Role of soluble inflammatory mediators and different immune cell populations in early control of symptomatic acute hepatitis C virus infection.

The natural course of acute Hepatitis C Virus (aHCV) infection is highly heterogeneous, and only few biomarkers have been identified to reliably predict the outcome of infection. We analysed a large panel of soluble inflammatory mediators, immune cell frequencies and phenotypes using peripheral blood samples from 26 patients with symptomatic aHCV infection from a controlled randomized clinical trial (ISRCTN88729946, www.isrctn.com). We found that patients with a spontaneous early HCV control demonstrated a distinct expression pattern of various soluble immune mediators including IFNα and IL-16. Immune cell phenotype and frequency differed between patients who cleared the viral infection early (n=13) and those who remained HCV RNA positive after 12 weeks of observation (n=13) with a reduced ratio of CD4+ T cells to NK cells in the non-early clearer. Further, correlation analyses of 50 cytokines and chemokines revealed more positive correlations in between the distinct cytokines, especially for IFNα and IL-16, and between the cytokines and HCV RNA levels in spontaneous early clearer patients. Beyond that, in vitro stimulation of CD4+ T cells with IL-16 reduced the susceptibility of these cells to killing by IFNα-activated NK cells. These data indicate that the immune cell composition and cytokine pattern varies considerably in patients with symptomatic aHCV infection. NK cell-mediated killing of CD4+ T cells might affect early control of HCV infection.

Systemic inflammation and immune cell phenotypes are associated with neuro-psychiatric symptoms in patients with chronic inflammatory liver diseases.

BACKGROUND & AIMS: Chronic inflammatory liver diseases are frequently associated with neuropsychiatric and cognitive dysfunctions. We hypothesized that symptomatic patients may show altered levels of soluble inflammatory mediators (SIMs) as well as changes in immune cell phenotypes. METHODS: A comprehensive immune-phenotyping including investigation of 50 SIMs as well as ex-vivo phenotypes of NK-cells, CD3+, CD4+, CD8+ and regulatory T cells in 40 patients with viral and autoimmune chronic liver diseases was performed. The patients' cognitive functions were assessed using an extensive battery of neuropsychological testing. RESULTS AND CONCLUSION: Overall, our data indicate that while SIMs are significantly up-regulated, NK- and T-cells are less-activated in patients with neuropsychiatric symptoms accompanying chronic inflammatory liver diseases compared to patients without these symptoms. Moreover, HCV patients showed a unique pattern of immune alterations as compared to patients with HBV, autoimmune hepatitis and primary biliary cirrhosis. These findings hint towards potential mechanisms explaining these symptoms in patients with chronic liver diseases.

Hyperferritinemia and hypergammaglobulinemia predict the treatment response to standard therapy in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic hepatitis with an increasing incidence. The majority of patients require life-long immunosuppression and incomplete treatment response is associated with a disease progression. An abnormal iron homeostasis or hyperferritinemia is associated with worse outcome in other chronic liver diseases and after liver transplantation. We assessed the capacity of baseline parameters including the iron status to predict the treatment response upon standard therapy in 109 patients with untreated AIH type 1 (AIH-1) in a retrospective single center study. Thereby, a hyperferritinemia (> 2.09 times upper limit of normal; Odds ratio (OR) = 8.82; 95% confidence interval (CI): 2.25-34.52) and lower immunoglobulins (<1.89 times upper limit of normal; OR = 6.78; CI: 1.87-24.59) at baseline were independently associated with the achievement of complete biochemical remission upon standard therapy. The predictive value increased when both variables were combined to a single treatment response score, when the cohort was randomly split into a training (area under the curve (AUC) = 0.749; CI 0.635-0.863) and internal validation cohort (AUC = 0.741; CI 0.558-0.924). Patients with a low treatment response score (<1) had significantly higher cumulative remission rates in the training (p<0.001) and the validation cohort (p = 0.024). The baseline hyperferritinemia was accompanied by a high serum iron, elevated transferrin saturations and mild hepatic iron depositions in the majority of patients. However, the abnormal iron status was quickly reversible under therapy. Mechanistically, the iron parameters were not stringently related to a hepatocellular damage. Ferritin rather seems deregulated from the master regulator hepcidin, which was down regulated, potentially mediated by the elevated hepatocyte growth factor. In conclusion, baseline levels of serum ferritin and immunoglobulins, which are part of the diagnostic work-up of AIH, can be used to predict the treatment response upon standard therapy in AIH-1, although confirmation from larger multicenter studies is pending.

