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

doi:10.1371/journal.pone.0136086

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.

Highlights

To assess the efficacy of cancer immunotherapy, identification and quantification of antigenspecific T cell responses during the course of treatment is required [1,2]
Patient TK-f01 was a high responder; a robust and sustained CD8+ T cell response to NY-ESO-1 was detected after initiating vaccination [14,15]
CD8+ T cells had to be enriched from Peripheral blood mononuclear cells (PBMCs) and cultured for 12 days with irradiated autologous CD4- and CD8-depleted PBMC in the presence of overlapping NY-ESO-1 peptides

Summary

Introduction

To assess the efficacy of cancer immunotherapy, identification and quantification of antigenspecific T cell responses during the course of treatment is required [1,2]. 0.1% of antigen-specific T cells in the sample is required for optimal analysis and an in vitro stimulation step may be needed to reliably perform this assay. Intracellular cytokine assays [5] and cytokine capture assays [6] are flow cytometry-based and similar to ELISPOT both depend on cytokine production by the T cells. The sensitivity of both assays is comparable to that of the tetramer assay. To determine frequencies of antigen-specific T cells, greater sensitivity is desirable in assays not depending on cytokine production or any in vitro expansion that could cause bias by selecting for T cells with greater proliferative potential

Methods

Results

Conclusion