LBP18: HLA-PEPTIDE BINDING ANALYSIS BY THE CELL-SURFACE EXPRESSINO ASSAY

The interaction of HLAproteins with peptides is usually analyzed by the in vitro binding assay or the T-cell activation assay. Although widely used, there are some limitations to these assays, such as the difficulty to handle hydrophobic peptides and the potential inter-assay variations of the binding parameters. In this study, we have devised a cell-surface expression assay, which estimates the peptide- HLAinteraction by measuring the cell-surface expression levels of HLA. This assay utilizes the general property of MHC to be expressed at high levels when stabilized by peptides. Methods HLA-DQα -stable cells were established using retroviral vector pMXs-puro (Kitamura et al. (2003) Exp Hematol 31:1007) and packaging cells PLAT-E (Morita et al. (2000) Gene Ther 7:1063). The HLA-DQα -stable cells were transduced with pMXs-IG/ HLA-DQβ , in which the model peptide was inserted between the signal sequence and the mature protein sequence of HLA-DQB1(modified from Kozono, et al. (1994) Nature 369:151). The cell-surface HLA-DQexpression was measured by flow cytometry using GFP as an internal control. Results Using this assay, we validated the interactions of HLA-DQwith the known high- and low-affinity peptides. The cell-surface HLA-DQincreased greatly in the presences of the high-affinity peptides such as insulin B (1-15) for DQ0602 (Ettinger et al. (1998) J Immunol 160: 2365) compared to the low-affinity peptides or to the potential non-binders. The interaction of HLA-DQwith a series of self- and non-self-peptides such as CLIP and insulin B were also analyzed. The surface-expression assay will be useful to estimate the HLA-peptide interactions for a large numbers of alleles and peptides, and to identify the self- and non-self-peptides that bind to the disease-associated HLAallele products.