Elucidating Germinal Center-Mediated Antibody Evolution

项目摘要 抗体通过两种不同的途径多样化。第一种是组合装配， 免疫球蛋白（IG）可变区（V）外显子在B细胞发育过程中。第二个涉及V外显子体细胞 超突变（SHM）和基于亲和力的选择在生殖中心（GC）。存在着根本性的差距， 了解这些系统如何协作以识别、适应和消除各种致病威胁。 长期的目标是阐明基础GC B细胞生物学和阐明潜在的机制 保护性抗体的发展。这项建议的目的是阐明GC的机制 可塑性-特别是GC多样化的程度，以及允许B细胞GC进入的参数， 抗体不断进化。中心假设是GC系统提供了关于 抗体的发展。一方面，GC反应增强了可从主要组分容易获得的亲和力， 保留曲目。另一方面，GC可塑性足够灵活，可以招募极低的 亲和/非同源B细胞克隆，其BCR最初不具有足够的亲和力以在GC中良好竞争，但 其可能具有识别关键表位的独特潜力（给定几个所需的突变），而不是其他 被主要剧目很好地瞄准。更深入地了解这些GC功能是如何调节的 有望揭示如何更有效地招募低频率/低亲和力B细胞的新见解， 潜在的成为广泛中和，并引导他们走向保护功效。这一假设将是 探索有两个具体目标：1）表征GC介导的多样性的能力和局限性 SHM多样化系统;和2）定义调节B细胞参与生发中心的特征。下 第一个目标，GC多样化系统的亲和力发展和特异性潜力的灵活性将 使用极低的BCR/抗原亲和力（Ka<102 M-1）和BCR阴性模型系统进行检查， 竞争环境的生理环境。在第二个目标下，调节经济增长的可变因素 将定义SHM介导的IG进化在生理环境中的灵活性。这种方法是创新的， 因为申请人最近发表的工作和初步数据表明，GC介导的多样化 可以提供对在生理学内原代IG库中不存在的新表位的特异性， 竞争环境的多样化主要IG剧目。创新的小鼠模型将用于探测 亲和力和IG频率参与GC成熟的作用的参数和机制方面 和在表达多种免疫调节因子的动物模型的情况下对保护性抗体应答的贡献。 人IG库。发现极低亲和力的B细胞克隆如何进入GC并继续 在竞争激烈的GC环境中成熟-通常积累极高水平的SHM， 用疫苗复制-将是对基础B细胞知识的重大贡献。等 知识也有可能为疫苗接种和抗体生物工程战略提供信息。

PROJECT SUMMARY Antibodies diversify through two distinct pathways. The first involves combinatorial assembly of Immunoglobulin (Ig) variable region (V) exons during B cell development. The second involves V exon somatic hypermutation (SHM) and affinity-based selection in germinal centers (GCs). There are fundamental gaps in understanding how these systems collaborate to recognize, adapt, and neutralize diverse pathogenic threats. The long-term goal is to shed light onto fundamental GC B cell biology and elucidate underlying mechanisms of protective antibody development. The objective for this proposal is to elucidate mechanisms underlying GC plasticity—in particular, the extent of GC diversification, and the parameters that allow B cell GC entry and continued antibody evolution. The central hypothesis is that the GC system provides dual function with regard to antibody development. On one hand, the GC reaction intensifies affinities readily available from the primary repertoire. On the other hand, GC plasticity is flexible enough to allow for the recruitment of extremely low affinity/non-cognate B cell clones whose BCR is not initially of sufficient affinity to compete well in the GC, but which may have unique potential (given a few needed mutations) to recognize critical epitopes not otherwise targeted well by the primary repertoire. A deeper understanding of how these GC functions are regulated promises to reveal new insights into how to more effectively recruit low frequency/low affinity B cells with potential to become broadly neutralizing, and shepherd them toward protective efficacy. This hypothesis will be explored with two specific aims: 1) Characterize the capacity and limitations of diversity mediated by the GC SHM diversification system; and 2) Define features that regulate B cell participation in germinal centers. Under the first aim, the flexibility of affinity development and specificity potential by the GC diversification system will be examined using an extremely low BCR/antigen affinity (Ka<102 M-1), and BCR-negative model systems in the physiologic context of competitive settings. Under the second aim, modifiable factors that regulate the flexibility of SHM-mediated Ig evolution in physiologic contexts will be defined. The approach is innovative, because the applicant's recent published work and preliminary data indicate that GC-mediated diversification can provide specificities to new epitopes not otherwise present in the primary Ig repertoire within a physiologic, competitive environment of a diverse primary Ig repertoire. Innovative mouse models will be used to probe the parameters and mechanistic aspects of the roles of affinity and Ig frequency on participation in GC maturation and contribution to protective antibody responses in the context of an animal model expressing a diverse human Ig repertoire. Discovering how extremely low affinity B cell clones gain access to the GC and continue to mature in the highly competitive GC environment—often accumulating extremely high levels of SHM not yet replicated with vaccines—would be a significant contribution to fundamental B cell knowledge. Such knowledge also has the potential to inform strategies for vaccination as well as antibody bioengineering.