Mechanisms of endogenous antigen processing by the MHC class II pathway: studies with viral and cellular proteins

The immune system?s T cells recognise short protein fragments of proteins (called epitopes) that are made by various processing pathways and then displayed on the surface of the body?s cells. If a cell is infected with a virus or is cancerous then epitopes from viral or cancer-specific cellular proteins are displayed. T cells specific for these epitopes can then destroy the affected cell. Vaccines and immunotherapies are being developed that aim to harness T cells to selectively destroy virus-infected or cancerous cells in this way. It therefore becomes important to understand the processing pathways how epitopes are made, since these control the supply and composition of the epitopes displayed on the cell surface.

To study how epitopes are made from proteins found in different locations within cells I have used Epstein-Barr virus (EBV) as a model. I have made T cells specific for different types of EBV protein and used these to study processing. This virus is associated with several different cancers therefore the lessons learnt from this system have direct medical impact as well as teaching us more about the immune system in general. I have found that some proteins are transferred between cells before processing while others are processed within the cell itself even though these are all located in the same cellular compartment, the nucleus. Furthermore my work also suggests that proteins within the nucleus are unavailable for processing by an important internal processing pathway called macro-autophagy. Here I intend to continue working in the EBV system to learn more about the rules governing how proteins are processed within cells. I will then extend the work to study how cellular proteins expressed in cancer cells are processed and in turn recognised by T cells and how these processes differ from viral proteins. Finally, I will study a range of inherited fatal neurological conditions, including Huntington?s disease, caused by proteins that form toxic aggregates. These aggregates may be cleared by some of the processing pathways and I wish to establish whether the rules learned from the EBV system will apply here also, whether nuclear-localised aggregates, like EBNA1, evade processing by macro-autophagy and if an alternative pathway called chaperone mediated autophagy can compensate.

While exogenously-acquired proteins are the main source of antigens presented to CD4+ T cells, it is known that many proteins expressed endogenously within MHC class II-positive cells can also access the MHC II presentation pathway. This proposal seeks to further our understanding of different pathways that govern the processing of such proteins, in particular the influence that intracellular location might have on processing route.

Firstly, I plan to build on my recent work studying the processing for CD4+ T cell recognition of Epstein-Barr virus (EBV) antigens within EBV-transformed lymphoblastoid cell lines (LCLs). I have CD4+ T cell clones to a range of epitopes in the virus-coded nuclear antigens EBNAs, in the latent membrane proteins LMPs 1 and 2, and in a cytoplasmic protein BHRF1 now known to be a latent cycle antigen. Our evidence shows that EBNA 2 and the EBNA3 proteins are processed via the inter-cellular transfer of as-yet-poorly-defined antigenic species, whereas EBNA1, the LMPs and BHRF1 are processed by different intracellular pathways. We have found that nuclear-localised EBNA1 is only processed by the intracellular macro-autophagy (MA) pathway when it is experimentally relocalised into the cytoplasm. The work will focus on determining the relative importance of MA, chaperone-mediated autophagy (CMA) and plasma membrane recycling to the endo/lysosome system as MHC II access routes for these proteins, and the effect of antigen or epitope re-location on that process.

Secondly, I shall move on to study the processing of cellular proteins of medical importance. I will investigate the processing of WT1 and STEAP, two tumour-associated antigens expressed in a wide range of malignancies that are good CD4 targets and compare their processing with the EBV proteins. I will go on to study antigen processing in the context of neurological diseases caused by poly-glutamine (polyQ) containing proteins forming toxic aggregates within cells. Degradative pathways such as MA, which can clear such aggregates, are of interest as potential therapeutic interventions for such conditions. In animal models disease severity is reduced when naturally nuclear-resident polyQ proteins are relocalised out of the nucleus. The objective will be to test the notion that MA cannot clear nuclear-localised aggregates and determine whether CMA can act as a substitute pathway to clear nuclear-localised aggregates.