Suppressive function of DC-induced Treg's. (A) T cells were cultured with DCs for 7 days, and the cocultures were labeled with CD25 PE and CD11c FITC. Cell sorting was used to obtain CD25^high and CD25^- fractions of CD11c^- cells. The plot shows the postsort purity of the fractions used as suppressors in the MLR. (B) Suppression of the allogeneic MLR by Treg's induced by mature DCs. Mature DCs (from the same donor as used to generate the CD25⁺ FOXP3⁺ T cells) were cultured with CFSE labeled allogeneic responder T cells at a ratio of 1:10. CD25⁺ suppressors (middle panel) and CD25^- cells (bottom panel) obtained via cell sorting were added to the cultures at suppressor-responder ratios of 1:3 to 1:30. Three to 4 days later, flow cytometry was performed to determine the proliferation in the cultures. Top panel shows control MLR without any cells added. (C) FOXP3⁺ Treg's were generated by culturing T cells with either Cyt-DCs or FcγRIIB blockade-matured DCs (RIIB-DC). Seven days later, DC-expanded T cells were subjected to cell-sorting as in panel A, and CD25⁺ and CD25^- populations were obtained and used as suppressors. The MLR was set up with mature DCs (stimulators) and CFSE-labeled allogeneic T cells as responders as in panel B. The sorted CD25⁺ and CD25^- T cells were added to compare the suppressive ability of the Treg's generated by DCs matured by 2 different maturation stimuli. (D) Bulk CD3⁺ T cells and sorted CD3⁺ CD25^- T cells were cocultured with Cyt-DCs. After 7 days of coculture, T cells from both conditions were flow sorted to obtain CD25^high as well as CD25^- T cells as described in panel A. The CD25⁺ and CD25^- cells were used as suppressors in an MLR. The MLR was set up with mature DCs and CFSE-labeled allogeneic T cells as responders as in panel B, and the CD25⁺ and CD25^- populations were added to compare the suppressive ability of the Treg's generated from either bulk T cells or CD25^- T cells (de novo Treg's).