BioMed Research International

Volume 2014 (2014), Article ID 903764, 9 pages

http://dx.doi.org/10.1155/2014/903764

Review Article

Natural Killer Cell Function and Dysfunction in Hepatitis C Virus Infection

Kayla A. Holder, Rodney S. Russell, and Michael D. Grant

Immunology and Infectious Diseases Program, Division of BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Phillip Drive, St. John’s, NL, Canada, A1B 3V6

Received 3 January 2014; Accepted 14 April 2014; Published 25 June 2014

Academic Editor: Naohiko Masaki

Copyright © 2014 Kayla A. Holder et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Viruses must continually adapt against dynamic innate and adaptive responses of the host immune system to establish chronic infection. Only a small minority (~20%) of those exposed to hepatitis C virus (HCV) spontaneously clear infection, leaving approximately 200 million people worldwide chronically infected with HCV. A number of recent research studies suggest that establishment and maintenance of chronic HCV infection involve natural killer (NK) cell dysfunction. This relationship is illustrated in vitro by disruption of typical NK cell responses including both cell-mediated cytotoxicity and cytokine production. Expression of a number of activating NK cell receptors in vivo is also affected in chronic HCV infection. Thus, direct in vivo and in vitro evidence of compromised NK function in chronic HCV infection in conjunction with significant epidemiological associations between the outcome of HCV infection and certain combinations of NK cell regulatory receptor and class I human histocompatibility linked antigen (HLA) genotypes indicate that NK cells are important in the immune response against HCV infection. In this review, we highlight evidence suggesting that selective impairment of NK cell activity is related to establishment of chronic HCV infection.

1. Host Invasion and Immune Evasion

Human immunity is classically divided into innate and adaptive components. The adaptive immune response is generally regarded as being uniquely mediated by B and T lymphocytes, as it is only progenitors of these cells that undergo somatic recombination-activating gene- (Rag-) dependent variable (V) gene rearrangement in order to produce a diverse clonotypic repertoire of antigen-specific receptors [1]. Antigen-mediated clonal selection, leading to expansion and persistence of particular cells or their products at elevated levels, provides the adaptive immune system with specificity and memory. In contrast, innate immune responses offer a first line of defense, stemming from cells and mechanisms that recognize pathogen-associated molecular patterns (PAMPs) in a generic, nonspecific, and noninstructive manner [2]. Coexistence of persistent viruses and their hosts exerts selective pressures on both the host immune system and on viral genomes, forcing viruses to continually evolve mechanisms through which host immune defenses are evaded.

Viral evasion strategies can include antigenic variation, synthesis of decoy proteins that inactivate immune responses, production of proteins (immunoevasins) that compromise antigen presentation, and production or induction of proteins that disrupt host humoral and cellular immune responses and/or effector functions [2, 3]. While T-cell-mediated immune responses provide long-term control of viral infections, initial management of these infections by natural killer (NK) cells, prior to development of the adaptive immune response, is thought to be crucial. In humans, depressed NK cell function is associated with sensitivity to viral infections [4]. Of particular note, Biron et al. described the case of a patient with genetic NK cell deficiency and extreme sensitivity to herpes virus infections, despite having normal numbers of B and T lymphocytes [5]. Multiple NK cell studies in the context of viral infection indicate that viruses evade immune pressure by generating variants that modulate recognition of infected cells by NK cells. Furthermore, NK cells are not only important for direct early control of viral infections, but they also contribute to induction of the adaptive antiviral immune response by releasing immunomodulatory cytokines and chemokines [6] and through bidirectional interactions with dendritic cells (DC) (reviewed in [7, 8]). These reciprocal interactions ultimately drive the T-cell immune response and, in some cases, culminate in reduced viral replication or even clearance of viral infection [9]. Recent studies also demonstrate that murine and possibly human NK cells have receptors specific for cytomegalovirus (CMV) that enable selective proliferation and expansion of NK subsets, thus endowing NK cells with limited properties previously attributed exclusively to T and B lymphocytes [10–13].

Epidemiological studies suggest that NK cells play a role in determining the outcome of hepatitis C virus (HCV) infection [14, 15]. Here, we will consider the effects HCV infection has upon NK cells by reviewing the epidemiological associations, noting in vivo evidence of NK cell dysfunction in chronic HCV infection and discussing recent in vitro experiments indicating that direct interaction between circulating NK cells and HCV-infected cells impairs NK cell function.

2. Hepatitis C Virus

Approximately 3% of the world’s population is infected with HCV [16], an enveloped, positive-sense RNA virus of the Hepacivirus genus within the Flaviviridae family [3]. The HCV RNA genome is encased by core protein multimers to form the viral nucleocapsid that is surrounded by an endoplasmic reticulum (ER) membrane-derived envelope studded with HCV envelope proteins 1 and 2 (E1/E2) [17, 18]. Host cell infection with HCV occurs through the interaction of HCV E1 and/or E2 with multiple cellular coreceptors including CD81 (also termed target of antiproliferative antibody 1 (TAPA1)) [19–23], scavenger receptor class B type I (SRBI) [24–26], occludin (OCLN) [27–29], and claudin-1 (CLDN1) [30, 31]. In the absence of effective treatment, approximately 80% of individuals infected with HCV fail to mount an immune response adequate for viral clearance and, consequently, develop chronic infection and suffer an increased risk for liver fibrosis and hepatocellular carcinoma [32–34]. While approximately 20% of HCV-infected individuals spontaneously clear infection, the mechanism of spontaneous clearance remains poorly defined and a greater understanding of both the viral clearance process and of viral strategies underlying immune escape is necessary for future vaccine development and more effective management of infection. There is mounting evidence that NK cells are involved in the clearance and control of HCV infection, including associations between NK cell dysfunction, chronic HCV infection, and the pathogenesis of liver disease [14, 35, 36].

