Natural killer cells in hepatitis C virus infection
Author(s): Federica Bozzano, Francesco Marras, Roberto Biassoni and Andrea De Maria
Source: Expert Review of Clinical Immunology. 8.8 (Nov. 2012): p775.
Document Type: Article
DOI: http://dx.doi.org.ezproxy.fiu.edu/10.1586/eci.12.71
Copyright: COPYRIGHT 2012 Expert Reviews Ltd.
http://www.expert-reviews.com/loi/eci
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Related Subjects
• Antiviral agents
• Hepatitis C virus
• Immune response
• Immunogenetics
• Infection
• Killer cells
• Liver cancer
• Liver cirrhosis
• Liver transplantation
• Virus diseases
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Author(s): Federica Bozzano 1 1 , Francesco Marras 2 2 , Roberto Biassoni 2 2 , Andrea De Maria [*] 3 3 3 3 4 4
Keywords
:
directly acting antivirals; HCV; HLA; IFN-λ; IFN-[alpha]; IL-28B; KIR; natural cytotoxicity receptors; NK cell; protease inhibitors; standard treatment; treatment outcome
Hepatitis C virus (HCV) infection affects approximately 170 million people worldwide and is fraught with high morbidity and few predictive tests for disease progression. The major complication of chronic hepatitis C is advanced fibrosis and cirrhosis, which develops in 20-35% of patients over 20-30 years [1] . Once cirrhosis is present, HCV-associated hepatocellular carcinoma (HCC) may develop at a rate of 3-6% per year with HCC developing in only a fraction of patients without fibrosis (0.4%) and a cumulative lifetime risk of approximately 5% of chronically infected patients [2,3] . The incidence of HCC in the western world is clearly rising because of chronic HCV infection [4] . HCV is also associated with cryoglobulinemia and non-Hodgkin’s lymphoma, and may worsen with the prognosis of any other disease with increased non-HCV related mortality [5] and is one of the leading causes of liver transplantation [6] . HCV reinfection occurs in 70-80% of graft recipients with 10-20% of them ultimately developing fibrosis, cirrhosis and higher risk of allograft failure and death [7] .
Few, if any, tools in everyday clinical practice have so far been available to identify the minor proportion of chronically infected patients who will ultimately develop an HCV-associated condition (i.e., cirrhosis, HCC and non-Hodgkin’s lymphoma) and who would most benefit from intensive treatment. This leads, in the absence of an effective vaccine, to the clinical need to treat/monitor all patients, even the majority who do not develop HCV-associated adverse events. This represents an unsatisfactory compromise, but a necessary one. We are also treating those patients who would probably never develop a critical HCV-associated illness in their lifetime, are not sure that those who respond to treatment are really those who would need it, nor do we have evidence that standard treatment nonresponders are those who actually ultimately would develop end-organ failure or disease.
HCV is a remarkably successful single (+)-strand RNA virus that belongs to the Flaviviridae family. Humans represent the only reservoir of HCV and the virus naturally targets hepatocytes for replication, determining an acute viral hepatitis. A significant body of molecular and clinical evidence indicates that HCV also invades, at least partially replicates, and ultimately persists in vivo in cells of the immune system, both T and B lymphocytes, and monocytes [8-13] . These cells may in turn serve as a reservoir in which biologically competent virus persists and is compartmentalized. Immune cell targeting by HCV implies the possibility that different entry receptors may be involved in lymphocytes, compared with those necessary for hepatocyte entry. In fact, the range for HCV receptors so far identified is wide, including CD81 and possibly other tetraspanins and scavenger receptors (SR-B1 [14]) , glycosaminoglycans and very low-density lipoprotein [15] and CD5 [8] , thus supporting the notion of a wide range of target cells that can be infected. HCV compartmentalization to immune cells also contributes towards explaining the increased frequency of mixed cryoglobulinemia and lymphoproliferative disorders such as non-Hodgkin’s B cell lymphoma in chronically infected patients, and also graft reinfection after liver transplant with HCV residing in lymphocyte reservoirs.
Viral replication is extremely robust, even in the chronic phase of infection. Replication occurs through an RNA-dependent RNA polymerase that lacks a ‘proofreading’ function, which results in the introduction of base mutations and possible amino acid substitutions with each round of replication. Hypervariability in HCV sequence occurs mainly in the region coding for envelope proteins, and results in rapid evolution of diverse but related quasispecies within an infected person. This presents a major challenge with respect to immune-mediated control of HCV. In addition to individual variability, six HCV genotypes and multiple subtypes have been identified, whose distribution worldwide may be dishomogeneous.
The natural history of HCV infection is widely divergent among infected patients in terms of virus clearance upon acute infection, of eventual development of cirrhosis, end-stage liver disease and HCC, and of response to standard treatment. Several clinical and virologic factors may determine the eventual outcome of HCV disease or of its response to treatment, including age at infection, drug abuse, gender or physiological conditions, alcohol intake, coinfection or superinfection with other viruses (HIV, HBV and HAV), and HCV genotype [1,16] . HCV genotype plays a relevant role in the response to treatment, with some genotypes being easier to eradicate. A combination of pegylated IFN-[alpha]2a or -[alpha]2b and ribavirin (RBV) results in only 40-50% sustained virological responses (SVRs) in patients infected with HCV-1a/b genotypes, compared with a more favorable probability of response (80%) in patients infected with HCV-2 or -3 [16] . Newer compounds directly inhibiting viral replication steps have been developed, also referred to as directly acting antiviral agents, which include the HCV protease inhibitors telapravir and bocepravir, and more are in the pipeline [17,18] . Use of telaprevir and boceprevir is effective only in association with standard drugs (pegylated IFN-[alpha] and ribavirin). Treatment with these HCV nonstructural protein NS3-4A serine protease inhibitors associated with pegylated IFN-[alpha] and ribavirin results in considerably higher response rates – up to 75% from 40% – even in patients who previously failed standard treatment [19-22] albeit at the cost of higher rates of side effects [23] .
