Antibody quality and germinal center requirements for peroxisomal function in lymphocytes

点击翻译按钮获取中文摘要

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.