The Role of Intrathecal Plasma Cell Clones in MS Pathogenesis

描述(申请人提供)：多发性硬化症(MS)是一种原因不明的人类中枢神经系统脱髓鞘疾病。B细胞免疫在疾病发病机制中的作用尚不清楚，但由于病理显示免疫球蛋白介导的脱髓鞘和抗B细胞治疗多发性硬化症的有效治疗，已重新引起人们的兴趣。鞘内持续增加的免疫球蛋白合成和寡克隆条带(OCB)的存在表明了疾病本质的线索。OCB并不是MS所特有的，在已知的中枢神经系统感染性疾病中，OCB代表的是对致病因子的抗体，这为我们的假设提供了理论基础，即MS中的寡头抗体是针对疾病相关抗原的。无论B细胞在MS发病机制中的主要作用是通过它们的效应性抗体分子、细胞因子的产生还是作为抗原提呈细胞，它都是通过与独特的疾病特异性B细胞表面Ig受体结合来发挥作用的。该提案的目的是确定MS CSF OCB的抗原靶点，并确定靶向这些抗原的B细胞如何在疾病发病机制中发挥作用。我们建立了一种单细胞RT-PCR方法，可以有效地扩增MS-CSF CD138+浆细胞表达的重链(VH)和轻链(VL)可变区。从MS患者的CD138+细胞产生的谱系都显示出T细胞依赖的、抗原驱动的反应的精液特征，包括克隆性扩增、体细胞超突变和偏向使用VH4生殖系片段。脑脊液CD138+细胞的表型是短暂的浆母细胞，连续的脑脊液谱系显示持续的动态B细胞反应，表明持续的抗原刺激而不是旁观者的激活。我们已经验证了一种哺乳动物表达系统来生产重组IgG1Abs，该系统能够忠实地复制体内血浆细胞重链和轻链V区序列的配对。多个MS RAB已被证明与少突胶质细胞抗原和富含髓鞘的糖脂结合。目的1将鉴定MS-CSF血浆母细胞来源的抗体识别的表位，并将发展抗原特异性试验来筛选其他MS-RABS、MS-CSF和炎症控制CSF的特异性。将发展包括共聚焦显微镜、FACS分类和ELISA法在内的免疫分析方法来评估抗体对推测的MS抗原的特异性和敏感性。目的2研究部分糖脂和胶质细胞特异性RABS对中枢神经系统病理的长期和急性影响，重点是炎症、脱髓鞘、少突胶质细胞死亡和轴突损伤的测量。目的3将使用细胞培养和发育中的小脑外植体来评估髓鞘和神经胶质细胞特异性RABS对少突胶质细胞分化和髓鞘形成的影响。鉴定致病的MS特异性抗原(S)将有广泛的应用，不仅在早期确诊方面，而且在制定调控和可能预防疾病的策略方面也有广泛的应用。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a human CNS demyelinating disease of unknown cause. The role of B cell immunity in disease pathogenesis is unknown, but has gained renewed interest because of pathology indicating IgG- mediated demyelination and the effective treatment of MS with anti-B cell therapies. Clues to the nature of disease are indicated by increased and persistent intrathecal IgG synthesis and the presence of oligoclonal bands (OCBs). OCBs are not unique to MS and in known infectious diseases of the CNS represent antibody (Ab) to the disease-causing agent, providing a rationale for our hypothesis that the oligoconal IgG in MS is directed against disease-relevant antigens. Whether the primary role of B cells in MS pathogenesis is through their effector antibody molecules, in cytokine production or as antigen-presenting cells, it operates through antigen binding to unique and disease-specific B cell surface Ig receptors. The goals of this proposal are to identify the antigenic targets of MS CSF OCBs and to define how B cells targeting these antigens contribute to disease pathogenesis. We developed a single cell RT-PCR protocol to efficiently amplify the heavy (VH) and light (VL) chain variable regions expressed by sorted MS CSF CD138+ plasma cells. Repertoires generated from CD138+ cells of MS patients each displayed seminal features of a T cell-dependent, antigen-driven response including clonal expansion, somatic hypermutation and biased use of VH4 germline segments. The phenotype of CSF CD138+ cells is that of short-lived plasma blasts and serial CSF repertoires show a persistent dynamic B cell response indicative of ongoing antigenic stimulation and not bystander activation. We have verified a mammalian expression system to produce recombinant IgG1 Abs that faithfully duplicates the in vivo pairings of plasma cell heavy- and light-chain V region sequences. Multiple MS rAbs have been demonstrated to bind oligodendrocyte antigens and myelin- enriched glycolipids. Aim 1 will identify epitopes recognized by Abs derived from MS CSF plasma blasts and will develop antigen-specific assays to screen other MS rAbs, MS CSF and inflammatory control CSF for specificity. Immunoassays including confocal microscopy, FACS sorting, and ELISA will be developed to assess the specificity and sensitivity of Ab responses to putative MS antigens. Aim 2 will investigate both long-term and acute effects of selected glycolipid- and glial cell-specific rAbs on CNS pathology with an emphasis on the measurement of inflammation, demyelination, oligodendroglial cell death, and axonal damage. Aim 3 will assess the effect of myelin- and glial cell-specific rAbs on oligodendrocyte cell differentiation and myelination using both cell culture and developing cerebellar explants. Identification of pathogenic MS-specific antigen(s) will have wide application, not only for early definitive diagnosis, but also for developing strategies to modulate and possibly prevent disease.