Trafficking of spontaneously endocytosed MHC proteins

Class I MHC protein primarily presents endogenous antigen but also may pres ent exogenous antigen. Here, we investigated the intracellular pathway of s pontaneously internalized class I MHC protein by confocal microscopy. beta( 2)-microglobulin (beta(2)m), labeled with a single fluorophore, was exchang ed at the surface of B cell transfectants to specifically mark cell surface and endocytosed class I MHC protein. Intracellular beta(2)m colocalized wi th fluorophore-conjugated transferrin, implying that class I MHC protein en docytosed into early endosomes. These endosomes containing fluorescent beta (2)m were found close to or within the Golgi apparatus, marked by fluoresce nt ceramide. Even after 24 hr of incubation, very little fluorescent Plm wa s found in intracellular organelles stained by DiOC(6), marking the endopla smic reticulum, or fluorophore-conjugated low density lipoprotein, marking late endosomes and lysosomes. Fluorophore-conjugated superantigens (staphyl ococcal enterotoxin A and B), presumed to enter cells hound to class Il MHC protein, also were found to endocytose into beta(2)m-containing early endo somes. Staining with mAb and use of transfectants expressing MHC protein at tached to green fluorescent protein confirmed the presence of intracellular compartments rich in both class I and rt MHC protein and demonstrated that class I and II MHC protein also colocalize in discrete microdomains at the cell surface. These cell surface microdomains also contained transferrin r eceptor and often were juxtaposed to cholesterol-rich lipid rafts. Thus, cl ass I and II MHC protein meet in microdomains of the plasma membrane and en docytose into early endosomes, where both may acquire and present exogenous antigen.