Natural killer cells are lymphocytes that develop in the bone marrow, differentiate in lymphoid tissues, and migrate into various tissues [37]. The environment in which a NK cell matures impacts NK cell phenotype and function giving rise to a heterogeneous population. Although NK cells can be prominent in nonlymphoid tissues such as the liver and decidua [38–40], the functions of these NK cells can differ greatly from the functions of those in peripheral blood. The NK cells that comprise between 5 and 20% of peripheral blood lymphocytes provide inherent defense against some transformed cells and a variety of pathogens, including viruses [5, 41–43]. First identified in 1975 and classically defined as cytolytic lymphocytes, NK cells remain categorized as innate immune effector cells since they do not undergo receptor gene rearrangement [41, 44]. Thus, selective effector function against infected or transformed cells is mediated by composite integration of signals received through a diverse assortment of germ-line encoded activating and inhibitory receptors interacting with their respective ligands [45] (Figure 1).

Figure 1: Natural killer cell natural cytotoxicity. NK cell inhibitory receptors recognize “self” MHC-I molecules which can prevent cytolysis of healthy cells even when activating receptors interact with their ligands. Transformed or virus-infected cells often reduce MHC-I expression; thus when a NK cell activating receptor engages its ligand, there is no negative signal to overcome the positive activating signal and the target cell is destroyed through a perforin and granzyme apoptosis-inducing mechanism.

3. Natural Killer Cell Functions

Peripheral NK cells primarily fall into two major functionally distinct subsets. The highly cytotoxic “mature” NK cells are more responsive to cell surface ligands than the “immature” NK cells, which are more responsive to soluble factors [6, 46]. The NK cells are less cytotoxic but predominantly secrete cytokines, such as proinflammatory interferon- (IFN-) γ and tumour necrosis factor- (TNF-) α, or immunoregulatory interleukin- (IL-) 10 [47, 48].

Through their constellation of cell surface receptors, NK cells can detect variations in cell surface composition manifest through a multitude of ligands and integrate this information to determine the type and intensity of response that is appropriate [49]. With no prior exposure, NK cells can recognize and kill target cells that have downregulated expression of class I human histocompatibility-linked leukocyte antigen- (HLA-) I molecules, a process that occurs during many viral infections [45]. In humans, the NK receptors that recognize class I HLA molecules, predominantly killer cell immunoglobulin-like receptors (KIR), can convey NK cell activating signals through short (S) cytoplasmic domains or NK cell inhibitory signals if the KIR possesses a long (L) cytoplasmic domain [50] (see Figure 3). Inhibitory NKG2A receptors can also prevent cytolysis of healthy cells by recognizing expression of nonclassical class I HLA-E complexes [45, 51–53]. As per the “missing self” hypothesis, healthy cells expressing normal amounts of class I HLA complexes are less sensitive to NK cell lysis than are transformed or virus-infected cells with decreased HLA-I expression [54] (Figure 2). Virus-infected cells expressing adequate amounts of class I HLA molecules generally remain protected from NK cell-mediated lysis. Therefore, maintaining expression of class I HLA molecules, especially HLA-E, is one mechanism by which pathogens can thwart NK cell surveillance.

Figure 2: Natural killer cell receptor repertoire. Natural killer cells express some fraction of a diverse set of germ-line encoded receptors, many of which display allelic polymorphism. They are variously associated with different adaptor molecules that convey either activating or inhibitory signals. Two of the three natural cytotoxicity receptors, NKp30, NKp46, and also the CD16 FcγRIII receptor, mediate signaling through ITAM-containing ζ chain homodimers, FcεRIγ homodimers, or FcεRIγ/ζ heterodimers. Activating receptors depicted here are CD16, NKp30, NKp46, NKp44, KIR3DS1, CD160, and NKG2D; inhibitory receptors depicted are TIM-3 and NKG2A/CD94. The CD56 molecule is comprised of five immunoglobulin-like domains and, in the absence of CD3, is a reliable marker for NK cells.

Figure 3: Natural killer cell antibody-dependent cell-mediated cytotoxicity (ADCC). Natural killer cells can recognize and kill antibody-coated target cells. When antibodies bind antigens displayed on the surface of transformed or infected target cells, NK CD16 receptors bind the Fc portion of bound antibodies (most IgG subclasses) and mediate cytotoxicity against the target cell by degranulation and directed release of cytotoxins.

