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.
| Original language | English |
|---|---|
| Pages (from-to) | 311-318 |
| Number of pages | 8 |
| Journal | Biotechnology and Bioengineering |
| Volume | 106 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2010 Jun 1 |
| Externally published | Yes |
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Keywords
- Gene expression analysis
- Microarray analytical devices
- Single-cell
- T-cell antigen receptor specificity
- T-cell receptor beta genes
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology
Cite this
TCR-β repertoire analysis of antigen-specific single T cells using a high-density microcavity array. / Arakaki, Atsushi; Ooya, Kaori; Akiyama, Yasuto; Hosokawa, Masahito; Komiyama, Masaru; Iizuka, Akira; Yamaguchi, Ken; Matsunaga, Tadashi.
In: Biotechnology and Bioengineering, Vol. 106, No. 2, 01.06.2010, p. 311-318.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - TCR-β repertoire analysis of antigen-specific single T cells using a high-density microcavity array
AU - Arakaki, Atsushi
AU - Ooya, Kaori
AU - Akiyama, Yasuto
AU - Hosokawa, Masahito
AU - Komiyama, Masaru
AU - Iizuka, Akira
AU - Yamaguchi, Ken
AU - Matsunaga, Tadashi
PY - 2010/6/1
Y1 - 2010/6/1
N2 - 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.
AB - 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.
KW - Gene expression analysis
KW - Microarray analytical devices
KW - Single-cell
KW - T-cell antigen receptor specificity
KW - T-cell receptor beta genes
UR - http://www.scopus.com/inward/record.url?scp=77953064476&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77953064476&partnerID=8YFLogxK
U2 - 10.1002/bit.22663
DO - 10.1002/bit.22663
M3 - Article
C2 - 20073087
AN - SCOPUS:77953064476
VL - 106
SP - 311
EP - 318
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 2
ER -