Control of antigen cross-presentation through selective engagement of the mannose receptor

Dendritic cells (DC) are professional antigen presenting cells (APCs). They have a sentinel role in peripheral tissues whereby they capture and process antigens. These antigens within the DC are degraded into short peptide fragments and are loaded onto class I or class II major histocompatibility complex (MHC) molecules to be presented on the cell surface. This in turn activates naïve antigen specific T cells in the secondary lymphoid organs and thus initiates the adaptive immune response. Intracellular antigens are processed and presented on MHC class I molecules to activate cytotoxic T lymphocytes, whilst exogenous antigens are generally presented on MHC class II molecules which stimulates T helper cells. Remarkably DC are capable of presenting exogenous antigens such as those originating from cancer cells and viruse that don't readily infect cells, and present them through MHC class I molecules. This process is known as antigen cross-presentation. The DC ability to bridge the gap between exogenous antigen and the simulation of antigen-specific CTL via cross presentation highlight their central role in the induction of adaptive immunity. This makes DC an attractive therapeutic target for vaccine development, especially against viruses and tumours.

Mannosylated antigens endocytosed via the mannose receptor (MR) are efficiently cross-presented. Current evidence indicate that MR is polyubiquitinated upon internalisation of a mannosylated antigen, and recruits p97 AAA, a member of the endoplasmic reticulum-associated degradation (ERAD) machinery. Recruitment of p97 AAA facilitates the translocation of the exogenous antigen from the endosomes to the cytosol for processing through the MHC class I pathway. Although the overall process of MR-mediated cross-presentation is well established, its understanding at the cellular and molecular level is still poor. In our laboratory we have developed libraries of glycopolymers which can target the MR. Specifically, we have designed sulfated polymers which can target the cysteine rich domain (CR) of MR and mannosylated polymers which can target the 8 C-type lectin like domains (CTLDs) of MR. This will allow us to determine whether MR ligation by CR or CTLDs ligands will affect the trafficking of these polymers and MR within the cell and if MR polyubiquitination was differentially affected. Differential MR polyubiquitination could have consequences on MR-mediated cross-presentation. Furthermore, we can exploit these polymers to specifically target antigens to the MR on DCs for cross-presentation. If successful these polymers have the potential to be used in therapeutics, as novel ways of vaccine delivery. To explore this possibility we will conjugate fluorescent proteins such as GFP to the polymers. These polymer-GFP conjugates provide us with two specific benefits; primarily by using GFP we will be able to follow the uptake and processing of the antigen up to the point where it is degraded allowing us to uncover the cellular trafficking of MR mediated cross presentation. Secondly the GFP can act as a surrogate antigen as it can be modified through the addition of a specific epitope to stimulate CTLs. In addition we have access to different MR constructs that differ in their ability to be ubiquitinated and polyubiquitinated. These constructs will enable us to truly determine the role of MR polyubiquitination in cross-presentation.

Overall our investigation primarily involves in vitro work and biochemical analysis, microscopy and flow cytometry techniques to answer our questions. Our results will build a platform for in vivo work to look at how we can use these polymers as an innovative way of raising an immune response against a specific virus.