Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B D. Vassilopoulos, 1 I. Rapti, 2 M. Nikolaou, 1 E. Hadziyannis 1 and S. J. Hadziyannis 2 1 Academic Department of Medicine, Athens University School of Medicine, Hippokration General Hospital, Athens, Greece; and 2 Department of Medicine and Hepatology, Henry Dunant Hospital, Athens, Greece Received February 2008; accepted for publication March 2008 SUMMARY. The immunopathogenesis of hepatitis B e anti- gen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-c (IFN-c) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with over- lapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182–191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-c producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-c assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained. Keywords: acute hepatitis B, cellular immunity, chronic hepatitis B, hepatitis B surface antigen, hepatitis B virus, interferon c. INTRODUCTION The course of chronic hepatitis B virus (HBV) infection is determined by the continuous interplay between viral replication and localized host immune responses in the liver [1–3]. A number of studies over the last two decades have clearly defined the successive phases of chronic HBV infection [4,5]. Most chronically infected HBV patients after the loss of hepatitis B e antigen (HBeAg), enter a phase of low viral replication and minimal liver inflammation, termed Ôinactive HBsAg carrier stateÕ [6,7]. A subset of these HBeAg negative patients (20–30%), shortly after or, more com- monly, years after HBeAg loss, demonstrate enhanced HBV replication (mainly form variants with decreased or absent capability of HBeAg production) associated with liver necroinflammation defined as ÔHBeAg negative chronic hepatitis B (CHB)Õ [8]. Currently, HBeAg negative infection is the predominant form of chronic HBV infection worldwide [9,10]. Despite the significant progress in our understanding of the epidemiology and clinical course of HBeAg negative chronic HBV infection, the immunopathogenetic mechanisms that are responsible for liver necro-inflam- mation, i.e. CHB, in approximately one third of chronically infected HBeAg negative patients have not been eluci- dated. Studies examining the immune responses either in the periphery or in the liver in this subset of patients are limited [11]. Similarly, it is unclear how most HBsAg inactive carriers are able to maintain low HBV replication and minimal hepatocyte damage for decades and even a lifetime. Abbreviations: BFA, brefeldin-A; CHB, chronic hepatitis B; cpm, counts per minute; EBV, Epstein–Barr virus; HAI, histologic activity index; HBeAg, hepatitis B e antigen; FBS, foetal bovine serum; HBV, hepatitis B virus; ICS, intracellular cytokine staining; IFN-c, interferon-c; MHC, major histocompatibility class; PBS, phosphate buffered saline; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin; SI, stimulation index; SFC, spot forming cells. Correspondence: Stephanos J. Hadziyannis, Professor of Medicine, Department of Medicine and Hepatology, Henry Dunant Hospital, 107 Messogion Ave., 115 26 Athens, Greece. E-mail: hadziyannis@ ath.forthnet.gr Journal of Viral Hepatitis, 2008, 15, 817–826 doi:10.1111/j.1365-2893.2008.00996.x Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd