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

• 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

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.

这项研究旨在分析食物抗原接受抗原表现的地方，并使用T细胞受体（TCR）摄入后T细胞识别。这项研究的最终目的是阐明食品抗原免疫识别的机制。首先，使用β-乳球蛋白（β-LG）和椭圆蛋白，原代食物过敏原作为模型抗原，我们分析了当TCR衍生的信号因变化的结构和用于刺激T细胞的抗原的变化结构和剂量而改变时，TCR衍生的信号的改变。使用杆状病毒表达系统制备了源自β-LG鼠的鼠CD4^+ T细胞克隆G1.19的固体类型的T细胞接收器（STCR）。通过生物素化酶（BIRA）识别的序列在STCRβ链恒定区域的下游加入。我们证实，在生物素存在下，纯化的STCR被BIRA生物素化，并通过添加链霉亲和素形成多聚体STCR。 G1.19细胞识别出与I-A^B分子复合物中的β-LG（P119-133）区域相对应的肽。通过将STCR添加到培养物中，抑制了G1.19细胞对抗原的增殖反应，这表明STCR竞争性地抑制了TCR对G1.19细胞与配体的结合。当细胞用STCR染色并在存在抗MHC II类分子的情况下用抗TCRCβ抗体标记的荧光染色时，通过流式细胞仪检测到特异性信号，表明STCR可以特异性结合抗卵皮胡椒和MHC II类Molecule的复合物。

项目成果

戸塚護(分担執筆): "食品と免疫・アレルギー(上野川修一編)"朝倉書店. (2003)

户冢守（合著者）：《食物与免疫/过敏（上野川秀一编辑）》朝仓书店（2003）。

戸塚 護: "抗原アナログによるアレルギー応答の制御"臨床免疫. 36・5. 771-776 (2001)

Mamoru Totsuka：“抗原类似物控制过敏反应”临床免疫学36・5（2001）。

戸塚 護: "アレルギーの発症メカニズムと予防の考え方"FOOD Style 21. 5・3. 43-46 (2001)

户冢守：“过敏发生的机制及预防的思路” FOOD Style 21. 5・3. (2001)

Ryuji Murakami, Kiyoshi Yamada, Shinya Nagafuchi, Satoshi Hachimura, Takeshi Takahashi, Shuichi Kaminogawa, and Mamoru Totsuka: "Nucleotides enhance the secretion of interleukin 7 from primary-cultured murine intestinal epithelial cells"Cytotechnology. in

Ryuji Murakami、Kiyoshi Yamada、Shinya Nagafuchi、Satoshi Hachimura、Takeshi Takahashi、Shuichi Kaminokawa 和 Mamoru Totsuka：“核苷酸增强原代培养的小鼠肠上皮细胞分泌白细胞介素 7”细胞技术。

Wataru Ise, Mamoru Totsuka, Yoshitaka Sogawa, Akio Ametani, Satoshi Hachimura, Takehito Sato, Yoshihiro Kumagai, Sonoko Habu, and Shuichi Kaminogawa: "Naive CD4^+ T cells exhibit distinct expression patterns of cytokines and cell-surface molecules on thei

Wataru Ise、Mamoru Totsuka、Yoshitaka Sogawa、Akio Ametani、Satoshi Hachimura、Takehito Sato、Yoshihiro Kumagai、Sonoko Habu 和 Shuichi Kaminokawa：“幼稚 CD4^ T 细胞在其上表现出独特的细胞因子和细胞表面分子表达模式。