Peripheral NK Cells Research Articles

MicroRNA-1 Negatively Regulates Peripheral NK Cell Function via Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) Signaling Pathways During PPRV Infection.

Peste des petits ruminants virus (PPRV) has emerged as a significant threat to the productivity of small ruminants worldwide. PPRV is lymphotropic in nature and induces in the hosts a transient but severe immunosuppression, especially innate immunity. However, it remains largely unknown how NK cells respond and are regulated at the earliest time points after an acute viral PPRV infection in goats. In this study, we revealed that multiple immune responses of goat peripheral NK cells were compromised during PPRV infection, including the cytolytic effector molecule expression and cytokine production. Importantly, we demonstrated that PPRV infection stimulated the expression of TWEAK, a negative regulator of cytotoxic function of NK cells, which may be involved in the suppression of cytotoxicity as well as cytokine production in infected goat NK cells. Furthermore, we found that PPRV infection induced TWEAK expression in goat NK cells involving post-transcription by suppressing miR-1, a novel negative miRNA directly targeting the TWEAK gene. Moreover, replication of virus is required for inhibition of miR-1 expression during PPRV infection, and the non-structural V protein of PPRV plays an important role in miR-1 mediated TWEAK upregulation. Additionally, we revealed that the regulation of NK cell immune responses by TWEAK is mediated by MyD88, SOCS1, and STAT3. Taken together, our results demonstrated that TWEAK may play a key role in regulating goat peripheral NK cell cytotoxicity and cytokine expression levels during PPRV infection.

Altered Frequency of NK Cells and Treg Cells by Astragalus Polysaccharide Combined with Budesonide in Asthma Model Mice

Objective. We investigated the efficacy of astragalus polysaccharide (APS) combined with budesonide and the effect on expressions of peripheral NK cells and Treg cells and the molecular mechanism in mice with bronchial asthma. Methods. In this study, we established a mouse model of asthma. Four groups of BaLB/C mice were developed; control group had no asthma induction, and the other three groups of mice were sensitized by OVA (Ovalbumin), OVA + budesonide, and OVA + APS + budesonide. Flow cytometry was used to determine the proportion of NK cells and Treg cells. Levels of cytokines IL-4 and IL-10 were detected using RT-PCR and ELISA. Results. Asthma mice treated with APS + budesonide showed alleviated airway resistance compared to model mice (P<0.05). The percentage of dendritic cells (DCs) was reduced (P<0.05), while anti-inflammatory NK cells and Treg cells significantly increased after APS + budesonide treatment (P<0.05). Further, APS + budesonide treatment resulted in improvements in IL-4 and IL-10 mRNA and protein levels (P<0.05). Conclusion. APS combined with budesonide medication may regulate expressions of DCs cells and related cytokines, reliving clinical symptom of bronchial asthma.

NK cells in pancreatic cancer demonstrate impaired cytotoxicity and a regulatory IL-10 phenotype

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the most common tumor subtypes and remains associated with very poor survival. T cell infiltration into tumor tissue is associated with improved clinical outcome but little is known regarding the potential role of NK cells in disease control. Here we analyze the phenotype and function of NK cells in the blood and tumor tissue from patients with PDAC. Peripheral NK cells are present in normal numbers but display a CD16hiCD57hi phenotype with marked downregulation of NKG2D. Importantly, these cells demonstrate reduced cytotoxic activity and low levels of IFN-γ expression but instead produce high levels of intracellular IL-10, an immunoregulatory cytokine found at increased levels in the blood of PDAC patients. In contrast, NK cells are largely excluded from tumor tissue where they display strong downregulation of both CD16 and CD57, a phenotype that was recapitulated in primary NK cells following co-culture with PDAC organoids. Moreover, expression of activatory proteins, including DNAM-1 and NKP30, was markedly suppressed and the DNAM-1 ligand PVR was strongly expressed on tumor cells. As such, in situ and peripheral NK cells display differential features in patients with PDAC and indicate local and systemic mechanisms by which the tumor can evade immune control. These findings offer a number of potential options for NK-based immunotherapy in the management of patients with PDAC.

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia.

