Distinct Functional Outcomes of BCR/TLR7 and BCR/TLR9 Co-engagement

 描述(由申请人提供)：SLE是一种原因不明的破坏性系统性自身免疫性疾病，呈现一系列不同的临床症状，困扰着超过150万美国人。许多危险因素已经被确定，人们普遍认为系统性红斑狼疮可以由一系列免疫调节缺陷引起。目前的治疗方法可能包括免疫抑制方案，与衰弱的不良副作用相关。鉴于疾病的异质性，值得注意的是，核酸结合的Toll样受体TLR7和TLR9已被发现在所有动物模型的自身抗体的产生和疾病的发展中发挥关键作用，并可能被证明是未来治疗的关键靶点。有趣的是，TLR9似乎在疾病的发生和发展中既起到了保护作用，又起到了促进疾病的作用--TLR9是产生针对DNA相关自身抗原的自身抗体所必需的，但令人费解的是，TLR9缺乏的自身免疫倾向小鼠患上的临床疾病比TLR9充足的小鼠严重得多。我们最近的研究表明，TLR9在清除由BCR/TLR9激活的自身反应性B细胞系-B细胞方面具有独特的作用，首先经历几轮分裂，然后经历相当深刻的增殖后死亡。相比之下，BCR/TLR7激活的B细胞存活并很容易转化为浆母细胞。这个项目的目的是为了更好地了解：(1)在疾病过程中，TLR9在哪里可以介导这种保护作用；(2)在疾病过程中，TLR9在B细胞激活中发挥关键作用；(3)TLR9是否在任何其他类型的细胞中发挥抑制作用；(4)BCR/TLR9与BCR/TLR7共参与下游转录网络的哪些独特特征解释了这些激活方案的不同功能结果；(5)TLR9和TLR7如何促进自噬小体的形成；(6)Unc93b翻译后修饰如何优先促进原代B细胞、树突状细胞和巨噬细胞中TLR9与TLR7的激活。我们的研究基于一个独特的实验系统，在该系统中，B细胞被生理上相关的自身抗原免疫复合物(IC)激活，使用针对IgG2a的转基因B细胞或使用新型抗IgM/IC双变构域多克隆靶向抗体。结合了DNA和RNA的ICS将被用来通过改变应答B细胞群体的遗传背景(TLR7或TLR9缺失)来解析BCR/TLR9和BCR/TLR7激活途径的独特特征。先进的基因组和计算分析将用于阐明基因调控网络。一种高效的慢病毒逆转录策略将被用于快速筛选Unc93b变体，以了解它们在特定细胞类型中支持TLR9与TLR7驱动的反应的能力。这些研究应该为设计有效的基于TLR的疗法治疗被诊断为SLE和其他相关系统性自身免疫性疾病的患者提供重要的见解。

 DESCRIPTION (provided by applicant): SLE is a devastating systemic autoimmune disease of unknown etiology that presents with a diverse array of clinical symptoms and afflicts over 1.5 million Americans. Numerous risk factors have been identified, and it is generally accepted that SLE can result from a spectrum of immunoregulatory defects. Current treatments can involve immunosuppressive regimens associated with debilitating adverse side effects. Given the heterogeneous nature of the disease, it is remarkable that nucleic acid binding Toll-like receptors TLR7 and TLR9 has been found to play a critical role in the production of autoantibodies and disease development in all animal models of disease, and may prove to be critical targets for future therapies. Intriguingly, TLR9 appears to play both a protective and disease promoting role in the disease onset and progression - TLR9 is required for the production of autoantibodies against DNA-associated autoantigens but, quite inexplicably, TLR9-deficient autoimmune-prone mice develop much more severe clinical disease than their TLR9- sufficient counterparts. Our recent studies have now demonstrated a unique role for TLR9 in purging the autoreactive B cell repertoire - B cells activated by BCR/TLR9 engagement first undergo several rounds of division and then undergo a quite profound post-proliferative death. By contrast, BCR/TLR7 activated B cells survive and readily convert to plasmablasts. The goals of this project are to gain a better understanding of: (1) where in the course of disease TLR9 can mediate this protective effect; (2) when in the course of disease TLRs play a key role in B cell activation; (3) whether TLR9 mediates a suppressive role in any other cell types; (4) what unique features of the transcriptional networks downstream of BCR/TLR9 vs BCR/TLR7 co- engagement account for the distinct functional outcomes of these activation schemes, (5) how TLR9 and TLR7 contribute to autophagosome formation, and (6) how specific Unc93b post translational modifications preferentially promote TLR9 vs TLR7 activation in primary B cells, dendritic cells and macrophages. Our studies are based on a unique experimental system in which B cells are activated by physiologically relevant autoantigen immune complexes (IC), by using either IgG2a-specific transgenic B cells or by using a novel anti-IgM/IC dual variable domain polyclonal targeting antibody. ICs that incorporate both DNA and RNA will be used to parse the unique features of BCR/TLR9 and BCR/TLR7 activation pathways by varying the genetic background (TLR7- or TLR9-deficient) of the responder B cell populations. Advanced genomic and computational analyses will be used to elucidate gene regulatory networks. A highly efficient lentiviral retrogenesis strategy will be used to rapidly screen Unc93b variants for their capacity to support TLR9 vs TLR7 driven responses in specific cell types. These studies should provide important insights regarding the best strategies for designing effect TLR-based therapeutics for the treatment of patients diagnosed with SLE and other related systemic autoimmune diseases.