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

因子 VIII 免疫反应的体内机制：项目 2

• 批准号: 10406334
• 负责人: Roland W. Herzog
• 金额: $ 27.5万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406334
• 关键词: Agonist; Animal Model; Anti-Inflammatory Agents; Antibodies; Antibody Formation; Antibody Response; Antibody titer measurement; Antigen Presentation; Antigen-Presenting Cells; Antigens; Architecture; Area; Autoimmune Diseases; B-Cell Activation; B-Lymphocyte Epitopes; B-Lymphocytes; Biological Assay; Biology; Blood Coagulation Disorders; CD4 Positive T Lymphocytes; CD80 Antigens; Cellular Immunology; Coagulation Process; Collaborations; Complication; Confocal Microscopy; DNA; Dendritic Cells; Dioxygenases; Dose; Epitopes; Event; Experimental Models; F8 gene; Factor VIII; Flow Cytometry; Fluorescence; Generations; Goals; Helper-Inducer T-Lymphocyte; Hemophilia A; Imaging technology; Immune; Immune response; Immune signaling; Immune system; In Vitro; Inflammatory; Innate Immune System; Integration Host Factors; Intravenous; Knock-out; Label; Lead; Link; Location; Malignant Neoplasms; Memory B-Lymphocyte; Molecular; Molecular Structure; Morbidity - disease rate; Neurobiology; Patients; Pattern recognition receptor; Pharmaceutical Preparations; Plasma; Play; Positioning Attribute; Proteins; Pyrroles; Regulatory T-Lymphocyte; Reporting; Research; Research Personnel; Risk; Role; Signal Transduction; Structure; System; T cell therapy; T-Cell Activation; T-Lymphocyte; TALL-1 protein; TLR9 gene; Treatment Cost; Viral; Work; adaptive immune response; antibody inhibitor; base; cell motility; cell type; cytokine; enzyme replacement therapy; experimental study; immunogenicity; in vivo; indoleamine; inhibitor; innate immune pathways; innate immune sensing; intravital microscopy; lymphoid organ; microbiome; microbiome alteration; microbiome research; monocyte; mortality; neutralizing antibody; novel; recruit; response; sensor; two photon microscopy; two-photon; uptake; von Willebrand Factor

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.

项目2：摘要 抗凝结因子VIII（FVIII）的抗体形成是当前蛋白质的主要且严重的并发症 X连锁出血障碍血友病的替代疗法A。大约20-30％的患者 开发中和抗体（“抑制剂”），以抑制凝血活性，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可以引起非常高的抗体 在低抗原剂量下静脉注射地层目的地。 FVIII特异性B细胞反应是 取决于CD4+ T辅助细胞，需要共刺激。但是，令人惊讶的是关于In的知之甚少 FVIII抗原表现为T细胞或B细胞活化的体内机制。在此提案中，我们试图回答 MHC II向CD4+ T细胞表示哪些抗原呈递细胞（APC）（导致FVIII- 特定的B细胞激活），这些APC如何与prime FVIII特异性CD4+ T细胞相互作用，CD4+的子集 诱导T细胞促进B细胞激活（包括T卵泡辅助器，TFH，细胞），以及如何先天 免疫信号传导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 实验模型，以确定来自微生物组的信号的影响以及改变的影响 FVIII的分子结构。我们提出了三个具体目标。目标1是定义体内MHC的机制 II介绍了FVIII抗原与CD4+ T细胞的抗原。我们将利用荧光标记的FVIII在体内研究 抗原吸收，并基于表位标记的FVIII建立体内MHC II演示分析。我们将使用 特定APC，共聚焦显微镜和插入式2光子显微镜的耗竭的组合到 确定关键APC的要求和作用，它们在淋巴机构中的位置及其位置 互动。我们将可视化的树突状细胞和T细胞迁移以及APC周围特定T细胞的聚类， 并询问增加B细胞激活的先天和细胞因子反应。目标2是定义机制 FVIII特异性CD4+ T细胞和B细胞的体内激活。结合部署 实验/敲除菌株，自适应T细胞转移研究，流式细胞仪和细胞因子刺激，我们将 确定T和B细胞激活的需求以及激活B细胞的CD4+ T细胞的子集 及其对共同刺激的要求（特别强调了TFH细胞在原发性和 内存B细胞激活）。 AIM 3是确定来自先天免疫的信号的影响 针对FVIII的B和T细胞激活上的传感和微生物组。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 改变抑制剂形成。在与项目3合作的 抑制性微生物组改变了FVIII抗原表现，CD4+ T细胞激活和TFH反应。