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

Impaired circulating CD56dim NK cells are associated with decompensation of HBV-related cirrhosis

NK cells play an important role in immune regulation and defense of infection, but their characteristics in patients with decompensated cirrhosis and their relationship with liver function remain unclear. We studied the functional properties of NK cells (including CD56dim NK and CD56bright NK cells) in patients with HBV-related decompensated liver cirrhosis (HBV-DLC) and analyzed their relationship with decompensation of liver function. Thirty patients with HBV-DLC and 25 patients with HBV-related compensated liver cirrhosis (HBV-CLC) were recruited in this study. Twenty five age- and sex-matched healthy individuals were recruited as healthy controls (HCs). The phenotypical and functional characteristics of NK cell subsets were detected by flow cytometry, and the correlation between NK cells and decompensation of liver function was analyzed. The frequency of circulating CD56bright NK cells was significantly increased while circulating CD56dim NK cells was significantly decreased in HBV-DLC patients as compared with HCs and HBV-CLC patients. Peripheral activated-CD56bright NK cells from HBV-DLC patients might express lower levels of inhibitory receptor CD158b1/2 and higher levels of activating receptor NKG2D and their expression of perforin and granzyme A/B also increased significantly compared with HCs, suggesting a high immune activation status of peripheral CD56bright NK cells in HBV-DLC patients. In HBV-DLC patients, the expression of CD107a and perforin in circulating CD56dim NK cells was positively correlated with cytolytic capacity while CD107a and perforin expression in circulating CD56dim NK cells were significantly decreased, suggesting an impaired cytolytic capacity of circulating CD56dim NK cells. Besides, we found that the perforin expression of circulating CD56dim NK cells correlated negatively with child-pugh classification in HBV-DLC patients. The functional properties of circulating NK cell subsets in HBV-DLC patients have changed significantly, especially of CD56dim NK cells which closely related to decompensation of liver function. These findings may help provide new perspectives and theoretical basis for the treatment of patients with HBV-DLC.

The clinical expression and significance of inhibitory receptor TIGIT gene on peripheral NK cells in rheumatoid arthritis

Objective To investigate the expression of inhibitory receptor TIGIT gene in peripheral NK cells of patients with rheumatoid arthritis (RA) and its clinical significance. Methods A case control study was conducted of 58 RA patients(30 patients with active RA disease, 28 patients with remission of RA) and 22 healthy controls (HC) in the department of rheumatology and immunology from Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine during December 2018 to June 2019, the related clinical data were collected. Flow cytometry was used to compare the expression of TIGIT gene on peripheral NK cells. The IFN-γ secretion level of cytokines in peripheral blood was detected by ELISA, and analyzed the correlations between TIGIT gene and disease activity and IFN-γ level. T-test or non-parametric test was used for comparison between the two groups, and Pearson correlation analysis was used for correlation between the two variables. Results Compared with HC group, the number of NK cells in RA disease group was reduced, which was (13.88±4.56) ×107 cells/L in RA disease group and (25.69± 2.48) ×107 cells/L in HC group (t=-2.036, P=0.041). The percentage of TIGIT gene expression in peripheral blood NK cells was not statistically different between RA disease group (39.73±9.37)% and HC group (45.64±9.91)% (t=-1.241, P=0.218). However, the average fluorescence intensity (MFI) of TIGIT gene expression was decreased, which was (7.21±2.03) in RA group and (9.01±3.29) in HC group (t=-2.947, P=0.004).MFI of TIGIT gene in NK cells of the disease active subgroup was (6.72±2.01), lower than that of the disease remission subgroup (8.75±2.64), (t=-3.316, P=0.002), and MFI of TIGIT gene in NK cells of the disease active subgroup was negatively correlated with DAS28 score (r2=0.649 6, P<0.000 1).The secretion level of IFN-γ cytokine in the RA disease group was (67.13±14.84) pg/ml, higher than that in the HC group (57.21±14.23) pg/ml (t=2.757, P=0.017), and the secretion level of IFN-γ cytokine in the disease active subgroup was negatively correlated with the MFI of TIGIT gene on NK cells (r2=0.662 2, P<0.000 1). Experimental results of peripheral blood mononuclear cell stimulation in the RA disease activity subgroup showed that the secretion level of cytokines IFN-γ was reduced after stimulation compared with that before stimulation (t=11.38, P<0.000 1). Conclusion The abnormal expression of TIGIT gene on peripheral NK cells are observed in patients with RA, which correlate with disease activity and IFN-γ secretion level. Key words: Arthritis, rheumatoid; Receptors, immunologic; Killer cells, natural; Interferon-gamma

Liver cirrhosis in HIV/HCV-coinfected individuals is related to NK cell dysfunction and exhaustion, but not to an impaired NK cell modulation by CD4+ T-cells.

