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.
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.
Department of Immunotherapeutics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan.
Manami
M
Miyai
Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Okayama, Japan.
Shingo
S
Eikawa
Department of Immunotherapeutics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan.
Akihiro
A
Hosoi
Department of Immunotherapeutics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan.
Tamaki
T
Iino
Department of Immunotherapeutics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan.
Hirokazu
H
Matsushita
Faculty of Health and Welfare, Kawasaki University of Medical Welfare, Kurashiki, Okayama, Japan.
Midori
M
Isobe
Faculty of Health and Welfare, Kawasaki University of Medical Welfare, Kurashiki, Okayama, Japan.
Akiko
A
Uenaka
Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Okayama, Japan.
Heiichiro
H
Udono
Department of Thoracic Surgery, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan.
Jun
J
Nakajima
Faculty of Health and Welfare, Kawasaki University of Medical Welfare, Kurashiki, Okayama, Japan.
Eiichi
E
Nakayama
Department of Immunotherapeutics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan.
Kazuhiro
K
Kakimi
D000951:Antigens, Neoplasm / Q000276:immunology* / Q000494:pharmacology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D019496:Cancer Vaccines / Q000276:immunology* / Q000494:pharmacology; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D059425:Interferon-gamma Release Tests; D008175:Lung Neoplasms / Q000276:immunology / Q000628:therapy*; D008565:Membrane Proteins / Q000276:immunology* / Q000494:pharmacology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D016896:Treatment Outcome; D014611:Vaccination / Q000379:methods
D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; D019496:Cancer Vaccines; D008565:Membrane Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta