REGULATION OF IGA B CELL ISOTYPE DIFFERENTIATION

伊加缺乏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 血清或粘膜分泌物中几乎完全不存在可检测到的伊加 并且与感染的发病率增加有关， 自身免疫性疾病 粘膜免疫系统，其中伊加是中枢免疫系统。 组成部分，代表了抵御病原体的第一道防线 接触身体的粘膜表面。 更好地理解 对伊加B细胞个体发育的研究不仅可以加深我们对伊加 缺乏和其他体液免疫缺陷，而且我们的能力， 设计诱导强烈粘膜伊加应答的有效口服疫苗， 从而提供针对各种病原体的有效口服免疫， 包括艾滋病毒。 这项建议的直接目标是界定 参与伊加类转换的分子机制，特别是 I-α外显子和生殖细胞系α转录本在这个过程中的作用。 这 将通过以下具体目标实现这一目标： 1)表征来自以下的B细胞的细胞和分子表型： 最近产生的I-alpha外显子敲除小鼠，以便更好地定义 I-α基因座在伊加类别转换中的作用， I-alpha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;通过确定伊加类转换和 I-α外显子敲除小鼠的伊加表达通过经典的免疫调节机制发生。 机制，即，通过DNA缺失重排和评估 插入的HPRT的组成性或诱导性转录的程度 在I-α外显子敲除小鼠的B细胞中存在的小基因， 该位点的转录活性与伊加类之间的关系 开关. 2)利用基因靶向技术开发体外 用于定义调节伊加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外显子进行类别转换 具有新霉素抗性基因的细胞，无论是在相同或相反的 转录方向作为内源性I-α外显子;通过评估 这些改变对靶向CH12.LX中伊加类别转换的影响 克隆;通过确定这些改变或转录的影响， 在I-α基因座和伊加类别转换的活性;以及通过使 在CH 12.LX细胞中I-α基因座的另外的改变，例如， 插入诱导型启动子或缺失I-α外显子 启动子，为了更精确地定义转录的作用， se与I-α启动子元件在伊加类别转换中的作用。 3)确认在 CH12.LX体内研究的体外结果，通过引入适当的 使用基因在小鼠中I-α基因座的改变或缺失 ES细胞的靶向。 如果先前在CH12.LX细胞中的研究表明 I-α启动子区域在调节伊加类中起关键作用， 开关，在ES的I-α位点引入适当的改变 细胞，例如，删除I-α外显子启动子区域，以证实这些 体内发现。 如果先前在CH12.LX细胞中的研究表明， I-α基因座的转录调节伊加类别转换， 在ES细胞的I-α位点引入适当的改变， LX细胞中进行，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.