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

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

Ali Ahmad*,‡,1 and Fernando Alvarez†,‡

Departments of *Microbiology & Immunology and †Pediatrics and ‡Center of Research, Ste. Justine Hospital,University of Montreal, Quebec, Canada

Abstract: Natural killer (NK) cells constitute thefirst line of host defense against invading patho-gens. They usually become activated in an earlyphase of a viral infection. Liver is particularly en-riched in NK cells, which are activated by hepato-tropic viruses such as hepatitis C virus (HCV). Theactivated NK cells play an essential role in recruit-ing virus-specific T cells and in inducing antiviralimmunity in liver. They also eliminate virus-in-fected hepatocytes directly by cytolytic mecha-nisms and indirectly by secreting cytokines, whichinduce an antiviral state in host cells. Therefore,optimally activated NK cells are important in lim-iting viral replication in this organ. This notion issupported by the observations that interferontreatment is effective in HCV-infected persons inwhom it increases NK cell activity. Not surpris-ingly, HCV has evolved multiple strategies tocounter host’s NK cell response. Compromised NKcell functions have been reported in chronic HCV-infected individuals. It is ironic that activated NKcells may also contribute toward liver injury. Fur-ther studies are needed to understand the role ofthese cells in host defense and in liver pathology inHCV infections. Recent advances in understandingNK cell biology have opened new avenues forboosting innate and adaptive antiviral immune re-sponses in HCV-infected individuals. J. Leukoc.Biol. 76: 743–759; 2004.

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

HEPATITIS C VIRUS (HCV) INFECTION

HCV infection has assumed the proportion of a global pan-demic. Approximately 170 million people are infected world-wide with this virus. They make up �3% of the world popu-lation. Their number is approximately five times more thanthose infected with human immunodeficiency virus type 1(HIV-1) [1]. HCV was the leading cause of post-transfusionand community-acquired non-A, non-B hepatitis until the in-troduction of blood screening in 1990. The institution of bloodscreening for HCV has markedly reduced its incidence. How-ever, it still remains a significant problem in intravenous drugabusers (reviewed in ref. [2]). A significant proportion of the

infected persons develops chronic hepatitis, cirrhosis, and liverdysfunction. The infection is the most common cause for livertransplantation in adults. Three to 4% of the chronically in-fected individuals develop fatal hepatocellular carcinoma(HCC). HCV and HIV-1 frequently coinfect humans: It hasbeen estimated that as high as 18% of HIV-infected personsare also infected with HCV [3]. The coinfected persons expe-rience more severe hepatitis and progress more rapidly to thedevelopment of AIDS.

HCV was identified as a cause of non-A, non-B hepatitis bymolecular 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 becauseof its association with Herpes simplex virus type 2. The use ofcontaminated needles, blood, and blood products representsthe major route of its transmission. Acute HCV infectionsusually remain undiagnosed, as they present mild or no clinicalsymptoms. Approximately 15% of the infected persons undergospontaneous recovery. These individuals may be important forunderstanding the immune mechanisms that resist and elimi-nate a natural HCV infection in humans. It is important that anoverwhelming majority of the infected persons fails to controlthe infection and develops a chronic infection with a variabledegree of hepatitis and viremia [1, 6]. Molecular mimicrybetween host and viral antigens has been implicated in liverautoimmunity in HCV-infected patients. A fraction of the pa-tients positive for HCV RNA and/or anti-HCV antibodiesshows the presence of type I antiliver kidney microsome anti-bodies, which also recognize cytochrome P450 (CYP)2D6. Thepatient livers are infiltrated with autoreactive mononuclearcells, which recognize CYP2D6. It is interesting that the viralcore protein residues 178–187 bear sequence homology withhuman cytochrome P450 (CYP2A6 and CYP2A7) residues8–17 [7]. Although HCV is a hepatotropic virus and infectshepatocytes, viral genome and its replicative intermediates arefrequently present in the peripheral blood mononuclear cells(PBMC) and lymphoid tissues of chronically infected persons.The infection has also been associated with several extrahe-patic manifestations, e.g., among others, type II mixed cryo-globulinemia (MC), non-Hodgkin’s lymphomas, and rheumatic

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

Received March 25, 2004; accepted May 24, 2004; doi: 10.1189/jlb.0304197.

Journal of Leukocyte Biology Volume 76, October 2004 743

and cutaneomucous symptoms. MC is a benign, B cell-prolif-erative disorder accompanied by the presence of monoclonalimmunoglobulin (Ig)M with rheumatoid factor activity, which isencoded by a restricted set of variable-region, germ-line genes,VH 1-69 and VK A27 [8, 9]. It is believed that HCV prefer-entially infects B cells expressing these Ig genes. HCV-in-fected individuals also have a higher incidence of non-Hodgkin’s lymphomas. These lymphomas have been shown toexpress the same restricted repertoire of the germ-line Iggenes, as in the case of MC IgM [10]. The viral glycoprotein E2has been implicated in the oligoclonal expansion of theselymphoma cells [8]. The most common rheumatic and cutaneo-mucous symptoms in HCV-infected patients include fatigue,arthralgia, paraestheisa, myalgia, pruritis, and the sicca syn-drome (reviewed in ref. [11]).

HCV-infected persons are usually treated with interferon(IFN)-�, with or without ribavirin. Usually recombinant humanIFN-�2a or -2b is used. Only less than half of the patientsrespond to this treatment. The treatment is usually accompa-nied with toxic side-effects, which deteriorate the quality of lifein these persons. The recent use of pegylated IFN-� with orwithout ribavirin has significantly improved the treatment ef-ficacy [12, 13]. The effect of highly active antiretroviral therapy(HAART) in HCV and HIV-1-coinfected patients is controver-sial. The HAART-mediated restoration of immune responses inthese patients often leads to hepatotoxicity and increased HCVdiversity [14]. As yet, there is no anti-HCV vaccine available,and the prospects of such a vaccine are also dim. The onlyspecies in which HCV replicates are humans and chimpan-zees. The virus does not replicate efficiently in vitro in cellculture [6]. The lack of a suitable in vitro viral replicationsystem is a major hindrance in studying immunobiology of thisvirus.

HCV is an enveloped, positive-strand RNA virus of �50 nmdiameter and belongs to the genus Hepacivirus in the Flavi-viridae family. The lipid bilayer HCV envelope is derived fromhost cell membranes, into which viral envelope glycoproteinsE1 and E2 are inserted. The envelope contains nucleocapsidwith a 9.6-kb-positive strand RNA. The viral genome containsone open-reading frame (ORF) and two (a 5�- and a 3�-)untranslated regions (UTR; ref. [1]). It is translated from aninternal ribosome entry site (IRES) in the 5�-UTR into a singlepolyprotein of �3011 amino acids (aa). The polyprotein isprocessed by viral and host-signal proteases into four structuralproteins (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 inref. [2]). The viral envelope glycoproteins E1 and E2 areusually targeted to the endoplasmic reticulum (ER), where theyare retained in the pre-Golgi compartment instead of beingsecreted [15]. A ribosomal frame-shift into –2/�1 readingframe at or near codon 11 results into a novel HCV protein ofunknown function, F or core � 1 protein, which reacts withsera from HCV-infected patients [16].

A member of the tetraspanin family of proteins, CD81 hasbeen shown to act as a viral receptor, which appears to beessential but not sufficient for viral entry [17]. Its transgenicexpression in mice does not confer susceptibility to HCV

infection [18]. It is expressed on the surface of almost allnucleated cells as a complex with a variety of other cell-surfacereceptors; e.g., it occurs as a complex with CD19 and CD21 onB cells and sends a costimulatory signal to the cells (reviewedin ref. [19]). It has four membrane-spanning domains and twoextracellular loops. The viral glycoprotein E2 binds to themajor extracellular loop of CD81 [20]. However, this may notbe the only viral receptor, as HCV can also bind to severalother molecules: the receptor for low-density lipoprotein, thedendritic cell (DC)-specific intercellular adhesion molecule3-grabbing nonintegrin (DC-SIGN), and its liver counterpart(L-SIGN), a scavenger receptor of class B type 1, asialoglyco-protein receptor [21–24]. E2 is the most variable viral envelopeglycoprotein; therefore, it is not surprising that E2 interactionswith its putative ligands, e.g., CD81, have been reported to bestrain-specific [25]. It has two HVRs (HVR-1 and -2). Muta-tions occur mostly in these regions, probably as a result ofpressure from virus-neutralizing antibodies and HCV-specificcytolytic T lymphocytes (CTL). The HVR-1 variants have beenshown to act as T cell receptor (TCR) antagonists for HCV-specific CD4� and CD8� T cells specific for HVR-1. Theyinduce inhibition of proliferation, cytokine production, andearly signaling events [26]. HCV has a high mutation rate as aresult of a lack of proofreading ability of its RNA-dependentRNA polymerase. Therefore, it exists in several distinct butclosely related virus species within an infected individual.They are called HCV quasispecies. Based on the nucleotidesequences of the conserved and nonconserved regions, thesequasispecies have been grouped into six major genotypes (ge-notypes 1–6) and more than 100 subtypes. Of these, genotypes1a and 1b are prevalent in North America and Western Europeand are relatively resistant to the IFN therapy [1].

