Introduction Efforts to identify the immune-correlates responsible for resolution of hepatitis C virus (HCV) infection are fundamental to develop new treatment strategies and an effective vaccine against HCV. We have previously shown that imbalanced natural killer (NK)-cell subsets, with hyper-expression of co-inhibitory markers, are associated with chronic HCV infection.

Aim In this study we aim to assess the role of NK-cells in determining the outcome of acute HCV infection in a cohort of well characterised patients.

Method We analysed 12 patients with acute HCV infection who met the following criteria: ALT>10xULN, exposure to HCV within previous 4 months and HCV-RNA(+). Viral load was determined by qPCR. Peripheral blood mononuclear cells collected at 3 time-points (baseline, BL; month 1, M1; month 6, M6) were stained with fluorochrome-labelled antibodies to NK-cells (CD3/CD56/CD16). Proportions of CD56dim(CD16bright) and CD56bright(CD16dim) subsets and expression of PD-1/PD-L1 were evaluated by 6-colour flow cytometry and correlated with HCV-RNA and ALT at each time-point and over-time. Supernatants from cell-cultures in the presence of HCV-antigens were collected for cytokine analysis and quantification.

Results Six patients resolved HCV spontaneously (Resolvers), whilst six developed chronic infection (Chronics). At presentation, mean viraemia and ALT levels did not differ between Resolved and Chronic patients. In Resolvers HCV-RNA became undetectable at M3, which was then followed by ALT normalisation. Overall, Resolvers had higher proportions of total NKs than Chronics (p=0.023). Cytotoxic CD56dim NK cells were also higher in Resolvers (p=0.001), and became progressively predominant within their total NK pool, as shown by their progressive increase of the CD56dim/CD56bright ratio, which differentiated Resolvers from Chronics after M1 (p=0.01). In Chronic patients’ cytotoxic CD56dim NK cells had, however, an overall greater expression of the cytotoxicity marker CD16 (p=0.008). Chronic patients also had higher proportions of both subsets of NK cells expressing the immunoinhibitory marker PD-1 (p=0.02), and stronger per-cell expression of the immunoinhibitory ligand PD-L1 on CD56dim NK cells (p=0.001). Analysis of clinical parameters revealed that in Resolvers the progressive increase of CD56dim/CD56bright ratio correlated with the decline of HCV-RNA over-time (r=−0.997, p=0.042), due to reduction of CD56bright (r=0.999, p=0.011) and expansion of CD56dim (r=−0.997, p=0.041). At M1, Resolvers’ CD56dim were positively correlated with ALT (r=0.820, p=0.046).

Conclusion In patients with acute HCV infection the proportions and evolution of the two functionally distinct NK cell subsets differ in patients that resolve the infection compared to those who become chronically infected. A favourable outcome of infection is associated with the establishment of a defined NK profile, with predominance of CD56dim (cytotoxic) NK cells with low expression of the immunoinhibitory markers PD-1 and PD-L1, which may be linked to an improved early clearance of virus-infected hepatocytes, as shown by the correlation of this subset with serum HCV-RNA and ALT decline.