Humoral Immunity, Astrocyte Injury, and Demyelination in Neuromyelitis Optica

项目摘要 视神经肌萎缩症谱系障碍（NMOSD）是一种严重的自身免疫性疾病， 水通道蛋白4（AQP 4）。NMOSD中的中枢神经系统（CNS）损伤是通过结合 AQP 4自身抗体（AQP 4-IgG）靶向星形胶质细胞和抗体效应子功能的激活; 然而，血清AQP 4-IgG滴度与疾病复发或严重程度无关。因此，识别 影响CNS通路、鞘内产生或AQP 4-IgG效应子功能的因素对于 了解NMOSD的发病机制利用单细胞分选、重组抗体技术， 重链可变区库分析，我们已经重建了鞘内AQP 4-IgG NMOSD反应，激活脑微血管内皮细胞的分离抗内皮抗体 细胞（BMEC），并鉴定了扩增的CD 27-IgD-双阴性（DN）记忆B细胞群 与鞘内AQP 4特异性浆母细胞克隆相关。我们现在准备好测试我们的 假设AQP 4-IgG、针对BMEC的自身抗体和促炎性DN B细胞在 音乐会，以推动NMOSD活动和病理。 在目标1中，我们将研究葡萄糖调节蛋白78（GRP 78）自身抗体对 NMOSD疾病活动我们将研究NMOSD抗GRP 78的丰度和表位特异性， 自身抗体，评估BMEC中激活的细胞信号传导途径，并测量其增加BMEC的能力。 大脑和视网膜中的血管通透性。在目标2中，我们将讨论如何结合特异性的个人 抗AQP 4自身抗体影响NMOSD中的损伤形成和CNS损伤。我们发现了多个 对靶点产生不同作用的患者来源的AQP 4特异性重组抗体 星形胶质细胞我们将评估AQP 4 rAb激活补体依赖性细胞毒性的能力，启动 抗体依赖性细胞介导的细胞毒性，并调节星形胶质细胞趋化因子和细胞因子的产生， 体外然后，我们将评估这些参数如何影响动物模型中的NMOSD病变形成， 受影响患者的疾病活动。在目标3中，我们将分析一个扩大的群体， CD 27-IgD-DN B细胞在NMOSD患者中的表达，并研究其在疾病活动中的作用。丰度和 将在NMOSD复发和缓解期间比较DN B细胞的免疫球蛋白库， 评估NMOSD DN B细胞响应于各种免疫抑制剂的细胞因子应答和免疫球蛋白产生。 刺激。此外，我们将研究NMOSD DN B细胞向自体T细胞呈递AQP 4的潜力。 细胞这些研究的结果将阐明AQP 4-IgG如何进入中枢神经系统，鞘内B如何进入中枢神经系统， 细胞有助于NMO病变的启动，以及AQP 4-IgG的离散亚群如何有助于CNS 损伤我们的研究成果将消除该领域取得进展的障碍，并推进 NMOSD和其他影响CNS和眼睛的自身免疫性疾病的诊断和治疗。

Project Summary Neuromyelitis optica spectrum disorder (NMOSD) is a severe autoimmune disorder targeted against the aquaporin-4 (AQP4) water channel. Central nervous system (CNS) injury in NMOSD is initiated by the binding of AQP4 autoantibodies (AQP4-IgG) to target astrocytes and the activation of antibody effector functions; however, serum AQP4-IgG titers do not correlate with disease relapse or severity. Therefore, identifying factors that influence CNS access, intrathecal production, or effector function of AQP4-IgG is essential for understanding NMOSD pathognesis. Using single-cell sorting, recombinant antibody technology, and heavy-chain variable region repertoire analysis, we have reconstructed the intrathecal AQP4-IgG response in NMOSD, isolated anti-endothelial antibodies that activate brain microvascular endothelial cells (BMECs), and identified an expanded CD27-IgD- double negative (DN) memory B cell population clonally related to intrathecal AQP4-specific plasmablasts. We are now uniquely prepared to test our hypothesis that AQP4-IgG, autoantibodies against BMECs, and pro-inflammatory DN B cells act in concert to propel NMOSD activity and pathology. In Aim 1, we will examine the contribution of glucose regulated protein-78 (GRP78) autoantibodies to NMOSD disease activity. We will investigate the abundance and epitope specificity of NMOSD anti-GRP78 autoantibodies, evaluate the cell signaling pathways activated in BMECs, and measure their ability to increase vascular permeability in the brain and retina. In Aim 2, we will address how the binding specificity of individual anti-AQP4 autoantibodies affect lesion formation and CNS injury in NMOSD. We have identified multiple species of patient-derived AQP4-specific recombinant antibodies that produce distinct effects on target astrocytes. We will gauge AQP4 rAbs on their ability to activate complement dependent cytotoxicity, initiate antibody-dependent cell-mediated cytotoxicity, and modulate astrocyte chemokine and cytokine production in vitro. We will then evaluate how these parameters affect NMOSD lesion formation in animal models and disease activity in affected patients. And in Aim 3, we will analyze an expanded population of class-switched CD27-IgD- DN B cells in NMOSD patients and investigate their role in disease activity. The abundance and immunoglobulin repertoire of DN B cells will be compared during NMOSD relapse and remission, and we will assess the cytokine response and immunoglobulin production of NMOSD DN B cells in response to various stimuli. In addition, we will investigate the potential for NMOSD DN B cells to present AQP4 to autologous T cells. The results of these investigations will elucidate how AQP4-IgG accesses the CNS, how intrathecal B cells contribute to NMO lesion initiation, and how discrete subpopulations of AQP4-IgG contribute to CNS injury. The outcomes of our research will remove barriers to progress in the field and advance approaches to the diagnosis and treatment of NMOSD and other autoimmune disorders impacting the CNS and eye.