Center for AIDS Research, Kumamoto University, Honjo, Kumamoto, Japan.
PLoS One. 2012;7(7):e40386. doi: 10.1371/journal.pone.0040386. Epub 2012 Jul 6.
T-cell receptor (TCR) α/β chains are expressed on the surface of CD8(+) T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5'-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8(+) subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8(+) subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8(+) T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains.
T 细胞受体 (TCR) α/β 链表达在 CD8(+) T 细胞的表面，参与抗原识别、激活和增殖。然而，由于其结构的复杂性，即广泛的基因重排，人类 TCRα/β 链的特征描述方法尚未得到很好的建立。在这里，我们报告了一种集成的 5'-RACE 和多重 PCR 方法的发展，用于在人类 CD8(+)亚群的单细胞水平上扩增 TCRα/β 的全长转录本，包括幼稚、中央记忆、早期效应记忆、晚期效应记忆和效应表型细胞。使用这种方法，TCRα 和 TCRβ 链的 PCR 成功率分别约为 47%和 62%，我们能够分析每个 TCR 链的超过 1000 个转录本的读数。我们的综合分析揭示了以下几点：(1)TCRδ-α 的嵌合重排，(2) 多个转录起始位点对 TCRα/β 转录的控制，(3)CD8(+)亚群中 TCRα/β 链的改变利用，以及 (4)TCRα/β 链的克隆大小与 CD8(+)T 细胞的效应表型之间的强烈关联。基于这些发现，我们得出结论，我们的方法是识别 TCRα/β 库动力学的有用工具，并为人类 TCRα/β 链的研究提供了新的见解。
