IDO-competent DCs, even when not suppressing the allogeneic T-cell response, induce regulatory activity in allogeneic CD4⁺ T cells. (A) Differently activated DCs were used as stimulators of an MLR as in Figure 3C. At the end of the coculture T cells were recovered and were examined for CD25, CTLA-4, and FOXP3 expression (shaded histograms indicate CD4⁺CD25⁻ T cells; open histograms, CD4⁺CD25⁺ T cells). Results of 1 experiment, representative of 4 experiments are shown. (B) CD4⁺ T cells were recovered from MLRs, in which the differently activated DCs were used as stimulators as in panel A, and were sorted by flow cytometry into a CD4⁺CD25⁻ and a CD4⁺CD25⁺ population (left). These separate T-cell populations were subsequently cocultured for 5 days with fresh autologous T cells (1:1; suppressor assay). The latter were stained with CFSE. The proliferative responses of these T cells to stimulation with immobilized anti-CD3 mAb were assessed as the percentage of CFSE-negative cells. One representative experiment is shown. (C) Cumulative results: indicates the median percentage of CFSE-negative T cells only without (left) or with anti-CD3–mediated stimulation (right); , median percentage of CFSE-negative T cells cocultured with CD4⁺CD25⁻ T cells retrieved from MLRs stimulated by differently activated DCs, as indicated; , median percentage of CFSE-negative fresh T cells cocultured with CD4⁺CD25⁺ T cells retrieved from the same MLRs. Error bars indicate the range of experimental results of 15 consecutively tested donor/responder pairs. **P < .01. (D) DCs were activated with LPS/IFN-γ for 48 hours and used as stimulators of an MLR as in panel A, in the absence (left) or presence (right) of MTHT. After the MLR, the CD4⁺ T cells were recovered, sorted, and subjected to suppressor assays as in panel B. Proliferative responses are shown by CFSE dilution of fresh T cells (open histograms with thick black line) on coculture with the sorted autologous CD4⁺CD25⁻ (top) and CD4⁺CD25⁺ (bottom) T cells. Filled gray histograms indicate CFSE dilution of anti-CD3–stimulated fresh T cell only; open histograms with thin line, CFSE dilution of unstimulated fresh T cells only. The numbers indicate the percentage of CFSE-negative cells in suppressor assays. One representative experiment is shown. (E) Cumulative results: indicates the median percentage of CFSE-negative fresh T cells only in the absence (left) or presence (right) of stimulation with anti-CD3 mAb (controls); , median percentage of CFSE-negative fresh T cells after coculture with CD4⁺CD25⁺ T cells retrieved from MLRs stimulated by DCs activated with LPS/IFN-γ for 4 hours or for 48 hours, as indicated, in the absence or presence of the IDO inhibitor MTHT. Error bars indicate the range of a total of 6 consecutive experiments. *P < .05.