Repertoire selection of AQP4-specific T cells that cause CNS autoimmunedisease

项目摘要/摘要 根据观察到水通道蛋白4(AQP4)特异性抗体是IgG1，一种T细胞依赖的亚型，它是 推测水通道蛋白4(AQP4)特异性T细胞在视神经脊髓炎(NMO)发病机制中起关键作用。 已经在NMO患者中发现了AQP4特异性T细胞，与健康对照组(HC)相比，AQP4反应 T细胞扩增并表现出Th17极化，这一发现进一步支持Th17细胞在NMO中的作用。 不幸的是，评估AQP4反应性T细胞如何直接参与NMO的中枢神经系统炎症是不可行的 病人。因此，重要的是要建立模型来评估AQP4特异性T细胞在NMO中的潜在作用。 在野生型(WT)小鼠和大鼠体内建立基于AQP4的NMO模型的初步尝试尚未遇到 成功。近年来，研究发现AQP4基因缺陷(AQP4-/-)小鼠体内存在致病的AQP4特异性T细胞。那些T 细胞识别两个新的决定因素。与在WT小鼠中发现的其他AQP4决定因素相比，新的 决定簇在AQP4-/-小鼠中诱导强烈的增殖，但在WT小鼠中仅有微弱的反应。高反应性是 AQP4特异的，但不是表位特异的，因为我们发现了第二个AQP4表位诱导AQP4- /-，但不是WT，小鼠。识别AQP4-/-和WT中这些决定因素的T细胞受体(TCR)谱系 老鼠是不同的。从AQP4/-供体小鼠分离出对这些决定因素有反应的T细胞，诱导临床和组织学 受体WT小鼠中枢神经系统自身免疫性疾病的受试率为100%。总的来说，这些发现代表着第一个成功的 诱导临床靶向AQP4的中枢神经系统自身免疫。我们的发现提示AQP4-/-小鼠对这些表位的反应 反映了中枢T细胞耐受性的丧失。对只有T细胞或B细胞缺陷的小鼠的研究结果表明， 外周T细胞调节也改变了致病的AQP4特异性T细胞的表达。 我们建议验证我们的假设，即小鼠胸腺对AQP4特异性T细胞的负选择存在缺陷， 这种可能性可能与NMO的发病机制有关。我们将研究外周T细胞调节如何影响 致病性AQP4特异性免疫反应。在具体目标1中，通过使用独特的方法和新的老鼠，我们将 鉴定致病AQP4特异性T细胞的表型，并产生AQP4特异性TCR转基因小鼠。在……里面 特定目标2，我们将描述表达HLA4的AQP4-/-和WT小鼠的AQP4特异性T细胞的谱系。 DR17(DRB1*0301)，与NMO关联最高的MHC II等位基因。另外，我们将研究AQP4特定的T细胞 NMO患者的细胞反应以确定DR17限制性AQP4特异性T细胞表位是否识别这些小鼠 与NMO患者的AQP4 T细胞表位相对应。在初步数据中，使用这些小鼠，我们发现了一种新的人类白细胞抗原- DR17限制性的AQP4决定簇，并观察到它被来自HLA-DR17+NMO患者的T细胞识别， 支持这一目标的可行性的研究结果。在这个项目中获得的发现应该阐明控制机制 NMO中致病的AQP4特异性T细胞的发展，为促进体内NMO的发展提供了基础 模型，并可能为选择性NMO免疫疗法的发展提供见解。

PROJECT SUMMARY / ABSTRACT Based upon the observation that aquaporin-4 (AQP4)-specific antibodies are IgG1, a T cell-dependent isotype, it was hypothesized that aquaporin-4 (AQP4)-specific T cells have a key role in neuromyelitis optica (NMO) pathogenesis. AQP4-specific T cells have been identified in NMO patients and, in comparison to healthy controls (HC), AQP4-reactive T cells are expanded and exhibit Th17 polarization, findings that further support the role of Th17 cells in NMO. Unfortunately, it is not feasible to evaluate how AQP4-reactive T cells participate directly in CNS inflammation in NMO patients. Thus, it is important to develop models to evaluate the potential role of AQP4-specific T cells in NMO. Initial attempts in generating an in vivo AQP4-based NMO model in wild-type (WT) mice and rats have not met with success. Recently, it was observed that pathogenic AQP4-specific T cells exist in AQP4-deficient (AQP4-/-) mice. Those T cells recognize two novel determinants. In comparison to other AQP4 determinants identified in WT mice, the novel determinants induce robust proliferation in AQP4-/- mice, but only a weak response in WT mice. Hyper-reactivity is AQP4-specific, but not epitope-specific as we discovered a second AQP4 epitope induced vigorous proliferation in AQP4- /-, but not WT, mice. The T cell receptor (TCR) repertoires used for recognition of these determinants in AQP4-/- and WT mice are distinct. T cells reactive to these determinants isolated from AQP4-/- donor mice induced clinical and histologic CNS autoimmune disease in 100% of recipient WT mice tested. Collectively, these findings represent the first successful induction of clinical AQP4-targeted CNS autoimmunity. Our findings suggest responses to those epitopes in AQP4-/- mice reflect a loss of central T cell tolerance. Findings from studying mice deficient in T cells only or B cells only indicate that peripheral T cell regulation also alters expression of pathogenic AQP4-specific T cells. We propose to test our hypothesis that there is a defect in thymic negative selection of AQP4-specific T cells in mice, a possibility that may be relevant to NMO pathogenesis. We will examine how peripheral T cell regulation may influence pathogenic AQP4-specific immune responses. In Specific Aim 1, by using unique approaches and novel mice, we will characterize the phenotype of pathogenic AQP4-specific T cells and generate AQP4-specific TCR transgenic mice. In Specific Aim 2, we will characterize the repertoire of AQP4-specific T cells in AQP4-/- and WT mice that express HLA- DR17 (DRB1*0301), the MHC II allele most highly associated with NMO. Separately, we will examine AQP4-specific T cell responses in NMO patients to determine if DR17-restricted AQP4-specific T cell epitopes identified those mice correspond to AQP4 T cell epitopes in NMO patients. In preliminary data, using these mice we discovered a novel HLA- DR17-restricted AQP4 determinant, and observed that it is recognized by T cells from HLA-DR17+ NMO patients, findings that support feasibility of this aim. Findings obtained in this program should elucidate mechanisms controlling development of pathogenic AQP4-specific T cells in NMO, provide a foundation advancing development of in vivo NMO models, and may provide insight for development of selective NMO immunotherapy.