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Originally published online as doi:10.1189/jlb.0304197 on June 24, 2004

Published online before print June 24, 2004
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(Journal of Leukocyte Biology. 2004;76:743-759.)
© 2004 by Society for Leukocyte Biology

Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis

Ali Ahmad*,{ddagger},1 and Fernando Alvarez{dagger},{ddagger}

* Departments of Microbiology & Immunology and
{dagger} Pediatrics and
{ddagger} Center of Research, Ste. Justine Hospital, University of Montreal, Quebec, Canada

1Correspondence: Laboratory of Immunovirology, Center of Research, Ste. Justine Hospital, 3175 Côte Ste-Catherine, Montreal, Qc, H3T 1C5, Canada. E-mail: ahmada{at}justine.umontreal.ca


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ABSTRACT
 
Natural killer (NK) cells constitute the first line of host defense against invading pathogens. They usually become activated in an early phase of a viral infection. Liver is particularly enriched in NK cells, which are activated by hepatotropic viruses such as hepatitis C virus (HCV). The activated NK cells play an essential role in recruiting virus-specific T cells and in inducing antiviral immunity in liver. They also eliminate virus-infected hepatocytes directly by cytolytic mechanisms and indirectly by secreting cytokines, which induce an antiviral state in host cells. Therefore, optimally activated NK cells are important in limiting viral replication in this organ. This notion is supported by the observations that interferon treatment is effective in HCV-infected persons in whom it increases NK cell activity. Not surprisingly, HCV has evolved multiple strategies to counter host’s NK cell response. Compromised NK cell functions have been reported in chronic HCV-infected individuals. It is ironic that activated NK cells may also contribute toward liver injury. Further studies are needed to understand the role of these cells in host defense and in liver pathology in HCV infections. Recent advances in understanding NK cell biology have opened new avenues for boosting innate and adaptive antiviral immune responses in HCV-infected individuals.

Key Words: hepatitis C virus • natural killer cells • NK cell receptors • NK cell co-receptors


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HEPATITIS C VIRUS (HCV) INFECTION
 
HCV infection has assumed the proportion of a global pandemic. Approximately 170 million people are infected world-wide with this virus. They make up ~3% of the world population. Their number is approximately five times more than those infected with human immunodeficiency virus type 1 (HIV-1) [1 ]. HCV was the leading cause of post-transfusion and community-acquired non-A, non-B hepatitis until the introduction of blood screening in 1990. The institution of blood screening for HCV has markedly reduced its incidence. However, it still remains a significant problem in intravenous drug abusers (reviewed in ref. [2 ]). A significant proportion of the infected persons develops chronic hepatitis, cirrhosis, and liver dysfunction. The infection is the most common cause for liver transplantation in adults. Three to 4% of the chronically infected individuals develop fatal hepatocellular carcinoma (HCC). HCV and HIV-1 frequently coinfect humans: It has been estimated that as high as 18% of HIV-infected persons are also infected with HCV [3 ]. The coinfected persons experience more severe hepatitis and progress more rapidly to the development of AIDS.

HCV was identified as a cause of non-A, non-B hepatitis by molecular cloning in 1989 (ref. [4 ]; reviewed in refs. [1 , 5 ]). The virus transmission via sexual route is rare; however, high-risk sexual behavior may increase chances of infection because of its association with Herpes simplex virus type 2. The use of contaminated needles, blood, and blood products represents the major route of its transmission. Acute HCV infections usually remain undiagnosed, as they present mild or no clinical symptoms. Approximately 15% of the infected persons undergo spontaneous recovery. These individuals may be important for understanding the immune mechanisms that resist and eliminate a natural HCV infection in humans. It is important that an overwhelming majority of the infected persons fails to control the infection and develops a chronic infection with a variable degree of hepatitis and viremia [1 , 6 ]. Molecular mimicry between host and viral antigens has been implicated in liver autoimmunity in HCV-infected patients. A fraction of the patients positive for HCV RNA and/or anti-HCV antibodies shows the presence of type I antiliver kidney microsome antibodies, which also recognize cytochrome P450 (CYP)2D6. The patient livers are infiltrated with autoreactive mononuclear cells, which recognize CYP2D6. It is interesting that the viral core protein residues 178–187 bear sequence homology with human cytochrome P450 (CYP2A6 and CYP2A7) residues 8–17 [7 ]. Although HCV is a hepatotropic virus and infects hepatocytes, viral genome and its replicative intermediates are frequently present in the peripheral blood mononuclear cells (PBMC) and lymphoid tissues of chronically infected persons. The infection has also been associated with several extrahepatic manifestations, e.g., among others, type II mixed cryoglobulinemia (MC), non-Hodgkin’s lymphomas, and rheumatic and cutaneomucous symptoms. MC is a benign, B cell-proliferative disorder accompanied by the presence of monoclonal immunoglobulin (Ig)M with rheumatoid factor activity, which is encoded by a restricted set of variable-region, germ-line genes, VH 1-69 and VK A27 [8 , 9 ]. It is believed that HCV preferentially infects B cells expressing these Ig genes. HCV-infected individuals also have a higher incidence of non-Hodgkin’s lymphomas. These lymphomas have been shown to express the same restricted repertoire of the germ-line Ig genes, as in the case of MC IgM [10 ]. The viral glycoprotein E2 has been implicated in the oligoclonal expansion of these lymphoma cells [8 ]. The most common rheumatic and cutaneomucous symptoms in HCV-infected patients include fatigue, arthralgia, paraestheisa, myalgia, pruritis, and the sicca syndrome (reviewed in ref. [11 ]).

HCV-infected persons are usually treated with interferon (IFN)-{alpha}, with or without ribavirin. Usually recombinant human IFN-{alpha}2a or -2b is used. Only less than half of the patients respond to this treatment. The treatment is usually accompanied with toxic side-effects, which deteriorate the quality of life in these persons. The recent use of pegylated IFN-{alpha} with or without ribavirin has significantly improved the treatment efficacy [12 , 13 ]. The effect of highly active antiretroviral therapy (HAART) in HCV and HIV-1-coinfected patients is controversial. The HAART-mediated restoration of immune responses in these patients often leads to hepatotoxicity and increased HCV diversity [14 ]. As yet, there is no anti-HCV vaccine available, and the prospects of such a vaccine are also dim. The only species in which HCV replicates are humans and chimpanzees. The virus does not replicate efficiently in vitro in cell culture [6 ]. The lack of a suitable in vitro viral replication system is a major hindrance in studying immunobiology of this virus.

HCV is an enveloped, positive-strand RNA virus of ~50 nm diameter and belongs to the genus Hepacivirus in the Flaviviridae family. The lipid bilayer HCV envelope is derived from host cell membranes, into which viral envelope glycoproteins E1 and E2 are inserted. The envelope contains nucleocapsid with a 9.6-kb-positive strand RNA. The viral genome contains one open-reading frame (ORF) and two (a 5'- and a 3'-) untranslated regions (UTR; ref. [1 ]). It is translated from an internal ribosome entry site (IRES) in the 5'-UTR into a single polyprotein of ~3011 amino acids (aa). The polyprotein is processed by viral and host-signal proteases into four structural proteins (nucleocapsid or core, envelope proteins E1 and E2, and p7) and six nonstructural proteins (NS2, -3, -4A, -4B, -5A, and -5B) with various enzymatic activities (Fig. 1 ; reviewed in ref. [2 ]). The viral envelope glycoproteins E1 and E2 are usually targeted to the endoplasmic reticulum (ER), where they are retained in the pre-Golgi compartment instead of being secreted [15 ]. A ribosomal frame-shift into –2/+1 reading frame at or near codon 11 results into a novel HCV protein of unknown function, F or core + 1 protein, which reacts with sera from HCV-infected patients [16 ].



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  		Figure 1. A schematic diagram showing HCV genome, polyprotein, and its cleaved components. S and NS, Structural and nonstructural proteins, respectively; HVR, hypervariable region; ISDR, IFN sensitivity-determining region. Not drawn to the scale.

