The broad goals of this proposal are to determine the roles for integrin receptors in the development and function of T-lymphocytes. A combination of in vivo and in vitro approaches have been chosen. In order to test the role of integrin receptor function in T cell development, transgenic mice have been produced with a dominant negative form of integrin. Specifically, we are using a construct with the extracellular domain of the Tac antigen connected to the active integrin beta1 cytoplasmic domain, which has been shown by us and many other laboratories to interfere with integrin function in tissue culture cells. The proximal lck promoter has been chosen to direct thymic specific expression of this construct in transgenic mice, leading to an increase in the proportion and number of thymocyte precursors, suggesting a block in the normal differentiation of CD4- CD8- (so called double negative cells (DN)). We now show that the Tac-beta1 construct is acting as a transdominant inhibitor of integrin activation in the DN population. We specifically show that there is a reduction in the expression of an activation epitope in the integrin alpha4beta1. Our hypothesis is that this construct has decreased the activation of a number of integrin receptors, particularly in the DN population, whose integrins are normally the most active in the thymus. Independently, we will test the role of integrins in the differentiation of DN to DP cells using antibody and peptide perturbation experiments, performed in fetal thymic organ culture (FTOC), in which embryonic day 14 thymic lobes are isolated and cultured for several days, during which the thymocytes emerge from a completely DN population and differentiate into DP. Lastly, we will test the Tac-beta1 construct as a transdominant inhibitor of integrin activation via the T cell receptor in cultured mouse TH2 cells. If this is successful, then using a modified CD4 promoter, we will express Tac-beta1 at high levels in the peripheral T cells of transgenic mice to determine the role of integrin activation in T cell function.
