B CELL ACTIVATION INDUCED BY OLIGODEOXYNUCLEOTIDES CONTAINING CPG MOTIFS

含有未甲基化的CpG二核苷酸并带有两个嘌呤的DNA 在5‘端和3’端的两个嘧啶(CpG基序“)引起 强大的B细胞激活能力。这表现在早期的表达上 活化基因，如EGR-1和c-fos，免疫球蛋白分泌和 IL-6，并在体外和体内进入细胞周期。这 激活通过B细胞抗原受体与信号协同作用 (Bcr)。这种未甲基化的CpG基序出现的频率是 在微生物DNA中，例如在脊椎动物中。细菌DNA可以激活B细胞，但 脊椎动物的DNA并非如此。因此，由CpG基序激活的淋巴细胞可能 是一种重要的免疫防御机制，因为它区分了 微生物和自身DNA，似乎有效地促进了 抗原特异性免疫。 这个CpG基序与CREB/ATF的结合位点几乎相同 转录因子家族，Cre。CREB/ATF蛋白可以 转录调控许多基因，包括EGR-1、c-fos和IL-6。 CpG ODN可以结合一个或多个CREB/ATF蛋白，并特异性竞争 CREB/ATF与Cre的结合。这些数据增加了一种可能性 CpG ODN的作用可能是由于它们与一个或多个 更多的CREB/ATF蛋白。 本提案的广泛目标首先是确定如何 CpG DNA和BCR信号通路之间存在协同作用； 第二，确定CpG DNA通过的分子机制 诱导EGR-1、c-fos和IL-6的转录。第一个具体 AIM将确定CpG DNA和BCR信号通路 通过近端或更远端的激活步骤相互作用。第二 目的明确CpG的分子机制(S)。 ODN体外诱导EGR-1、c-fos和IL-6转录 启动子报告基因的转录检测和转染 构建成B细胞。第三个具体目标是，首先，确定， 鉴定并克隆CREB/ATF或其他B细胞蛋白(如有必要 绑定CpG ODN；第二，确定此绑定如何影响 用凝胶位移和超位移分析这些蛋白质的性质， 西部和西南斑点，免疫沉淀和评估 磷酸化、相关蛋白质或DNA结合活性的变化。 这些研究的完成将提高人们对 调节淋巴细胞激活的机制。这些研究还包括 对可能的意外免疫激活的影响 反义ODN在人类基因治疗和DNA疫苗中的应用 含有CpG基序。这些研究将确定其作用机制。 一类有希望的新型免疫调节剂的免疫调节。

DNA containing an unmethylated CpG dinucleotide flanked by two purines on the 5' side and two pyrimidines on the 3' side (CpG motif") causes potent B cell activation. This is manifested by expression of early activation genes such as egr-1 and c-fos, secretion of immunoglobulin and IL-6, and entry into the cell cycle both in vitro and in vivo. This activation synergizes with signals through the B cell antigen receptor (BCR). Such unmethylated CpG motifs occur more than twenty times as often in microbial DNA as in vertebrates. Bacterial DNA activates B cells, but vertebrate DNA does not. Thus, lymphocyte activation by CpG motifs may be an important immune defense mechanism since it distinguishes between microbial and self DNA and appears to effectively promote antigen-specific immunity. This CpG motif is nearly identical to the binding site for the CREB/ATF family of transcription factors, the CRE. CREB/ATF proteins can transcriptionally regulate many genes, including egr-1, c-fos, and IL-6. CpG ODN can bind one or more CREB/ATF proteins, and specifically compete the binding of CREB/ATF to the CRE. These data raise the possibility that the effects of CpG ODN may result from their interactions with one or more CREB/ATF proteins. The broad goals of the present proposal are first, to determine how synergy occurs between the CpG DNA and the BCR signaling pathways; and second, to determine the molecular mechanism through which CpG DNA induces the transcription of egr-1, c-fos, and IL-6. The first specific aim will determine whether the CpG DNA and BCR signaling pathways interact through proximal or more distal activation steps. The second specific aim will elucidate the molecular mechanism(s) through which CpG ODN induces egr-1, c-fos, and IL-6 transcription using in vitro transcription assays, and transfection of promoter reporter gene constructs into B cells. The third specific aim will first, identify, characterize, and, if necessary, clone CREB/ATF or other B cell proteins that bind CpG ODN; and second, determine how this binding affects the properties of these proteins using gel shift and supershift assays, western and southwestern blots, immunoprecipitation, and assessment of changes in phosphorylation, associated proteins, or DNA binding activity. Completion of these studies will improve the understanding of the mechanisms regulating lymphocyte activation. These studies also have implications for possible unintended immune activation resulting from the use of "antisense' ODN and for human gene therapy and DNA vaccines containing CpG motifs. These studies will determine the mechanism of immune regulation by a promising new class of immunomodulators.