Figure 5.

The conversion of Foxp3⁺Tregs from CD4⁺CD25⁻T cells through coculture with isolated splenic CD11b⁺IDO⁺DCs. (a) CD4⁺CD25⁻T cells (10⁵) stimulated with mDCs (2 × 10⁴) were cocultured for 4 days with isolated splenic CD11b⁺DCs (10⁴) with or without 1-MT pretreatment. The cells were surface-stained with anti-CD4 mAbs, followed by intracellular staining with anti-Foxp3 mAbs to determine the frequency of CD4⁺Foxp3⁺Tregs. The dates are presented using a dot plot, expressed as the % positive cells. The results are representative of three experiments showing similar results. (b) After cocultivation for 4 days, as described in (a), CD11b⁺DC-induced CD4⁺CD25⁺T cells were isolated and used as inhibitors at different doses to suppress the proliferation of new CD4⁺CD25⁻T cells in response to stimulation with anti-CD3/28 mAbs. The suppressive activity of freshly isolated CD4⁺CD25⁺T cells was also examined as a control. The proliferative responses were measured after 4 days. The data are representative of three experiments with similar results, reported as the means ± SEM. ^# P > 0.05 compared with the indicated groups using unpaired t-tests. (c) CD4⁺T cells from the spleens of CIA mice treated with CD11b⁺DC with or without 1-MT on the third week after the onset of arthritis were collected and intracellularly stained with anti-Foxp3 and anti-IL-17A mAbs. FACS was used to measure the percent frequency of positively stained cells, and the frequencies of Treg/Th17 cells are expressed as the means ± SEM of four independent experiments. ^# P > 0.05, ^* P < 0.05 compared with the indicated groups using unpaired t-tests.