Entry of antigen-presenting B cells into the follicle directed by IFNa and IL-17

描述（由申请方提供）：本修订提案的总体目标是确定IL-17和IFN 1协同作用启动B细胞发育的机制，该细胞在自身免疫BXD 2模型中产生自身抗体。我们已经发现IL-17和IFN 1都是形成生发中心（GC）所必需的，所述生发中心能够在自身免疫BXD 2小鼠中产生致病性自身抗体，即，这些细胞因子中的任一种的缺乏消除了这些小鼠中的GC形成、自身抗体形成和自身免疫性疾病。IL-17对BXD 2小鼠中GC形成的作用的分析表明，IL-17促进CD 4 T细胞和B细胞的相互作用，从而增强自发性生发中心（GC）的形成和稳定，并且该作用是通过上调B细胞中G蛋白信号传导（Rgs）13和Rgs 16的调节剂介导的。主要研究者的最新结果表明，在BXD 2小鼠的边缘窦中浆细胞样DC（pDC）的数量增加，并且这些pDC表现出IFN 1的表达显著升高。值得注意的是，存在稳健的边缘区前体B细胞群（MZP），其表现出增强的将抗原（Ag）直接转运到GC轻区区域中的能力。基于我们积累的初步数据，我们已经制定了一个假设的模型，其中IL-17和IFNa以协调的方式通过调节Ag转运MZP B细胞的迁移行为来促进自发性GC反应。我们建议通过解决其两个基本组成部分来测试这个假设模型：（1）IFNa是否调节MZP在GC LZ区的内流和IL-17是否调节MZP在GC LZ区的保留？(2)IFN α和IL-17是否调节MZP B细胞的Ag转运导致GC的发展？为了回答第一个问题，IL-17和IFNa调节趋化性的能力将在体外使用transwell室测定，并且在体内通过分析GFP+ MZP B细胞在其中IL-17 R、IFNa R或Rgs基因已被调节的BXD 2小鼠中的归巢来测定。这将通过使用从BXD 2-Il-17 r-/-、BXD 2-Ifnar-/-、BXD 2-Rgs 13-/-和BXD 2-Rgs 16-/-小鼠获得的B细胞来实现。为了回答第二个问题，将通过FACS和共聚焦成像分析确定MZP B细胞的体内Ag捕获和转运。将使用鸡蛋溶菌酶（HEL）或HEL-卵清蛋白（OVA）缀合物策略与表达MD 4 B细胞受体转基因和OVA T细胞受体转基因的BXD 2小鼠组合来研究MZP B细胞的Ag转运功能。相关性：该项目的成功完成将有助于确定关键的迁移信号和上游介质，促进Ag的运输，以启动GC响应。这些结果将为目前使用的基于IFN 1调节的疗法的作用机制提供新的见解，以及在自身免疫性疾病中靶向Th 17细胞的建议，并为治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): The overall goal of this revised proposal is to identify the mechanisms by which IL-17 and IFN1 act in concert to initiate the development of B cells that produce autoantibodies in the BXD2 model of autoimmunity. We have found that both IL-17 and IFN1 are required for the formation of germinal centers (GCs) that enable the production of pathogenic autoantibodies in autoimmune BXD2 mice, i.e., a deficiency of either of these cytokines abrogates GC formation, autoantibody formation, and autoimmune disease in these mice. Analysis of the effects of IL-17 on GC formation in the BXD2 mice suggest that IL-17 facilitates the interaction of CD4 T cells and B cells thereby enhancing the formation and stabilization of spontaneous germinal centers (GCs), and that this effect is mediated by upregulation of regulators of G-protein signaling (Rgs)13 and Rgs16 in the B cells. The principal investigator's most recent results indicate that there is an increased number of plasmacytoid DCs (pDCs) in the marginal sinus in BXD2 mice and that these pDCs exhibit significantly elevated expression of IFN1. Notably, there is a robust marginal zone precursor B cell population (MZP) that exhibits an enhanced ability to transport antigen (Ag) directly into a GC light zone area. Based on our accumulated preliminary data, we have formulated a hypothetical model in which IL-17 and IFNa act in a coordinated manner to facilitate the spontaneous GC response through regulating the migration behavior of Ag-transporting MZP B cells. We propose to test this hypothetical model by addressing two of its essential components: (1) Does IFNa regulate influx and IL-17 regulate the retention of MZP in the GC LZ area? (2) Do IFNa and IL-17 regulate Ag transport by MZP B cells leading to GC development? To answer the first question, the ability of IL-17 and IFNa to regulate chemotaxis will be determined in vitro using a transwell chamber, and in vivo by analyzing homing of GFP+ MZP B cells in BXD2 mice in which IL-17R, IFNaR, or Rgs genes has been modulated. This will be achieved by using B cells obtained from BXD2-Il-17r-/-, BXD2-Ifnar-/-, BXD2-Rgs13-/- and BXD2-Rgs16-/- mice. To answer the second question, in vivo Ag capture and transportation by MZP B cells will be determined by FACS and confocal imaging analyses. The Ag transport function of MZP B cells will be studied using the hen egg lysozyme (HEL) or HEL-ovalbumin (OVA) conjugate strategies in combination with BXD2 mice that express the MD4 B-cell receptor transgene and the OVA T-cell receptor transgene. RELEVANCE: Successful accomplishment of the project will help to identify critical migratory signals and upstream mediators that facilitate the transportation of Ag to initiate a GC response. The results will provide new insights into the mechanisms of action of currently used therapies based on modulation of IFN1 as well as the proposed targeting of Th17 cells in autoimmune disease as well as suggesting novel targets for therapeutic intervention.