Introduction HIV worsens HCV‐related liver disease by accelerating fibrosis progression; however, progression rates are extremely variable among HIV/HCV‐coinfected individuals. NK cells are associated with modulation of liver fibrosis and are profoundly altered during HCV and HIV infections. CD4+ T‐cells modulate NK cell function, and are also affected by HIV infection. Here, we aim to characterize the association of hepatic fibrosis with both the phenotype and function of peripheral NK cells and their regulation by CD4+ T‐cells, in HIV/HCV‐coinfected individuals.MethodsThirty‐four HIV/HCV‐coinfected individuals with minimal (n = 16) and advanced (n = 18) fibrosis (METAVIR F0/F1 and F4 scores respectively) and 20 healthy volunteers were enrolled. PBMC were obtained from peripheral blood samples and NK and CD4+ T‐cells were isolated and analysed. NK cell phenotype (CD25, CD69, Nkp46, NKG2D, PD‐1), degranulation (CD107a) and IFN‐γ and TNF‐α production, as well as CD4+ T‐cell activation (CD69, CD25 and CD38) were measured by flow cytometry. CD4+ T‐cell conditioned medium (CM) derived from F0/F1 or F4 individuals was assessed for IL‐2 levels by ELISA. Modulation of NK cell functionality by these CMs was also analysed.ResultsWhen comparing to NK cells from individuals with minimal fibrosis, degranulation and cytokine secretion by NK cells from subjects with F4 scores was significantly impaired, while PD‐1 expression was augmented. On the one hand, neither the expression of activation markers nor IL‐2 secretion was distinctly induced in CD4+ T‐cells from subjects with F0/F1 or F4 METAVIR scores. Finally, NK cell degranulation and cytokine secretion were not differentially modulated by CD4+ T‐cell CM, whether CD4+ T‐cells derived from subjects with minimal or advanced fibrosis.ConclusionsLow levels of NK and CD4+ T‐cells in HIV/HCV‐coinfected individuals with advanced liver fibrosis have been previously described. Here, we show that advanced liver fibrosis in coinfected individuals is associated to a defective function of NK cells and an increased expression of the exhaustion/senescence marker PD‐1. This NK signature could not be attributed to changes in the ability of CD4+ T‐cells to modulate NK cell function.

Human peripheral blood DNAM-1neg NK cells are a terminally differentiated subset with limited effector functions

AbstractNatural killer (NK) cells are a heterogeneous population of innate lymphocytes whose potent anticancer properties make them ideal candidates for cellular therapeutic application. However, our lack of understanding of the role of NK cell diversity in antitumor responses has hindered advances in this area. In this study, we describe a new CD56dim NK cell subset characterized by the lack of expression of DNAX accessory molecule-1 (DNAM-1). Compared with CD56bright and CD56dimDNAM-1pos NK cell subsets, CD56dimDNAM-1neg NK cells displayed reduced motility, poor proliferation, lower production of interferon-γ, and limited killing capacities. Soluble factors secreted by CD56dimDNAM-1neg NK cells impaired CD56dimDNAM-1pos NK cell–mediated killing, indicating a potential inhibitory role for the CD56dimDNAM-1neg NK cell subset. Transcriptome analysis revealed that CD56dimDNAM-1neg NK cells constitute a new mature NK cell subset with a specific gene signature. Upon in vitro cytokine stimulation, CD56dimDNAM-1neg NK cells were found to differentiate from CD56dimDNAM-1pos NK cells. Finally, we report a dysregulation of NK cell subsets in the blood of patients diagnosed with Hodgkin lymphoma and diffuse large B-cell lymphoma, characterized by decreased CD56dimDNAM-1pos/CD56dimDNAM-1neg NK cell ratios and reduced cytotoxic activity of CD56dimDNAM-1pos NK cells. Altogether, our data offer a better understanding of human peripheral blood NK cell populations and have important clinical implications for the design of NK cell–targeting therapies.