The Third Signal Cytokine Interleukin 12 Rather Than Immune Checkpoint Inhibitors Contributes to the Functional Restoration of Hepatitis D Virus-Specific T Cells.

BACKGROUND: Hepatitis D virus (HDV) infection affects 15-20 million individuals worldwide and causes severely progressive hepatitis. It is unknown to what extent cellular immune responses contribute to liver disease and control of viral replication in HDV infection. METHODS: Immune cell frequencies and phenotypes were determined in 49 HDV-infected patients, 25 individuals with hepatitis B virus monoinfection and 18 healthy controls. T-cell proliferative and cytokine-producing capacities were analyzed by stimulation with overlapping peptides spanning the large HDV antigen. To restore T-cell responses, blocking antibodies (anti-cytotoxic T-lymphocyte antigen 4, anti-programmed death ligand 1) or proinflammatory cytokines (interleukin [IL] 12) were used. RESULTS: Immune cell frequencies and phenotypes did not vary between the groups. Exclusively, the senescence marker CD57 was significantly up-regulated in CD8+ T cells from patients with hepatitis delta. HDV-specific T-cell proliferation and cytokine production were weak and could only partly be rescued by blockade of the programmed death 1 pathway. However, a more robust and consistent increase in HDV-specific CD4+ and CD8+ T-cell responses was evident when the third signal cytokine IL-12 was added, which also affected cytomegalovirus- and Epstein-Barr virus-specific T cells. CONCLUSIONS: This investigation of virus-specific T-cell immunity in patients with HDV infection, the largest to date, revealed premature aging of immune cells and impaired T-cell functionality. This could be restored by blocking inhibitory pathways and, in particular, by supplementing with IL-12.

Direct-Acting Antiviral-Induced Hepatitis C Virus Clearance Does Not Completely Restore the Altered Cytokine and Chemokine Milieu in Patients With Chronic Hepatitis C.

BACKGROUND: Persistent infection with hepatitis C virus (HCV) causes profound alterations of the cytokine and chemokine milieu in peripheral blood. However, it is unknown to what extend these alterations affect the progression of liver disease and whether HCV clearance normalizes soluble inflammatory mediators. METHODS: We performed multianalyte profiling of 50 plasma proteins in 28 patients with persistent HCV infection and advanced stages of liver fibrosis or cirrhosis and 20 controls with fatty liver disease. The patients were treated for 24 weeks with sofosbuvir and ribavirin and underwent sampling longitudinally. Ten patients experienced viral relapse after treatment cessation. RESULTS: The cytokine and chemokine expression pattern was markedly altered in patients with chronic HCV infection as compared to healthy controls and patients with nonalcoholic steatohepatitis. Distinct soluble factors were associated with the level of fibrosis/cirrhosis, viral replication, or treatment outcome. The baseline expression level of 10 cytokines distinguished patients with a sustained viral response from those who experienced viral relapse. While the majority of upregulated analytes declined during and after successful therapy, HCV clearance did not lead to a restoration of parameters that were suppressed. CONCLUSIONS: Chronic HCV infection appears to disrupt the milieu of soluble inflammatory mediators even after viral clearance. Thus, HCV cure does not lead to complete immunological restitution.

Symptoms of anxiety and depression are frequent in patients with acute hepatitis C and are not associated with disease severity.

