The purpose of the proposed research is to gain knowledge regarding the function of the ecto enzyme, 5 prime-nucleotidase (ecto-5 prime-NT) on human B and T cells. The proposal is divided into three related projects. First, we will develop methods to separate ecto-5 prime-NT+ from ecto-5 prime-NT- lymphocytes. A monoclonal antibody will be produced after immunization of mice with 5 prime-NT purified from human placenta. Using anti-ecto-5 prime-NT antibodies, human peripheral T and B cells will be separated into ecto-5 prime-NT+ and ecto-5 prime-NT- populations by cell sorting, panning, complement mediated lysis of ecto-5 prime-NT+ cells, and/or rosetting. In the second project, we will conduct basic studies of purine metabolism in ecto-5 prime-NT+ and ecto-5 prime-NT-T cells. The ability of these two populations to proliferate in response to mitogens will be compared in purine-free medium (in which the cells must meet their purine requirements by de novo synthesis), in medium supplemented with hypoxathine (which can meet all purine requirements via salvage), and in medium supplemented with inosine 5 prime-monophosphate (which can meet all purine requirements via salvage only after conversion to inosine by ecto-5 prime-NT). We will learn whether the presence or absence of ecto-5 prime-NT activity influences the routes by which human T cells meet their purine requirements and whether ecto-5 prime-NT lymphocytes might have a selective advantage over ecto-5 prime-NT-lymphocytes in specific microenvironments such as the thymus and bone marrow where there is considerable cell death and purines might be salvaged from exogenous nucleotides. Finally, we will compare the functional capabilities of ecto-5 prime-NT+ vs. ecto-5-NT-T and B cells. The results of these experiments in combination with those of the second project will reveal whether specifc patterns of purine metabolism are adapted for specific types of lymphocyte functions; i.e., those which require proliferation as compared to those which do not. The purified populations will also be characterized for the co-expression of other cell surface antigens using commercially available monoclonal antibodies to determine whether the expression of ecto-5 prime-NT activity, in conjunction with other cell surface markers, can be used to define functional subsets of human T and B lymphocytes. Our goal is to understand the significance of the reduced numbers of ecto-5 prime-NT+ lymphocytes (and increased numbers of ecto-5 prime-NT- lymphocytes) in patients with immunodeficiency diseases.
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