STAT3 directly regulates NKp46 transcription in NK cells of HBeAg-negative CHB patients.

NK cells play an important role in early control of HBV infection. The function of NK cells is inhibited in chronic hepatitis B virus (CHB) infection, although the underlying mechanism remains unknown. We found that the expression of STAT3 decreased in peripheral NK cells of CHB patients, and was associated with low levels of degranulation and IFN-γ secretion. In addition, STAT3 levels were positively correlated with cytolysis-associated molecules and antiviral cytokines, such as CD107a, granzyme B, perforin, and IFN-γ. HBsAg directly inhibited the expression and activation of STAT3 in NK cells, and knocking down STAT3 expression in NK cells inhibited proliferation, decreased cyclin d1 levels, and suppressed responsiveness to IL-21 stimulation. Furthermore, STAT3 directly bound to the promoter of NKp46, an important activating receptor of NK cells, to regulate its transcription and expression. Taken together, our findings indicate that STAT3 is an important positive regulator of NK cells, and provide a new mechanism of NK cell dysfunction in CHB.

Identification of NK Cell Subpopulations That Differentiate HIV-Infected Subject Cohorts with Diverse Levels of Virus Control.

HIV infection is controlled immunologically in a small subset of infected individuals without antiretroviral therapy (ART), though the mechanism of control is unclear. CD8+ T cells are a critical component of HIV control in many immunological controllers. NK cells are also believed to have a role in controlling HIV infection, though their role is less well characterized. We used mass cytometry to simultaneously measure the levels of expression of 24 surface markers on peripheral NK cells from HIV-infected subjects with various degrees of HIV natural control; we then used machine learning to identify NK cell subpopulations that differentiate HIV controllers from noncontrollers. Using CITRUS (cluster identification, characterization, and regression), we identified 3 NK cell subpopulations that differentiated subjects with chronic HIV viremia (viremic noncontrollers [VNC]) from individuals with undetectable HIV viremia without ART (elite controllers [EC]). In a parallel approach, we identified 11 NK cell subpopulations that differentiated HIV-infected subject groups using k-means clustering after dimensionality reduction by t-neighbor stochastic neighbor embedding (tSNE) or linear discriminant analysis (LDA). Among these additional 11 subpopulations, the frequencies of 5 correlated with HIV DNA levels; importantly, significance was retained in 2 subpopulations in analyses that included only cohorts without detectable viremia. By comparing the surface marker expression patterns of all identified subpopulations, we revealed that the CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells are more abundant in EC and HIV-negative controls than in VNC and that the frequency of these cells correlated with HIV DNA levels. We hypothesize that this population may have a role in immunological control of HIV infection.IMPORTANCE HIV infection results in the establishment of a stable reservoir of latently infected cells; ART is usually required to keep viral replication under control and disease progression at bay, though a small subset of HIV-infected subjects can control HIV infection without ART through immunological mechanisms. In this study, we sought to identify subpopulations of NK cells that may be involved in the natural immunological control of HIV infection. We used mass cytometry to measure surface marker expression on peripheral NK cells. Using two distinct semisupervised machine learning approaches, we identified a CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells that differentiates HIV controllers from noncontrollers. These cells can be sorted out for future functional studies to assess their potential role in the immunological control of HIV infection.

Anti-PD1 up-regulates PD-L1 expression and inhibits T-cell lymphoma progression: possible involvement of an IFN-γ-associated JAK-STAT pathway.

