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Figure S2. The cAMP-elevating agent forskolin is a potent inhibitor of IL-2 expression and proliferation of conventional CD4+ T cells. (A) CD4+ T cells were stimulated for 20 hours in the absence or presence of the cAMP-elevating agent forskolin. qRT-PCR for the expression of IL-2 mRNA was performed and data were normalized according to the expression of EF1alpha mRNA and expressed as percent IL-2 mRNA by setting the relative IL-2 mRNA expression of the CD4 single culture to 100%. Shown are the means +/- SD of three independent experiments each performed in triplicates. (B) CD4+ T cells were stimulated as described in (A). After 20 hours, IL-2 was analyzed in the supernatants by ELISA. One representative of three experiments performed in triplicates +/- SD is shown. n.d., not detectable. (C) CD4+ T cells were stimulated either in the presence of different amounts of forskolin or in co-culture with nTregs at variable ratios. Proliferation was measured after four days and %-inhibition of proliferation was calculated as follows: 100-[(cpm of CD4+ + Forskolin or nTregs) / (cpm of CD4+) x 100)]. (D) CD4+ T cells were stimulated with forskolin as described in (C) in the presence or absence of 10 ng/ml mrIL-2. In (C) and (D) one representative result performed in triplicates +/- SD of three independent experiments is shown.