Unraveling Human T Follicular Helper Cell Development

摘要 中和抗体对于预防传染病至关重要。淋巴结(LN)生发中心 (GC)是B细胞经历抗体亲和力成熟并发育成长寿浆细胞的部位--KEY 感染或感染后产生高效中和抗体所需的事件 接种疫苗。T滤泡辅助细胞(TFH)是CD4+T细胞亚群，负责在体内提供B细胞帮助 正在进行的GC。TFH对于GC的形成和维持是绝对必要的。推而广之，TFH是必要的 在抗原暴露后产生有效的中和抗体。虽然TFH反应的许多方面 和促甲状腺激素释放激素的功能已经在动物模型中得到了检验，人类促甲状腺激素释放激素在引流性LN中的反应只有 最近通过一种已建立的技术的新应用进行了探索：连续超声引导下的精细 接种疫苗后引流层粘连的针吸。通过这项技术，我们最近展示了 针对SARS-CoV-2尖峰蛋白(S)的抗原特异性TFH免疫在GC中持续更多 在接种疫苗后4个月内，与S特异性GC B细胞的存在相关。此外，我们 已经注意到LN TFH转录表型的显著变化提示在此基础上功能成熟 在较小的初步队列中使用单细胞RNA-seq延长GC时间间隔。在这项提案中，我们将 对这些发现进行扩展以解决我们的主要假设：人类TFH表型成熟发生在 接种疫苗后引流LN的时间和这些表型变化与TFH的变化有关 功能。为了探索这一假设，我们提出了三个具体的目标：1)我们首先建立TFH的表型 随着时间的推移，成熟发生在多个抗原特异性TFH群体中，我们将从这些群体中定义和表征 使用单细胞rna-seq和体外表位的14名新冠肺炎基因疫苗接受者的队列 鉴定方法。2)我们将确定这些抗原特异的TFH种群在第三代中如何变化 新冠肺炎基因疫苗“Boost”后的免疫应答及其连续LN采样 队列参与者。我们还将确定是否有新的抗原特异性TFH群体招募到GC 召回回应。3)最后，我们将验证我们观察到的深刻的表型变化导致了 利用分选的人体外系统检测抗原特异性TFH为B细胞提供帮助的功能 Ln TFH和小鼠体内过继移植模型。通过实现这些目标，我们将显著增强 我们对人类TFH在引导GC B细胞对疫苗的反应中所起的作用的理解。

ABSTRACT Neutralizing antibodies are critical for protection from infectious diseases. The lymph node (LN) germinal center (GC) is the site where B cells undergo antibody affinity maturation and develop into long-lived plasma cells – key events that are required for the development of highly effective neutralizing antibodies following infection or vaccination. T follicular helper cells (TFH) are the CD4+ T cell subset responsible for providing B cell help during an ongoing GC. TFH are absolutely required for GC formation and maintenance. By extension, TFH are necessary to produce effective neutralizing antibodies following antigen exposure. While many aspects of the TFH response and TFH function have been examined in animal models, human TFH responses in the draining LN have only recently been explored with the novel application of an established technique: serial ultrasound-guided fine needle aspiration of draining LN following vaccination. With this technique, we recently demonstrated that the antigen-specific TFH response to SARS-CoV-2 spike (S) protein mRNA vaccination persists in the GC for more than 4 months following vaccination and correlates with the presence of S-specific GC B cells. Furthermore, we have noted substantial LN TFH transcriptional phenotypic changes suggestive of functional maturation over this prolonged GC time interval using single cell RNA-seq in a small preliminary cohort. In this proposal, we will expand upon these findings to address our primary hypothesis: human TFH phenotypic maturation occurs over time in the draining LN following vaccination and these phenotypic changes are associated with changes in TFH function. To explore this hypothesis, we propose three specific aims: 1) We will first establish that TFH phenotypic maturation occurs over time in multiple antigen-specific TFH populations that we will define and characterize from a cohort of fourteen COVID-19 mRNA vaccine recipients using single cell RNA-seq and ex vivo epitope identification methods. 2) We will determine how these antigen-specific TFH populations change in a tertiary immune response following COVID-19 mRNA vaccine “boost” and continued serial LN sampling of the same cohort participants. We will also ascertain if new antigen-specific TFH populations are recruited to the GC during a recall response. 3) Finally, we will verify that the profound phenotypic changes we observe lead to changes in the functional capacity of antigen-specific TFH to provide help to B cells using an ex vivo system of sorted human LN TFH and an in vivo murine adoptive transfer model. By addressing these aims, we will significantly enhance our understanding of the role that human TFH play in directing GC B cell responses to vaccination.