Profiling the T-cell Receptor Beta-chain Repertoire by Massively Parallel Sequencing. Homo sapiens

T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 5'-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3(beta) diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR(beta) clonotypes and provides precise measurements of CDR3(beta) length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3(beta) nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

T细胞受体（T-cell receptor, TCR）基因组位点会发生体细胞V(D)J重排，并在重组接头处添加或移除非模板化碱基，以此生成抗原识别所需的结构多样性T细胞库。T细胞受体β亚基可通过其高变的CDR3（互补决定区3, Complement Determining Region 3）序列进行明确鉴定，该区域正是编码抗原接触主要位点的V(D)J重排发生位置。由于潜在T细胞库的规模极大，传统序列分析方法难以开展相关研究，因此T细胞库的复杂性与动态特性至今仍未被完全阐明。本研究利用5'-RACE技术、Illumina测序技术以及一种新型短读长组装策略，对人外周血T淋巴细胞中的CDR3β多样性进行了系统性采样分析。通过对4050万条短读长序列进行组装，本研究共鉴定出33664个独特的TCRβ克隆型，并精准测定了CDR3β的长度多样性、非模板化碱基使用模式、序列趋同现象，以及TRBV（T细胞受体β可变基因, T-cell receptor beta variable gene）和TRBJ（T细胞受体β连接基因, T-cell receptor beta joining gene）的基因使用偏好与配对偏好。TRBV与TRBJ保守残基之间的CDR3长度范围为21至81个核苷酸（nt）。TRBV基因的使用率范围从TRBV17的0.01%到TRBV20-1的24.6%不等；TRBJ基因的使用率范围则从TRBJ2-6的1.6%到TRBJ2-1的17.2%不等。本研究共鉴定出1573例序列趋同事件，即不同的CDR3β核苷酸序列可编码得到相同的氨基酸翻译产物。无需预先知晓抗原信息，基于直接序列的免疫谱分析有望成为理解免疫应激下T细胞库动态变化的有效工具。