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

项目总结/摘要 基于水通道蛋白-4（AQP4）特异性抗体是IgG 1（一种T细胞依赖性同种型）的观察， 假设水通道蛋白-4（AQP4）特异性T细胞在视神经肌萎缩症（NMO）发病机制中具有关键作用。 已经在NMO患者中鉴定了AQP4特异性T细胞，并且与健康对照（HC）相比， T细胞扩增并表现出Th17极化，这一发现进一步支持了Th17细胞在NMO中的作用。 不幸的是，在NMO中评估AQP4反应性T细胞如何直接参与CNS炎症是不可行的。 患者因此，重要的是开发模型来评估AQP4特异性T细胞在NMO中的潜在作用。 在野生型（WT）小鼠和大鼠中产生体内基于AQP4的NMO模型的初始尝试尚未满足要求。 成功最近，观察到致病性AQP4特异性T细胞存在于AQP4缺陷（AQP4-/-）小鼠中。那些t 细胞识别两个新的决定因素。与WT小鼠中鉴定的其他AQP4决定簇相比， 决定簇在AQP4-/-小鼠中诱导稳健的增殖，但在WT小鼠中仅诱导弱反应。高反应性是 AQP4特异性，但不是表位特异性的，因为我们发现第二个AQP4表位诱导AQP4- /-，但不是WT小鼠。用于识别AQP4-/-和WT中这些决定簇的T细胞受体（TCR）库 老鼠是不同的。从AQP4-/-供体小鼠中分离的对这些决定簇反应的T细胞诱导了临床和组织学 在100%的受试WT小鼠中发生CNS自身免疫性疾病。总的来说，这些发现代表了第一个成功的 诱导临床AQP4靶向CNS自身免疫。我们的研究结果提示AQP4-/-小鼠对这些表位的反应 反映了中央T细胞耐受性的丧失。研究仅缺乏T细胞或仅缺乏B细胞的小鼠的结果表明， 外周T细胞调节也改变致病性AQP4特异性T细胞的表达。 我们建议验证我们的假设，即小鼠中AQP4特异性T细胞的胸腺阴性选择存在缺陷， 这可能与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特异性免疫应答。在具体目标1中，通过使用独特的方法和新型小鼠，我们将 表征致病性AQP4特异性T细胞的表型并产生AQP4特异性TCR转基因小鼠。在 具体目标2，我们将表征AQP4-/-和WT小鼠中表达HLA-1的AQP4特异性T细胞的库。 DR17（DRB1 * 0301），与NMO最高度相关的MHC II等位基因。另外，我们将研究AQP4特异性T 细胞反应，以确定DR17限制性AQP4特异性T细胞表位是否识别了这些小鼠 对应于NMO患者中的AQP4 T细胞表位。在初步数据中，使用这些小鼠，我们发现了一种新的HLA- DR17限制性AQP4决定簇，并观察到它被来自HLA-DR17 + NMO患者的T细胞识别， 这些发现支持这一目标的可行性。在这个项目中获得的发现应该阐明控制 致病性AQP4特异性T细胞在NMO中的发育，为推进体内NMO的发育提供了基础 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.