Multidimensional Single Cell Analysis of the Germinal Center Reaction

 描述（由申请人提供）：生产可以中和靶病原体的强效高亲和力抗体仍然是疫苗研究的中心目标。虽然我们对抗体分子本身了解很多，但对产生抗体的高亲和力记忆B细胞的编程仍然知之甚少。许多有前途的疫苗抗原不能使用当代疫苗制剂引发保护性抗体。因此，在我们对抗体亲和力成熟的基本理解以及如何使用合理的蛋白质疫苗接种来增强不良免疫原性方面仍然存在主要差距。研究重点：GC是暴露于外源抗原后在次级淋巴组织内出现的动态微环境。这些GC反应中抗体特异性的进化是产生高亲和力记忆B细胞和长寿浆细胞所必需的。滤泡辅助性T（TFH）细胞为GC的启动和GC反应中正在进行的进化机制的控制提供了分子线索。了解控制记忆BCR进化和GC B细胞功能的调节机制仍然是该领域的重要基本目标，并且是当前应用的主要焦点。具体目标：我们已经开发了多维的单细胞策略来跟踪这些复杂的细胞行为在克隆和亚克隆水平在体内。我们将使用这种方法：SA-1：研究次级GC诱导的分子控制和回忆SA-2的功能;剖析GC转录编程SA-3的阶段特异性循环进展;修改GC B细胞进化和GC退出的分子控制。影响：我们使用蛋白质疫苗接种的多克隆小鼠模型，采用最先进的抗原特异性单细胞分析， 免疫功能这些模型抗原的高分辨率研究有能力改变围绕现有疫苗范式的基本概念框架。重要的是，这些基本的新原理和测定法可用于重新设计当代疫苗制剂，以优化对更复杂抗原的高亲和力B细胞免疫。

 DESCRIPTION (provided by applicant): Producing potent high-affinity antibodies that can neutralize target pathogens remains a central goal for vaccine research. While a great deal is known about antibody molecules themselves, still too little is understood about the programming of high-affinity memory B cells that produce them. Many promising vaccine antigens fail to elicit protective antibodies using contemporary vaccine formulations. Thus, major gaps remain in our basic understanding of antibody affinity maturation and how to enhance poor immunogenicity using rational protein vaccination. Research Focus: GCs are dynamic microenvironments that emerge within secondary lymphoid tissues following exposure to foreign antigens. The evolution of antibody specificity within these GC reactions is required to generate high-affinity memory B cells and long-lived plasma cells. Follicular helper T (TFH) cells provide the molecular cues for initiation of GCs and control of ongoing evolutionary mechanisms within the GC reaction. Understanding the regulatory mechanisms that control memory BCR evolution and GC B cell function remain an important basic goal for the field and is the major focus of the current application. Specific Aims: We have developed multi-dimensional single cell strategies to track these complex cellular behaviors at the clonal and sub-clonal levels in vivo. We will use this approach: SA-1: to study the molecular control of secondary GC induction and function at recall SA-2: to dissect the stage-specific cyclic progression of GC transcriptional programming SA-3: to modify the molecular control of GC B cell evolution and GC exit. Impact: We use polyclonal murine models of protein vaccination with state-of-the-art single cell analyses of antigen-specific immune function. These high-resolution studies of model antigens have the capacity to shift the basic conceptual framework that surrounds existing vaccine paradigms. Importantly, these basic new principles and assays can be used to re-design contemporary vaccine formulations that optimize high-affinity B cell immunity to more complex antigens.