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

为了了解什么样的T细胞群参与了肾移植排斥反应，本研究对肾脏浸润性淋巴细胞(RIL)表达的T细胞受体基因进行了序列分析。首先，我建立了一种TCR序列分析的方法，即连接在TCR基因上游的人工合成锚点作为靶序列进行聚合酶链式反应。利用所建立的方法，我接下来分析了在混合淋巴细胞反应(MLR)中参与识别人类白细胞抗原-DR分子的TCR，这是一种体外同种异体免疫反应模型。我发现TCR的多样性并不取决于应答细胞和刺激细胞之间DR分子的氨基酸差异的数量，甚至不取决于参与MLR的DR的单个氨基酸差异，这是一组差异巨大的TCR。最后，我用Percoll密度梯度离心法和胶原酶处理从排斥和肾切除的同种异体移植物中分离出RIL，分析了RIL的TCR。分离CD25^+(活化T标记)组分，进行TCRbeta序列分析。在31个克隆测序中，共检测到7个不同的Vbeta基因家族和8个Jbeta片段。有几个克隆的CDR3序列完全相同，即6个克隆Vbeta6.9-Jbeta1.2，3个克隆Vbeta3.1-Jbeta2.1，2个克隆Vbeta3.1-Jbeta2.7，2个克隆Vbeta5.6-Jbeta2.5，2个克隆Vbeta12b-Jbeta2.3。考虑到在从体外MLR获得的约30个TCR克隆中很少观察到具有相同CDR3的cDNA，CD25^+RIL被认为是由表达相对有限的TCR多样性的T细胞群组成的。

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.