Sequence analysis of T-cell receptor participating in rejection of renal allografts.

参与同种肾移植排斥的T细胞受体的序列分析。

• 批准号: 02670718
• 负责人: OBATA Fumiya
• 金额: $ 0.58万
• 依托单位: Kitasato University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670718/
• 关键词: Renal Transplantation; Rejection; Infiltrating Lymphocytes; T-Cell Receptor; PCR; 浸潤T細胞; T細胞抗原受容体; HLAーDR; MLR; 同種免疫応答; PCR

In order to know what kind of T cell population participate in rejection of renal allografts, in this research project, I carried out sequence analysis of T-cell receptor cDNA expressed by renal infiltrating lymphocytes (RIL). First, I established the method for TCR sequence analysis, called Linker-Ligated PCR, in which synthetic anchor ligated at the upstream of TCR cDNA is utilized as target sequence for PCR-amplification. By utilizing the method established, I next analyzed TCR involved in recognition of HLA-DR molecules in mixed lymphocyte reaction (MLR), a in vitro model for allogeneic immune reaction. I found that diversity of TCR does not depend on the number of amino acid difference of DR molecules between responder and stimulator cells and even for a single amino acid difference of DR, enormously diverse set of TCR involved in MLR. Finally, I analyzed TCR in RIL isolated from rejected and nephrectomized allograft by treatment with collagenase and density-gradient centrifugation in Percoll. CD25^+(activated T marker) fraction was isolated and subjected to TCRbeta sequence analysis. Seven different Vbeta gene families and 8 Jbeta segments were detected among 31 cDNA clones sequenced. Several cDNA clones were found to have completely identical CDR3 sequences, i. e., 6 clones with Vbeta6.9-Jbeta1.2, 3 clones with Vbeta3.1-Jbeta2.1, 2 clones with Vbeta3.1-Jbeta2.7, 2 clones with Vbeta5.6-Jbeta2.5, 2 clones with Vbeta12b-Jbeta2.3. Considering that cDNA with identical CDR3 is rarely observed in the analysis of around 30 TCR cDNA clones obtained from in vitro MLR, CD25^+ RIL was thought to composed of T cell population expressing relatively limited TCR diversity.

为了知道哪种T细胞群参与肾脏同种异体移植的排斥，在该研究项目中，我对肾脏浸润淋巴细胞（RIL）表达的T细胞受体cDNA进行了序列分析。首先，我建立了TCR序列分析的方法，称为接头绑扎的PCR，其中在TCR cDNA上游的合成锚定为PCR扩增的目标序列。通过利用所建立的方法，我接下来分析了识别混合淋巴细胞反应中HLA-DR分子的TCR，这是同种异体免疫反应的体外模型。我发现TCR的多样性不取决于响应者和刺激器细胞之间DR分子的氨基酸差异，甚至对于DR的单个氨基酸差异，MLR涉及的TCR的单个氨基酸差异。最后，我通过用胶原酶和密度梯度离心在Percoll中的拒绝和肾切除术分离的RIL中分析了TCR。 CD25^+（激活的T标记）分离并进行TCRBETA序列分析。在测序的31个cDNA克隆中检测到了7个不同的VBETA基因家族和8个JBETA段。发现几个cDNA克隆具有完全相同的CDR3序列，即。 e。，6个带有VBETA6.9-JBETA1.2的克隆，3个带有VBETA3.1-JBETA2.1的克隆，2个带有VBETA3.1-JBETA2.7的克隆，2个克隆，2个带有VBETA5.6-JBETA2.5的克隆，带有VBETA5.6-JBETA2.5，2个带有VBETA12B-JBETA2.3的克隆。考虑到与从体外MLR获得的大约30个TCR cDNA克隆分析中很少观察到具有相同CDR3的cDNA，因此认为CD25^+ RIL被认为由表达相对有限的TCR多样性的T细胞群组成。

项目成果

Obata F,Tsunoda M,Kaneko T,Ito K,Masewicz S,Mickelson EM,Ollier WER,Pawelec G,Cella M,et al.: "Human T-cell receptor TCRAV,TCRBV,and TCRAJ sequences newly found in T-cell clones reactive with allogeneic HLA-class II antignes." Immunogenetics.

Obata F,Tsunoda M,Kaneko T,Ito K,Masewicz S,Mickelson EM,Ollier WER,Pawelec G,Cella M,et al.：“在 T 细胞中新发现的人类 T 细胞受体 TCRAV、TCRBV 和 TCRAJ 序列

Watanabe K, Yuge K, Sato K, Sonoda K, Masaki K, Maruyama S, Okubo M, Obata F, Otani F, Kaneko T, Takahara H, Matsubayashi T, Yago K, and Kashiwagi N: "Donor bone marrow cell facilitates induction of tolerance to kidney allografts in dogs treated with frac

Watanabe K、Yuge K、Sato K、Sonoda K、Masaki K、Maruyama S、Okubo M、Obata F、Otani F、Kaneko T、Takahara H、Matsubayashi T、Yago K 和 Kashiwagi N：“供体骨髓细胞促进诱导

Obata F.,Ito K,Ito I,and Kashiwagi N: "Linkage between HLA-DRBI and -DRB3 types in the Japanese population analyzed by oligonucleotide genotyping." Human Immunology. 33. 284-288 (1992)

Obata F.、Ito K、Ito I 和 Kashiwagi N：“通过寡核苷酸基因分型分析日本人群中 HLA-DRBI 和 -DRB3 类型之间的关联。”

Watanabe K.et al.: "Donor bone marrow cell facilitates induction of tolerance to kidney allografts in dogs treated with fractionated lymphoid irradiation and FK 506." Transplantation Proceedings. 23. 568-572 (1991)

Watanabe K. 等人：“供体骨髓细胞有助于诱导接受分次淋巴照射和 FK 506 治疗的狗对肾同种异体移植物的耐受性。”

Okubo H, Ito K, Tanaka S, Watanabe N, Kashiwagi N, and Obata F: "Analysis of the HLA-DR gene frequencies in Japanese cases of juvenile rheumatoid arthritis and rheumatoid arthritis by oligonucleotide DNA typing." Rheumatology International.

Okubo H、Ito K、Tanaka S、Watanabe N、Kashiwagi N 和 Obata F：“通过寡核苷酸 DNA 分型分析日本幼年类风湿性关节炎和类风湿性关节炎病例中的 HLA-DR 基因频率。”