A novel form of light chain gene replacement

项目总结 B和T淋巴细胞构成了我们适应性免疫系统的基础，它基于特定的 通过结构不同的表面抗原受体识别外源分子。这些地区的结构多样性 受体起源于淋巴细胞中抗原受体基因的特异性重排 发展。这种重排过程被称为V(D)J重组，当RAG1/2蛋白启动时 通过缺口-发夹在重组信号序列(RSS)上切割抗原受体基因片段 机制，当DNA断裂被感知并通过非同源末端连接修复时完成。 传统上，B细胞系的多样性被认为受到功能基因V、D和J的数量的限制 免疫球蛋白(Ig)重链和轻链基因座的片段。然而，次要V(D)J的形式 重排已被报道，其中未重排的Ig重链V(VH)基因片段取代了 通过与嵌入在VH基因3‘端的隐蔽RSS重组来重排VH基因(称为VH 基因替换)。对这一过程的机械性洞察一直受到这些事件的低频率的阻碍 事件和通常情况下，使用种系预先重排的VH等位基因。光中是否发生了类似的事件 链上的基因定位尚不清楚。我们最近对来自中国的B细胞进行了大量轻链测序。 表达Ig VH12重链的转基因小鼠，根据对 卵磷脂。有趣的是，我们的分析发现了罕见但反复出现的内源杂交 重排的Ig kappa V(KV)基因序列，其中精选的KV4-91基因的3‘端是 被另一种KV基因取代。基于这个初步数据，我们的工作假设是RAG蛋白 通过切割位于VH12小鼠KV基因骨架区域3的隐匿RSS来介导KV基因替换 KV基因(KV FR3 CRSS)。拟议的项目将扩展我们的初步发现，以确认KV基因 替换导致单个B细胞产生生产性抗体，测试KV FR3 CRSS是否支持 RAG介导的体外和细胞内的切割和重排，也排除了激活诱导的胞苷 脱氨酶(AID)作为VH12小鼠KV基因替换的替代机制。这个项目将 挑战我们目前对抗体结构多样性的理论限制的理解，并导致 为合理设计治疗性抗体考虑KV基因嵌合体。这项工作还将突出一个 自动KV序列分析的重要潜在警告，因为此类事件(无论起源如何) 可能在KV基因通话中被遗漏，或被误认为体细胞过度突变。这个项目的潜力将发生变化 现有的范例将该项目归入“高风险-高回报”类别，而R21正是为此而设计的。

PROJECT SUMMARY B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates through site-specific rearrangement of antigen receptor genes during lymphocyte development. This rearrangement process, called V(D)J recombination, is initiated when the RAG1/2 proteins cleave antigen receptor gene segments at recombination signal sequences (RSS) through a nick-hairpin mechanism, and is completed when the DNA breaks are sensed and repaired by non-homologous end-joining. Classically, B cell repertoire diversity is considered restricted by the number of functional V, D, and J gene segments in the immunoglobulin (Ig) heavy and light chain gene loci. However, forms of secondary V(D)J rearrangement have been reported in which an unrearranged Ig heavy chain V (VH) gene segment replaces a rearranged VH gene via recombination with a cryptic RSS embedded in the 3’ end of the VH gene (called VH gene replacement). Mechanistic insight into this process has been hampered by the low frequency of these events and, typically, the use of germline pre-rearranged VH alleles. Whether similar events occur in the light chain loci remains unclear. We recently performed bulk light chain repertoire sequencing of B cells from transgenic mice expressing the Ig VH12 heavy chain, sorted based on immunoreactivity to phosphatidylcholine. Interestingly, our analysis uncovered infrequent, but recurrent, endogenous hybrid rearranged Ig kappa V (KV) gene sequences, in which the 3’ end of the highly selected KV4-91 gene was replaced by another KV gene. Based on this preliminary data, our working hypothesis is that the RAG proteins mediate KV gene replacement in VH12 mice via cleavage of a cryptic RSS identified in framework region 3 of the KV gene (KV FR3 cRSS). The proposed project will extend our preliminary findings to confirm KV gene replacement leads to productive antibody generation in single B cells, test whether the KV FR3 cRSS supports RAG-mediated cleavage and rearrangement in vitro and in cells, and also exclude activation-induced cytidine deaminase (AID) as an alternative mechanism for KV gene replacement in VH12 mice. This project will challenge our current understanding of the theoretical constraints on antibody structural diversity, and lead to considering KV gene chimeras for rational design of therapeutic antibodies. This work will also highlight an important potential caveat of automated KV sequence analysis, because such events (regardless of origin) may be missed in KV gene calls or be mistaken for somatic hypermutation. The potential of this project to shift existing paradigms places the project in the “high risk-high reward” category for which the R21 was designed.