A member of the tetraspanin family of proteins, CD81 has been shown to act as a viral receptor, which appears to be essential but not sufficient for viral entry [17 ]. Its transgenic expression in mice does not confer susceptibility to HCV infection [18 ]. It is expressed on the surface of almost all nucleated cells as a complex with a variety of other cell-surface receptors; e.g., it occurs as a complex with CD19 and CD21 on B cells and sends a costimulatory signal to the cells (reviewed in ref. [19 ]). It has four membrane-spanning domains and two extracellular loops. The viral glycoprotein E2 binds to the major extracellular loop of CD81 [20 ]. However, this may not be the only viral receptor, as HCV can also bind to several other molecules: the receptor for low-density lipoprotein, the dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and its liver counterpart (L-SIGN), a scavenger receptor of class B type 1, asialoglycoprotein receptor [21 22 23 24 ]. E2 is the most variable viral envelope glycoprotein; therefore, it is not surprising that E2 interactions with its putative ligands, e.g., CD81, have been reported to be strain-specific [25 ]. It has two HVRs (HVR-1 and -2). Mutations occur mostly in these regions, probably as a result of pressure from virus-neutralizing antibodies and HCV-specific cytolytic T lymphocytes (CTL). The HVR-1 variants have been shown to act as T cell receptor (TCR) antagonists for HCV-specific CD4+ and CD8+ T cells specific for HVR-1. They induce inhibition of proliferation, cytokine production, and early signaling events [26 ]. HCV has a high mutation rate as a result of a lack of proofreading ability of its RNA-dependent RNA polymerase. Therefore, it exists in several distinct but closely related virus species within an infected individual. They are called HCV quasispecies. Based on the nucleotide sequences of the conserved and nonconserved regions, these quasispecies have been grouped into six major genotypes (genotypes 1–6) and more than 100 subtypes. Of these, genotypes 1a and 1b are prevalent in North America and Western Europe and are relatively resistant to the IFN therapy [1 ].


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THE INDUCTION OF IMMUNE RESPONSE IN LIVER
 
Liver is an organ interposed between systemic circulation and gastrointestinal tract. It is constantly exposed to numerous antigens and potentially toxic molecules, which we ingest every day from our food. The internal microenvironment of normal liver is maintained in a relatively "immuno-silent" state. This state is maintained in many ways. For example, activated CD8+ T cells are trapped in the liver, where they undergo apoptosis. The antigen presentation to naïve CD8+ T cells within this organ is incomplete, as antigen-presenting cells (APC) in liver lack costimulatory molecules. So antigen presentation to these cells also results in tolerance. Furthermore, liver microenvironment biases CD4+ T cells toward the T helper cell type 2 (TH-2) phenotype. The production of interleukin (IL)-4 and IL-10 by the liver sinusoidal endothelial cells (LSEC) plays a significant role in this bias. The induction of tolerance and biased TH-2 responses prevent unnecessary immune responses to thousands of innocuous antigens to which liver is constantly exposed from the gastrointestinal tract (reviewed in ref. [27 ]). It is noteworthy that in a normal liver, lymphocytes remain in sinusoids and do not infiltrate the liver parenchyma. However, slow blood flow in the sinusoids, the lack of a basement membrane, and the presence of fenestrations in their lining endothelium allow blood lymphocytes to interact directly with hepatic cells, i.e., LSEC, liver-resident macrophages [Kupffer cells (KC)], and even with hepatocytes (Fig. 2 ). These hepatic cells also act as APC but lack essential costimulatory molecules. Because of its ability to trap and induce apoptosis in CD8+ T cells, the liver has been dubbed as a "graveyard" for CD8+ T cells (ref. [28 ]; reviewed in ref. [2 ]). The LSEC are constantly exposed to lipopolysaccharides (LPS) from intestines and constitutively express intercellular adhesion molecule-1 (ICAM-1), -2, and vascular adhesion protein 1 but not costimulatory molecules. They can actively recruit activated CD8+ T cells and NK cells from circulation. The activated CD8+ T cells may divide a few times but undergo apoptosis as a result of neglect. However, the situation changes when an individual is infected with hepatotropic viruses. These viruses induce production of type I IFN from hepatocytes and other cells in the liver (e.g., among others, immature plamacytoid DC), which, in turn, induce production of CC chemokine ligand [macrophage-inflammatory protein-1{alpha} (MIP-1{alpha})] in KC. This chemokine promotes infiltration of NK cells in virus-infected livers [29 ]. Molecular interactions between vascular cell adhesion molecule 1 (VCAM-1) on the vascular endothelial cells and very late antigen 4 (VLA-4) on NK cells are also involved in this infiltration [30 ]. The production of type I IFN and other cytokines (including IL-12, IL-15, IL-18) from hepatocytes activates NK cells and induces IFN-{gamma} production from them. The IFN-{gamma} produced by NK cells induces expression of CXC chemokine ligand 9 [CXCL-9; the monokine-induced by IFN-{gamma} (MIG)] and CXCL-10 [the IFN-{gamma}-inducible protein-10 (IP-10)] from LSEC. The LSEC-produced CXCL-9 and CXCL-10 bind to and recruit CC chemokine receptor 5 (CCR-5) and CXC chemokine receptor 3 (CXCR-3)-positive, activated T cells to the liver. See Table 1 for a summary of the events involved in the induction of an adaptive immune response to a hepatotropic virus. It is noteworthy that IFN-{gamma} produced by NK cells plays a major role in liver infiltration of CD4+ and CD8+ T cells. It has been shown in animal models that the depletion of NK cells before a hepatotropic viral infection leads to inhibition of a virus-specific T cell response as well as inhibition of liver injury [31 ]. In addition to activating NK cells, type I IFN also induces expression of costimulatory molecules on the liver APC and causes maturation of DC. This ensures activation of CD4+ and CD8+ naïve T cells in the liver. Thus, upon infection, the internal environment of the liver becomes poised for mounting an immune response.



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  		Figure 2. A schematic representation of a liver sinusoid showing different cell types. SC, Stellate or Ito cell; T, T cell; IMDC, immature DC; NK, natural killer cell. Cells not drawn to the scale or number. Adapted from reference 27 .


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  		Table 1. The Molecular and Cellular Events Involved in Inducing Immune Response in the Liver


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ANTIVIRAL IMMUNE RESPONSE AND HCV-INDUCED HEPATITIS
 
Being a hepatotropic virus, HCV preferentially induces the expression of antigen processing and IFN-stimulated genes in the infected livers as determined by genomic analyses of livers of acutely infected chimpanzees [32 , 33 ]. Consequently, HCV induces a strong humoral and cellular antiviral immunity in the host. Virus-specific antibodies and CD4+ and CD8+ T cells can be demonstrated in the peripheral blood of the infected persons. These cells, however, occur at higher frequencies in the infected liver than in the peripheral blood (ref. [34 ]; reviewed in ref. [35 ]). Various studies have suggested that antiviral cellular immune responses rather than antibodies are important in controlling HCV infection [36 37 38 ]. In agreement with these studies, similar to the normal children, ~15% of the children with agammaglobulinemia can spontaneously control HCV infection.

The HCV-specific CD8+ and CD4+ T cells have been demonstrated to recognize viral epitopes in the conserved and variable regions of all structural and nonstructural viral proteins. The CD4+ TH cells are considered important in maintaining antiviral CD8+ CTL responses. The virus-specific CTL kill virus-infected cells. They also contribute to virus control by noncytolytic mechanisms by secreting cytokines, e.g., IFN-{gamma}, IFN-{alpha}/ß, and tumor necrosis factor {alpha} (TNF-{alpha}), which induce an antiviral state in host cells. The state makes uninfected cells resistant to infection and frees or "cures" the infected ones from the virus by stopping viral replication. The noncytolytic mechanisms have been shown to be important in controlling several different viruses (reviewed in ref. [39 ]). It is interesting that IFN-{gamma} and not perforin was shown to be important in the control of murine cytomegalovirus (MCMV) in the livers of mice [40 ]. Moreover, it was also shown to control hepatitis B virus (HBV) in the livers of transgenic mice [41 , 42 ]. In in vitro studies, IFN-{gamma} inhibits amplification of HCV replicons in Huh-7 liver cells [43 ]. In humans, the induction of IFN-{gamma}-producing, antiviral CTL corresponds with the successful clearance of the HCV infection [44 ]. Furthermore, the degree of viremia correlates inversely with the expression of IFN-{gamma} in the livers of HCV-infected persons, suggesting that the IFN-{gamma}-induced antiviral state may be important in CTL-mediated control of HCV replication in human liver [45 ]. The progression of the majority of the infected persons to chronic infection suggests the inability of the antiviral immunity to contain this infection. There may be several reasons for this failure, including emergence of escape variants as a result of a high rate of virus mutations, a decreased production of antiviral cytokines or "stunning" of HCV-specific CTL, a compromised cytolytic potential of the CTL, and antagonistic peptides. Consequently, the antiviral immune response may be causing more liver damage in these individuals.