4. Natural Killer Cells in HCV Infection

Natural history studies have revealed that HCV-exposed individuals with coordinate expression of certain KIR variants and their corresponding HLA-I ligand gain a higher probability of spontaneous HCV clearance than those without [14, 55–58]. To maintain functional recognition of rapidly evolving class I HLA complexes, the NK cell KIR genes must also evolve under pathogen-mediated pressure [59, 60]. Genotyping of those exposed to HCV demonstrated that coordinate expression of NK cell receptor KIR2DL3 and its cognate class I HLA C group 1 (HLA-C1) ligand confers an increased likelihood of spontaneous HCV clearance or of a sustained virological response (SVR) to treatment when spontaneous HCV clearance is not achieved [14, 56, 61]. One functional interpretation of this association is that as the interaction between KIR2DL3 and HLA-C1 is relatively low affinity, it generates a weaker inhibitory signal than other KIR/ligand interactions allowing a greater functional response by NK cells [57, 58]. Other KIRs that have been identified as relevant to the outcome of HCV infection and treatment efficacy are 2DL2, 2DL3, 2DS1, 2DS2, and 3DL1 [61, 62]. The expression of KIR3DL1 is decreased in individuals with HCV infection, suggesting that this receptor may also be involved in the regulation of chronic HCV infection [55]. Expression of KIRs with short cytoplasmic tails promotes target cell cytolysis and IFN-γ production through DAP12 signaling (see Figure 2) upon recognition of cognate class I HLA ligands [50, 63, 64]. Although the precise nature of its HLA ligand has yet to be identified, the activating receptor KIR3DS1 appears to support protection against hepatocellular carcinoma in those chronically infected with HCV and favors HCV genotype 1a clearance in response to combination treatment with ribavirin and pegylated IFN-α [63, 65]. Favourable genetic combinations of KIR and class I HLA complexes contribute to the NK cell response not only in acute HCV infection, but also towards controlling disease progression in those chronically infected and towards sustained virological responses in those receiving treatment. Viruses can evade the NK cell immune response through the generation of variants that can escape stimulatory and/or enhance inhibitory NK cell receptor (NKR) recognition.

Some studies indicate that the HLA-E molecule, which is a ligand for the NK inhibitory receptor NKG2A, is upregulated on hepatocytes in complex with an HCV core protein peptide (aa35–44) during chronic HCV infection [66]. Nattermann et al. reported an increased expression of NKG2A/CD94 inhibitory receptors on circulating NK cells in patients with chronic HCV infection; however, in an in vitro model representative of acute HCV infection, neither NKG2A nor HLA-E expression increased on NK or HCV-infected cells, respectively [67–69]. Nonetheless, to combat NK cell-mediated control of infection, HCV may employ multiple means of increasing expression of natural or decoy ligands for inhibitory NKRs as chronic infection develops and disease progresses.

Since viral infection often results in reduced cell surface class I HLA expression, NK cell inhibitory signals will be dampened, allowing NK cells to lyse altered autologous target cells if activating receptors such as the natural cytotoxicity receptors (NCRs) NKp46, NKp44, NKp30, or NKG2D or nonclassical class I HLA-E-specific NCRs CD94/NKG2C and CD94/NKG2E are engaged (see Figure 2) [45, 70–75]. In addition, CD16 (FcγRIII) is a low-affinity activating receptor specific for the constant (Fc) region of immunoglobulin G (IgG) that triggers antibody-dependent cell-mediated cytotoxicity (ADCC) upon recognition of antibody-coated cells (Figure 3) [45]. The only NCR ubiquitously expressed on NK cells is NKp46, and while NKp30 and NKp46 are constitutively expressed on subsets of NK cells, NCR NKp44 expression is restricted to activated NK cells [72, 76]. Many ligands of viral origin have been identified for NK activating receptors, which can serve as decoys and disrupt positive NK cell stimuli. Of note, human (H)CMV capsid protein pp65 disrupts NKp30 expression and influenza hemagglutinin (HA) can interact with, and inhibit, NK cells through NKp30, NKp44, and NKp46 [77–79]. Although no NCR ligands resulting from HCV infection have yet been identified, studies have reported decreased NK cell activating receptor expression on NK cells from chronically infected individuals. A progressive increase in the proportion of NKG2C⁺ NK cells was reported to occur following liver transplantation for chronic hepatitis C and was initially thought to be associated with HCV recurrence [80]. A similar NK subset observed in human immunodeficiency virus infection expressed reduced levels of the NCRs NKp30, NKp44, and NKp46 [81]. Expansion of this CD57⁺NKG2C⁺ NK subset has since been shown to relate primarily to CMV infection and involve selection of NK cells expressing KIR capable of licensing NK effector function and enforcing self-tolerance [82, 83]. Despite evidence for downregulation of NCRs on this NK subset, they are polyfunctional in terms of cytokine expression and demonstrate enhanced cytotoxicity against antibody-coated target cells [82, 84].

Freshly isolated peripheral blood and liver-derived NK cells from chronically infected HCV patients exhibited significantly lower levels of NCR NKp30 and NKp46 expression than the NK cells of healthy controls [67]. Although no differences in NKG2C or NKG2D surface expression were noted, some in vitro experimental data suggested downregulated NKG2D and/or NKp30 surface expression after ex vivo exposure of NK cells to HCV-infected cells [68, 69]. Direct contact between NK cells and HCV-infected cells in vitro promotes downregulation of NKp30, suggesting that HCV can affect NK cell recognition and function through upregulation of an as yet unidentified ligand that physically interacts with NKp30 [69]. This possibility is supported by the demonstration of increased binding of recombinant NKp30 protein to HCV-infected Huh-7.5 cells [69]. Cytotoxicity and IFN-γ production by NK cells are also reduced following short (5 hours) or extended (18 hours) exposure to HCV-infected Huh-7.5 cells [67–69, 85, 86].

