Molecular Regulation of VH Gene Replacement in Human Immature B Cells

描述(申请人提供)：虽然VH替换的概念有很长的历史，但我们最近的研究才认识到VH替换在人类B细胞中的自然发生和分子基础。VH替换通过RAG介导的二级重组发生，涉及重排的VHDJH区域内的隐蔽RSS(CRSS)和来自VH基因上游的23bpRSS。在人类B细胞发育过程中，VH替换发生在骨髓未成熟B细胞中，约占人类外周B细胞库的5%。我们最近的研究表明，在自身免疫性疾病和编码抗病毒抗体的IgH基因中，VH替换产物的频率显著升高，提示VH替换具有尚未实现的重要功能。目前的建议侧重于人类未成熟B细胞中VH替换的分子调控。我们发现，交联型BCR强烈诱导Eu12&lt；HC+细胞和原代未成熟B细胞中的VH替换。已有研究表明，在小鼠未成熟B细胞中，核因子-kB的激活是诱导RAG基因表达和Ig：编辑所必需的。我们发现交联型BCR可诱导核因子-kB的DNA结合活性，用CAPE阻断核因子-kB的激活可抑制EU12&lt；HC+细胞中的VH替换。基于这些观察，我们假设VH替换是由BCR介导的人类未成熟B细胞中的信号调节的，并可能因自身免疫性疾病中炎性细胞因子或共刺激分子水平的升高而进一步增强。(1)我们将使用实时荧光定量聚合酶链式反应方法来确定VH替换的相对发生率和发生阶段，并与来自健康供者和类风湿关节炎(RA)或系统性红斑狼疮(SLE)患者的Ig：基因编辑的未成熟B细胞进行比较。(2)我们将通过使用特定的siRNA来下调BLNK的表达，并通过激活或阻断NF-kB活性来调节BCR信号，以确定诱导VH替换的负责信号事件。(3)我们将通过检测生殖系转录、组蛋白乙酰化和RAG介导的体外切割试验来分析不同B细胞亚群中VH基因的可及性，以确定VH替换是否通过调节IgH基因的可及性来控制；我们还将确定在正常的未成熟B细胞中，VH基因上游的可及性是否受BCR或共刺激信号的调节，以及这些可及性在RA和SLE未成熟B细胞中是否受到异常调控。了解VH替代的分子调控具有深远的临床意义，因为VH替代产品在自身免疫性疾病和抗病毒反应中高度丰富。公共卫生相关声明：VH替换通过RAG介导的二次重组发生，涉及重排的VH基因内的隐蔽重组信号序列(CRSS)和上游VH基因的23bpRSS。虽然VH替换的概念有很长的历史，但我们最近的研究才认识到VH替换在人类B系细胞中的分子基础和自然发生。正常情况下，VH替代约占健康供者外周B细胞IgH谱系的5%。值得注意的是，在来自不同自身免疫性疾病的IgH基因和编码各种抗病毒抗体的IgH基因中，VH替换产物的频率显著增加。目前的建议侧重于人类未成熟B细胞中VH替换的分子调控。我们发现交联型B细胞抗原受体(BCR)强烈地诱导人未成熟B细胞中的VH替换。了解VH替换的分子调控将具有重要的临床意义，因为VH替换产品在自身免疫性疾病和抗病毒反应中的频率显著升高。

DESCRIPTION (provided by applicant): Although the concept of VH replacement has a lengthy history, the natural occurrence and molecular basis of VH replacement in human B cells have just been realized from our recent studies. VH replacement occurs through RAG-mediated secondary recombination involving the cryptic RSS (cRSS) within the rearranged VHDJH region and the 23 bp RSS from an upstream VH gene. VH replacement occurs in bone marrow immature B cells during human B cell development and contributes to about 5% of the periphery B cell repertoire in humans. Our recent studies showed that the frequencies of VH replacement products are significantly elevated in autoimmune diseases and in IgH genes encoding anti-viral antibodies, suggesting an unrealized important function of VH replacement. The current proposal focuses on the molecular regulation of VH replacement in human immature B cells. We found that crosslinking BCR strongly induces VH replacement in the EU12 <HC+ cells and in primary immature B cells. It has been shown that activation of NF-kB is required for the induction of RAG gene expression and Ig: editing in murine immature B cells. We found that crosslinking BCR induces NF-kB DNA binding activities and blocking NF-kB activation with CAPE inhibits VH replacement in the EU12 <HC+ cells. Based on these observations, we hypothesize that VH replacement is regulated by BCR-mediated signaling in human immature B cells and might be further enhanced by elevated levels of inflammatory cytokines or costimulatory molecules in autoimmune diseases. (1) We will use real time LM-PCR method to determine the relative rate and occurrence stage of VH replacement in comparison with Ig: gene editing in immature B cells from healthy donors and rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) patients. (2) We will modulate BCR signaling by knocking-down BLNK expression using specific siRNA and by activating or blocking NF-kB activities to determine the responsible signaling events for inducing VH replacement. (3) We will analyze the accessibilities to VH genes in different B cell subsets by detection of germline transcription, histone acetylation, and by RAG-mediated in vitro cleavage assays to determine if VH replacement is controlled through regulating the IgH locus accessibility; we will also determine if the accessibilities to upstream VH genes are regulated by BCR or co-stimulatory signaling in normal immature B cells and if these accessibilities are abnormally regulated in RA and SLE immature B cells. Understanding the molecular regulation of VH replacement has profound clinical implications, because VH replacement products are highly enriched in autoimmune diseases and anti-viral responses. PUBLIC HEALTH RELEVANCE STATEMENT: VH replacement occurs through RAG-mediated secondary recombination involving the cryptic recombination signal sequence (cRSS) within the rearranged VH gene and a 23 bp RSS of an upstream VH gene. Although the concept of VH replacement has a length history, the molecular basis and natural occurrence of VH replacement in human B lineage cells have just been realized from our recent studies. Normally, VH replacement contributes to about 5% of the IgH repertoire in the periphery B cells of healthy donors. Strikingly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different autoimmune diseases and in IgH genes encoding various anti-viral antibodies. The current proposal focuses on the molecular regulation of VH replacement in human immature B cells. We found that crosslinking B cell antigen receptor (BCR) strongly induces VH replacement in human immature B cells. Understanding the molecular regulation of VH replacement will have important clinical implications, because the frequencies of VH replacement products are significantly elevated in autoimmune diseases and anti-viral responses.