Overall, several clinical questions riddle everyday clinical practice for who cares for HCV infected patients. HCV patients may have diverging responses, starting from their chance to clear virus, over to their relative lifetime risk of progressing to cirrhosis, liver failure or HCC, and their likelihood to achieve SVR upon specific treatment. Until recently, few and mostly viral or clinical parameters were available to help identify or describe those who would have had unfavorable responses [1,24] . Their use, however, would not help understand which patient would clear acute HCV, or which would respond to standard treatment. In recent years it has become clear that the efficiency of the immune system in confronting the virus relies mostly on innate mechanisms including interferon stimulated gene regulation [25,26] , IFN-& lambda; (IL-28B ) polymorphisms and NK cell immunogenotype and function. Considerable advances have been made particularly in understanding the role of NK cells in the pathophysiology of HCV infection and their possible use for clinical purposes. There has so far been little translation of NK cell parameters in clinical practice. This may be due to the difficulty in understanding NK cell function and biology, the perception that results might be conflicting, the difficulty in setting the results in a clinical perspective or simply by the prevalence in the field of old habits that do not evolve (similar to the use of CD4+ cells in the HIV arena) mixed with resistance of introducing simple new tests in large registrative trials in order to validate the results [27] .
NK cell receptors & function
NK cells participate in innate immune responses and have been originally identified as an efficient direct antitumor and antiviral host defense. Over the years, their complex interaction with downstream adaptive responses and with the regulation of immune responses against tumor or invading pathogens has been increasingly recognized [28] .
Two major subsets of NK cells have been described in vivo in peripheral blood, characterized by different CD56 and CD16 expression: CD56dim CD16+ and CD56bright CD16+/− . A third subset of NK cells described as ‘exhausted’, and defined by absence of CD56 but expressing CD16 has been originally described in HIV-1 infected patients, but the mechanism(s) underlying its functional differentiation are unclear [28] . NK cells derive from CD34+ precursors that are found in bone marrow and subsequently migrate to lymph nodes, where they further develop into CD56bright NK cells. Although they can be found in peripheral blood lymphocytes as a minor subset (5-10% of NK cells), CD56bright NK cells are thought to represent the wide majority of NK cells in the body and reside mainly in lymph nodes and secondary lymphoid organs, while CD56 dim NK cells represent a subsequent developmental stage of CD56bright NK cells, which are bound towards peripheral tissues [29] and may undergo further differentiation with the loss of NKG2A, expression of killer cell immunoglobulin-like receptor (KIR) and of CD57 [30,31] . Until recently, a functional differentiation between the two principal subsets of peripheral NK cells described CD56dim as cytotoxic and CD56bright mainly as IFN-[gamma] producers and less cytotoxic. However, recently it was shown that upon natural cytotoxicity receptor (NCR) stimulation CD56 dim NK cells are capable of relevant, early, transient IFN-[gamma] production as opposed to late protracted IFN-[gamma] production by CD56bright [32] . In addition, using activating receptor stimuli different from NCRs (NKG2D, DNAM-1 and 2B4) CD56dim NK cells secrete chemokines, while these receptors require additional costimulus for IFN-[gamma] or TNF-[alpha] production. This confirms that a hierarchy of early chemokine and cytokine production can be observed in peripheral NK cells, which may exert within the same early time frame both cytotoxic functions via perforin/granzyme release and cytokine production (e.g., TNF-[alpha]), and at the same time induce immune cell recruitment (via chemokines) and activation (via IFN-[gamma]), thus shaping downstream immune responses [32,33] .
NK cell function is regulated by a finely tuned balance of stimuli deriving from activating and inhibitory receptors expressed on their surface (Table 1). Activating stimuli may be delivered to NK cells through triggering via cytokine receptors (IL-2, IL-12, IL-15, IL-18 and IFN-[alpha]) and combinations thereof (e.g., IL-2+IL-15, IL-2+IL-12, IL-12+IL-18), via Toll-like receptors (TLR) including TLR2, TLR3, TLR7/8, TLR9, either alone or rather in combination with cytokines (e.g., IL-12, IFN-[alpha] and IL-18), or via activating receptors representing an array of different molecules expressed on their surface including NCR, NKG2D, NKG2C (a lectin-type triggering receptor that dimerizes with CD94), 2B4 (CD244), NKp80, DNAM-1, NTB-A and the receptor for IgFc (CD16 [28]) . Among NCRs, NKp30, NKp46, NKp44 and NKG2D are those involved in the natural cytotoxicity process [34] . NCRs play a major role in NK-mediated killing of most tumor cell lines [28,35] and their surface density on NK cells correlates with the magnitude of cytolytic activity against NK-susceptible target cells [36] . Their expression is mostly restricted to NK cells and, particularly in the case of NKp46, they represent the most accurate surface markers for human NK cell identification. Exceptions for NK cell identification have been documented [37-39] . Other NK cell activating receptors have been shown to be involved in tumor recognition (see [28,40,41] for a review). In addition, some major triggering receptors such as NKG2D and other activating coreceptors including DNAM-1 and CD94/NKG2C may be also expressed by T cells [42] and also monocytes and B cells [43] .
Ligands recognized by NCRs have so far been actively sought and characterized; however, for some NCRs they still lack molecular definition (e.g., NKp46 and NKp44). In addition, given ligands may show variable expression on different cells [44] , and cells of different histotype may share the same activating receptor ligand [45,46] (e.g., stress-inducible polymorphic MHC class I-related chains [MIC-A, MIC-B [47] ] or ULBPs [48] for NKG2D or poliovirus receptor [PVR], and Nectin-2 for DNAM-1). In other instances, ligands may be associated with neoplastic transformation of the cell (e.g., B7-H6 for NKp30 [49]) or may be represented by viral glycoproteins as in the case of influenza, Dengue, West Nile Virus [50,51] or as yet undefined ligand(s) for HIV [52] .
