Mature T cells with autoreactive specificities will, at times, encounter self-antigens in the peripheral tissues. tolerance may be maintained, despite this, because such T cells can be induced into a state of functional inactivation (clonal anergy). In this state, T cells are incapable of mounting a proliferative response upon re-exposure to antigen. an examination of the biochemistry underlying the development of anergy could serve to answer several fundamental questions related to the control of T-cell proliferation in peripheral tolerance--namely, a) how to antigen receptor-mediated signals induce clonal anergy, b) how dow an accessory cell-derived costimulatory signal block this induction of anergy, and c) what is the molecular basis for the functional unresponsiveness observed in the anergic state? This study will investigate these questions using an in vitro model of cloned murine Th1 T cells that are induces into an anergic state by antigen-receptor stimulation in the absence of an adequate accessory cell-derived costimulatory signal. Specifically, the study will examine the role of protein phosphorylation in the development of clonal anergy. Phosphorylation experiments will be performed to characterize the level of phosphorylation on a number of well-defined T-cell phosphoproteins immunoprecipitated with specific antibodies, in order to determine a relationship between protein phosphorylation and the development of anergy. newly phosphorylated proteins will be detected by a) in situ labelling of 32P-orthophosphate loaded intact T cells, b) in vitro labelling of immunoprecipitates with [gamma-32P] ATP in a kinase reaction, or c) transfer to nitrocellulose and western blotting with an antiphosphotyrosine antiserum. Other experiments will examine protein phosphorylations occurring in total cell lysates using a 2-dimensional gel electrophoresis analysis. Finally, novel phosphoproteins detected in this investigation will be biochemically characterized by a combination of 2-dimensional PAGE, phosphopeptide mapping, phospho-amino acid analysis, and carbohydrate analysis. Because anergy represents one of the major potential mechanisms for producing peripheral tolerance at the level of the T cell, information gained in these studies could further our understanding of the pathogenesis of autoimmune disease, as well a s provide a rationale for the development of protocols for the induction of antigen-specific tolerance to allogeneic tissue grafts.
