Phenotype of IL-2–JES6-1–expanded T reg cells. Mice were treated on days 0, 1, and 2 with 1 µg/5 µg IL-2–JES6-1, 1 µg IL-2, or PBS, and the phenotype of the expanded T reg cells was analyzed on day 3 or 7, as described. (A) Splenic CD4⁺CD25⁺Foxp3⁺ T reg cells were analyzed on day 3 by flow cytometry for expression of cell-surface molecules, CD25, GITR, TGF-β, ICOS, CD44, CD103, ICAM-1, and PD-1, and intracellular molecules, Foxp3, and CTLA-4. (B) Quantification of IL-10 and TGF-β mRNA after in vitro restimulation of FACS-sorted CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells isolated from mice on day 3 after treatment (reference gene, 18s RNA). (C) In vitro suppression of CD4⁺CD25⁻ T cells (effectors) by FACS-sorted CD4⁺CD25⁺ T reg cells (suppressors; in indicated ratios) isolated from mice on day 3 or 7 after treatment. Proliferation of effectors was measured by incorporation of [³H]thymidine. (D) In vivo suppression of homeostatic proliferation of CD4⁺CD25⁻ Thy1.1⁺ T cells adoptively transferred into RAG^−/− hosts in a 1:1 ratio with CD4⁺CD25⁺ T reg cells isolated from C57BL/6 mice on day 3 after treatment. Thy1.1⁺ cell numbers were determined on day 7 after transfer. Data are representative of two to three independent experiments. Data are means ± SEM. Flow cytometry plots depict log₁₀ fluorescence.