TCR-beta repertoire analysis of antigen-specific single T cells using a high-density microcavity array.
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.
Arakaki
Atsushi
A
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho Koganei, Tokyo 184-8588, Japan.
Ooya
Kaori
K
Akiyama
Yasuto
Y
Hosokawa
Masahito
M
Komiyama
Masaru
M
Iizuka
Akira
A
Yamaguchi
Ken
K
Matsunaga
Tadashi
T
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*
D011948:Receptors, Antigen, T-Cell