Molecular Regulation of VH Gene Replacement in Human Immature B Cells

描述（由申请人提供）：虽然VH置换的概念有很长的历史，但我们最近的研究才认识到VH置换在人B细胞中的自然发生和分子基础。VH置换通过RAG介导的二级重组发生，所述二级重组涉及重排的VHDJH区内的隐蔽RSS（cRSS）和来自上游VH基因的23 bp RSS。在人B细胞发育期间，VH置换发生在骨髓未成熟B细胞中，并占人外周B细胞库的约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置换产物的频率显著升高，这表明VH置换的重要功能尚未实现。目前的建议集中在人未成熟B细胞中VH置换的分子调控上。我们发现，交联BCR强烈诱导EU 12 <HC+细胞和原代未成熟B细胞中的VH置换。已经表明，在鼠未成熟B细胞中，诱导RAG基因表达和IG编辑需要NF-κ B的活化。我们发现，交联BCR诱导NF-κ B DNA结合活性，并且用CAPE阻断NF-κ B活化抑制EU 12 <HC+细胞中的VH置换。基于这些观察结果，我们假设VH置换受人类未成熟B细胞中BCR介导的信号转导的调节，并且可能通过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)我们将使用真实的时间LM-PCR方法来确定与IG相比，VH替换的相对率和发生阶段：来自健康供体和类风湿性关节炎（RA）或系统性红斑狼疮（SLE）患者的未成熟B细胞中的基因编辑。(2)我们将通过使用特异性siRNA敲低BLNK表达和通过激活或阻断NF-κ B活性来调节BCR信号传导，以确定诱导VH置换的负责信号传导事件。(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基因的可接近性，以确定VH替换是否通过调节IgH位点可接近性来控制;我们还将确定上游VH基因的获得是否受BCR或共调控，正常未成熟B细胞中的刺激性信号传导，以及这些辅助信号在RA和SLE未成熟B细胞中是否受到异常调节。了解VH置换的分子调控具有深远的临床意义，因为VH置换产物在自身免疫性疾病和抗病毒应答中高度富集。公共卫生相关声明：VH置换通过涉及重排的VH基因内的隐蔽重组信号序列（cRSS）和上游VH基因的23 bp RSS的RAG介导的二级重组发生。虽然VH置换的概念有很长的历史，但从我们最近的研究中才认识到VH置换在人B谱系细胞中的分子基础和自然发生。正常情况下，VH替代在健康供体的外周B细胞中贡献约5%的IgH库。引人注目的是，VH置换产物的频率在来源于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基因中显著升高。目前的建议集中在人未成熟B细胞中VH置换的分子调控上。我们发现交联B细胞抗原受体（BCR）强烈诱导人未成熟B细胞中的VH置换。了解VH置换的分子调控将具有重要的临床意义，因为VH置换产物的频率在自身免疫性疾病和抗病毒应答中显著升高。

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.