Class switch recombination during early B cell development

描述（由申请人提供）：免疫球蛋白（Ig）基因是独特的，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B细胞发育早期（BM）， 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。离开骨髓后，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）和类开关重组（CSR），这两种情况都是由激活诱导脱氨酶（AID）启动的。目前的范式描述了一个严格的分割VDJ重组到BM和CSR和SHM到次要淋巴器官。然而，随着时间的推移，一些证据表明CSR和SHM可以在B细胞个体发生的早期阶段以低频率发生。我们已经开发了新的数据，表明在体外用IL7扩增的Rag1-/-或Mb1-/- pro-B细胞中，以及在小鼠体内注射LPS后，可以诱导出强大的CSR。我们观察到，在VDJ或VJ加入前的前b细胞中，CSR诱导剂可以刺激CSR所需的AID和种系转录本的表达。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的工具。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连接可以发生在使用Abelson细胞系的CSR经历细胞中。我们打算确定在¿MT小鼠中检测到的次生Ig同型是否可能来自骨髓前b细胞中的CSR。Rag1-/-或Mb1-/- pro-B细胞诱导AID+和IgE+ pro-B细胞的体内频率将通过两种小鼠报告菌株进行测定。AID+-EYFP前b细胞的富集将使我们能够表征CSR并确定VDJ谱是否在这一过程中被选择。最后，我们将确定high -myc结片段是否与高频T12相关；成熟B细胞中的15个易位在AID+前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细胞肿瘤发生发展的新见解的基础。

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.