Class switch recombination during early B cell development

描述（由申请方提供）：免疫球蛋白（IG）基因的独特之处在于，它们经历三种不同类型的基因改变以实现全功能的体液免疫应答。在骨髓（BM）中的早期B细胞发育期间，V（D）J或VJ连接分别发生在IgH和L链基因上，并且由RAG重组酶介导。从骨髓中排出后，B细胞迁移到次级淋巴器官，在那里它们经历体细胞超突变（SHM）和类别转换重组（CSR），在这两种情况下都是由活化诱导的脱氨酶（AID）引发的。目前的范例描述了一个严格的分区VDJ重组的BM和CSR和SHM的次级淋巴器官。然而，随着时间的推移出现了几条证据，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。我们已经开发了新的数据，显示在用IL 7扩增的离体培养物中的Rag 1-/-或Mb 1-/- pro-B细胞中以及在用LPS注射小鼠后的体内可以诱导稳健的CSR。我们观察到在VDJ或VJ加入前的pro-B细胞中，CSR诱导剂可以刺激AID和生殖系转录物的表达，这是CSR所必需的。我们发现，CSR可以在一系列Abelson转化细胞系中诱导，然后诱导VDJ加入，从而为更好地分析这些观察结果提供了重要工具。引人注目的是，我们发现AID和Rag基因表达可以重叠，这可能是人类白血病和淋巴瘤中大部分pro-B和pre-B细胞染色体易位的原因。基于这些有趣的新研究，我们建议在早期B细胞发育过程中更全面地表征CSR。我们已经进行了体外研究，并直接表明，VDJ连接可以发生在CSR经验的细胞使用Abelson细胞系。我们建议确定在MT小鼠中检测到的次级IG同种型是否可能来自骨髓pro-B细胞中的CSR。将使用两种小鼠报告菌株测定在Rag 1-/-或Mb 1-/- pro- B细胞中诱导的AID+和IgE+ pro-B细胞的体内频率。AID+-EYFP pro-B细胞的富集将使我们能够表征CSR并确定在该过程中是否选择了VDJ库。最后，我们将确定与成熟B细胞中的高频率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。这些研究将形成关于体液免疫和早期B细胞肿瘤发生的新见解的基础。 公共卫生相关性：骨髓中的B淋巴细胞发育的特征在于称为VDJ连接的DNA重排，导致IgM抗体的表达。基于新的证据表明，从IgM到其他免疫球蛋白同种型的开关可以发生在VDJ加入之前，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.