In Vivo Mechanism of Immune Response to Factor VIII: Project 2

项目2：摘要 抗凝血因子VIII(FVIII)抗体的形成是当前蛋白质中一个主要和严重的并发症 X连锁出血性疾病血友病A的替代疗法约20%-30%的患者 产生中和抗体(“抑制物”)，抑制凝血活性，从而使治疗复杂化， 增加发病率和死亡率的风险，并提高治疗成本。FVIII可以诱导出非常高滴度的抗体 尽管静脉注射的抗原剂量较低，但仍可形成。FVIII特异的B细胞反应是 依赖于CD4+T辅助细胞，需要协同刺激。然而，令人惊讶的是，人们对IN知之甚少 FVIII抗原呈递给T细胞或B细胞激活的体内机制。在这项提议中，我们试图回答 MHC II呈递到CD4+T细胞需要哪些抗原提呈细胞(导致FVIII- 特异的B细胞激活)，这些APC如何与FVIII特异性的CD4+T细胞相互作用，哪些亚群的CD4+ 诱导T细胞促进B细胞活化(包括T滤泡辅助细胞，TFH，细胞)，以及如何先天 免疫信号可能会改变这些事件。与其他项目合作，我们将使用这些新建立的项目 确定来自微生物组的信号的影响和改变效果的实验模型 FVIII的分子结构。我们提出了三个具体目标。目的1是定义体内MHC的机制 II FVIII抗原呈递给CD4+T细胞。我们将利用荧光标记的FVIII进行体内研究 抗原摄取，并建立基于表位标记的FVIII的体内MHC II递呈试验。我们将使用 特定APC的耗尽、共聚焦显微镜和活体双光子显微镜的组合 确定关键APC的需求和作用，它们在淋巴器官中的位置，以及它们的 互动。我们将可视化树突状细胞和T细胞在APC周围的特定T细胞的迁移和聚集， 并询问增加B细胞激活的先天反应和细胞因子反应。目标2是定义机制 在体内激活FVIII特异性的CD4+T细胞和B细胞。使用耗尽的组合 实验/基因敲除菌株，过继T细胞转移研究，流式细胞仪，和细胞因子分析，我们将 确定T和B细胞激活的要求，以及激活B细胞的CD4+T细胞亚群 以及它们对共刺激的要求(特别强调TFH细胞在原代和 记忆B细胞激活)。目标3是确定来自先天免疫的信号的影响。 感受和微生物群对B细胞和T细胞激活抗FVIII的作用。在这里，我们将研究TLR9的影响 树突状细胞亚群上的激动剂和抗炎药作为炎症信号的一个例子 改变抑制剂的形成。在与项目3的合作中，我们将确定免疫刺激剂或 抑制微生物群改变FVIII抗原提呈、CD4+T细胞活化和TFH反应。

Project 2: Abstract Antibody formation against coagulation factor VIII (FVIII) is a major and serious complication in current protein replacement therapy for the X-linked bleeding disorder hemophilia A. Approximately 20-30% of patients develop neutralizing antibodies (“inhibitors”) that inhibit coagulation activity, thereby complicating treatment, increasing risks of morbidity and mortality, and raising treatment costs. FVIII can elicit very high-titer antibody formation despite being given intravenously at low antigen doses. FVIII-specific B cell responses are dependent on CD4+ T helper cells and require co-stimulation. However, surprisingly little is known about the in vivo mechanism of FVIII antigen presentation to T cells or B cell activation. In this proposal, we seek to answer which antigen presenting cells (APCs) are required for MHC II presentation to CD4+ T cells (leading to FVIII- specific B cell activation), how these APCs interact to prime FVIII-specific CD4+ T cells, which subsets of CD4+ T cells are induced to promote B cell activation (including T follicular helper, Tfh, cells), and how innate immune signaling may alter these events. Working with the other projects, we will use these newly established experimental models to determine the impact of signals derived from the microbiome and the effect of altered molecular structure of FVIII. We propose three specific aims. Aim 1 is to define the mechanism of in vivo MHC II presentation of FVIII antigen to CD4+ T cells. We will utilize fluorescently labeled FVIII to study in vivo antigen uptake, and establish an in vivo MHC II presentation assay based on epitope-tagged FVIII. We will use a combination of depletion of specific APCs, confocal microscopy, and intravital 2-photon microscopy to determine the requirements and roles of the critical APCs, their location in lymphoid organs, and their interactions. We will visualize dendritic cell and T cell migration and clustering of specific T cells around APCs, and interrogate innate and cytokine responses that increase B cell activation. Aim 2 is to define the mechanism of in vivo activation of FVIII-specific CD4+ T cells and B cells. Using a combination of depletion experiments/knock-out strains, adoptive T cell transfer studies, flow cytometry, and cytokine assays, we will determine the requirements for T and B cell activation, and the subsets of CD4+ T cells that activate B cells and their requirements for co-stimulation (with particular emphasis on the role of Tfh cells in primary and memory B cell activation). Aim 3 is to determine the effects of signals derived from innate immune sensing and the microbiome on B and T cell activation against FVIII. Here, we will study the effects of TLR9 agonists and anti-inflammatory drugs on dendritic cell subsets as an example of a how inflammatory signals alter inhibitor formation. In collaboration with Project 3, we will determine how an immune stimulatory or suppressive microbiome alters FVIII antigen presentation, CD4+ T cell activation, and Tfh responses.