Detection and tracking of NY-ESO-1-Specific CD8+ T cells by high-throughput T cell receptor β (TCRB) gene rearrangements sequencing in a peptide-vaccinated patient

Manami Miyai, Shingo Eikawa, Akihiro Hosoi, Tamaki Iino, Hirokazu Matsushita, Midori Isobe, Akiko Uenaka, Heiichiro Udono, Jun Nakajima, Eiichi Nakayama, Kazuhiro Kakimi

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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-1fspecific 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 NYESO- 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.

Original languageEnglish
Article numbere0136086
JournalPloS one
Volume10
Issue number8
DOIs
Publication statusPublished - Aug 20 2015

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Miyai, M., Eikawa, S., Hosoi, A., Iino, T., Matsushita, H., Isobe, M., Uenaka, A., Udono, H., Nakajima, J., Nakayama, E., & Kakimi, K. (2015). Detection and tracking of NY-ESO-1-Specific CD8+ T cells by high-throughput T cell receptor β (TCRB) gene rearrangements sequencing in a peptide-vaccinated patient. PloS one, 10(8), [e0136086]. https://doi.org/10.1371/journal.pone.0136086