TCR-β repertoire analysis of antigen-specific single T cells using a high-density microcavity array

Authors

  • Atsushi Arakaki,

    1. Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho Koganei, Tokyo 184-8588, Japan; telephone: 81-42-388-7020; fax: 81-42-385-7713
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  • Kaori Ooya,

    1. Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho Koganei, Tokyo 184-8588, Japan; telephone: 81-42-388-7020; fax: 81-42-385-7713
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  • Yasuto Akiyama,

    1. Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007, Shimo-nagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan
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  • Masahito Hosokawa,

    1. Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho Koganei, Tokyo 184-8588, Japan; telephone: 81-42-388-7020; fax: 81-42-385-7713
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  • Masaru Komiyama,

    1. Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007, Shimo-nagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan
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  • Akira Iizuka,

    1. Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007, Shimo-nagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan
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  • Ken Yamaguchi,

    1. Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007, Shimo-nagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan
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  • Tadashi Matsunaga

    Corresponding author
    1. Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho Koganei, Tokyo 184-8588, Japan; telephone: 81-42-388-7020; fax: 81-42-385-7713
    • Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho Koganei, Tokyo 184-8588, Japan; telephone: 81-42-388-7020; fax: 81-42-385-7713.
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Abstract

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-β 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-β 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. © 2010 Wiley Periodicals, Inc.

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