Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Following acute infection a majority (50%-85%) of healthy adults will develop persistent viremia. Effective clearance of an acute viral infection typically requires the coordinated function of multiple arms of the immune system, including the innate immune system (interferons (IFN), natural killer (NK) and natural killer T (NKT) cells), as well as the adaptive or acquired immune response specific to a given pathogen (CD4+ and CD8+ T cells). Numerous studies have examined the failure of both endogenous IFN system, as well as failure of adaptive T cells due to evasion or exhaustion in HCV (reviewed in Bowen and Walker1 and Gale and Foy2) In order to understand this early suboptimal development of the immune response, increasing attention has been paid to components of the innate immune system, including NK cells.
NK cells constitute an important role in early effector function against foreign antigens and play an important role in the activation and maintenance of subsequent adaptive immune responses, with their primary role being the ability to rapidly produce large amounts of cytokines. When activated, NK cells are able to initiate a response that involves both cellular cytotoxicity and secretion of cytokines such as IFNγ, which may have direct antiviral effects as well as serve to recruit other cell types involved in host defenses. NK cells may play a role in both the differentiation of adaptive T cells as well as their subsequent proliferation.3 NK cells play important roles in the defense against viral infection,4 as well as in tumor surveillance.5
Phenotypic characterization of intrahepatic mononuclear cell populations show NK cells are relatively enriched compared to peripheral blood, comprising 10% to 15% of PBMC and 20% to 30% of mononuclear cells in normal human liver. Given their known importance in resolution of viral diseases in general and their relative concentration within the liver, it is perhaps surprising that there is not more data on this immune response in HCV. However, due to the extremely rapid nature of the NK response, it has been difficult to measure NK function in acutely infected persons and even chimpanzees. The study by Khakoo et al. suggested that a genetic element predisposing to enhanced NK function contributes to spontaneous clearance of HCV,6 but this was a retrospective study and clear functional differences have not been described in the setting of acute infection. Therefore, most studies have focused on measurement of NK function in the setting of longstanding chronic infection.
Most studies to date in HCV have defined NK on the basis of lack of CD3 (the prototypic T cell marker) and CD56 positivity; with function assessed as cytolysis of target cell lines such as K652 that are relatively deficient in HLA class I expression, as well as production of cytokines such as IFNγ. Unfortunately, there has been no clear consensus about either the frequency or function of NK cells in chronic HCV. In this issue, Morishima et al. reports that there is a decreased frequency of NK cells,7 similar to what others have reported previously in both blood8–10 and liver,11, 12 particularly in livers with cirrhosis. However, Corado et al. reported that while there were no differences in the frequency of CD3-CD56+ cells, persons with chronic HCV had a four fold lower level of spontaneous cytotoxicity than normal controls.13 This functional difference has been confirmed by some9, 10 but not all groups.14 In some cases, rather than cytolysis of target cells, the potential for cytolysis was addressed using flow cytometric analysis of perforin, a major effector molecule for cytolysis in NK cells,9 and similarly found reductions in perforin expression although cytotoxicity of target cell lines was preserved. The present study found that when the input number of NK cells was corrected for in the analysis, there was no difference in cytolytic function between HCV+ persons and healthy controls.
One limitation of all these studies is that they cannot recapitulate the hepatic microenvironment, particularly with regard to local concentrations of virus. HCV envelope 2 (E2) may play a role in inhibiting both NK cytotoxicity and production of cytokines through binding to and cross-linking of CD81, which is expressed on most nucleated cells.15, 16 While this cross-linking of E2 to CD81 activates conventional T cells, it serves to inhibit the activity of NK cells through mechanisms distinct from NK inhibitory receptors.15 Therefore, it remains possible that high local concentrations of envelope protein may alter NK function, an effect that would be difficult to discern ex vivo.
Recently it has been appreciated that NK cells may have substantial functional diversity, and the phenotypic differences of the different NK subsets are still being defined. Currently, two major populations of NK cells are recognized based on the relative level of CD56 expression, low (CD56dim) or high (CD56bright).17 CD56dim cells, which comprise approximately 90% of all NK cells, are primarily cytotoxic and express relatively low levels of cytokines compared to CD56bright cells, which produce cytokines such as IFNγ upon activation. There is still some controversy about whether these are separate lineages of NK cells or whether the CD56dim population represents a terminally differentiated cell that is poised to undergo apoptosis upon stimulation.18 This failure to distinguish among the different NK subsets may explain discrepancies among the published studies, since variable numbers of different NK subsets, as well as measurement of different effector functions, might lead to different results. At least 3 reports, including that of Morishima et al., have found a reduction in the CD56dim population.7–9 This finding may be linked to a decrease in serum interleukin (IL)-15, a key cytokine in the development and maintenance of NK homeostasis,9 although liver expression of IL-15 may be increased.19 However, even if one focuses on the CD56dim population it is not clear that there are clear differences in the cytolytic capabilities of these cells when the number of effector cells is taken into account, as the present study and others demonstrate.7, 14
Part of the reason to define the role of different subsets of NK cells is that it is not clear which function of NK — cytolysis or production of cytokines such as IFNγ — may be more relevant in control of HCV. Certainly ex vivo IFNγ production by NK cells can inhibit HCV replication in the replicon system20; whether this is operative in vivo is unclear. Although production of IFNγ is associated temporally with viral clearance,21 since there are abundant amounts of IFNγ message in the liver of chronically infected chimpanzees and humans,22–24 the question remains whether or not this cytokine is truly effective at control of HCV in vivo. A recent report by Shin and colleagues demonstrated that high level expression of IFNγ in the liver of HCV-infected chimpanzees failed to result in reduction in HCV titers despite increased HCV-specific and non-specific immunity,25 suggesting that either there are additional cytokines involved in the control of HCV or there is some intrinsic block to IFNγ in chronic HCV. If the cytolytic capability of these cells is the primary effector mechanism, then the increase in MHC class I expression observed on the surface of infected hepatocytes may serve to render infected hepatocytes relatively resistant to NK-mediated cytolysis.26 This increase in class I expression may be independent of inflammation, as HCV core protein has been shown to upregulate MHC-Class I expression on hepatocytes and results in impairment of NK activation.27
Furthermore, different NK subsets may have different roles in the pathogenesis of liver injury, but this too is an area that needs further development. Lin et al. found that activated CD56bright correlated with inflammation but not fibrosis,8 whereas there was no relationship between grade or stage and CD56dim. Because CD56bright can produce a plethora of immunoregulatory cytokines in addition to IFNγ, including tumor necrosis factor, granulocyte macrophage colony stimulating factor (GM-CSF), IL-10 and IL-13, this subset of cells may be activating hepatic stellate cells directly or enhancing nonspecific inflammation. However, the present study found that CD56dim correlated inversely with fibrosis stage. Whether low levels of CD56dim cells are the cause of increased fibrosis, by failing to protect the liver, or are simply associated with longstanding disease, as there may be apoptosis and loss of these cells over time, will require prospective studies.
Better analytic tools are also needed in this field, as analysis by flow cytometry of phenotypic markers does not necessarily translate into quantitative or qualitative data about the function of NK cells, and chromium release is a relatively insensitive assay. For example, the use of tetramers allows investigators to determine whether or not a given CD8+ T cell is antigen-specific or not, and whether that cell is capable of producing cytokines; similarly, the use of ELIspot facilitates accurate quantitation of antigen-specific CD4+ and CD8+ T cells within the larger population of T cells. Clearly more studies are needed to understand the role of NK cells in both spontaneous clearance of HCV and the role of these cells in the pathogenesis of liver injury.