THE INDUCTION OF IMMUNE RESPONSEIN LIVER

Liver is an organ interposed between systemic circulation andgastrointestinal tract. It is constantly exposed to numerousantigens and potentially toxic molecules, which we ingest everyday from our food. The internal microenvironment of normalliver is maintained in a relatively “immuno-silent” state. Thisstate is maintained in many ways. For example, activatedCD8� T cells are trapped in the liver, where they undergoapoptosis. The antigen presentation to naıve CD8� T cellswithin this organ is incomplete, as antigen-presenting cells(APC) in liver lack costimulatory molecules. So antigen pre-sentation to these cells also results in tolerance. Furthermore,liver microenvironment biases CD4� T cells toward the Thelper cell type 2 (TH-2) phenotype. The production of inter-leukin (IL)-4 and IL-10 by the liver sinusoidal endothelial cells(LSEC) plays a significant role in this bias. The induction oftolerance and biased TH-2 responses prevent unnecessaryimmune responses to thousands of innocuous antigens to whichliver is constantly exposed from the gastrointestinal tract (re-viewed in ref. [27]). It is noteworthy that in a normal liver,lymphocytes remain in sinusoids and do not infiltrate the liverparenchyma. However, slow blood flow in the sinusoids, thelack of a basement membrane, and the presence of fenestra-

744 Journal of Leukocyte Biology Volume 76, October 2004 http://www.jleukbio.org

tions in their lining endothelium allow blood lymphocytes tointeract directly with hepatic cells, i.e., LSEC, liver-residentmacrophages [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 totrap and induce apoptosis in CD8� T cells, the liver hasbeen dubbed as a “graveyard” for CD8� T cells (ref. [28];reviewed in ref. [2]). The LSEC are constantly exposed tolipopolysaccharides (LPS) from intestines and constitutivelyexpress intercellular adhesion molecule-1 (ICAM-1), -2,and vascular adhesion protein 1 but not costimulatory mol-ecules. They can actively recruit activated CD8� T cellsand NK cells from circulation. The activated CD8� T cellsmay divide a few times but undergo apoptosis as a result ofneglect. However, the situation changes when an individualis infected with hepatotropic viruses. These viruses induceproduction of type I IFN from hepatocytes and other cells inthe liver (e.g., among others, immature plamacytoid DC),which, in turn, induce production of CC chemokine ligand[macrophage-inflammatory protein-1� (MIP-1�)] in KC.This chemokine promotes infiltration of NK cells in virus-infected livers [29]. Molecular interactions between vascu-lar cell adhesion molecule 1 (VCAM-1) on the vascularendothelial cells and very late antigen 4 (VLA-4) on NKcells are also involved in this infiltration [30]. The produc-tion of type I IFN and other cytokines (including IL-12,IL-15, IL-18) from hepatocytes activates NK cells and in-duces IFN-� production from them. The IFN-� produced byNK cells induces expression of CXC chemokine ligand 9[CXCL-9; the monokine-induced by IFN-� (MIG)] andCXCL-10 [the IFN-�-inducible protein-10 (IP-10)] fromLSEC. The LSEC-produced CXCL-9 and CXCL-10 bind toand recruit CC chemokine receptor 5 (CCR-5) and CXCchemokine receptor 3 (CXCR-3)-positive, activated T cellsto the liver. See Table 1 for a summary of the eventsinvolved in the induction of an adaptive immune response toa hepatotropic virus. It is noteworthy that IFN-� producedby NK cells plays a major role in liver infiltration of CD4�and CD8� T cells. It has been shown in animal models thatthe depletion of NK cells before a hepatotropic viral infec-tion leads to inhibition of a virus-specific T cell response aswell as inhibition of liver injury [31]. In addition to acti-vating NK cells, type I IFN also induces expression ofcostimulatory molecules on the liver APC and causes mat-uration of DC. This ensures activation of CD4� and CD8�naıve T cells in the liver. Thus, upon infection, the internalenvironment of the liver becomes poised for mounting animmune response.

Fig. 1. A schematic diagram showing HCV genome, polyprotein, and itscleaved components. S and NS, Structural and nonstructural proteins, respec-tively; HVR, hypervariable region; ISDR, IFN sensitivity-determining region.Not drawn to the scale.

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

TABLE 1. The Molecular and Cellular Events Involved inInducing Immune Response in the Liver

1. The virus induces IFN-�/�, IL-12, IL-18, and IL-15 fromhepatocytes and DC.

2. IFN-�/� induces MIP-1� from KC.3. MIP-1� causes liver infiltration of NK cells.4. IFN-�/�, IL-12, IL-18, and IL-15 activate NK cells.5. NK cells secrete IFN-�.6. IFN-� induces MIG and IP-10 from LSEC.7. MIG and IP-10 recruit activated T cells to liver.8. IFN induces costimulatory molecules on liver APC, which then

activate naıve T cells.

See text for details.

Ahmad and Alvarez NK and NKT cells in HCV-induced hepatitis 745

ANTIVIRAL IMMUNE RESPONSE ANDHCV-INDUCED HEPATITIS

Being a hepatotropic virus, HCV preferentially induces theexpression of antigen processing and IFN-stimulated genes inthe infected livers as determined by genomic analyses of liversof acutely infected chimpanzees [32, 33]. Consequently, HCVinduces a strong humoral and cellular antiviral immunity in thehost. Virus-specific antibodies and CD4� and CD8� T cellscan be demonstrated in the peripheral blood of the infectedpersons. These cells, however, occur at higher frequencies inthe infected liver than in the peripheral blood (ref. [34]; re-viewed in ref. [35]). Various studies have suggested that anti-viral cellular immune responses rather than antibodies areimportant in controlling HCV infection [36–38]. In agreementwith these studies, similar to the normal children, �15% of thechildren with agammaglobulinemia can spontaneously controlHCV infection.

The HCV-specific CD8� and CD4� T cells have beendemonstrated to recognize viral epitopes in the conserved andvariable regions of all structural and nonstructural viral pro-teins. The CD4� TH cells are considered important in main-taining antiviral CD8� CTL responses. The virus-specific CTLkill virus-infected cells. They also contribute to virus controlby noncytolytic mechanisms by secreting cytokines, e.g.,IFN-�, IFN-�/�, and tumor necrosis factor � (TNF-�), whichinduce an antiviral state in host cells. The state makes unin-fected cells resistant to infection and frees or “cures” theinfected ones from the virus by stopping viral replication. Thenoncytolytic mechanisms have been shown to be important incontrolling several different viruses (reviewed in ref. [39]). It isinteresting that IFN-� and not perforin was shown to be im-portant in the control of murine cytomegalovirus (MCMV) inthe livers of mice [40]. Moreover, it was also shown to controlhepatitis B virus (HBV) in the livers of transgenic mice [41,42]. In in vitro studies, IFN-� inhibits amplification of HCVreplicons in Huh-7 liver cells [43]. In humans, the induction ofIFN-�-producing, antiviral CTL corresponds with the success-ful clearance of the HCV infection [44]. Furthermore, thedegree of viremia correlates inversely with the expression ofIFN-� in the livers of HCV-infected persons, suggesting thatthe IFN-�-induced antiviral state may be important in CTL-mediated control of HCV replication in human liver [45]. Theprogression of the majority of the infected persons to chronicinfection suggests the inability of the antiviral immunity tocontain this infection. There may be several reasons for thisfailure, including emergence of escape variants as a result of ahigh rate of virus mutations, a decreased production of antiviralcytokines or “stunning” of HCV-specific CTL, a compromisedcytolytic potential of the CTL, and antagonistic peptides. Con-sequently, the antiviral immune response may be causing moreliver damage in these individuals.

