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.
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.
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DOI：
10.1371/journal.pone.0136086
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kakimi K
中科院分区：
文献类型：
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作者：
Miyai M;Eikawa S;Hosoi A;Iino T;Matsushita H;Isobe M;Uenaka A;Udono H;Nakajima J;Nakayama E;Kakimi K
全面的免疫学评估对于监测接受抗原特异性癌症免疫治疗的患者至关重要。T细胞应答的识别和量化对于进一步发展这类疗法是最重要的。利用NY-ESO-1f多肽疫苗研究中一名高应答患者(TK-F01)的临床样本，我们进行了高通量T细胞受体β链基因下一代测序，以监测NY-ESO-1特异性CD8+T细胞的频率。我们将这些结果与传统的免疫学检测方法，如干扰素-γ捕获法、四聚体结合法和有限稀释克隆法进行了比较。我们测序了两个NY-ESO-1f特异性CD8+T细胞克隆6-8L和2F6的人TCRB互补决定区3(CDR3)重排，以及在多肽疫苗接种过程中的PBMC。克隆6-8L含有TCRB CDR3基因TCRBV11-03*01和BJ02-01*01，氨基酸序列为CASSLRGNEQFF；2F6含有TCRBV05-08*01和BJ02-04*01(CASSLVGTNIQYF)。以这两个序列为模型，我们评估了NY-ESO-1特异性CD8+T细胞在体外PBMC中的频率。6-8L CDR3序列是外周血单核细胞中出现频率第二高的序列，甚至在接种疫苗之前就出现了很高的频率(0.7133%)，并在接种过程中持续存在。尽管通过四聚体染色和干扰素-γ捕获试验检测到NY-ESO1特异性CD8+T细胞在第一次到第六次免疫后显著扩张，但通过CDR3DNA测序评估，频率并没有随着多肽疫苗接种次数的增加而增加。经12天体外刺激克隆分析，B*52：01限制性NY-ESO-1f多肽特异性CD8+T细胞在PBMC中的频率仅为0.0023%，远低于NGS测序法的0.7133%。因此，需要体外刺激的分析可能低估了具有较低增殖潜力的克隆的频率。使用NGS的高通量TCRB测序可能更好地估计抗原特异性T细胞的实际频率，从而提供更准确的患者监测。
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.