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Analysis of food-antigen recognition by immune system using soluble T-cell receptor

使用可溶性 T 细胞受体分析免疫系统对食物抗原的识别

基本信息

批准号：
12660109
负责人：
TOTSUKA Mamoru
金额：
$ 0.9万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660109/
关键词：
T cell receptor β-Lactoglobulin Ovalbumin Food allergen Baculovirus expression system MHC class II molecule Antigen presentation Flow cytometry 食品アレルゲン昆虫細胞分泌発現腸管免疫抗原認識

项目摘要

This study aimed to analyze where food antigens are subjected to antigen presentation and recognized by T cells after ingested, using a soluble type of T cell receptor (TCR). The final aim of this study was to elucidate the mechanisms of immune recognition of food antigens. First, using β-lactoglobulin (β-Lg) and ovalbumin, primary food allergens, as model antigens, we analyzed the alteration of T cell functions when TCR-derived signals are altered by varying structure and dose of antigens used to stimulate the T cells. A soluble type of T cell receptor (sTCR) derived from a murine CD4^+ T cell clone specific for β-Lg, G1.19, was prepared using a baculovirus expression system. The sequence recognized by a biotinylation enzyme (BirA) was added downstream of the constant region of sTCR β chain. We confirmed that purified sTCR were biotinylated by BirA in the presence of biotin, and that multimer sTCR were formed by adding streptavidin. G1.19 cells recognize a peptide corresponding to 119-133 region of β-Lg(p119-133)in complex with I-A^b molecule. Proliferative response of G1.19 cells to the antigen was inhibited by adding sTCR to the culture, indicating that sTCR competitively inhibited the binding of TCR on G1.19 cells to the ligand. When the cells were stained with sTCR and fluorescence labeled anti-TCR Cβ antibodies in the presence of anti-MHC class II molecule, specific signals were detected by flow cytometry, indicating sTCR can specifically bind to the complex of the antigenic peptide and a MHC class II molecule.

本研究旨在利用可溶性 T 细胞受体 (TCR) 分析食物抗原在摄入后在何处进行抗原呈递并被 T 细胞识别。本研究的最终目的是阐明食物抗原的免疫识别机制。首先，使用主要食物过敏原 β-乳球蛋白 (β-Lg) 和卵清蛋白作为模型抗原，我们分析了当用于刺激 T 细胞的不同结构和剂量的抗原改变 TCR 衍生信号时，T 细胞功能的变化。使用杆状病毒表达系统制备源自对 β-Lg (G1.19) 具有特异性的鼠 CD4^+ T 细胞克隆的可溶型 T 细胞受体 (sTCR)。将生物素化酶 (BirA) 识别的序列添加到 sTCR β 链恒定区的下游。我们证实纯化的 sTCR 在生物素存在下被 BirA 生物素化，并且通过添加链霉亲和素形成多聚体 sTCR。 G1.19 细胞识别与 I-A^b 分子复合的 β-Lg (p119-133) 的 119-133 区域对应的肽。通过向培养物中添加sTCR，G1.19细胞对抗原的增殖反应受到抑制，表明sTCR竞争性抑制G1.19细胞上的TCR与配体的结合。当细胞在抗MHC II类分子存在下用sTCR和荧光标记的抗TCR Cβ抗体染色时，流式细胞术检测到特异性信号，表明sTCR可以特异性结合抗原肽和MHC II类分子的复合物。