Both NKR expression itself and/or variation in NKR ligand expression can impact NK cell functions. The T-cell immunoglobulin mucin-3 (TIM-3) receptor was recently shown to inhibit cytotoxic and cytokine responses of NK cells upon interaction with galectin-9 (Gal-9) or phosphatidylserine (PtdSer) on target cells [87–92]. As Gal-9 binding depends on recognition of certain carbohydrates and the extent of NK cell receptor ectodomain glycosylation can vary, the degree of ligand recognition and binding may also vary, thereby providing another level of regulatory control over NK cell functions [93–95]. Circulating levels of the Gal-9 ligand for NK cell TIM-3 receptors are significantly increased in HCV-infected individuals compared to uninfected controls [96]. As this interaction mediates an inhibitory response, this is another potential mechanism through which HCV can inhibit typical NK functions to favor establishment of persistent infection. While many studies have investigated the suppressive effect of TIM-3 in the context of effector T-cell functions on HCV infection, the impact of TIM-3 on NK cells with respect to HCV infection has not yet been extensively studied [84, 85].

Several previous studies investigating the role of NK cells in HCV infection focused on interactions between HCV E2 and CD81 on NK cells as a potential mechanism underlying NK cell dysfunction [97–99]. These studies exposed IL-2-stimulated or purified NK cells to soluble and immobilized recombinant HCV E2, anti-CD81 monoclonal antibodies, or plate-bound cell culture-derived HCV (HCVcc) [97, 98]. Engaging NK CD81 in these ways decreased IFN-γ production and NK cell cytotoxicity; therefore, cross-linking of NK CD81 by HCV E2 has been proposed as an NK inhibitory mechanism promoting HCV persistence [100–103]. However, more recent studies reported no significant change in NK cell cytotoxicity when NK cells were exposed to intact, infectious cell-free HCVcc at supraphysiological levels [69, 99]. These results suggest that although HCV E2 may specifically bind to CD81 on NK cells, the configuration of E2 within an intact infectious virus particle does not facilitate the degree of NK CD81 receptor cross-linking necessary to mediate an inhibitory signal [97, 98]. Most of these systems used peripheral NK cells stimulated in vitro with recombinant IL-2 (rIL-2), rIL-12, or IFN-α combined with extended coculture with HCV-infected cells, introducing additional complications to interpreting in vivo relevance of the reported effects [67, 68, 97, 98, 104].

One recent study used the monoclonal antibody AP33, a broadly neutralizing antibody against the CD81-binding site on HCV E2 (amino acid residues 412–423) [105–107], in conjunction with HCV-infected human hepatoma (Huh-7.5) and unadulterated NK cells to prevent E2/CD81 interactions and probe the significance of NK CD81/HCV E2 interactions in the context of NK cell inhibition. Firstly, there was no evidence of cell surface expression of HCV E2 on HCV-infected Huh-7.5 cells, and secondly, blocking potential HCV E2/NK CD81 interactions with either AP33 or soluble anti-CD81 had no significant effect on NK cell inhibition mediated by HCV-infected cells. These data provide compelling evidence that an HCV E2/NK CD81 interaction does not underlie the inhibition of NK cytotoxicity mediated by HCV-infected cells in vitro [69, 99].

Chronic viral infection requires successful evasion of innate and adaptive host immune responses. Through interaction with host innate and adaptive immune responses, HCV has evolved mechanisms to circumvent immune pressures and to establish chronic infection in the majority of cases. Numerous experimental studies demonstrate NK cell dysfunction in HCV infection and epidemiological data suggests that NK cells play some role in clearance or control of HCV infection [14, 35, 36, 69]. Since NK cells uniquely bridge innate and adaptive immune responses, their sufficient function may be especially important in initial and lasting control of HCV infection.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by research operating grants from the Canadian Institutes of Health Research (CIHR) to Rodney S. Russell and Michael D. Grant. Rodney S. Russell holds a CIHR New Investigator Award. Kayla A. Holder held a CIHR Banting and Best Canada Graduate Student Master of Science (MSc) fellowship and an MSc training award from the National CIHR Hepatitis C Training Program. Kayla A. Holder was also supported by the Memorial University of Newfoundland Faculty of Medicine and School of Graduate Studies.

References

C. H. Bassing, W. Swat, and F. W. Alt, “The mechanism and regulation of chromosomal V(D)J recombination,” Cell, vol. 109, no. 2, pp. S45–S55, 2002.

K. Murphy, P. Travers, and M. Walport, Janeway's Immunobiology, Garland Science, New York, NY, USA, 2007.

B. D. Lindenbach, H. J. Thiel, and C. M. Rice, Flaviviridae: The Viruses and their Replication, Lippincott Williams and Wilkins, Philadelphia, Pa, USA, 5th edition, 2007.

G. V. Quinnan Jr., N. Kirmani, A. H. Rook et al., “Cytotoxic T cells in cytomegalovirus infection. HLA-restricted T-lymphocytes and non-T-lymphocyte cytotoxic responses correlate with recovery from cytomegalovirus infection in bone-marrow-transplant recipients,” The New England Journal of Medicine, vol. 307, no. 1, pp. 7–13, 1982.

C. A. Biron, K. S. Byron, and J. L. Sullivan, “Severe herpesvirus infections in an adolescent without natural killer cells,” The New England Journal of Medicine, vol. 320, no. 26, pp. 1731–1735, 1989.

M. A. Cooper, T. A. Fehniger, S. C. Turner et al., “Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset,” Blood, vol. 97, no. 10, pp. 3146–3151, 2001.

