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

描述(由申请人提供)：这项修订提案的总体目标是确定IL-17和IFN1协同作用的机制，以启动在Bxd2自身免疫模型中产生自身抗体的B细胞的发展。我们发现，IL-17和IFN1都是形成生发中心(GCs)所必需的，这些GCs能够在自身免疫的Bxd2小鼠中产生致病性自身抗体，即这两种细胞因子中的任何一种的缺乏都会导致GC的形成、自身抗体的形成和自身免疫性疾病的发生。IL-17对Bxd2小鼠GC形成的影响分析表明，IL-17促进T细胞和B细胞之间的相互作用，从而促进自发性生发中心(GC)的形成和稳定，这一作用是通过上调B细胞中G蛋白信号转导(RGS)13和RGS16的调节来实现的。主要研究人员的最新结果表明，Bxd2小鼠边缘窦中的浆细胞样树突状细胞(PDC)数量增加，并且这些pDC表现出显著的IFN1表达。值得注意的是，存在一个强大的边缘带前体B细胞群(MZP)，它显示出增强的将抗原(Ag)直接运送到GC轻区的能力。基于我们积累的初步数据，我们建立了一个假设模型，在该模型中，IL-17和IFNA协同作用，通过调节转运Ag的MZP B细胞的迁移行为来促进自发的GC反应。我们建议通过解决它的两个基本组成部分来检验这个假设模型：(1)IFNA调节内流，IL-17调节MZP在GC LZ区的滞留吗？(2)IFNA和IL-17是否调节MZP B细胞的Ag转运，从而导致GC的发生？为了回答第一个问题，将使用Transwell小室在体外确定IL-17和IFNA调节趋化的能力，并通过分析IL-17R、IFNaR或RGS基因已被调控的Bxd2小鼠中GFP+MZP B细胞的归巢来确定其在体内的能力。这将通过使用从Bxd2-Il-17r-/-、Bxd2-Ifnar-/-、Bxd2-Rgs13-/-和Bxd2-Rgs16-/-小鼠获得的B细胞来实现。为了回答第二个问题，MZP B细胞体内的Ag捕获和转运将通过FACS和共聚焦成像分析来确定。利用鸡蛋溶菌酶(HEL)或HEL-卵清蛋白(OVA)结合表达MD4 B细胞受体转基因和OVA T细胞受体转基因的Bxd2小鼠，将研究MZP B细胞的抗原转运功能。相关性：该项目的成功完成将有助于确定关键的迁徙信号和上游媒介，促进银的运输，以启动GC反应。这一结果将为目前使用的基于IFN1调节的治疗方法的作用机制提供新的见解，以及拟议的自身免疫性疾病中Th17细胞的靶向，并为治疗干预提供新的靶点。 公共卫生相关性：在这个项目中，我们提出，在次级淋巴器官中的局部免疫介质的产生促进了特殊的B细胞，这些细胞将抗原输送到自身免疫的Bxd2小鼠中称为生发中心的抗体形成区域。这些事件的结合导致链式反应的启动，从而导致致病性自身抗体的产生。对这一过程的了解将有助于确定一种新的治疗干预措施，以防止致病性自身抗体的产生，并建议更有效地利用目前可用的治疗策略来治疗各种自身免疫性疾病，包括狼疮和类风湿关节炎。

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. PUBLIC HEALTH RELEVANCE: In this project, we propose that local production of immune mediators in the secondary lymphoid organ facilitates specialized B cells that transport antigens into the antibody formation area called germinal centers in autoimmune BXD2 mice. The combination of these events results in the initiation of the chain reaction that leads to the production of pathogenic autoantibodies. An understanding of this process will help to identify a novel therapeutic intervention to prevent the generation of pathogenic autoantibodies as well as suggesting more effective utilization of currently available therapeutic strategies for various autoimmune diseases including lupus and rheumatoid arthritis.