NK cell inhibitory receptors, recognizing mostly HLA class I molecules on ‘self’ cells (notable exceptions to this concept are represented among others by Siglec-7 and IRP60 recognizing non-HLA-related structures), turn NK cells ‘off’ and represent the major fail-safe device to prevent NK-mediated attack of normal HLA class I+ autologous cells.
NK cells detect compromised expression of MHC class I on target cell by KIRs, CD94/NKG2A and leukocyte immunoglobulin-like receptor (LIR-1/ILT2A, CD85j). CD85j is a glycoprotein with immunoglobulin superfamily structure distinct from, but related to, known KIRs that binds cellular MHC class I antigens [53] . KIRs are a family of activating and inhibitory receptors expressed on NK cells that are encoded by a family of genes highly polymorphic for allele and content found on human chromosome 19 that are specific to HLA-A, HLA-B and HLA-C allotypes [54] .
KIR that display long cytoplasmatic tails (KIR-L) contain immunoreceptor tyrosine-based inhibitory motif receptors (ITIMs) and two or three extracellular Ig-domains (KIR2D and KIR3D) specific for HLA-C or HLA-A/B allotypes, respectively. KIR2DL1 recognizes HLA-Cw2, HLA-Cw4, HLA-Cw5 and HLA-Cw6 (allotype C2), and KIR2DL2 and KIR2DL3 bind to HLA-Cw1, HLA-Cw3, HLA-Cw7 and HLA-Cw8 (allotype C1). KIR3DL1 binds to HLA-B alleles expressing Bw4 epitope and KIR3DL2 binds HLA-A3 and HLA-A11 [54] . KIR-S genes are mixed with KIR-L genes, are highly polymorphic in extracellular domain and show a short intracytoplasmatic tail without the ITIM motif but are associated with DAP12 signaling adaptor.
Notably, not only NK cells but also CD8+ T cells may express KIRs [55] . Importantly, their expression has been shown to be oligoclonal or monoclonal and to impair antigen-specific CD8+ cytotoxic T-lymphocyte function during viral infection [56,57] . Thus, not only NK cells but also T cells may be involved in KIR:HLA engagement that may modulate their function.
NK cells need to express at least one inhibitory receptor to be functionally active and ‘licensed’ or ‘educated’ to function [28,58,59] . In addition, the balance of KIR carriage:HLA class I expression and NKG2A/CD94:HLA-E or CD85j(LIR-1/ILT2):HLA class I expression needs also to be considered to define NK cell reactivity. In the presence of a given KIR:HLA carriage and similar densities of KIR molecules on licensed NK cells, variable degrees of activating receptor expression are likely to influence NK cell function. Indeed, although KIR:HLA carriage may influence NK cell activation, this pathway alone does not exhaustively represent overall NK cell function, as represented in KIR-negative CD56bright NK cells and in KIR-negative NKG2A-positive NK cell precursors.
NK cells during viral infections
NK cells play a pivotal role in the control of tumors and viruses, bacterial, fungal and other intracellular parasite infections [60] . NK cell cytokine production, such as IFN-[gamma] and TNF-[alpha], induce immediate activation of antimicrobial pathways in infected cells with following modulation of adaptive response to the pathogen [61,62] . Over recent years, accumulating evidence has linked NCRs on NK cells with direct or indirect recognition of pathogen-associated structures.
NK cells are involved in the immune response to Epstein-Barr virus, murine and human cytomegalovirus (MCMV and HCMV, respectively) and herpes simplex virus. Clinically, the importance of the role of NK cells in antiviral immunity has been shown in NK-deficient patients with recurrent herpes virus infections [63,64] , or in patients with low NK cell responses [65] and in mice engineered to lack NCRs [66] . Recently, NK cells have been shown to provide enhanced responses to recall pathogens/antigens with persistence of this reactivity for periods extending beyond the period of innate immune responses, providing emerging evidence for some sort of immunological memory and the capacity for self-renewal of mature NK cells ([28] ; for reviews, see [67,68]) . An additional area that is quickly moving into sight is the possibility of interaction of NK cells with T cells (either CD8+ or CD4+ ) in the regulation of T-cell responses and in the determination of chronic persistent infection, which has been shown for LCMV infection in rodents [69-71] . Although thus far no evidence has been provided for human infections, this might be a relevant issue in future and might possibly also include human chronic HCV infection.
NK cells have a fundamental role in the continuous editing of adaptive immune responses through crosstalk with dendritic cells (DCs) and thus contribute to protect not only from overwhelming infection by new pathogens but also from rechallenge with previously known pathogens and from reactivation of latent pathogens. For example, successfully Ebola-virus vaccinated mice using virus-like particles survive after virus challenge, but succumb if NK cells are depleted before challenge [72] .
Identification and disposal of virus infected cells by NK cells requires a fail-safe system using two codes, where the first step is represented by recognition of viral-associated structures in the infected cell. This step is provided by TLR engagement (usually TLR3, TLR7/8 and TLR9) with participation of cytokines active on NK cells [33,73] , or by NCR engagement when virus-associated structures or virus-induced endogenous ligands are recognized possibly in the presence of other NK cell activating stimuli [34] . A second step is provided by failure of inhibitory NK-cell receptors (e.g., KIRs, CD85J and NKG2A) in engaging HLA molecules on infected cells. An indirect proof of the relevance of NK cell responses in viral infection is provided by the rich array of countermeasures that have been developed by viruses to evade NK cell function.
