In vivo approaches to dissecting B cell-T cell cooperation in germinal centers

描述(申请人提供)：生发中心(GC)对人类健康非常重要。它们不仅产生高亲和力的抗体，使接种成为可能，而且是过敏和体液自身免疫性疾病的原因，而且也是引发大多数B细胞恶性肿瘤的遗传损伤的来源。对于GC反应中选择B细胞克隆如何产生高度突变的抗体，例如广泛中和HIV所需的抗体，我们的理解存在一个根本的差距。这一差距是开发针对许多人类疾病的有效疫苗的主要障碍。研究人员的长期目标是在细胞和分子水平上详细了解B细胞反应的各个方面，从第一次接触抗原到产生高亲和力抗体。最近的工作由 这位研究人员和其他人强调了T滤泡辅助细胞(TFH)和B细胞在GC选择中相互作用的重要性。此应用程序的目标是调查 这种相互作用的具体方面，特别是关于它对B细胞命运选择的影响。提出这项研究的基本原理是，由于TFH细胞控制GC的关键方面--如大小、持久性和选择性压力--了解B细胞和TFH细胞之间的关系将提高我们诱使GCs产生广泛中和所需的高度突变抗体的能力。在这方面，提出了三个具体目标。首先，研究人员开发的一种方法--多光子显微镜在体内的光激活将被用来确定GC与传入的TFH细胞的可及性在确定GC中T细胞特异性如何控制方面的作用。这一目标的假设是，GC对TFH细胞的进出是开放的，这种开放影响GC T细胞的克隆性和特异性以及GC反应的寿命。其次，研究人员还开发了一种在体内触发Tfh细胞对GC B细胞的选择的方法，该方法将用于探索B细胞在选择时诱导的基因表达变化。这个目的的假设是TFH细胞帮助触发特定的基因表达程序，这些程序决定了GC B细胞随后做出的命运选择。最后，研究人员建议开发一种新的方法来测量体内TFH细胞和B细胞之间的相互作用历史。这项技术将使免疫细胞之间的相互作用能够以一种技术上简单得多的方式进行探测和量化，而且比目前可用的方法更具生理学意义。这项拟议的研究具有重要意义，因为它将有助于我们在细胞和分子水平上理解GC选择的关键方面，特别是关于它对T细胞帮助的依赖。这一知识应该可以更好地控制GC反应，特别是关于其寿命和产量。能够控制TE GC的这些方面是通过接种疫苗实现广泛中和的先决条件。

DESCRIPTION (provided by applicant): Germinal centers (GCs) are of tremendous importance to human health. Not only do they generate the high-affinity antibodies that make vaccination possible and are the cause of allergies and humoral autoimmune diseases, but they are also the source of the genetic lesions that trigger most B cell malignancies. There is a fundamental gap in our understanding of how the selection of B cell clones during the GC reaction can generate the highly mutated antibodies necessary, for example, for broad neutralization of HIV. This gap represents a major obstacle to the development of effective vaccines for many human diseases. The long-term goal of the investigator's research is to develop a detailed understanding, at the cellular and molecular levels, of all aspects of the B cel response, from first contact with antigen to production of high-affinity antibodies. Recent work by the investigator and others has underscored the importance of interaction between T follicular helper (Tfh) cells and B cells in GC selection. The objective of this application is to investigate specific aspects of this interaction, especially with regard to its influence on B cell fate choice. The rationale for the proposed research is that, because Tfh cells control key aspects of the GC-such as magnitude, persistence, and selective pressure-understanding the relationship between B cells and Tfh cells should improve our ability to coax GCs into producing the highly mutated antibodies required for broad neutralization. In this context, three specific aims are proposed. First, in vivo photoactivation by multiphoton microscopy, a method developed by the investigator, will be used to determine the role of accessibility of GCs to incoming Tfh cells in determining how T cell specificity in the GC is controlled. The hypothesis in this aim is that GCs are open to the ingress and egress of Tfh cells, and that this openness impacts the clonality and specificity of GC T cells and the longevity of GC reactions. Second, a method to trigger selection of GC B cells by Tfh cells in vivo, also developed by the investigator, will be used to probe the gene expression changes that are induced in B cells upon selection. The hypothesis in this aim is that Tfh cell help triggers specific gene expression programs that determine the subsequent fate choices made by of GC B cells. Finally, the investigator proposes to develop a novel approach to measuring the history of interactions between Tfh cells and B cells in vivo. This technique will allow for interactions between immune cells to be probed and quantified in a technically much simpler manner, and in a more physiological context, than by currently available methods. The proposed research is significant because it will contribute to our understanding of key aspects of GC selection at the cellular and molecular levels, especially with regard to its reliance on T cell help. This knowledge should allow better control over the GC reaction, especially with regard to its longevity and output. Ability to control these aspects of te GC is a prerequisite for achieving broad neutralization by vaccination.