Figure 4

Generation of CD4⁺ CD25⁺Foxp3⁺ Treg in the presence of HCCsp. A, Nonadherent PBMCs were stimulated with unirradiated DCs/allo-HCC in the absence of HCCsp (right panel) or unirradiated DCs/allo-HCC/sp in the presence of HCCsp (left panel). On day 4, T cells were purified, cultured, and analyzed by 3-color flow cytometry for expression of CD4, CD25, and Foxp3. Three different populations; a) CD4⁺CD25^high T cells; b) CD4⁺CD25^low T cells; c) CD4⁺CD25^- T cells were gated to analyze Foxp3 expression. Numbers represent cells positively staining for the indicated surface markers. Similar results were obtained in three individual experiments. B, Nonadherent PBMCs from three healthy donors were stimulated with unirradiated DCs/allo-HCC in the absence or presence of HCCsp. Naive PBMCs from three healthy donors were also cultured in the absence or presence of HCCsp. CD4⁺ T cells were gated to analyze CD25^high Foxp3⁺ expression and the percentage of CD25^highFoxp3⁺ in CD4⁺ population was shown. For each group, the mean ± SD is shown. *, Significant differences.