The HCV-induced immune response brings about profound changes in the liver microenvironment. It may be important in controlling HCV replication; however, it is not without a price. The HCV-induced hepatitis is mediated by the antiviral cellular immune response. Although HCV infects and replicates in the cytoplasm of hepatocytes and possibly of B cells, the virus per se does not seem to be cytopathic. The viral genotype as well as the viral load in the infected persons do not correlate with the degree of liver disease [46 ]. Data from chimpanzees and mouse models of viral hepatitis have suggested the involvement of virus-specific CTL and their cytokines (e.g., TNF-{alpha}) in this hepatitis (ref. [47 ]; reviewed in refs. [34 , 35 ]). It has been demonstrated in animal models that high-affinity peptides trigger activation and migration of CTL to liver, where they undergo apoptosis and cause liver damage [48 ]. CTL have also been implicated in liver damage in HBV infection; the infusion of virus-specific CTL in transgenic HBV mice causes necroinflammatory hepatitis (reviewed in ref. [49 ]). It is interesting that the HCV-specific CTL can also kill uninfected hepatocytes in vitro [50 , 51 ]. The molecular mechanism(s) underlying this potentially pathogenic, autoreactive activity of CTL in HCV-infected individuals are not known. At least in part, it may be caused by Fas–FasL interactions. In vitro studies show that a few HCV-infected cells can provoke a few HCV-specific CTL to mediate killing of many bystander, uninfected hepatocytes [52 ]. The importance of Fas–FasL interactions in inducing liver damage was also demonstrated in a mouse model of hepatitis, in which the use of antisense oligonucleotides that inhibited Fas expression on hepatocytes also inhibited liver damage [53 ]. It is noteworthy that Fas is constitutively expressed on hepatocytes in mice. This expression is weak on human heptocytes under physiological conditions but is readily induced by proinflammatory cytokines. More importantly, hepatocytes can also express FasL under inflammatory conditions and cause their own death. It may be relevant to mention that HCV core protein induces FasL expression in hepatoblastoma cells [54 ]. The antiviral CTL may also cause liver damage indirectly via cytokines, e.g., TNF-{alpha}. The intrahepatic T cells from the individuals with chronic HCV infection produce almost 50 times more TNF-{alpha} than the ones who control this infection [37 ]. Furthermore, TNF-related apoptosis-inducing ligand (TRAIL) kills hepatocytes from virus-infected, inflamed livers via death receptors (DR)-4 and DR-5 but not from healthy ones [55 ]. Yet, other evidence for the immune-mediated liver damage comes from HAART-treated HCV and HIV-1-coinfected persons. HAART restores anti-HCV immunity in these patients and consequently causes hepatotoxicity [14 ].

The antiviral immune response disturbs the normal anti-inflammatory cytokine milieu of the liver, which may activate stellate cells (SC) to produce matrix proteins and fibrosis-promoting cytokine transforming growth factor-ß (TGF-ß). The destruction of hepatocytes promotes their regeneration and susceptibility to cancer-inducing genetic changes.

It is noteworthy that NK cells and NK T (NKT) cells are enriched in the liver. They could potentially play a role in HCV control and the pathogenesis of HCV-induced hepatitis. In the last decade, a great deal has been learned about the molecular structures that are used by these cells to recognize ligands on target cells. Consequently, we are better able to appreciate the role of these cells in host defense against viral infections. This knowledge has also enabled us to understand many aspects of virus-specific T cells that may be involved in the HCV-induced pathogenesis. Therefore, we will give a brief account of NK and NKT cell biology and review our current knowledge about NK cell receptors (NKR) and their ligands.


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NK CELLS AND THEIR RECEPTORS
 
NK cells constitute a population of bone marrow (BM)-derived, low-density, large granular lymphocytes that make up 10–15% of the PBMC. They play an important role in host defense from pathogens and malignancy [56 57 58 59 60 ]. They are capable of spontaneously killing tumor and virus-infected cells, which have down-regulated one or more major histocompatibility complex (MHC) molecules and/or expressed certain stress antigens on their surface. They kill target cells without MHC restriction and prior activation. By killing virus-infected cells and causing the release of proinflammatory substances, e.g., heat shock proteins and TNF-{alpha}, NK cells provide the necessary "danger signal" to the immune system for inducing virus-specific immunity [61 , 62 ]. NK cells secrete several cytokines and chemokines, e.g., IFN-{gamma}, TNF-{alpha}, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-5, IL-13, IL-10, TGF-ß, MIP-1{alpha}, MIP-1ß, and regulated on activation, normal T expressed and secreted (RANTES). The production of these cytokines and chemokines is important in initiating an inflammatory response and in determining the nature and strength of the ensuing pathogen-specific immunity. NK cells represent a major source of IFN-{gamma} other than activated T cells. An immediate production of this cytokine from NK cells is a crucial factor in inducing effective antiviral, cellular immunity in the host. In addition to directly killing virus-infected cells by releasing cytotoxic molecules such as perforin and granzymes, NK cells also kill target cells by Fas/FasL, TNF-{alpha}, and TRAIL/DR-4 and DR-5 interactions [63 64 65 ]. The NK cell-secreted, soluble factors such as IFN-{gamma} and TNF-{alpha} also play a role in inhibiting virus replication by inducing an antiviral state in host cells [39 ]. Furthermore, NK cells play important immunoregulatory roles by interacting with T and B cells and APC.

In vivo studies in animal models have demonstrated that depletion of NK cells may result in the inability of the host to control viral infections. NK cell-deficient persons experience repeated recurrences of herpesvirus infections [58 , 59 ]. The infected host usually responds to a viral infection by enhanced NK cell activity. Viruses may directly activate NK cells by encoding a viral protein that is recognized by an activating receptor on the host NK cells; e.g., MCMV encodes a viral protein m157, which binds and activates LY49H+ NK cells in MCMV-resistant mice, and influenza virus encodes haemagglutinin, which binds to NKp46 and NKp44 on human NK cells (see below for details on NKR). Viruses can also activate NK cells indirectly by inducing expression of stress-inducible proteins or cytokines in the host. The stress-inducible proteins activate NKG2D-bearing NK and other cells. The virus-induced cytokines that activate NK cells include type 1 IFN (or IFN-{alpha} and -ß), IL-15, IL-18, IL-12, and IL-21 [59 , 66 67 68 ]. These cytokines positively affect different aspects of NK cell activation, proliferation, and survival [69 ].

The activity of NK cells is regulated by so-called NKR, which are a variety of molecular structures that are expressed on the surface of NK cells and bind specific ligands on target cells. The NKR are of inhibitory or of stimulatory types, and their triggering sends inhibitory or stimulatory signals to NK cells, respectively. Individual NK cells express inhibitory and stimulatory NKR and may kill or spare a target cell depending on the balance between inhibitory and stimulatory signals that it receives from the target cell via NKR. In humans, the NKR belong to four groups, which are briefly described here.

