Enrichment of Foxp3⁺ cells in the IFN-γ protocol involves IL-2-dependent proliferation and conversion from Foxp3^– precursors. (A) CBA CD25⁻ cells were flow sorted, CFSE labeled and stimulated with B10 GM-CSF/TGF-β-differentiated BM DC in the presence of IFN-γ. Cells were harvested at day 3 to give a surrogate zero time point (CFSE intensity precluded analysis at day 0) or restimulated under the same conditions on day 7, harvested on day 14 and stained for Foxp3. (B) Purified naive CBA CD4⁺ T cells were co-cultured with B10 GM-CSF/TGF-β-differentiated BM DC in the presence of 5 ng/mL IFN-γ without or with 1 μg/mL anti-IL2 antibody (JES6-1A12), restimulated under the same conditions on day 7, harvested on day 14 and stained for CD25 and Foxp3. (C) FACS-sorted Foxp3/GFP^–CD4⁺ T cells from naive Foxp3-GFP knock-in B6 mice were co-cultured with DBA/2 GM-CSF/TGF-β-differentiated BM DC in the presence of IFN-γ (5 ng/mL). Cells were restimulated on day 7 under the same conditions, harvested on day 14, and intracellular Foxp3/GFP expression was examined in the resultant population. In parallel, cells were stimulated in the absence of IFN-γ but the presence of anti-IFN-γ antibody. Data are representative of two independent experiments. Numbers in each dot plot indicate the frequency of cells in the quadrants shown. (D) Purified naive CBA CD4⁺ T cells were cultured with GM-CSF- and TGF-β-differentiated B10 BM DC (GT-DC) in the absence (left panel) or presence (right panel) of 5 ng/mL IFN-γ. Cells were harvested on days 3 and 7 (before restimulation on day 7) and on days 10 and 14 (after restimulation), stained for annexin V, washed, then fixed for analysis of intracellular Foxp3 expression. Numbers in each dot plot indicate the frequency of cells in each quadrant. Data are representative of four independent experiments. The histogram shows an analysis (means±SD) of four independent experiments gated on Foxp3^– and Foxp3⁺ cells; p values were calculated with the Mann–Whitney test.

The ex vivo IFN-γ-conditioning protocol extinguishes Th2 and Th17 responses. Upper panel: Purified naive CBA CD4⁺ T cells were co-cultured with B10 GM-CSF differentiated BM DC (G-DC) without additional cytokines (neutral conditions). Middle panel: Purified naive CBA CD4⁺ T cells were co-cultured with B10 GM-CSF/TGF-β-differentiated BM DC (GT-DC) under neutral conditions. Lower panel: Purified naive CBA CD4⁺ T cells were co-cultured with B10 GM-CSF/TGF-β-differentiated BM DC in the presence of IFN-γ (5 ng/mL). Cells were restimulated under the same conditions on day 7, harvested on day 14, treated for 4 h with PMA and ionomycin, and stained for intracellular IFN-γ, IL-4 and IL-17 expression. Plots are gated on T cells and numbers in the dot plots indicate the frequency of cells in each region. Data are representative of two to three independent experiments.

IFN-dependent selection of Foxp3⁺ cells is mediated via T-cell STAT1 signaling. (A) Purified naive wild-type (WT) 129 CD4⁺ T cells were co-cultured with wild-type B6 GM-CSF/TGF-β-differentiated BM DC in the absence or presence of IFN-γ (5 ng/mL). (B) Purified naive STAT1 knockout (KO) 129 CD4⁺ T cells were co-cultured with IFN-γ receptor knockout (IFNGR KO) B6 GM-CSF/TGF-β-differentiated BM DC in the absence or presence of IFN-γ (5 ng/mL). (C) Purified naive wild-type (WT) 129 CD4⁺ T cells were co-cultured with IFN-γ receptor knockout (IFNGR KO) B6 GM-CSF/TGF-β-differentiated BM DC in the absence or presence of IFN-γ (5 ng/mL). (D) Purified naive STAT1 knockout (KO) 129 CD4⁺ T cells were co-cultured with wild-type (WT) B6 GM-CSF/TGF-β-differentiated BM DC in the absence or presence of IFN-γ (5 ng/mL). Cells were restimulated on day 7, harvested on day 14, and intracellular Foxp3 expression was analyzed. Numbers in each dot plot indicate the frequency of cells in the quadrant; data are representative of two to three independent experiments. (E) Purified naive wild-type (WT) 129 CD4⁺ T cells were co-cultured with GM-CSF/TGF-β-differentiated CBA BM DC in the absence or presence of IFN-γ (0.5–50 ng/mL). (F) Purified naive STAT1 knockout (KO) 129 CD4⁺ T cells were co-cultured with GM-CSF/TGF-β-differentiated CBA BM DC in the absence or presence of IFN-γ (0.5–50 ng/mL). Cells were restimulated on day 7, harvested on day 14, and intracellular Foxp3 expression was analyzed.

IFN-γ-conditioning results in NO-mediated Foxp3 upregulation. (A) Purified naive CBA CD4⁺ T cells were co-cultured with GM-CSF/TGF-β-differentiated B10 BM DC+5ng/mL IFN-γ in the absence or presence of the NOS inhibitor l-NMMA (0.1–1.0 mM). Cells were restimulated on day 7, harvested on day 14 and stained for TCR-β and intracellular Foxp3 expression. (B) Purified naive CBA CD4⁺ T cells were co-cultured with GM-CSF/TGF-β-differentiated B10 BM DC in the absence or presence of the NO donor SNAP (0.1–0.3 mM). No exogenous IFN-γ was added. Cells were restimulated on day 7, harvested on day 14 and stained for TCR-β and intracellular Foxp3 expression. Figures in representative dot plots indicate the frequency of Foxp3⁺ cells and histograms show means and SD of three independent experiments.

