The helper T cell recognition of protein antigens requires that the antigen be processed and presented by an Ia expressing antigen presenting cell (APC). This involves the internalization of the antigen into an acidic intracellular vesicle where the antigen is denatured, often involving proteolysis, and the resulting processed antigen transported to and held on the APC surface where it is recognized by the specific T cell along with Ia. While the requirement for APC function in helper T cell activation is well documented, the molecular mechanisms underlying the phenomenon remain to be elucidated. This proposal represents an approach to further our understanding of these processes, taking advantage of recently developed native antigen- and peptide antigen- antibody conjugates which allow antigens to be targeted to APC surfaces. Such conjugates synthesized using the well characterized soluble globular protein antigen, cytochrome c, and a variety of antibodies specific for APC surface structures will provide a means of introducing appropriately radiolabeled native antigens into the APC in a directed fashion allowing an elucidation of the intracellular processing pathway. Several key questions will be address which include: Into which intracellular compartment is antigen transported? Where does the initial proteolytic cleavage event occur? What are the proteolytic products of that cleavage? What is the content of such vesicles with regard to proteolytic enzymes, Ia, and other potential accessory molecules? Can antigen enter the processing pathway by binding to any APC surface structure? Can intracellular cytoplasmically synthesized proteins enter the processing pathway? Peptides-antibody conjugates will be used to target the processed form of the antigen to the APC surface to investigate the mechanism by which peptides become associated with Ia and the T cell receptor. Both native antigen-and peptide- antibody conjugates will be tested for their efficacy as immunogens in vivo and the mechanisms underlying increased immunogenicity investigated. New information gained from the proposed studies will be applied toward the design of immunogens and therapeutic immunoconjugates for use in vivo.
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