OBJECTIVE: Neuropsychiatric symptoms of hepatitis C virus (HCV) infection and during peginterferon α therapy have been investigated in the chronic stage of the infection, but have not been described during the acute phase of the disease so far. We therefore evaluated anxiety and depression in patients with acute hepatitis C by the Hospital Anxiety and Depression Scale (HADS) within a clinical trial. METHODS: Data were analysed from the German Hep-Net Acute HCV-III study. Anxiety and depression were characterized by an anxiety (HADS-A) and a depression subscale (HADS-D). More than eight points in each subscale were considered clinically relevant. Data were prospectively collected at baseline, end of treatment and at the end of the study. RESULTS: At baseline, a HADS-A above eight points was observed significantly more frequently than a HADS-D above eight points [n=23/103 (22%) vs. n=12/103 (12%); P=0.041].A pathological HADS-A or HADS-D score did not correlate with age, sex, IL28B genotype, the probable mode of infection, HCV genotype or severity of disease as investigated by alanine aminotransferase and bilirubin levels.Antiviral therapy did not influence anxiety as 12/50 (24%) of patients had HADS-A above 8 at the end of therapy. The proportion of patients with HADS-D above eight points increased from 12% at baseline to 24% (n=12/50) at the end of therapy (P=0.06). HADS results were not associated with lost to follow-up or sustained virological response rates. CONCLUSION: HADS data in acute HCV infection indicate that anxiety and depression do not correlate with severity of the disease, mode of acquisition, lost to follow-up and sustained virological response rates.

Type I Interferon Elevates Co-Regulatory Receptor Expression on CMV- and EBV-Specific CD8 T Cells in Chronic Hepatitis C.

Hepatitis C virus (HCV) readily sets up persistence in a large fraction of infected hosts. Mounting epidemiological and immunological evidence suggest that HCV's persistence could influence immune responses toward unrelated pathogens and vaccines. Nonetheless, the fundamental contribution of the inflammatory milieu during persistent HCV infection in impacting immune cells specific for common pathogens such as CMV and EBV has not been fully studied. As the co-regulatory receptors PD-1, Tim-3, and 2B4 have all been shown to be vital in regulating CD8(+) T cell function, we assessed their expression on CMV/EBV-specific CD8(+) T cells from patients with chronic hepatitis C (CHC) and healthy controls ex vivo and upon stimulation with virus-specific peptides in vitro. Total and CMV/EBV-specific CD8(+) T cells expressing PD-1, Tim-3, and 2B4 were highly enriched in patients with CHC compared to healthy individuals ex vivo. In vitro peptide stimulation further potentiated the differential co-regulatory receptor expression of PD-1, Tim-3, and 2B4, which then culminated in an enhanced functionality of CMV/EBV-specific CD8(+) T cells in CHC patients. Comprehensively analyzing plasma cytokines between the two cohorts, we observed that not only was IFNα-2a dominant among 21 other inflammatory mediators elevated in CHC patients but it also correlated with PD-1 and Tim-3 expressions ex vivo. Importantly, IFNα-2a further caused upregulation of these markers upon in vitro peptide stimulation. Finally, we could prospectively study patients receiving novel IFN-free antiviral therapy. Here, we observed that treatment-induced clearance of HCV resulted in a partial reversion of the phenotype of CMV/EBV-specific CD8(+) T cells in patients with CHC. These data reveal an alteration of the plasma concentrations of IFNα-2a together with other inflammatory mediators during CHC, which appeared to pervasively influence co-regulatory receptor expression on CMV/EBV-specific CD8(+) T cells.

Altered effector functions of NK cells in chronic hepatitis C are associated with IFNL3 polymorphism.

Interferon α-mediated effector functions of NK cells may contribute to the control of HCV replication and the pathogenesis of liver disease. The single-nucleotide polymorphism rs12979860 near IFNL3 (previously known as IL28B) is important in response to IFN-α treatment and in spontaneous resolution of acute hepatitis C. The role of the IFNL3 polymorphism in NK cell function is unclear. Thus, we investigated the role of IFNL3 polymorphism in type I IFN-dependent regulation of NK cell functions in patients with cHC and healthy control subjects. We demonstrated a marked polarization of NK cells toward cytotoxicity in response to IFN-α stimulation in patients with hepatitis C. That TRAIL up-regulation was present, particularly in patients with the IFNL3-TT allele, was supported by a shift in the pSTAT-1:pSTAT-4 ratios toward pSTAT-1. In patients bearing the IFNL3-TT allele, NK cell effector function correlated with liver disease activity. In contrast, higher cytokine production of NK cells was observed in healthy individuals with the IFNL3-CC genotype, which may support spontaneous HCV clearance in acute infection. Overall, these findings show that the role of NK cells may differ in chronic infection vs. early antiviral defense and that the IFNL3 genotype differentially influences NK cell function.

Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+ T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses.

