The liver is a site at which antigen encounter can result in either T cell immunity or tolerance, but the influences that determine the outcome of T cell priming are not well understood. In this exploratory/developmental proposal, we will create a set of novel tools based on recombinant adeno- associated virus (rAAV) to explore this issue. Specifically, we will modify the rAAV capsid and promoter to alter cell tropism, we will engineer in a series of fluorescent reporters that will contrast with liver autofluorescence, and we will buld vectors using either a high-affinity antigenic peptides, or sequence variants, or a control peptide Other vectors will promote or inhibit direct antigen presentation by the transduced hepatocytes, or cause the death of transduced hepatocytes thus rendering their antigens available for cross-presentation. Using all of these tools, we will test the impact of antigen density, affinity, cell ype-specific expression and direct versus cross-presentation on primary CD8+ and CD4+ T cell activation, on the development of effector cells, and on memory. These studies will create novel tools of wider usefulness. In particular, the red fluorophores such as mCherry are optimized for in vivo microscopy, Furthermore, if in this study we are able to define optimized priming conditions for memory T cell priming, in future studies we will engineer in pathogen-encoded antigens to test whether AAV could be used as a vaccine vehicle against hepatocellular pathogens.
Infections of liver cells impose a large disease burden both in the USA and globally. The way the immune system responds to such infections is poorly understood. In the project we will use techniques from the field of gene therapy to create new tools that will increase understanding of immune responses to infections of the liver.
|Crispe, Ian N (2016) Hepatocytes as Immunological Agents. J Immunol 196:17-21|
|Mohar, Isaac; Brempelis, Katherine J; Murray, Sara A et al. (2015) Isolation of Non-parenchymal Cells from the Mouse Liver. Methods Mol Biol 1325:3-17|