PurposeNK/T-cell neoplasms are rare, highly aggressive, and insensitive to chemotherapy. These lymphomas have a poor prognosis, with patients being vulnerable to relapse. Hence, there is a need for alternative treatments. The purpose of this study is to investigate whether anti-PD1 takes effect on NK/T cell lymphoma.MethodsThe expression of PD-L1 in NK/T cell lines was investigated by flow cytometry and by Western blot. In vivo, overall survival and median survival time of mice bearing an NK/T cell line tumor was assessed. Tumor-infiltrating T cells and monocyte-derived suppressor cells were evaluated by flow cytometry. Levels of PD-L1 and components of the JAK-STAT pathway were assessed in tumor tissues by immunohistochemistry.ResultsNK/T cell lines had greater expression of PD-L1 than normal peripheral blood human NK cells. In vivo, anti-PD1 treatment improved overall survival and median survival time of mice bearing an NK/T cell line. Furthermore, anti-PD1 treatment increased levels of PD-L1. Cultured tumor-infiltrating lymphocytes from mice treated with anti-PD1 had greater levels of IFN-γ than cultured lymphocytes from untreated animals. Further, levels of JAK2 and STAT1 were greater in mice treated with anti-PD1.ConclusionIn vivo, anti-PD1 inhibited the progression of an NK/T-cell lymphoma and up-regulated PD-L1 expression. This up-regulation may be through the IFN-γ-associated JAK-STAT pathway.

CD56bright natural killer cells exhibit abnormal phenotype and function in severe aplastic anemia.

CD56bright NK cells have been highlighted to serve immunoregulatory functions. However, their roles in severe aplastic anemia (SAA) have not been elucidated. Here, we investigated the quantities, phenotypes, cytokine secretion abilities, and the cytotoxicities of peripheral CD56bright NK cells along with CD56dim NK cells obtained from patients with SAA, SAA in remission (R-SAA), and healthy controls (HC). We observed the decreased quantities of CD56bright NK cells in SAA compared with in R-SAA and HC. In SAA, the quantities of CD56bright NK cells correlated with the disease severity. Activating receptors NKp46 and NKp44 on CD56bright NK cells were upregulated while inhibiting receptor NKG2A was downregulated in SAA. CD56bright NK cells obtained from SAA patients produced increased IL-10 and decreased IFN-γ in vitro compared with cells obtained from HC, while TNF-α and IL-13 productions were not different between two groups. Under a 7-day prestimulation with IL-2 and IL-12, the serum concentrations of which were higher in SAA patients, CD56bright NK obtained from HC also produced increased IL-10 mRNAs. There were no differences of cytotoxicites between CD56bright NK cells in SAA and in HC. We discovered that CD56bright NK cells exhibited abnormal receptor expressions and cytokine production in SAA, and they were related with the severity of illness.

Abstract B157: Modulation of natural killer cell dysfunction in human bladder cancer

Abstract T-cells undergo a progressive program of dysfunction termed “exhaustion” during chronic inflammatory pathologies such as cancer. T-cell exhaustion is characterized by diminished effector functionality and inefficient tumor immunosurveillance. However, it is unknown whether an analogous program undermines the contribution of innate lymphocytes like natural killer cells (NK) to tumor-surveillance, and what phenotype might define it. Therefore, we examined expression of inhibitory receptors and the corresponding functional potential of NK and T-cells present in peripheral blood mononuclear cells (PBMC) and tumor tissue of 59 individuals representative of the clinical spectrum of human bladder cancer (BC). PBMC and cell suspensions from freshly dissociated primary tumors were analyzed by flow cytometry to measure changes in the expression of the inhibitory receptors Tim-3, TIGIT, and PD-1, and to determine the composition of hematopoietic lineages in each tissue. NK effector function was assessed via activation with IL-2 or IL-15, followed by co-culture with Class I HLA-deficient targeT-cells to measure cytokine production and degranulation. Additionally, we determined the effect on NK cell function of ex vivo blockade of Tim-3 and TIGIT by the addition of monoclonal antibodies prior to functional assays. We found that NK cells significantly up-regulate expression of Tim-3 and TIGIT, but not PD-1, in both the PBMC and tumors of BC patients. T-cells demonstrate a similar pattern of expression. but with a far lower frequency of positive cells. The magnitude of Tim-3 expression by NK cells is a barometer of tumor invasiveness on cells in both PBMC and tumor tissue, while TIGIT is induced equivalently in BC patients independently of tumor stage. Importantly, both molecules are expressed at similar frequencies on NK cells isolated from blood or tumor, independent of the magnitude of overall expression, yet define NK with different functional potential. NK cells in PBMC from BC patients are functionally comparable to NK cells from healthy donors in their ability to produce IFNγ/TNFα and degranulate in response to targeT-cells, while tumor NK are refractory to both stimuli. NK cells from tumor tissue are not terminally exhausted as effector functions are restored after “resting” ex vivo prior to stimulation. Ex vivo blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK and T-cells from BC patients, but is ineffective for NK cells in tumor tissue, implicating suppressive factors specific to tumor in mediating NK dysfunction. Tim-3 blockade was most efficient in peripheral NK cells from BC patients that were activated with IL-15 versus IL-2, suggesting that local cytokine milieu can affect responsiveness to subsequent checkpoint inhibition. Overall, our data suggest that both NK and T-cells from patients with BC are enriched for expression of the shared inhibitory receptors Tim-3 and TIGIT, and that expression in the peripheral blood mirrors expression in the tumor. However, tissue-specific cues present in bladder tumors, but not in the periphery, ultimately confer effector dysfunction. Furthermore, because blockade of Tim-3 enhances IFNγ and TNFα production by NK cells present in the peripheral blood of BC patients, it may represent a new strategy to modulate innate, antitumor immunity while simultaneously conferring benefit in the adaptive compartment. Citation Format: Adam M. Farkas, Francois Audenet, Harry Anastos, Matthew Galsky, John P. Sfakianos, Nina Bhardwaj. Modulation of natural killer cell dysfunction in human bladder cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B157.

