Dynamics of Antigen-Driven Selection in Germinal Centers

7.项目总结/摘要 对感染具有保护作用的高亲和力抗体是从胚芽中的低亲和力前体进化而来的 中心（GC）。这种进化被认为是通过高亲和力B细胞突变体的选择性扩增而发生的， 较低亲和力的通过细胞凋亡消除。然而，基于亲和力的选择限制了 抗体反应中的克隆多样性是未知的，克隆的精确动力学和强度也是未知的。 GC的演变因此，我们不知道GC如何（或者是否）在以下两者之间产生理想的平衡： 保护性反应所必需的抗体亲和力和克隆多样性。此参数对于 在制定疫苗接种策略时，特别是那些旨在促进罕见B发展的策略时， 细胞克隆具有“广泛中和”的潜力，对艾滋病毒和流感。 这种知识上的差距很大程度上是由于我们在技术上无法精确测量克隆多样性， 在各个GC中随时间的选择强度。我们最近开发了两种基于显微镜的 这些技术大大提高了我们在GC响应期间测量克隆多样性进化的能力 在小鼠中：（i）单核苷酸作图，其依赖于“Brainbow”等位基因的随机重组来定量 通过成像确定单个GC中克隆选择的程度;以及（ii）原位光活化，这使我们能够 通过流式细胞术从单个GC分离数百个B和T细胞。我们建议使用这些方法， 进一步了解T细胞在控制GC克隆多样性中的作用， 克隆动态的差异之间的主要和回忆GC，并探讨如何克隆动态 逆转录病毒感染诱导的慢性GC随时间的变化。为了实现我们的目标，我们 期望增加我们对GC中克隆竞争如何影响多样性的理解， 抗体反应的免疫优势。我们希望我们的发现能为未来的疫苗提供信息， 发展，特别是对高度多样化的病原体，如艾滋病毒和流感。

7. PROJECT SUMMARY/ABSTRACT High-affinity antibodies that are protective against infection evolve from lower-affinity precursors in the germinal center (GC). This evolution is thought to occur by selective expansion of higher-affinity B cell mutants while lower-affinity ones are eliminated by apoptosis. However, the degree to which affinity-based selection restricts clonal diversity in the antibody response is unknown, as are the precise kinetics and strength of clonal evolution in GCs. Thus, we do not know how (or indeed whether) the GC generates the ideal balance between antibody affinity and clonal diversity necessary for a protective response. This parameter is important to consider when devising vaccination strategies, especially those aimed at fostering the development of rare B cell clones with “broadly-neutralizing” potential against HIV and influenza. This gap in knowledge is largely due to our technical inability to precisely measure clonal diversity and the strength of selection over time in individual GCs. We have recently developed two microscopy-based techniques that greatly improve our ability to measure the evolution of clonal diversity during the GC response in mice: (i) multicolor fate-mapping, which relies on stochastic recombination of a “Brainbow” allele to quantify the extent of clonal selection in individual GCs by imaging; and (ii) in situ photoactivation, which allows us to isolate hundreds of B and T cells from individual GCs by flow cytometry. We propose to use these methods to further our understanding of the role of T cell help in controlling GC clonal diversity, to understand the difference in clonal dynamics between primary and recall GCs, and to investigate the how clonal dynamics change over time in chronic GCs induced by retroviral infection. Upon fulfillment of our specific aims, we expect to have increased our understanding of how clonal competition in GCs affects the diversity and immunodominance of the antibody response. We expect that our findings will inform future vaccine development, especially against highly diverse pathogens such as HIV and influenza.