Kinetics and intracellular pathways required for major histocompatibility complex II-peptide loading and surface expression of a fluorescent hapten-protein conjugate in murine macrophage

A fluorescent antigen, FITC(10)BSA, that is sensitive to several of the bio chemical processes involved in antigen processing was constructed. In combi nation with both flow cytometry and subcellular fractionation, the unique p robe provided new details regarding the kinetics and intracellular pathways involved in antigen processing in murine macrophage. These studies suggest ed that macrophage utilized multiple vesicles as opposed to a few specific organelles for major histocompatibility complex (MHC) type II-peptide loadi ng and transport. Although newly formed MI-IC II-peptide complexes were det ected in cathepsin D-positive, lysosomal associated membrane glycoprotein ( LAMP-1)-positive lysosomes, MHC II-peptide loading also occurred in transfe rrin receptor-positive endosomes. Interestingly, MHC II-fluoresceinated com plexes were only observed in transferrin receptor-positive organelles as op posed to MHC II-unlabelled peptide complexes which were detected in traditi onal early lysosomal compartments. More importantly, MHC II-peptide complex es were monitored in light transferrin receptor-positive fractions followin g their initial appearance in dense endosomal/lysosomal fractions. Control experiments suggested that these complexes represented intermediates in the process of migrating to the cell surface through a retrograde pathway with in the macrophage.