This grant proposes continued studies on the organization and expression of IgA heavy chain genes in rabbit. The studies will focus on two major areas, (1) to investigate the molecular genetic basis for trans-IgA molecules in which the V/H region of the alpha-heavy chains is encoded by a V/H gene from one chromosome and the C/a region is encoded by a C/a gene from the homologous chromosome, and (2) to examine how the expression of C/a genes in mucosal tissues is regulated. The molecular genetic basis for the trans IgA heavy chains will be determined by developing IgA- secreting rabbit X rabbit hybridomas from heterozygous rabbits and cloning their VDJ and C/a genes. From restriction mapping and nucleotide sequence analysis of these genes one can determine whether the trans molecules result from interchromosomal DNA recombination or from trans-splicing. These studies will be the first to clearly demonstrate that either interchromosomal recombination or trans-splicing can occur in mammals, in vivo. Rabbits have 13 non-allelic C/a genes and thus potentially, 13 different IgA subclasses, or isotypes. The multiple isotypes are differentially expressed in various mucosal tissues, making this mucosal immune system the most complex and perhaps the most interesting of any studied. By RNase protection assays, most of the 13 C/a genes are known to be expressed in gut-associated lymphoid tissue, but in lung and tonsil of the respiratory tract, only one C/a gene, C/a4 is expressed. These studies will be extended to investigate the mechanism responsible for the differential expression and to determine at the protein level whether IgA is differentially expressed in different parts of the gut, correlating with potential differences in the microbial flora.
The specific aims for analyzing C/a gene expression are (1) to clone the remainder of the C/a chromosomal region and search for 3'alpha enhancer regions, (2) to test whether the differential expression of IgA among tissues is due to tissue- specific isotype switching, by searching for germline 1/a-C/a transcripts and IgA secreted by IgM+IgA- cells following activation with T cells/factors from gut or respiratory tract, in order to test whether the differential expression of IgA among tissues is due to tissue-specific isotype switching, and (3) to prepare isotype-specific monoclonal antibodies and perform immunofluorescence on plasma cells in gut from the duodenum to the colon. The studies proposed are important not only because they will shed light on the regulation of expression of a complex multi-gene family but also because they will begin to identify factors that result in isotype- specific induction of IgA in mucosal tissues. The importance of understanding how immunity of mucosal surfaces is regulated cannot be overestimated because of its role as the primary immunologic barrier against external pathogens.
