The long-range goals of this project are to understand the biochemistry and biology of antigen presentation by class II MHC molecules. The project centers on the presentation of peptides from the protein hen-egg white lysozyme (HEL). The investigator's laboratory has extensive experience studying HEL and a large technical armamentarium for this purpose. The study is primarily based on analysis of the dominant peptide from HEL selected by I-Ak (=Ak) molecules of antigen presenting cells, during intracellular HEL processing. The features that make the 48-62 peptide dominant (=DGSTDYGILQINSRW) have been identified. These include a robust anchor residue (D52) and main chain interactions between the peptide and residues in the Ak peptide-combining site. Using this information, how changes in the anchor residue of the peptide influence a number of binding parameters and immunogenicity will be analyzed. Analysis of APC bearing 48-62 tethered to the Akb chain is included. Based on this information, HEL molecules bearing changes in the main anchor position will be engineered and will be examined both in cell lines, and in transgenic mice expressing HEL in their APC, for the expression of dominant, and sub-dominant peptides. These peptides will be examined biochemically, including the use of new approaches for identifying minor epitopes. This manipulation will inform us whether dominant peptides control the expression of other HEL peptides. The experiments will be extended using transgenic mice expressing HEL in their APC as a transmembrane protein. These experiments should make it possible to examine how the display of major epitopes influence immunological and tolerogenic parameters. The strength of this goal is that it is based on a combination of biochemical and immunological parameters. The last goal is to examine different conformational states of peptide-MHC class II complexes that are relevant for T cell recognition. T cells that recognize peptides derived from HEL processing (type A); and unique ones that exclusively recognize MHC-peptide complexes formed by direct binding of peptide (type B) have been identified. These distinct types of recognition are important to understand because of their relevance for vaccination, autoimmunity and processing. In brief, the goal is to interpret and point out the significance of the biochemical parameters that underlie peptide-MHC interactions.
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