Class switch recombination during early B cell development

早期 B 细胞发育过程中的类别转换重组

• 批准号: 8594576
• 负责人: Amy L Kenter
• 金额: $ 22.49万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-24 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8594576
• 关键词: Accounting; Affinity; Anti-Bacterial Agents; Antibodies; Autoimmune Diseases; Autoimmunity; B-Cell Development; B-Lymphocytes; Bacterial Infections; Biological Assay; Bone Marrow; Breeding; Cell Line; Cell Ontogeny; Cells; Chromosomal translocation; Data; Development; Exons; Frequencies; Future; Gene Expression; Gene Mutation; Gene Rearrangement; Genes; Helminths; Human; Humoral Immunities; IL7 gene; IgE; Immune; Immune response; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Isotypes; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Immunologic Deficiency Syndromes; In Vitro; Infection; Injection of therapeutic agent; Laboratories; Lymphoid; Mature B-Lymphocyte; Mediating; Mus; Organ; Parasitic infection; Phase; Phenotype; Play; Process; Production; Rag1 Mouse; Regulation; Reporter; Role; Secondary to; Series; Shapes; Staging; Structure; Structure of germinal center of lymph node; Testing; Time; Transcript; Transformed Cell Line; V(D)J Recombination; activation-induced cytidine deaminase; antigen binding; base; congenital immunodeficiency; design; experience; in vivo; insight; leukemia/lymphoma; programs; public health relevance; recombinase; response; tool; tumorigenesis

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.

描述（由申请人提供）：免疫球蛋白（Ig）基因是独特的，因为它们经历三种不同类型的基因改变以实现功能齐全的体液免疫反应。在骨髓 (BM) 的早期 B 细胞发育过程中，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。从骨髓中退出后，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)，这两种情况都是由激活诱导脱氨酶 (AID) 启动的。当前的范式描述了对 BM 和 CSR 的严格分区 VDJ 重组以及对次级淋巴器官的 SHM。然而，随着时间的推移，出现了一些证据表明 CSR 和 SHM 可能在 B 细胞个体发育的早期阶段以较低的频率发生。我们开发的新数据表明，在用 IL7 扩增的离体培养物中以及在给小鼠注射 LPS 后的体内培养物中，Rag1-/- 或 Mb1-/- pro-B 细胞中可以诱导强大的 CSR。我们观察到，在加入 VDJ 或 VJ 之前的 pro-B 细胞中，CSR 诱导剂可以刺激 CSR 所必需的 AID 和种系转录物的表达。我们发现可以在一系列 Abelson 转化细胞系中诱导 CSR，然后诱导 VDJ 连接，从而为更好地分析这些观察结果提供重要工具。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 和前 B 细胞染色体易位的原因。基于这些有趣的新研究，我们建议更全面地表征早期 B 细胞发育过程中的 CSR。我们进行了体外研究，并直接表明，使用 Abelson 细胞系，VDJ 加入可以发生在经历过 CSR 的细胞中。我们建议确定在 ¿MT 小鼠中检测到的次级 Ig 同种型是否可能源自骨髓亲 B 细胞中的 CSR。 Rag1-/- 或 Mb1-/- pro-B 细胞中诱导的 AID+ 和 IgE+ pro-B 细胞的体内频率将使用两种小鼠报告品系测定。 AID+-EYFP pro-B 细胞的富集将使我们能够表征 CSR 并确定在此过程中是否选择 VDJ 库。最后，我们将确定与成熟 B 细胞中高频 T12;15 易位相关的 Igh-myc 连接片段是否可以在 AID+ pro-B 细胞中检测到。这些研究将为有关体液免疫发展和早期 B 细胞肿瘤发生的新见解奠定基础。