FK778 treatment results in the induction of regulatory function in CD4⁺CD25⁻ T cells independent of CD4⁺CD25⁺ regulatory T cells. (A) CD25⁺ cells were depleted from total CD4⁺ T cells (top panel) using magnetic cell isolation; the protocol included 2 passages. Typically, the resultant CD25⁻ population (bottom panel) was more than 99% pure. (B) CD4⁺CD25⁻ T cells (5 × 10⁴) were stimulated with allogeneic stimulator cells (5 × 10⁴) in the presence or absence of 50 μg/mL FK778 for 7 days. Viable cells were recovered by density gradient centrifugation. Proliferation of recovered T cells after treatment with 0 μg/mL (♦), 10 μg/mL (▵), or 50 μg/mL (○) FK778 during the primary MLC, was examined in a secondary MLC in the absence (left panel) or presence (right panel) of 12.5 U/mL IL-2. (C) The suppressive capacity was investigated in a coculture assay. At day 6, proliferation (y-axis) of allogeneic stimulated naive CD4⁺CD25⁻ responder T cells (5 × 10⁴) was studied in the absence or presence of increasing numbers of untreated (•) or FK778 (50 μg/mL)–treated (○) cells. One representative experiment of 5 is presented. (D) The inhibitory effect on proliferation by the FK778-treated cells was confirmed by CFSE labeling (x-axis) of the responder cells. The percentage of divided cells was assessed at day 6 of coculture assays and is depicted in each plot. One of 2 independent experiments is shown. (E) The potential of FK778-treated cells to inhibit the production of the effector cytokine IFN-γ, produced by responder cells, was analyzed in supernatants of coculture assays by ELISA. At day 5, the concentration of IFN-γ (y-axis) is shown at increasing numbers of untreated (•) or FK778-treated (○) cells. (F) Cell-cell contact dependency of the FK778-treated cells to mediate suppression was analyzed in transwell coculture assays. The FK778-treated cells were added either directly to the responder cells in the lower compartment (○) or, to disrupt cell-cell contact, to the upper transwell chamber (•). ³H incorporation (y-axis) was measured at day 6. (G) The suppressive capacity of anti-CD3/anti-CD28–stimulated CD4⁺CD25⁻ T cells that were primed in the presence (○) or absence (•) of FK778. One representative experiment of 3 is shown. *P < .05; **P < 0.01 for differences between untreated and FK778-treated cells. In panels B, C, E, and G, error bars represent SD counts.