Killer cell Ig-like receptor (KIR) family
KIR are type I integral membrane glycoproteins that are usually expressed as monomers on the cell surface [70 , 71 ]. At present, more than 12 KIR genes have been discovered (Table 2 ). Diversity in these receptors is further enhanced as a result of gene polymorphism and alternate splicing. For example, the KIR2DL1 gene has at least eight alleles. The KIR may have short or long cytoplasmic tails (Fig. 3 ). The ones with long cytoplasmic tails have two ITIMs and are inhibitory in function. The receptors with a short cytoplasmic tail possess a charged aa in their transmembrane domains and associate noncovalently with a dimer of an adaptor protein, KARAP/DAP-12 [72 ]. The adaptor protein has ITAMs in its cytoplasmic tail. Upon binding to the MHC ligand, these receptors send activating signals to NK cells to kill target cells and secrete cytokines. As depicted in Figure 3 , KIR have been divided into four major groups: KIR2DS, KIR2DL, KIR3DL, and KIR3DS. This grouping is based on the number of Ig domains in the extracellular parts (2D for two domains and 3D for three domains) and the length of cytoplasmic tails (L for long and S for short tails). The receptors are further numbered differently if they show more than 2% sequence divergence within the group. Each KIR recognizes shared determinants present in a set of related MHC class I alleles on target cells. Major human KIR and their ligands are shown in Table 2 .


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  		Table 2. Major Human NKR, Their Functions, Distribution, and Ligands



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  		Figure 3. A schematic representation of the structure of major NKR. N and C, N and C termini of the receptors. {gamma} and {zeta}, Respective chains of the CD3 and Fc{epsilon}R1. ITAM, Immunoreceptor tyrosine-based activating motif; ITIM, immunoreceptor tyrosine-based inhibitory motif. Not drawn to the scale.

NKG2/CD94 receptors
These are type II C-type, lectin-like, integral membrane glycoproteins (Table 2 ; Fig. 3 ). They are expressed on the cell surface as heterodimers with CD94, which is an invariant type II C-type, lectin-like polypeptide. CD94 lacks a cytoplasmic tail and therefore, cannot transduce signals. It is however essential for the expression of NKG2 receptors. Four distinct genes, A/B, C, E/H, and F, encode the NKG2 receptors [70 , 71 , 73 ]. Of these receptors, CD94/NKG2A is an inhibitory one, as it contains a long cytoplasmic tail with two ITIMs. Others have short cytoplasmic tails, and each associates noncovalently with a homodimer of DAP-12, as in the case of activating KIRs [72 ]. NKG2 receptors bind HLA-E [74 75 76 ], whose expression requires peptides derived from signal sequences of HLA-A, -B, -C, and -G [77 ]. If the expression of these MHC antigens decreases in a cell, it will also result in a decreased expression of HLA-E. Thus, NK cells have developed a clever strategy of monitoring the overall expression of MHC class I antigens on target cells by simply monitoring the expression of the HLA-E antigen via NKG2/CD94 receptors [77 ].

Natural cytotoxicity receptors (NCR)
NKp46, NKp30, and NKp44 are NCR (see Table 2 and Fig. 3 ). They belong to Ig superfamily and trigger NK cell-mediated functions upon their engagement. NKp46 and NKp30 are expressed on resting and activated NK cells, whereas NKp44 is expressed on cytokine-activated NK cells (reviewed in refs. [78 , 79 ]). NKp46 bind the sialic acid-binding glycoproteins, e.g., haemagglutinin and haemagglutinin-neuraminidase of the influenza and parainfluenza viruses, respectively [80 ]. The ligands for other NCR are not yet known.

NKG2D receptors
The NKG2D differs from other members of the NKG2 family in significant ways. They do not form heterodimers with CD94 on the cell surface (Fig. 3) . Instead, they are expressed as homodimers, and each homodimer associates noncovalently with a homodimer of the adaptor protein DAP-10. The cytoplasmic tail of DAP-10 carries a YxxM motif (similar to the one present in the cytoplasmic tail of the costimulatory molecule CD28), which can recruit the regulatory subunit p85 of phosphatidylinositol-3 kinase and Grb2. NKG2D exist in two isoforms, which differ from each other in the length of their cytoplasmic tails by about one dozen aa. The shorter-tailed (S) form can associate with DAP-10 and DAP-12, whereas the longer-tailed (L) form mediates signals only via DAP-12 [81 , 82 ]. Resting NK cells express only the L form of the receptor. However, they also express its S form upon activation. The same is true for other immune cells, e.g., {gamma}{delta} TCR+ T cells and macrophages. Thus, activated immune cells can receive triggering signals and costimulatory signals via NKG2D, whose receptors recognize and bind stress-inducible proteins, ULBP1-4, MICA, and MICB [83 , 84 ]. In mouse, NKG2D bind H-60 (a minor histocompatibilty antigen) and the retinoic acid early inducible protein (and to a murine, ULBP-like transcript; reviewed in [82 ]). The NKG2D-specific ligands are usually expressed little in normal tissues but are induced on host cells by stress, transformation, and viral infections [85 86 87 ].

In addition to the above-mentioned receptors, NK cells also express a number of molecules, which are often called co-receptors. They bind to their cognate ligands on target cells. They are listed in Table 3 . CD16 is the low-affinity receptor for IgG. In conjunction with antigen-specific antibodies, NK cells can kill virus-infected cells, which express the antigen. This phenomenon is called antibody-dependent cellular cytotoxicity and may play a role in controlling virus replication in the host [88 ]. CD56 is an isoform of the neural cell-adhesion molecule and is involved in homotypic intercellular adhesions. About 10% of the peripheral blood NK cells express high levels of CD56 and low levels of CD16 on their surface, and the reverse is true for the remaining 90% of the NK cells [89 90 91 ]. The CD56+ NK cells are less cytotoxic as compared with the CD16+ ones. The coengagement of NK cell coreceptors by cognate ligands on target cells adds to the strength of the overall stimulating signal. In the absence of inhibitory signals, their coengagement may be sufficient to cause lysis of the target cells.


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  		Table 3. Human NK Cell Coreceptors, Their Ligands, Expression, and Functions


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REGULATION OF NK CELL FUNCTIONS BY NKR
 
The functional activity of NK cells is regulated by inhibitory and activating receptors. The MHC class I-binding receptors, KIR are expressed clonally on overlapping subsets of NK cells and function independently of each other. An individual NK cell may express two to nine receptors (average, five) [92 93 94 ]. These receptors are usually of inhibitory and stimulatory types. Under normal conditions, each NK cell in an individual expresses at least one inhibitory receptor capable of binding to a self-MHC class I antigen. This makes all NK cells of the individual self-tolerant. The existence of MHC class I- and HLA-E-binding, inhibitory NKR and their dominant role over their activatory counterparts explain why NK cells do not kill target cells that express normal levels of these MHC molecules, a phenomenon that was observed by Karre, et al. (ref. [95 ]; reviewed in ref. [96 ]) in the mid-1980s and prompted a proposal for the missing self-hypothesis for explaining NK cell-mediated killing. It is noteworthy that a common strategy used by viruses is to down-regulate the expression of MHC class I antigens on the surface of infected cells to evade antiviral CTL responses. This makes the infected cells susceptible to killing by NK cells (reviewed in refs. [97 , 98 ]). Although KIR-mediated, inhibitory signals are dominant over stimulatory signals, they are unable to inhibit NKG2D-triggered NK cell cytotoxicity. This means that NK cells will kill the target cells, which express stress-inducible ligands, even if they express normal levels of MHC antigens. If such target cells further down-regulate their expression of MHC antigens, they would become super-susceptible to NK cell-mediated killing.

The rules governing the expression of NKR on NK cells are not fully known. It has been observed that each individual NK cells rarely expresses inhibitory receptors to more than one self-MHC class I antigens. This enables individual NK cell to sense a decrease in even single MHC antigens. Studies conducted in mice suggest that the functional orthologs of KIR, Ly49, are acquired by developing NK cells in a stochastic and cumulative manner (reviewed in refs. [99 , 100 ]). Certain cytokines may also modulate their expression. For example, IL-15, IL-10, and TGF-ß1 induce expression of CD94/NK2GA on NK and CD8+ T cells. IL-15 also induces expression of NKG2D receptors on NK and CTL. IL-21 increases the expression of NCR on developing NK cells [101 ]. Viral infections, e.g., lymphocytic choriomeningitis virus, may also induce expression of inhibitory receptors on CTL [102 ]. A deregulated expression of NKR has important implications for the functional activity of NK cells; it may lead to the emergence of autoimmune NK cell clones if inhibitory receptors on NK cells are reduced and/or activating receptors are overexpressed. NK cells may also become immunodeficient if inhibitory receptors to MHC antigens are overexpressed on them. The autoimmune cells may kill normal autologous cells, whereas the immunodeficient ones may not be able to kill otherwise susceptible, malignant or virus-infected cells. Transgenic expression of the Ly49A, an inhibitory NKR, impaired antiviral cellular responses in mice [103 ]. In addition to changes in the expression of NKR, changes in the expression of MHC class I antigens or other ligands for NKR and coreceptors may also affect the susceptibility of target cells to NK cell-mediated lysis. For example, stress, transformation, or viral infections induce the expression of MICA, MICB, and ULBP on host cells and make them susceptible to NK cell-mediated killing via NKG2D ([85 , 86 ]; reviewed in ref. [103 ]).


