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.

项目摘要 产生抗体，改进它们的质量，创造持久的体液记忆是适应性免疫的关键部分。 免疫力保护免受微生物的侵害并与它们保持密切的关系， 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。 的天性.在开发一种绘制营养物质和代谢产物分布图的方法时， 淋巴器官的显微解剖特征，我们使用IMS（成像质量）进行了无偏倚的脂质组学分析， 光谱法）。这项研究表明，一组以非酰基键为特征的相互关联的磷脂 - 也就是说，醚连接的和缩醛磷脂种类-其富集在生发中心（GC）中。值得注意的是，重新 醚和缩醛磷脂和磷脂的合成需要几种专门定位于 过氧化物酶体一种这样的酶是PexRAP，由基因Dhrs 7 b编码，其编码醚脂质中的晚期步骤。 合成.与此同时，其他人最近的工作提供了证据，证明过氧化物酶体和脂肪酸氧化（粮农组织） 在活化和GC B细胞中，由它们执行的蛋白质含量增加。然而，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细胞或对Ab应答的功能影响。过氧化物酶体也是 在体液反应的任何分支中起重要作用吗这个探索/发展计划的第一个目标是 应用程序是为了测试高风险的假设，即这些物种的局部细胞内代是 在GC B细胞中除了在Ab应答中更广泛的作用外，还具有重要的功能，即，而不是替代品 循环缩醛磷脂和其他醚脂质充分结合的模型。具体假设 与该目标相关的包括（1）PexRAP的B细胞类型限制性消耗不仅将减少IgG 2c和伊加 输出，也是生殖中心。Dhrs 7 b（该基因）功能广泛丧失的初步发现 编码PexRAP）支持对Ab应答和GC的影响，但可能反映B细胞外在功能，或 独立于GC维持的生化步骤。(2)GC B细胞内Dhrs 7 B的条件性破坏 和/或Tfh细胞将破坏高亲和力、体细胞超突变Ab以及体液抗体的形成 记忆;（3）这种过氧化物酶体生物合成功能的缺乏不仅会减少特定的醚脂质最 在我们的脂质组学结果中突出，而且还有生殖系转录物诱导和IG类转换。以太的功能 以前没有研究过淋巴细胞中的脂质。因此，第二个目标是测试 这些醚脂质促进GC部分是因为它们增强BCR信号转导，部分是因为 它们帮助活性氧（ROS）解毒以保护B细胞免受脂质过氧化。预计 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 活化的和GC B细胞的过氧化物酶体的增加促进体液免疫，（ii）鉴定一种新的 过氧化物酶体酶PexRAP在调节抗体应答中的功能（iii）开发以下作用的证据： 醚脂在B细胞合成中对GC的功能作用及机理的深入了解。