CD25⁺ cells are dispensable for the differentiation of Tr cells by immature DCs. (A) Levels of CD25 expression on peripheral blood CD4⁺CD45RO^– or CD4⁺CD45RO^–CD25^– T cells were analyzed by flow cytometry. (B) Subsequently, CD4⁺CD45RO^– or CD4⁺CD45RO^–CD25^– T cells were stimulated with immature allogeneic DCs. At the end of 3 rounds of activation, T cells were collected and tested for their ability to suppress responses of autologous CD4⁺ T cells. Thawed CD4⁺ T cells were stimulated with mature DCs alone (MLR) or in the presence of a 1:1 ratio of T_(imm) or CD25^– T_(imm) cells. [³H]-Thymidine was added at the indicated time for an additional 16 hours. (C) T_(imm) cell lines were compared with T_(mat) cell lines for the expression of mRNA for FoxP3. Relative levels of FoxP3 expression were determined by quantitative real-time PCR. The amounts of FoxP3 mRNA are expressed as relative to CD4⁺CD25^– T cells (which were given an arbitrary value of 1). mRNA from freshly isolated CD4⁺CD25⁺ Tr cells was used as positive control. Results are representative of 3 independent experiments.