Supporting Information
Figure S1  Phenotype of In Vitro Differentiated T Cells
After two round of differentiation cultures, T cells were stimulated by plate-immobilized anti-CD3/CD28 and 3H-thymidine incorporation as measurement of proliferation was analyzed after 3 d of culture (A). In parallel, T cells were analyzed for Treg relevant surface receptor expression as indicated on the x-axis (B).
(1.0 MB AI). Click here for additional data file.   Figure S2  In Vivo Treatment of Mice with IL-4 Antibody–Cytokine Complexes
B6 mice were given every other day ip injections of phosphate-buffered saline (PBS), recombinant mouse IL-4 (rmIL-4), anti-IL-4 mAb (anti-IL-4 mAb, 11B11, or MAB404), or a mixture of rmIL-4 plus anti-IL-4 mAbs (11B11 or MAB404). Mice were analyzed on day 7 by flow cytometry for CD3, CD4, and CD25 expression. Shown is CD25 versus CD4 expression in CD3+ CD4+ spleen cells (A–F). Numbers indicate percentages of CD4+ CD25high CD3+ cells. Total cell counts (G) of CD4+ CD25high cells in spleen from mice in (A–F) are shown as mean ± SD. The data are representative of three independent experiments.
(369 KB AI). Click here for additional data file.   Figure S3  Effect of IL-4 on Already Existing Natural or Inducible Treg Cells
(A) CD4+CD25high nTreg cells were FACS-sorted and activated with plate-bound anti-CD3/CD28 plus IL-2 during 3 d and in the presence or absence of IL-4 (100 ng/ml) and harvested for real-time PCR analysis. The results shown represent the mean ± SD of three independent experiments.
(B) iTreg cells were induced in vitro. FOXP3 espression was assessed by real-time PCR analysis in resting cells, in cells re-stimulated with plate-bound anti-CD3/CD28, with or without TGF-β, plus IL-2 during 3 d and in the presence (black bar) or absence (white bar) of IL-4 (100 ng/ml).
(C) Activation dramatically increases CD4+CD25+ Treg cells suppressive capacity of CD4+CD25+ nTreg cells. CD4+CD25+ nTreg cells were preactivated during 2 d in the presence or absence of an increasing IL-4 concentration. After vigorous washing, their suppressive capacity on responder CD4+CD25− was tested. IL-4 pretreatment did not affect the suppressive capacity of FACS-sorted CD4+CD25high cells. 1 × 104 CD4+CD25+ nTreg cells were added to 5 × 104 CD4+CD25– and 5 × 104 irradiated PBMCs. The results are representative of three independent experiments.
(269 KB AI). Click here for additional data file.   Figure S4  Schematic Structure of the FOXP3 Gene and Location of the GATA3 Sites
The scheme shows the location of the 11 exons spaced by a large intron (6000 bp) from the 5′untranslated region (UTR). Human, murine, and rat sequences are aligned and transcription start site (TSS) is indicated with an arrow.
(529 KB AI). Click here for additional data file.