HIV profoundly affects NK cell phenotype and function early during the disease course, with relevant reduced NCR expression, NK cell activation and increases in ‘exhausted’ NK cell populations [74-76] . Similarly, proof of defective NK-DC editing with the generation of less mature and more tolerogenic DC, is provided during chronic viral infection [77-79] . Other viruses downregulate ligand expression by target cells or may downmodulate HLA molecules to evade T cells. In some cases they additionally developed production of decoy molecules that mimic HLA molecules to evade NK cells, as for example is the case for CMV.
Overall, selection and persistent preservation of these strategies to evade NK cell surveillance during virus-host coevolution underscores the vital role of NK cells in the defense against invading viruses.
NK cells in HCV infection
During HCV infection, most of the replication takes place in the liver, however the possibility of minor involvement of extrahepatic immune reservoirs should not be underestimated. Although monocytes, T lymphocytes and B lymphocytes may be infected by HCV (see above), there has so far been no evidence of direct NK cell infection, and culture of NK cells with HCV strain JFH-1 does not induce their impairment [80] .
In rodents, the liver is reportedly enriched with hyporesponsive NK cells [81] . In addition, there is evidence that in rodents at least a fraction of liver NK cells might have ‘memory’ characteristics not shared by peripheral or spleen NK cells [82] . Liver residing cells in humans are less accessible to conventional evaluation during HCV infection and possible differences are yet uncharted. So far, a good correlation between the proportion of peripheral blood versus liver NK cell subsets has been reported in HCV infected patients to support conclusions from studies on peripheral blood mononuclear cells [83] .
Although in vitro exposure of NK cells to cell-free HCV does not result in impaired NK cell function [84] , cell-contact dependent reduction of NKp30 and NKG2D and of NK cell cytotoxicity has been recently reported in an in vitro model using infected hepatoma cell lines and peripheral NK cells [85] . Upon acute infection in vivo , however, NK cells have been found to upregulate expression of NKG2D as well as cytotoxic function and IFN-[gamma] production, irrespective of subsequent disease course in the patients (i.e., clearance vs chronic replication [86]) . Thus, while in vitro cell-associated (but not cell-free) HCV affects NK cell function and NKp30 or NKG2D expression, no effect is seen in vivo during acute infection. This might be related to the district where virus replication is taking place (liver vs peripheral blood), or to specific characteristics of the in vitro systems used. Direct participation of NK cells in a different clinical setting, namely exposed-uninfected patients, was also suggested in another study where upregulation of NKp30, and not of NKG2D, was observed in peripheral NK cells [87] . Differences in patients, with the potential bias of opiate use in exposed-uninfected intravenous drug users, with uncertain timing of exposure and probably differences in HCV replication (with lower or inconsistent replication in exposed-uninfected patients) needs to be further addressed. The apparent discrepant triggering receptor upmodulation in studies in vivo with, on the other hand, decreased receptor expression upon NK cell challenge with HCV-infected hepatoma cell lines [84] is difficult to reconcile. However, this might represent two aspects of NK cell-HCV interaction in different compartments, different timing during infection (acute vs chronic replication) or both. At any rate, these studies altogether prove that upon encountering HCV, NK cell responses take place as shown by the detection of both phenotypic and functional changes
None of these studies was designed or powered to identify NK cell parameters that might help predict subsequent HCV outcome. The interaction of KIR gene receptors with HLA molecules on target cells has however been previously shown to affect the outcome of acute infection in a landmark genetic study [88] . In this work, polymorphic KIR genes and HLA-C genes, which encode for the natural ligands of KIR molecules, were studied in over 1000 HCV infected patients. Carriage of KIR2DL3 genes and of genes encoding for its natural ligand HLA-C group 1 genes ( HLA-C1 ) was found to be associated with increased protection (2.3-fold) from chronic replication with clearance of acute infection. This association was true when gene carriage was homozygous and was limited to patients infected by accidental needlestick or intravenous drug use after exclusion of patients with transfusion of blood or blood products (with presumably high titer overwhelming infectious events) This KIR2DL3-HLA-C1 association has been proposed to have a lower affinity compared with the interaction of other inhibitory KIRs with HLA-C1 or C2, resulting in a weaker degree of inhibition of NK cells with a lower threshold for activation and possibly increased surveillance and HCV clearance [89] . Direct measurements confirm lower affinity between KIR2DL3-HLA-C1 compared with KIR2DL2-HLA-C1 [90] . This association has been confirmed in another elegant study showing that NK-cells inhibited by KIR2DL3 responded in vitro to influenza A virus in HLA-C1 homozygous donors compared with HLA-C2 homozygous donors [91] . KIR-HLA-C typing has the additional advantage of widespread availability of the methodology, accessible and limited sample size (e.g., 1-2 × 10 6 peripheral blood mononuclear cells) and increasing standardization for routine KIRotyping and HLA-C supratyping [92,93] . Physiopathological limits of immunogenotypic characterization are represented by the description of only DNA gene carriage without characterization of actual transcription and of final protein surface expression. Indeed, in addition to NK cells, KIRs are expressed also by CD8 + CTLs specific for viral antigens presented in the context of MHC class I [56,57] . The relative contribution of KIR-expressing CD8+ CTL-target cell interaction in DNA genetic studies of HCV infection has been so far not explored nor excluded. Although NK cell KIR-HLA-C interactions may provide major contribution to clinical divergence during HCV infection, differences in functional impairment of KIR-expressing CD8+ CTL for patients with KIR2DL3-HLA-C1 versus KIR2DL2-HLA-C2 haplotypes may also contribute to viral clearance through less efficient CTL inhibition. This has recently been implied at the genomic level by the analysis of KIR and HLA carriage in patients with HTLV I infection or with HCV infection [94] . Similarly, there is no information so far on the relevance of gene carriage compared with surface protein expression on NK cells in vivo , and this might in future help account for those patients that progress to chronic disease even in the presence of favorable genetic carriage of KIR2DL3-HLA-C1.
