Antibody quality and germinal center requirements for peroxisomal function in lymphocytes

淋巴细胞过氧化物酶体功能的抗体质量和生发中心要求

• 批准号: 10318012
• 负责人: Mark R Boothby
• 金额: $ 25.95万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-16 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318012
• 关键词: Affect; Affinity; Alleles; Anatomy; Antibodies; Antibody Response; Antioxidants; B-Cell Activation; B-Lymphocytes; Benchmarking; Binding; Biochemical; Biology; Cell Survival; Cell physiology; Cells; Chimera organism; Data; Defect; Development; Enzymes; Erythrocytes; Ethers; Excision; FRAP1 gene; Generations; Genes; Germ Lines; Goals; Humoral Immunities; Image; Immune; Immune response; Immunization; Immunize; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Individual; Intervention; Ions; Knock-out; Leukocytes; Limb structure; Link; Lipid Peroxidation; Lipids; Lymphocyte; Maintenance; Maps; Mass Spectrum Analysis; Measurement; Memory; Memory B-Lymphocyte; Metabolism; Microbe; Modeling; Molecular; Mucous Membrane; Mus; Mutate; Nutrient; Organelles; Outcome; Output; Pathway interactions; Pattern; Performance; Phospholipid Ethers; Phospholipids; Plasma Cells; Plasmalogens; Positioning Attribute; Process; Property; Proteins; Reaction; Reactive Oxygen Species; Regulation; Reporter; Reporting; Role; Shapes; Sheep; Signal Transduction; Spleen; Structure; Structure of germinal center of lymph node; Superoxides; Surface; Tamoxifen; Technology; Testing; Tissues; Transcript; Transgenes; Work; adaptive immunity; base; cell type; experimental study; fatty acid oxidation; fluorescence imaging; fluorophore; high risk; improved; in vivo; insight; lipid biosynthesis; lipidomics; loss of function; lymphoid organ; multimodality; novel; pathogen; peroxisome; programs; receptor; response; vaccine efficacy

Project Summary Generating antibodies, refining their qualities, and creating durable humoral memory are crucial parts of adaptive immunity. Protecting against microbes and maintaining commensal relationships with them draw on each facet of antibody regulation. As such, it is vital to decipher key cellular and molecular processes that affect antibody (Ab) qualities. In developing a means of mapping the distributions of nutrients and products of metabolism relative to micro-anatomic features of lymphoid organs, we performed unbiased lipidomic analyses using IMS (imaging mass spectrometry). This study revealed that an interconnected set of phospholipids characterized by non-acyl linkages - i.e., ether-linked and plasmalogen species – that were enriched in germinal centers (GC). Of note, de novo synthesis of ether and plasmalogen lipids and phospholipids requires several enzymes localized exclusively to peroxisomes. One such enzyme is PexRAP, encoded by the gene Dhrs7b, which encodes a late step in ether lipid synthesis. In parallel, recent work of others provided evidence that peroxisomes and fatty acid oxidation (FAO) executed by them are increased in both activated and GC B cells. However, there is as yet no direct evidence of a functional impact of peroxisomes or their FAO in vivo on GC B cells or on Ab responses. So are peroxisomal functions important in any limb of humoral response? A first goal of this exploratory / developmental program application is to test the high-risk hypothesis that the local cell-intrinsic generation of these species is functionally important in GC B cells in addition to a broader role in Ab responses, i.e., rather than the alternate model in which incorporation of circulating plasmalogens and other ether lipids is sufficient. Specific hypotheses connected to this goal include (1) B cell type-restricted depletion of PexRAP will reduce not only IgG2c and IgA output but also germinal centers. Preliminary findings with widespread loss-of-function for Dhrs7b (the gene encoding PexRAP) support an impact on Ab responses and GC but might reflect B cell extrinsic functions or biochemical steps independent from maintenance of GC. (2) Conditional disruption of Dhrs7b within GC B cells and/or Tfh cells will undermine the formation of high-affinity, somatically hyper-mutated Ab as well as humoral memory; (3) that the lack of this peroxisomal biosynthetic function will reduce not only the specific ether lipids most prominent in our lipidomic results, but also germ line transcript induction and Ig class switching. Functions of ether lipids in lymphocytes have not previously been studied. Accordingly, a second objective is to test the possibility that these ether lipids promote GC in part because they enhance BCR signal transduction, and in part because they assist in detoxifying reactive oxygen species (ROS) to protect the B cells from lipid peroxidation. Expected outcomes & impact of the proposed studies are that we will (i) provide the first direct evidence of a mechanism by which the increased peroxisomes of activated and GC B cells promote humoral immunity, (ii) identify a novel function of the peroxisomal enzyme PexRAP in regulating antibody responses (iii) develop evidence of an effect of ether lipid synthesis in B cells on GC and insight into the mechanism for the functional role.

