FUNCTIONAL-CHARACTERIZATION OF NOVEL IL-2 TRANSCRIPTIONAL INHIBITORS

IL-2-mediated T cell proliferation is a critical early event in the in flammatory process. Formation of the NFAT-1 transcriptional complex on the IL-2 promoter is essential for IL-2 transcription. Using a cell l ine that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated beta-galactosidase activity. WIN 61058 and WIN 53071 were identified as mu M inhibitors. These compounds also inhibited bet a-galactosidase mRNA levels. Similar inhibition of NFAT-1-mediated gen e expression was observed in a second cell line, which is stably trans fected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 mu M, both compounds inhibited IL-2 mRNA and protein leve ls in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhi bition was reversed by exogenous IL-2. WIN 53071 inhibited IL-2 produc tion induced in the calcium-dependent PMA and ionomycin pathway. Conve rsely, calcium-independent anti-CD28 Ab and PMA-induced IL-2 productio n was resistant. Both compounds altered the NFAT-1 transcriptional com plex, causing its retarded mobility on gels. By these functional crite ria, we believe we have identified two structurally distinct, novel in hibitors oi NFAT-1-mediated transcription.