title: TCR-beta repertoire analysis of antigen-specific single T cells using a high-density microcavity array.
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-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.
authors.affiliation: Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho Koganei, Tokyo 184-8588, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Arakaki
authors.forename: Kaori
authors.initials: K
authors.lastname: Ooya
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Akiyama
authors.forename: Masahito
authors.initials: M
authors.lastname: Hosokawa
authors.forename: Masaru
authors.initials: M
authors.lastname: Komiyama
authors.forename: Akira
authors.initials: A
authors.lastname: Iizuka
authors.forename: Ken
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsunaga
mesh_terms: D002469:Cell Separation / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation*
chemical_list: D011948:Receptors, Antigen, T-Cell