Serum HBV pregenomic RNA exhibited opposite associations with NKdim and NKbright cell immunity in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus (HBV) pregenomic RNA (pgRNA) is a new biomarker that reflects HBV replication, but its relationship with natural killer (NK) cell immunity in chronic hepatitis B (CHB) is unknown. We assessed serum HBV pgRNA levels in 323 CHB patients by reverse transcription-polymerase chain reaction, assessed cytokine production and activation and inhibitory markers of NK cells by flow cytometry, and measured serum cytokines by enzyme-linked immunosorbent assays (ELISAs). Among the different CHB phases, the serum HBV pgRNA level was highest in the immune-tolerant (IT) and immune-active (IA) phases. Regarding NK and NKdim cells, HBV pgRNA was negatively associated with frequencies, but positively associated with NKp44 and NKp46 expression (activation markers). Regarding NKbright cells, serum HBV pgRNA was positively associated with frequency and programmed cell death protein 1 (PD1) expression (inhibitory marker), but negatively associated with NKp44 and NKp46. Serum HBV pgRNA was not associated with NKp30 (activation marker) on NK cells or subsets. Lastly, serum HBV pgRNA was positively correlated with the levels of serum IL-7 and IL-12P40 (NK cell-promoting cytokines) and negatively correlated with serum prostaglandin E2 (PGE2) level (which negatively regulates NK cells). In conclusion, we found varied relationships between serum HBV pgRNA and NK cells and subsets, indicating that HBV pgRNA may play a complicated role in NK cell-related immunity, providing new information on HBV and host immunity.

Changes in NK Cell Subsets and Receptor Expressions in HIV-1 Infected Chronic Patients and HIV Controllers.

Natural killer (NK) cells are major effectors of the innate immune response and purported to play an influential role in the spontaneous control of HIV infection. In the present study, we compared the phenotypes of NK cells in the peripheral blood of three groups of subjects with chronic HIV-1 infection, HIV controllers, and healthy donors. The results showed that CD56+/CD16- NK cell subsets decreased in chronic patients and remained unchanged in controllers. Notably, we found that people living with chronic HIV-1 infection had suppressed NKp80, NKp46, and NKG2D expressions on NK cells compared to healthy donors, while HIV controllers remained unchanged. In contrast, NKG2D expression was substantially higher in controllers than in chronic patients (M=97.67, p<0.001). There were no significant differences in inhibitory receptors KIR3DL1 and KIR2DL1 expressions. In addition, plasma cytokine IFN-γ, TNF-α and IL-12showed higher levels in HIV controllers compared to chronic patients. Overall, our study revealed that, as compared to chronic patients, HIV controllers show an increased activating receptors expression and higher number ofCD56+/CD16-NK cell subset, with increased expression levels of plasma cytokines, suggesting that higher immune activation in controllers may have a key role in killing and suppressing HIV.

Enumeration of peripheral blood NKp46 positive NK lymphocytes reflects NK cytotoxic activity in vitro.

Natural killer (NK) cells are the predominant innate lymphocyte subsets that mediate anti-tumor and anti-viral responses. The monitoring of NK cells function is important in various physiological and pathological conditions. Different approaches have been used to directly or indirectly evaluate NK cells activities. The purpose of this study was to investigate the correlation between the number of NK cells and cytotoxic activity of NK cells and to determine whether NKp46+NK cells reflect NK cytotoxicity status. In our study, we retrospectively analyzed laboratory data on NK cytotoxicity and NK lymphocyte levels of 4896 infertile women which underwent routine immunology investigation after IVF failures. In healthy women, NKp46 expression was assessed on NK cells (n = 214) and cytotoxicity activity was evaluated with regard to NKp46 expression. We found that despite a significant correlation coefficient (n = 4689, r = 0.447), the correlation with cytotoxicity is maintained only within the zones with a low or high NK cells frequency. NK cells frequency has no significant prognostic value for their cytotoxicity - within the medium NK frequency zone the samples may have any cytotoxicity, both reduced and elevated. However, our data demonstrate that NKp46+NK cells frequency correlates with cytotoxicity activity even more significantly than the NK cells frequency (n = 214, r = 0.67 and r = 0.62, respectively) and has significant prognostic value for the abnormal NK cytotoxicity status indications, both low and increased. Our results further support an important role of NKp46 in NK cells killing and afford grounds for using the measurement of the NKp46+NK cells frequency as an alternative method for abnormal NK cytotoxicity status indication, which is responsive, simple and reliable.

Immunomodulatory and Metabolic Changes after Gnetin-C Supplementation in Humans.

