Tsuruta Y, Iwagami S, Furue S, Teraoka H, Yoshida T, Sakata T, Suzuki R
Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan.
J Immunol Methods. 1993 May 5;161(1):7-21. doi: 10.1016/0022-1759(93)90193-b.
The study of T cell receptor (TCR) genes has been hampered by their large repertoires and elusive methods for gene amplification. We have developed a new method for amplification of all human TCR genes (alpha, beta, gamma, and delta) with the ligation of a universal adaptor to the leader sequence of variable (V) regions, which permitted effective and reproducible amplification of all four types of TCR genes. cDNA sequencing of TCR-gamma, -delta, -alpha, -beta was carried out in respectively 15, 13, 28, and 26 T cell clones from human peripheral blood T cells using a newly developed universal adaptor and these methods. TCR-gamma V-II (V gamma 9) was a major population, and V-I (V gamma 2 and 3) and V-III (V gamma 10) were next major populations among TCR-gamma subfamilies, and confirmed the previous observations determined using mAbs specific to TCR-gamma. All five clones of TCR-gamma V-II and three of five clones of TCR-gamma V-I subfamilies had in-frame V-N-J junctions. In contrast, sequences from both TCR-gamma V-III (4/4 clones) and V-IV (1/1 clones) subfamilies had intron-like regions that caused out-of-frame cDNA, suggesting that most of TCR-gamma V-III and V-IV in PBL are not functional. V delta 2 was a major population and V delta 1 was a next predominant population among TCR-delta subfamilies, also confirming the previous observations determined using mAbs to TCR-delta. With regards to TCR-alpha and -beta, this new method randomly amplified TCR cDNAs. In addition, the sequences of 5' portions of three TCR-V-alpha and one TCR-V beta were extended. Two new TCR-alpha subfamilies and one new TCR-beta family were also identified. In summary, this new method will provide a scientific tool for understanding structures of the human TCR genes involved in specific immune responses.
T细胞受体（TCR）基因的研究一直受到其庞大的基因库以及难以捉摸的基因扩增方法的阻碍。我们开发了一种新方法，通过将通用接头连接到可变（V）区的前导序列来扩增所有人类TCR基因（α、β、γ和δ），该方法能够有效且可重复地扩增所有四种类型的TCR基因。使用新开发的通用接头和这些方法，分别对来自人外周血T细胞的15、13、28和26个T细胞克隆进行了TCR-γ、-δ、-α、-β的cDNA测序。TCR-γ V-II（Vγ9）是主要群体，V-I（Vγ2和3）和V-III（Vγ10）是TCR-γ亚家族中的次主要群体，这证实了先前使用针对TCR-γ的单克隆抗体所确定的观察结果。TCR-γ V-II的所有五个克隆和TCR-γ V-I亚家族的五个克隆中的三个具有框内V-N-J连接。相比之下，TCR-γ V-III（4/4克隆）和V-IV（1/1克隆）亚家族的序列都有导致框外cDNA的内含子样区域，这表明外周血淋巴细胞（PBL）中大多数TCR-γ V-III和V-IV无功能。Vδ2是TCR-δ亚家族中的主要群体，Vδ1是次主要群体，这也证实了先前使用针对TCR-δ的单克隆抗体所确定的观察结果。关于TCR-α和-β，这种新方法随机扩增TCR cDNA。此外，三个TCR-V-α和一个TCR-Vβ的5'部分序列得到了延伸。还鉴定出两个新的TCR-α亚家族和一个新的TCR-β家族。总之，这种新方法将为理解参与特异性免疫反应的人类TCR基因结构提供一种科学工具。