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NKT AND NATURAL T (NT) CELLS
 
NKT cells represent a heterogeneous group of immunoregulatory and effector cells, which express both NK and T cell markers (reviewed in refs. [105 , 106 ]). NKT and conventional T cells arise from common CD4+CD8+ precursor lymphocytes within the thymus. The classical NKT cells are NK1.1+ (CD161 or NKR-P1) and CD3+ CD56+/– and are of the CD4+CD8– or CD4–CD8– phenotype. The expression of the marker NK1.1 is not essential for NKT cells, as the mice that do not express this marker [e.g., BALB/c, nonobese diabetic (NOD), and SJL] also contain these cells. NKT cells may also express other NK cell markers, e.g., CD94, Ly49, or KIR (in humans). They express a very restricted repertoire of TCR (usually a monoclonal TCR that comprises the invariant V{alpha}24-J{alpha}18 chain in association with a limited repertoire of Vß11 or -8 genes in humans). The mouse classical NKT cells express an invariant TCR {alpha} chain V14{alpha}-J{alpha}128 in association with a restricted number of polyclonal Vß genes (Vß8, Vß7, or Vß2). A common feature of these NKT cells is their positive selection by CD1d, nonpolymorphic MHC class Ib molecules, which are expressed on the cell surface with ß2-microglobulin (ß2M) but are transporter associated with antigen processing (TAP)-independent. The CD1d bind lipid antigens. The natural ligands for classical NKT cells are not known. In vitro, they are activated by {alpha}-galactosylceramide (GalCer), a natural anticancer glycolipid obtained from a sea sponge. NKT cells produce IL-4 and IFN-{gamma} abundantly upon their stimulation. However, they can also produce GM-CSF, IL-5, IL-13, RANTES, IL-8, IL-10, and TGF-ß1.

The classical NKT cells are abundant in liver and thymus. Murine liver is particularly enriched for these cells: ~50% of the intrahepatic lymphocytes (IHL) are NKT cells. They reside in sinusoids and play an essential role in preventing liver metastasis and in clearing a viral infection. It was demonstrated in the HBV transgenic mouse model that activation of NKT by a single injection of GalCer induced IFN-{gamma} production, which was sufficient in controlling viral replication noncytopathically without the need of further lymphocyte infiltration. It is interesting that NKT and NK cells and not CD4 or CD8+ T cells were involved in this viral control in liver [41 , 42 ]. The NKT cells were also shown to mediate antitumor effects of IL-12 in mice [107 ]. However, NKT cells may also cause liver damage, as demonstrated in murine hepatitis models induced by concanavalin A or LPS plus IL-12. NKT cells disappear from livers following their activation, probably as a result of apoptosis. The newly emerging NKT cells are biased and produce only IL-4 upon restimulation. Because of their ability to produce large quantities of IL-4, it was initially thought that NKT cells might be essential for inducing TH-2 immune responses. However, the experimental induction of these types of responses in CD1d–/– and ß2M–/– mice (which lack NKT cells) shows that these cells are not absolutely necessary for these responses. It is important to note that NKT cell activation is accompanied by NK cell activation, which is essential for the effector functions of NKT cells [108 , 109 ]. Furthermore, NK cells also play a role in the migration and retention of NKT cells in the liver of mice (see review, ref. [106 ]).

NKT cells protect mice from autoimmune diseases, e.g., among others, autoimmune diabetes, multiple sclerosis, and rheumatoid arthritis (reviewed in ref. [110 ]). They are less frequent and are defective in IL-4 production in NOD mice and in individuals that are at risk for type I diabetes (T1D). It has been demonstrated that in vivo activation of NKT cells by GalCer protects NOD from the onset of T1D and prolongs survival of pancreatic islets transplanted into newly diabetic mice. These effects are a result of induction of TH-2 responses in spleens and pancreas of these mice, which prevent B and T cell-mediated autoimmunity to islet B cells. IL-7 acts synergistically with GalCer in this protection by inducing enhanced production of IL-4 and IL-10 from NKT cells (refs. [111 , 112 ]; reviewed in ref. [110 ]).

Nonclassical NK1.1+ CD8+ NKT cells have variant TCR and are not restricted by CD1d. The spleen and BM are the main sites where these cells reside. Intestinal, intraepithelial lymphocytes usually contain CD8{alpha}{alpha}+ NKT cells. In humans, nonclassical NKT cells may express variant {alpha}ß or {gamma}{delta} TCR, CD161, and may be restricted by CD1a, -b, or -c. They produce IFN-{gamma} and not IL-4. It is noteworthy that CD1a, -b, and -c are not expressed in mice.

The conventional CD8+ T cells with variant {alpha}ß TCR acquire NK1.1 on activation. They may also express other inhibitory NKR. The cells expressing inhibitory KIR usually represent oligo- and monoclonally expanded antigen-specific CD8+ T cells of the effector memory phenotype [113 , 114 ]. They are usually expanded in chronic viral infections [115 ]. The expression of KIR promotes their survival by preventing them from undergoing activation-induced cell death. It also increases their antigen threshold and prevents them from their effector functions. It has been demonstrated in vitro and in vivo that viral infections and certain cytokines, e.g., IL-2 and IL-15, induce the expression of CD56 and NKG2A/CD94 on CTL. This expression coincides with the acquisition of NK-like cytolytic activities in these CD3+CD56+ NKT cells. They have also been named as NT cells. The distinction between NT and nonclassical NKT cells is not very clear, and many authors have used them interchangeably.


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NKR AS REGULATORS OF ANTIVIRAL AND ANTITUMOR IMMUNITY
 
In recent years, it has become quite clear that NKR play an important role in regulating immune responses and effector functions of NK cells and NKT cells [71 ]. We and others have shown an enhanced expression of KIR receptors on HIV-specific CTL in HIV-infected AIDS patients (reviewed in ref. [116 ]). The blockage of these receptors with specific monoclonal antibodies (mAb) markedly increases the cytotoxic functions of the HIV-specific CTL from these patients against HIV-infected cells in in vitro assays [117 ]. Data from animal models have shown that in vivo blockage of these receptors also enhances antitumor as well as antiviral cytolytic activities of NK and CTL [118 ]. Furthermore, they have opened novel avenues of increasing the efficacy of antiviral and antitumor vaccinations; e.g., in vivo expression of NKR ligands such as MICA, MICB, or ULBP may be used to activate NK and CTL, and the administration of GalCer as an adjuvant may increase antiviral immunity induced by a vaccine. At least in theory, the blocking of inhibitory NKR in vivo may augment antiviral activity of NK and CTL in virus-infected individuals.

Viral infections tend to induce the expression of stress-inducible proteins, e.g., MICA, MICB, and ULBP. These proteins act as ligands for the NKG2D receptor, which is expressed on the NK cells, {gamma}{delta} receptor-positive T cells, and the CD28– subset of CD8+ T cells [87 , 119 ]. The CD28– CTL of the memory phenotype as well as {gamma}{delta} TCR+ T cells occur in unusually high frequencies in the peripheral blood of individuals that are suffering from chronic virus infections [120 , 121 ]. Certain cytokines, e.g., IL-15, are also known to induce the expression of NKG2D on antigen-specific CTL and convert them into lymphokine-activated killer cells, which can kill not only virus-infected, antigen-expressing target cells but also uninfected, "stressed" host cells [122 ]. An induced expression of NKG2D ligands on host cells may make them susceptible to killing by the NKG2D receptor-positive T and NK cells. This type of lysis may cause tissue damage and contribute toward pathogenesis of the infection, as these ligands are also induced on uninfected host cells as a result of stress and inflammatory mediators [85 , 87 ].