Gnetin-C is a naturally occurring stilbene derived from the seeds of Gnetum gnemon L., an edible plant native to Southeast Asia that is called melinjo. Although the biological properties and safety of G. gnemon extract, which contains nearly 3% Gnetin-C, have been confirmed in various human studies, whether or not pure Gnetin-C is safe for humans is unclear at present. We conducted a randomized, double-blind, placebo-controlled trial. Healthy subjects were randomly divided into two groups. The interventional group (n = 6) was given Gnetin-C, and the control group (n = 6) was provided a placebo, for 14 days. Lipid profiles, biomarkers of oxidative stress and circulating blood cells were assessed before and after the intervention. All subjects completed the study, with no side effects reported across the study duration. Gnetin-C supplementation demonstrated a statistically significant increase in the absolute number of circulating natural killer (NK) cells expressing the activating receptors NKG2D and NKp46. NK cells derived from subjects who received Gnetin-C for two weeks showed higher cytotoxicity against K562 target cells than those before receiving Gnetin-C. In addition, Gnetin-C also resulted in a significant decrease in the absolute neutrophil count in the blood compared with the placebo. Furthermore, Gnetin-C significantly reduced the levels of uric acid, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total adiponectin, and high-molecular-weight adiponectin. These data indicate that Gnetin-C has biological effects of enhancing the NK activity on circulating human immune cells. The immunomodulatory effects are consistent with a putative improvement in cancer immunosurveillance via the upregulation of the NKG2D receptor. The study was registered with UMIN-CTR, number 000030364, on 12 December 2017.

Direct antiviral agents upregulate natural killer cell potential activity in chronic hepatitis C patients.

Direct antiviral agents (DAAs) can eliminate hepatitis C virus rapidly and make chronic hepatitis C (CHC) curable. The changes in the innate immune system during treatment with DAAs are still in dispute. To investigate how the functions of natural killer (NK) cells change during and after treatment with DAAs in each NK cell subset. Thirteen CHC patients were treated with sofosbuvir/ledipasvir, and the expression levels of NKp46 and NKG2A were tested via flow cytometry at baseline, at 2, 4, 8 and 12 weeks during the therapy and 12 and 24 weeks after the end of treatment; expression levels were compared between CHC patients and 13 healthy controls. A redirected killing assay was used to detect the cytotoxicity of NK cells. After coculturing NK cells with JFH-Huh7 cells for 72 h, HCV RNA was tested to analyze the inhibition ability of NK cells. All patients achieved sustained virologic response. The expression of the activating receptor NKp46 was decreased first at week 8 during therapy with DAAs and then increased and normalized to levels in healthy controls after treatment with DAAs. The expression of the inhibitory receptor NKG2A was decreased during and after treatment with DAAs. Each NK cell subset has a similar changing trend during and after treatment with DAAs, although some differences can be found earlier and later. The ratio of NKp46 and NKG2A was upregulated after treatment with DAAs. CD56bright NK cells have less amplitude in the frequency ratio changes after treatment with DAAs. The coculture results showed that both the specific lysis and the inhibition of HCV replication were significantly upregulated after treatment with DAAs. DAA treatments can affect patients' NK cell function. After DAA treatments, the expression of functional markers is downregulated, but the potential activity of NK cells is upregulated. The function of NK cells is normalized to levels in healthy controls. CD56bright NK cells play an important role in this process.

Accelerated cell cycle progression of human regulatory T cell-like cell line caused by continuous exposure to asbestos fibers.

Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. Based on our hypothesis in which continuous exposure to asbestos of immune cells cause reduction of antitumor immunity, the decrease of natural killer cell killing activity with reduction of NKp46 activating receptor expression, inhibition of cytotoxic T cell clonal expansion, reduced CXCR3 chemokine receptor expression and production of interferon-γ production in CD4+ T cells were reported using cell line models, freshly isolated peripheral blood immune cells from health donors as well as asbestos exposed patients such as pleural plaque and mesothelioma. In addition to these findings, regulatory T cells (Treg) showed enhanced function through cell-cell contact and increased secretion of typical soluble factors, interleukin (IL)-10 and transforming growth factor (TGF)-ß, in a cell line model using the MT-2 human polyclonal T cells and its sublines exposed continuously to asbestos fibers. Since these sublines showed a remarkable reduction of FoxO1 transcription factor, which regulates various cell cycle regulators in asbestos-exposed sublines, the cell cycle progression in these sublines was examined and compared with that of the original MT-2 cells. Results showed that cyclin D1 expression was markedly enhanced, and various cyclin-dependent kinase-inhibitors were reduced with increased S phases in the sublines. Furthermore, the increase of cyclin D1 expression was regulated by FoxO1. The overall findings indicate that antitumor immunity in asbestos-exposed individuals may be reduced in Treg through changes in the function and volume of Treg.

