Neuronal antigen surveillance and autoimmunity in CNS demyelinating disease

有一个关键的需求尚未得到满足，那就是确定推动患者疾病进展的致病机制 患有多发性硬化症(MS)。MS中轴突损伤和功能障碍的积聚不充分 受当前治疗方法的影响。这项工作的长期目标是发现新的策略来预防或 逆转MS的疾病进展，尽管有证据表明CD8 T细胞与轴突损伤和 多发性硬化症的进展，这些细胞的功能作用以及招募 这些细胞到脱髓鞘的大脑是未知的。神经元特异性CD8 T细胞的抗原靶点是 也是未知的。这项研究的总体目标是测试神经元抗原特异性的机制作用。 探讨CD8T细胞在损伤脱髓鞘轴突中的作用，并确定MS患者是否存在此类细胞。 其基本原理是轴突损伤是MS进展的主要底物，轴突MHC I类表达 炎症和脱髓鞘以及针对神经元特异性的细胞毒性CD8 T细胞上调 抗原损伤脱髓鞘轴突。这一提议的中心假设是神经元抗原特异性 CD8T细胞损伤脱髓鞘轴突。在强有力的初步证据的指导下，这一假设将得到检验 利用AAV介导的神经元转导驱动新抗原OVA在脑内的表达 铜酮毒性或免疫髓鞘少突胶质细胞所致中枢神经系统脱髓鞘的研究 抗OVA转基因CD8 T细胞宿主体内的糖蛋白衍生肽(MOG-EAE) 衍生多肽SIINFEKL(OT-I)。这项研究还将使用自体T细胞和成纤维细胞来源的IPSC- 在微流控室内培养衍生神经元以确定MS患者CD8 T细胞是否损伤自身 轴突。将追求三个具体目标：1)确定CD8 T细胞介导的轴突损伤的机制 在脱髓鞘的中枢神经系统中；2)确定轴突损伤所需的MHC-I类表达的细胞位置和 确定脱髓鞘如何促进中枢神经系统神经元抗原特异性CD8 T细胞的渗透；3)确定 MS患者是否有神经抗原特异性CD8T细胞。这种方法在概念上是创新的。 因为有观点认为脱髓鞘和炎症会导致自体多肽在 MHC I类。该方法在技术上是创新的，基于使用新的AAV载体来驱动 脱髓鞘CNS内神经元中的新抗原，选择性地缺失脑内常驻抗原递呈 细胞(APC)与外周APC相比，使用多个宿主操纵耦合采用可追踪转移 效应细胞在时间和空间上的分布，以及患者来源的抗神经元T细胞运输的使用 神经元和自体CD8 T细胞。这项工作将对该领域产生重大而强大的影响 CD8T细胞针对神经元特异性抗原损伤脱髓鞘轴突的能力 并通过鉴定多发性硬化症患者的T细胞。

There is a critical unmet need to identify the pathogenic mechanisms that drive disease progression in patients with multiple sclerosis (MS). Accumulation of axon injury and functional disability in MS are not adequately impacted by current therapies. The long-term goal of this work is to discover new strategies to prevent or reverse disease progression in MS. Despite evidence that CD8+ T cells are associated with axon injury and progression in MS, the functional role for these cells and the relevant mechanisms required for recruitment of these cells to the demyelinated brain are unknown. The antigenic targets of neuron-specific CD8+ T cells are also unknown. The overall objectives of this study are to test the mechanistic role of neuron antigen-specific CD8+ T cells in the injury of demyelinated axons and to determine whether patients with MS have such cells. The rationale is that axon injury is the primary substrate of progression in MS, axonal MHC class I expression is upregulated by inflammation and demyelination, and cytotoxic CD8+ T cells directed against neuron-specific antigens injure demyelinated axons. The central hypothesis of this proposal is that neuron antigen-specific CD8+ T cells injure demyelinated axons. Guided by strong preliminary evidence, the hypothesis will be tested using AAV-mediated transduction of neurons to drive expression of the neoantigen ovalbumin (OVA) within the context of CNS demyelination induced by cuprizone toxicity or immunization against a myelin oligodendrocyte glycoprotein-derived peptide (MOG-EAE) in hosts that have transgenic CD8+ T cells directed against the OVA- derived peptide SIINFEKL (OT-I). The study will also use autologous T cells and fibroblast-derived iPSC- derived neurons grown in microfluidic chambers to determine whether MS patient CD8+ T cells injure their own axons. Three specific aims will be pursued: 1) determine the mechanisms of CD8+ T cell-mediated axon injury in the demyelinated CNS; 2) identify the cellular locus of MHC class I expression required for axon injury and determine how demyelination drives CNS infiltration of neuron antigen-specific CD8+ T cells; 3) determine whether MS patients have neuron-antigen specific CD8+ T cells. This approach is conceptually innovative because of the proposal that demyelination and inflammation induce axonal presentation of self-peptides on MHC class I. The approach is technically innovative based on the use of novel AAV vectors to drive neoantigens in neurons within the demyelinated CNS, selective deletion of brain-resident antigen presenting cells (APCs) vs peripheral APCs, use of multiple host manipulations coupled to adoptive transfer of traceable effector cells to temporally and spatially profile anti-neuronal T cell trafficking, and the use of patient-derived neurons and autologous CD8+ T cells. This work will make a significant, powerful impact on the field by revealing the capacity of CD8+ T cells directed against neuron-specific antigens to injure demyelinated axons and by identifying such T cells in patients with MS.