Ontogenetic niche of B cells at the CNS borders in homeostasis, aging and autoimmunity

项目摘要 B细胞在神经系统自身免疫性疾病如多发性硬化（MS）中具有重要的致病作用 以及神经精神系统性红斑狼疮（NP-SLE），其是SLE最致残的表现之一。 B细胞在中枢神经系统（CNS）自身免疫中的相关性被治疗性免疫增强剂所强调。 单克隆抗体介导的B细胞耗竭在MS中的功效。自身反应性B细胞是如何产生并浸润CNS的 仍然是个谜。中枢神经系统被包裹在三层膜内：软脑膜、蛛网膜和硬脑膜。之间 蛛网膜和软脑膜位于蛛网膜下腔，其中含有含有各种免疫球蛋白的脑脊液。 包括B细胞。在神经炎症过程中，血淋巴细胞浸润脑膜， 体液和/或细胞反应。因此，脑膜B细胞被认为仅来源于全身性的 流通然而，我们最近发现，在年轻的成年小鼠中，脑膜B细胞主要来源于骨骼 颅骨扁平骨的骨髓，称为头盖骨，通过特殊的血管通道。相反，在衰老过程中， 年龄相关的B细胞（ABC）从外周迁移到硬脑膜中，在硬脑膜中它们可以分化成IG- 分泌浆细胞我们假设脑膜B细胞来源于颅骨并在局部分化， 对CNS-Ag有耐受性。相比之下，起源于外周的B细胞，因此不受外周血淋巴细胞的训练。 局部抗原环境，可分化成自身反应性浆细胞后，CNS-Ag遇到。在目标1中，我们 研究稳态下脑膜B细胞对局部抗原耐受机制。初步数据 表明在B细胞发育过程中自身Ag经历诱导脑膜B细胞耗竭。在旁边，我们 将检查脑膜B细胞在外来Ag接触后的活化。最后，我们将研究 硬脑膜B细胞发育中的微环境，重点关注硬脑膜成纤维细胞产生的CXCL 12。在目标2中， 将研究SWAP-70/DEF 6双敲除中脑膜B细胞和浆细胞的自身反应性 (DKO)狼疮模型初步数据显示DKO小鼠脑膜中浆母细胞的积累。我们 将比较转录谱和B细胞和浆细胞的B细胞受体（BCR）库， 硬脑膜和脾脏，以确定系统性B细胞克隆是否在淋巴器官中均匀分布， 脑膜，或中枢神经系统环境是否招募进一步分化为浆细胞的特定克隆。在 平行地，检查DKO小鼠的行为改变和CNS病理。我们还将确定 在DKO小鼠的硬脑膜中最大限度地扩增BCR克隆，并产生单克隆抗体，以确定 自身抗原特异性。在目标3中，我们将获得人硬脑膜免疫球蛋白的单细胞转录组谱， 从尸体解剖标本中分离的细胞，填补了我们对人类脑膜知识的关键空白。总体而言，这 该提案将促进我们对中枢神经系统中B细胞和促进神经炎症机制的理解。 为了实现这一目标，我们将利用科隆纳实验室的互补专业知识， 神经炎症和Pernis实验室，研究人类和小鼠模型的自身免疫。

PROJECT SUMMARY B cells have an important pathogenic role in neurological autoimmune disorders, such as multiple sclerosis (MS) and neuropsychiatric systemic lupus erythematosus (NP-SLE), one of the most disabling manifestations of SLE. The relevance of B cells in central nervous system (CNS) autoimmunity is underscored by the therapeutic efficacy of mAb-mediated B cell depletion in MS. How autoreactive B cells are generated and infiltrate the CNS remains enigmatical. The CNS is enclosed within three membranes: pia, arachnoid, and dura. Between the arachnoid and pia lies the subarachnoid space, which contains cerebrospinal fluid that harbors assorted immune cells, including B cells. During neuroinflammation, blood lymphocytes infiltrate the meninges to mount local humoral and/or cellular responses. Thus, meningeal B cells are thought to exclusively derive from the systemic circulation. However, we recently showed that in young adult mice meningeal B cells mainly derive from the bone marrow of cranial flat bones, known as calvaria, through special vascular channels. During aging, in contrast, age-associated-B cells (ABC) migrate from the periphery into the dura, where they may differentiate into Ig- secreting plasma cells. We hypothesize that meningeal B cells that derive from calvaria and differentiate locally are tolerant to CNS-Ag. By contrast, B cells that originate from the periphery and hence are not educated by the local antigenic milieu, may differentiate into autoreactive plasma cells upon CNS-Ag encounter. In Aim 1, we will investigate mechanisms of meningeal B cell tolerance to local antigens under steady-state. Preliminary data suggest that self-Ag experience during B cell development induces meningeal B cell depletion. Alongside, we will examine meningeal B cell activation upon foreign Ag encounter. Finally, we will investigate the impact of the microenvironment in dura B cell development, focusing on CXCL12 produced by dura fibroblasts. In Aim 2, we will investigate autoreactivity of meningeal B cells and plasma cells in the SWAP-70/DEF6 double knock-out (DKO) model of lupus. Preliminary data show accumulation of plasmablasts in the meninges of DKO mice. We will compare the transcriptional profiles and B cell receptor (BCR) repertoires of B cells and plasma cells from the dura and spleen to determine whether systemic B cells clones disseminate equally in lymphoid organs and meninges, or whether the CNS environment recruits specific clones that further differentiate into plasma cells. In parallel, DKO mice will be examined for behavioral alterations and CNS pathology. We will also identify the utmost expanded BCR clones in the dura of DKO mice and generate monoclonal antibodies to ascertain specificity for autoantigens. In Aim 3, we will obtain a single-cell transcriptomic profile of human dura immune cells isolated from autoptic specimens, filling a critical gap in our knowledge of human meninges. Overall, this proposal will advance our understanding of B cells in the CNS and mechanisms that promote neuroinflammation. To achieve this, we will leverage the complementary expertise of the Colonna lab, which studies neuroinflammation, and the Pernis lab, which studies autoimmunity in both humans and mouse models.