Immune complexes: Origins and effects in HCV infection

描述（由申请人提供）：丙型肝炎病毒（HCV）感染与延迟和不充分的体液免疫反应有关，也与自身免疫性疾病混合冷球蛋白血症（MC）有关。MC患者发展为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细胞克隆或寡克隆扩增，通常由VH1-69、JH4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。RFs与在对HIV、流感和HCV感染有反应的患者中观察到的广泛活性中和抗体有关。我们认为携带VH1-69和V:3-20编码IgM的B细胞被病毒抗原识别激活，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，体细胞超突变使这些抗体产生自身反应，限制了它们在体内的效力。HCV+MC+患者血液中携带rf的B细胞的主要部分是无能的。在这个应用中，我们建议通过（目的1）定义HCV反应性抗体在有和没有MC的HCV患者中的库来测试这个模型；（目的2）验证vh1 -69编码抗体与HCV病毒粒子脂质成分反应的假设；以及（目标3）确定HCV+MC+患者携带rf的无能B细胞的激活需求。在Aim 1中，我们将从患有和不患有MC的HCV患者中制备长寿的B细胞系。B细胞将被选择用于对特定HCV蛋白抗原的反应性，或表达VH1-69免疫球蛋白。表达HCV特异性抗体或HCV中和抗体的B细胞系将通过克隆和测序重排表达的免疫球蛋白基因来表征。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Aim 2，我们将利用我们已经确定的HCV+MC+患者的一组RFs。这些rf在转染的细胞中以五聚体人IgM的形式表达。体细胞超突变和非突变RFs（种系对应体）均可用于研究。我们将评估这些igm与一组脂质片段结合的能力。在第3项研究中，我们将验证一个假设，即体外携带rf的B细胞的无能行为代表了一种限制自身抗体产生的稳态机制；我们提出，HCV RNA存在于免疫复合物中，作为一种有效的信号来驱动携带rf的B细胞的增殖和分化。这一机制将通过体外刺激携带rf的B细胞进行测试，刺激包括细胞培养产生的HCV、由HCV和抗HCV IgG构建的免疫复合物、细胞因子和T细胞帮助。测量结果包括B细胞Ca++的动员、增殖和抗体产生。这些研究将为MC的发病机制和可能限制产生有效的hcv特异性免疫反应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用于研究的病毒、病毒蛋白和患者，我们小组具有开展这项研究的独特资格。！

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. !