UNLABELLED: T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8(+) T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show that in vitro HCV NS3-1073-specific CD8(+) T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073-specific CD8(+) T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8(+) T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8(+) T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed an in vitro response toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine. IMPORTANCE: Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8(+) T cells are abundant in HCV-seronegative individuals but that their repertoire is highly diverse due to the involvement of both naive precursors and cross-reactive memory cells of different specificities, which can influence the response to vaccines. The data may emphasize the need to personalize immune-based therapies based on the individual's T cell repertoire that is present before the immune intervention.

Primary biliary acids inhibit hepatitis D virus (HDV) entry into human hepatoma cells expressing the sodium-taurocholate cotransporting polypeptide (NTCP).

BACKGROUND: The sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry. METHODS: HuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR. RESULTS: Addition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA. CONCLUSIONS: Primary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes.

In vitro stimulation with HBV therapeutic vaccine candidate Nasvac activates B and T cells from chronic hepatitis B patients and healthy donors.

Hepatitis B virus (HBV) chronic infections remain a considerable health problem worldwide. The standard therapies have demonstrated limited efficacy, side effects or need life-long treatments. Nowadays therapeutic vaccination is a promising option. Recently, we developed a new vaccine formulation called Nasvac, based on the combination of surface and core antigens from HBV. Clinical trials already performed showed good efficacy in virus control. However, the exact mode of action of Nasvac formulation remains unclear. So far the functional impairment of DCs during persistent HBV infection is a controversial issue. On the other hand, it is known that B cells may function as antigen presenting cells (APC) activating T cells. The hepatitis B core antigen contained in Nasvac vaccine is able to bind and activate a high frequency of naive human B cells. In the present study the surface expression of activation and exhaustion markers on B cells and the subsequent activation of T cells after in vitro stimulation with Nasvac antigens were evaluated in chronic HBV patients and healthy donors. B- and T-cell phenotype and proliferation were assessed by flow cytometry. Our results indicate that in contrast to exhaustions markers B cell activation markers were increased on both study groups after Nasvac stimulation. A shift toward an activation phenotype was observed for both B and T cells. The present work suggests that B cells could act as efficient APCs for Nasvac antigens in humans, which might suggest the use of activated B cells as immunotherapeutic strategy for chronic hepatitis B.

A heterogeneous hierarchy of co-regulatory receptors regulates exhaustion of HCV-specific CD8 T cells in patients with chronic hepatitis C.

BACKGROUND & AIMS: The functionality of virus-specific T cells is regulated by a sophisticated network of an expanding repertoire of co-regulatory receptors, which could be harnessed for immunotherapeutic applications. However, targeting particular pathways during persistent virus infections has resulted in variable outcomes. The extent to which T cell exhaustion can be reversed, by targeting multiple co-regulatory pathways, still remains not fully investigated. METHODS: We analysed the phenotype and in vitro functionality of HCV-specific CD8(+) T cells expressing PD-1, CTLA-4, TIM-3 or 2B4 either alone or in various combinations and compared expression levels to those of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) specific T cells in peripheral blood mononuclear cells (PBMCs) from the same cohort of patients with chronic hepatitis C (CHC) infection. RESULTS: Blockade and/or crosslinking of distinct co-regulatory pathways in exhausted HCV-specific CD8(+) T cells resulted in rather diverse and individualized T cell responses, irrespective of the type and number of receptors targeted. Overall, in vitro manipulations of these pathways yielded three response possibilities: (i) total non-response (ii) good single blockade response and (iii) good dual/multiple blockade response, with each comprising approximately one-third of the patients tested. The diversity of the in vitro responsiveness of HCV-specific T cells was reflected by an enormous ex vivo phenotypic heterogeneity. Despite this broad heterogeneity, HCV-specific CD8(+) T cells differed from EBV- and CMV-specific T cells in particular by TIM-3 expression, which also correlated with liver disease activity and viral load. CONCLUSIONS: HCV-specific CD8(+) T cell functionality, upon co-regulatory receptor manipulations, was characterized by an individual pattern of responses in patients with CHC, suggesting that treatment approaches, targeting these receptors, should consider inter-individual differences and be personalized.

PEG-IFN alpha but not ribavirin alters NK cell phenotype and function in patients with chronic hepatitis C.

