The Role of Intrathecal Plasma Cell Clones in MS Pathogenesis

鞘内浆细胞克隆在多发性硬化症发病机制中的作用

• 批准号: 8321989
• 负责人: Gregory Parks Owens
• 金额: $ 26.78万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321989
• 关键词: Acute; Affect; American; Animals; Antibodies; Antibody Formation; Antigen Targeting; Antigen-Presenting Cells; Antigens; Asses; Astrocytes; Autoantibodies; Autoantigens; B-Lymphocytes; Binding; Biological Assay; Biological Markers; Blast Cell; Brain; Cell Culture Techniques; Cell Death; Cell Differentiation process; Cell Line; Cell Therapy; Cell surface; Cells; Cerebrospinal Fluid; Chronic; Clinic; Clonal Expansion; Collection; Communicable Diseases; Complement; Complex; Confocal Microscopy; Demyelinating Diseases; Demyelinations; Diagnosis; Disease; Enzyme-Linked Immunosorbent Assay; Epitopes; Frequencies; Galactosylceramides; Genes; Glycolipids; Goals; Human; IgG1; Immunity; Immunoassay; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; Individual; Inflammation; Inflammatory; Life; Light; Lipids; Measles virus; Measurement; Mediating; Memory B-Lymphocyte; Molecular and Cellular Biology; Multiple Sclerosis; Mus; Myelin; Nature; Neuroglia; Neurology; Neuromyelitis Optica; Neurons; Oligoclonal Bands; Oligodendroglia; Optic Nerve; Pathogenesis; Pathogenicity; Pathology; Patients; Phagocytosis; Phenotype; Physiology; Plasma; Plasma Cells; Population; Production; Proteins; Protocols documentation; Recombinant Antibody; Recombinants; Relapse; Research Project Grants; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Rodent Model; Role; Seminal; Sensitivity and Specificity; Sorting - Cell Movement; Specificity; Staining method; Stains; Subacute Sclerosing Panencephalitis; Sulfoglycosphingolipids; Surface Immunoglobulins; System; T-Lymphocyte; Technology; University Hospitals; antigen binding; aquaporin 4; cytokine; disease phenotype; effective therapy; in vivo; interest; myelination; nervous system disorder; population based; prevent; receptor; response; rituximab; tissue/cell culture; white matter

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.

描述（由申请人提供）：多发性硬化症（MS）是一种原因不明的人类CNS脱髓鞘疾病。B细胞免疫在疾病发病机制中的作用是未知的，但由于病理学表明IgG介导的脱髓鞘和用抗B细胞疗法有效治疗MS而重新引起关注。鞘内IgG合成增加和持续存在以及寡克隆带（OCB）的存在表明了疾病性质的线索。OCB不是MS所独有的，在已知的CNS感染性疾病中，OCB代表致病因子的抗体（Ab），这为我们的假设提供了理论基础，即MS中的寡聚IgG针对疾病相关抗原。无论B细胞在MS发病机制中的主要作用是通过其效应抗体分子、在细胞因子产生中还是作为抗原呈递细胞，它都是通过抗原与独特的和疾病特异性的B细胞表面IG受体结合来起作用的。本提案的目的是确定MS CSF OCB的抗原靶点，并确定靶向这些抗原的B细胞如何促进疾病发病机制。我们开发了一种单细胞RT-PCR方案，以有效扩增分选的MS CSF CD 138+浆细胞表达的重链（VH）和轻链（VL）可变区。从MS患者的CD 138+细胞产生的库各自显示T细胞依赖性的、抗原驱动的应答的种子特征，包括克隆扩增、体细胞超突变和VH 4种系区段的偏向使用。CSF CD 138+细胞的表型是短寿命血浆母细胞的表型，并且系列CSF库显示持续的动态B细胞应答，表明正在进行的抗原刺激而不是旁观者活化。我们已经验证了哺乳动物表达系统，以产生重组IgG 1抗体，忠实地复制浆细胞重链和轻链V区序列的体内配对。已证明多种MS rAb结合少突胶质细胞抗原和富含髓鞘的糖脂。目的1将鉴定由MS CSF血浆母细胞衍生的Ab识别的表位，并将开发抗原特异性测定以筛选其他MS rAb、MS CSF和炎症对照CSF的特异性。将开发包括共聚焦显微镜、FACS分选和ELISA在内的免疫测定法，以评估Ab对推定MS抗原反应的特异性和灵敏度。目的2将研究选定的糖脂和神经胶质细胞特异性rAbs对CNS病理学的长期和急性影响，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评估髓鞘和胶质细胞特异性rAbs对少突胶质细胞分化和髓鞘形成的影响。致病性MS特异性抗原的鉴定将具有广泛的应用，不仅用于早期确定性诊断，而且用于开发调节和可能预防疾病的策略。