Antibody quality and germinal center requirements for peroxisomal function in lymphocytes

项目摘要 产生抗体，提炼抗体的性质，创造持久的体液记忆是适应性的关键部分 豁免权。对微生物的保护和与它们的共生关系从每个方面都得到了利用 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。在开发一种方法来绘制营养物质和代谢产物相对于 淋巴器官的显微解剖特征，我们使用IMS(成像质量)进行了无偏向的脂肪组学分析 光谱分析)。这项研究揭示了一组相互连接的以非酰基键为特征的磷脂 -即乙醚连接和血浆原物种--在生发中心(GC)中富含。值得注意的是，从头开始 乙醚和血浆蛋白原类脂和磷脂的合成需要几种酶，这些酶专门定位于 过氧酶体。其中一种酶是PexRAP，由Dhrs7b基因编码，它编码乙醚类脂的晚期步骤 综合。与此同时，其他人最近的工作提供了证据，证明过氧化物体和脂肪酸氧化(粮农组织) 由它们执行的在激活的和GC B细胞中都增加。然而，到目前为止还没有直接证据表明 体内过氧化体或其粮农组织对GC B细胞或抗体反应的功能影响。过氧化体也是如此 在任何一种体液反应中都有重要的作用？这一探索性/发展计划的第一个目标 应用是测试高风险假设，即这些物种的局部细胞固有生成是 除了在抗体反应中发挥更广泛的作用外，在GC B细胞中功能上也很重要，即，而不是替代 循环中的血浆原和其他乙醚类脂充分结合的模型。具体假设 与这一目标相关的包括：(1)B细胞类型-限制PexRAP的耗尽不仅会降低IgG2c和IgA 输出，也包括生发中心。Dhrs7b(基因)广泛功能丧失的初步发现 编码PexRAP)支持对抗体反应和GC的影响，但可能反映B细胞的外部功能或 独立于GC维护的生化步骤。(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的形成 记忆；(3)这种过氧化体生物合成功能的缺乏不仅会最大程度地降低特定的乙醚类脂类 在我们的脂肪组学结果中很突出，但也包括生殖系转录诱导和Ig类转换。乙醚的功能 淋巴细胞中的脂类以前还没有被研究过。因此，第二个目标是测试这种可能性 这些乙醚类脂类促进GC的部分原因是它们增强了BCR信号转导，部分原因是 它们有助于解毒活性氧物种(ROS)，以保护B细胞免受脂质过氧化。预期 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 通过激活的和GC B细胞中增加的过氧酶体促进体液免疫，(Ii)鉴定一种新的 过氧化物酶PexRAP在调节抗体反应中的作用(III)证据表明 乙醚对B细胞脂肪合成的影响及对GC的作用机制。

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.