Mark A.A. Claassen

Abundant numbers of regulatory T cells localize to the liver of chronic hepatitis C infected patients and limit the extent of fibrosis

BACKGROUND & AIMS Weak hepatitis C virus (HCV) specific immunity in peripheral blood has been shown to be partially controlled by regulatory T cells (Treg). However, little is known about Treg present in livers of HCV-infected patients, their association with clinical parameters, and immunopathology resulting in disease progression. METHODS The frequency and phenotype of CD4+FoxP3+ Treg, conventional CD4+ T cells, and the distribution of lymphocytes and leukocytes were studied by multi-color flowcytometry in liver and peripheral blood of 43 chronic HCV patients at different phases of liver disease. Comparisons between healthy blood and liver and correlations with disease parameters were made. RESULTS An extensive lymphocyte infiltration containing abundant numbers of CD4+FoxP3+ Treg was present in HCV-infected livers, while absent from healthy liver. Moreover, in all patients, intrahepatic CD4+FoxP3+ Treg showed a fully differentiated and highly activated phenotype on the basis of the surface markers CD45RO, CCR7, CTLA-4 and HLA-DR. These Treg were more numerous in those HCV-infected livers showing only limited fibrosis. However, HCV RNA loads or alanine transaminase levels did not correlate with CD4+FoxP3+ Treg frequencies. CONCLUSIONS Our data demonstrate that large numbers of highly activated and differentiated CD4+FoxP3+ Treg localize to the infiltrated chronic HCV-infected liver and may result in limiting the extent of fibrosis. This suggests that CD4+FoxP3+ Treg play a pivotal role in limiting collateral damage by suppressing excessive HCV-induced immune activation.

Intrahepatic regulatory T cells are phenotypically distinct from their peripheral counterparts in chronic HBV patients

Peripheral blood CD4+CD25+ regulatory T cells (Treg) prevent the development of strong HBV-specific T cell responses in vitro. In this study, we examined the phenotype of FoxP3+ regulatory T cells in the liver of patients with a chronic HBV infection. We showed that the liver contained a population of CD4+FoxP3+ cells that did not express CD25, while these cells were absent from peripheral blood. Interestingly, intrahepatic CD25-FoxP3+CD4+ T cells demonstrated lower expression of HLA-DR and CTLA-4 as compared to their CD25+ counterparts. Patients with a high viral load have a higher proportion of regulatory T cells in the liver, but not in blood, compared to patients with a low viral load. In conclusion, the intrahepatic Treg are phenotypically distinct from peripheral blood Treg. Our data suggest that the higher proportion of intrahepatic Treg observed in patients with a high viral load may explain the lack of control of viral replication.

Retention of CD4+ CD25+ FoxP3+ Regulatory T Cells in the Liver after Therapy-Induced Hepatitis C Virus Eradication in Humans

ABSTRACT Following infection with the hepatitis C virus (HCV), in most cases immunity fails to eradicate the virus, resulting in slowly progressing immunopathology in the HCV-infected liver. We are the first to examine intrahepatic T cells and CD4+ CD25+ FoxP3+ regulatory T cells (Treg) in patients chronically infected with HCV (chronic HCV patients) during and after antiviral therapy by collecting multiple aspiration biopsy samples from the liver at different time points. We found that intrahepatic Treg frequencies were increased upon alpha interferon and ribavirin administration in about 50% of chronic HCV patients, suggesting stronger regulation of intrahepatic immunity by Treg during antiviral therapy. After cessation of antiviral therapy, the frequency of intrahepatic Treg remained above baseline in the large majority of livers of individuals who successfully cleared the virus. The phenotype of those Treg that were retained in the liver months after therapy-induced clearance of HCV RNA indicated a reduced contribution of effector memory cells. Our findings, gathered by multiple samplings of the liver, indicate that successful antiviral therapy of chronic HCV patients does not lead to normalization of the local immune response to a resting state comparable to that for healthy livers. The continuous presence of high numbers of Treg, with a phenotype reflecting a relatively weak suppressive activity, suggests ongoing residual regulation of immunopathology. These findings provide important insight into the dynamics of the immune response to HCV, as well as the effect of therapy on intrahepatic immunity.

Role of T cell immunity in hepatitis C virus infections.

Chronic infections with the hepatitis C virus (HCV) are a major global health issue. Viral replication is restricted to hepatocytes, and occurs for decades at high replication rates. Over the last decade, it became accepted that HCV-specific CD4(+) and CD8(+) T cells are crucial for protective immunity to HCV. However, a characteristic feature of persistent HCV infection is the dysfunctional T cell response, and over recent years enormous progress has been made in understanding the mechanisms that dampen the antiviral T cell responses in blood and liver of chronic HCV patients and also impact disease progression.

