The rejection of vascularized organ allografts is initiated and sustained by T lymphocytes. Alloreactive T cells develop in the thymus, where they must successfully complete both positive and negative selection prior to maturation and release into the peripheral lymphoid compartment. Although selection appears to be based on the T cell receptor (TCR) specificity of a thymocyte, the mechanisms underlying this selective elimination or promotion of immature T cells are not fully understood, and the goals of this proposal are to investigate the role of accessory molecules in these processes. First, the role of signals transduced by the CD4 and CD8 molecules will be studied. CD4 and CD8 can augment TCR/CD3-induced signals in mature T cells, and signals delivered through TCR/CD3 have been shown to lead to programmed cell death in immature CD4+CD8+ (double positive) thymocytes. It is thus proposed that co-stimulation of CD4 and/or CD8 can enhance the process of TCR/CD3-induced cell death. Double positive thymocytes will be isolated and stimulated with heteroconjugate antibodies to crosslink TCR/CD3 to itself, to CD4, or to CD8. The influence of co- ligation of CD4 or CD8 on programmed cell death will be assessed by electron microscopy and by propidium iodide exclusion as measured by flow cytometry. The role of tyrosine phosphorylation in this process will be determined using pharmacologic inhibitors of tyrosine kinases, whose activity will be monitored by immunoblotting. Signals delivered via another accessory molecule, CD28, will next be examined. CD28 stimulation is a potent co-mitogen for both mature and CD4+CD8+ thymocytes, and it is hypothesized that stimulation of CD28 rescues CD4+CD8+ cells from TCR/CD3- induced programmed cell death. We will examine the requisite timing of CD28 costimulation and the intrathymic distribution of BB-1/B7, a natural ligand for CD28. We will also explore additional possibilities which may regulate the response to the thymocyte to TCR/CD3 stimulation (i.e., positive or negative selection). These include TCR/CD3 density, use of the zeta-zeta or zeta-eta dimer, and CD45 isoform expression. Finally, we will examine whether signals transduced by the CD28 pathway are required for in vivo thymocyte development. for these studies, soluble recombinant CD28 protein will be injected into both balb/c and nude mice in order to block the interaction of native CD28 with its ligand, and the effect of this manipulation on thymic and extrathymic T cell development will be assessed. These studies should further define the role of the CD4, CD8, and CD28 accessory molecules in thymic selection. Ultimately this should lead to a clearer understanding of the alloimmune response and may help develop ways to manipulate it.
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