A. R. French and W. M. Yokoyama, “Natural killer cells and viral infections,” Current Opinion in Immunology, vol. 15, no. 1, pp. 45–51, 2003.

M. Altfeld, L. Fadda, D. Frleta, and N. Bhardwaj, “DCs and NK cells: critical effectors in the immune response to HIV-1,” Nature Reviews Immunology, vol. 11, no. 3, pp. 176–186, 2011.

C. A. Biron, K. B. Nguyen, G. C. Pien, L. P. Cousens, and T. P. Salazar-Mather, “Natural killer cells in antiviral defense: function and regulation by innate cytokines,” Annual Review of Immunology, vol. 17, pp. 189–220, 1999.

H. Arase, E. S. Mocarski, A. E. Campbell, A. B. Hill, and L. L. Lanier, “Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors,” Science, vol. 296, no. 5571, pp. 1323–1326, 2002.

K. A. Daniels, G. Devora, W. C. Lai, C. L. O'Donnell, M. Bennett, and R. M. Welsh, “Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H,” Journal of Experimental Medicine, vol. 194, no. 1, pp. 29–44, 2001.

M. Gumá, A. Angulo, C. Vilches, N. Gómez-Lozano, N. Malats, and M. López-Botet, “Imprint of human cytomegalovirus infection on the NK cell receptor repertoire,” Blood, vol. 104, no. 12, pp. 3664–3671, 2004.

J. G. O'Leary, M. Goodarzi, D. L. Drayton, and U. H. von Andrian, “T cell- and B cell-independent adaptive immunity mediated by natural killer cells,” Nature Immunology, vol. 7, no. 5, pp. 507–516, 2006.

S. I. Khakoo, C. L. Thio, M. P. Martin et al., “HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection,” Science, vol. 305, no. 5685, pp. 872–874, 2004.

G. Alter, S. Jost, S. Rihn et al., “Reduced frequencies of NKp30+NKp46+, CD161+, and NKG2D+ NK cells in acute HCV infection may predict viral clearance,” Journal of Hepatology, vol. 55, no. 2, pp. 278–288, 2011.

WHO, WHO Report on Global Surveillance of Epidemic-Prone Infectios Diseases-Dengue and Dengue Haemorrhagic Fever, 2011.

A. Ploss and J. Dubuisson, “New advances in the molecular biology of hepatitis C virus infection: towards the identification of new treatment targets,” Gut, vol. 61, no. 1, pp. i25–i35, 2012.

V. Deleersnyder, A. Pillez, C. Wychowski et al., “Formation of native hepatitis C virus glycoprotein complexes,” Journal of Virology, vol. 71, no. 1, pp. 697–704, 1997.

B. Bartosch, A. Vitelli, C. Granier et al., “Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor,” The Journal of Biological Chemistry, vol. 278, no. 43, pp. 41624–41630, 2003.

J. A. McKeating, L. Q. Zhang, C. Logvinoff et al., “Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner,” Journal of Virology, vol. 78, no. 16, pp. 8496–8505, 2004.

J. Zhang, G. Randall, A. Higginbottom, P. Monk, C. M. Rice, and J. A. McKeating, “CD81 is required for hepatitis C virus glycoprotein-mediated viral infection,” Journal of Virology, vol. 78, no. 3, pp. 1448–1455, 2004.

M. Flint, C. Maidens, L. D. Loomis-Price et al., “Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81,” Journal of Virology, vol. 73, no. 8, pp. 6235–6244, 1999.

P. Pileri, Y. Uematsu, S. Campagnoli et al., “Binding of hepatitis C virus to CD81,” Science, vol. 282, no. 5390, pp. 938–941, 1998.

J. Grove, T. Huby, Z. Stamataki et al., “Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity,” Journal of Virology, vol. 81, no. 7, pp. 3162–3169, 2007.

B. Bartosch, G. Verney, M. Dreux et al., “An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies,” Journal of Virology, vol. 79, no. 13, pp. 8217–8229, 2005.

E. Scarselli, H. Ansuini, R. Cerino et al., “The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus,” EMBO Journal, vol. 21, no. 19, pp. 5017–5025, 2002.

S. Ciesek, S. Westhaus, M. Wicht et al., “Impact of intra- and interspecies variation of occludin on its function as coreceptor for authentic hepatitis C virus particles,” Journal of Virology, vol. 85, no. 15, pp. 7613–7621, 2011.

I. Benedicto, F. Molina-Jiménez, B. Bartosch et al., “The tight junction-associated protein occludin is required for a postbinding step in hepatitis C virus entry and infection,” Journal of Virology, vol. 83, no. 16, pp. 8012–8020, 2009.

A. Ploss, M. J. Evans, V. A. Gaysinskaya et al., “Human occludin is a hepatitis C virus entry factor required for infection of mouse cells,” Nature, vol. 457, no. 7231, pp. 882–886, 2009.

L. Meertens, C. Bertaux, L. Cukierman et al., “The tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis C virus,” Journal of Virology, vol. 82, no. 7, pp. 3555–3560, 2008.

M. J. Evans, T. Von Hahn, D. M. Tscherne et al., “Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry,” Nature, vol. 446, no. 7137, pp. 801–805, 2007.

T. L. Tellinghuisen, M. J. Evans, T. Von Hahn, S. You, and C. M. Rice, “Studying hepatitis C virus: making the best of a bad virus,” Journal of Virology, vol. 81, no. 17, pp. 8853–8867, 2007.

