The release of lymphokines like IL-2 and interferon-Gamma from T lymphocytes is controlled at the level of gene transcription. The induction of lymphokine mRNA has been monitored with an oocyte translation assay and with northern blotting. Primary populations of mouse and human T lymphoblasts, which release large amounts of lymphokine upon restimulation with lectin, have been used throughout. Application of cyclosporin A (CsA) blocks the production of lymphokine mRNA, but the induction of several other genes (c-fos, IL-2 receptor, heat shock protein) is not inhibited. Since these genes are induced by the same agents (lectin, phorbol esters, OKT3 antibody) as lymphokine genes, CSA probably does not inhibit signalling at the cell surface. This grant will continue to study the control of lymphokine release and the effects of CsA. The selectivity of CsA for lymphokine gene activity will be tested with: a large panel of DNA probes, unprimed and freshly sensitized T lymphocytes, and nuclear transcription and northern blotting assays. The function of accessory cells in lumphokine release will be monitored at the level of gene transcription. Different kinds of accessory cells (dendritic cells, macrophages, B lymphocytes) and different populations of responding T cells (resting vs. lymphoblasts; T4 vs. T8) will be tested. The possibility that accessory function can be replaced by phorbol esters, and that accessory cells can be directly inhibited by CsA, will be considered. The mechanism of action of CsA will be studied in two principal experiments. The preliminary observation that IL-2 can increase interferon mRNA will be pursued, since the increase is CsA-resistant. Conditions for the induction of interferon by IL-2 will be optimized and then evaluated on isolated nuclei to test if IL-2 stimulated cells exhibit CsA-insensitive, interferon gene transcription. Direct CsA-DNA interactions will be evaluated using footprinting assays on DNA segments obtained from the 5 flanking region of lymphokine genes. DNA binding regions should be protected from DNAase I and methidiumpropyl EDTA sensitivity. Nonimmunosuppressive analogues of CsA, provided by Sandoz, will be used as controls in this and all other experiments. Should these experiments indicate that CsA does not act directly on lymphokine genes, experiments will be initiated to identify proteins that are pertinent to lymphokine transcription.
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