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.