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LIVER NK AND NKT CELLS
 
A normal human liver contains lymphocytes that are usually enriched for NK and NKT cells. In contrast to the peripheral blood that contains ~13% NK and 4% NKT cells, intra-hepatic lymphocytes (IHL) contain 37% NK cells and 26% NKT cells. The percentage of NK cells in the IHL pool may increase to 90% in hepatic diseases [2 ]. The liver NK cells are the classical CD3–CD56+ cells and were first recognized as "pit cells" [123 ]. In contrast to the peripheral blood NK cells, which are predominantly (90%) CD16+, few of the liver NK cells express this molecule. The human liver NKT cells comprise mostly CD3+CD56+ cells with variant TCR. They also express other NKR, e.g., CD94/NKG2 and KIR, and may express low levels of {alpha}ß or {gamma}{delta} TCR. Liver NK and NKT cells produce cytokines and chemokines and can mediate MHC-unrestricted killing of target cells with or without stimulation with cytokines, e.g., IL-2 [2 ]. NKT cells can also produce IFN-{gamma}, TNF-{alpha}, IL-2, and/or IL-4 upon stimulation; however, NK cells produce IFN-{gamma} and TNF-{alpha}. Some NKT (5–6%) produced IFN-{gamma} and IL-4 simultaneously [124 ].

Unlike mouse liver, human livers contain few classical NKT cells with invariant TCR. Instead, they are enriched for CD3+CD56+ nonclassical NKT cells [125 ]. In healthy adult humans, 2% of the peripheral blood CD3+ T cells are CD56+, but in liver, they are 30%. They may be CD4+, CD4–CD8–, or CD8+ and may have {alpha}ß or {gamma}{delta} TCR. They may represent a functional counterpart of NKT cells in human livers. However, they mainly produce IFN-{gamma} and not IL-4 upon their stimulation. They are restricted by CD1d and are TH-1-biased in HCV-infected livers [125 ].

The human classical NKT (V{alpha}24/Vß11) can be readily detected in livers of HCV-infected persons; however, their frequencies are decreased as compared with healthy persons. A similar situation has been described in the circulation of HIV-infected persons [65 , 126 ]. Human classical NKT cells express high levels of CCR-5 and CXCR-6 and are susceptible to infection with monocytotropic or R5 strains of HIV [126 ]. Their depletion in HIV-infected persons may be caused, at least in part, by direct infection of these cells by HIV. We do not know whether these cells per se can be infected with HCV or whether they are depleted by other mechanisms, e.g., apoptosis, or migration to other compartments. Their depletion may predispose infected persons to autoimmune conditions as well as skew the liver microenvironment toward TH-1 cytokines.


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NK CELLS IN HCV INFECTION
 
As stated earlier, infections with hepatotropic viruses activate liver NK cells, which play a critical role in the recruitment of T cells to liver. Activated NK cells can kill virus-infected cells via perforin/granzyme, and FasL pathways produce proinflammatory cytokines, which can induce antiviral state host cells. Human NK cells cocultured with HCV replicon-containing hepatic cells in Transwell microplates inhibit the replicon expression at protein and RNA levels by secreting antiviral factors including IFN-{gamma} [127 ]. Thus, NK cells could also potentially contribute toward HCV control. It would not be surprising that in the face of an adequate NK cell response, hepatotropic viruses such as HCV may be controlled even in the absence of virus-specific immune responses. This notion is supported by the observations that as in humans, a certain percentage of HCV-infected chimpanzees can clear infection spontaneously, and this clearance does not correlate with the appearance of acquired immunity [128 ]. However, the potential importance of NK cells in the control of HCV was discounted in a study that demonstrated that depletion of CD8+ T cells by mAb aggravates HCV infections in animal models [129 ]. It is noteworthy that about one-third of human and chimpanzee NK cells express CD8. The depletion of CD8+ T cells by anti-CD8 antibodies would also deplete CD8+ NK cells. Therefore, the results from such studies should be interpreted with a caveat. The very fact that HCV has developed multiple strategies to evade the host’s NK cell response testifies to the importance of this arm of innate immunity in controlling this infection. Paradoxically, NK cells, too, act as a double-edged sword and might contribute toward pathogenesis and cause liver damage by killing hepatocytes and by secreting proinflammatory cytokines.


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HOW HCV EVADES HOST’S NK CELL RESPONSES
 
HCV seems to have evolved several mechanisms to evade the host’s NK cell response (summarized in Table 4 ). One of the strategies is to inhibit production of type 1 IFN from the host cells in response to the infection. As stated above, this cytokine protects the host in multiple ways, e.g., by activating NK cells, inducing DC maturation, and inhibiting viral replication. It also suppresses translation from the viral IRES [130 ]. The viral serine protease (NS3/NS4A complex) inhibits the IRF-3 activation [131 ]. This factor is known to be essential for virus-induced production of type I IFN. Dominant-negative IRF-3 mutants enhance, and constitutively active ones suppress HCV RNA replication in hepatoma cells. Moreover, the viral glycoprotein E2 and the nonstructural viral protein NS5A can bind and inactivate PKR (refs. [132 133 134 ]; reviewed in ref. [135 ]); PKR is a kinase that plays an important role in the production of type 1 IFNs from host cells by RNA viruses. Its activity is also essential for the IFN-induced host resistance to viral replication. The nonstructural HCV protein NS5A contains a region (aa 2209–2248), the ISDR. NS5A from IFN-resistant genotypes 1a and 1b can physically bind and inhibit PKR in an ISDR-dependent manner. The binding of NS5A to the kinase prevents the latter’s dimerization and activation (ref. [134 ]; reviewed in ref. [135 ]). Recent studies have shown that ISDR is necessary but not sufficient to inactivate PKR; an additional 26 residues on its c-terminus are also needed to form complex with the kinase [134 ]. ISDR from IFN-sensitive HCV strains do not bind with PKR. The viral glycoprotein E2 also contains a 12-aa sequence that is identical to the phosphorylation sites of the PKR in its target substrate, the translation initiation factor eIF2{alpha} [133 ]. By virtue of this sequence, E2 blocks kinase activity of PKR and consequently, its inhibitory effect on protein synthesis and cell growth. The E2 sequences of the IFN-resistant HCV genotypes 1a, 1b, 4, 5, and 6 more closely resemble the phosphorylation site of PKR than the corresponding sequences from less resistant 2a, 2b, and 3 genotypes. Thus, these effects correlate with the relative resistance of the virus to IFN-{alpha}. The inhibition of PKR by viral proteins frees host cells from its growth inhibitory effects, which favor cell growth and the development of HCC. Furthermore, the viral core protein induces the expression of the SOCS-3 protein [122 ], which is known to abrogate the IFN-induced signaling in human cells. It also mediates inhibitory effects of IL-10 on LPS-mediated activation of macrophages, and interferes with activation of Janus tyrosine kinase–signal transducer and activator of transcription signaling mediated by gp130 cytokines, leptin, growth hormone, and prolactin (refs. [133 , 136 ]; reviewed in ref. [137 ]). More importantly, SOCS-3 negatively regulates insulin-mediated signaling, and therefore, its chronic expression may promote type II diabetes [138 ].