BACKGROUND: Ribavirin (RBV) remains part of several interferon-free treatment strategies even though its mechanisms of action are still not fully understood. One hypothesis is that RBV increases responsiveness to type I interferons. Pegylated Interferon alpha (PEG-IFNa) has recently been shown to alter natural killer (NK) cell function possibly contributing to control of hepatitis C virus (HCV) infection. However, the effects of ribavirin alone or in combination with IFNa on NK cells are unknown. METHODS: Extensive ex vivo phenotyping and functional analysis of NK cells from hepatitis C patients was performed during antiviral therapy. Patients were treated for 6 weeks with RBV monotherapy (n = 11), placebo (n = 13) or PEG-IFNa-2a alone (n = 6) followed by PEG-IFNa/RBV combination therapy. The effects of RBV and PEG-IFNa-2a on NK cells were also studied in vitro after co-culture with K562 or Huh7.5 cells. RESULTS: Ribavirin monotherapy had no obvious effects on NK cell phenotype or function, neither ex vivo in patients nor in vitro. In contrast, PEG-IFNa-2a therapy was associated with an increase of CD56bright cells and distinct changes in expression profiles leading to an activated NK cell phenotype, increased functionality and decline of terminally differentiated NK cells. Ribavirin combination therapy reduced some of the IFN effects. An activated NK cell phenotype during therapy was inversely correlated with HCV viral load. CONCLUSIONS: PEG-IFNa activates NK cells possibly contributing to virological responses independently of RBV. The role of NK cells during future IFN-free combination therapies including RBV remains to be determined.

Compromised function of natural killer cells in acute and chronic viral hepatitis.

BACKGROUND: Natural killer (NK) cells are an integral part of the innate immune system. They have been suggested to play an important role in both defense against viral hepatitis and the pathogenesis of other liver diseases. METHODS: NK cells from 134 individuals including patients with acute hepatitis B and C as well as chronic hepatitis B, C, and delta (D) patients were studied. RESULTS: Infection with viral hepatitis was associated with increased frequencies of NK cells in the peripheral blood; that NK cells showed a less activated phenotype and were compromised in cytolotytic function and cytokine production in all viral hepatitis infections: Hepatitis virus infections did not alter NK cell differentiation, and the activity and severity of liver disease were reflected by alterations of NK cell surface receptors as demonstrated by principal component analysis. CONCLUSION: NK cell phenotypic and functional alterations can equally be observed in HBV, HCV, and HDV infections. Instead, patterns of NK cell alterations differ in acute and chronic infections. Thus, our data suggest a common mechanism in the alteration of NK cell phenotype and function with unique variations that depend on disease activity rather than virus-specific factors.

Delayed versus immediate treatment for patients with acute hepatitis C: a randomised controlled non-inferiority trial.

BACKGROUND: Early treatment of acute hepatitis C virus (HCV) infection with interferon alfa monotherapy is very effective, with cure rates of greater than 85%. However, spontaneous clearance of HCV occurs in 10-50% of cases. We aimed to assess an alternative treatment strategy of delayed antiviral therapy in patients who do not eliminate the virus spontaneously compared with immediate treatment. METHODS: In our open-label phase 3 non-inferiority trial, we enrolled adults (≥18 years) with acute hepatitis C but no HIV or hepatitis B co-infection at 72 centres in Germany. We randomly allocated patients with symptomatic acute hepatitis C (1:1) to receive immediate pegylated interferon alfa-2b treatment for 24 weeks or delayed treatment with pegylated interferon alfa-2b plus ribavirin (for 24 weeks) starting 12 weeks after randomisation if HCV RNA remained positive. We used a computer-generated randomisation sequence and block sizes of eight, stratified by bilirubin concentration. We assigned all asymptomatic patients to immediate treatment with pegylated interferon alfa-2b for 24 weeks. The primary endpoint was sustained HCV RNA negativity in all randomly allocated participants who completed screening (intention-to-treat analysis), with a non-inferiority margin of 10%. For the primary analysis, we calculated the virological response of patients in the immediate and delayed treatment groups and an absolute risk difference stratified by bilirubin status. The trial was stopped early on advice from the study advisory committee because of slow recruitment of participants. This study is registered, number ISRCTN88729946. FINDINGS: Between April, 2004, and February, 2010, we recruited 107 symptomatic and 25 asymptomatic patients. 37 (67%) of 55 symptomatic patients randomly allocated to receive immediate treatment and 28 (54%) of 52 symptomatic patients randomly allocated to receive delayed treatment had a sustained virological response (difference 13·7%, 95% CI -4·6 to 32·0; p=0·071). 18 (72%) of 25 asymptomatic patients had a sustained virological response. 22 (42%) of 52 symptomatic patients allocated to receive delayed treatment did not complete follow-up compared with 20 (25%) of 80 symptomatic or asymptomatic patients assigned immediate treatment (p=0·037). 11 symptomatic patients (21%) assigned delayed treatment had spontaneous HCV clearance. 14 patients who received delayed pegylated interferon alfa-2b plus ribavirin treatment and completed follow-up achieved sustained virological response. INTERPRETATION: Delayed treatment is effective although not of equal efficacy to immediate treatment; coupled with the rate of spontaneous clearance it can reduce unnecessary treatment in closely monitored populations. Immediate treatment seems preferable in populations where loss to follow-up is great. FUNDING: German Network of Competence on Viral Hepatitis (HepNet, funded by the German Federal Ministry of Education and Research, grants 01KI0102, 01KI0401, and 01KI0601), MSD, Schering-Plough.

