Dominance of cytolytic effector functions in hepatitis B virus-specific CD8+ T cell and NK cell mediated antiviral efficacy using hepatitis B virus infected hepatocytes overexpressing human sodium taurocholate cotransporting polypeptide

It is well accepted that virus-specific CD8+ T cells play an important role in the pathogenesis and clearance of hepatitis B virus (HBV) infection. However, so far little is known about the relative contribution of cytolytic versus non-cytolytic effector mechanisms to the inhibition of viral replication due to the lack of suitable cell culture models. Recently, hepatoma cells transduced with the HBV entry receptor human sodium taurocholate cotransporting polypeptide (hNTCP) were found to be susceptible to HBV. Thus, for the first time, this model allows the in vitro analysis of the interplay of virus-specific CD8+ T cells, NK cells and infected hepatocytes. Specifically, virus-specific CD8+ T cells were co-cultured with HBV infected HepG2 cells overexpressing hNTCP. Antiviral effects were determined by measuring cytoplasmic viral load, cytokine production and transaminase levels.

The co-culture experiments of effector and target cells revealed that HBV-specific effector functions of CD8+ T cells were strongly induced by infected hepatocytes transduced with hNTCP. These results indicate an efficient endogenous processing and presentation of viral proteins to virus-specific CD8+ T cells by HBV infected HepG2 cells. Importantly, viral replication was significantly inhibited by virus-specific CD8+ T cells (reduction of viral titres of more than 1 log scale after 4 days of co-culture) in concert with elevated transaminase levels suggesting the presence of cytolytic effector functions. The relative contribution of non-cytolytic effector functions, e.g. mediated by the secretion of interferon-γ, was evaluated by transwell experiments. Importantly, inhibition of viral replication was strongly reduced by the separation of virus-specific CD8+ T cells from infected target cells. Furthermore, no increases in transaminase levels were observed indicating the absence of CD8+ T cell induced cell lysis. Similar effects were observed in co-culture experiments performed with NK cells.

In sum, our results indicate that hNTCP overexpressing hepatocytes represent a unique tool to directly study virus-specific CD8+ T and NK cell mediated antiviral efficacy in vitro for the first time. Our results demonstrate that inhibition of HBV replication primarily occurs by cytotoxic effector mechanisms of HBV-specific CD8+ T cells which require direct cell-cell contact.