FIG. 6.

dnNFAT inhibits IL-2 production. (A) The activity of the IL-2 promoter is inhibited by dnNFAT. Jurkat T cells were cotransfected with an IL-2 promoter-luciferase reporter plasmid and various amounts (1 and 10 μg) of dnNFAT expression vector. The effect of mutation of the PxIxIT motif by replacement of the Pro, Ile, and Thr residues with Ala (AxAxAA) was investigated. Luciferase activity was measured in cultures incubated without (−) or with (+) ionomycin (2 μM) and PMA (100 nM) (I+P). The data are presented as fold activation compared to an untreated control. (B) IL-2 secretion is inhibited by dnNFAT. Jurkat T cells were cotransfected with expression vectors for GFP and dnNFAT (either wild-type PxIxIT or mutated AxAxAA). Transfected cells expressing GFP were selected by flow cytometry and treated without (Untreated) or with ionomycin (2 μM) and PMA (100 nM) (I+P), and the amount of IL-2 secreted in the culture medium was measured. (C) IL-2 expression is inhibited by dnNFAT. Jurkat T cells were cotransfected without (Control) and with expression vectors for GFP and dnNFAT (either wild-type PxIxIT or mutated AxAxAA). The cells were treated without (thin line) or with (thick line) ionomycin (2 μM) and PMA (100 nM) (I+P). The intracellular IL-2 and GFP was measured by flow cytometry. IL-2 expression (mean fluorescence intensity) of the transfected GFP positive (+) and untransfected GFP negative (−) cells in each culture is shown. (D) Thymocytes from dnNFAT transgenic mice have reduced IL-2 expression. Thymocytes were isolated from dnNFAT transgenic mice (Tg+) and control nontransgenic littermates (NLC). Expression of dnNFAT was detected by protein immunoblot analysis using MAb M2, specific to the Flag epitope. Cells were stimulated with ionomycin (2 μM) and PMA (100 nM), and the amount of IL-2 secreted in the culture medium was measured. hGH, human growth hormone.