NK cells in hepatitis C: role in disease susceptibility and therapy.

Hepatitis C is caused by infection with the hepatitis C virus (HCV), a single-stranded RNA virus, which was first described in 1989. Hepatitis C is a major global health burden with approximately 150 million people chronically infected worldwide. Chronic HCV infection is a main cause of liver cirrhosis and hepatocellular carcinoma. Current treatment of hepatitis C is based on the administration of peg-IFN-α and ribavirin. However, this therapy can be associated with severe side effects. The role of NK cells in the pathogenesis of acute and chronic liver disease in HCV infection has only been studied in recent years, but those studies indicate an important role for this cell type. NK cells are a major component of the innate arm of the immune system. We summarize the current knowledge on NK cell phenotype and function in acute and chronic HCV infection. Moreover, we discuss the role of NK cells in IFN-α-based antiviral therapy. Understanding the mode of function and role of NK cells during HCV infection and therapy will become even more important in the near future, when new IFN-α-free treatment regimens become available.

Hepatitis E virus (HEV)-specific T-cell responses are associated with control of HEV infection.

Hepatitis E virus (HEV) infection is usually self-limited but may lead to acute hepatitis and rarely to fulminant hepatic failure. Persistent HEV infections have recently been described in organ transplant recipients receiving immunosuppressive medications, suggesting that HEV is controlled by adaptive immune responses. However, only few studies have investigated HEV-specific T-cell responses and immune correlates for the failure to clear HEV infection have not been established so far. We investigated T-cell responses against HEV in 38 subjects including anti-HEV-positive (exposed, n = 9) and anti-HEV-negative (n = 10) healthy controls, 12 anti-HEV-positive but HEV RNA-negative organ transplant recipients, and seven transplant recipients with chronic hepatitis E. Proliferation as well as cytokine production of CD4+ and CD8+ T cells was studied after stimulation with overlapping peptides spanning all proteins encoded by HEV-open reading frame (ORF)2 and HEV-ORF3. We show that (1) strong and multispecific HEV-specific T-cell responses are present in exposed healthy controls, and to a lesser extent also in recovered patients after transplantation; (2) that these responses are absent in patients with chronic hepatitis E but become detectable after viral clearance; and (3) that HEV-specific T-cell responses can be restored in vitro by blocking the PD-1 or CTLA-4 pathways. However, a combination of PD-1 and CTLA-4 blockade had no synergistic effects. We conclude that chronic hepatitis E is associated with impaired HEV-specific T-cell responses and suggest that enhancing adaptive cellular immunity against HEV might prevent persistent HEV infections.

Hepatitis D virus-specific cytokine responses in patients with chronic hepatitis delta before and during interferon alfa-treatment.

