PMC

Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4⁺ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector. Bars show the mean luciferase activity ± SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4⁺ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene. Bars show the mean luciferase activity ± SD measured as arbitrary light units of three independent experiments.

RUNX1 and RUNX3 expression in Foxp3⁺ T reg cells and in human CD4⁺, CD127⁻, CD25^high cells. (A) Real-time PCR analysis of CD4⁺, CD127⁻, and CD25^high T reg cells and CD4⁺, CD127⁺, and CD25^neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-β, FOXP3, and RUNX3 mRNA in CD25⁺ compared with CD25⁻ cells. Bars show the mean ± SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy. Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls. HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining. Data shown are representative from one of the three tissue samples with similar results. Bars, 5 µm. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4⁺ T cells after alone or combined anti-CD2/3/28 mAb and TGF-β stimulation in the presence of IL-2. Real-time PCR of human naive CD4⁺ T cells after 48 h of culture. Bars show the mean ± SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-β in human naive CD4⁺ T cells is reduced after siRNA-mediated RUNX1/3 knockdown. Real-time PCR of RNA from human naive CD4⁺ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-β and IL-2. Bars show the mean ± SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3. Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4⁺ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d. Bars show the mean ± SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown. Human naive CD4⁺ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-β in the presence of IL-2. CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h. One of three independent experiments is shown. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3⁺ CD4⁺ T cells. (A) FACS-purified naive CD4⁺ CD8⁻ T cells from Cbfb^F/F CD4-cre and control Cbfb^F/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, ± 10 nM retinoic acid (RA), and increasing concentrations of TGF-β. After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-γ expression. One of five experiments is shown. (B) Naive CD4⁺ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 × 10⁶ cells per transfer). 6 wk later, TCRβ⁺CD4⁺ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression. Results from one of four Cbfb^F/F CD4-cre and control Cbfb^F/+ CD4-cre mice with same findings are shown. The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.

Cbfb^F/F CD4-cre mouse cells and iT reg cells generated from human naive CD4⁺ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4⁺8⁻ T cells from Cbfb^F/F CD4-cre (left) and control Cbfb^F/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-β. After 3 d, Foxp3-GFP⁺ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1⁺ CD4⁺ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1⁺ cells were analyzed for CFSE dilution. (C) As a control, Cbfb^F/+ CD4-cre CD4⁺ T cells activated in absence of TGF-β were mixed with CFSE-loaded naive CD45.1⁺ CD4⁺ cells at the indicated ratios. Four days later CD45.1⁺ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1⁺ CD4⁺ cells in the cultures containing Foxp3-GFP⁺ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4⁺ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4⁺ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4⁺ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4⁺ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown.

Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4⁺ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean ± SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P < 0.05