Palindromic Nucleotide Analysis in Human T Cell Receptor Rearrangements

Diversity of T cell receptor (TCR) genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3) of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8+ and CD4+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8+ naïve T cells. The naïve CD8+ T cell clones with P nucleotides are more highly expanded.T细胞受体（T cell receptor, TCR）基因的多样性主要源自其从种系编码的V、D、J基因片段重排过程中引入的核苷酸插入。V-D与D-J连接区的核苷酸插入本为随机事件，但其中存在少数特殊插入片段：1至3个碱基会反向重复种系DNA序列，这类短互补回文序列被称为P核苷酸。本研究采用ImmunoSeq深度测序检测技术，对T细胞受体β链的第三互补决定区（complementarity determining region, CDR3）进行分析，并依托所得数据研究初始与记忆性CD8+、CD4+ T细胞库中的P核苷酸特征。我们对健康成年人横断面群体及不同T细胞亚型的P核苷酸分布进行了估算，结果显示所有T细胞亚型中的P核苷酸频率均介于1%至2%之间，且其分布相对于基因片段的编码端存在显著偏倚。通过最大条件概率模型将观测到的回文序列归类为P核苷酸后发现，单碱基P核苷酸在VDJ重组中极为罕见，P核苷酸主要为2碱基长度。为探究P核苷酸在胸腺选择中的作用，我们对比了CD8+初始T细胞功能性与非功能性序列中的P核苷酸，发现携带P核苷酸的初始CD8+ T细胞克隆扩增程度更高。