Proliferation and phenotype of IL-2– and IL-4–cultured naive CD4⁺CD25⁺ T cells. (A) Representative MLC experiment showing proliferation of naive CD4⁺CD25⁺ T cells cultured for 4 days with self- (DA, □) or allogeneic (PVG, ■) APCs and different Th1 and Th2 cytokines. Proliferation induced by IL-2 and IL-4 was greater to allo-Ag than to self. CTL indicates cultured with no cytokine added. Data are mean plus or minus SD (n = 4). *P < .001 compared with CTL. (B) Representative fluorescence-activated cell sorter profiles showing CD4⁺CD25⁺ T cells retained CD25 and Foxp3 expression after culture. Expression of surface CD4 (Cy5), CD25 (PE), and intracellular Foxp3 (FITC) analyzed by 3-color staining. (Top panel) Naive unfractionated peripheral lymph node and spleen and freshly isolated CD4⁺CD25⁺ T cells preculture. (Middle and bottom panels) CD4⁺CD25⁺ T cells cultured with allo-Ag and either IL-2 (left) or IL-4 (right). (C) Expression of cytokines and cytokine receptors by CD4⁺CD25⁺ T cells: fresh (□) or cultured with allogeneic APCs (/PVG) with no cytokines (CTL, ▩), IL-2 (▨), or IL-4 (■) for 2 to 6 days. Representative quantitative real-time RT-PCR data expressed as ratio of specific gene copy number per 10³ copies of Gapdh. Culture with IL-2 and allo-Ag increased expression of Ifnγ and Il-5 mRNA, whereas IL-4 and allo-Ag induced IL-5Rα and enhanced IFN-γ expression. (D) Composite semiquantitative RT-PCR of mRNA from CD4⁺CD25⁺ T cells cultured for 2 to 4 days with allogeneic APCs (/PVG) and no cytokine (□), IL-2 (▨), or IL-4 (■) (n = 4-5). Y-axis shows cDNA dilutions (near to 1 in 100) after normalization for Gapdh. PCRs were at 40 cycles, except for GAPDH (30 cycles).