Up-regulation of NKG2A inhibitory receptor on circulating NK cells contributes to transfusion-induced immunodepression in patients with ß-thalassemia major.

Accumulating evidence has shown that allogeneic blood transfusions can induce significant immunosuppression in recipients, and thereby increase the risk of postoperative infection and/or tumor relapse. Although it is well known that natural killer (NK) cells are responsible for the immunodepression effects of transfusion, the underlying mechanisms remain obscure. In this study, we investigated the role of NK cells in transfusion-induced immunodepression in ß-thalassemia major. The proportion of circulating NK cells and the expression of NK receptors (NKG2A, CD158a, NKP30, NKP46 and NKG2D) as well as CD107a were detected by multicolor flow cytometry. IFN-γ production by circulating NK cells was detected by intracellular cytokine staining. Our results showed that the proportion and cytotoxicity (CD107a expression) of circulating NK cells in transfusion-dependent ß-thalassemia major patients were remarkably lower than those of ß-thalassemia minor patients or healthy volunteers. Expression of NKG2A inhibitory receptor on circulating NK cells in patients with ß-thalassemia major was remarkably up-regulated, but there were no significant differences in the expression levels of NKP30, NKP46, NKG2D, CD158a and IFN-γ. These results indicate NKG2A inhibitory receptor may play a key role in transfusion-induced immunodepression of NK cells in patients with ß-thalassemia major.

Functional Behavior of NKp46-Positive Intrahepatic Natural Killer Cells Against Hepatitis C Virus Reinfection After Liver Transplantation.

BACKGROUND: NKp46 expression in natural killer (NK) cells has recently been shown to affect the responsiveness to antiviral treatment in hepatitis C virus (HCV)-infected patients. However, the density of NKp46 on intrahepatic NK cells is remarkably higher than that on peripherally circulating NK cells, whereas the biophylactic function of intrahepatic NK cells against HCV reinfection remains unclear. METHODS: We analyzed the phenotypic and functional properties of intrahepatic NK cells using mononuclear cells extracted from ex vivo liver perfusates from living liver transplantation donors. To investigate the role of intrahepatic NK cells in relation to HCV infection, we evaluated posttransplant HCV load kinetics in HCV-related patients. RESULTS: Intrahepatic NK cells from healthy donors showed a distinctive phenotype even in each of the CD56 and CD56 fractions compared with peripheral blood NK cells. In the assays using a Huh7-HCV replicon system, anti-HCV activity was induced via recognition of the NK cell receptors, including NKp46, NKp30, and NKG2D, which was demonstrated by the use of monoclonal antibodies that neutralized neutralizing molecules. Unexpectedly, the density of NKp46 on intrahepatic NK cells varied considerably among individuals, allowing us to demonstrate that HCV reload in the early posttransplant period was delayed in recipients of liver allografts containing a higher proportion of NKp46 NK cells. CONCLUSIONS: Intrahepatic NKp46 NK cells exhibited anti-HCV activity via cell-to-cell contact. The variation of the NKp46 proportion in individuals could be attributed to the diversity of HCV resistance observed in these individuals, which possibly reflects the clinical outcome of infection in patients.

Expression of NKp30, NKp46 and DNAM-1 activating receptors on resting and IL-2 activated NK cells from healthy donors according to CMV-serostatus and age.

Human natural killer (NK) cells are innate lymphoid cells with capacity to kill tumor cells and virus-infected cells. According to the expression of CD56 and CD16 several NK cell subsets have been identified, a major CD56dimCD16+ subpopulation characterized by higher cytotoxic capacity, two CD56bright subsets (CD16-and CD16+) that represent different maturation stages and the fourth CD56-CD16+ subset that correspond to activated dysfunctional NK cells. Previous studies have shown quantitative changes in the frequency, phenotype and distribution of NK cell subsets depending on CMV-serostatus and age. We have analyzed the expression of NKp30, NKp46 and DNAM-1 NK activating receptors on resting and IL-2 activated NK cells from CMV-seronegative and seropositive healthy young donors and from CMV-seropositive elderly individuals. Our results showed that CMV-serostatus of healthy young donors is associated with phenotypic differences on both CD56bright and CD56dim NK cells with an increase of NKp46 and a decrease of NKp30 expression respectively. A reduced expression of DNAM-1 related to ageing and a lower NKp30 expression associated with CMV-seropositivity were observed. The expression of NKp46 and NKp30 was lower in CD57+ NK cells while the expression of DNAM-1 was increased. In vitro NK cell activation by IL-2 increased the expression of NKp46 and NKp30. In summary, both age and CMV-serostatus influence the expression of these cytotoxicity activating receptors that will have functional consequences. In elderly donors is difficult to isolate age from the effect of chronic CMV infection since in our study all elderly donors were CMV-seropositive. The possibility of modulating the expression of these activating receptors by cytokines such as IL-2 may open new opportunities for improving age-associated deterioration of NK cell function.

