A novel form of light chain gene replacement

项目摘要 B和T淋巴细胞形成我们的适应性免疫系统的基础，这是基于特定的 通过结构多样的表面抗原受体识别外来分子。结构多样性在这些 受体起源于淋巴细胞增殖过程中抗原受体基因位点特异性重排 发展这种重排过程称为V（D）J重组，当RAG 1/2蛋白 通过切口发夹在重组信号序列（RSS）处切割抗原受体基因片段 当DNA断裂被感应到并通过非同源末端连接修复时，DNA断裂完成。 传统上，B细胞库多样性被认为受到功能性V、D和J基因数量的限制 免疫球蛋白（IG）重链和轻链基因座中的片段。然而，次级V（D）J的形式 已经报道了重排，其中未重排的IG重链V（VH）基因区段取代了未重排的Ig重链V（VH）基因区段。 通过与嵌在VH基因3'端的隐蔽RSS重组重排的VH基因（称为VH 基因替换）。对这一过程的机械洞察力受到这些低频率的阻碍， 事件，并且通常使用种系预重排的VH等位基因。类似的事件是否发生在 链位点仍不清楚。我们最近进行了大量的轻链库测序的B细胞从 表达IG VH 12重链的转基因小鼠，根据免疫反应性进行分类， 卵磷脂有趣的是，我们的分析揭示了不常见的，但经常性的，内源性杂交， 重排的IG kappa V（KV）基因序列，其中高度选择的KV 4 -91基因的3'末端是 被另一个KV基因取代基于这些初步数据，我们的工作假设是RAG蛋白 在VH 12小鼠中通过切割在VH 12小鼠的框架区3中鉴定的隐蔽RSS介导KV基因置换， KV基因（KV FR 3 cRSS）。该项目将扩展我们的初步研究结果，以确认KV基因 替换导致单个B细胞中产生生产性抗体，测试KV FR 3 cRSS是否支持 RAG介导的体外和细胞内裂解和重排，也排除激活诱导的胞苷 脱氨酶（AID）作为VH 12小鼠中KV基因置换的替代机制。该项目将 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 考虑KV基因嵌合体用于合理设计治疗性抗体。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基因调用中丢失或被误认为体细胞超突变。这个项目的潜力在于 现有的范例将该项目置于“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.