Class switch recombination during early B cell development

说明(申请人提供)：免疫球蛋白(Ig)基因是独一无二的，因为它们受到三种不同类型的基因改变的影响，以实现完全功能的体液免疫反应。在骨髓中B细胞发育的早期，V(D)J或VJ连接分别发生在IgH和L链基因上，并由RAG重组酶介导。B细胞从骨髓中走出后，迁移到次级淋巴器官，在那里它们经历了由激活诱导脱氨酶(AID)启动的体细胞超突变(SHM)和类切换重组(CSR)。目前的范例描述了一种严格的分割VDJ到BM和CSR和SHM到次级淋巴器官的重组。然而，随着时间的推移，已经出现了几条证据表明，CSR和SHM可以在B细胞个体发育的非常早期阶段以低频率发生。我们开发的新数据表明，在体外用IL7扩增的Rag1-/-或Mb1-/-Pro-B细胞和注射内毒素的小鼠体内都可以诱导出强大的CSR。我们观察到，在VDJ或VJ加入之前的PRO-B细胞中，CSR诱导剂可以刺激CSR至关重要的AID和生殖系转录物的表达。我们发现，在一系列Abelson转化的细胞系中可以诱导CSR，随后VDJ连接的诱导，从而为更好地分析这些观察到的结果提供了重要的工具。值得注意的是，我们发现AID和RAG基因的表达可以重叠，这可能是人类白血病和淋巴瘤中前B细胞和前B细胞染色体易位的主要原因。基于这些有趣的新研究，我们建议更全面地描述早期B细胞发育过程中的CSR。我们已经进行了体外研究，并直接证明了使用Abelson细胞系在经历过CSR的细胞中可以发生VDJ连接。我们建议确定在MT小鼠中检测到的次级Ig亚型是否来自骨髓Pro-B细胞中的CSR。在Rag1-/-或Mb1-/-Pro-B细胞中诱导的AID和IgE Pro-B细胞的体内频率将用两个小鼠报告株来测定。AID-EYFP PRO-B细胞的丰富将使我们能够表征CSR并确定是否在这一过程中选择了VDJ谱系。最后，我们将确定成熟B细胞中与高频T12；15易位相关的IgH-myc连接片段是否在AID Pro-B细胞中可检测到。这些研究将形成对体液免疫发展和早期B细胞肿瘤发生的新见解的基础。 公共卫生相关性：骨髓中B淋巴细胞的发育以DNA重排为特征，称为VDJ连接，导致IgM抗体的表达。基于新的证据表明，在加入VDJ之前，可能会发生从IgM到其他免疫球蛋白同型的转换，我们将研究抗体基因重排的时间顺序及其与免疫缺陷和自身免疫性疾病的关系。

DESCRIPTION (provided by applicant): Immunoglobulin (Ig) genes are unique in that they are subject to three different types of gene alterations to achieve a fully functional humoral immune response. During early B cell development in the bone marrow (BM), V(D)J or VJ joining occurs on the IgH and L chain genes, respectively and is mediated by the RAG recombinase. Following exit from the bone marrow, B cells migrate to the secondary lymphoid organs where they undergo somatic hypermutation (SHM) and class switch recombination (CSR) that is initiated in both cases by activation induced deaminase (AID). Current paradigms describe a strict partition VDJ recombination to the BM and CSR and SHM to the secondary lymphoid organs. However, several lines of evidence have emerged over time suggesting that CSR and SHM can occur at low frequency at very early stages of B cell ontogeny. We have developed new data showing that robust CSR can be induced in Rag1-/- or Mb1-/- pro-B cells in ex vivo cultures expanded with IL7 and in vivo following injection of mice with LPS. We observe that in pro-B cells prior to VDJ or VJ joining, CSR inducers can stimulate the expression of AID and germline transcripts that are critically required for CSR. We find that CSR can be induced in a series of Abelson transformed cell lines followed by the induction of VDJ joining, thereby providing important tools to better analyze these observations. Strikingly, we find that AID and Rag gene expression can overlap which may account for the bulk of pro-B and pre-B cell chromosomal translocations in human leukemias and lymphomas. Based on these intriguing new studies we propose to more fully characterize CSR during early B cell development. We have carried out in vitro studies and show directly that VDJ joining can occur in CSR experienced cells using Abelson cell lines. We propose to determine whether secondary Ig isotypes detected in the ¿MT mouse may derive from CSR in bone marrow pro-B cells. The in vivo frequency of AID+ and IgE+ pro-B cells induced in Rag1-/- or Mb1-/- pro- B cells will be determined using two mouse reporter strains. Enrichment of AID+-EYFP pro-B cells will enable us to characterize CSR and to determine whether VDJ repertoires are selected in this process. Finally we will determine whether Igh-myc junction fragments associated with high frequency T12;15 translocations in mature B cells are detectable in AID+ pro-B cells. These studies will form the basis for new insights regarding development of humoral immunity and early B cell oncogenesis. Public Health Relevance: B lymphocyte development in the bone marrow is characterized by DNA rearrangements termed VDJ joining, leading to expression of IgM antibodies. Based on new evidence that a switch from IgM to other immunoglobulin isotypes can occur prior to VDJ joining we will study the temporal ordering of antibody gene rearrangements and their relationship to immunodeficiency and autoimmune diseases.