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

项目2：摘要 针对凝血因子VIII（FVIII）的抗体形成是当前蛋白质治疗中的主要且严重的并发症， X连锁出血性疾病血友病A的替代疗法。大约20-30%的患者 产生抑制凝血活性的中和抗体（“抑制剂”），从而使治疗复杂化， 发病率和死亡率风险增加，治疗费用增加。FVIII可引发极高滴度抗体 尽管在低抗原剂量下静脉内给予，FVIII特异性B细胞应答是 依赖于CD 4 + T辅助细胞并需要共刺激。然而，令人惊讶的是， FVIII抗原呈递给T细胞或B细胞活化的体内机制。在这份提案中，我们试图回答 其中抗原呈递细胞（APC）是MHC II呈递给CD 4 + T细胞所需的（导致FVIII-1）。 特异性B细胞活化），这些APC如何相互作用以引发FVIII特异性CD 4 + T细胞，哪些CD 4 + T细胞亚群 T细胞被诱导以促进B细胞活化（包括T滤泡辅助细胞，Tfh细胞），以及如何先天地 免疫信号可以改变这些事件。与其他项目合作，我们将使用这些新建立的 实验模型，以确定来自微生物组的信号的影响和改变的微生物的影响。 FVIII的分子结构我们提出三个具体目标。目的1：明确MHC在体内的作用机制 II将FVIII抗原呈递给CD 4 + 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呈递测定。我们将使用 结合特定APC的耗竭、共聚焦显微镜和活体双光子显微镜， 确定关键APC的要求和作用，它们在淋巴器官中的位置， 交互.我们将观察树突状细胞和T细胞的迁移以及特定T细胞在APC周围的聚集， 并询问增加B细胞活化的先天性和细胞因子应答。目标二是明确机制 FVIII特异性CD 4 + T细胞和B细胞的体内活化。使用消耗和 实验/敲除菌株，过继性T细胞转移研究，流式细胞术和细胞因子测定，我们将 确定T和B细胞活化的要求，以及活化B细胞的CD 4 + 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）。目的3是确定来自先天免疫的信号的作用， 感测和微生物组对B和T细胞针对FVIII的活化的影响。在这里，我们将研究TLR 9 激动剂和抗炎药物对树突状细胞亚群的影响，作为炎症信号如何的一个例子， 改变抑制剂形成。与项目3合作，我们将确定免疫刺激或 抑制性微生物组改变FVIII抗原呈递、CD 4 + T细胞活化和Tfh应答。

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.