Kitaura Kazutaka, Yamashita Hiroshi, Ayabe Hitomi, Shini Tadasu, Matsutani Takaji, Suzuki Ryuji
Repertoire Genesis Incorporation, Ibaraki, Japan.
BITS Co. Ltd., Tokyo, Japan.
Front Immunol. 2017 May 3;8:389. doi: 10.3389/fimmu.2017.00389. eCollection 2017.
A diverse antibody repertoire is primarily generated by the rearrangement of V, D, and J genes and subsequent somatic hypermutation (SHM). Class-switch recombination (CSR) produces various isotypes and subclasses with different functional properties. Although antibody isotypes and subclasses are considered to be produced by both direct and sequential CSR, it is still not fully understood how SHMs accumulate during the process in which antibody subclasses are generated. Here, we developed a new next-generation sequencing (NGS)-based antibody repertoire analysis capable of identifying all antibody isotype and subclass genes and used it to examine the peripheral blood mononuclear cells of 12 healthy individuals. Using a total of 5,480,040 sequences, we compared percentage frequency of variable (V), junctional (J) sequence, and a combination of V and J, diversity, length, and amino acid compositions of CDR3, SHM, and shared clones in the IgM, IgD, IgG3, IgG1, IgG2, IgG4, IgA1, IgE, and IgA2 genes. The usage and diversity were similar among the immunoglobulin (Ig) subclasses. Clonally related sequences sharing identical V, D, J, and CDR3 amino acid sequences were frequently found within multiple Ig subclasses, especially between IgG1 and IgG2 or IgA1 and IgA2. SHM occurred most frequently in IgG4, while IgG3 genes were the least mutated among all IgG subclasses. The shared clones had almost the same SHM levels among Ig subclasses, while subclass-specific clones had different levels of SHM dependent on the genomic location. Given the sequential CSR, these results suggest that CSR occurs sequentially over multiple subclasses in the order corresponding to the genomic location of IGHCs, but CSR is likely to occur more quickly than SHMs accumulate within Ig genes under physiological conditions. NGS-based antibody repertoire analysis should provide critical information on how various antibodies are generated in the immune system.
多样化的抗体库主要由V、D和J基因重排以及随后的体细胞超突变（SHM）产生。类别转换重排（CSR）产生具有不同功能特性的各种同种型和亚类。虽然抗体同种型和亚类被认为是由直接和顺序CSR产生的，但在抗体亚类产生过程中SHM如何积累仍未完全了解。在这里，我们开发了一种基于新一代测序（NGS）的新型抗体库分析方法，能够识别所有抗体同种型和亚类基因，并用于检测12名健康个体的外周血单个核细胞。使用总共5480040个序列，我们比较了可变（V）、连接（J）序列以及V和J组合的百分比频率、多样性、长度以及IgM、IgD、IgG3、IgG1、IgG2、IgG4、IgA1、IgE和IgA2基因中CDR3、SHM和共享克隆的氨基酸组成。免疫球蛋白（Ig）亚类之间的使用情况和多样性相似。在多个Ig亚类中经常发现共享相同V、D、J和CDR3氨基酸序列的克隆相关序列，特别是在IgG1和IgG2之间或IgA1和IgA2之间。SHM在IgG4中发生频率最高，而IgG3基因在所有IgG亚类中突变最少。共享克隆在Ig亚类中的SHM水平几乎相同，而亚类特异性克隆的SHM水平因基因组位置而异。考虑到顺序CSR，这些结果表明CSR按照与IGHCs基因组位置相对应的顺序在多个亚类中依次发生，但在生理条件下，CSR可能比Ig基因内SHM积累得更快。基于NGS的抗体库分析应能提供关于免疫系统中各种抗体如何产生的关键信息。