WHO, Global Alert and Response, Hepatitis C, 2012.

K. Li and S. M. Lemon, Innate Immune Responses in Hepatitis C Virus Infection, Semin Immunopathol.

F. Bozzano, A. Picciotto, P. Costa et al., “Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment,” European Journal of Immunology, vol. 41, no. 10, pp. 2905–2914, 2011.

G. Ahlenstiel, B. Edlich, L. J. Hogdal et al., “Early changes in natural killer cell function indicate virologic response to interferon therapy for hepatitis C,” Gastroenterology, vol. 141, no. 4, pp. 1231–e2, 2011.

P. Carrega and G. Ferlazzo, “Natural killer cell distribution and trafficking in human tissues,” Frontiers in Immunology, vol. 3, Article ID Article 347, 2012.

C. Grégoire, L. Chasson, C. Luci et al., “The trafficking of natural killer cells,” Immunological Reviews, vol. 220, no. 1, pp. 169–182, 2007.

D. G. Doherty and C. O'Farrelly, “Innate and adaptive lymphoid cells in the human liver,” Immunological Reviews, vol. 174, pp. 5–20, 2000.

B. Gao, S. Radaeva, and O. Park, “Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases,” Journal of Leukocyte Biology, vol. 86, no. 3, pp. 513–528, 2009.

R. B. Herberman, M. E. Nunn, and D. H. Lavrin, “Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. I. Distribution of reactivity and specificity,” International Journal of Cancer, vol. 16, no. 2, pp. 216–229, 1975.

E. De Vries, H. R. Koene, J. M. Vossen et al., “Identification of an unusual Fcγ receptor IIIa (CD16) on natural killer cells in a patient with recurrent infections,” Blood, vol. 88, no. 8, pp. 3022–3027, 1996.

S. Jawahar, C. Moody, M. Chan, R. Finberg, R. Geha, and T. Chatila, “Natural killer (NK) cell deficiency associated with an epitope-deficient Fc receptor type IIIA (CD16-II),” Clinical and Experimental Immunology, vol. 103, no. 3, pp. 408–413, 1996.

R. Kiessling, E. Klein, H. Pross, and H. Wigzell, “‘Natural’ killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell,” European Journal of Immunology, vol. 5, no. 2, pp. 117–121, 1975.

L. L. Lanier, “NK cell recognition,” Annual Review of Immunology, vol. 23, pp. 225–274, 2005.

C. Fauriat, E. O. Long, H.-G. Ljunggren, and Y. T. Bryceson, “Regulation of human NK-cell cytokine and chemokine production by target cell recognition,” Blood, vol. 115, no. 11, pp. 2167–2176, 2010.

M. U. Mondelli, S. Varchetta, and B. Oliviero, “Natural killer cells in viral hepatitis: facts and controversies,” European Journal of Clinical Investigation, vol. 40, no. 9, pp. 851–863, 2010.

A. S. Fauci, D. Mavilio, and S. Kottilil, “NK cells in HIV infection: paradigm for protection or targets for ambush,” Nature Reviews Immunology, vol. 5, no. 11, pp. 835–843, 2005.

L. L. Lanier, “NK cell receptors,” Annual Review of Immunology, vol. 16, pp. 359–393, 1998.

F. Borrego, J. Kabat, D.-K. Kim et al., “Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells,” Molecular Immunology, vol. 38, no. 9, pp. 637–660, 2002.

M. Yawata, N. Yawata, M. Draghi, F. Partheniou, A.-M. Little, and P. Parham, “MHC class I specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response,” Blood, vol. 112, no. 6, pp. 2369–2380, 2008.

N. Anfossi, P. André, S. Guia et al., “Human NK Cell education by inhibitory receptors for MHC class I,” Immunity, vol. 25, no. 2, pp. 331–342, 2006.

S. Kim, J. Poursine-Laurent, S. M. Truscott et al., “Licensing of natural killer cells by host major histocompatibility complex class I molecules,” Nature, vol. 436, no. 7051, pp. 709–713, 2005.

K. Karre, H. G. Ljunggren, G. Piontek, and R. Kiessling, “Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy,” Nature, vol. 319, no. 6055, pp. 675–678, 1986.

B. Oliviero, S. Varchetta, E. Paudice et al., “Natural killer cell functional dichotomy in chronic hepatitis B and chronic hepatitis C virus infections,” Gastroenterology, vol. 137, no. 3, pp. 1151.e7–1160.e7, 2009.

S. Knapp, D. Hegazy, L. Brackenbury et al., “Consistent beneficial effects of killer cell immunoglobulin-like receptor 2dl3 and group 1 human leukocyte antigen-c following exposure to hepatitis c virus,” Hepatology, vol. 51, no. 4, pp. 1168–1175, 2010.

A. K. Moesta, P. J. Norman, M. Yawata, N. Yawata, M. Gleimer, and P. Parham, “Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C Than KIR2DL3,” Journal of Immunology, vol. 180, no. 6, pp. 3969–3979, 2008.

P. Parham, “NK cell lose their inhibition,” Science, vol. 305, no. 5685, pp. 786–787, 2004.

M. Uhrberg, N. M. Valiante, B. P. Shum et al., “Human diversity in killer cell inhibitory receptor genes,” Immunity, vol. 7, no. 6, pp. 753–763, 1997.

S. I. Khakoo, R. Rajalingam, B. P. Shum et al., “Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans,” Immunity, vol. 12, no. 6, pp. 687–698, 2000.

