Effective vaccination requires stimulation of both the cytotoxic T cell and antibody-mediated immune response. To achieve stimulation of these responses, the immunogen must be processed into fragments that bind to molecules of the major histocompatibility complex (MHC), for recognition by specific T-cell receptors. The Class I and Class II MHC molecules bind fragments of antigen from different intracellular sources. Class I molecules generally bind peptides derived from proteins synthesized by the presenting cell and thereby present fragments of viral proteins being produced in an infected cell. In contrast, Class II molecules are capable of presenting peptides derived from exogenous, internalized proteins as well as peptides derived from endogenously synthesized proteins. T cell receptors on CD8+ cytotoxic T cells recognize antigenic peptides bound to Class I MHC molecules, while Class II-peptide complexes are recognized by CD4+ helper cells, correlating with the diversification of the peptide-binding properties of Class I and Class II molecules. The goal of this proposal are to 1) define the pathways of intracellular transport of MHC molecules that contribute to their binding of antigenic peptides, 2) determine the cellular mechanisms which control the intracellular distribution of MHC molecules, antigens and proteases, 3) identify the intracellular locations in which antigenic peptides associate with MHC molecules and 4) define the molecular processes involved in antigen binding. The proposed studies use both morphological and biochemical approaches to address these questions. Preliminary results from electron microscopy studies have indicated potential pathways for the processing of antigen and its association with Class II histocompatibility molecules. To determine the kinetics of Class II molecule transport through the endocytic pathway and the site(s) where antigenic peptides are bound, endosomes will be isolated from human B cell lines and analyzed for the presence of newly synthesized Class II molecules by immunoprecipitation. In addition, endosomal Class II molecules will be tested for their ability to present antigenic peptides to responding T cells, to determine where functional antigenic complexes form. The uptake of a defined antigen and the role of surface Ig in stimulating processing in the endocytic pathway will be investigated with assays for proteolytic activity in intracellular compartments and by further microscopy studies. The presentation of antigen by Class I MHC molecules will be studied using in vitro assays. An assay that measures the transport of peptides across microsomal membranes will be used to characterize the transporter that allows antigenic peptides generated in the cytoplasm to have access to assembling Class I molecules. In vitro translation and translocation of Class I molecules into microsomes will be used to identify the molecules involved in their folding and assembly, during which they associate with antigenic peptides. The information derived from these studies will contribute to understanding where and how MHC molecules bind antigenic peptides and is critical for the design and development of vaccines. In particular, these studies will identify specific routes that should be used for delivery of antigen depending on whether a T cell or B cell response (or both) is desired. In addition, MHC molecules that are not occupied by foreign antigen, bind self-peptides and present them at the cell surface. Normally, these self-peptides are not recognized, due to mechanisms of tolerance. When this tolerance breaks down, destructive autoimmune reactions develop. Identifying the intracellular sources of peptides presented by MHC molecules will therefore also contribute to understanding how autoimmune reactions might be prevented.
| Bresnahan, P A; Barber, L D; Brodsky, F M (1997) Localization of class I histocompatibility molecule assembly by subfractionation of the early secretory pathway. Hum Immunol 53:129-39 |
| Zia-Amirhosseini, P Z; Harris, R Z; Brodsky, F M et al. (1995) Hypersensitivity to nonsteroidal anti-inflammatory drugs. Nat Med 1:2-4 |