Clinical implications
Clinical usefulness of KIR-HLA-C supertype (C1 or C2) characterization during acute symptomatic HCV infection could be envisaged if the test would be introduced in clinical practice. Treatment of acute HCV infection with pegylated IFN-[alpha] results in virus control and clearance in 71-95% of treated patients as opposed to spontaneous clearance in 20-40% of untreated patients [95,96] . It is recommended to delay treatment initiation for 12 weeks from clinical presentation, in order to avoid treatment to patients spontaneously clearing the virus [97] . In some cases, however, HCV RNA may not disappear until 2-3 months after clinical presentation, and no clinical or virological parameters were found to predict likelihood of spontaneous resolution of infection [97] . In this context, KIR-HLA-C assessment would provide additional clinical information, patient reassurance and best treatment planning. In addition, it would provide useful information, that is thus far lacking, on the KIR-HLA-C haplotype in patients that fail early treatment during acute HCV infection.
NK cells in chronic HCV infection & response to standard treatment
During chronic HCV infection, early reports concentrated on circulating NK cell numbers and NK-cell subset analysis [98-101] . Reduced NK cell numbers in HCV infection could be secondary to increased NK cell turnover or reduced survival – possibly due to decreased IL-15 levels and production by DCs [102,103] – or increased compartmentalization away from the bloodstream. The evaluation of whether NK cell numbers are reduced or rather increased during acute or chronic HCV infection may be of clinical interest, but it is in general biased by the analysis of only one minor – peripheral blood – compartment whereas the majority of the body NK cell pool is contained in secondary lymphoid organs where compartmentalization could take place. In fact, this possibility is supported by NK cell number increases after successful treatment with IFN-[alpha] [101,104] and by the finding that CD3-CD56+ cells, operatively defined as NK cells, were found to be decreased in the liver during chronic HCV infection [99] .
Imbalances in cytokine production either produced or activated by NK cells have been observed. In addition to decreased IL-15 production by DCs [102] , which would decrease the support in survival, activity and proliferation of NK cells, peripheral NK cells have been found to produce IL-10, IL-6 and IL-8 [105,106] thus potentially skewing the crosstalk with DC further toward immune tolerance and dampening of adaptive responses. IFN-[gamma] production by NK cells has been reported to be defective in several instances. Hepatic NK cells express increased TRAIL levels compared with healthy controls, express NKp44, which is a marker of NK cell activation, and are polarized toward CD107a expression as a marker of cytotoxic activity without increases in IFN-[gamma] production [107] . Defective IFN-[gamma] production by IL-12 activated peripheral NK cells has also been observed using immobilized HCV viral particles (HCVcc), while this is not observed using free HCVcc [106] . Thus, CD81 crosslinking affects IFN-[gamma] production and CD16-mediated or TCR-mediated functional activation [108] , while natural cytotoxicity is not affected [105] . Recently it has been reported that NK cells recognize HCV-infected hepatoma cell lines after IFN-[alpha] administration in vitro via DNAM-1 and NKG2D, and that HCV would not modify DNAM-1 and NKG2D ligand expression in infected hepatoma cell lines [109] .
During chronic HCV infection, activating NK cell receptor expression is modified reflecting potential imbalances in NK cell function and regulation of their natural cytotoxicity receptors (e.g., NKp30 and NKp46). Initially conflicting results from different studies showing either reduced [110] or increased [105] expression of NKp30, NKp46 NCRs and NKG2D [111] during chronic HCV infection have been possibly reconciled by the finding that patients with HCV-1 genotype who will respond to ribavirin + peg-IFN-[alpha] treatment with SVRs display significantly lower NKp30 expression compared with those who will not respond to treatment [112] . Conversely, nonresponder (NR) patients have increased expression of NKp46, which is not further affected by IFN-[alpha] treatment. Thus, variable findings in previous works are likely to depend on the patient groups that were considered. In studies that collect(ed) predominantly naive HCV-1 infected patients, lower NKp30 levels would be expected, while increased NKp30 and NKp46 could be observed in patient groups enriched for patients who failed – or would fail – treatment.
Interestingly, patients who would respond prospectively to treatment not only had lower NKp30 and NKp46 molecule density on peripheral NK cells, but were also able to increase its expression in vivo after 12 weeks of IFN-[alpha] treatment, while NR patients had stably high NCR expression that could not be upregulated during IFN-[alpha] treatment in vivo [112] . Observations by other groups are in line with these reported effects of IFN-[alpha] on NKp30 and also showed that other receptors including inhibitory receptors (e.g., NKG2A) might be modulated in the context of IFN-[alpha] treatment in vivo [113] . These effects of IFN-[alpha] treatment were not due to CD56bright or CD56dim imbalances, and could be reproduced in vitro on purified NK cells cultured in the presence of rIL-2 and IFN-[alpha] [112] thus suggesting that receptor inducibility is a protective feature, identifying patients who will respond to standard treatment with pegylated IFN-[alpha] + RBV. These findings are reminiscent of the observation that in NR patients interferon-stimulated genes in hepatic biopsies result in upregulation and do not undergo further upregulation during IFN-[alpha] treatment, while SVR patients have lower interferon stimulated genes (ISG) expression with still conserved inducibility [25] . Since IFN-[alpha] administration in vitro upregulates NCRs in SVR patients the overall picture suggests that different immunogenetic regulation occurs in NR and SVR patients. Already maximal ISG [25] and NCR expression is observed in NR patients, and neither can be further increased by exogenous administration of IFN-[alpha], thus leading to treatment failure. On the other hand, patients with intrinsically low expression of ISG and NCRs despite chronic HCV replication have conserved ability to upregulate NCRs upon treatment thus efficiently clearing the virus and then returning to baseline low-level expression [25] . Finally in this context, the recent finding that breast cancer patients with recurrent disease have a distinct pattern of NKp30 expression and function associated with alternative NKp30 splicing isoforms [114] is in line with the possibility that NKp30 expression is immunogenotypically regulated and may contribute to determining at least in part differential control and outcome of diseases including HCV clearance upon treatment or acute infection in addition to tumor recurrence.
