Research Project
3193430
The Epstein-Barr Virus (EBV) Latent Infection Membrane Protein (LMP) is expressed on the surface of latently infected and appears to play a role in the T lymphocyte recognition of these cells. The purpose of this proposal is to define the regions of the EBV-LMP that are recognized by MHC class I-and class II-restricted mouse and human T cells and to further define the antigen processing pathways that lead to the expression of T cell epitopes for recognition in the context of MHC class I vs. class II molecules. As a model system LMP gene-transfected mouse M12 B lymphoma cells (expressing MHC-class I and II and LMP) will be used to produce and characterize a panel of class I and class II-restricted LMP-specific T cells clones. The regions of the EBV-LMP molecule that are recognized by the class I and class II restricted T cells will be determined by their ability to respond to a series of synthetic 8-24 amino acid peptides that correspond to the entire EBV-LMP molecule. Experiments will be conducted to determine if similar portions of the EBV-LMP molecule are recognized when animals are immunized with the isolated LMP molecule rather than with cells expressing the LMP on their cell surface. We will determine by virtue of the ability of the synthetic LMP-peptides to bind to isolated MHC molecules, which peptides contain potential T cell epitopes that were not detected by their ability to stimulate the LMP-specific T cell clones isolated. Peptides found to contain non-expressed T cell epitopes will be utilized in studies to determine why these epitopes are not expressed. Using LMP transfected HLA-A2 and A11 bearing American Burkett's lymphoma lines similar studies will be carried out using LMP-specific human T cell clones by testing their ability to respond to the battery of synthetic LMP peptides. Using the mouse system and an LMP gene-containing herpes simplex vector, we will employ chemical inhibitors of proteolytic enzyme activity as well as microinjection of isolated LMP or antibodies that inhibit protein degradation in order to more clearly define the pathways for antigen processing leading to class I vs. class II restricted T cell responses. In summary, the results of this study will provide comprehensive information about the regions of the EBV-LMP molecule that are recognized by class I and class II-restricted T cells as well as provide a better definition of the similarities and differences in the antigen processing pathways leading to the expression of class I and class II-restricted T cell epitodes.
