HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

Non-random distribution of interleukin receptors on the cell surface.

Spatial organization of cell surface proteins plays a key role in the process of transmembrane signalling. Receptor clustering and changes in their cell surface distribution are often determining factors in the final outcome of ligand-receptor interactions. There are several techniques for assessing the distribution of protein molecules. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining distance relationships of cell surface molecules. However, it does not provide information on the distribution of molecular clusters. Different kinds of microscopies fill this gap. The evaluation of the images provided by the listed techniques is often questionable. Herein we show the applicability of Ripley's K(t) function as a tool for analyzing the cell surface receptor patterns (Y. Nakamura, et al., Nature 1994, 369, 330-333). We have implemented an effective image processing algorithm for fast localization of gold labels on biological samples. We investigated spatial organization of Interleukin-2R alpha and -15R alpha (IL-2R alpha and IL-15R alpha) on a human CD4+leukaemia T-cell line, Kit225 FT7.10 by using transmission electron microscopy (TEM). TEM analysis showed co-clustering of the two types of alpha-chains even on the few-hundred-nanometer scale. The analysis of our data may contribute to our understanding the action of the IL-2/IL-15 receptor system in T-cell function.