B CELL RESPONSES IN MULTIPLE SCLEROSIS

IgG和寡克隆条带（OGB）的增加仅见于 CNS感染性和炎性疾病患者的CSF， 特别是多发性硬化症（MS）。尽管它们在MS中的特异性 未知，在CNS感染性疾病中，OGB特异于 致病因子，从而为我们的假设提供了一个理论基础 MS脑和CSF中的OGB是针对导致 疾病为了区分特异性和随机性体液 响应，我们测序了IgG重链（V/H）和轻链（V/L）可变区， 在多个急性MS斑块中表达的区域，并发现了一个限制性的 主要由V/H4生殖系基因组成的应答。某些V/H序列 被过度代表，克隆扩展，并显示非随机 体细胞突变的积累，指示抗原的特征 驱动的B细胞应答。亚急性硬化症的平行分析 全脑炎（SSPE）大脑也显示过度代表， 体细胞突变的V/H和V/L序列。我们共表达了这些序列 在哺乳动物表达载体中，并显示重组IgG是 对感染组织培养物中的麻疹病毒（SSPE的原因）具有特异性 细胞我们将利用重组麻疹病毒特异性 SSPE脑内麻疹抗原的抗体鉴定。信息 从SSPE中积累的蛋白质将用于合成重组抗体 从候选MS序列中筛选以证明它们的免疫特异性， 蛋白质或碳水化合物抗原。一旦特异性 建立，我们将筛选噬菌体展示cDNA文库从MS脑， 鉴定和表征其蛋白产物与MS反应cDNA 重组抗体或从急性MS斑块中纯化的IgG。我们将 还继续对急性MS斑块的V/H区域进行测序，以确定 如果限制使用特定的V/H4或其他家族生殖系， 我们已经做好了充分的准备来进行这些研究 因为我们有：（a）病理证实的急性MS脑和非MS脑 （B）经证实的识别疾病的能力， 相关的IgG序列，并使用这些序列， 产生对引起疾病的试剂特异性的重组IgG;（c） 分子生物学专业知识，构建和筛选复杂的cDNA （d）含有来自MS和其它CNS患者的OGB的CSF 炎症性疾病。MS特异性抗原的鉴定将具有 广泛的应用，不仅为早期明确诊断，而且为 制定疾病调节（如果不是预防）战略。

Increased IgG and oligoclonal bands (OGBs) are found exclusively in the CSF of patients with infectious and inflammatory diseases of the CNS, particularly multiple sclerosis (MS). Although their specificity in MS is unknown, in infectious diseases of the CNS, OGBs are specific for the agent that causes disease, thus providing a rationale for our hypothesis that OGBs in MS brain and CSF are directed against the antigen that causes disease. To distinguish between a specific versus a random humoral response, we sequenced IgG heavy (V/H) and light (V/L) chain variable regions expressed in multiple acute MS plaques and found a restricted response consisting primarily of V/H4 germline genes. Some V/H sequences were over-represented, clonally expanded, and displayed a non-random accumulation of somatic mutations, features indicative of an antigen driven B cell response. A parallel analysis of subacute sclerosing panencephalitis (SSPE) brain also revealed over-represented and somatically mutated V/H and V/L sequences. We co-expressed these sequences in mammalian expression vectors and showed that the recombinant IgG was specific for measles virus (the cause of SSPE) in infected tissue culture cells. We will exploit our success with recombinant measles virus specific antibody to identify measles antigen in SSPE brain. Information accumulated from SSPE will be used to synthesize recombinant antibodies from candidate MS sequences to demonstrate their immunologic specificity, for either protein or carbohydrate antigens. Once specificity is established, we will screen phage display cDNA libraries from MS brain to identify and characterize cDNAs whose protein products react with MS recombinant antibodies or with IgG purified from acute MS plaques. We will also continue to sequence V/H regions from acute MS plaques to determine if restricted use of specific V/H4 or other family germlines is characteristic of MS. We are well-prepared to conduct these studies because we have: (a) pathologically verified acute MS brains and non-MS neurologic disease brains; (b) a demonstrated ability to identify disease- relevant IgG sequences from human brain, and to use these sequences to generate recombinant IgG specific for the agent that causes disease; (c) the expertise in molecular biology to construct and screen complex cDNA libraries; and (d) CSF containing OGBs from patients with MS and other CNS inflammatory diseases. Identification of an MS-specific antigen will have wide application, not only for early definitive diagnosis, but also for developing strategies for modulation, if not prevention, of disease.