Immune complexes: Origins and effects in HCV infection

描述（由申请人提供）：丙型肝炎病毒（HCV）感染与体液免疫应答延迟和不足相关，也与自身免疫性疾病混合性冷球蛋白血症（MC）相关。MC患者发生B细胞非霍奇金淋巴瘤的风险增加。我们发现MC患者有表达类风湿因子（RF）样IgM的B细胞的克隆或寡克隆扩增，通常由VH 1 -69、JH 4和V：3-20 IG基因片段编码。这种IgM的RF活性是体细胞超突变的结果。RF与在对HIV、流感和HCV感染有应答的患者中观察到的广泛活性中和抗体有关。我们认为携带VH 1 -69和V：3-20编码IgM的B细胞通过识别病毒抗原而被激活，这些IgM的多反应性可能有助于它们控制HCV感染的能力。然而，体细胞超突变使得这些抗体具有自身反应性，并限制了它们在体内的功效。HCV+MC+患者血液中携带RF的B细胞的主要部分是无反应性的。在本申请中，我们建议通过（目的1）定义有和无MC的HCV患者中HCV反应性抗体的库来测试该模型;（目的2）测试VH 1 -69编码的抗体与HCV病毒体的脂质组分反应的假设;以及（目的3）定义HCV+MC+患者中无反应性RF承载B细胞的活化要求。对于目标1，我们将从有和没有MC的HCV患者制备长寿命的B细胞系。选择B细胞以使其对特异性HCV蛋白抗原具有反应性，或表达VH 1 -69免疫球蛋白。表达HCV特异性或HCV中和抗体的B细胞系将通过重排的表达的免疫球蛋白基因的克隆和测序来表征。这将使我们能够确定与HCV反应性相关的免疫球蛋白库。对于目标2，我们将利用我们已经表征的来自HCV+MC+患者的一组RF。这些RF在转染的细胞中表达为五聚体人IgM。体细胞超突变和非突变（生殖系对应物）RF均可用于研究。我们将评估这些IgM与一组脂质部分结合的能力。对于目标3，我们将测试的假设，即无变应性行为的RF轴承B细胞在体外代表了一种稳态机制，以限制自身抗体的产生，我们提出，HCV RNA存在于免疫复合物作为一个有力的信号，以驱动RF轴承B细胞的增殖和分化。将通过用一组刺激物体外刺激携带RF的B细胞来测试该机制，所述刺激物包括细胞培养物产生的HCV、由HCV和抗HCV IgG构建的免疫复合物、细胞因子和T细胞辅助。待测量的结果包括B细胞Ca++动员、增殖和抗体产生。这些研究将为MC的发病机制和可能限制有效HCV特异性免疫应答产生的机制提供新的见解。由于我们在HCV免疫学和病毒学方面的专业知识，以及我们随时可以获得病毒，病毒蛋白和患者进行研究，我们的团队是唯一有资格进行这项研究的。! 公共卫生相关性：丙型肝炎病毒感染导致肝癌和肝硬化，一些患者还发展出由产生抗体的B细胞的异常生长或功能引起的疾病。我们的工作旨在了解这种肝脏特异性病毒如何与B细胞相互作用并破坏其功能。我们希望了解正常的抗体反应如何控制丙型肝炎病毒感染，以及如何提高对丙型肝炎和其他导致持续感染的病毒的抗体反应。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection is associated with a delayed and inadequate humoral immune response, and also with an autoimmune condition, mixed cryoglobulinemia (MC). MC patients are at increased risk for development of B cell non-Hodgkin lymphoma. We found that patients with MC have clonal or oligoclonal expansions of B cells expressing rheumatoid factor (RF)-like IgM, frequently encoded by VH1-69, JH4, and V:3-20 Ig gene segments. This IgM's RF activity arises as a result of somatic hypermutation. The RFs are re- lated to broadly active neutralizing antibodies observed in patients responding to HIV, influenza, and HCV infections. We propose that B cells bearing VH1-69 and V:3-20-coded IgM are activated by recognition of a viral antigen, and that polyreactivity of these IgMs may contribute to their ability control HCV infection. However, somatic hypermutation renders these antibodies autoreactive and limits their efficacy in vivo. A major fraction of the RF-bearing B cells in the blood of HCV+MC+ patients is anergic. In this application, we propose to test this model by (Aim 1) defining the repertoire of HCV-reactive antibodies in HCV patients with and without MC; (Aim 2) testing the hypothesis that VH1-69-coded antibodies react to lipid components of the HCV virion; and (Aim 3) defining the activation requirements for anergic RF-bearing B cells in HCV+MC+ patients. For Aim 1, we will prepare long-lived B cell lines from HCV patients with and without MC. B cells will be selected for reactivity to specific HCV protein antigens, or for expression of VH1-69 immunoglobulin. B cell lines expressing HCV- specific or HCV-neutralizing antibody will be characterized by cloning and sequencing of the rearranged expressed immunoglobulin genes. This will permit us to define the immunoglobulin repertoire associated with reactivity to HCV. For Aim 2, we will take advantage of a panel of RFs from HCV+MC+ patients that we have already characterized. These RFs are expressed in transfected cells as pentameric human IgM. Both the somatically-hypermutated and non-mutated (germline counterpart) RFs are available for study. We will evaluate the ability of these IgMs to bind to a panel of lipid moieties. For Aim 3, we will test the hypothesis that the anergic behavior of RF-bearing B cells in vitro represents a homeostatic mechanism to limit autoantibody production; we propose that HCV RNA present in immune complexes serves as a potent signal to drive the proliferation and differentiation of RF-bearing B cells. This mechanism will be tested by in vitro stimulation of RF-bearing B cells with a panel of stimuli including cell culture-produced HCV, immune complexes built from HCV and anti-HCV IgG, cytokines, and T cell help. Outcomes to be measured include B cell Ca++ mobilization, proliferation, and antibody production. These studies will provide new insights into the pathogenesis of MC and the mechanisms that may limit generation of effective HCV-specific immune responses. Our group is uniquely qualified to perform this study because of our expertise in HCV immunology and virology, and our ready access to viruses, viral proteins, and patients for study. ! PUBLIC HEALTH RELEVANCE: Hepatitis C virus infection causes liver cancer and cirrhosis, and some patients also develop disease caused by abnormal growth or function of antibody-producing B cells. Our work is directed at understanding how this liver-specific virus interacts with B cells and disrupts their function. We hope to learn how normal antibody responses can control hepatitis C virus infection, and how to improve the antibody response to hepatitis C and other viruses that cause persistent infection.