Use of NK cell parameters to help explain, or predict, response to treatment is not limited to ISGs or NCR-activating receptor inducibility. Homozygosity for KI2DL3-HLA-C1 has been recently shown, in addition to its role in contributing to virus clearance during acute HCV infection (discussed earlier), it has been convincingly shown to also significantly associate with individual patient chances in responding to standard treatment (pegylated IFN-[alpha] + RBV [115]) . In a study evaluating 186 consecutive patients with different genotypes, a favorable response to treatment was found in patients carrying homozygous KIR2DL3-HLAC-1 DNA haplotypes, irrespective of HCV genotype. Although this association was significant and convincing, not all the patients with the favorable genotype had SVR, and also the converse could be observed, namely favorable responses to treatment in the absence of KIR2DL3-HLAC-1 homozygous carriage.
With regard to the high priority quest for determinants of response to HCV treatment, another genetic target for potentially useful polymorphism characterization has been pinpointed in association with IFN-λ (IL-28B). A first report of an association of two genetic single nucleotide polymorphisms (SNPs; rs8099917 and rs12979860) near the IL-28B gene that were associated with a twofold higher chance of responding (SVR) to standard treatment (pegylated IFN-[alpha] + RBV) in those who carried the favorable polymorphism (C/C) and also explaining reduced response in patients of African origin – who have lower frequency of these SNPs – compared with those of European origin [116,117] . At the same time, one of the two SNPs (rs12979860) was also shown to confer a twofold to 2.5-fold chance of clearing HCV upon acute infection [118] . In spite of the usefulness of the observations, which have been subsequently corroborated by other groups in different clinical settings, it should be noted that they do not directly affect IFN-λ as the SNPs are 3 kb upstream of the coding region of the gene itself. Thus, it is still unknown how these genetic polymorphism(s) might functionally affect IL-28B function, or whether they might alternatively affect other parts of the immune system that may be otherwise responsive to IFN-[alpha] (e.g., ISGs, NK cells and KIR expression). Efforts in this direction have appeared very recently in two prospective cohorts (HCV monoinfected or HIV/HCV coinfected). Among the 88 patients, an association is reported between favorable SNPs with overall innate immunity transcriptional function as assessed by microarray technology, and of no association with ISG baseline expression or IFN-mediated induction [119] . Contrary to what has been reported for KIR-HLA haplotypes where the favorable association (KIR2DL3-HLA-C1) confers increased chances of clearing HCV with standard treatment, the reported associations between IL-28B SNPs and SVR have been shown only for HCV-1, but not for HCV-3 [120] .
As evidenced in a study attempting to correlate multiple SNPs near the IFN-λ (IL-28B ) coding region with other clinical and virologic parameters, including rapid virological response, early virological response, SVR, BMI, virus load and ALT/AST, in a structured study of 191 treatment-naive patients, rs12979860 was the critical predictor for viral responses, and the only predictor for SVR in patients without rapid virological response. It should be noted however that it was not possible to identify all SVR or NR just by analysis of SNP carriage.
Clinical implications
So far, the quest for parameters associated with response to standard treatment in HCV infected patients has resulted in the identification of two different types of parameters, namely those that are useful only once treatment has begun (e.g., early decrease in viral load, early increase in NK cell CD107a expression or TRAIL in virological responders [113]) as opposed to those that can be used before treatment to define the best management choices for patients. It should be noted that the critical issue in clinical medicine is represented by the correct prediction/identification of treatment responders before a treatment is administered. This allows avoidance of the administration of ineffective treatment, choice of definite treatment course, optimization of available treatment options, and avoidance of unnecessary adverse effects and containment of costs of treatment. Based on the presently available information, the critical issue of identification of responders to standard treatment has added at least three different predictive determinants over the last few years. Clinical usefulness is indicated for IL-28B SNP rs12979860, which might identify patients infected with HCV-1 who have a twofold to fourfold chance of SVR response, with some limits for HCV-2b and HCV-3 SVR prediction. Although the exact physiopathologic significance is still lagging behind, the possible association with ISG regulation and inducibility and with innate immune responses may soon provide a rationale for its use. The usefulness of this test alone is however limited by its failure to identify up to 25-25% of HCV-1 patients who will fail standard treatment and up to 10-20% of those who will fail protease inhibitors, including triple treatment, despite favorable C/C polymorphism. Similarly, a fraction of patients (20-35% and up to 60%, in standard and protease inhibitor triple treatment regimens, respectively) will clear HCV despite unfavorable SNPs (C/T or T/T). KIR-HLA-C1 analysis has also been shown to be useful as a determinant for response to treatment, and could possibly help define a twofold increase in the chances in up to 65-70% of responders, with the advantage of being useful irrespective of HCV genotype, as compared with the IL-28B polymorphism. However, in this case attention should be drawn to the incomplete predictivity of this test alone. Indeed, successful SVR can be obtained in 20-30% of HCV-1 patients despite unfavorable KIR2DL2-HLA-C2 carriage and failure has been observed in up to 30-40% of those with a favorable KIR2DL3-HLA-C1 polymorphism. In this context, activating receptor regulation on NK cells and their inducibility at baseline is likely to provide integrated information on the chances of response to treatment in HCV-1 infected patients, potentially explaining, for example, SVR in patients with unfavorable KIR-HLA or IL-28B SNPs. Lack of rapid genetic tests to identify NCR regulation may represent only an apparent limitation of its applicability, as flow cytometric and cell culture laboratories are widely available worldwide.
