Project 2

项目2

• 批准号: 10428168
• 负责人: JEREMY M. BOSS
• 金额: $ 52.16万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-25 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10428168
• 关键词: Address; Affinity; Antibodies; Antigens; Attention; Autoimmune; B cell differentiation; B-Lymphocyte Subsets; B-Lymphocytes; BACH2 gene; Binding; Biological Assay; CRISPR/Cas technology; Cell Differentiation process; Cell Lineage; Cells; Chlorides; Chromatin; DNA Methylation; Data; Development; Disease; Epigenetic Process; Generations; Genes; Genetic Transcription; Health; Heme; Heme Iron; Hemin; Heterogeneity; Human; Humoral Immunities; Immune response; Immunity; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Switch Recombination; Immunoglobulin-Secreting Cells; Infection; Influenza; Iron; Kinetics; Knowledge; Link; Mediating; Memory; Memory B-Lymphocyte; Metabolic; Metabolism; Modeling; Molecular; Mus; Mutagenesis; Mutation; Outcome; Pathway Analysis; Pathway interactions; Plasma Cells; Play; Process; Protocols documentation; Role; Sampling; Secondary to; Signal Transduction; Structure of germinal center of lymph node; Surface; System; Systemic Lupus Erythematosus; Technology; Testing; Up-Regulation; Vaccination; Work; base; cellular engineering; epigenome; epigenomics; experience; functional outcomes; in vivo; influenza infection; iron metabolism; mitochondrial metabolism; novel; pandemic disease; plasma cell differentiation; programs; response; synergism; transcription factor; transcriptome

Summary Antibody based humoral immunity arises following the initial activation of naïve B cells and their subsequent differentiation to antibody secreting cells (ASC), as well as memory B cells (MBC). Antigen-experienced MBC provide a more rapid and robust immune response through their differentiation to ASC. The current pandemic has focused the world’s attention on the generation of antibodies and B cell protective humoral immunity, which is derived from MBC and ASC. Yet, there are significant gaps in our basic knowledge of the cell fate heterogeneity and epigenetic choices that B cells take to become ASC. For example, how do MBC respond more quickly to recall challenge, are there underlying pathways in MBC that do not exist in naïve cells; and how do these processes define the heterogeneity observed in the transcriptomes and epigenomes of MBC? Our recent work and preliminary data suggest that: 1) epigenetic and transcriptional programming of multiple pathways, including the use of iron/heme pathways is important as heme-treated ex vivo differentiation cultures results in sharp increases in the number of ASCs formed; 2) MBC are epigenetically primed to respond to secondary challenges; and 3) isotype specific plasma cell and MBC display heterogeneous programming at the transcriptional and also at the epigenetic level for MBC. How this programming is established is not fully understood. To address these gaps in our knowledge, Aim 1 will determine how iron/heme dependent pathways modulate ASC formation and test the hypothesis that heme content alters the kinetics and programming of ASC differentiation through multiple mechanisms and pathways, including mitochondrial metabolism, modulation of transcription factor activity, and the activity of epigenetic modifiers that require iron. We will take advantage of a lentigenic CRISRP/Cas9 gene editing system that we established to introduce mutations and probe the roles of factors influencing iron/heme metabolism. In conjunction with Projects 3 and 4, we will examine human B cell subsets from healthy and SLE subjects to characterize their responses to heme and heterogeneity. Aim 2 will determine the epigenetic bases and plasticity for distinct transcriptomes associated with IgM, IgG, and IgA MBC and ASC using an influenza-infection model developed with Project 1. We hypothesize that distinct isotype- specific epigenetic programs are initiated during MBC formation that are maintained as the cells differentiate to ASC of the same isotype, and that these programs may be plastic as cells undergo additional class switch recombination. To test these hypotheses, we will determine the epigenetic basis of isotype-switched ASC transcriptomes; the heterogeneity of the ASC response to influenza; and determine if the epigenome is plastic when MBC and ASC are further class switched from IgG to IgA. Understanding the molecular and epigenetic underpinnings of how MBC differentiate would ultimately aid our ability to develop treatments that result in superior B cell memory, ASC generation, and protection. We will collaborate with each of the projects in the role of heme in heterogeneous responses, epigenetic pathway analysis, and the use of our gene editing experience.

摘要 基于抗体的体液免疫产生于幼稚B细胞的初始激活及其随后的 分化为抗体分泌细胞(ASC)和记忆B细胞(MBC)。有抗原经验的MBC 通过分化为ASC，提供更快速和更强大的免疫反应。当前的大流行 将世界的注意力集中在抗体的产生和B细胞保护性体液免疫上，这 是从MBC和ASC派生出来的。然而，在我们对细胞命运的基本知识上存在着重大的差距 异质性和表观遗传选择，B细胞采取成为ASC。例如，MBC如何响应 更快地回忆起挑战，在MBC中是否存在幼稚细胞中不存在的潜在途径；以及如何 这些过程是否定义了在转录本和表观基因组中观察到的异质性？我们的 最近的工作和初步数据表明：1)表观遗传和转录编程 途径，包括铁/血红素途径的使用，对于经血红素处理的体外分化培养是重要的 导致形成的ASCs数量急剧增加；2)MBC具有表观遗传反应能力 次要挑战；以及3)同种类型特定的浆细胞和MBC在 MBC的转录水平和表观遗传水平。这一计划是如何建立的还不完全 明白了。为了解决我们知识中的这些差距，目标1将确定铁/血红素依赖的途径 调节ASC的形成并检验血红素含量改变ASC的动力学和程序的假设 通过多种机制和途径分化，包括线粒体代谢，调节 转录因子活性，以及需要铁的表观遗传修饰物的活性。我们将利用 我们建立的慢基因CRISRP/Cas9基因编辑系统用于引入突变并探讨其作用 影响铁/血红素代谢的因素。结合项目3和4，我们将检查人类B细胞 来自健康受试者和系统性红斑狼疮受试者的亚群，以表征他们对血红素和异质性的反应。目标2将 确定与Ig M、Ig G和Ig A MBC相关的不同转录体的表观遗传基础和可塑性 和ASC使用与项目1一起开发的流感感染模型。我们假设不同的同型- 特定的表观遗传程序在MBC形成期间启动，并在细胞分化为 相同同种类型的ASC，这些程序可能是可塑性的，因为细胞经历了额外的类别切换 重组。为了检验这些假设，我们将确定同型转换型ASC的表观遗传学基础 转录本；ASC对流感反应的异质性；以及确定表观基因组是否为塑料 当MBC和ASC进一步从免疫球蛋白转换为免疫球蛋白时。理解分子和表观遗传学 MBC差异化的基础最终将有助于我们开发治疗方法，从而导致 卓越的B细胞存储器、ASC生成和保护。我们将与该角色中的每个项目进行协作 血红素在异质反应、表观遗传途径分析和我们的基因编辑经验的使用。