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, FITC10BSA, that is sensitive to several of the biochemical processes involved in antigen processing was constructed. In combination with both flow cytometry and subcellular fractionation, the unique probe provided new details regarding the kinetics and intracellular pathways involved in antigen processing in murine macrophage. These studies suggested that macrophage utilized multiple vesicles as opposed to a few specific organelles for major histocompatibility complex (MHC) type II-peptide loading and transport. Although newly formed MHC II-peptide complexes were detected in cathepsin D-positive, lysosomal associated membrane glycoprotein (LAMP-1)-positive lysosomes, MHC II-peptide loading also occurred in transferrin receptor-positive endosomes. Interestingly, MHC II-fluoresceinated complexes were only observed in transferrin receptor-positive organelles as opposed to MHC II-unlabelled peptide complexes which were detected in traditional early lysosomal compartments. More importantly, MHC II-peptide complexes were monitored in light transferrin receptor-positive fractions following 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 within the macrophage.