The pivotal role of target cells in modulating efficacy of antiviral CD8 T cell immunity in the liver

Question:

CD8 T cells are essential for successful control of viral infections in the liver. Alongside direct MHC-I-dependent recognition and elimination of infected target cells, CD8 T cells can eliminate infected cells via a non-canonical CD8 T cell effector function. Thereby antigen-specific CD8 T cells recognize antigen on non-infected, but antigen cross-presenting liver sinusoidal endothelial cells leading to secretion of TNF. Soluble TNF induces selectively apoptosis in infected hepatocytes indicating a decisive role of the infected target cells. Here, we aimed to investigate the role of infected target cells during antiviral immunity in the liver.

Methods:

We used xCELLigence technology to determine killing of primary human and murine hepatocytes in real-time after infection with recombinant adenoviruses and/or hepatitis B virus after treatment with the CD8 T cell effector molecules TNF and FasL. Impedance measurement via xCELLigence technology was supplemented by live cell microscopy and bioluminescent caspase assays. We verified the results in elaborated co-culture systems of virus-infected hepatocytes and CD8 effector cells enabling to dissect antigen recognition from hepatocyte sensitization. To demonstrate the sensitization of virus-infected hepatocytes towards CD8 T cell effector molecules in vivowe treated virus-infected mice with recombinant TNF and FasL.

Results:

We detected that murine as well as human primary hepatocytes are more susceptible to CD8 T cell effector molecules after adenoviral infection. Also in co-culture systems, we observed enhanced killing of virus-infected hepatocytes by CD8 T cells compared to non-infected hepatocytes, despite similar antigen presentation. To our surprise, HBV infected hepatocytes were not sensitized towards death induced by CD8 T cell effector molecules (TNF and FasL). Furthermore, by co-infection of primary human hepatocytes with HBV and adenoviruses we were not able to detect induction of hepatocyte death anymore. Therefore we conclude that HBV counteracts the sensitization as active immune escape mechanism. Treatment of adenovirus infected mice with recombinant TNF or FasL led to hepatocyte death shown by elevated serum ALT levels and immunohistochemistry, thereby proving thein vivorelevance of the cell culture results. Mice infected with an adenovirus, expressing full-length HBV genome, did not show signs of liver pathology after injection of TNF or FasL confirming the active immune escape of HBV from non-canonical CD8 T cell effector function.

Conclusion:

Our results have far reaching consequences for understanding CD8 T cell mediated immunity towards hepatocytes. First, our results demonstrate that low numbers of virus-specific CD8 T effector cells are able to eliminate high numbers of infected target cells that are highly sensitive to killing. Second, the non-responsiveness of non-infected cells towards death-inducing signaling pathways reduces collateral damage and auto-immunity, but at the same time may enable escape of tumor cells that also lack this sensitization. Third, we demonstrate here that HBV has developed an immune escape for this target cell sensitization Our results will be instrumental to develop mechanism-based strategies to overcome persistent HBV infection.