Cumulative evidence suggests that NK cells contribute to the control of HCV infection. In this article we show the following 3 things: (1) NK cells from healthy controls as well as patients with acute hepatitis C can efficiently recognize HCV-infected hepatoma cells when stimulated with IFN-α and suppress replication of the virus; (2) HCV infection does not alter the expression of NK cell receptor ligands on hepatoma cells; and (3) the induction of effector functions in IFN-α–stimulated NK cells, following coculture with hepatoma cells, depends on the activation receptor DNAM-1.
We show here that efficient recognition of hepatoma cells by NK cells is dependent on IFN-α stimulation. This finding is rather surprising, because previous studies investigating the interaction between NK cells and various malignant cell types have indicated that stimulation of NK cells with IL-2 is sufficient [29
]. Thus, we conclude that immunomodulatory effects of IFN-α in the treatment of HCV infection can be attributed, at least partly, to induction of functional changes of NK cells enabling them to recognize hepatoma cells. Of note, a significant proportion of IFN-α–stimulated NK cells cocultured with hepatoma cells was polyfunctional. The reduction of HCV replication could be due to lysis of infected cells [23
] as well as noncytotoxic mechanisms [21
Overall, HCV infection did not induce any obvious changes in expression levels of various NK cell receptor ligands. This could suggest that functional impairment of NK cells in HCV infection is mediated mainly by direct effects on the NK cells rather than by interference of HCV with NK cell receptor ligands on the target cells. Support for this probability comes from earlier descriptions of the inhibitory effects of HCV-E2 protein on NK cell function [31
]. However, a more recent study did not confirm these findings using full infectious particles instead of HCV-E2 protein [33
]. In our hands, NK cells from patients with acute hepatitis C were functional and responded to IFN-α stimulation in vitro equally well as NK cells from healthy controls. However, we did not have the possibility to directly compare the functional characteristics of NK cells from patients with acute hepatitis C and healthy controls. There might be interindividual differences in the intrinsic capacity to respond to IFN-α, because PBMCs from patients who respond to treatment with IFN-α show higher up-regulation of TRAIL upon IFN-α stimulation in vitro than do PBMCs from patients who do not respond to such treatment [23
NKp30 expression may be altered in chronic hepatitis C [17
], and the molecule has been designated as a mediator of interactions between NK cells and antigen-presenting cells [35
]. Recently, it was shown that the inhibitory effects of myeloid-derived suppressor cells on NK cells are mediated via NKp30 [36
]. The role of NKp46 in hepatitis C remains controversial [17
]; nevertheless, this receptor is involved in interactions between NK cells and antigen-presenting cells and tumor cells [35
]. However, neither NKp30 nor NKp46 seemed to be involved in NK cell recognition of Huh-7.5 cells. NKG2D partially contributed to the recognition of hepatoma cells by IFN-α–stimulated NK cells, yet the most dominant effect was evident in the presence of DNAM-1. DNAM-1 is expressed by almost all human NK cells, T cells, and monocytes [39
]. To the best of our knowledge, no data are available on the role of DNAM-1 on NK cells in either acute or chronic hepatitis C. However, the DNAM-1 is involved in the interactions between NK cells and numerous tumor cells, including melanoma, ovarian carcinoma, leukemia, and neuroblastoma cells [40
]. Moreover, DNAM-1 is involved in the killing of dendritic cells [44
]. The ligands for DNAM-1, CD112, and CD155 are 2 related molecules belonging to the nectin family [45
]. Importantly, CD155 is highly expressed on Huh-7.5 cells, indicating that hepatoma cells are indeed delivering signals to NK cells via CD155:DNAM-1 interaction. However, our results cannot exclude the possibility that ligation of additional NK cell receptors is required to induce full NK cell effector functions against HCV-infected hepatoma cells.
Because the recognition of hepatoma cells by NK cells required stimulation with IFN-α, and because this recognition was mediated by DNAM-1, one could speculate that the effect of IFN-α was mediated via changes in DNAM-1 receptor expression. However, this was not the case. Stimulation of NK cells with IFN-α in vitro did not affect DNAM-1 expression on NK cells. Although NK cells from patients with acute hepatitis C exhibit an activated phenotype, DNAM-1 expression was not altered, as compared with healthy controls. Thus, these data suggest that IFN-α may alter the intracellular signaling of DNAM-1. Alternatively, IFN-α may regulate another yet unknown receptor on NK cells, with DNAM-1 acting as a coreceptor.
DNAM-1 has been shown to amplify effector functions of immune cells as a stimulatory molecule in various contexts, including allergic inflammation [46
], autoimmune diseases [47
], and viral infections such as human immunodeficiency virus infection [48
]. Thus, DNAM-1 has already been suggested as a therapeutic target for modulating immune responses [12
]. For example, NK cell responses against tumors and pathogens might be augmented by DNAM-1 activation. Therefore, understanding in detail how IFN-α enhances DNAM-1–dependent recognition of hepatoma cells might lead to the identification of potential therapeutic targets to enhance innate immunity against not only HCV infection but also other hepatotropic pathogens and primary hepatocellular carcinomas. Conversely, blocking DNAM-1 could down-regulate immune responses against hepatocytes, which may have implications in the context of autoimmune hepatitis.
It is important to bear in mind that results reported here were obtained using a tumor cell line as a model system and not primary hepatocytes. Recently, however, persistent infection with HCV has been demonstrated in primary human hepatocytes [49
], which will provide an interesting ground to substantiate the present results.
In conclusion, IFN-α increases recognition of HCV-infected and uninfected hepatoma cells by NK cells from patients with acute hepatitis C, potentially contributing to the control of HCV infection. This effect is mediated via DNAM-1. Understanding the detailed molecular mechanism by which IFN-α modulates DNAM-1–dependent interactions of NK cells with hepatoma cells may help identify novel immunomodulatory treatment approaches for hepatitis C.