Frequencies of circulating MAIT cells are diminished in chronic hCV, HIV and HCV/ HIV Co-Infection and do not recover during therapy

Objective Mucosal-associated invariant T (MAIT) cells comprise a subpopulation of T cells that can be activated by bacterial products and cytokines to produce IFN-γ. Since little is known on MAIT cells during HCV infection, we compared their phenotype and function in comparison to HIV and HCV/HIV co-infected patients, and determined the effect of IFN-α-based and direct-acting antiviral therapy on MAIT cells of HCV patients. Methods Blood samples from patients with chronic HCV (CHCV), virologically suppressed HIV, acute HCV/HIV co-infection (AHCV/HIV) and healthy individuals were examined by flowcytometry for phenotype and function of MAIT and NK cells. Results and Conclusions Compared to healthy individuals, the frequency of CD161+Vα7.2+ MAIT cells was significantly decreased in patients with CHCV, HIV and AHCV/HIV co-infection. CD38 expression on MAIT cells was increased in AHCV/HIV patients. MAIT cells were responsive to IFN-α in vitro as evidenced by enhanced frequencies of IFN-γ producing cells. IFN-α-based therapy for CHCV decreased the frequency of IFN-γ+ MAIT cells, which was still observed 24 weeks after successful therapy. Importantly, even after successful IFN-α-based as well as IFN-α-free therapy for CHCV, decreased frequencies of MAIT cells persisted. We show that the frequencies of MAIT cells are reduced in blood of patients with CHCV, HIV and in AHCV/HIV co-infection compared to healthy individuals. Successful therapy for CHCV did not normalize MAIT cell frequencies at 24 weeks follow up. The impact of HIV and HCV infection on the numbers and function of MAIT cells warrant further studies on the impact of viral infections and the antimicrobial function of MAIT cells.

Declining Hepatitis C Virus (HCV) Incidence in Dutch Human Immunodeficiency Virus-Positive Men Who Have Sex With Men After Unrestricted Access to HCV Therapy

Background Direct-acting antivirals (DAAa) cure hepatitis C virus (HCV) infections in 95% of infected patients. Modeling studies predict that universal HCV treatment will lead to a decrease in the incidence of new infections but real-life data are lacking. The incidence of HCV among Dutch human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) has been high for >10 years. In 2015 DAAs became available to all Dutch HCV patients and resulted in a rapid treatment uptake in HIV-positive MSM. We assessed whether this uptake was followed by a decrease in the incidence of HCV infections. Methods Two prospective studies of treatment for acute HCV infection enrolled patients in 17 Dutch HIV centers, having 76% of the total HIV-positive MSM population in care in the Netherlands. Patients were recruited in 2014 and 2016, the years before and after unrestricted DAA availability. We compared the HCV incidence in both years. Results The incidence of acute HCV infection decreased from 93 infections during 8290 person-years of follow-up (PYFU) in 2014 (11.2/1000 PYFU; 95% confidence interval [CI], 9.1-13.7) to 49 during 8961 PYFU in 2016 (5.5/1000 PYFU; 4.1-7.2). The incidence rate ratio of 2016 compared with 2014 was 0.49 (95% CI, .35-.69). Simultaneously, a significant increase in the percentage positive syphilis (+2.2%) and gonorrhea (+2.8%) tests in HIV-positive MSM was observed at sexual health clinics across the Netherlands and contradicts a decrease in risk behavior as an alternative explanation. Conclusions Unrestricted DAA availability in the Netherlands was followed by a 51% decrease in acute HCV infections among HIV-positive MSM.

Boceprevir, peginterferon and ribavirin for acute hepatitis C in HIV infected patients

BACKGROUND & AIMS Acute hepatitis C virus infections (AHCV) are prevalent among HIV positive men having sex with men and generally treated with pegylated interferon-alpha (PegIFN) and ribavirin (RBV) during 24weeks. The addition of a protease inhibitor could shorten therapy without loss of efficacy. METHODS We performed an open-label, single arm study to investigate the efficacy and safety of a 12-week course of boceprevir, PegIFN and RBV for AHCV genotype 1 infections in 10 Dutch HIV treatment centers. The primary endpoint of the study was achievement of sustained virological response rate at week 12 (SVR12) in patients reaching a rapid viral response at week 4 (RVR4) and SVR12 in the intent to treat (ITT) entire study population was the most relevant secondary endpoint. RESULTS One hundred twenty-seven AHCV patients were screened in 16 months, of which 65 AHCV genotype 1 patients were included. After spontaneous clearance in six patients and withdrawal before treatment initiation in two, 57 started therapy within 26 weeks after infection. RVR4 rate was 72%. SVR12 rate was 100% in the RVR4 group. SVR12 rate in the ITT group was 86% and comparable to the SVR12 rate of 84% in 73 historical controls treated for 24 weeks with PegIFN and RBV in the same study centers. CONCLUSION With the addition of boceprevir to PegIFN and RBV, treatment duration of AHCV genotype 1 can be reduced to 12 weeks without loss of efficacy. Given the high drug costs and limited availability of interferon-free regimens, boceprevir PegIFN and RBV can be a considered a valid treatment option for AHCV. ClinicalTrials.gov, number NCT01912495.

