REGULATION OF IGA B CELL ISOTYPE DIFFERENTIATION

IGA缺乏症是人类最常见的体液免疫缺陷， 大约每600人中就有1人受到影响。这一缺陷导致了 血清或粘膜分泌物中几乎完全不存在可检测到的IgA 并与感染发生率的增加和 自身免疫性疾病。以免疫球蛋白A为中枢的粘膜免疫系统 组件，代表对抗病原体的第一道防线。 遇到身体的粘膜表面。更好地理解 对免疫球蛋白A B细胞个体发育的研究不仅会增进我们对免疫球蛋白A的理解 缺陷和其他体液免疫缺陷，也是我们的能力 设计有效的口服疫苗，诱导强烈的粘膜IgA反应， 从而提供对各种病原体的有效口服免疫力， 包括艾滋病毒。这项提案的直接目标是定义 免疫球蛋白A类转换的分子机制，特别是其作用 I-α外显子和生殖系α转录本在这一过程中的作用。这 目标将通过以下具体目标实现： 1)鉴定B细胞的细胞和分子表型 最近产生的I-α外显子敲除小鼠，以便更好地定义 I-α基因座在免疫球蛋白A类转换中的作用 I-α外显子敲除小鼠中的类开关以一种方式被调节 类似于野生型小鼠；通过确定IgA类开关和 在I-α外显子敲除小鼠中IGA的表达通过经典的 机制，即通过DNA缺失重排和通过评估 插入的HPRT的构成转录或诱导转录的程度 I-α基因外显子敲除小鼠B细胞中的微量基因 该基因座转录活性与免疫球蛋白A类的关系 换个位置。2)利用基因打靶技术开发一种体外 用于确定调节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-α外显子来实现类开关 带有新霉素抗性基因的细胞，处于相同或相反的位置 转录定位作为内源性I-α外显子；通过评估 这些改变对靶向CH12.LX免疫球蛋白A类转换的影响 克隆；通过确定这些改变或转录的影响 在I-α基因座和IgA类开关上的活性； CH12.LX细胞中I-α基因座的额外改变，例如， 可诱导启动子的插入或I-α外显子的缺失 启动子，为了更准确地定义转录因子的作用 Ig A类开关中Se与I-α启动子元件的比较。3)确认 CH12.LX体内研究的体外发现 利用基因检测小鼠I-α基因座的改变或缺失 以ES细胞为靶点。如果之前对CH12.LX细胞的研究表明 I-α启动子区域在调节IgA类中起关键作用 开关，在ES的I-α轨迹上引入适当的改变 细胞，例如，删除I-α外显子启动子区域，以确认这些 活体内的发现。如果之前对CH12.LX细胞的研究表明 I-α基因上的转录调节IgA类的转换，然后 在ES细胞的I-α基因座引入适当的改变，如 在CH12.LX细胞中完成，以在体内证实这些发现。

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.