The HCV-induced immune response brings about profoundchanges in the liver microenvironment. It may be important incontrolling HCV replication; however, it is not without a price.The HCV-induced hepatitis is mediated by the antiviral cel-lular immune response. Although HCV infects and replicatesin the cytoplasm of hepatocytes and possibly of B cells, thevirus per se does not seem to be cytopathic. The viral genotype

as well as the viral load in the infected persons do not correlatewith the degree of liver disease [46]. Data from chimpanzeesand mouse models of viral hepatitis have suggested the in-volvement of virus-specific CTL and their cytokines (e.g.,TNF-�) in this hepatitis (ref. [47]; reviewed in refs. [34, 35]).It has been demonstrated in animal models that high-affinitypeptides trigger activation and migration of CTL to liver, wherethey undergo apoptosis and cause liver damage [48]. CTL havealso been implicated in liver damage in HBV infection; theinfusion of virus-specific CTL in transgenic HBV mice causesnecroinflammatory hepatitis (reviewed in ref. [49]). It is inter-esting that the HCV-specific CTL can also kill uninfectedhepatocytes in vitro [50, 51]. The molecular mechanism(s)underlying this potentially pathogenic, autoreactive activity ofCTL in HCV-infected individuals are not known. At least inpart, it may be caused by Fas–FasL interactions. In vitrostudies show that a few HCV-infected cells can provoke a fewHCV-specific CTL to mediate killing of many bystander, un-infected hepatocytes [52]. The importance of Fas–FasL inter-actions in inducing liver damage was also demonstrated in amouse model of hepatitis, in which the use of antisense oligo-nucleotides that inhibited Fas expression on hepatocytes alsoinhibited liver damage [53]. It is noteworthy that Fas is con-stitutively expressed on hepatocytes in mice. This expression isweak on human heptocytes under physiological conditions butis readily induced by proinflammatory cytokines. More impor-tantly, hepatocytes can also express FasL under inflammatoryconditions and cause their own death. It may be relevant tomention that HCV core protein induces FasL expression inhepatoblastoma cells [54]. The antiviral CTL may also causeliver damage indirectly via cytokines, e.g., TNF-�. The intra-hepatic T cells from the individuals with chronic HCV infec-tion produce almost 50 times more TNF-� than the ones whocontrol this infection [37]. Furthermore, TNF-related apopto-sis-inducing ligand (TRAIL) kills hepatocytes from virus-in-fected, inflamed livers via death receptors (DR)-4 and DR-5but not from healthy ones [55]. Yet, other evidence for theimmune-mediated liver damage comes from HAART-treatedHCV and HIV-1-coinfected persons. HAART restores anti-HCV immunity in these patients and consequently causeshepatotoxicity [14].

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

It is noteworthy that NK cells and NK T (NKT) cells areenriched in the liver. They could potentially play a role in HCVcontrol and the pathogenesis of HCV-induced hepatitis. In thelast decade, a great deal has been learned about the molecularstructures that are used by these cells to recognize ligands ontarget cells. Consequently, we are better able to appreciate therole of these cells in host defense against viral infections. Thisknowledge has also enabled us to understand many aspects ofvirus-specific T cells that may be involved in the HCV-inducedpathogenesis. Therefore, we will give a brief account of NK andNKT cell biology and review our current knowledge about NKcell receptors (NKR) and their ligands.

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 frompathogens and malignancy [56–60]. They are capable of spon-taneously killing tumor and virus-infected cells, which havedown-regulated one or more major histocompatibility complex(MHC) molecules and/or expressed certain stress antigens ontheir surface. They kill target cells without MHC restrictionand prior activation. By killing virus-infected cells and causingthe release of proinflammatory substances, e.g., heat shockproteins and TNF-�, NK cells provide the necessary “dangersignal” to the immune system for inducing virus-specific im-munity [61, 62]. NK cells secrete several cytokines and che-mokines, e.g., IFN-�, TNF-�, granulocyte macrophage-colonystimulating factor (GM-CSF), IL-5, IL-13, IL-10, TGF-�, MIP-1�, MIP-1�, and regulated on activation, normal T expressedand secreted (RANTES). The production of these cytokinesand chemokines is important in initiating an inflammatoryresponse and in determining the nature and strength of theensuing pathogen-specific immunity. NK cells represent a ma-jor source of IFN-� other than activated T cells. An immediateproduction of this cytokine from NK cells is a crucial factor ininducing effective antiviral, cellular immunity in the host. Inaddition to directly killing virus-infected cells by releasingcytotoxic molecules such as perforin and granzymes, NK cellsalso kill target cells by Fas/FasL, TNF-�, and TRAIL/DR-4and DR-5 interactions [63–65]. The NK cell-secreted, solublefactors such as IFN-� and TNF-� also play a role in inhibitingvirus replication by inducing an antiviral state in host cells[39]. Furthermore, NK cells play important immunoregulatoryroles by interacting with T and B cells and APC.

In vivo studies in animal models have demonstrated thatdepletion of NK cells may result in the inability of the host tocontrol viral infections. NK cell-deficient persons experiencerepeated recurrences of herpesvirus infections [58, 59]. Theinfected host usually responds to a viral infection by enhancedNK cell activity. Viruses may directly activate NK cells byencoding a viral protein that is recognized by an activatingreceptor on the host NK cells; e.g., MCMV encodes a viralprotein m157, which binds and activates LY49H� NK cells inMCMV-resistant mice, and influenza virus encodes haemag-glutinin, which binds to NKp46 and NKp44 on human NKcells (see below for details on NKR). Viruses can also activateNK cells indirectly by inducing expression of stress-inducibleproteins or cytokines in the host. The stress-inducible proteinsactivate NKG2D-bearing NK and other cells. The virus-in-duced cytokines that activate NK cells include type 1 IFN (orIFN-� and -�), IL-15, IL-18, IL-12, and IL-21 [59, 66–68].These cytokines positively affect different aspects of NK cellactivation, proliferation, and survival [69].

The activity of NK cells is regulated by so-called NKR,which are a variety of molecular structures that are expressedon the surface of NK cells and bind specific ligands on targetcells. The NKR are of inhibitory or of stimulatory types, andtheir triggering sends inhibitory or stimulatory signals to NKcells, respectively. Individual NK cells express inhibitory andstimulatory NKR and may kill or spare a target cell depending

on the balance between inhibitory and stimulatory signals thatit receives from the target cell via NKR. In humans, the NKRbelong to four groups, which are briefly described here.

Killer cell Ig-like receptor (KIR) family

KIR are type I integral membrane glycoproteins that are usu-ally expressed as monomers on the cell surface [70, 71]. Atpresent, more than 12 KIR genes have been discovered (Table2). Diversity in these receptors is further enhanced as a resultof gene polymorphism and alternate splicing. For example, theKIR2DL1 gene has at least eight alleles. The KIR may haveshort or long cytoplasmic tails (Fig. 3). The ones with longcytoplasmic tails have two ITIMs and are inhibitory in func-tion. The receptors with a short cytoplasmic tail possess acharged aa in their transmembrane domains and associatenoncovalently with a dimer of an adaptor protein, KARAP/DAP-12 [72]. The adaptor protein has ITAMs in its cytoplas-mic tail. Upon binding to the MHC ligand, these receptors sendactivating signals to NK cells to kill target cells and secretecytokines. As depicted in Figure 3, KIR have been divided intofour major groups: KIR2DS, KIR2DL, KIR3DL, and KIR3DS.This grouping is based on the number of Ig domains in theextracellular parts (2D for two domains and 3D for threedomains) and the length of cytoplasmic tails (L for long and Sfor short tails). The receptors are further numbered differentlyif they show more than 2% sequence divergence within thegroup. Each KIR recognizes shared determinants present in aset of related MHC class I alleles on target cells. Major humanKIR and their ligands are shown in Table 2.

NKG2/CD94 receptors

These are type II C-type, lectin-like, integral membrane gly-coproteins (Table 2; Fig. 3). They are expressed on the cellsurface as heterodimers with CD94, which is an invariant typeII C-type, lectin-like polypeptide. CD94 lacks a cytoplasmictail and therefore, cannot transduce signals. It is howeveressential for the expression of NKG2 receptors. Four distinctgenes, A/B, C, E/H, and F, encode the NKG2 receptors [70, 71,73]. Of these receptors, CD94/NKG2A is an inhibitory one, asit contains a long cytoplasmic tail with two ITIMs. Others haveshort cytoplasmic tails, and each associates noncovalently witha homodimer of DAP-12, as in the case of activating KIRs [72].NKG2 receptors bind HLA-E [74–76], whose expression re-quires peptides derived from signal sequences of HLA-A, -B,-C, and -G [77]. If the expression of these MHC antigensdecreases in a cell, it will also result in a decreased expressionof HLA-E. Thus, NK cells have developed a clever strategy ofmonitoring the overall expression of MHC class I antigens ontarget cells by simply monitoring the expression of the HLA-Eantigen 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-mediatedfunctions upon their engagement. NKp46 and NKp30 are ex-pressed on resting and activated NK cells, whereas NKp44 isexpressed on cytokine-activated NK cells (reviewed in refs.[78, 79]). NKp46 bind the sialic acid-binding glycoproteins,

e.g., haemagglutinin and haemagglutinin-neuraminidase of theinfluenza and parainfluenza viruses, respectively [80]. Theligands for other NCR are not yet known.

