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

Atsushi Arakaki, Kaori Ooya, Yasuto Akiyama, Masahito Hosokawa, Masaru Komiyama, Akira Iizuka, Ken Yamaguchi, Tadashi Matsunaga

研究成果: Article査読

11 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)311-318
ページ数8
ジャーナルBiotechnology and Bioengineering
106
2
DOI
出版ステータスPublished - 2010 6 1
外部発表はい

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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