Considering the limited effectiveness of each single method in identifying 100% of SVR in all HCV genotypes, simultaneous assessment of more than one determinant of response should be recommended and could increase positive and negative predictivity of innate immune balance and of response to treatment with standard or with new drug combinations (Figure 1). Prospective studies assessing the relative predictive weight and the best combination(s) of these determinants to identify all SVR patients will represent a major clinical breakthrough that could allow full individualization of patient treatment.
Conclusion
Analysis of NK cell function and immunogenetic characteristics over the last few years has produced mayor breakthroughs in our understanding of the mechanisms underlying HCV escape from immune control and of the successful determinants of successful virus clearance upon acute infection and upon standard treatment. These implications extend also to progression to cirrhosis and to development of hepatocellular carcinoma or to liver transplantation in HCV infected patients, which lie beyond the scope of the present review.
Wide availability of techniques used to assess NK cell immunogenotype, phenotype or function and genetic testing for SNPs provide convincing support to the possibility of using the so far acquired knowledge in clinical practice. It is time for a translational effort in this area, in order to provide individualized treatment to patients. In this regard, additional work is needed in institutional and in industry-driven clinical trials embedding these assays and thus offering the opportunity for extended clinical validation, for integrated evaluation of multiple predictive tests, and for shedding light on relevant clinical gray areas. Several points are in fact still open and should be discussed and focused on by national and international societies based on trial results to offer improved clinical guidelines. These points include, for example, the role of immunogenotype and NK cell testing/activating receptor expression-modulation in guiding access to standard or new drug treatment, the use of one or multiple assays to determine the likelihood of SVR, the optimal assay combination to identify SVR-to-be, the best determinant of response assay combination depending on the drug regimen or the most suitable sequence of drug treatment and determinant of response assessment.
In summary, clinicians need to be ready to offer analysis of NK cell evaluation and of individual immunogenotypic aspects to HCV patients (e.g., KIR:HLA-C1, NCR expression/modulation and IL28B SNPs), providing comprehensive explanation of useful determinants of clinical response. However, so far there is little pressure to routinely apply these tests in everyday clinical decisions. Improved trial designs with scientific society assistance are needed to monitor and guide development in the area of determinants of clinical response assessment. Increased awareness of NK cell immunopathology, wide assay availability, and prospective evaluation of NK cell and IL28B -related assays in nonresponder patients to standard treatment entering new drug trials are needed in parallel to increasing therapeutic efforts with future waves of new drug classes including NS3-4A serine protease inhibitors, IFN-λ, polymerase inhibitors or cyclophyllin inhibitors. Learning to frame NK cell function in HCV infection and to use assays to determine the individual likelihood of responding to treatment will help us learn how to best use the new drugs and drug classes that will soon be available and how to individualize HCV treatment.
Five-year view
Greater effort will occur in the field of HCV infection over the next few years. The innate immune compartment and the host immunogenetic association would be investigated widely. Embedding new acquisitions on viral, immune and immunogenetic (e.g., gene transcript signatures) parameters into clinical practice will lead to individualization of clinical strategies, favoring tailored treatment and clinical management. Fingerprinting of predictive immunogenetic and viral parameters will result in decreased chances of viral escape from host control, and also in predictive identification of patients at higher risk of developing cirrhosis and HCC who particularly need aggressive treatments upfront.
Next-generation drugs raise hopes of easier drug combination and virus eradication, exploiting new and fewer drug regimens. The high replication and mutant generation time of HCV however might result in the appearance of surprising resistance patterns.
The regular embedding of multiple immune and genetic individual characteristics will teach us how to optimally select patients with reduced host defense pressure toward improved virus control using protease inhibitors and next-generation drugs. Learning how to optimize translational adaptation of basic tests will spread also to the HIV coinfected HCV patient who presently suffers from a high burden of HCV associated disease even in the presence of successful antiretroviral treatment.
Identifying prediction markers also has great importance not only for HCV monoinfected but also for HIV/HCV coinfected patients who have significantly lower responses to treatment and high drug-drug interactions if simultaneously treated with antiretrovirals. The next few years will also see efforts in improving our knowledge of the functional characteristics of liver-residing NK cells and in understanding their role in the successful eradication of HCV.
Relevance of gene carriage compared with surface protein expression on NK cells in vivo (i.e., KIR gene carriage, and KIR surface expression) will undergo further investigation to help account for those patients that progress to chronic disease even in the presence of favorable genetic carriage. Clarifying the role of NK cells and of NK cell-activating receptor modulation in the complex network of cell-to-cell interaction (NK-DC, NK-T cell and NK-monocyte) that contributes to the shaping of adaptive immune responses will provide additional understanding of pathologic processes that could help translate into improved clinical management optimized use of resources and treatment individualization.
Table 1.Natural killer cell receptors, function and ligand characteristics and their expression with regard to hepatitis C virus.