NKG2D receptors

The NKG2D differs from other members of the NKG2 family insignificant ways. They do not form heterodimers with CD94 onthe cell surface (Fig. 3). Instead, they are expressed as ho-modimers, and each homodimer associates noncovalently witha homodimer of the adaptor protein DAP-10. The cytoplasmictail of DAP-10 carries a YxxM motif (similar to the one presentin the cytoplasmic tail of the costimulatory molecule CD28),which can recruit the regulatory subunit p85 of phosphatidyl-inositol-3 kinase and Grb2. NKG2D exist in two isoforms,which differ from each other in the length of their cytoplasmictails by about one dozen aa. The shorter-tailed (S) form canassociate with DAP-10 and DAP-12, whereas the longer-tailed(L) form mediates signals only via DAP-12 [81, 82]. RestingNK cells express only the L form of the receptor. However, theyalso express its S form upon activation. The same is true forother immune cells, e.g., �� TCR� T cells and macrophages.Thus, activated immune cells can receive triggering signalsand costimulatory signals via NKG2D, whose receptors recog-nize and bind stress-inducible proteins, ULBP1-4, MICA, andMICB [83, 84]. In mouse, NKG2D bind H-60 (a minor histo-compatibilty antigen) and the retinoic acid early inducibleprotein (and to a murine, ULBP-like transcript; reviewed in[82]). The NKG2D-specific ligands are usually expressed littlein normal tissues but are induced on host cells by stress,transformation, and viral infections [85–87].

In addition to the above-mentioned receptors, NK cells alsoexpress 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 receptorfor IgG. In conjunction with antigen-specific antibodies, NKcells can kill virus-infected cells, which express the antigen.This phenomenon is called antibody-dependent cellular cyto-toxicity and may play a role in controlling virus replication inthe host [88]. CD56 is an isoform of the neural cell-adhesionmolecule and is involved in homotypic intercellular adhesions.About 10% of the peripheral blood NK cells express highlevels of CD56 and low levels of CD16 on their surface, and thereverse is true for the remaining 90% of the NK cells [89–91].The CD56� NK cells are less cytotoxic as compared with theCD16� ones. The coengagement of NK cell coreceptors bycognate ligands on target cells adds to the strength of theoverall stimulating signal. In the absence of inhibitory signals,their coengagement may be sufficient to cause lysis of thetarget cells.

REGULATION OF NK CELL FUNCTIONSBY NKR

The functional activity of NK cells is regulated by inhibitoryand activating receptors. The MHC class I-binding receptors,KIR are expressed clonally on overlapping subsets of NK cellsand function independently of each other. An individual NKcell may express two to nine receptors (average, five) [92–94].These receptors are usually of inhibitory and stimulatory types.Under normal conditions, each NK cell in an individual ex-presses at least one inhibitory receptor capable of binding to aself-MHC class I antigen. This makes all NK cells of theindividual self-tolerant. The existence of MHC class I- andHLA-E-binding, inhibitory NKR and their dominant role overtheir activatory counterparts explain why NK cells do not kill

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

Receptor Function Signaling partner Distribution Ligand

1. NKp46 � FcεR1�, CD3� chains All NK cells HA2. NKp44 � - - - - - - - - - - - - - - - - - - - - ?3. NKp30 � - - - - - - - - - - ANK ?4. NKG2D � DAP-10/12 NK, CTL (C) MICA, MICB, ULBP5. KIR2DS1 � DAP-12 NK, CTL (C) HLA-C Lys.p80G2

6. KIR2DS2 � - - - - - - - - - - - - - - - - - - - - HLA-C Asn.p80G1

7. KIR2DS4 � - - - - - - - - - - - - - - - - - - - - HLA-C?8. KIR2DS3, 5 � - - - - - - - - - - - - - - - - - - - - ?9. KIR2DS6 � - - - - - - - - - - - - - - - - - - - - ?

10. KIR3DS1 � - - - - - - - - - - - - - - - - - - - - ?11. KIR2DL1 – SHP-1, 2 NK, CTL (C) HLA-C Lys.p80G2

12. KIR2DL2/3 – - - - - - - - - - - - - - - - - - - - - HLA-C Asn.p80G1

13. KIR3DL1 – - - - - - - - - - - - - - - - - - - - - HLA-B Bw4.Ile.p8014. KIR3DL2 – - - - - - - - - - - - - - - - - - - - - HLA-A3, ?15. KIR2DL4* � ? - - - - - - - - - - HLA-G16. KIR2DL5 – SHP-1, 2 - - - - - - - - - - ?17. KIR2DL7 – - - - - - - - - - - - - - - - - - - - - ?18. NKG2A – - - - - - - - - - - NK, CTL (C) HLA-E19. NKG2C, E � DAP-12 - - - - - - - - - - - - - - - - - - - -

FcεR1�, gamma chain of the high affinity receptor for Fc region of IgE; HA, haemagglutinin; ANK, activated NK cells; DAP, DNAX accessory protein; MICA,MICB, MHC class I chain-related A, B; ULBP, UL16-binding protein; HLA, human leukocyte antigen; Lys, lysine; Asn, asparagine; SHP-1, 2, Src homology-2-containing tyrosine phosphatase 1, 2; Ile, isoleucine. *KIR2DL4 has a charged residue in its transmembrane region and functions as an activating receptor. �,Activating; –, Inhibitory function. (C), A clonal and variegated distribution. Dashed lines, same as above. The superscripts G1 and G2, Group 1 HLA-C (Cw1, -3,-7, -8 and related) and group 2 HLA-C (Cw2, -4, -5, -6 and related) alleles, respectively.

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 pro-posal for the missing self-hypothesis for explaining NK cell-

mediated killing. It is noteworthy that a common strategy usedby viruses is to down-regulate the expression of MHC class Iantigens on the surface of infected cells to evade antiviral CTLresponses. This makes the infected cells susceptible to killingby NK cells (reviewed in refs. [97, 98]). Although KIR-medi-ated, inhibitory signals are dominant over stimulatory signals,they are unable to inhibit NKG2D-triggered NK cell cytotox-icity. This means that NK cells will kill the target cells, whichexpress stress-inducible ligands, even if they express normallevels of MHC antigens. If such target cells further down-regulate their expression of MHC antigens, they would becomesuper-susceptible to NK cell-mediated killing.

The rules governing the expression of NKR on NK cells arenot fully known. It has been observed that each individual NKcells rarely expresses inhibitory receptors to more than oneself-MHC class I antigens. This enables individual NK cell tosense a decrease in even single MHC antigens. Studies con-ducted in mice suggest that the functional orthologs of KIR,Ly49, are acquired by developing NK cells in a stochastic andcumulative manner (reviewed in refs. [99, 100]). Certain cyto-kines may also modulate their expression. For example, IL-15,IL-10, and TGF-�1 induce expression of CD94/NK2GA on NKand CD8� T cells. IL-15 also induces expression of NKG2Dreceptors on NK and CTL. IL-21 increases the expression ofNCR on developing NK cells [101]. Viral infections, e.g.,lymphocytic choriomeningitis virus, may also induce expres-sion of inhibitory receptors on CTL [102]. A deregulated ex-pression of NKR has important implications for the functionalactivity of NK cells; it may lead to the emergence of autoim-mune NK cell clones if inhibitory receptors on NK cells arereduced and/or activating receptors are overexpressed. NKcells may also become immunodeficient if inhibitory receptorsto MHC antigens are overexpressed on them. The autoimmunecells may kill normal autologous cells, whereas the immuno-deficient ones may not be able to kill otherwise susceptible,malignant or virus-infected cells. Transgenic expression of theLy49A, an inhibitory NKR, impaired antiviral cellular re-sponses in mice [103]. In addition to changes in the expressionof NKR, changes in the expression of MHC class I antigens orother ligands for NKR and coreceptors may also affect thesusceptibility of target cells to NK cell-mediated lysis. Forexample, stress, transformation, or viral infections induce the

Fig. 3. A schematic representation of the structure of major NKR. N and C,N and C termini of the receptors. � and �, Respective chains of the CD3 andFcεR1. ITAM, Immunoreceptor tyrosine-based activating motif; ITIM, immu-noreceptor tyrosine-based inhibitory motif. Not drawn to the scale.