High-dimensional single-cell proteomics analysis identifies immune checkpoint signatures and therapeutic targets in ulcerative colitis.

Immune checkpoints are regulators of immune cells and play key roles in the modulation of immune responses. The role of checkpoints in autoimmune disease is poorly understood but likely to be central since checkpoint inhibition during cancer treatment can cause autoimmunity. We generated a high-dimensional single-cell proteomics data set from PBMCs of healthy individuals and patients with ulcerative colitis (UC) by mass cytometry, enabling systems-wide analyses of immune cell frequencies and cell type-specific expression patterns of 12 immune checkpoints. Subtle but significant changes in immune cell frequencies and checkpoint expression were observed between UC patients on different treatment regimens and between patients and healthy controls. Most strikingly, UC patients showed a reduced number of peripheral NK-cells and those cells showed an altered phenotype including increased TIGIT expression. Based on these results, we modulated NK-cell function ex vivo through targeting of TIGIT pathway members. In summary, we describe a pattern of changes in immune cell abundance and checkpoint expression as a basis for UC patient stratification and we show modulation of a corresponding immune cell subset through checkpoint targeting. Our approach can be used for the identification of pathogenic immune cell subsets and guide target selection in autoimmunity and chronic inflammation.

MicroRNA-378 protects human umbilical vein endothelial cells from injuries by soluble CD226 through down-regulating the expression of soluble CD226 in natural killer cells

This study aimed to determine the expression of microRNA (miRNA or miR)-378 in peripheral blood from patients with pregnancy-induced hypertension (PIH), and to try to understand its effect on vascular endothelial injuries induced by PIH. The study included 33 PIH patients. Quantitative real-time polymerase chain reaction was used to determine the expression of miR-378 in peripheral blood and NK cells. NK cells were transfected with miR-378 mimics and its negative control. Bioinformatics was used to predict the potential target gene of miR-378. Flow cytometry and Western blotting were performed to identify changes in the expression of CD226 protein. By co-culturing HUVECs with NK cells, the effects of miR-378 and CD226 on the proliferation, migration and apoptosis of HUVECs were evaluated. The results showed that the expression of miR-378 in peripheral blood was down-regulated with the progression of PIH. The expression of miR-378 was reduced in innate immune NK cells from peripheral blood of PIH patients. miR-378 negatively regulated the expression of CD226 in NK cells, and CD226 is a target gene of miR-378. miR-378 inhibited the expression and secretion of soluble CD226 by NK cells. miR-378 regulated the proliferation, migration and apoptosis of HUVECs by affecting the levels of soluble CD226 expressed by NK cells. The present study demonstrates that miR-378 is down-regulated in peripheral blood and NK cells from patients with PIH, and negatively correlated with the development of PIH. miR-378 protects HUVECs from injuries by soluble CD226 through down-regulating the expression of soluble CD226 in NK cells.

