Hepatitis C Virus (HCV) Eradication With Interferon-Free Direct-Acting Antiviral-Based Therapy Results in KLRG1+ HCV-Specific Memory Natural Killer Cells.

Direct acting antiviral therapies rapidly clear chronic hepatitis C virus (HCV) infection and restore natural killer (NK) cell function. We investigated NK-cell memory formation following HCV clearance by examining NK-cell phenotype and responses from control and chronic HCV patients before and after therapy following sustained virologic response at 12 weeks post therapy (SVR12). NK-cell phenotype at SVR12 differed significantly from paired pretreatment samples, with an increase in maturation markers CD16, CD57, and KLRG1. HCV patients possessed stronger cytotoxic responses against HCV-infected cells as compared to healthy controls; a response that further increased following SVR12. The antigen-specific response was mediated by KLRG1+ NK cells, as demonstrated by increased degranulation and proliferation in response to HCV antigen only. Our data suggest that KLRG1+ HCV-specific memory NK cells develop following viral infection, providing insight into their role in HCV clearance and relevance with regard to vaccine design.

HBV vaccination and HBV infection induces HBV-specific natural killer cell memory.

OBJECTIVE: Vaccination against hepatitis B virus (HBV) confers protection from subsequent infection through immunological memory that is traditionally considered the domain of the adaptive immune system. This view has been challenged following the identification of antigen-specific memory natural killer cells (mNKs) in mice and non-human primates. While the presence of mNKs has been suggested in humans based on the expansion of NK cells following pathogen exposure, evidence regarding antigen-specificity is lacking. Here, we demonstrate the existence of HBV-specific mNKs in humans after vaccination and in chronic HBV infection. DESIGN: NK cell responses were evaluated by flow cytometry and ELISA following challenge with HBV antigens in HBV vaccinated, non-vaccinated and chronic HBV-infected individuals. RESULTS: NK cells from vaccinated subjects demonstrated higher cytotoxic and proliferative responses against autologous hepatitis B surface antigen (HBsAg)-pulsed monocyte-derived dendritic cells (moDCs) compared with unvaccinated subjects. Moreover, NK cell lysis of HBsAg-pulsed moDCs was significantly higher than that of hepatitis B core antigen (HBcAg)-pulsed moDCs (non-vaccine antigen) or tumour necrosis factor α-activated moDCs in a NKG2D-dependent manner. The mNKs response was mediated by CD56dim NK cells coexpressing CD57, CD69 and KLRG1. Further, mNKs from chronic hepatitis B patients exhibited greater degranulation against HBcAg-pulsed moDCs compared with unvaccinated or vaccinated patients. Notably, mNK activity was negatively correlated with HBV DNA levels. CONCLUSIONS: Our data support the presence of a mature mNKs following HBV antigen exposure either through vaccination or infection. Harnessing these antigen specific, functionally active mNKs provides an opportunity to develop novel treatments targeting HBV in chronic infection.

Expansion of dysfunctional CD56-CD16+ NK cells in chronic hepatitis B patients.

BACKGROUND & AIMS: Natural killer (NK) cells are primary innate effector cells that play an important role in the control of human viral infections. During chronic viral infection, NK cells undergo significant changes in phenotype, function and subset distribution, including the appearance of CD56-CD16+ (CD56-) NK cells, previously identified in chronic human immunodeficiency virus (HIV) and hepatitis C virus infection. However, the presence of CD56- NK cells in the pathogenesis of chronic hepatitis B (CHB) remains unknown. METHODS: Phenotype and function of CD56- NK cells from patients with CHB (n = 28) were assessed using flow cytometry and in vitro stimulation with HBV antigen. RESULTS: CHB patients had a higher frequency of CD56- NK cells compared to healthy controls in peripheral blood (6.2% vs 1.4%, P < .0001). Compared to CD56+ NK cells, CD56- NK cells had increased expression of inhibitory receptors, and reduced expression of activating receptors, as measured by MFI and qPCR. CD56- NK cells were less responsive to target cell and cytokine stimulation compared to their CD56+ counterparts. In addition, CD56- NK cells demonstrated defective dendritic cells (DCs) interactions resulting in reduced DCs maturation, lower expression of NK CD69 and impaired capacity of NK cells to eliminate immature DCs in co-culture studies. Finally, frequency of CD56- NK cells was positively correlated with serum HBV DNA levels. CONCLUSION: Chronic HBV infection induces the expansion of highly dysfunctional of CD56- NK cells that likely contribute to inefficient innate and adaptive antiviral immune response in chronic HBV infection.

KLRG1+ natural killer cells exert a novel antifibrotic function in chronic hepatitis B.

BACKGROUND & AIMS: Natural killer (NK) cells are known to exert strong antiviral activity. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is expressed by terminally differentiated NK cells and KLRG1-expressing lymphocytes are known to expand following chronic viral infections. We aimed to elucidate the previously unknown role of KLRG1 in the pathogenesis of chronic hepatitis B (CHB). METHODS: KLRG1+ NK cells were taken from the blood and liver of healthy individuals and patients with CHB. The phenotype and function of these cells was assessed using flow cytometry and in vitro stimulation. RESULTS: Patients with CHB had a higher frequency of KLRG1+ NK cells compared to healthy controls (blood 13.4 vs. 2.3%, p <0.0001 and liver 23.4 vs. 2.6%, p <0.01). KLRG1+ NK cells were less responsive to K562 and cytokine stimulation, but demonstrated enhanced cytotoxicity (9.0 vs. 4.8%, p <0.05) and IFN-γ release (8.0 vs. 1.5%, p <0.05) via antibody dependent cellular cytotoxicity compared to their KLRG1- counterparts. KLRG1+ NK cells possessed a mature phenotype, demonstrating stronger cytolytic activity and IFN-γ secretion against hepatic stellate cells (HSCs) than KLRG1- NK cells. Moreover, KLRG1+ NK cells more effectively induced primary HSC apoptosis in a TRAIL-dependent manner. Increased KLRG1+ NK cell frequency in the liver and blood was associated with lower fibrosis stage (F0/F1) in patients with CHB. Finally, the expression of CD44, degranulation and IFN-γ production were all increased in KLRG1+ NK cells following stimulation with osteopontin, the CD44 ligand, suggesting that HSC-derived osteopontin may cause KLRG1+ NK cell activation. CONCLUSIONS: KLRG1+ NK cells likely play an antifibrotic role during the natural course of CHB infection. Harnessing this antifibrotic function may provide a novel therapeutic approach to treat liver fibrosis in patients with CHB. LAY SUMMARY: Individuals that are chronically infected with hepatitis B virus (HBV) possess an increased number of immune cells, called natural killer (NK) cells expressing the surface marker KLRG1 in the blood and liver. Here, we demonstrate that these specific NK cells are able to kill activated stellate cells in the liver. Because activated stellate cells contribute to liver scarring, i.e. fibrosis, and subsequent liver dysfunction in individuals with chronic HBV infection, KLRG1+ NK cells are a novel immune cell type that can limit liver scarring.