This application examines antigen presentation restricted by the Major Histocompatibility Gene Complex. We use primarily mononuclear phagocytes as antigen-presenting cells and T cell hybridomas that interact with hen egg lysozyme (HEL) in the context of I-A k molecule. We have selected hybrids that recognize an epitope of HEL contained in a linear 16-amino acid peptide (positions 46-61). The 46-61 epitope is normally hidden in native HEL, but is subsequently exposed during handling by the macrophage and is then recognized by T cells. Previous studies indicated that HEL was presented in a temperature-dependent process sensitive to lysosomotropic agents (""""""""processing stage""""""""); peptide 46-61 did not require processing and, moreover, was presented by macrophages lightly fixed in formaldehyde. Our purpose is to find how, and in what site in the macrophage, the HEL molecule is processed, whether there is recycling of immunogenic molecules from intracellular vesicles to the plasma membrane of the macrophage, what portion of the HEL molecule is presented and then recognized by the T cell, and the relationship between the immunogen and the Ia molecules. We describe three interrelated technical approaches. The first is an analysis of a panel of monoclonal antibodies directed to 46-61 and to denatured HEL (both currently available) and to a putative HEL-Ia complex. These monoclonal will be used to follow the biochemical changes in the HEL molecule in the macrophage and the topology of the changes, to detect a possible Ia-antigen association, and to attempt blocking T cell recognition. The second technical approach uses the 46-61 peptide presented by fixed macrophages. To study the interaction with membrane proteins and/or I-A molecules, we will use 125-I-labeled peptide or peptide linked to a photoactivatable crosslinker or incorporated with membrane proteins/I-A in liposomes. We hope to distinguish if there is a direct peptide-Ia linkage or if the 46-61 peptide first binds to protein which eventually interacts or associates with I-A. Finally, synthetic peptides are being produced in order to investigate the structural requirements for presentation. Our initial studies suggest that the peptide may require two features: a hydrophobic sequence required for anchorage and a polar region. Different synthetic peptides should tell us about the minimal length, critical amino acid residues, whether there is competition for a macrophage site or the T cell receptor, and about the fine specificity of our large panels of monoclonal antibodies.
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