CD8⁺ T cells cocultured with MPA-DCs had no regulatory capacity in vitro. Purified CD8⁺ T cells were cocultured for 6 days with allogeneic MPA-DCs. (A) CD8⁺ MPA-DCs did not suppress the proliferation of responder CD4⁺ T cells. At day 6, CD8⁺ MPA-DCs were tested for their suppressive activity in MLR. Responder CD4⁺ T cells (designated naive CD4⁺) were cocultured with allogeneic mature DCs for 5 days at a DC:T ratio of 1:3 and were used as positive control. CD8⁺ MPA-DCs were added to the MLR at different ratios. T-cell proliferation was assessed by 3H-thymidine incorporation during the last 18 hours of the 5 day MLR (mean cpm ± SD of triplicate wells). Data are representative of 1 of 6 experiments. (B) CD8⁺ MPA-DCs did not inhibit the secretion of IFN-γ in MLR. At the end of the second MLR, the level of IFN-γ (picograms per milliliter) was detected by ELISA in supernatants. Each point represents 1 experiment and the black bars represent the mean of 5 independent experiments. Wilcoxon test was used to calculate statistical differences between groups. *P < .05 and NS = Not Significant. (C) CD8⁺ MPA-DCs did not inhibit intracellular IFN-γ expression of CD4⁺ T cells. Responder CD4⁺ T cells were cocultured with allogeneic mDC in the presence (right dot plot) or absence (left dot plot) of CD8⁺ MPA-DCs at a ratio of 1:1 for 5 days. At the end of coculture, cells were harvested and CD4 FITC/IFN-γ APC double staining was assessed by FACS analysis for the 2 conditions. The percentage of IFN-γ-positive cells among CD4-positive cells is shown in panel C. Results are representative of 1 of 4 independent experiments.