V. Suppiah, S. Gaudieri, N. J. Armstrong et al., “IL28B, HLA-C, and KIR variants additively predict response to therapy in chronic hepatitis C virus infection in a European cohort: a cross-sectional study,” PLoS Medicine, vol. 8, no. 9, Article ID e1001092, 2011.

L. Golden-Mason, K. M. Bambha, L. Cheng et al., “Natural killer inhibitory receptor expression associated with treatment failure and interleukin-28B genotype in patients with chronic hepatitis C,” Hepatology, vol. 54, no. 5, pp. 1559–1569, 2011.

W. H. Carr, D. B. Rosen, H. Arase, D. F. Nixon, J. Michaelsson, and L. L. Lanier, “Cutting edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation,” Journal of Immunology, vol. 178, no. 2, pp. 647–651, 2007.

A. López-Vázquez, L. Rodrigo, J. Martínez-Borra et al., “Protective effect of the HLA-Bw4I80 epitope and the killer cell immunoglobulin-like receptor 3DS1 gene against the development of hepatocellular carcinoma in patients with hepatitis C virus infection,” Journal of Infectious Diseases, vol. 192, no. 1, pp. 162–165, 2005.

A. Rivero-Juarez, R. Gonzalez, A. Camacho et al., “Natural killer KIR3DS1 is closely associated with HCV viral clearance and sustained virological response in HIV/HCV patients,” PLoS ONE, vol. 8, no. 4, Article ID e61992, 2013.

J. Nattermann, H. D. Nischalke, V. Hofmeister et al., “The HLA-A2 restricted T cell epitope HCV core35-44 stabilizes HLA-E expression and inhibits cytolysis mediated by natural killer cells,” American Journal of Pathology, vol. 166, no. 2, pp. 443–453, 2005.

J. Nattermann, G. Feldmann, G. Ahlenstiel, B. Langhans, T. Sauerbruch, and U. Spengler, “Surface expression and cytolytic function of natural killer cell receptors is altered in chronic hepatitis C,” Gut, vol. 55, no. 6, pp. 869–877, 2006.

J. C. Yoon, J.-B. Lim, J. H. Park, and J. M. Lee, “Cell-to-cell contact with hepatitis C virus-infected cells reduces functional capacity of natural killer cells,” Journal of Virology, vol. 85, no. 23, pp. 12557–12569, 2011.

K. A. Holder, S. N. Stapleton, M. E. Gallant, R. S. Russell, and M. D. Grant, “Hepatitis C Virus-Infected Cells Downregulate NKp30 and Inhibit Ex Vivo NK Cell Functions,” The Journal of Immunology, vol. 191, pp. 3308–3318, 2013.

S. Sivori, M. Vitale, L. Morelli et al., “p46, a novel natural killer cell-specific surface molecule that mediates cell activation,” Journal of Experimental Medicine, vol. 186, no. 7, pp. 1129–1136, 1997.

A. Pessino, S. Sivori, C. Bottino et al., “Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity,” Journal of Experimental Medicine, vol. 188, no. 5, pp. 953–960, 1998.

M. Vitale, C. Bottino, S. Sivori et al., “NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis,” Journal of Experimental Medicine, vol. 187, no. 12, pp. 2065–2072, 1998.

D. Pende, S. Parolini, A. Pessino et al., “Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells,” Journal of Experimental Medicine, vol. 190, no. 10, pp. 1505–1516, 1999.

J. Wu, Y. Song, A. B. H. Bakker et al., “An activating immunoreceptor complex formed by NKG2D and DAP10,” Science, vol. 285, no. 5428, pp. 730–732, 1999.

N. Lee, M. Llano, M. Carretero, F. Navarro, M. López-Botet, and D. E. Geraghty, “HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 9, pp. 5199–5204, 1998.

C. Cantoni, C. Bottino, M. Vitale et al., “NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily,” Journal of Experimental Medicine, vol. 189, no. 5, pp. 787–795, 1999.

T. I. Arnon, H. Achdout, O. Levi et al., “Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus,” Nature Immunology, vol. 6, no. 5, pp. 515–523, 2005.

Y. Bar-On, A. Glasner, T. Meningher et al., “Neuraminidase-mediated, NKp46-dependent immune-evasion mechanism of influenza viruses,” Cell Reports, vol. 3, no. 4, pp. 1044–1050, 2013.

E. Seidel, A. Glasner, and O. Mandelboim, “Virus-mediated inhibition of natural cytotoxicity receptor recognition,” Cellular and Molecular Life Sciences, vol. 69, no. 23, pp. 3911–3920, 2012.

S. Varchetta, B. Oliviero, M. Francesca Donato et al., “Prospective study of natural killer cell phenotype in recurrent hepatitis C virus infection following liver transplantation,” Journal of Hepatology, vol. 50, no. 2, pp. 314–322, 2009.

C. M. Mela, C. T. Burton, N. Imami et al., “Switch from inhibitory to activating NKG2 receptor expression in HIV-1 infection: lack of reversion with highly active antiretroviral therapy,” AIDS, vol. 19, no. 16, pp. 1761–1769, 2005.

V. Béziat, O. Dalgard, T. Asselah et al., “CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients,” European Journal of Immunology, vol. 42, no. 2, pp. 447–457, 2012.

