Defining the role of T cell help in germinal centers by intercellular enzymatic labeling

项目总结 在生发中心(GC)产生高亲和力抗体是多种临床疾病的关键步骤。 相关过程，从先前感染或接种疫苗对病原体的保护到发展 过敏和自身免疫性疾病。抗体亲和力成熟遵循典型的达尔文主义框架，在 哪些GC B细胞将随机突变引入其免疫球蛋白(Ig)的抗原结合部分 基因，在它们的后代中产生亲和力的变化。罕见的获得亲和力增加突变的B细胞 然后在GC群体中选择性地扩增，从而增加GC B细胞作为 整个过程，我们称之为正向选择。 尽管进行了数十年的研究，但正向选择的确切细胞机制--换句话说，GC如何“挑选” Out“具有最高亲和力的B细胞--仍然是一个争论的话题。十多年前，我们提供了第一个 在小鼠中的活体证据表明T滤泡辅助细胞(TFH)作为这一选择性过程的仲裁者。在我们的模型中， TFH细胞会感觉到B细胞表面能呈现多少肽(这反过来又取决于B细胞 细胞亲和力)，选择性地为亲和力最高的B细胞提供帮助。然而，尽管积累了泛函 这一模型的证据是，基于B细胞的亲和力，选择性地将T细胞帮助传递到B细胞从来没有直接 在生理环境中表现出来。为了实现这一点，我们开发了LIPSTIC，这是一种允许我们 直接记录T细胞有助于在体内高精度地将B细胞转化为B细胞。在本项目的目标1中，我们建议使用 LIPSTIC作为一种手段，在经典的半抗原载体诱导的GC选择模型中直接测试T细胞帮助模型。 在目标2中，我们将通过在人类疫苗诱导的GCs中测试我们在小鼠LIPSTIC中的发现来跟进这一点。 在目标3中，我们使用原始的LIPSTIC和关于这一策略的两个新版本来研究 流感诱导的GC中多抗原驱动的选择的动力学。

PROJECT SUMMARY Generation of high affinity antibodies in germinal centers (GCs) is a critical step in a wide variety of clinically relevant processes, from protection against pathogens by prior infection or vaccination to the development of allergies and autoimmune diseases. Antibody affinity maturation follows a prototypical Darwinian framework, in which GC B cells introduce random mutations into the antigen-binding portions of their immunoglobulin (Ig) genes, generating variations in affinity within their progeny. Rare B cells that acquire affinity-increasing mutation are then selectively expanded within the GC population, thus increasing the average affinity of GC B cells as a whole, in a process we refer to as positive selection. Despite decades of work, the precise cellular mechanisms of positive selection—in other words, how GCs “pick out” B cells with the highest affinity—remains a topic of debate. More than 10 years ago, we provided the first in vivo evidence in mice for a role for T follicular helper (Tfh) cells as arbiters of this selective process. In our model, Tfh cells would sense how much peptide a B cell could present on its surface (which in turn depended on the B cell’s affinity), providing help selectively to the highest-affinity B cells. However, despite accumulating functional evidence for this model, selective delivery of T cell help to B cells based on their affinity has never been directly demonstrated in physiological settings. To achieve this, we developed LIPSTIC, a method that allows us to directly record T cell help to B cells with great precision in vivo. In Aim 1 of this project, we propose to use LIPSTIC as a means to directly test the T cell help model in classic hapten-carrier induced GC selection models. In Aim 2, we will follow up on this by testing our findings from mouse LIPSTIC in human vaccine-induced GCs. In Aim 3, we use the original LIPSTIC in conjunction with two novel versions on this strategy to investigate the dynamics of multi-antigen driven selection in influenza-induced GCs.