Immune complexes: Origins and effects in HCV infection

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)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，混合性冷球蛋白血症(MC)有关。MC患者患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细胞的克隆性或寡克隆性扩增，通常由VH1-69、JH4和V：3-20Ig基因片段编码。这种IgM的RF活性是体细胞超突变的结果。RFs与在对艾滋病毒、流感和丙型肝炎病毒感染有反应的患者中观察到的广泛活跃的中和抗体有关。我们认为，携带VH1-69和V：3-20编码的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丙型肝炎病毒感染。然而，体细胞的超突变使这些抗体产生自身反应，并限制了它们在体内的效力。丙型肝炎病毒+MC+患者血液中携带RF的B细胞的大部分是无能的。在这一应用中，我们建议通过(目标1)定义患有和不患有MC的丙型肝炎患者的丙型肝炎病毒反应性抗体的库；(目的2)测试VH1-69编码的抗体与丙型肝炎病毒粒子的脂质成分发生反应的假设；以及(目标3)定义丙型肝炎病毒+MC+患者中无能的RF承载B细胞的激活要求来测试这一模型。对于目标1，我们将从患有和不患有MC的丙型肝炎患者中制备长期存活的B细胞株。B细胞将被选择用于对特定的丙型肝炎病毒蛋白抗原的反应，或用于表达VH1-69免疫球蛋白。表达丙型肝炎病毒特异性抗体或中和抗体的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鉴定。这将使我们能够确定与丙型肝炎病毒反应性相关的免疫球蛋白谱系。对于目标2，我们将利用我们已经确定的一组来自丙型肝炎病毒+MC+患者的RF。这些RFs在转基因细胞中以五聚体人IgM的形式表达。体细胞高突变和非突变(生殖系对应)RFs均可用于研究。我们将评估这些免疫球蛋白与一组脂质部分结合的能力。对于目标3，我们将检验一种假设，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动态平衡机制；我们认为免疫复合体中存在的HCVRNA是驱动携带RF的B细胞增殖和分化的有力信号。这一机制将通过一组刺激物体外刺激携带RF的B细胞来检验，这些刺激物包括细胞培养产生的丙型肝炎病毒、丙型肝炎病毒免疫复合物和抗丙型肝炎病毒免疫球蛋白、细胞因子和T细胞帮助。测量的结果包括B细胞钙离子动员、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的丙型肝炎病毒特异性免疫反应产生的机制提供新的见解。我们的团队是唯一有资格进行这项研究的人，因为我们在丙型肝炎病毒免疫学和病毒学方面的专业知识，以及我们随时可以接触到病毒、病毒蛋白和患者进行研究。

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.