NK22 Cells in the Uterine Mid-Secretory Endometrium and Peripheral Blood of Women with Recurrent Pregnancy Loss and Unexplained Infertility.

PROBLEM: We aimed to investigate natural killer 22 (NK22) cells in the peripheral blood and the uterine endometrium of women with unexplained recurrent pregnancy loss (URPL) and unexplained infertility (UI). METHOD OF STUDY: Peripheral blood and endometrial samples were collected from women with URPL (n = 43) and UI (n = 38). Intracellular cytokine production, such as IL-22, IFN-γ and TNF-α, and the expression of NKp46 on NK cells were analyzed by three-color flow cytometry. RESULTS: The percentages of endometrial CD56(+) /IL-22(+) and CD56(dim) /IL-22(+) cells in women with URPL were significantly higher than those of UI (P < 0.05, respectively). In addition, the percentage of CD56(bright) /IL-22(+) cells in women with RPL was negatively correlated with those of CD56(bright) /IFN-γ(+) and CD56(bright) /TNF-α(+) in both peripheral blood and endometrial NK cells. This was not seen in women with UI. The percentage of CD56(bright) /IL-22(+) cells was negatively correlated with CD56(bright) /NKp46 expressing NK cells in peripheral blood. CONCLUSION: Endometrial NK22 cells are differently regulated in women with URPL and UI. Women with URPL have higher level of NK22 cells with a potential to induce NK2 shift than women with UI.

High frequencies of CD158b+ NK cells are associated with persistent hepatitis C virus infections.

BACKGROUND: During the early phases of a hepatitis C virus (HCV) infection, NK cell activation appears to be critical to the induction of adaptive immune responses that have the potential of clearing the infection. This study aimed to investigate the phenotype and function of NK cells in chronic HCV (CHC) patients, particularly patients who cleared HCV infections spontaneously (SR-HCV). MATERIAL AND METHODS: Peripheral blood NK cells were compared between 36 CHC patients, 12 SR-HCV patients, and 14 healthy controls (HC). The phenotype and function of NK cells were characterized by flow cytometry. In addition, the potential associations between the frequency of NK cell subsets and ALT, AST and HCV viral loads were also analyzed. RESULTS: Our data revealed that the population of CD3-CD56+ NK cells was significantly decreased in CHC and SR-HCV patients compared to levels in HC (P = 0.031, P = 0.014). Interestingly, we found that the levels of the CD158b inhibitory receptor were higher in CHC patients compared to levels observed in HCand SR-HCV subjects (P = 0.018, P = 0.036). In addition, the percentages of the activation receptors NKp30 and NKp46 were significantly decreased in CHC and SR-HCV patients compared to their expression levels in HC (P < 0.05). Moreover, the frequencies of inducible CD107a (but not IFN-γ-secreting) NK cellsfrom both CHC and SR-HCV patients were significantly lower than frequencies observed in controls (P = 0.018, P = 0.027). CONCLUSION: Our data indicated that the higher frequency of inhibitory NK cells combined with fewer activated NK cells may be associated with HCV-related chronic inflammation involved in CHC pathogenesis.

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age.

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3(-)CD2(+) and NKp46(+) largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3(-)CD2(+)NKp46(+). The remainder of the NK-like cells comprised two minor populations, CD3(-)CD2(+)NKp46(-) and CD3(-)CD2(-)NKp46(+); the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3(-)CD2(+) and NKp46(+) NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8alphaalpha and CD8alphabeta and did not express CD21, WC1, CD14 or gammadelta TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3(-)CD2(-)NKp46(+) population expressed CD8alpha compared to CD3(-)CD2(+)NKp46(+) cells. Furthermore, a significantly greater proportion of the CD3(-)CD2(+)NKp46(-) population expressed CD8 compared to total CD3(-)CD2(+) cells. Adult cattle had a significantly higher proportion of perforin(+) cells compared to calves aged