LYMPHOKINE PRODUCTION--EFFECT OF CYCLOSPORINE

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Lymphokines, the specialized products of stimulated T cells, mediate the growth and function of lymphocytes and other cell types. Lymphokine synthesis is controlled at the transcriptional level and is induced by stimulation with antigens or mitogens. The production of certain lymphokine mRNAs [IL-2, IFN-gamma, IL-4] but not the induction of other mRNAs [p55 IL-2 receptor, c-fos, a heat shock protein, IL-6, GM-CSF] is blocked selectively by the immunosuppressive drugs cyclosporin A [CSA] and FK-506. Several regulatory sequences have been identified in the promoter of the human IL-2 gene, using in vivo functional analysis and DNA-binding [electromobility shift assays EMSA]. The characterization of the factors that interact with these regulatory sites varies greatly: some have been purified and cloned while others are defined primarily by EMSA. The nuclear factor for activated T cells [NF-AT], which binds at position -289 to -269 in the 5' region of the IL-2 enhancer, remains uncharacterized. This gap is significant since NF-AT is the only known factor that is T cell restricted, requires "two signals" for induction, and is sensitive to CSA and FK-506. Other sites on the IL-2 enhancer, such as NF-KB and OCT-1, are activated by only one signal and are insensitive to CSA and FK-506. To pursue these and my preliminary findings we will: a) isolate, clone, and generate antibodies to the inducible, CSA:FK-506 sensitive factor that binds to oligonucleotide columns made with the wild-type but not mutant NF- AT site; b) similarly characterize a noninducible, CSA:FK-506 resistant modulator which was identified in the effluent of NF-AT columns and which markedly increases the activity of the NF-AT binding protein; and c) conduct a series of experiments to assess the role of these two factors in lymphokine gene expression. The latter experiments will describe the subcellular localization of the corresponding proteins and mRNA's following application of different T cell stimuli, the role of inhibitors and posttranslational modification such as tyrosine kinase dependent phosphorylation, the interaction of the two proteins with the NF-AT site as revealed by UV-crosslinking and in vitro transcription assays, the presence of comparable binding and modulator proteins in other CSA-sensitive and CSA-resistant genes, and the effects of immunosuppressive drugs on each of the above aspects of NF-AT activity.