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© The Rockefeller University Press, 0021-9525/1998//71 $5.00
The Journal of Cell Biology, Volume 140, Number 1, , 1998 71-79

Article

Detection of Dimers of Dimers of Human Leukocyte Antigen (HLA)–DR on the Surface of Living Cells by Single-Particle Fluorescence Imaging



Richard J. Cherry, Keith M. Wilson, Kathy Triantafilou, Peter O'Toole, Ian E.G. Morrison, Patricia R. Smith, and Nelson Fernández

Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom

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°C than at 22°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.

Abbreviations used in this paper: CLIP, class II–associated invariant peptide; Cy3.29-OSu, 5,5'-disulphato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1'-ethyl-3,3,3',3'-tetramethylindocarbocyanine; HLA, human leukocyte antigen; LDL, low-density lipoprotein; MHC, major histocompatibility complex; PE, R-phycoerythrin; SMCC, succinimidyl trans- 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; TCR, T cell receptor.

This research was supported by Biotechnology and Biological Sciences Research Council and the University of Essex Research Promotion Fund.


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