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  		Table 4. List of HCV-Adopted Strategies to Counter and Evade the Host’s NK Cell Responses

HCV has also developed the strategy of directly inhibiting NK cell responses; the viral E2 glycoprotein binds to CD81 on NK cells and inhibits their effector functions [17 , 139 ]. It is interesting that CD81 also is expressed on the surface of CTL, but E2 does not inhibit the function of these cells, it rather stimulates them [140 ]. As E2 is highly variable, and not all HCV strains possess E2 that can bind CD81, it will be interesting to know whether the HCV strains that are spontaneously eliminated by some infected individuals have E2 that can inhibit NK cell functions. Another mechanism by which HCV evades NK cell responses is by stabilizing the expression of MHC class I molecules on the surface of the infected cells. Viruses, in general, tend to decrease the expression of MHC class I molecules on the surface of infected cells. This helps them escape antiviral CTL responses. However, HCV does not decrease the expression of these antigens on the surface of infected cells; it rather increases it [141 , 142 ]. The viral core protein plays a role in this increased expression. It enhances DNA binding affinity and transcriptional activity of p53, without affecting its mRNA or protein levels. The activated p53, in turn, activates the transporter associated with antigen procecessing-1 or TAP-1 [143 , 144 ]. The activated TAP-1 increases the available pool of peptides that bind MHC class I molecules in the lumen of the ER. The resulting increased expression of MHC antigens on the surface of HCV-infected cells may enhance their resistance to their killing by NK cells. It is noteworthy that increased MHC expression on hepatocytes negatively correlates with susceptibility to IFN-{alpha} treatment [142 ]. The mature DC derived in vitro from monocytes activate NK cells [145 ]. The DC-activated NK cells express CD69, produce IFN-{gamma}, and efficiently kill K562 cells. The DC matured by LPS activated NK cells via IL-12, whereas those activated by IFN-{alpha} activated NK cells via MICA and MICB. The DC recovered from the monocytes of HCV-infected patients did not activate NK cells, as IFN-{alpha} was unable to induce expression of MICA and MICB on these DC [145 ]. This suggests that DC may be unresponsive to IFN-{alpha} in these patients. It is interesting that the IFN-{alpha} receptor is also down-regulated in livers in patients that are chronically infected with HCV, and the efficacy of the IFN treatment is related to the expression of the receptor mRNA in the livers of the patients [146 ].

Several researchers have reported that NK cell activity decreases in HCV-infected individuals [147 , 148 ]. The decrease probably depends on the stage of the disease. Although, CD3–CD56+ NK cells are enriched in liver, their number decreases with disease progression, and this may predispose this organ to malignancy [149 ]. An increased expression of the MHC class I antigens (A, B, or C) in HCV-infected cells may make them resistant to NK cell-mediated lysis. Apart from increasing the expression of MHC class I antigens, HCV also seems to enhance the expression of stress-inducible proteins, e.g., MICA. Increased expressions of these proteins on hepatocytes have been reported in HCC individuals [150 ]. As mentioned above, these proteins act as ligands for NKG2D receptors, which are present on NK cells and activated CTL. Thus, heptocytes from HCV-infected livers may be predisposed to killing by NK cells and CTL even if they are not infected by HCV. This may also explain, at least in part, the CTL-mediated killing of uninfected bystander hepatocytes in HCV-infected individuals.

Furthermore, like other chronic infections, NK cells and CD3+CD56+ NKT cells from the livers of HCV-infected individuals express a dysregulated expression of KIR [151 ]. The expression of inhibitory receptors decreases on these cells, which may render them autoreactive and promote killing of hepatocytes and other bystander host cells. It still remains to be determined how the expression of NKG2D and other activating NKR, e.g., NCR, are regulated in HCV-infected livers. It is noteworthy that HCV-specific CTL have been well documented to kill the bystander host cell in HCV-infected patients [50 ]. This type of killing may play an important role in HCV-induced hepatitis.

It is well known that IFN-{alpha} is used as treatment for HCV. This cytokine induces NK cell blastogenesis and cytotoxicity [69 ]. It can also suppress HCV replication and cure the virus-infected cells [130 , 151 ]. It was reported that an effective IFN therapy in HCV-infected individuals correlated to their increase in NK activity. In the individuals in whom the therapy failed to increase NK cell response, no decrease in viremia was observed [142 , 152 , 153 ]. As stated earlier, HCV has evolved a strategy to counter the IFN-mediated host response by encoding a core protein that can induce SOCS-3 protein [154 ]. Certain other cytokines, e.g., IL-1ß, can also attenuate the effects of IFN on host cells, and there is an abundance of this cytokine in the circulation of chronic HCV-infected patients [155 ]. Thus, the host’s NK cell response could play a role in suppressing HCV replication; however, the virus has evolved strategies to counter this response. In the presence of these viral strategies, this host response may be contributing toward pathogenesis of HCV-induced hepatitis by killing uninfected hepatocytes.


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A UNIFIED HYPOTHESIS FOR THE ROLE OF NK CELLS AND ANTIVIRAL CELLULAR IMMUNE RESPONSE IN HCV-INDUCED HEPATITIS
 
The available data on innate and adaptive immune responses in HCV-infected patients point to a unified hypothesis of HCV-induced hepatitis. According to this hypothesis, HCV infection changes the relatively immuno-silent status of normal liver and induces adaptive and innate immune responses in the organ. The anti-inflammatory TH-2 cytokine milieu of the liver is skewed toward a highly proinflammatory TH-1. This is further aggravated by a selective depletion of NKT cells. Although NK cells and CTL may be important in clearing viral infection, they also cause liver destruction by a variety of mechanisms. They may be rather killing uninfected but inflamed hepatocytes, as the viral strategies may prevent destruction of the infected cells. It is commonly believed that an antiviral immune response in chronically infected individuals is too weak to eradicate HCV but strong enough to cause liver damage. It is quite conceivable that a strategy used to augment this response may also be accompanied by enhanced liver damage in these patients. Therefore, our attempts to boost antiviral immune responses may further damage the liver in the infected patients unless we counter the viral strategies that prevent effectiveness of the boosted immune responses. As HCV per se is not cytopathic, and viral loads do not correlate with the degree of liver fibrosis, a strategy worth testing may involve calming down the proinflammatory microenvironment of the infected livers by depleting NK cells or CTL and/or by neutralizing one or more key proinflammatory cytokines, e.g., IFN-{gamma}, TNF-{alpha}, and MIP-1{alpha}. Another strategy may involve inhibiting liver infiltration of NK cells by blocking VCAM-1/VLA-4 interactions by anti-VCAM-1 antibodies. In support of this notion are the reports in literature showing that the depletion of CTL and long-term IL-10 therapy in HCV-infected persons results in significant improvement in their clinical condition [156 , 157 ]. In this regard, it is noteworthy that in addition to its antiviral effects, IFN-{alpha} (the standard therapy for HCV-infected persons) is well known for its cytostatic effects and its ability to down-regulate IFN-{gamma} production from IL-12-stimulated NK and T cells [158 ]. Further studies are required to learn more about these aspects of this disease. Finally, recent advances made in understanding the biology of NK cells, their receptors, and their role in regulating antiviral immune responses have provided novel avenues for countering the immune evasion of HCV (see below).


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FUTURE DIRECTIONS
 
To better understand the potential role of NK cells in the pathogenesis of HCV-induced hepatitis, it would be important to direct our future research on the following issues.

The future studies should be carried out to determine how E2/CD81 interactions lead to NK cell inactivation and how this inactivation could be overcome. It seems that on the surface of NK cells, CD81 complexes with some inhibitory receptor, which becomes triggered when E2 interacts with CD81. Antibodies or peptides that inhibit this interaction may be useful in increasing the efficacy of NK cells against HCV-infected hepatocytes.

As stated earlier, NK cells modulate immune responses by secreting a variety of cytokines and chemokines. The production of these soluble mediators from NK cells depends on the milieu in which they differentiate. In analogy to TH-1 or TH-2 CD4+ helper T cells, NK cells could also differentiate into NK1 or NK2 types depending on the cytokine milieu. NK1 cells predominantly produce IFN-{gamma}, whereas NK2 ones produce IL-5 and IL-13 cytokines [159 , 160 ]. These two types of NK cells occur in vivo in humans and have been implicated in the pathogenesis of multiple sclerosis [161 ]. It has been well documented that liver damage in HCV-infected persons is correlated with TH-1 cytokine response, whereas there is increased production of TGF-ß in liver cirrhosis [162 , 163 ]. Thus, NK cells, in the livers of HCV-infected patients, may be strongly biased to produce a TH-1 cytokine profile. They may represent a major source of proinflammatory cytokines, which may be involved in liver damage. Because of their abundance in the liver, the cytokines produced by NK cells may be a major factor in determining the fate of antiviral immune responses as well as in liver injury. Therefore, cytokine profiles of intra-hepatic and peripheral blood NK and NKT cells from HCV-infected persons should be investigated.