Gene Expression Profiling To Predict and Assess the Consequences of Therapy-Induced Virus Eradication in Chronic Hepatitis C Virus Infection

ABSTRACT Systems biology has proven to be a powerful tool to identify reliable predictors of treatment response in chronic hepatitis C virus (HCV) infection. In the present study, we studied patients with chronic HCV infection who responded to interferon (IFN)-based therapy, as evidenced by an absence of HCV RNA at the end of treatment, and focused on two issues that have not received much attention. First, we evaluated whether specific genes or gene expression patterns in blood were able to distinguish responder patients with a viral relapse from responder patients who remained virus negative after cessation of treatment. We found that patients with chronic HCV infection who were sustained responders and relapsers after IFN-based therapy showed comparable baseline clinical parameters and immune compositions in blood. However, at baseline, the gene expression profiles of a set of 18 genes predicted treatment outcome with an accuracy of 94%. Second, we examined whether patients with successful therapy-induced clearance of HCV still exhibited gene expression patterns characteristic of HCV or whether normalization of their transcriptome was observed. We observed that the relatively high expression levels of IFN-stimulated genes (ISGs) in patients with chronic HCV infection prior to therapy were reduced after successful IFN-based antiviral therapy (at 24 weeks of follow-up). These ISGs included the CXCL10, OAS1, IFI6, DDX60, TRIM5, and STAT1 genes. In addition, 1,428 differentially expressed non-ISGs were identified in paired pre- and posttreatment samples from sustained responders, which included genes involved in transforming growth factor beta (TGF-β) signaling, apoptosis, autophagy, and nucleic acid and protein metabolism. Interestingly, 1,424 genes with altered expression levels in responder patients after viral eradication were identified, in comparison to normal expression levels in healthy individuals. Additionally, aberrant expression levels of a subset of these genes, including the interleukin-32 (IL-32), IL-16, CCND3, and RASSF1 genes, were also observed at baseline. Our findings indicate that successful antiviral therapy for patients with chronic HCV infection does not lead to normalization of their blood transcriptional signature. The altered transcriptional activity may reflect HCV-induced liver damage in previously infected individuals. IMPORTANCE Tools to predict the efficacy of antiviral therapy for patients with HCV infection are important to select the optimal therapeutic strategy. Using a systems biology approach, we identify a set of 18 genes expressed in blood that predicts the recurrence of HCV RNA after cessation of therapy consisting of peginterferon and ribavirin. This set of genes may be applicable as a useful biomarker in clinical decision-making, since the number of genes included in the predictor is small and the correct prediction rate is high (94%). In addition, we observed that the blood transcriptional profile in patients with chronic HCV infection who were successfully treated is not normalized to the status observed in healthy individuals. Even 6 months after therapy-induced elimination of HCV RNA, gene expression profiles in blood are still altered in these patients with chronic HCV infection, strongly suggesting long-term modulation of immune parameters in previously infected patients.

The Intrahepatic T Cell Compartment Does Not Normalize Years After Therapy-Induced Hepatitis C Virus Eradication

Little is known about the immune status in liver and blood of patients with chronic hepatitis C virus (HCV) infection long after therapy-induced viral clearance. In this study, we demonstrate that, 4 years after clearance, regulation of HCV-specific immunity in blood by regulatory T cells (Tregs) and the immunosuppressive cytokines interleukin 10 and transforming growth factor β is still ongoing. Importantly, analysis of liver specimens collected 4 years after HCV clearance shows that intrahepatic Tregs are still present in all patients, suggesting that liver T cells remain regulated. Identifying mechanisms that regulate HCV-specific memory T-cell responses after clearance is highly relevant for the development of protective vaccines, especially in patients at high risk of reinfection.

Negative Regulation of Hepatitis C Virus Specific Immunity Is Highly Heterogeneous and Modulated by Pegylated Interferon-Alpha/Ribavirin Therapy

Specific inhibitory mechanisms suppress the T-cell response against the hepatitis C virus (HCV) in chronically infected patients. However, the relative importance of suppression by IL-10, TGF-β and regulatory T-cells and the impact of pegylated interferon-alpha and ribavirin (PegIFN-α/ribavirin) therapy on these inhibitory mechanisms are still unclear. We revealed that coregulation of the HCV-specific T-cell responses in blood of 43 chronic HCV patients showed a highly heterogeneous pattern before, during and after PegIFN-α/ribavirin. Prior to treatment, IL-10 mediated suppression of HCV-specific IFN-γ production in therapy-naive chronic HCV patients was associated with higher HCV-RNA loads, which suggests that protective antiviral immunity is controlled by IL-10. In addition, as a consequence of PegIFN-α/ribavirin therapy, negative regulation of especially HCV-specific IFN-γ production by TGF-β and IL-10 changed dramatically. Our findings emphasize the importance of negative regulation for the dysfunctional HCV-specific immunity, which should be considered in the design of future immunomodulatory therapies.