Abstract |
Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.
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Authors | Manami Miyai, Shingo Eikawa, Akihiro Hosoi, Tamaki Iino, Hirokazu Matsushita, Midori Isobe, Akiko Uenaka, Heiichiro Udono, Jun Nakajima, Eiichi Nakayama, Kazuhiro Kakimi |
Journal | PloS one
(PLoS One)
Vol. 10
Issue 8
Pg. e0136086
( 2015)
ISSN: 1932-6203 [Electronic] United States |
PMID | 26291626
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Antigens, Neoplasm
- CTAG1B protein, human
- Cancer Vaccines
- Membrane Proteins
- Receptors, Antigen, T-Cell, alpha-beta
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Topics |
- Antigens, Neoplasm
(immunology, pharmacology)
- CD8-Positive T-Lymphocytes
(immunology)
- Cancer Vaccines
(immunology, pharmacology)
- Gene Rearrangement, beta-Chain T-Cell Antigen Receptor
(genetics)
- High-Throughput Nucleotide Sequencing
(methods)
- Humans
- Interferon-gamma Release Tests
- Lung Neoplasms
(immunology, therapy)
- Membrane Proteins
(immunology, pharmacology)
- Receptors, Antigen, T-Cell, alpha-beta
(genetics)
- Treatment Outcome
- Vaccination
(methods)
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