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

Coreceptor Ligand Expression Function

1. LFA-1*,c ICAM-1 All NK cells Costimulation2. LFA-2*,c LFA-3, CD48 - - - - - - - - - - - - - - - - - - - -3. CD8 MHC class I Subset - - - - - - - - - -4. CD69 Unknown ANK - - - - - - - - - -5. CD56 Self Subset Homotypic adhesion6. CD16* IgG, IgE Most cells Activation and triggering7. 2B4*,p CD48 All NK cells Activation or inhibition8. NTB-A*,p ? - - - - - - - - - - - - - - - - - - - -9. NKR-P1a Ocil - - - - - - - - - - Costimulation or inhibition

10. DNAM CD155, CD112 - - - - - - - - - - Costimulation

LFA, Lymphocyte function antigen; Ocil, osteoclast inhibitory lectin; DNAM, DNAX accessory molecule. *The coreceptor may also trigger NK cells if engagedsufficiently; cInvolvement of the coreceptor in conjugate formation with target cells; pThe function is dependent upon the availability of adaptor molecules; aThefunction is dependent on the allele. None of these molecules is exclusively expressed on NK cells. A dashed line indicates same as above.

expression of MICA, MICB, and ULBP on host cells and makethem susceptible to NK cell-mediated killing via NKG2D ([85,86]; reviewed in ref. [103]).

NKT AND NATURAL T (NT) CELLS

NKT cells represent a heterogeneous group of immunoregula-tory and effector cells, which express both NK and T cellmarkers (reviewed in refs. [105, 106]). NKT and conventionalT cells arise from common CD4�CD8� precursor lympho-cytes within the thymus. The classical NKT cells are NK1.1�(CD161 or NKR-P1) and CD3� CD56�/ and are of theCD4�CD8– or CD4–CD8– phenotype. The expression of themarker NK1.1 is not essential for NKT cells, as the mice thatdo not express this marker [e.g., BALB/c, nonobese diabetic(NOD), and SJL] also contain these cells. NKT cells may alsoexpress other NK cell markers, e.g., CD94, Ly49, or KIR (inhumans). They express a very restricted repertoire of TCR(usually a monoclonal TCR that comprises the invariant V�24-J�18 chain in association with a limited repertoire of V�11 or-8 genes in humans). The mouse classical NKT cells expressan invariant TCR � chain V14�-J�128 in association with arestricted number of polyclonal V� genes (V�8, V�7, or V�2).A common feature of these NKT cells is their positive selectionby CD1d, nonpolymorphic MHC class Ib molecules, which areexpressed on the cell surface with �2-microglobulin (�2M) butare transporter associated with antigen processing (TAP)-inde-pendent. The CD1d bind lipid antigens. The natural ligands forclassical NKT cells are not known. In vitro, they are activatedby �-galactosylceramide (GalCer), a natural anticancer glyco-lipid obtained from a sea sponge. NKT cells produce IL-4 andIFN-� abundantly upon their stimulation. However, they canalso produce GM-CSF, IL-5, IL-13, RANTES, IL-8, IL-10, andTGF-�1.

The classical NKT cells are abundant in liver and thymus.Murine liver is particularly enriched for these cells: �50% ofthe intrahepatic lymphocytes (IHL) are NKT cells. They residein sinusoids and play an essential role in preventing livermetastasis and in clearing a viral infection. It was demon-strated in the HBV transgenic mouse model that activation ofNKT by a single injection of GalCer induced IFN-� produc-tion, which was sufficient in controlling viral replication non-cytopathically without the need of further lymphocyte infiltra-tion. It is interesting that NKT and NK cells and not CD4 orCD8� T cells were involved in this viral control in liver [41,42]. The NKT cells were also shown to mediate antitumoreffects of IL-12 in mice [107]. However, NKT cells may alsocause liver damage, as demonstrated in murine hepatitis mod-els induced by concanavalin A or LPS plus IL-12. NKT cellsdisappear from livers following their activation, probably as aresult of apoptosis. The newly emerging NKT cells are biasedand produce only IL-4 upon restimulation. Because of theirability to produce large quantities of IL-4, it was initiallythought that NKT cells might be essential for inducing TH-2immune responses. However, the experimental induction ofthese types of responses in CD1d/ and �2M/ mice(which lack NKT cells) shows that these cells are not abso-lutely 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 migrationand 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, andrheumatoid arthritis (reviewed in ref. [110]). They are lessfrequent and are defective in IL-4 production in NOD mice andin individuals that are at risk for type I diabetes (T1D). It hasbeen demonstrated that in vivo activation of NKT cells byGalCer protects NOD from the onset of T1D and prolongssurvival of pancreatic islets transplanted into newly diabeticmice. These effects are a result of induction of TH-2 responsesin spleens and pancreas of these mice, which prevent B and Tcell-mediated autoimmunity to islet B cells. IL-7 acts syner-gistically with GalCer in this protection by inducing enhancedproduction of IL-4 and IL-10 from NKT cells (refs. [111, 112];reviewed in ref. [110]).

Nonclassical NK1.1� CD8� NKT cells have variant TCRand are not restricted by CD1d. The spleen and BM are themain sites where these cells reside. Intestinal, intraepitheliallymphocytes usually contain CD8�� NKT cells. In humans,nonclassical NKT cells may express variant �� or �� TCR,CD161, and may be restricted by CD1a, -b, or -c. They produceIFN-� and not IL-4. It is noteworthy that CD1a, -b, and -c arenot expressed in mice.

The conventional CD8� T cells with variant �� TCR ac-quire NK1.1 on activation. They may also express other inhib-itory NKR. The cells expressing inhibitory KIR usually repre-sent oligo- and monoclonally expanded antigen-specific CD8�T cells of the effector memory phenotype [113, 114]. They areusually expanded in chronic viral infections [115]. The expres-sion of KIR promotes their survival by preventing them fromundergoing activation-induced cell death. It also increasestheir antigen threshold and prevents them from their effectorfunctions. It has been demonstrated in vitro and in vivo thatviral 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-likecytolytic activities in these CD3�CD56� NKT cells. Theyhave also been named as NT cells. The distinction between NTand nonclassical NKT cells is not very clear, and many authorshave used them interchangeably.

NKR AS REGULATORS OF ANTIVIRAL ANDANTITUMOR IMMUNITY

In recent years, it has become quite clear that NKR play animportant role in regulating immune responses and effectorfunctions of NK cells and NKT cells [71]. We and others haveshown 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 monoclo-nal antibodies (mAb) markedly increases the cytotoxic func-tions of the HIV-specific CTL from these patients againstHIV-infected cells in in vitro assays [117]. Data from animalmodels have shown that in vivo blockage of these receptors also

enhances antitumor as well as antiviral cytolytic activities ofNK and CTL [118]. Furthermore, they have opened novelavenues of increasing the efficacy of antiviral and antitumorvaccinations; e.g., in vivo expression of NKR ligands such asMICA, MICB, or ULBP may be used to activate NK and CTL,and the administration of GalCer as an adjuvant may increaseantiviral immunity induced by a vaccine. At least in theory, theblocking of inhibitory NKR in vivo may augment antiviralactivity 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 pro-teins act as ligands for the NKG2D receptor, which is ex-pressed on the NK cells, �� receptor-positive T cells, and theCD28– subset of CD8� T cells [87, 119]. The CD28– CTL ofthe memory phenotype as well as �� TCR� T cells occur inunusually high frequencies in the peripheral blood of individ-uals that are suffering from chronic virus infections [120, 121].Certain cytokines, e.g., IL-15, are also known to induce theexpression of NKG2D on antigen-specific CTL and convertthem into lymphokine-activated killer cells, which can kill notonly virus-infected, antigen-expressing target cells but alsouninfected, “stressed” host cells [122]. An induced expressionof NKG2D ligands on host cells may make them susceptible tokilling by the NKG2D receptor-positive T and NK cells. Thistype of lysis may cause tissue damage and contribute towardpathogenesis of the infection, as these ligands are also inducedon uninfected host cells as a result of stress and inflammatorymediators [85, 87].

