REGULATION OF IGA B CELL ISOTYPE DIFFERENTIATION

IGA B 细胞同种型分化的调节

• 批准号: 2068498
• 负责人: GREGORY R HARRIMAN
• 金额: $ 20.81万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2068498
• 关键词: B lymphocyte; animal genetic material tag; antibody formation; cell differentiation; clone cells; embryonic stem cell; gene rearrangement; gene targeting; genetic transcription; immunoglobulin A; immunoglobulin genes; laboratory mouse; polymerase chain reaction; southern blotting; tissue /cell culture; transfection

IgA deficiency is the most common humoral immunodeficiency in humans, affecting approximately 1 in 600 people. This deficiency results in the virtual complete absence of detectable IgA in serum or mucosal secretions and is associated with an increased incidence of infections and autoimmune disease. The mucosal immune system, of which IgA is a central component, represent the first line of defense against pathogens encountering the mucosal surfaces of the body. A better understanding of IgA B cell ontogeny would enhance not only our understanding of IgA deficiency and other humoral immunodeficiencies, but also our ability to design effective oral vaccines which induce strong mucosal IgA responses, thereby providing effective oral immunity against various pathogens, including HIV. The immediate objective of this proposal is to define the molecular mechanisms involved in IgA class switch, particularly the role of I-alpha exons and germline alpha transcripts in this process. This objective will be achieved through the following specific aims: 1) Characterize the cellular and molecular phenotype of B cells from recently generated I-alpha exon knockout mice, so as to better define the role of the I-alpha locus in IgA class switch by determining whether IgA class switch in I-alpha exon knock-out mice is regulated in a manner analogous to wild type mice; by determining whether IgA class switch and IgA expression in I-alpha exon knock-out mice occurs through classical mechanisms, i.e., through DNA deletional rearrangement and by assessing the extent of constitutive or induced transcription by the inserted hprt minigene occurring in B cells from I-alpha exon knockout mice and the relationship between transcriptional activity at this locus and IgA class switch. 2) Utilize gene-targeting techniques to develop an in vitro model system for defining the molecular mechanisms which regulate IgA class switch by replacing the I-alpha exon on both alleles in CH12.LX cells with a neomycin resistance gene, either in the same or opposite transcriptional orientation as the endogenous I-alpha exon; by assessing the effect of these alterations on IgA class switch in targeted CH12.LX clones; by determining the effect of these alterations or transcriptional activity at the I-alpha locus and IgA class switch; and by making additional alterations at the I-alpha locus in CH12.LX cells, e.g., insertion of an inducible promoter or deletion of the I-alpha exon promoter, in order to more precisely define the role of transcription per se versus I-alpha promoter elements in IgA class switch. 3) Confirm in vitro findings of CH12.LX studies in vivo by introducing appropriate alterations or deletions at the I-alpha locus in mice using gene targeting of ES cells. If preceding studies in CH12.LX cells suggest that the I-alpha promoter region plays a key role in regulating IgA class switch, introduce appropriate alterations at the I-alpha locus of ES cells, e.g., deleting the I-alpha exon promoter region, to confirm these findings in vivo. If preceding studies in CH12.LX cells suggest that transcription at the I-alpha locus regulates IgA class switch, then introduce appropriate alterations at the I-alpha locus of ES cells, as done in CH12.LX cells, to confirm these findings in vivo.

IgA 缺乏症是人类最常见的体液免疫缺陷， 影响大约六百分之一的人。 这一缺陷导致 血清或粘膜分泌物中几乎完全不存在可检测到的 IgA 并与感染发生率增加有关 自身免疫性疾病。 以IgA为核心的粘膜免疫系统 成分，代表抵御病原体的第一道防线 接触身体的粘膜表面。 更好的理解 IgA B 细胞个体发育的研究不仅会增强我们对 IgA 的理解 缺乏和其他体液免疫缺陷，还有我们的能力 设计有效的口服疫苗，诱导强烈的粘膜 IgA 反应， 从而提供针对各种病原体的有效口腔免疫力， 包括艾滋病毒。 该提案的直接目标是定义 IgA 类别转换涉及的分子机制，特别是作用 在此过程中，I-α 外显子和种系 α 转录本的相关性。 这 目标将通过以下具体目标来实现： 1) 表征 B 细胞的细胞和分子表型 最近生成了I-α外显子敲除小鼠，以便更好地定义 通过确定 IgA 是否存在来研究 I-α 基因座在 IgA 类别转换中的作用 I-α 外显子敲除小鼠中的类别转换以某种方式受到调节 类似于野生型小鼠；通过确定 IgA 类别是否转换以及 I-α 外显子敲除小鼠中的 IgA 表达通过经典方法发生 机制，即通过 DNA 缺失重排和评估 插入的 hprt 组成型或诱导转录的程度 存在于 I-α 外显子敲除小鼠 B 细胞中的小基因 该位点的转录活性与 IgA 类别之间的关系 转变。 2）利用基因打靶技术开发体外 定义调节 IgA 的分子机制的模型系统 通过替换 CH12.LX 中两个等位基因上的 I-alpha 外显子来进行类别转换 具有新霉素抗性基因的细胞，无论是相同的还是相反的 转录方向为内源性 I-α 外显子；通过评估 这些改变对目标 CH12.LX 中 IgA 类别转换的影响 克隆；通过确定这些改变或转录的影响 I-alpha 基因座和 IgA 类别转换的活性；并通过使 CH12.LX 细胞中 I-α 基因座的其他改变，例如， 插入诱导型启动子或删除 I-α 外显子 启动子，以便更精确地定义转录的作用 Se 与 IgA 类开关中的 I-α 启动子元件。 3) 确认 CH12.LX 的体外研究结果通过引入适当的体内研究 使用基因对小鼠 I-α 基因座进行改变或缺失 ES细胞的靶向。 如果之前对 CH12.LX 细胞的研究表明 I-α启动子区在调节IgA类别中起着关键作用 开关，在 ES 的 I-alpha 基因座上引入适当的改变 细胞，例如，删除 I-α 外显子启动子区域，以确认这些 体内发现。 如果之前对 CH12.LX 细胞的研究表明 I-alpha 基因座的转录调节 IgA 类别转换，然后 在 ES 细胞的 I-α 基因座上引入适当的改变，如 在 CH12.LX 细胞中进行，以在体内证实这些发现。