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.

概括 基于抗体的体液免疫在幼稚 B 细胞最初激活及其随后的激活后产生 分化为抗体分泌细胞 (ASC) 以及记忆 B 细胞 (MBC)。有抗原经历的MBC 通过分化为 ASC，提供更快速、更强大的免疫反应。当前的疫情 抗体的产生和B细胞保护性体液免疫引起了全世界的关注， 源自 MBC 和 ASC。然而，我们对细胞命运的基础知识存在重大差距 B 细胞成为 ASC 的异质性和表观遗传选择。例如MBC如何应对 更快地回想起挑战，MBC 中是否存在初始细胞中不存在的潜在途径；以及如何 这些过程是否定义了 MBC 转录组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）多个基因的表观遗传和转录编程 途径，包括铁/血红素途径的使用很重要，因为血红素处理的离体分化培养物 导致形成的 ASC 数量急剧增加； 2) MBC 在表观遗传学上已准备好响应 次要挑战； 3) 同型特异性等离子体细胞和 MBC 在 MBC 的转录水平和表观遗传水平。这种编程是如何建立的尚不完全 明白了。为了解决我们知识中的这些空白，目标 1 将确定铁/血红素依赖性途径如何 调节 ASC 形成并检验血红素含量改变 ASC 动力学和编程的假设 通过多种机制和途径进行分化，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剂的活性。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究其作用 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 来自健康和 SLE 受试者的子集来表征他们对血红素和异质性的反应。目标2将 确定与 IgM、IgG 和 IgA MBC 相关的不同转录组的表观遗传基础和可塑性 和 ASC 使用项目 1 开发的流感感染模型。我们假设不同的同种型 特定的表观遗传程序在 MBC 形成过程中启动，并随着细胞分化而维持 相同同种型的 ASC，并且这些程序可能是可塑的，因为细胞会经历额外的类别转换 重组。为了检验这些假设，我们将确定同种型转换 ASC 的表观遗传基础 转录组； ASC 对流感反应的异质性；并确定表观基因组是否是可塑的 当 MBC 和 ASC 进一步从 IgG 类别切换为 IgA 时。了解分子和表观遗传学 MBC 如何区分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 细胞记忆、ASC 生成和保护。我们将与该角色中的每个项目进行合作 血红素在异质反应、表观遗传途径分析以及我们的基因编辑经验的运用。