The Journal of Immunology CD8 + T Cell Control of Hepatitis B Virus Replication: Direct Comparison between Cytolytic and Noncytolytic Functions Sandra Phillips, Shilpa Chokshi, Antonio Riva, Alexander Evans, Roger Williams, and Nikolai V. Naoumov 1 Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell–mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8 + T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8 + T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus- specific CD8 + T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8 + T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8 + T cells produce an array of cytokines, among which IFN-g and TNF-a are responsible for HBV inactivation in the target cells. Blockade of IFN-g and TNF-a abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-g and rTNF- a resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8 + T cells efficiently control HBV replication by non- cytolytic mechanisms, and this effect is mediated by IFN-g and TNF-a. The Journal of Immunology, 2010, 184: 287–295. H epatitis B virus (HBV) is a hepatotropic, noncytopathic virus that can cause hepatitis, cirrhosis, and hepatocel- lular carcinoma. Approximately 350 million people worldwide are chronically infected with HBV with various levels of viral replication (1–3). It is now established that the host immune response plays a major role in the outcome of HBV infection (4– 7). During acute HBV infection, the development of a strong cellular immune response, directed to multiple viral Ags, is as- sociated with the resolution of HBV infection and life-long anti- viral immunity. In contrast, the presence of a weak and narrowly focused cellular immune response is unable to control HBV rep- lication, leading to viral persistence and progressive liver injury. HBV-specific CD8 + T cells are believed to play a critical role in the control of HBV replication but are also implicated in the pathogenesis of the disease by destruction of infected liver cells (1, 3, 8, 9). This Ag- specific killing of infected hepatocytes was initially believed to be the main mechanism by which CD8 + T cells control HBV infection. However, this concept was chal- lenged by a series of studies in HBV transgenic mice (10, 11) and HBV-infected chimpanzees (12, 13), which revealed the mecha- nism of noncytolytic inhibition of HBV replication, i.e., that HBV- specific CD8 + T cells could inhibit HBV replication without lysis of infected hepatocytes. Upon activation, these immune cells were shown to produce cytokines, such as IFN-g and TNF-a, which suppressed HBV gene expression and replication without de- stroying the infected hepatocytes. This key antiviral mechanism of noncytolytic HBV control, mediated by CD8 + T cells, has not been fully examined using human effector and target cells; in particular, the relative involvement of the two processes, a direct killing of HBV-infected cells and the intracellular HBV in- activation without cell lysis, is not well understood. In this study, we established an in vitro coculture model to determine the rel- ative contribution and balance between the cytolytic and non- cytolytic effector functions that HBV-specific CD8 + T cells use to control HBV replication. Materials and Methods Culture of 2.2.15 cells The human hepatoma cell line 2.2.15, derived from HepG2 cells, is stably transfected with HBV DNA and supports full HBV replication with pro- duction and secretion of viral Ags and infectious virions (14). In a pre- liminary experiment, we confirmed that 2.2.15 cells are HLA-A2 positive and, therefore, can express A2-restricted HBV peptides within MHC class I. For this purpose, 2.2.15 cells were stained with FITC-labeled Ab to A2 (Serotec, Oxford, U.K.) and analyzed by FACSort using Cell Quest soft- ware (BD Biosciences, Oxford, U.K.). For cell culture, 2.2.15 cells were seeded in 24-well tissue culture plates and grown for 3 d to reach con- fluency under positive selection with 0.4 mg/ml Geneticin (Invitrogen, Paisley, U.K.) in DMEM (Invitrogen) containing 10% FCS (Invitrogen), 500 U/ml penicillin, 500 mg/ml streptomycin (Invitrogen), and 2 mM L- glutamine (Invitrogen). Generation of HBV-specific CD8 1 T cell line and clone PBMCs were isolated by standard density centrifugation from patients who were positive for HLA-A2 and who spontaneously cleared hepatitis B surface Ag after acute hepatitis B. The HLA-A2–restricted epitope between amino acids 18–27 of the HBV nucleocapsid has been established as the key immunodominant epitope associated with control of HBV replication Institute of Hepatology, University College London, London, U.K. 1 Current address: Immunology and Infectious Diseases, Novartis Pharma AG, Basel, Switzerland. Received for publication August 20, 2009. Accepted for publication November 1, 2009. Address correspondence and reprint requests to Sandra Phillips, Institute of Hepatol- ogy, University College London, 69-75 Chenies Mews, London WC1E 6HX, U.K. E- mail address: s.phillips@ucl.ac.uk The online version of this article contains supplemental material. Abbreviations used in this paper: HBV, hepatitis B virus; HBc 18–27 , HBcore 18–27 epitope; ICS, intracellular cytokine staining; LDH, lactate dehydrogenase; PD-1, programmed death-1. 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