LIVER NK AND NKT CELLS

A normal human liver contains lymphocytes that are usuallyenriched for NK and NKT cells. In contrast to the peripheralblood 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 mayincrease to 90% in hepatic diseases [2]. The liver NK cells arethe 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 NKcells express this molecule. The human liver NKT cells com-prise mostly CD3�CD56� cells with variant TCR. They alsoexpress other NKR, e.g., CD94/NKG2 and KIR, and mayexpress low levels of �� or �� TCR. Liver NK and NKT cellsproduce cytokines and chemokines and can mediate MHC-unrestricted killing of target cells with or without stimulationwith cytokines, e.g., IL-2 [2]. NKT cells can also produceIFN-�, TNF-�, IL-2, and/or IL-4 upon stimulation; however,NK cells produce IFN-� and TNF-�. Some NKT (5–6%)produced IFN-� and IL-4 simultaneously [124].

Unlike mouse liver, human livers contain few classical NKTcells with invariant TCR. Instead, they are enriched forCD3�CD56� nonclassical NKT cells [125]. In healthy adulthumans, 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 �� or �� TCR. They may represent afunctional counterpart of NKT cells in human livers. However,they mainly produce IFN-� and not IL-4 upon their stimula-

tion. They are restricted by CD1d and are TH-1-biased inHCV-infected livers [125].

The human classical NKT (V�24/V�11) can be readilydetected in livers of HCV-infected persons; however, theirfrequencies are decreased as compared with healthy persons.A similar situation has been described in the circulation ofHIV-infected persons [65, 126]. Human classical NKT cellsexpress high levels of CCR-5 and CXCR-6 and are susceptibleto infection with monocytotropic or R5 strains of HIV [126].Their depletion in HIV-infected persons may be caused, atleast in part, by direct infection of these cells by HIV. We donot know whether these cells per se can be infected with HCVor whether they are depleted by other mechanisms, e.g., apo-ptosis, or migration to other compartments. Their depletionmay predispose infected persons to autoimmune conditions aswell as skew the liver microenvironment toward TH-1 cyto-kines.

NK CELLS IN HCV INFECTION

As stated earlier, infections with hepatotropic viruses activateliver NK cells, which play a critical role in the recruitment ofT cells to liver. Activated NK cells can kill virus-infected cellsvia perforin/granzyme, and FasL pathways produce proinflam-matory cytokines, which can induce antiviral state host cells.Human NK cells cocultured with HCV replicon-containinghepatic cells in Transwell microplates inhibit the repliconexpression at protein and RNA levels by secreting antiviralfactors including IFN-� [127]. Thus, NK cells could alsopotentially contribute toward HCV control. It would not besurprising that in the face of an adequate NK cell response,hepatotropic viruses such as HCV may be controlled even inthe absence of virus-specific immune responses. This notion issupported by the observations that as in humans, a certainpercentage of HCV-infected chimpanzees can clear infectionspontaneously, and this clearance does not correlate with theappearance of acquired immunity [128]. However, the potentialimportance of NK cells in the control of HCV was discountedin a study that demonstrated that depletion of CD8� T cells bymAb aggravates HCV infections in animal models [129]. It isnoteworthy that about one-third of human and chimpanzee NKcells express CD8. The depletion of CD8� T cells by anti-CD8antibodies would also deplete CD8� NK cells. Therefore, theresults from such studies should be interpreted with a caveat.The very fact that HCV has developed multiple strategies toevade the host’s NK cell response testifies to the importance ofthis arm of innate immunity in controlling this infection. Par-adoxically, NK cells, too, act as a double-edged sword andmight contribute toward pathogenesis and cause liver damageby killing hepatocytes and by secreting proinflammatory cyto-kines.

HOW HCV EVADES HOST’S NK CELLRESPONSES

HCV seems to have evolved several mechanisms to evade thehost’s NK cell response (summarized in Table 4). One of the

strategies is to inhibit production of type 1 IFN from the hostcells in response to the infection. As stated above, this cytokineprotects the host in multiple ways, e.g., by activating NK cells,inducing DC maturation, and inhibiting viral replication. Italso suppresses translation from the viral IRES [130]. The viralserine protease (NS3/NS4A complex) inhibits the IRF-3 acti-vation [131]. This factor is known to be essential for virus-induced production of type I IFN. Dominant-negative IRF-3mutants enhance, and constitutively active ones suppress HCVRNA replication in hepatoma cells. Moreover, the viral glyco-protein E2 and the nonstructural viral protein NS5A can bindand inactivate PKR (refs. [132–134]; reviewed in ref. [135]);PKR is a kinase that plays an important role in the productionof type 1 IFNs from host cells by RNA viruses. Its activity isalso essential for the IFN-induced host resistance to viralreplication. The nonstructural HCV protein NS5A contains aregion (aa 2209–2248), the ISDR. NS5A from IFN-resistantgenotypes 1a and 1b can physically bind and inhibit PKR in anISDR-dependent manner. The binding of NS5A to the kinaseprevents the latter’s dimerization and activation (ref. [134];reviewed in ref. [135]). Recent studies have shown that ISDRis necessary but not sufficient to inactivate PKR; an additional26 residues on its c-terminus are also needed to form complexwith the kinase [134]. ISDR from IFN-sensitive HCV strains donot bind with PKR. The viral glycoprotein E2 also contains a12-aa sequence that is identical to the phosphorylation sites ofthe PKR in its target substrate, the translation initiation factoreIF2� [133]. By virtue of this sequence, E2 blocks kinaseactivity of PKR and consequently, its inhibitory effect onprotein synthesis and cell growth. The E2 sequences of theIFN-resistant HCV genotypes 1a, 1b, 4, 5, and 6 more closelyresemble the phosphorylation site of PKR than the correspond-ing sequences from less resistant 2a, 2b, and 3 genotypes.Thus, these effects correlate with the relative resistance of thevirus to IFN-�. The inhibition of PKR by viral proteins freeshost cells from its growth inhibitory effects, which favor cellgrowth and the development of HCC. Furthermore, the viralcore protein induces the expression of the SOCS-3 protein[122], which is known to abrogate the IFN-induced signaling inhuman cells. It also mediates inhibitory effects of IL-10 onLPS-mediated activation of macrophages, and interferes withactivation of Janus tyrosine kinase–signal transducer and ac-tivator of transcription signaling mediated by gp130 cytokines,leptin, growth hormone, and prolactin (refs. [133, 136]; re-

viewed in ref. [137]). More importantly, SOCS-3 negativelyregulates insulin-mediated signaling, and therefore, its chronicexpression may promote type II diabetes [138].

HCV has also developed the strategy of directly inhibitingNK cell responses; the viral E2 glycoprotein binds to CD81 onNK cells and inhibits their effector functions [17, 139]. It isinteresting that CD81 also is expressed on the surface of CTL,but E2 does not inhibit the function of these cells, it ratherstimulates them [140]. As E2 is highly variable, and not allHCV strains possess E2 that can bind CD81, it will be inter-esting to know whether the HCV strains that are spontaneouslyeliminated by some infected individuals have E2 that caninhibit NK cell functions. Another mechanism by which HCVevades NK cell responses is by stabilizing the expression ofMHC class I molecules on the surface of the infected cells.Viruses, in general, tend to decrease the expression of MHCclass I molecules on the surface of infected cells. This helpsthem escape antiviral CTL responses. However, HCV does notdecrease the expression of these antigens on the surface ofinfected cells; it rather increases it [141, 142]. The viral coreprotein plays a role in this increased expression. It enhancesDNA binding affinity and transcriptional activity of p53, with-out affecting its mRNA or protein levels. The activated p53, inturn, activates the transporter associated with antigen proce-cessing-1 or TAP-1 [143, 144]. The activated TAP-1 increasesthe available pool of peptides that bind MHC class I moleculesin the lumen of the ER. The resulting increased expression ofMHC antigens on the surface of HCV-infected cells may en-hance their resistance to their killing by NK cells. It is note-worthy that increased MHC expression on hepatocytes nega-tively correlates with susceptibility to IFN-� treatment [142].The mature DC derived in vitro from monocytes activate NKcells [145]. The DC-activated NK cells express CD69, produceIFN-�, and efficiently kill K562 cells. The DC matured by LPSactivated NK cells via IL-12, whereas those activated by IFN-�activated NK cells via MICA and MICB. The DC recoveredfrom the monocytes of HCV-infected patients did not activateNK cells, as IFN-� was unable to induce expression of MICAand MICB on these DC [145]. This suggests that DC may beunresponsive to IFN-� in these patients. It is interesting thatthe IFN-� receptor is also down-regulated in livers in patientsthat are chronically infected with HCV, and the efficacy of theIFN treatment is related to the expression of the receptormRNA in the livers of the patients [146].

