The induction of regulatory activity by FK778 is critically dependent on inhibition of pyrimidine synthesis, and CD4⁺CD25⁻ T cells treated with FK778 show impaired p27^kip1 degradation and enhanced STAT3 phosphorylation. (A) Exogenous uridine was added to primary cultures to restore the pyrimidine pool in the presence of FK778. At day 7, the cells were harvested and subsequently restimulated in a secondary MLC in the absence of FK778 and uridine. The restimulation pattern of CD4⁺CD25⁻ T cells primed in the presence (○) or absence (•) of FK778 alone (left panel) or in combination with exogenous uridine (right panel). One representative experiment of 2 is presented. (B) The suppressive capacity of CD4⁺CD25⁻ T cells primed in the presence of FK778 alone (○) or combined FK778 and uridine (•) was analyzed in coculture suppression assays. ³H incorporation was measured at day 6. (C) p27^kip1 degradation in FK778-treated CD4⁺CD25⁻ T cells. Western blot analysis of p27^kip1 expression in CD4⁺CD25⁻ T cells stimulated with anti-CD3/anti-CD28–coated beads in the presence or absence of 50 μg/mL FK778. One representative experiment of 2 is presented. (D) Phosphorylation of STAT3 was analyzed in cell lysates using a bead-based multiplex assay. The level of fluorescence, as marker for phosphorylation, is shown for the CD25^intermediate and the CD25^high cell populations of the untreated control (left panel) and the FK778-treated setting (right panel). One representative experiment of 2 is presented. *P < .05; **P < .01 for differences between untreated and FK778-treated cells. In panels A and B, error bars represent SD counts.