Apoptosis of activated CD8+ T cells in the liver has been well documented (ref. [28 ]; reviewed in refs. [2 , 27 ]). It is not yet clear whether activated NK cells also undergo apoptosis in this organ. It is noteworthy that activated NK cells, like activated T cells, are prone to apoptosis. A subset of NK cells from healthy persons also undergoes apoptosis when they come in contact with NK-sensitive target cells in in vitro assays [164 , 165 ]. It has been shown that IL-2 or IL-12-activated NK cells undergo apoptosis when they are incubated with anti-CD16, -CD2, or -CD94 antibodies [166 167 168 ]. They also undergo apoptosis when incubated in high concentrations of certain proinflammatory cytokines, e.g., IL-18 or IL-15 and IL-12 [169 ]. The production of TNF-{alpha} from the cytokine-stimulated NK cells has been implicated in this type of killing. It may represent a negative feedback mechanism to control the secretion of IFN-{gamma} from NK cells. In line with these observations, high serum concentrations of the proinflammatory cytokines have been found to correlate inversely with the peripheral blood NK cell loss in patients suffering from various systemic autoimmune disorders [170 ]. It is noteworthy that the concentration of the NK activity enhancing cytokines, e.g., IL-18, IL-12, is elevated in the sera of HCV-infected patients [171 , 172 ]. It is not known whether NK cells are undergoing enhanced apoptosis in these patients. The cells may also have increased turnover in this organ. Apoptosing NK cells still may cause liver damage as do apoptosing CTL in this organ [31 , 173 ]. Ultimately, NK cell turnover decreases and results in decreased NK cell activity and NK cells numbers, as reported in HCV-infected patients [147 , 149 ]. This decreased NK cell surveillance may contribute toward the development of HCC. Thus, we speculate that an inappropriate activation of NK cells as a result of overall hyperactivation of the immune system or increased amounts of antigen-antibody complexes in this infection may cause apoptosis of these cells.

The NKR repertoire of an individual may influence his or her ability to mount an effective antiviral response. In certain individuals, who inherit an activating allele of a KIR gene and also happen to express its MHC ligand, NK cells are in a relatively higher state of activation because of an epistatic interaction between these two genetically diverse loci. These individuals may be relatively resistant to viral infections. For example, Martin et al. [174 ] have shown that the individuals expressing KIR3DS1 (an activating form of the KIRDL1 gene) and its specific ligand (Bw4 specificity HLA-B molecules with isoleucine at position 80; HLA-B Bw4-80.Ile) are relatively resistant to the development of AIDS. HLA-B Bw4-80.Ile alleles were not associated with protection from AIDS in the absence of KIR3DS1. They rather showed a relatively rapid progression of AIDS. It is tempting to speculate that the individuals expressing KIR3DS1 and HLA-B Bw4-80.Ile may also be more resistant to other pathogenic infections as well as to the development of tumors. Because of a higher state of immune activation, they may be more prone to autoimmune diseases. As mentioned above, ~15% of HCV-infected individuals spontaneously recover from the infection. It is not known whether an epistatic interaction between NKR and MHC loci plays a role in the virus clearance in these individuals. The results presented at the 8th Annual Meeting of the Society for Natural Immunity at Noordwijkerhout (The Netherlands) [175 ] demonstrate that resolution of HCV infection is associated with the expression of two group 1 HLA-C alleles and homozygosity of the KIR2DL3 receptor. This beneficial effect of the inhibitory KIR and their HLA-C ligands with respect to HCV infection again underlies the role of the host’s immune response in the pathogenesis of HCV-induced hepatitis. It also suggests that activated NK cells may be involved in this pathogenetic process. Clearly, further studies are needed to verify these results. If proven so, manipulation of the NKR–ligand interactions may represent an important way of altering the course of the infection for the benefit of the host.

Chronic viral infections such as HCV are accompanied with an aberrant expression of NKR on the surface of NK cells as well as on other immunocytes, e.g., monocyte/macrophages, DC, and B and T cells (CD4+ and CD8+ subsets). Knowledge of the NKR genes that become up- or down-regulated in these infections should be helpful in designing rational therapies to modulate immune responses in the infected patients. Unfortunately, at present, mAb are not available for all of these receptors, and the ones that exist may not distinguish between the activating and inhibitory forms of these receptors. Therefore, it may be difficult to study their expression on infected host cells. However, alternate methods, such as real-time reverse transcriptase-polymerase chain reaction and/or oligonucleotide microarrays with appropriate controls, may give a fair idea of the genes whose expression may be dysregulated in HCV-infected individuals. Furthermore, as the level of expression of coreceptors on NK cells and of their cognate ligands on target cells (see Table 3 ) influences NK cell functions, and these levels frequently change in chronic viral infections, the question of how HCV infection modulates their expression in the infected host is worth addressing.

At mentioned above, MHC-specific NKR are expressed clonally on overlapping subsets of NK cells independently of each other. Therefore, changes in the expression of NKR will also be manifested on the clonal level and may give rise to immunodeficient and/or self-reactive, autoimmune NK cell clones. The detection of these clones may not be possible with the use of polyclonal NK cell preparations, which are normally used in NK cell assays. One may have to use NK cell clones derived from the infected patients. Furthermore, one cannot use traditional K562 cells as target cells in these assays. These target cells do not express MHC antigens and are killed by NK cells via NKp46 and NKp30 receptors (reviewed in ref. [78 ]). These assays merely determine the overall killing potential of NK cells via these two receptors and give no information as to the expression of MHC-specific receptors. The cytotoxic activities of NK cell clones may be tested against a panel of target cells with distinct MHC haplotypes including self-phytohemagglutinin-activated T cell blasts. Alternately, one may also use MHC-deficient human target cells, which are engineered to express a single MHC allele (reviewed in ref. [116 ]).

It is noteworthy that the expression of NKR ligands on target cells is also an important factor in determining the functional activity of NKR+ immunocytes. Therefore, it would be important to know how HCV regulates the expression of these ligands on host cells, in particular, on hepatocytes. We know that HCV stabilizes the expression of MHC class I antigens. However, we do not know which of these antigens are stabilized. Furthermore, we need to know how HCV regulates the expression of HLA-E, HLA-G, and stress-inducible proteins, such as MICA, MICB, and ULBP. These are important NKR ligands, which may affect the functional activities of NK cells, CTL, DC, and macrophages. HCV may induce expression of the stress-inducible proteins on hepatocytes directly or indirectly via proinflammatory cytokines. Such host cells, whether infected or not, may be killed by NKG2D-expressing NK cells and T cells. It is tempting to speculate that NKG2D-type-activating NKR on cytolytic cells in these patients may be involved in the killing of uninfected hepatocytes. This is a testable hypothesis and, if proven, may lead to novel approaches for treating this infection.

A fortuitous outcome of the newly gained knowledge of NKR and their ligands is the availability of novel adjuvants that could increase the efficacy of antiviral vaccines. More specifically, the ligands that bind activating receptors and activate NK cells, e.g., among others, MICA, MICB, ULBP, and GalCer, may turn out to be effective adjuvants in antiviral vaccination formulations. By activating NK cells, these adjuvants should induce more powerful immune responses in the vaccinees.

Finally, one should investigate the effect of blocking interactions between NKR and their ligands on hepatitis in animal models. For this purpose, receptor-specific antibodies and/or their soluble ligands may be used. Soluble MHC molecules might have served as important tools to manipulate NKR-ligand interactions in vivo. As they have been shown to induce apoptosis of CD8+ NK and T cells via their interaction with the MHC-binding domain of CD8 molecules [176 , 177 ], their mutant versions lacking CD8-binding domains may be a better choice. If proven useful, they may represent novel tools in our fight against this formidable disease. It is noteworthy that some humanized anti-KIR antibodies are already being marketed as drugs.


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ACKNOWLEDGEMENTS
 
A. A. holds a "Chercheur-boursier senior" award from "Fonds de la recherche en Santé du Québec" (FRSQ.) We thank Ms. Sylvie Julien for her excellent secretarial help, our colleagues in the laboratory for insightful discussions, and the Canadian Institutes of Health Research (CIHR) for support. We regret that due to space limitations, all studies on the subject could not be cited.

Received March 25, 2004; accepted May 24, 2004.


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