Several researchers have reported that NK cell activitydecreases in HCV-infected individuals [147, 148]. The de-crease probably depends on the stage of the disease. Although,CD3–CD56� NK cells are enriched in liver, their numberdecreases with disease progression, and this may predisposethis organ to malignancy [149]. An increased expression of theMHC class I antigens (A, B, or C) in HCV-infected cells maymake them resistant to NK cell-mediated lysis. Apart fromincreasing the expression of MHC class I antigens, HCV alsoseems to enhance the expression of stress-inducible proteins,e.g., MICA. Increased expressions of these proteins on hepa-tocytes have been reported in HCC individuals [150]. Asmentioned above, these proteins act as ligands for NKG2Dreceptors, which are present on NK cells and activated CTL.Thus, heptocytes from HCV-infected livers may be predis-

TABLE 4. List of HCV-Adopted Strategies to Counter and Evadethe Host’s NK Cell Responses

1. E2 binds CD81 on NK cells and inhibits their activation.2. Core stabilizes expression of MHC class I on infected cells and

makes them resistant to NK cells.3. E2 and core bind to and inhibit activation of PKR.4. Core induces SOCS-3, which inhibits IFN-mediated signaling.5. The virus reduces the expression of receptor for type I IFN.6. The virus dysregulates the expression of NK cell-activating

cytokines.7. The viral protease inhibits activation of IRF, which is required

for IFN induction.

See text for details. PKR, double stranded RNA-dependent protein kinase;SOCS-3, suppressor of cytokine signaling-3; IRF, IFN regulatory factor.

posed to killing by NK cells and CTL even if they are notinfected by HCV. This may also explain, at least in part, theCTL-mediated killing of uninfected bystander hepatocytes inHCV-infected individuals.

Furthermore, like other chronic infections, NK cells andCD3�CD56� NKT cells from the livers of HCV-infectedindividuals express a dysregulated expression of KIR [151].The expression of inhibitory receptors decreases on these cells,which may render them autoreactive and promote killing ofhepatocytes and other bystander host cells. It still remains tobe determined how the expression of NKG2D and other acti-vating NKR, e.g., NCR, are regulated in HCV-infected livers.It is noteworthy that HCV-specific CTL have been well docu-mented 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-� 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 IFNtherapy in HCV-infected individuals correlated to their in-crease in NK activity. In the individuals in whom the therapyfailed to increase NK cell response, no decrease in viremia wasobserved [142, 152, 153]. As stated earlier, HCV has evolveda strategy to counter the IFN-mediated host response by en-coding a core protein that can induce SOCS-3 protein [154].Certain other cytokines, e.g., IL-1�, can also attenuate theeffects of IFN on host cells, and there is an abundance of thiscytokine in the circulation of chronic HCV-infected patients[155]. Thus, the host’s NK cell response could play a role insuppressing HCV replication; however, the virus has evolvedstrategies to counter this response. In the presence of theseviral strategies, this host response may be contributing towardpathogenesis of HCV-induced hepatitis by killing uninfectedhepatocytes.

A UNIFIED HYPOTHESIS FOR THE ROLE OFNK CELLS AND ANTIVIRAL CELLULARIMMUNE RESPONSE IN HCV-INDUCEDHEPATITIS

The available data on innate and adaptive immune responsesin HCV-infected patients point to a unified hypothesis ofHCV-induced hepatitis. According to this hypothesis, HCVinfection changes the relatively immuno-silent status of normalliver and induces adaptive and innate immune responses in theorgan. The anti-inflammatory TH-2 cytokine milieu of the liveris skewed toward a highly proinflammatory TH-1. This isfurther aggravated by a selective depletion of NKT cells.Although NK cells and CTL may be important in clearing viralinfection, they also cause liver destruction by a variety ofmechanisms. They may be rather killing uninfected but in-flamed hepatocytes, as the viral strategies may prevent destruc-tion of the infected cells. It is commonly believed that anantiviral immune response in chronically infected individualsis too weak to eradicate HCV but strong enough to cause liverdamage. It is quite conceivable that a strategy used to augmentthis response may also be accompanied by enhanced liver

damage in these patients. Therefore, our attempts to boostantiviral immune responses may further damage the liver in theinfected patients unless we counter the viral strategies thatprevent effectiveness of the boosted immune responses. AsHCV per se is not cytopathic, and viral loads do not correlatewith the degree of liver fibrosis, a strategy worth testing mayinvolve calming down the proinflammatory microenvironmentof the infected livers by depleting NK cells or CTL and/or byneutralizing one or more key proinflammatory cytokines, e.g.,IFN-�, TNF-�, and MIP-1�. Another strategy may involveinhibiting liver infiltration of NK cells by blocking VCAM-1/VLA-4 interactions by anti-VCAM-1 antibodies. In support ofthis notion are the reports in literature showing that the deple-tion of CTL and long-term IL-10 therapy in HCV-infectedpersons results in significant improvement in their clinicalcondition [156, 157]. In this regard, it is noteworthy that inaddition to its antiviral effects, IFN-� (the standard therapy forHCV-infected persons) is well known for its cytostatic effectsand its ability to down-regulate IFN-� production from IL-12-stimulated NK and T cells [158]. Further studies are requiredto learn more about these aspects of this disease. Finally,recent advances made in understanding the biology of NKcells, their receptors, and their role in regulating antiviralimmune responses have provided novel avenues for counteringthe immune evasion of HCV (see below).

FUTURE DIRECTIONS

To better understand the potential role of NK cells in thepathogenesis of HCV-induced hepatitis, it would be importantto direct our future research on the following issues.

The future studies should be carried out to determine howE2/CD81 interactions lead to NK cell inactivation and how thisinactivation could be overcome. It seems that on the surface ofNK cells, CD81 complexes with some inhibitory receptor,which becomes triggered when E2 interacts with CD81. Anti-bodies or peptides that inhibit this interaction may be useful inincreasing the efficacy of NK cells against HCV-infected hepa-tocytes.

As stated earlier, NK cells modulate immune responses bysecreting a variety of cytokines and chemokines. The produc-tion of these soluble mediators from NK cells depends on themilieu in which they differentiate. In analogy to TH-1 or TH-2CD4� helper T cells, NK cells could also differentiate intoNK1 or NK2 types depending on the cytokine milieu. NK1cells predominantly produce IFN-�, whereas NK2 ones pro-duce IL-5 and IL-13 cytokines [159, 160]. These two types ofNK cells occur in vivo in humans and have been implicated inthe pathogenesis of multiple sclerosis [161]. It has been welldocumented that liver damage in HCV-infected persons iscorrelated with TH-1 cytokine response, whereas there is in-creased production of TGF-� in liver cirrhosis [162, 163].Thus, NK cells, in the livers of HCV-infected patients, may bestrongly biased to produce a TH-1 cytokine profile. They mayrepresent a major source of proinflammatory cytokines, whichmay be involved in liver damage. Because of their abundancein the liver, the cytokines produced by NK cells may be a majorfactor 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 beenwell documented (ref. [28]; reviewed in refs. [2, 27]). It is notyet clear whether activated NK cells also undergo apoptosis inthis organ. It is noteworthy that activated NK cells, like acti-vated T cells, are prone to apoptosis. A subset of NK cells fromhealthy persons also undergoes apoptosis when they come incontact with NK-sensitive target cells in in vitro assays [164,165]. It has been shown that IL-2 or IL-12-activated NK cellsundergo apoptosis when they are incubated with anti-CD16,-CD2, or -CD94 antibodies [166–168]. They also undergoapoptosis when incubated in high concentrations of certainproinflammatory cytokines, e.g., IL-18 or IL-15 and IL-12[169]. The production of TNF-� from the cytokine-stimulatedNK cells has been implicated in this type of killing. It mayrepresent a negative feedback mechanism to control the secre-tion of IFN-� from NK cells. In line with these observations,high serum concentrations of the proinflammatory cytokineshave been found to correlate inversely with the peripheralblood NK cell loss in patients suffering from various systemicautoimmune disorders [170]. It is noteworthy that the concen-tration 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 en-hanced apoptosis in these patients. The cells may also haveincreased turnover in this organ. Apoptosing NK cells still maycause liver damage as do apoptosing CTL in this organ [31,173]. Ultimately, NK cell turnover decreases and results indecreased NK cell activity and NK cells numbers, as reportedin HCV-infected patients [147, 149]. This decreased NK cellsurveillance may contribute toward the development of HCC.Thus, we speculate that an inappropriate activation of NK cellsas a result of overall hyperactivation of the immune system orincreased amounts of antigen-antibody complexes in this in-fection may cause apoptosis of these cells.

