TLR/BCR Synergy in an Autoreactive B Cell Activation

描述(申请人提供)：从B细胞受体(BCR)转基因模型AM14分离的B细胞识别一种亲和力相对较低的原型自身抗原IgG2a，并且对大多数含有IgG2a的免疫复合体(IC)相对不敏感。然而，这些产生类风湿因子(RF)的B细胞在由IgG2a结合到染色质的IC(染色质IC)的反应下，通过一种涉及位于质膜上的BCR和位于内部隔室的Toll样受体9(TLR9)的顺序结合的机制，强烈地增殖。染色质(和其他蛋白质/核酸分子实体)是SLE中重要的自身抗体靶标，初步数据表明，这种为激活RF+B细胞而建立的序贯激活模式也可能适用于其他自身抗原反应性B细胞的激活。有必要进一步评估这一激活途径的功能结果，以确定是否有独特的特征将自身反应性B细胞的激活与仅通过BCR或TLR9信号传递的细胞区分开来。同样关键的是，确定同样的激活机制是否适用于非RF自身反应性B细胞，如果是的话，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来解决：1.使用含有纯化的染色质部分和定义的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BCR单独激活、TLR单独激活或BCR/TLR9顺序激活的B细胞在脂筏形成、特异性细胞表面分化抗原的表达、抗体和细胞因子的产生、对Fas配体促凋亡作用的敏感性、对自身反应性T细胞和/或树突状细胞产生的因子的敏感性以及与特定淋巴微环境的结合能力以及在体内存活的能力；3.通过在体外用非染色质相关自身抗原刺激AM14 RF+B细胞，并通过监测TLR缺陷的自身免疫倾向小鼠的自身抗体产生，确定BCR/TLR序列结合范例在多大程度上适用于一般的自身抗体反应；以及4.确定TLR9信号通路的抑制剂是否可以阻止SLE的发展和/或进展。研究结果将为开发治疗系统性红斑狼疮等系统性疾病的新疗法提供重要信息。

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.