Elsevier

Clinical Immunology

Volume 129, Issue 3, December 2008, Pages 419-427
Clinical Immunology

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

https://doi.org/10.1016/j.clim.2008.07.029Get rights and content

Abstract

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.

Introduction

Worldwide 400 million people suffer from a chronic hepatitis B virus (HBV) infection and approximately 1 million people die annually from HBV-related disease. In the majority of adult patients, infection with HBV manifests itself as a self-limiting acute hepatitis, which confers protective immunity and causes no further disease. However, in 10% of infected adults, HBV infection becomes persistent, which may result in severe liver disease and may lead to premature death as a consequence of decompensated liver failure or hepatocellular carcinoma [1], [2]. In patients with an acute self-limiting HBV infection, a multispecific CD4+ and CD8+ T cell response with a type 1 cytokine profile is important to control the infection [3], [4]. Importantly, patients with a chronic HBV infection lack such a vigorous multispecific T cell response [4], [5]. At present, it is still unclear why the immune system fails in chronic HBV infections.

Many mechanisms have been described by which pathogens can escape immune control to ensure conditions that allow their survival. One of the mechanisms that are exploited by pathogens, such as Leishmania major, Mycobacterium tuberculosis, Plasmodium spp., Cytomegalovirus and Human Immunodeficiency Virus, is by enhancing regulatory mechanisms that normally terminate an immune response (reviewed in [6]). In this, specific T cells with a regulatory function have been shown to suppress virus specific immune responses, and contribute to the development of persistence. Various populations of these regulatory T cells (Treg) have been described on the basis of the production of immunosuppressive cytokines, such as interleukin (IL)-10 or transforming growth factor (TGF)-beta, or on the basis of high expression of CD25, and the forkhead family transcription factor 3 (FoxP3) [7], [8], [9]. CD4+CD25+ Treg represent a stable population of human peripheral lymphocytes with a frequency between 3–10% of the total CD4+ population [10].

In recent years, Treg have been implicated in regulating the immune response during HBV infections. We and others showed that patients with a chronic HBV infection have increased percentages of CD4+CD25+FoxP3+ Treg in their peripheral blood compared to healthy controls and individuals who have resolved their HBV infection [11], [12], [13], [14]. CD4+CD25+ T cells isolated from peripheral blood of these patients are capable of inhibiting the HBV-specific CD4+ and CD8+ T cell response in vitro[11], [12], [15]. Furthermore, we showed that reduction of the viral load by potent antiviral therapy resulted in a decrease of the frequency of peripheral blood Treg [16]. Combined, these findings suggest an important role for Treg during either the establishment or the maintenance of chronic HBV infection. However, little information is available on the involvement of intrahepatic Treg in HBV infection. This information is crucial since the liver is the primary site of infection. Gaining more insight in the phenotype and function of intrahepatic Treg may provide valuable information for development of novel treatment strategies. In this study we performed an in depth analysis of Treg populations in the liver in chronic HBV patients, by assessing their phenotype and frequency in relation to disease parameters.

Section snippets

Patients

32 chronic HBV patients underwent percutaneous needle liver biopsy as part of their diagnostic evaluation (Table 1). All patients had detectable HBV DNA levels in serum. Patients co-infected with human immunodeficiency virus, hepatitis A virus, hepatitis C virus or hepatitis D virus, and patients with a resolved viral hepatitis were excluded from this study. Excess tissue from liver biopsies (not needed for histological examination) was used to isolate intrahepatic leukocytes. In addition, a

The liver contains a population of CD4+CD25-FoxP3+ cells which is not detected in peripheral blood

To determine the presence of FoxP3-expressing regulatory T cells in the liver, cell suspensions prepared from diagnostic liver samples were assessed by flow cytometry. On the basis of CD45 expression parenchymal cells were excluded from the analysis, as described before [18]. As shown in Fig. 1, CD3+CD4+FoxP3+ Treg are abundantly present in the liver of chronic HBV patients. We consistently found that the fluorescence intensity of the FoxP3 staining on intrahepatic Treg was higher than on

Discussion

The present study shows that regulatory T cells in the liver of chronic HBV patients display a different phenotype than their circulating counterparts. Higher FoxP3 expression is observed on intrahepatic Treg from chronic HBV patients and a population of CD25-FoxP3+Treg is observed in the liver but not in the blood. We could not detect CD8+FoxP3+ regulatory T cells in liver nor blood from chronic HBV patients (data not shown). Higher levels of serum HBV DNA correlated with a higher frequency of

Acknowledgments

We would like to thank Dr. R. de Knegt, Dr. P. Taimr, Dr. H. van Buuren and Dr. J. Conchillo for their help with obtaining the liver biopsy samples. H.L.A. Janssen is a clinical fellow of the Netherlands Organization for Scientific Research (NWO, grant nr. 907-00-021) and recipient of a ZonMW Vidi Grant (grant nr.:917.56.329).

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