Single particle imaging of cell-surface HLA-DR tetramers.

Single particle imaging is a method for studying the mobility and associations of receptors in cell surfaces of living cells. In principle, it involves labeling or tagging receptors with small fluorescent particles and imaging with a highly sensitive imaging system that allows resolution at nanometer scale. The fluorescent particle can be either unconjugated or conjugated to, for example, an antibody specific for a receptor. A variant of the method was originally applied to investigate low-density lipoprotein receptors (LDL) using LDL particles labeled with lipophilic fluorescent probes [l]. We have recently extended this approach to study the association state of immunoreceptors, primarily focused on Major Histocompatibility Complex (MHC) Class I1 molecules expressed on the surface of transfected human fibroblasts [2]. These fibroblasts are transfected with HLA-DR A and B genes and express non-covalently associated HLA DR ci and p chains at the cell surface; the cells were kindly supplied by Prof. R. Lechler (RPMS, Hammersmith Hospital, London). In order to specifically tag the Class I1 molecules, Fab fragments derived from a monoclonal antibody specific for a monophorphic epitope were conjugated to , the phycobiliprotein R-phycoerythrin. Phycoerythrin are small (1 1 x 8 nm) naturally occurring isolated from cyanobacteria and algae which hnction in harvesting light for photosynthesis [3]. The particles emit sufficiently bright fluorescence, to permit imaging individual molecules. The method for studying receptor associations by single particle imaging is described in detail elsewhere [4]. Briefly. the probe is first imaged after binding to polylysine-coated microscope slides in the absence of cells. The imaging system is a cooled slow-scan coupled charge device (CCD) camera attached to a fluorescence microscope. The image of an individual particle is a diffraction-limited spot which covers a number of pixels. The pixel intensities are fitted to a 2-dimensional Gaussian function which provides an accurate measurement of the spot intensity. Fig. l (A) shows a histogram of the intensities of spots measured in this way. The same procedure is then employed for probes bound to cells. Fig. I(B) shows a typical result obtained for cells at 22°C. The intensity distribution shown in Fig. l(a) corresponds to single particles. The width of the distribution is largely determined by noise in the measurement. The on-cell distribution clearly contains a significant number of spots of higher intensity than observed for single particles. These must therefore correspond to clusters of two or more particles arising from associated receptors To quantify the data, the histogram is deconvolved into components consisting of 1 -particle, 2-particle and 3-particle components. For the data shown in Fig. I(b) this yields 75 % of spots corresponding to single particles and 25 Yo to double particles. Higher size clusters are negligible. The receptors are labeled with saturating concentration of probe and thus these numbers are indicative of the proportion of HLA-DR heterodimers and tetramers on the cell surface. The antibody recognizes the heterodimers so that the tetramer binds two particles. Although the existence of MHC Class I1 tetramers is suggested by X-ray crystallography of the hydrophilic domain, the present experiments provide the first clear evidence that