T lymphocytes capable of responding to virtually any antigen on invading pathogens or transplanted tissue are produced in the thymus as a result of selection and intrathymic differentiation. These cells migrate to the peripheral lymphoid organs and upon encountering foreign antigen undergo an orderly sequence of events that results in proliferation and acquisition of immunologic function. During this process they coordinate the activity of other cells involved in the immune response by controlling cell fate decisions and proliferation of B cells, macrophages and granulocytes through the production of cytokines and cell-cell interactions. We propose to explore the pathways used by the T cell antigen receptor to communicate with genes necessary for immunologic activation. Most of our attention will be directed toward the least understood part of the pathway which connects Ca2+-dependent events and tyrosine kinase substrates to the activation of certain genes, such as IL-2 that are critical for proliferation. A comprehensive and unbiased genetic approach has been devised to produce mutations in the proteins carrying signals from the antigen receptor to the IL-2 gene. Cells having these mutations will be placed into complementation groups by analysis of heterokaryons formed between the different clones. Each complementation group will be characterized to localize the site of action of the defective protein. Genes able to complement the mutant phenotype will be cloned by transfecting cDNA expression libraries into the mutants and rescuing plasmids able to restore signal transmission. This genetic approach will be enhanced by an analysis of the nuclear events essential for IL-2 gene activation using an in vitro transcription system developed in our laboratory that faithfully reflects the complex requirements for the activation of the IL-2 gene. We will use this system to identify proteins (kinases, proteases, etc) necessary for the activation of two transcription factors, OAP (Oct-1 Associated Protein) and NF-AT (Nuclear Factor of Activated T cells), which are required for IL-2 gene activation and specifically activated by triggering the antigen receptor. By continuing this process of working backward from proteins essential IL-2 gene activation we expect, by the end of the 5 year granting period, to define the processes used by the antigen receptor to communicate with genes in the nucleus.
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