Expansion of FOXP3⁺ Treg's by DCs from patients with myeloma. (A) CD14^- cells were used as the source of T cells and either cultured alone or with autologous cytokine-matured DCs. Seven days later, FOXP3⁺ CD4⁺ T-cell numbers were examined by flow cytometry and compared with those seen in fresh blood at the time of blood draw. Numbers represent percentage of cells in that quadrant. (B) Results show mean (±SD) FOXP3⁺ CD4⁺ T-cell expansions from 3 different patients. Left panel shows expansion of CD4⁺ FOXP3⁺ T cells at the time of blood draw (day 0), and after 7 days of culturing T cells alone (T alone) and with cytokine matured DCs (T + Cyt-DCs). The right panel shows the geometric mean fluorescence of the FOXP3 expression in CD4⁺ FOXP3⁺ T cells before and after culture. (C) CD25⁺ and CD25^- T cells were sorted after 7 days of culture with cytokine-matured DCs as described in Figure 5. An MLR was set up using patient DCs as stimulators and CFSE-labeled allogeneic T cells as responder cells at a 1:10 ratio. Patient CD25⁺ and CD25^- cells were added as suppressors at a ratio of 1:3 to 1:30. Four days later, flow cytometry was performed to determine the proliferation of the CFSE-labeled responder cells. Figure represents 1 of 2 similar experiments. (D) Cytokine-matured DCs expand FOXP3⁺ CD4⁺ T cells in vivo. Flow cytometry was performed to examine FOXP3 expression in T cells before and after injection of cytokine-matured DCs in 4 patients. (E) The kinetics of FOXP3 expansion was examined in 3 patients who showed an increase in FOXP3⁺ CD4⁺ T cells. T cells were obtained before injection, 1 to 7 days after, and 28 days after injection of cytokine-matured DCs.