RUI: Approaches to Somatic Immunoglobulin Diversification in Vitro

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9405257 Goldsby Diversity is the hallmark of the antibody repertoire, and somatic mutation generates significant diversity during secondary immune responses in mice and humans. The V regions of genes encoding immunoglobulin in B cells undergo rates of somatic mutation as high as one base per thousand per generation. This is an extraordinary mutation rate and corresponds to the introduction of one new immunoglobulin mutation for every two cell divisions. The mechanism underlying hypermutation remains one of the major unsolved problems of immunology. The availability of culture systems and cell lines that would permit the observation and study of this phenomenon in vitro would provide a significant expansion of our capacity to analyze this process. Because the ileal Peyer's patch (IPP) of cattle contains more than one hundred million B cells and is the site of massive, developmentally regulated, extensive, somatic mutation of Ig genes, it has been chosen as a target tissue from which to establish in vitro systems that somatically diversify immunoglobulin V genes. A variety of approaches are planned to optimize the in vitro culture of IPP B cells, including the addition of mitogens and cytokines, culture on outgrowths of IPP follicular "stromal" cells and the culture of individual IPP follicles. The assay for occurrence of somatic diversification of bovine lambda V genes will employ RT-PCR and single strand conformational polymorphism (SSCP) as an assay. The development of a system that supports the transfer of this process of somatic immunoglobulin V gene diversification from tissue to tissue culture well facilitate the analysis of the dell and molecular biology of immunoglobulin gene diversification. %%% A major component of the mammalian immune response is the ability to produce a vast array of antibodies that are capable of specifically binding a wide range of foreign substances (antigens) and facilitating their removal from the blood. The range of s pecificities that the antibodies of an individual can recognize is termed the individual's antibody repertoire. The molecular mechanism(s) by which the vast diversity of an organism's antibody repertoire is generated remains one of the central questions of modern immunology. One of the major mechanisms that generates antibody diversity is by a rapid rate of mutation in the genes that encode the foreign antigen-binding portions of antibody molecules. The overall objective of this project is to develop cell culture systems that will support the continued diversification of antibody genes. This research will take advantage of certain unique properties of the development of the immune system in sheep and cattle which suggest that this project should be feasible. If this research is successful, the availability of this culture system will greatly facilitate the analysis of the cellular and molecular mechanisms of immunoglobulin gene diversification. The research will be done at an undergraduate institution and will directly involve undergraduate students in this cutting edge problem. ***