Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids.

Fluorescent lipid probes were used to track lipid trafficking between parent RBL cells and exosomes. We have checked the intracellular labeling of exosomes ("in vivo labeling") from parent cell incubated with either Bodipy-Cer, Bodipy-PC, or NBD-PC. Bodipy-PC labeled equally cells and exosomes, whereas Bodipy-Cer, a Golgi marker, was enriched in exosomes. Golgi membranes participated effectively in exosome biogenesis since cell incubation with brefeldin A leads to a modified phospholipid/protein ratio in exosomes. At the opposite, NBD-PC, a plasma membrane marker weakly labeled exosome membranes. Sorting of subpopulations indicated that the MHC-II containing exosomes were enriched in Bodipy-PC, whereas tetraspanin(CD 63 or CD81)-containing exosomes are essentially labeled with Bodipy-Cer and Bodipy-PC. These results indicated that RBL released two main subpopulations of exosomes that can be discriminated by their protein and lipid contents. When the bulk of exosomes was labeled after their purification ("in vitro labeling") with either of the above-mentioned lipid probes, the Bodipy-Cer was the only one to incorporate noticeably in all the subpopulations, indicating that the previous results obtained during "in vivo labeling" monitored real intracellular lipid trafficking between organelles and exosomes. Bodipy-Cer was further used as a tool to measure the respective amounts of each subpopulations. CD63, MHC II, and CD81-containing exosomes accounted for 47%, 32%, and 21%, respectively, of total exosomes.

Secretory granules of mast cells accumulate mature and immature MHC class II molecules.

Bone marrow-derived mast cells as well as dendritic cells, macrophages and B lymphocytes express major histocompatibility complex (MHC) class II molecules. In mast cells, the majority of MHC class II molecules reside in intracellular cell type-specific compartments, secretory granules. To understand the molecular basis for the localisation of MHC class II molecules in secretory granules, MHC class II molecules were expressed, together with the invariant chain, in the mast cell line, RBL-2H3. Using electron and confocal microscopy, we observed that in RBL-2H3 cells, mature and immature class II molecules accumulate in secretory granules. Two particular features of class II transport accounted for this intracellular localization: first, a large fraction of newly synthesized MHC class II molecules remained associated with invariant chain fragments. This defect, resulting in a slower rate of MHC class II maturation, was ascribed to a low cathepsin S activity. Second, although a small fraction of class II dimers matured (i.e. became free of invariant chain), allowing their association with antigenic peptides, they were retained in secretory granules. As a consequence of this intracellular localization, cell surface expression of class II molecules was strongly increased by cell activation stimuli which induced the release of the contents of secretory granules. Our results suggest that antigen presentation, and thereby antigen specific T cell stimulation, are regulated in mast cells by stimuli which induce mast cell activation.