项目概要 产生抗体、完善其品质以及创造持久的体液记忆是适应性的关键部分 免疫。预防微生物并与微生物保持共生关系需要考虑各个方面 抗体调节。因此，破译影响抗体 (Ab) 的关键细胞和分子过程至关重要 品质。开发一种绘制营养物质和代谢产物相对于 为了了解淋巴器官的微观解剖特征，我们使用 IMS（成像质量 光谱测定）。这项研究揭示了一组相互关联的磷脂，其特征是非酰基连接 - 即醚连接和缩醛磷脂种类 - 在生发中心 (GC) 中富集。值得注意的是，从头开始 醚和缩醛磷脂脂质和磷脂的合成需要几种专门定位于的酶 过氧化物酶体。其中一种酶是 PexRAP，由基因 Dhrs7b 编码，该基因编码醚脂质的后期步骤 合成。与此同时，其他人最近的工作提供了证据表明过氧化物酶体和脂肪酸氧化（FAO） 由它们执行的活化 B 细胞和 GC B 细胞均增加。但目前还没有直接证据表明 过氧化物酶体或其FAO在体内对GC B细胞或Ab反应的功能影响。过氧化物酶体也是如此 在体液反应的任何肢体中都有重要的功能吗？这个探索性/发展计划的第一个目标 应用程序是为了检验高风险假设，即这些物种的局部细胞内在生成是 除了在 Ab 反应中发挥更广泛的作用外，在 GC B 细胞中也具有重要的功能，即，而不是替代 该模型中加入循环缩醛磷脂和其他醚脂就足够了。具体假设 与此目标相关的因素包括 (1) PexRAP 的 B 细胞类型限制性消耗不仅会减少 IgG2c 和 IgA 输出还有生发中心。初步发现 Dhrs7b（基因）广泛丧失功能 编码 PexRAP）支持对 Ab 反应和 GC 的影响，但可能反映 B 细胞外在功能或 生化步骤独立于 GC 的维护。 (2) GC B细胞内Dhrs7b的条件性破坏 和/或 Tfh 细胞将破坏高亲和力、体细胞超突变抗体以及体液抗体的形成 记忆; (3)缺乏这种过氧化物酶体生物合成功能不仅会减少特定的醚脂类 在我们的脂质组学结果中很突出，而且在种系转录诱导和 Ig 类别转换中也很突出。乙醚的功能 此前尚未研究过淋巴细胞中的脂质。因此，第二个目标是测试可能性 这些醚脂促进 GC 的部分原因是它们增强了 BCR 信号转导，部分原因是 它们有助于活性氧 (ROS) 的解毒，以保护 B 细胞免受脂质过氧化的影响。预期的 拟议研究的结果和影响是，我们将（i）提供机制的第一个直接证据 活化 B 细胞和 GC B 细胞的过氧化物酶体增加可促进体液免疫，(ii) 鉴定出一种新的 过氧化物酶体酶 PexRAP 在调节抗体反应中的功能 (iii) 证据表明 B细胞中醚脂合成对GC的影响及其功能作用机制的深入探讨。