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

项目摘要 在生殖中心（GC）中产生高亲和力抗体是多种临床治疗中的关键步骤。 相关过程，从通过先前感染或疫苗接种预防病原体到 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框架， 其中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，在其后代中产生亲和力的变化。获得亲和力增加突变的罕见B细胞 然后在GC群体内选择性扩增，从而增加GC B细胞作为 在这个过程中，我们称之为积极选择。 尽管经过几十年的研究，积极选择的精确细胞机制-换句话说，GC如何“挑选” 然而，”淘汰“具有最高亲和力的B细胞仍然是一个争论的话题。10多年前，我们提供了第一个在 在小鼠中的体内证据表明，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细胞可以感知B细胞在其表面可以呈递多少肽（这反过来又取决于B 细胞的亲和力），选择性地向最高亲和力的B细胞提供帮助。然而，尽管积累了功能 该模型的证据是，基于T细胞的亲和性将T细胞辅助选择性递送至B细胞从未被直接地应用于治疗。 在生理环境中得到证实。为了实现这一目标，我们开发了LIPSTIC，这是一种允许我们 在体内以高精度直接记录T细胞对B细胞的帮助。在本项目的目标1中，我们建议使用 LIPSTIC作为在经典半抗原载体诱导的GC选择模型中直接测试T细胞辅助模型的手段。 在目标2中，我们将通过在人类疫苗诱导的GC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结合两个新版本来研究这种策略， 流感诱导的GC中多抗原驱动选择的动力学。

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.