Molecular Regulation of VH Gene Replacement in Human Immature B Cells

描述（由申请人提供）：虽然VH替代的概念有着悠久的历史，但我们最近的研究才刚刚认识到VH替代在人类B细胞中的自然发生和分子基础。VH的替换是通过rag介导的二次重组发生的，涉及重排VHDJH区域内的隐性RSS （cRSS）和来自上游VH基因的23 bp RSS。VH替代发生在人B细胞发育过程中的骨髓未成熟B细胞中，约占人外周血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替代产物的频率显著升高，这表明VH替代的重要功能尚未被认识到。目前的建议集中在人类未成熟B细胞中VH替代的分子调控。我们发现交联BCR在EU12 <HC+细胞和原代未成熟B细胞中强烈诱导VH替代。研究表明，在小鼠未成熟B细胞中，NF-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BCR诱导NF-kB DNA结合活性，用CAPE阻断NF-kB激活可抑制EU12 <HC+细胞中的VH替代。基于这些观察结果，我们假设在人类未成熟B细胞中，VH替代是由bcr介导的信号调节的，并且在自身免疫性疾病中，炎症细胞因子或共刺激分子水平的升高可能进一步增强VH替代。(1)我们将采用real time LM-PCR方法，与Ig：基因编辑在健康供体和类风湿关节炎（RA）或系统性红斑狼疮（SLE）患者未成熟B细胞中比较，确定VH替代的相对发生率和发生阶段。(2)我们将通过使用特定的siRNA敲低BLNK表达和激活或阻断NF-kB活性来调节BCR信号传导，以确定诱导VH替代的相关信号传导事件。(3)我们将通过检测种系转录、组蛋白乙酰化和rag介导的体外裂解实验来分析不同B细胞亚群中VH基因的可及性，以确定是否通过调节IgH位点可及性来控制VH的替代；我们还将确定正常未成熟B细胞中对上游VH基因的可及性是否受到BCR或共刺激信号的调节，以及这些可及性是否在RA和SLE未成熟B细胞中受到异常调节。了解VH替代的分子调控具有深远的临床意义，因为VH替代产物在自身免疫性疾病和抗病毒反应中高度富集。公共卫生相关性声明：VH置换发生在ragg介导的二次重组中，涉及重排VH基因内的隐性重组信号序列（cRSS）和上游VH基因的23bp RSS。虽然VH替代的概念由来已久，但我们最近的研究才刚刚认识到VH替代在人类B系细胞中的分子基础和自然发生。正常情况下，在健康供者的外周血B细胞中，VH替代贡献了约5%的IgH库。引人注目的是，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替代产物的频率显著升高。目前的建议集中在人类未成熟B细胞中VH替代的分子调控。我们发现交联B细胞抗原受体（BCR）在人未成熟B细胞中强烈诱导VH替代。了解VH替代的分子调控将具有重要的临床意义，因为VH替代产物的频率在自身免疫性疾病和抗病毒反应中显着升高。

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.