Receptor Function Cellular ligand Viral ligand NK cell subset expression Expression during HCV infection and response to Tx
NKp46 Activating Unknown Hemoagglutinin, influenza, West Nile, Dengue, poxvirus (ectromelia, vaccinia) CD56bright CD16+/− (100%)
CD56dim CD16+ (20-80%)
CD56− CD16+ (<5%) Highly expressed, noninducible with IFN-[alpha] (NR)
Low expression, inducible with IFN-[alpha] (SVR)
NKp30 B7H6, BAT-3, other, unknown CMV pp65 CD56bright CD16+/− (100%)
CD56dim CD16+ (20-80%)
CD56− CD16+ (70%) CD56dim
CD16+ (20-80%)CD56−
CD16+ (<5%)
NKG2C/CD94 HLA-E CMV? CD56bright CD16+/− (0-1%)
CD56dim CD16+ (5-30% variable)
CD56− CD16+ (expression high) NA
KIRs (3DS1) HLA-Bw4 NA NA
KIRs (2DL1) Inhibitory HLA-C2 Individual haplotype dependent,
CD56bright CD16+/− (5-30%, variable)
CD56− CD16+ (expression high) Low response rate to Tx, chronic evolution upon acute infection
KIRs (2DL2, 2DL3) HLA-C1 Individual haplotype dependent,
CD56bright CD16+/− (5-30%, variable)
CD56− CD16+ (expression high) Low response rate to Tx, chronic evolution upon acute infection
LIR-1/ILT2 HLA-G CD56bright CD16+/− (5-30%, variable)
CD56− CD16+ (expression high) Decreased expression in SVR
High expression in NR
NKG2A/CD94 HLA-E CD56bright CD16+/− (100%)
CD56dim CD16+ (0-30%)
CD56− CD16+ (0-25%) Increasing expression upon Tx with IFN-[alpha]-containing regimens
Increased in NR after Tx
CMV: Cytomegalovirus; HCV: Hepatitis C virus; NA: Not available; NK: Natural killer; NR: Nonresponder; SVR: Sustained virological response; Tx: Treatment.
Key issues
* Hepatitis C virus (HCV) affects 170 million people worldwide and the major complication is advanced fibrosis and cirrhosis and hepatocellular carcinoma.
* Pegylated interferon (PEG-IFN[alpha]) + RBV is the standard treatment for HCV infection, but new HCV protease inhibitors are emerging, and additional new direct-acting drugs will be available. So far, for difficult-to treat HCV genotypes, clinical cure is observed in only 40-55% of patients.
* Natural killer (NK) cells have an important role in innate immune responses. Two well-characterized major subsets of NK cells characterized by different CD56 and CD16 expression (CD56dim CD16+ and CD56bright CD16+/− ) are present in peripheral blood NK cells of healthy subjects. NK cell function results from balanced signaling via activating (e.g., natural cytotoxicity receptors, DNAM-1 and NKG2D) and inhibitory receptors (killer-cell immunoglobulin-like receptor [KIR], LIR-1 and NKG2A/CD94) and NK cells.
* KIRs are specific for HLA-A, HLA-B, HLA-C molecules. KIR2DL3:HLAC1 homozygosis is associated with increased protection from chronic replication with clearance of acute infection, and with best treatment results with standard treatment.
* Natural cytotoxicity receptors (NKp30, NKp46 and NKp44) are activating NK cell receptors. NKp30 and NKp46 expression and their modulation prior to treatment is associated to the outcome of standard PEG-IFN[alpha]+RBV regimens in drug-naive HCV-1 infected patients. High, fixed expression is associated with nonresponse.
* Interferon-stimulated genes in hepatic biopsies of NR patients are upregulated and do not undergo further upregulation during IFN-[alpha] treatment, while sustained virological response (SVR) patients have lower interferon stimulated genes expression with still conserved inducibility.
* Single nucleotide polimorphisms; rs8099917, rs12979860, near the IL-28B (IFN-λ) gene, have been associated with a several-fold higher chance of responding (SVR) to standard treatment (PEGIFN[alpha]+RBV).
* Each single method investigated to predict SVR patients in all HCV genotypes, falls short of identifying 100% of SVR.
* Simultaneous assessment of more than one determinant of response is recommended to be introduced in validation studies and in the next registrative trials for new drugs, in order to increase positive and negative predictivity of response, thus identifying the best patient candidates for HCV NS3 protease inhibitor drug use and for next-generation drug use.
CAPTION(S):
Figure 1. Representation of the presently described main clinical and immunogenetic parameters known to affect response to treatement in hepatitis C virus-infected patients and their possible use to predict responses to treatment and to plan individualized treatment strategies.
Bars on the sides of the central square represent the so far known clinical/virological (right), NK cell-related (top and bottom) and innate immune (left) correlates for differential response to treatment. Interferon stimulated gene expression (not shown [25] ) could parallel NCR expression/inducibility [112] . The panels show examples of simultaneous assessment of determinants of clinical response to treatment with potential for improved accuracy in individualized prediction do response. (A) A combination of favorable multiple immunogenotypic parameters predictive of high probability of response to treatment in a patient with unfavorable clinical and virological parameters. (B) A combination of mixed favorable (NK cell paraemters, NCR, KIR:HLA) and adverse (IL28B rs12979860 T/T polymorphism) immunogenotypic parameters predictive of high probability of response to treatment in a patient with unfavorable clinical and virological parameters. Such combinations could help explain positive responses to standard treatment in spite of adverse rs12979860 and clinical-virological parameters. (C) A combination of adverse multiple immunogenotypic parameters predictive of very low probability of response to treatment in a patient with unfavorable clinical and virological parameters. These patients represent those with T/T or with high noninducible NCR (30-40%) that do not respond to protease inhibitor combination therapy.
NCR: Natural cytotoxicity receptor.
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Author Affiliation(s):
[1] Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
[2] Istituto G Gaslini, Genova, Italy
[3] Department of Health Sciences, University of Genova, Genova, Italy. de-maria@unige.it
[4] IRCCS AOU San Martino – IST Genova, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
Author Note(s):
*Author for correspondence
Financial & competing interests disclosure
This work was supported by Istituto Superiore di Sanita` (I.S.S.): Programma Nazionale di Ricerca sull’AIDS, Accordi di Collaborazione Scientifica n. 40G.41 and 45G.11, Accordo di Collaborazione Scientifica n. 40D61. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed .
No writing assistance was utilized in the production of this manuscript .
Federica Bozzano, Francesco Marras, Roberto Biassoni, Andrea De Maria
Source Citation (MLA 7th Edition)
Bozzano, Federica, et al. “Natural killer cells in hepatitis C virus infection.” Expert Review of Clinical Immunology 8.8 (2012): 775+. Academic OneFile. Web. 5 Apr. 2015.
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