TCR-β Repertoire Analysis of Antigen-Specific Single T Cells Using a High-Density Microcavity Array
TCR-β Repertoire Analysis of Antigen-Specific Single T Cells Using a High-Density Microcavity Array
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DOI：
10.1002/bit.22663
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发表时间：
2010-06-01
影响因子：
3.8
通讯作者：
Matsunaga, Tadashi
中科院分区：
文献类型：
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作者：
Arakaki, Atsushi;Ooya, Kaori;Matsunaga, Tadashi
由T细胞受体（TCR）提供的细胞毒性T细胞的抗原特异性在人类自身免疫性疾病、感染和癌症中起核心作用。由于TCR库在单个细胞毒性T细胞中是独特的，因此需要在单细胞水平上分析其基因重排的策略。在这项研究中，我们应用了一种高密度微腔阵列，使靶细胞筛选的数千个单细胞的功能性TCR-β基因库特异性黑色素瘤（gp 100）和巨细胞病毒（CMV）抗原的鉴定。通过使用显微操作器在显微镜下分离表达具有识别荧光标记的抗原肽四聚体的能力的TCR的T细胞。在微腔阵列上规则排列的细胞便于从多克隆T细胞群中检测和分离靶单细胞。然后将分离的单细胞直接用于RT-PCR。通过对扩增的PCR产物进行测序，在单细胞水平上成功鉴定了gp 100和人巨细胞病毒抗原的抗原特异性TCR-β库。这种简单，准确，成本效益高的单细胞分析技术具有进一步的潜力，作为一个有价值的和广泛适用的工具，对各种细胞的基因筛选和表达分析的研究。Biotechnol. Bioeng. 2010;106：311-318. (C)2010 Wiley Periodicals，Inc.
The antigen specificity of cytotoxic T cells, provided by T-cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single-cell level is required. In this study, we applied a high-density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR-beta gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent-labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T-cell population. The isolated single cells were then directly utilized for RT-PCR. By sequencing the amplified PCR products, antigen-specific TCR-beta repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single-cell level. This simple, accurate, and cost-effective technique for single-cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells. Biotechnol. Bioeng. 2010;106: 311-318. (C) 2010 Wiley Periodicals, Inc.