TLR/BCR Synergy in an Autoreactive B Cell Activation

TLR/BCR 在自身反应性 B 细胞激活中的协同作用

• 批准号: 6704604
• 负责人: Ann Marshak-Rothstein
• 金额: $ 50.42万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2004-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6704604
• 关键词: B cell receptor; B lymphocyte; CD95 molecule; T cell receptor; apoptosis; autoantibody; autoantigens; autoimmunity; biological signal transduction; cell proliferation; cell surface receptors; chromatin; dendritic cells; enzyme linked immunosorbent assay; gene targeting; genetically modified animals; immune complex; laboratory mouse; leukocyte activation /transformation; receptor expression; systemic lupus erythematosus; toll like receptor

DESCRIPTION (provided by applicant): B cells isolated from the B cell receptor (BCR) trangenic model AM14 recognize a prototypic autoantigen, IgG2a with relatively low affinity, and are relatively unresponsive to most IgG2a-containing immune complexes (IC). However, these rheumatoid factor (RF) producing B cells proliferate vigorously in response to IC consisting of IgG2a bound to chromatin (chromatin-IC) via a mechanism that involves sequential engagement of the BCR located on the plasma membrane and Toll-like receptor 9 (TLR9) located in an internal compartment. Chromatin (and other protein/nucleic acid molecular entities) are prominent autoantibody targets in SLE and preliminary data suggests that this sequential activation paradigm, established for the activation of RF+ B cells, may also apply to the activation of other autoantigen reactive B cells. Further evaluation of the functional outcome of this activation pathway is necessary in order to determine whether there are unique features that distinguish the activation of autoreactive B cells from cells signaled through either the BCR or TLR9 alone. It will also be critical to determine whether the same activation mechanism applies to non-RF autoreactive B cells, and if so, whether this route of activation provides a unique therapeutic target. These issues will be addressed through the following specific aims: 1. Define the biochemical properties of immunostimulatory chromatin/DNA by using IC containing purified chromatin fractions and defined DNA fragments; monitor the binding, uptake and persistence of these DNA fragment IC; 2. Directly compare functional properties of B cells activated by BCR engagement alone, TLR engagement alone, or BCR/TLR9 sequential engagement with regard to parameters such as lipid raft formation, expression of specific cell surface differentiation antigens, antibody and cytokine production, sensitivity to pro-apoptotic effects of Fas-ligand, sensitivity to factors produced by autoreactive T cells and or dendritic cells, and capacity to home to particular lymphoid microenvironments and then survive in vivo; 3. Determine to what extent the BCR/TLR sequential engagement paradigm applies to autoantibody responses in general by stimulating AM14 RF+ B cells in vitro with non-chromatin-associated autoantigens and by monitoring autoantibody production in TLR-deficient autoimmune-prone mice; and 4. Determine whether the development and/or progression of SLE can be blocked by inhibitors of the TLR9 signaling pathway. The results of the studies will provide important information regarding the development of novel therapies for the treatment of systemic diseases such as SLE.

描述（由申请人提供）：从B细胞受体（BCR）转基因模型AM 14中分离的B细胞识别原型自身抗原，亲和力相对较低的IgG 2 a，并且对大多数含IgG 2 a的免疫复合物（IC）相对无应答。然而，这些产生类风湿因子（RF）的B细胞通过涉及位于质膜上的BCR和位于内部隔室中的Toll样受体9（TLR 9）的顺序接合的机制，响应于由与染色质结合的IgG 2a组成的IC（染色质-IC）而剧烈增殖。染色质（和其他蛋白质/核酸分子实体）是SLE中突出的自身抗体靶点，初步数据表明，为RF+ B细胞激活建立的这种顺序激活范例也可能适用于其他自身抗原反应性B细胞的激活。为了确定是否存在将自身反应性B细胞的活化与通过单独的BCR或TLR 9信号传导的细胞区分开的独特特征，需要进一步评估该活化途径的功能结果。同样重要的是确定相同的激活机制是否适用于非RF自身反应性B细胞，如果是，这种激活途径是否提供了独特的治疗靶点。这些问题将通过以下具体目标加以解决：1.通过使用含有纯化的染色质组分和确定的DNA片段的IC来确定免疫刺激染色质/DNA的生化性质;监测这些DNA片段IC的结合、摄取和持久性; 2.直接比较由单独的BCR接合、单独的TLR接合或BCR/TLR 9顺序接合激活的B细胞的功能特性，关于参数例如脂筏形成、特异性细胞表面分化抗原的表达、抗体和细胞因子产生、对Fas配体的促凋亡作用的敏感性、对自身反应性T细胞和/或树突细胞产生的因子的敏感性，以及归巢至特定淋巴微环境并随后在体内存活的能力; 3.通过在体外用非染色质相关的自身抗原刺激AM 14 RF+ B细胞并通过监测TLR缺陷的自身免疫易感小鼠中的自身抗体产生，确定BCR/TLR顺序接合范式在多大程度上适用于自身抗体应答;和4.确定SLE的发生和/或进展是否可以被TLR 9信号通路抑制剂阻断。这些研究的结果将为开发治疗系统性疾病（如SLE）的新疗法提供重要信息。