Alloreactive Foxp3⁺ T cells emerge in the presence of exogenous IFN-γ. (A) Purified naive CBA CD4⁺ T cells were co-cultured with B10 GM-CSF/TGF-β-differentiated BM DC without exogenous cytokine (neutral conditions) or in the presence of IFN-γ (5 ng/mL), restimulated under the same conditions on day 7 and harvested on day 14 for phenotypic and functional analysis. (B) Harvested populations were stained for TCR-β and Foxp3. Dot plots show representative data at 5 ng/mL IFN-γ, and the right-hand histogram shows IFN-γ dose–response data (means+SD of three independent experiments). (C) A single culture of CD4⁺ T cells driven by B10 GM-CSF/TGF-β-differentiated BM DC in the presence of IFN-γ was assayed for Foxp3 expression on day 7 after a single round of stimulation and on day 14 after restimulation on day 7. (D) Adoptive transfer protocol. All CBA-Rag^−/− mice were reconstituted with 1×10⁵ CD25⁻CD4⁺ cells from naive CBA mice as an effector population with or without ex vivo conditioned CD4 T cells (Tcon). The reconstituted mice then received a B10 skin graft the following day. Mice reconstituted with 10⁵ CD25⁻CD4⁺ cells alone acutely rejected B10 skin grafts (○; MST=22 days, n=4). Co-transfer of 2×10⁵ CD4 T cells conditioned with IFN-γ prevented rejection of B10 skin grafts (▪; MST>100 days, n=4; p<0.05, ▪ versus ○), whereas cotransfer of 2×10⁵ CD4 T cells conditioned without IFN-γ did not prevent rejection (▴; MST=19 days, n=4; p>0.05, ▴ versus ○). Data are representative of three independent experiments with similar group sizes.

Expression of IL-6 has a negative impact on IFN-dependent Treg development. (A) BM cells were cultured in the presence of 2 ng/mL GM-CSF with (GT-DC) or without (G-DC) 2 ng/mL TGF-β, harvested at day 6 and stimulated in the absence of T cells with recombinant CD40L for 24 h to mimic reverse stimulation via CD40-CD40L interactions. IL-6 RT-PCR data are normalized to a housekeeping gene GAPDH, and are the means±SD of two independent experiments (left panel). Supernatants were collected for IL-6 ELISA analysis and results normalized to 4×10⁵ input cells in 2 mL medium (right panel). (B) Purified naive CBA CD4⁺ T cells were co-cultured with wild-type B6 (WT B6) or IL-6 knockout B6 (IL-6 KO B6) GM-CSF/TGF-β-differentiated BM DC in the presence of IFN-γ (5 ng/mL). Cells were restimulated on day 7 under the same conditions, harvested on day 14, and intracellular Foxp3 expression was analyzed. Numbers in each dot plot indicate the frequency of cells in the quadrant. Data are representative of two independent experiments.

Enrichment of Foxp3⁺ cells depends on exogenous IFN-γ and endogenous TGF-β. (A) BM cells were cultured in the presence of 2 ng/mL GM-CSF with (GT-DC) or without (G-DC) 2 ng/mL TGF-β, harvested at day 6 and stimulated in the absence of T cells with recombinant CD40L for 24 h to mimic reverse stimulation by CD40–CD40L interactions. TGF-β RT-PCR data are normalized to the housekeeping gene GAPDH and are means±SD of two independent experiments (left panel). TGF-β production in supernatants was determined by ELISA and results were normalized to 4×10⁵ input cells in 2 mL medium (right panel). (B) Upper panel: Purified naive CBA CD4⁺ T cells were co-cultured with B10 GM-CSF/TGF-β-differentiated bone marrow DC in the presence of 5 ng/mL IFN-γ alone, or with IFN-γ+SB 431542 (10 mM), or anti-TGF-β antibody (10 mg/mL). Lower panel: Purified naive CBA CD4⁺ T cells were co-cultured with B10 GM-CSF/TGF-β-differentiated BM DC in the presence of TGF-β (2 ng/mL), without or with SB 431542 (10 mM). Cells were restimulated on day 7 under the same conditions, harvested on day 14, and intracellular Foxp3 expression was analyzed. Numbers in each dot plot indicate the frequency of cells in the quadrant. Data are representative of two independent experiments.

Non-redundant role for STAT1 signalling in the reciprocal development of regulatory and Th17 cells ex vivo. Purified naive CD4⁺ T cells from wild-type 129 mice (A) or STAT1 knockout 129 mice (B) were co-cultured with B6 GM-CSF/TGF-β-differentiated BM DC in the absence or presence of IFN-γ (5 ng/mL), or TGF-β (2 ng/mL). Cells were restimulated under the same conditions on day 7, harvested on day 14, treated for 4 h with PMA and ionomycin, and analyzed for intracellular Foxp3 and IL-17 expression in gated (TCR-β⁺) T cells. Figures in representative dot plots indicate the frequency of cells in each quadrant and summary histograms show means and SD of three independent experiments.