During the past five years, progress in understanding the signal transduction pathways initiated by the antigen receptor of T cells has resulted in a near molecule-by molecule description of events that bring information from the T cell membrane to the nucleus. During this same period, studies of lymphoid development have indicted that differences in TCR avidity for MHC complexed to self antigens determine the opposing cell fates of positive and negative selection nd hence shape the immune repertoire. These studies imply that signal intensity determines the two cell fates underlying positive and negative selection. This work has been paralleled by developmental studies of morphogens, such as activin and Hedgehog, that also imply that differences in signal intensity determine cell fates. These developments have set the stage for understanding a fundamental problem in signal transduction: How do different ligand concentrations (in the case of developmental morphogens), or differential avidity (in the case of the TCR) determine different cell fates? Over the next five years, we will attempt to answer this question by first filling essential gaps in our knowledge of the T cell signaling pathways. We will begin by defining the mechanism governing the function of the members of the NF-ATc family of transcription factors which respond to TCR signals in both developing and adult lymphocytes by rapid and reversible nuclear translocation. New members will be cloned, monoclonal antibodies produced, and characterized. These antibodies will be used to develop in situ assays of the cellular interactions that lead to NF-AT nuclear localization as a result of antigen receptor signaling. Since NF-AT is controlled by nuclear localization, we will define the mechanism underlying the calcineurin-dependent, cyclosporin-sensitive cytosolic import of NF-ATc family members. We will examine the means by which the vav protooncogene participates in the calcineurin-dependent functions of NF-ATc. The developmental functions of NF-ATc family members will be defined by studies of mice bearing introduced mutations and children with SCID or common variable immunodeficiency. In the final years of the granting period, we anticipate that the efforts of ourselves and others will have provided a near complete description of signal transduction in T lymphocytes, providing the opportunity to define the mechanisms by which graded signals are converted to discrete outcomes. Hence, we will reconstitute regulatory points in T cell signaling by gene replacement with alleles that can be activated in living animals using synthetic ligands that we have recently developed. Using the latter approach, we will test the hypothesis that differential signaling underlies positive and negative selection, and if correct, define this mechanism by an in vivo reconstitution. Understanding the biochemical mechanisms underlying signal transduction is essential to prevent or treat autoimmune disease and for the design of better, less toxic drugs to modulate the immune response to transplanted organs.
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