TLR/BCR Synergy in an Autoreactive B Cell Activation

描述（由申请人提供）：从B细胞受体（BCR）转基因模型AM14中分离的B细胞识别具有相对低亲和力的原型自身抗原IgG2a，并且对大多数含IgG2a的免疫复合物（IC）相对无反应。然而，这些产生类风湿因子（RF）的B细胞对由IgG2a结合到染色质（染色质-IC）组成的IC反应强烈增殖，其机制包括位于质膜上的BCR和位于内部隔室的toll样受体9 （TLR9）的顺序结合。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，这种为RF+ B细胞激活而建立的顺序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，以确定是否存在独特的特征来区分自身反应性B细胞的激活与单独通过BCR或TLR9信号传导的细胞。确定相同的激活机制是否适用于非rf自身反应性B细胞，以及如果适用，这种激活途径是否提供了独特的治疗靶点，也将是至关重要的。这些问题将通过以下具体目标加以解决：使用含有纯化染色质组分和定义的DNA片段的IC定义免疫刺激染色质/DNA的生化特性；监测这些DNA片段的结合、摄取和持久性；2. 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等参数方面，直接比较单独使用BCR、单独使用TLR或BCR/TLR9顺序结合激活的B细胞的功能特性。以及在特定淋巴细胞微环境中安家并在体内存活的能力；3. 通过在体外用非染色质相关自身抗原刺激AM14 RF+ B细胞和监测TLR缺陷自身免疫易感小鼠的自身抗体产生，确定BCR/TLR顺序参与范式在多大程度上适用于自身抗体反应；和4。确定是否可以通过TLR9信号通路抑制剂阻断SLE的发生和/或进展。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。

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.