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

 描述（由申请人提供）：SLE是一种病因不明的毁灭性全身性自身免疫性疾病，具有多种临床症状，困扰着150多万美国人。许多危险因素已被确定，它是普遍接受的SLE可以导致一系列的免疫调节缺陷。目前的治疗可能涉及与使人衰弱的不良副作用相关的免疫抑制方案。鉴于疾病的异质性，值得注意的是，已经发现核酸结合Toll样受体TLR 7和TLR 9在所有动物疾病模型中的自身抗体产生和疾病发展中起关键作用，并且可以证明是未来治疗的关键靶标。有趣的是，TLR 9似乎在疾病发作和进展中发挥保护和疾病促进作用-TLR 9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，TLR 9缺陷的自身免疫易感小鼠比其TLR 9充足的对应物发展更严重的临床疾病。我们最近的研究已经证明了TLR 9在清除自身反应性B细胞库中的独特作用-由BCR/TLR 9接合激活的B细胞首先经历几轮分裂，然后经历相当深刻的增殖后死亡。相比之下，BCR/TLR 7活化的B细胞存活并容易转化为浆母细胞。本项目的目的是更好地了解：（1）在疾病过程中，TLR 9可以介导这种保护作用;（2）在疾病过程中，TLR在B细胞活化中何时起关键作用;（3）TLR 9是否介导任何其他细胞类型的抑制作用;（4）TLR 9在疾病过程中的作用。（4）BCR/TLR 9与BCR/TLR 7共接合下游的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）TLR 9和TLR 7如何促进自噬体形成，和（6）特异性Unc 93 B b翻译后修饰如何优先促进原代B细胞、树突细胞和巨噬细胞中TLR 9相对于TLR 7的活化。我们的研究是基于一个独特的实验系统，其中B细胞被生理相关的自身抗原免疫复合物（IC）激活，通过使用IgG 2a特异性转基因B细胞或通过使用一种新的抗IgM/IC双可变结构域多克隆靶向抗体。通过改变应答B细胞群的遗传背景（TLR 7或TLR 9缺陷），将使用包含DNA和RNA的IC来解析BCR/TLR 9和BCR/TLR 7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。将使用高效的慢病毒逆转录策略来快速筛选Unc 93 b变体，以确定其在特定细胞类型中支持TLR 9与TLR 7驱动的应答的能力。这些研究应该提供重要的见解，为设计有效的TLR为基础的治疗方法，用于治疗诊断为SLE和其他相关的系统性自身免疫性疾病的患者的最佳策略。

 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.