Development of soluble TCR and soluble MHC/peptide compelx with high affinity for their ligands

在本项目中，我们试图开发具有高亲和力的可溶性MHC-II类/多肽复合体和可溶性TCR，以便直接鉴定和/或分离它们的配体。为了促进异源二聚体的形成，我们将亮氨酸拉链序列连接到TCR或MHC II类分子的α和β链胞外区域，并将抗原肽与MHC II类分子共价结合。在每种情况下，在一个亮氨酸拉链序列的C末端引入游离半胱氨酸，作为特定生物素化的靶标。最后，我们将生物素化的分子与链霉亲和素偶联，形成多价TCR或MHC II类/肽复合体。在Biaccore分析中，这种多组分的TCR或MHC II类/肽复合体的配体脱落率明显降低，并通过流式细胞仪检测到表达该配体的细胞。因此，该方法在T细胞发育、自身免疫、移植和肿瘤免疫等免疫学领域具有广泛的应用前景。

We tried in this project to develop soluble MHC class II/peptide complex and soluble TCR with so high affinity as to directly identify and/or isolate their ligands. To facilitate the heterodimer formation, we connected leucine zipper sequence to the extracellular domain of alpha and beta chain of TCR or MHC class II.In addition, the antigenic peptide was covalently bound to MHC class II molecules. In each case, free cysteine was introduced at the C-terminal of one of the leucine zipper sequence, which was used for the target for the specific biotinylation.Finally, we developed multivalent TCR or MHC class II/peptide complex by coupling the biotinylated molecule with streptavidin. Such multivatent TCR or MHC class II/peptide complex showed remarkably slow off-rate for their ligands in BIAcore analysis and detected the cells expressing the ligand by flow cytometry. Thus, this approach would have wide application for several immunological fields, including T cell development, autoimmunity, transplantation and tumor immunity.