Increased degranulation of natural killer cells during acute HCV correlates with the magnitude of virus-specific T cell responses

Background & Aims

Natural killer (NK) cells provide early defense against viral infections by killing infected cells and producing cytokines that inhibit viral replication. NK cells also interact with dendritic cells (DCs) and this reciprocal interaction regulates both innate and adaptive immunity. Genetic studies have suggested that NK cell activity is a determinant of HCV infectious outcome but a functional correlation has not been established. We hypothesized that increased NK cell activity during acute HCV infection correlates with spontaneous viral clearance.

Methods

We used multiparametric flow cytometry to monitor longitudinally the phenotype and the activity of NK cells in a cohort of intravenous drug users following HCV exposure. Three groups were studied: acute HCV with chronic evolution (n = 13), acute resolving HCV (n = 11), and exposed un-infected individuals (n = 10). We examined the expression of several NK cell-activating and -inhibiting receptors, IFN-γ production and CD107a degranulation upon stimulation, and the kinetics of NK cell responses relative to T cell responses.

Results

We observed decreased expression of the inhibitory NKG2A receptor in NK cells following spontaneous HCV clearance. In addition, we observed increased NK cell degranulation during acute HCV irrespective of infectious outcome. NK cell peak responses preceded or coincided with peak T cell responses. Furthermore, NK cell degranulation correlated with the magnitude of HCV-specific T cells.

Conclusions

Our results demonstrate that NK cells are activated during acute HCV regardless of infection outcome and may play an indirect role through induction and priming of T cell responses.

Introduction

The majority of individuals exposed to hepatitis C virus (HCV) develop persistent infection and chronic liver disease [1]. Acute HCV is characterized by a significant delay in the onset of adaptive T cell responses despite its active viral replication. This suggests a failure of innate immunity to contain viral replication and provide the necessary signals to prime an efficient adaptive immunity critical to spontaneous viral clearance [2], [3]. Natural killer (NK) cells are the most important effector population of the innate immune response. Two NK cell subsets can be distinguished based on their differential expression of CD56 and CD16: immunoregulatory CD3⁻CD56^brightCD16⁻ and cytolytic CD3⁻CD56^dimCD16⁺ [4]. NK cells provide an early defense line against viral infections by killing infected cells and producing cytokines that can directly inhibit viral replication and trigger the adaptive immune response. NK cells use inhibiting and activating receptors as a mean of controlling their activity. NK cells interact with dendritic cells (DCs) and this reciprocal interaction results in the regulation of both innate and adaptive immune responses [5], [6]. DCs can activate NK cells by binding to NKp30 on the surface of NK cells and by secreting numerous cytokines such as IL-12 [7]. NK cells, in turn, secrete IFN-γ and TNF-α which induce DC maturation and trigger the adaptive immune response [8]. In addition, NK cells can also kill immature DCs and inhibit their capacity to prime or tolerize adaptive T cell responses [5], [9].

Two observations highlighted the potential role of NK cells during the early phase of HCV infection. First, HCV surface glycoprotein E2 can bind CD81 on the surface of NK cells and inhibit cytotoxicity and IFN-γ production [10], [11]; however, Yoon et al. have recently demonstrated that the exposure of NK cells from healthy donors to in vitro-produced HCV virions did not influence their function [12]. Second, genes encoding the inhibitory NK cell receptor killer-cell immunoglobulin like receptor (KIR)2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand, directly influence the resolution of HCV infection in individuals homozygous for these genes [13], [14]. These observations suggest that the inhibition of NK function during the early phase of HCV may contribute to viral persistence.

Several groups have studied NK cells during chronic HCV infection but the results regarding NK cell frequency, cytotoxicity, cytokine production, and receptor expression are conflicting [15], [16], [17], [18], [19], [20]. This probably reflects the complexity of activating and inhibiting signals that control NK cells. Only one study has compared NK cell function in chronic HCV patients with spontaneous resolvers from a single source outbreak [15]. The authors of the study demonstrated that the frequency of the CD56^dim NK cell subset was decreased in individuals with chronic HCV, and that NK cells expressed the NKG2A/C/E receptors at higher frequency [15]; however, the activity of NK cells during acute HCV, when their role would be most prominent, and its correlation with the infectious outcome, were not studied.

In this study, we used multiparametric flow cytometry to monitor longitudinally the phenotypic and functional changes in NK cells from a unique cohort of intravenous drug users (IDUs) at high-risk of HCV infection before and during acute HCV infections that progressed to spontaneous resolution or viral persistence. In addition, we monitored NK cells activity in a group of HCV-exposed but un-infected individuals. We demonstrated that NK cell degranulation is increased during acute HCV, regardless of the infection outcome. We also observed a decline in NKG2A expression in NK cells following spontaneous viral clearance, and CD161 expression in infections progressing to chronicity. Finally, we showed that NK cell response peaks prior to T cell response and that NK cell degranulation correlate with the magnitude of the HCV-specific T cell response, suggesting an indirect role for NK cells in priming adaptive immune responses.