Z. Djaoud, G. David, C. Bressollette et al., “Amplified NKG2C+ NK cells in cytomegalovirus (CMV) infection preferentially express killer cell Ig-like receptor 2DL: functional impact in controlling CMV-infected dendritic cells,” Journal of Immunology, vol. 191, no. 5, pp. 2708–2716, 2013.

Z. Wu, C. Sinzger, G. Frascaroli et al., “Human cytomegalovirus-induced NKG2hi CD57hi natural killer cells are effectors dependent on humoral antiviral immunity,” Journal of Virology, vol. 87, no. 13, pp. 7717–7725, 2013.

U.-C. Meier, R. E. Owen, E. Taylor et al., “Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections,” Journal of Virology, vol. 79, no. 19, pp. 12365–12374, 2005.

J. Corado, F. Toro, H. Rivera, N. E. Bianco, L. Deibis, and J. B. De Sanctis, “Impairment of natural killer (NK) cytotoxic activity in hepatitis C virus (HCV) infection,” Clinical and Experimental Immunology, vol. 109, no. 3, pp. 451–457, 1997.

L. C. Ndhlovu, S. Lopez-Vergès, J. D. Barbour et al., “Tim-3 marks human natural killer cell maturation and suppresses cell-mediated cytotoxicity,” Blood, vol. 119, no. 16, pp. 3734–3743, 2012.

Y. Ju, N. Hou, J. Meng et al., “T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) mediates natural killer cell suppression in chronic hepatitis B,” Journal of Hepatology, vol. 52, no. 3, pp. 322–329, 2010.

L. Golden-Mason, R. H. McMahan, M. Strong et al., “Galectin-9 functionally impairs natural killer cells in humans and mice,” Journal of Virology, vol. 87, no. 9, pp. 4835–4845, 2013.

M. K. Gleason, T. R. Lenvik, V. McCullar et al., “Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9,” Blood, vol. 119, no. 13, pp. 3064–3072, 2012.

C. Zhu, A. C. Anderson, A. Schubart et al., “The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity,” Nature Immunology, vol. 6, no. 12, pp. 1245–1252, 2005.

R. H. DeKruyff, X. Bu, A. Ballesteros et al., “T cell/transmembrane, Ig, and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells,” Journal of Immunology, vol. 184, no. 4, pp. 1918–1930, 2010.

J. Hartmann, T.-V. Tran, J. Kaudeer et al., “The stalk domain and the glycosylation status of the activating natural killer cell receptor NKp30 are important for ligand binding,” The Journal of Biological Chemistry, vol. 287, no. 37, pp. 31527–31539, 2012.

S. Margraf-Schönfeld, C. Böhm, and C. Watzl, “Glycosylation affects ligand binding and function of the activating natural killer cell receptor 2B4 (CD244) protein,” The Journal of Biological Chemistry, vol. 286, no. 27, pp. 24142–24149, 2011.

L. H. Mason, J. Willette-Brown, S. K. Anderson et al., “Receptor glycosylation regulates Ly-49 binding to MHC class I,” Journal of Immunology, vol. 171, no. 8, pp. 4235–4242, 2003.

J. A. Mengshol, L. Golden-Mason, T. Arikawa et al., “A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection,” PLoS ONE, vol. 5, no. 3, Article ID e9504, 2010.

C.-T. K. Tseng and G. R. Klimpel, “Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions,” Journal of Experimental Medicine, vol. 195, no. 1, pp. 43–49, 2002.

S. Crotta, A. Stilla, A. Wack et al., “Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein,” Journal of Experimental Medicine, vol. 195, no. 1, pp. 35–41, 2002.

J. C. Yoon, M. Shiina, G. Ahlenstiel, and B. Rehermann, “Natural killer cell function is intact after direct exposure to infectious hepatitis C virions,” Hepatology, vol. 49, no. 1, pp. 12–21, 2009.

M. U. Mondelli, B. Oliviero, D. Mele, S. Mantovani, C. Gazzabin, and S. Varchetta, “Natural killer cell functional dichotomy: a feature of chronic viral hepatitis?” Frontiers in Immunology, vol. 3, Article ID Article 351, 6 pages, 2012.

A. Nellore and J. A. Fishman, “NK cells, innate immunity and hepatitis C infection after liver transplantation,” Clinical Infectious Diseases, vol. 52, no. 3, pp. 369–377, 2011.

L. B. Dustin and C. M. Rice, “Flying under the radar: the immunobiology of hepatitis C,” Annual Review of Immunology, vol. 25, pp. 71–99, 2007.

L. Golden-Mason and H. R. Rosen, “Natural killer cells: primary target for hepatitis C virus immune evasion strategies?” Liver Transplantation, vol. 12, no. 3, pp. 363–372, 2006.

S. Varchetta, D. Mele, S. Mantovani et al., “Impaired intrahepatic natural killer cell cytotoxic function in chronic hepatitis C virus infection,” Hepatology, vol. 56, no. 3, pp. 841–849, 2012.

A. Owsianka, A. W. Tarr, V. S. Juttla et al., “Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein,” Journal of Virology, vol. 79, no. 17, pp. 11095–11104, 2005.

A. M. Owsianka, J. M. Timms, A. W. Tarr et al., “Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding,” Journal of Virology, vol. 80, no. 17, pp. 8695–8704, 2006.

A. W. Tarr, A. M. Owsianka, J. M. Timms et al., “Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33,” Hepatology, vol. 43, no. 3, pp. 592–601, 2006.