Naive antigen-specific T lymphocytes require stimulation via their T cell receptor (TCR) and costimulatory molecules in order to become activated, secrete cytokines, clonally expand, and mount an immune response. When TCR is ligated by antigen alone without costimulation or IL-2, T cells become anergic and are incapable of clonally expanding and transcribing the IL-2 gene. In anergic cells many of the critical signals initiated by T cell activation do not occur, whereas other signals predominate. Anergic cells have defective activation of lck, ZAP 70, Ras, ERK, JKN and Trans activation of AP-1 and NF-AT. In contrast, these cells activate fyn, increase calcium levels, and activate Rap1, indicating that induction of anergy requires active signaling events. We have recently determined that anergizing signals result in increased intracellular cAMP that upregulates the cyclin dependent kinase (cdk) inhibitor p27kipl, rendering cyclin D2 cdk4 defective and preventing progression of T cells through the Gl restriction point of the cell cycle. In contrast, CD28 costimulation prevents p27kipl accumulation by decreasing the levels of intracellular cAMP and promoting ubiquitin-dependent degradation of p27kipl. p27kipl associates with JABi, a coactivator of cJun transcription factor, resulting in its cytoplasmic translocation and defective AP-1 transactivation. Better understanding of the biochemical and molecular basis of T cell anergy will facilitate the reversal of the anergic state of tumor-specific T cells isolated from cancer patients in order to make them capable of clonal expansion prior to the administration of tumor vaccines or immunotherapy. Understanding the molecular basis of T cell anergy will also provide targets to fashion more specific treatment approaches instead of global immunosuppression to prevent graft rejection and GVHD in patients undergoing allogeneic bone marrow and organ transplantation. Although our findings strongly suggest that p27kipl functions as an anergy factor, the regulation of p27kipl expression during T cell activation and the precise mechanism by which it mediates induction and maintenance of the anergic state remain unclear. To achieve these goals I propose three specific aims: 1) to identify the biochemical modification of p27kipl in T cell immunity and anergy 2) to identify molecules that associate with p27kipl and study their role in T cell immunity and anergy, and 3) to determine the role of p27kipl in the stimulation of naive and activated antigen-specific T cells in vitro and in vivo.
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