The NKR repertoire of an individual may influence his orher ability to mount an effective antiviral response. In certainindividuals, who inherit an activating allele of a KIR gene andalso happen to express its MHC ligand, NK cells are in arelatively higher state of activation because of an epistaticinteraction between these two genetically diverse loci. Theseindividuals may be relatively resistant to viral infections. Forexample, Martin et al. [174] have shown that the individualsexpressing KIR3DS1 (an activating form of the KIRDL1 gene)and its specific ligand (Bw4 specificity HLA-B molecules withisoleucine at position 80; HLA-B Bw4-80.Ile) are relativelyresistant to the development of AIDS. HLA-B Bw4-80.Ilealleles were not associated with protection from AIDS in theabsence of KIR3DS1. They rather showed a relatively rapidprogression of AIDS. It is tempting to speculate that theindividuals expressing KIR3DS1 and HLA-B Bw4-80.Ile mayalso be more resistant to other pathogenic infections as well asto the development of tumors. Because of a higher state ofimmune activation, they may be more prone to autoimmunediseases. As mentioned above, �15% of HCV-infected indi-viduals spontaneously recover from the infection. It is notknown whether an epistatic interaction between NKR and

MHC loci plays a role in the virus clearance in these individ-uals. The results presented at the 8th Annual Meeting of theSociety for Natural Immunity at Noordwijkerhout (The Neth-erlands) [175] demonstrate that resolution of HCV infection isassociated with the expression of two group 1 HLA-C allelesand homozygosity of the KIR2DL3 receptor. This beneficialeffect of the inhibitory KIR and their HLA-C ligands withrespect to HCV infection again underlies the role of the host’simmune response in the pathogenesis of HCV-induced hepa-titis. It also suggests that activated NK cells may be involvedin this pathogenetic process. Clearly, further studies areneeded to verify these results. If proven so, manipulation of theNKR–ligand interactions may represent an important way ofaltering the course of the infection for the benefit of the host.

Chronic viral infections such as HCV are accompanied withan aberrant expression of NKR on the surface of NK cells aswell as on other immunocytes, e.g., monocyte/macrophages,DC, and B and T cells (CD4� and CD8� subsets). Knowledgeof the NKR genes that become up- or down-regulated in theseinfections should be helpful in designing rational therapies tomodulate immune responses in the infected patients. Unfortu-nately, at present, mAb are not available for all of thesereceptors, and the ones that exist may not distinguish betweenthe activating and inhibitory forms of these receptors. There-fore, it may be difficult to study their expression on infectedhost cells. However, alternate methods, such as real-time re-verse transcriptase-polymerase chain reaction and/or oligonu-cleotide microarrays with appropriate controls, may give a fairidea of the genes whose expression may be dysregulated inHCV-infected individuals. Furthermore, as the level of expres-sion of coreceptors on NK cells and of their cognate ligands ontarget cells (see Table 3) influences NK cell functions, andthese levels frequently change in chronic viral infections, thequestion of how HCV infection modulates their expression inthe infected host is worth addressing.

At mentioned above, MHC-specific NKR are expressedclonally on overlapping subsets of NK cells independently ofeach other. Therefore, changes in the expression of NKR willalso be manifested on the clonal level and may give rise toimmunodeficient and/or self-reactive, autoimmune NK cellclones. The detection of these clones may not be possible withthe use of polyclonal NK cell preparations, which are normallyused in NK cell assays. One may have to use NK cell clonesderived from the infected patients. Furthermore, one cannotuse traditional K562 cells as target cells in these assays. Thesetarget cells do not express MHC antigens and are killed by NKcells via NKp46 and NKp30 receptors (reviewed in ref. [78]).These assays merely determine the overall killing potential ofNK cells via these two receptors and give no information as tothe expression of MHC-specific receptors. The cytotoxic activ-ities of NK cell clones may be tested against a panel of targetcells with distinct MHC haplotypes including self-phytohemag-glutinin-activated T cell blasts. Alternately, one may also useMHC-deficient human target cells, which are engineered toexpress a single MHC allele (reviewed in ref. [116]).

It is noteworthy that the expression of NKR ligands on targetcells is also an important factor in determining the functionalactivity of NKR� immunocytes. Therefore, it would be impor-tant to know how HCV regulates the expression of these ligands

on host cells, in particular, on hepatocytes. We know that HCVstabilizes the expression of MHC class I antigens. However, wedo not know which of these antigens are stabilized. Further-more, we need to know how HCV regulates the expression ofHLA-E, HLA-G, and stress-inducible proteins, such as MICA,MICB, and ULBP. These are important NKR ligands, whichmay affect the functional activities of NK cells, CTL, DC, andmacrophages. HCV may induce expression of the stress-induc-ible proteins on hepatocytes directly or indirectly via proin-flammatory cytokines. Such host cells, whether infected or not,may be killed by NKG2D-expressing NK cells and T cells. Itis tempting to speculate that NKG2D-type-activating NKR oncytolytic cells in these patients may be involved in the killingof uninfected hepatocytes. This is a testable hypothesis and, ifproven, may lead to novel approaches for treating this infec-tion.

A fortuitous outcome of the newly gained knowledge of NKRand their ligands is the availability of novel adjuvants thatcould increase the efficacy of antiviral vaccines. More specif-ically, the ligands that bind activating receptors and activateNK cells, e.g., among others, MICA, MICB, ULBP, and GalCer,may turn out to be effective adjuvants in antiviral vaccinationformulations. By activating NK cells, these adjuvants shouldinduce more powerful immune responses in the vaccinees.

Finally, one should investigate the effect of blocking inter-actions between NKR and their ligands on hepatitis in animalmodels. For this purpose, receptor-specific antibodies and/ortheir soluble ligands may be used. Soluble MHC moleculesmight have served as important tools to manipulate NKR-ligand interactions in vivo. As they have been shown to induceapoptosis of CD8� NK and T cells via their interaction withthe MHC-binding domain of CD8 molecules [176, 177], theirmutant versions lacking CD8-binding domains may be a betterchoice. If proven useful, they may represent novel tools in ourfight against this formidable disease. It is noteworthy that somehumanized anti-KIR antibodies are already being marketed asdrugs.

ACKNOWLEDGMENTS

A. A. holds a “Chercheur-boursier senior” award from ‘‘Fondsde la recherche en Sante du Quebec’’ (FRSQ.) We thank Ms.Sylvie Julien for her excellent secretarial help, our colleaguesin the laboratory for insightful discussions, and the CanadianInstitutes of Health Research (CIHR) for support. We regretthat due to space limitations, all studies on the subject couldnot be cited.

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Documents

Th2 bias of CD4+ NKT cells derived from multiple sclerosis in remission

Immunogenic Properties of a Chimeric Plant Virus Expressing a Hepatitis C Virus (HCV)-Derived Epitope: New Prospects for an HCV Vaccine

Measurements of HCV neutralizing antibodies and of HCV-specific CD4+ and CD8+ cells using hepatitis C virus pseudo-particles (HCVpp)

immunopathogenesis of granulomas in chronic inflammatory

Expression of CD1c enhances human invariant NKT cell activation by α-GalCer

The role of prostaglandin E2 in the immunopathogenesis of experimental pulmonary tuberculosis

HCV genotype 1a shows a better virological response to antiviral therapy than HCV genotype 1b

HCV Participant Briefing Book - Kentucky Housing Corporation

Invariant NKT cells are required for airway inflammation induced by environmental antigens

automated anti-HCV Cobas Möritz

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

Quantitative and Qualitative Differences in Proatherogenic NKT Cells in Apolipoprotein E-Deficient Mice

CD1d–lipid-antigen recognition by the semi-invariant NKT T-cell receptor

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

Hepatitis C Virus (HCV)-Induced Immunoglobulin Hypermutation Reduces the Affinity and Neutralizing Activities of Antibodies against HCV Envelope Protein

Interaction of the hepatitis C virus (HCV) core with cellular genes in the development of HCV-induced steatosis

NKT Photonics Supercontinuum - Optoprim

Seroprevalence and Risk Factors of HCV in Dialysis Patients

Detection of HCV Components and Pathological Reactions in Apoptotic Hepatocytes from Chronically HCV-infected Patients

Structure of HCV IRES domain II determined by NMR