Loss of Canonical Notch Signaling Affects Multiple Steps in NK Cell Development in Mice.

Within the hematopoietic system, the Notch pathway is critical for promoting thymic T cell development and suppressing the B and myeloid lineage fates; however, its impact on NK lymphopoiesis is less understood. To study the role of Notch during NK cell development in vivo, we investigated different NK cell compartments and function in Rbp-Jkfl/flVav-Cretg/+ mice, in which Rbp-Jk, the major transcriptional effector of canonical Notch signaling, was specifically deleted in all hematopoietic cells. Peripheral conventional cytotoxic NK cells in Rbp-Jk-deleted mice were significantly reduced and had an activated phenotype. Furthermore, the pool of early NK cell progenitors in the bone marrow was decreased, whereas immature NK cells were increased, leading to a block in NK cell maturation. These changes were cell intrinsic as the hematopoietic chimeras generated after transplantation of Rbp-Jk-deficient bone marrow cells had the same NK cell phenotype as the Rbp-Jk-deleted donor mice, whereas the wild-type competitors did not. The expression of several crucial NK cell regulatory pathways was significantly altered after Rbp-Jk deletion. Together, these results demonstrate the involvement of canonical Notch signaling in regulation of multiple stages of NK cell development.

The ETS1 Transcription Factor Is Implicated in Human and Murine Intermediate NK Cell Development Stages

Introduction: While key regulators of early NK cell development and differentiation have been identified, few studies have looked at transcription factor (TF) dynamics and regulatory interactions during subsequent stages of NK cell maturation. Epigenetic landscapes are highly dynamic during cellular differentiation, with TFs playing an important role in the establishment and activation of specific DNA elements, such as enhancers, and subsequent programming of gene expression. ETS1 is a TF that is expressed in adult immune tissues and is critical for the development of lymphoid cells. A role for ETS1 has been described in early NK cell development by activating core transcriptional regulators such as T-BET and ID2. However, despite its continual expression in subsequent stages of NK maturation, the role of ETS1 in NK maturation is not well characterized.Methods and Results: We used FACS to isolate purified human NK cells at various maturation stages as established previously (Freud et. al. Cell Reports, 2016, 16:379-91), ranging from intermediate precursors (Stage 3) through to fully developed and mature peripheral NK cells (Stage 6). Epigenetic programming of cells during lineage maturation allows us to identify critical TFs that are active at each stage of development. We employed Illumina EPIC/850K methylation arrays and RNA sequencing to interrogate epigenetic changes at regulatory elements and TF dynamics at multiple stages along the NK developmental axis. Analysis of TF recognition motifs within hypomethylated regions revealed strong enrichment of specific motif sequences implicating T-box (T-BET and Eomes), along with RUNX and ETS TF families in specific programming of epigenetic patterns during NK development. In studying the expression of TFs that potentially bind these motifs, ETS1 exhibited the highest and most consistent expression throughout NK development. Interestingly, despite consistently high expression, ETS motifs were continually programmed throughout NK maturation, including a significant degree of modification between tonsillar Stages 4A to 4B, where NK cells acquire the ability to produce IFN-γ and significantly gain cytotoxic capability and functional maturity. Among the genes that are upregulated at this stage is the NK-cell-specific gene, NKp46.The progressive hypomethylation of regulatory regions enriched in ETS motifs led us to believe that ETS1 has a continuous role in full NK cell maturation. To test our hypothesis, we developed a novel genetically engineered mouse line with a NK cell intermediate stage-specific conditional deletion of Ets1 mediated by NKp46-driven Cre expression, NKp46-Cre-Ets1fl/fl (NKp46-Ets1fl/fl). This allowed us to study the role of ETS1 in the transition between immature and mature NK cell stages in vivo. Using a comprehensive NK cell development panel for multi-color flow cytometry, we found a drastic reduction of total NK cells in NKp46-Ets1fl/fl mice (n=7) compared to the Ets1fl/fl (n=7) and the NKp46-Cre (n=7) controls in bone marrow (3.2x104 ± 5.9x103, 2.9x105 ± 5.7x104, 2.6x105 ± 8.0x104 total NK cells respectively; p= 0.0007), spleen (3.1x104 ± 7.2x103, 1.2x106 ± 2.4x105, 1.5x106 ± 7.7x105 total NK cells respectively; p= 0.0091) and blood (21 ± 6, 385 ± 35, 185 ± 35 NK cells/uL whole blood respectively; p= 0.0001). Supporting our hypothesis, we indeed observed that while CD11b-/CD27+/- immature NK cell populations in our model are unaltered, the loss of ETS1 is associated with a decrease in CD11b+/CD27+/- mature NK cell populations.Conclusions: Our findings demonstrate that in addition to its role in early NK establishment, persistent ETS1 expression is important in intermediate differentiation stages in both human and murine NK cell development. This constitutes a significant step forward in understanding the role of ETS1 as a master transcriptional regulator in the entire NK cell developmental axis. Current studies are ongoing to dissect the mechanism by which ETS1 affects NK cell development and function in the NKp46-Ets1fl/fl mice.(*EH and JW are recipients of Pelotonia Graduate and Undergraduate student fellowships respectively and contributed equally to this work. This work was partly supported by OCRA, NIH R01 CA159296, NIH R01 CA208353, P01CA95426, R35 CA197734 and OSUCCC Leukemia Tissue Bank and Genetically Engineered Mouse Modeling Core supported by P30CA016058) DisclosuresNo relevant conflicts of interest to declare.

