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 inve stigate 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 con sisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of sin gle and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers, The proportion of double par ticles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependen t equilibrium. These results are discussed in the context of a possibl e role for HLA-DR dimers of dimers in T cell receptor-MHC interactions , The technique is validated by demonstrating that fluorescence imagin g can distinguish between dimers and tetramers of human erythrocyte sp ectrin deposited from solution onto a solid substrate, The methodology will have broad applicability to investigation of the oligomeric stat e of immunological and other membrane-bound receptors in living cells.