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

本研究的目的是分析食物抗原在哪里进行抗原呈递，并在摄入后被T细胞识别，使用可溶性类型的T细胞受体（TCR）。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，我们分析了当通过改变用于刺激T细胞的抗原的结构和剂量来改变TCR来源的信号时T细胞功能的改变。利用杆状病毒表达系统制备了一种可溶性T细胞受体（sTCR），该受体来源于对β-Lg（G1.19）具有特异性的鼠CD 4 ^+ T细胞克隆。将生物素化酶（BirA）识别的序列添加到sTCR β链恒定区的下游。我们证实了纯化的sTCR在生物素存在下被BirA生物素化，并且通过加入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）119- 133区的肽段。G1.19细胞对抗原的免疫应答被sTCR抑制，表明sTCR竞争性地抑制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，通过流式细胞术检测到特异性信号，表明sTCR可以特异性地结合抗原肽和MHC II类分子的复合物。

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.