IN VIVO APPROACHES TO DISSECTING B CELL-T CELL COOPERATION IN GERMINAL CENTERS

描述（由申请人提供）：老年中心（GC）对人类健康至关重要。它们不仅产生高亲和力抗体，使疫苗接种成为可能，而且是过敏和体液自身免疫疾病的原因，而且它们也是引发大多数B细胞恶性肿瘤的遗传病变的来源。在GC反应过程中，B细胞克隆的选择如何产生高度突变的抗体，例如，广泛中和HIV所必需的抗体，在我们的理解中存在根本性的差距。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。研究者研究的长期目标是在细胞和分子水平上详细了解B细胞反应的所有方面，从第一次接触抗原到产生高亲和力抗体。最近的工作 研究者和其他人已经强调了T滤泡辅助（Tfh）细胞和B细胞之间的相互作用在GC选择中的重要性。本申请的目的是调查 这种相互作用的特定方面，特别是关于其对B细胞命运选择的影响。这项研究的基本原理是，由于Tfh细胞控制GC的关键方面，如幅度，持久性和选择性压力，理解B细胞和Tfh细胞之间的关系应该提高我们的能力，诱导GC产生广泛中和所需的高度突变抗体。在这方面，提出了三个具体目标。首先，在体内光活化的多光子显微镜，一种由研究者开发的方法，将被用来确定的GC的可及性的传入Tfh细胞在确定如何在GC中的T细胞特异性控制的作用。该目的的假设是GC对Tfh细胞的进出是开放的，并且这种开放性影响GC T细胞的克隆性和特异性以及GC反应的寿命。第二，也由研究者开发的触发Tfh细胞在体内选择GC B细胞的方法将用于探测选择时在B细胞中诱导的基因表达变化。该目标的假设是Tfh细胞帮助触发特定的基因表达程序，该程序决定GC B细胞随后做出的命运选择。最后，研究者建议开发一种新的方法来测量Tfh细胞和B细胞在体内的相互作用的历史。该技术将允许以技术上更简单的方式，并且在比目前可用的方法更生理的背景下，探测和量化免疫细胞之间的相互作用。这项研究意义重大，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，特别是关于其对T细胞帮助的依赖。这些知识应该允许更好地控制GC反应，特别是关于其寿命和输出。控制这些方面的能力是通过疫苗接种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.