Infiltration of CD160+ IL-21R+ NK cells with similar transcriptomic profile was detected in DSA+ ABMR allograft biopsies, potentially contributing to allograft injury. Thus, the IL-21/IL-21R axis, linking adaptive and innate humoral allo-immunity, or NK cells may represent appealing immunotherapy targets in DSA+ ABMR.