Fishing for CD4 positive cells in the zebrafish. (43.28)

Abstract

Abstract Differentiation of naïve CD4+ T-cells into functionally distinct subsets is essential for an effective immune response against different types of pathogens. The recent discovery of CD4 like molecules in teleost fish and many of the genes important in adaptive immunity, now gives us the opportunity to study the role of T-cells in lower vertebrate immune responses. Despite these discoveries the lack of molecular tools, such as antibodies, in organisms other than mammals has made this difficult to do. Due to this we have begun to develop a number of polyclonal and monoclonal antibodies to immune genes in zebrafish. Zebrafish has become a popular model to study immunity and is being used by several laboratories worldwide to study disease. Using the zebrafish genome we have discovered a locus that contains four CD4 like genes, which is different to the two discovered in other teleost genomes to date. Although related to other vertebrate CD4 molecules, these genes exhibit low identity to each other. Homology modelling of these genes identifies only one of these to be similar to mammalian CD4, as it is the only one predicted to contain 4 immunoglobulin domains. Using a peptide approach we have developed a polyclonal antibody towards this CD4 molecule and have begun using it to study CD4+ cells in the zebrafish using FACS and immunofluorescence analysis. In addition, we have looked by realtime PCR at the expression of all 4 genes in embryos and healthy or infected adult zebrafish.