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.

总结 基于抗体的体液免疫在幼稚B细胞的初始活化和随后的免疫应答之后产生。 分化为抗体分泌细胞（ASC）以及记忆B细胞（MBC）。抗原体验MBC 通过分化为ASC提供更快速和更强的免疫应答。目前的流行病 已经将世界的注意力集中在抗体和B细胞保护性体液免疫的产生上， 是由MBC和ASC衍生而来的。然而，在我们对细胞命运的基本知识方面， 异质性和表观遗传选择，B细胞成为ASC。比如，MBC如何应对 更快地回忆挑战，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MBC的转录组和表观基因组中观察到的异质性？我们 最近的工作和初步数据表明：1）表观遗传和转录编程的多个 途径，包括使用铁/血红素途径对于血红素处理的离体分化培养物很重要 导致形成的ASC数量急剧增加; 2）MBC在表观遗传学上被启动以响应 次级挑战;和3）同种型特异性浆细胞和MBC在细胞周期中显示异质性编程。 转录水平和表观遗传水平的MBC。如何建立这种编程并不完全 明白为了解决我们知识中的这些空白，Aim 1将确定铁/血红素依赖性途径 调节ASC的形成，并检验血红素含量改变ASC的动力学和编程的假设 分化通过多种机制和途径，包括线粒体代谢， 转录因子活性和需要铁的表观遗传修饰剂的活性。我们将利用 我们建立了一个慢基因CRISRP/Cas9基因编辑系统，用于引入突变并探测 影响铁/血红素代谢的因素。结合项目3和项目4，我们将研究人类B细胞 从健康和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和伊加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- 在MBC形成期间启动了特定的表观遗传程序，这些程序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，因为细胞经历额外的类别转换 重组为了验证这些假设，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具有可塑性 当MBC和ASC进一步从IgG转换为伊加时。了解分子和表观遗传 MBC如何分化的基础最终将有助于我们开发治疗方法的能力， 上级B细胞记忆、ASC生成和保护。我们将与每个项目合作， 血红素在异质性反应中的作用，表观遗传途径分析，以及我们基因编辑经验的使用。