STRUCTURAL HIERARCHY IN THE CLUSTERING OF HLA CLASS-I MOLECULES IN THE PLASMA-MEMBRANE OF HUMAN LYMPHOBLASTOID-CELLS

Major histocompatibility complex (MHC) class I antigens in the plasma membranes of human T (HUT-102B2) and B (JY) lymphoma cells were probed by immunochemical reagents using fluorescence, transmission electron, and scanning force microscopies. Fluorescent labels were attached to monoclonal antibodies W6/32 or KE-2 directed against the heavy chain o f HLA class I (A, B, C) and L368 or HB28 against the beta(2)-microglob ulin light chain. The topological distribution in the nanometer range was studied by photobleaching fluorescence resonance energy transfer ( pbFRET) on single cells. A nonrandom codistribution pattern of MHC cla ss I molecules was observed over distances of 2-10 nm. A second, nonra ndom, and larger-scale topological organization of the MHC class I ant igens was detected by indirect immunogold labeling and imaging by tran smission electron microscopy (TEM) and scanning force microscopy (SFM) . Although some differences in antigen distribution between the B- and T-cell lines were detected by pbFRET, both cell lines exhibited simil ar clustering patterns by TEM and SFM. Such defined molecular distribu tions on the surfaces of cells of the immune system may reflect an und erlying specialization of membrane lipid domains and fulfill important functional roles in cell-cell contacts and signal transduction.