Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)-DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers, The proportion of double particles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor-MHC interactions, The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate, The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.