IVIG induces apoptotic cell death in CD56dim NK cells resulting in inhibition of ADCC effector activity of human PBMC

The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56dim NK cells without affecting CD56bright NK cells. Induction of apoptosis in CD56dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab’)2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients.

CD56bright natural killer cells induce HBsAg reduction via cytolysis and cccDNA decay in long-term entecavir-treated patients switching to peginterferon alfa-2a.

HBV surface antigen (HBsAg) reduction is well observed in chronic hepatitis B (CHB) patients treated with pegylated interferon alpha-2a (PegIFNα). However, the mechanism of HBsAg suppression has not been fully elucidated. Twenty-seven of 55 entecavir-treated CHB e antigen positive patients were switched to PegIFNα treatment (Group A) whereas 28 patients continued entecavir treatment (Group B). The percentage or absolute number of CD56bright /CD56dim NK cells, expression of receptors and cytokines were evaluated by flow cytometry for 48weeks and correlated with treatment efficacy. In vitro, purified NK cells were co-cultured with HepAD38 cells for measurement of HBsAg, apoptosis and covalently closed circular DNA (cccDNA). In association with a reduction of HBsAg, the percentage and absolute number of CD56bright NK cells was significantly elevated in patients in group A, especially in Virologic Responders (VRs, HBsAg decreased). Furthermore, the percentage of NKp30+ , NKp46+ , TRAIL+ , TNF-α+ and IFNγ+ CD56bright NK cells were significantly expanded in Group A, which were positively correlated with the decline of HBsAg at week 48. In vitro, peripheral NK cells from Group A induced a decline of HBsAg in comparison with NK cells from Group B which was significantly inhibited by anti-TRAIL, anti-TNF-α and anti-IFNγ antibodies. Furthermore, apoptosis of HepAD38 cells and levels of cccDNA, were significantly reduced by TRAIL+ and TNF-α+ /IFNγ+ NK cells from Group A, respectively. A functional restoration of CD56bright NK cells in entecavir-treated patients who were switched to PegIFNα contributes to HBsAg and cccDNA clearance through TRAIL-induced cytolysis and TNF-α/IFNγ-mediated noncytolytic pathways.

NK Cells Differentiated from Hematopoietic Stem Cells Exert Anti-HCC and Anti-HCV Activities Similar to Those of Peripheral Blood-Propagated NK Cells

NK Cells Differentiated from Hematopoietic Stem Cells Exert Anti-HCC and Anti-HCV Activities Similar to Those of Peripheral Blood-Propagated NK Cells