Dynamics of Antigen-Driven Selection in Germinal Centers

7.项目摘要/摘要 防止感染的高亲和力抗体从生发组织中低亲和力的前体进化而来。 中心(GC)。这种进化被认为是通过选择性地扩增亲和力更高的B细胞突变体而发生的 亲和力较低的基因会被细胞凋亡所消除。然而，基于亲和力的选择限制的程度 抗体反应中的克隆多样性未知，克隆的精确动力学和强度也是未知的。 GC的进化。因此，我们不知道GC如何(或者是否)在 抗体亲和力和克隆多样性是保护性反应所必需的。此参数对以下各项很重要 在制定疫苗接种策略时，尤其是那些旨在促进罕见B型病毒发展的策略时，请考虑 具有“广泛中和”潜力的细胞克隆，以对抗艾滋病毒和流感。 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和 在单个GC中随着时间的推移而进行选择的强度。我们最近开发了两种基于显微镜的 极大地提高了我们在GC响应期间测量克隆多样性进化的能力的技术 在小鼠中：(I)多色命运映射，这依赖于“脑弓”等位基因的随机重组来量化 通过成像在单个GC中克隆选择的程度；以及(Ii)原位光激活，这使得我们能够 用流式细胞仪从单个GC中分离出数百个B和T细胞。我们建议使用这些方法来 进一步了解T细胞在控制GC克隆多样性中的作用，有助于理解 原发和回忆性GC克隆动力学的差异，以及克隆动力学是如何 逆转录病毒感染引起的慢性GCs随时间的变化。在实现我们的特定目标后，我们 期望增加我们对GC中的克隆竞争如何影响多样性和 抗体反应的免疫优势。我们希望我们的发现将为未来的疫苗提供参考 发展，特别是针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.