Mechanism of Action of Inhibitory CpG Oligonucleotides

描述（由申请人提供）：细菌DNA的CpG基序是被我们的先天免疫系统识别为“危险信号”的微生物分子模式之一，需要Toll样受体（TLR）9。短合成寡脱氧核苷酸（ODN）促进了结构-功能关系的研究。刺激性（ST-）ODN直接激活B细胞、巨噬细胞和树突状细胞的能力，从而一起触发体液免疫和TH 1免疫，使它们作为疫苗佐剂和作为癌症和变态反应的治疗具有有价值的潜在作用。改变少至2个碱基将强ST-ODN转化为阻断TLR 9但不阻断其它TLRs的抑制剂（IN-ODN）。在当前授权的支持下，已经定义了小鼠细胞中IN-ODN作用的序列要求。15个位点中只有3对决定IN-ODN活性。同一组IN-ODN可以阻断B细胞（B型）和非B细胞（A型）优选的ST-ODN。我们请求继续支持，以解决这些问题：1）IN-ODN是否与ST-ODN竞争TLR 9结合？2)ST-ODN和IN-ODN与TLR 9的结合强度是否决定了它们的生物活性？3)ST-ODN或IN-ODN是否将TLR 9分子连接在一起？ODN与自身或彼此聚合的能力是否允许它们这样做？4)ST-ODN和IN-ODN之间的竞争发生在小区的什么地方？5)IN-ODN能在体内阻断ST-ODN活性吗？同样微妙的结构要求是否适用？使用的实例是ODN诱导的“败血性休克”模型和小鼠中的炎性结肠炎。6)人类细胞的IN-ODN结构要求是什么？ODN类型之间有区别吗？这些问题的回答对于理解ODN的反应机制和开发其在人类疾病中的进一步应用，特别是用IN-ODN治疗狼疮和类风湿性关节炎的前景都是相关的。

DESCRIPTION (provided by applicant): The CpG motifs of bacterial DMA are among the microbial molecular patterns recognized as "danger signals" by our innate immune system, requiring Toll-Like Receptor (TLR) 9. Short synthetic oligodeoxyribonucleotides (ODN) have facilitated studies of structure-function relationships. The ability of stimulatory (ST-) ODN to activate B cells, macrophages, and dendritic cells directly, thus triggering humoral and TH1 immunity together, gives them valuable potential roles as vaccine adjuvants and as therapy in cancer and allergy. Changing as few as 2 bases converts a strong ST-ODN into an inhibitor (IN-ODN) that blocks TLR9 but not the other TLRs. With support from the current grant the sequence requirements for IN-ODN action in mouse cells have been defined. Only 3 pairs of positions out of 15 determine IN-ODN activity. The same panel of IN-ODN can block the ST-ODN preferred by B cells (Type B) and non-B cells (Type A). We request continued support in order to address these questions: 1) Do IN-ODN compete with ST-ODN for TLR9 binding? 2) Does the strength of binding of ST-ODN and IN-ODN for TLR9 determine their biologic activity? 3) Do ST-ODN or IN-ODN tie TLR9 molecules together? Does the ability of ODN to aggregate with themselves or each other allow them to do this? 4) Where in the cell does the competition between ST-ODN and IN-ODN take place? 5) Can IN-ODN block ST-ODN activity in vivo? Do the same subtle structural requirements apply? Examples to be used are a model of ODN-induced "septic shock" and an inflammatory colitis in mice. 6) What are the IN-ODN structural requirements for human cells? Do they differ between ODN types? The answers to these questions are pertinent both to understanding the mechanism of ODN responses and to developing further applications to human disease, especially the prospect of treating lupus and rheumatoid arthritis with IN-ODN.