BACKGROUND: Hepatitis delta is caused by infection with the hepatitis D virus (HDV) and is considered the most severe form of viral hepatitis. Treatment options for hepatitis delta are limited, with only 25% of patients responding to interferon (IFN)-alfa-based therapies. The role of the adaptive immune system in controlling HDV infection during spontaneous or treatment-induced viral clearance is not well understood. METHODS: We studied HDV-specific cytokine production of peripheral blood mononuclear cells stimulated with HDV peptide pools as well as serum cytokine levels in well-characterized patients with chronic HDV infection before and during pegylated-interferon-alfa±adefovir therapy. RESULTS: Hepatitis D virus-specific interleukin (IL)-2, IFN-γ-, interferon-inducible protein-10 and IL-10-responses were detectable in 53%, 35%, 65% and 6% of hepatitis delta patients. HDV-specific IFN-γ responses tended to be more common in patients with low HDV viral loads. HDV-specific cytokine responses declined during pegylated (PEG)-IFNa therapy and patterns of changes were associated with the treatment response. Serum cytokine levels also showed distinct changes during PEG-IFNa treatment. CONCLUSION: We suggest that cellular HDV-specific immune responses contribute to the control of HDV infection and that cytokine responses may indicate response to type-I-IFN-based antiviral therapy of hepatitis delta.

Dual function of the NK cell receptor 2B4 (CD244) in the regulation of HCV-specific CD8+ T cells.

The outcome of viral infections is dependent on the function of CD8+ T cells which are tightly regulated by costimulatory molecules. The NK cell receptor 2B4 (CD244) is a transmembrane protein belonging to the Ig superfamily which can also be expressed by CD8+ T cells. The aim of this study was to analyze the role of 2B4 as an additional costimulatory receptor regulating CD8+ T cell function and in particular to investigate its implication for exhaustion of hepatitis C virus (HCV)-specific CD8+ T cells during persistent infection. We demonstrate that (i) 2B4 is expressed on virus-specific CD8+ T cells during acute and chronic hepatitis C, (ii) that 2B4 cross-linking can lead to both inhibition and activation of HCV-specific CD8+ T cell function, depending on expression levels of 2B4 and the intracellular adaptor molecule SAP and (iii) that 2B4 stimulation may counteract enhanced proliferation of HCV-specific CD8+ T cells induced by PD1 blockade. We suggest that 2B4 is another important molecule within the network of costimulatory/inhibitory receptors regulating CD8+ T cell function in acute and chronic hepatitis C and that 2B4 expression levels could also be a marker of CD8+ T cell dysfunction. Understanding in more detail how 2B4 exerts its differential effects could have implications for the development of novel immunotherapies of HCV infection aiming to achieve immune control.

Interferon-alpha-induced TRAIL on natural killer cells is associated with control of hepatitis C virus infection.

BACKGROUND & AIMS: Pegylated interferon-alpha (PEG-IFNalpha), in combination with ribavirin, controls hepatitis C virus (HCV) infection in approximately 50% of patients by mechanisms that are not completely understood. Beside a direct antiviral effect, different immunomodulatory effects have been discussed. Natural killer (NK) cells might be associated with control of HCV infection. We examined the effects of IFNalpha on human NK cells and its relevance to HCV infection. METHODS: We performed gene expression profiling studies of NK cells following stimulation of peripheral blood mononuclear cells with IFNalpha. We evaluated IFNalpha-induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression using flow cytometry analyses of NK cells isolated from patients with acute or chronic hepatitis C that had received PEG-IFNalpha therapy. RESULTS: TRAIL was among the most up-regulated genes after IFNalpha stimulation of NK cells from healthy controls. After in vitro stimulation with IFNalpha, CD56(dim) NK cells from patients who had responded to PEG-IFNalpha therapy expressed higher levels of TRAIL than cells from patients with chronic hepatitis C. TRAIL expression, ex vivo, was inversely correlated with HCV-RNA levels during the early phase of PEG-IFNalpha therapy. In patients with acute hepatitis C, TRAIL expression on CD56(bright) NK cells increased significantly compared with cells from controls. In in vitro studies, IFNalpha-stimulated NK cells eliminated HCV-replicating hepatoma cells by a TRAIL-mediated mechanism. CONCLUSIONS: IFNalpha-induced expression of TRAIL on NK cells is associated with control of HCV infection; these observations might account for the second-phase decline in HCV-RNA levels during PEG-IFNalpha therapy.