Fig. 4
Hp-TGM induces T cell Foxp3 expression even in proinflammatory conditions. a, b CD4+CD25–GFP–CD62Lhi murine naive T cells were stimulated with plate-bound anti-CD3/CD28 for 4 days in culture with 100 U/ml IL-2 and variable concentrations of Hp-TGM or hTGF-β1, before flow cytometric analysis of CD4, CD25 and Foxp3 expression; 2 technical replicates per concentration; representative of 4 independent experiments;. a: percentage of CD25+Foxp3+ cells among total CD4+ cells; b, Mean fluorescence intensity (MFI) of Foxp3 among Foxp3+ cells. Gating strategy is shown in Supplementary Fig. 6. c–e Foxp3 induction in the same conditions as a, in the presence of SB431542 inhibitor c or pan-vertebrate anti-TGF-β d, e; 2 technical replicates per concentration; representative of 3 independent experiments. Analysis by multiple t tests showed no significant difference in responses to TGM when anti-TGF-β was present, but signicant effects (p < 0.01) on responses to all concentrations of TGF-β. f, g Magnetically sorted murine CD4+ T cells were cultured for 5 days with Th17-promoting ligands (100 ng/ml IL-6, 5 ng/ml IL-23, 10 ug anti-IFN-γ), together with the indicated concentrations of Hp-TGM or TGF-β, then stimulated with PMA/ionomycin in the presence of Brefeldin A for 4.5 h before staining for intracellular Foxp3 and IL-17. Data are mean ± SEM of triplicate replicates from 1 of 2 independent experiments; data were analysed by multiple t tests which showed no significant difference at any concentration of TGF-β v TGM for percentage IL-17+, and p < 0.001 and p < 0.01, respectively for 10 and 50 ng/ml TGM v TGF-β for percentage Foxp3+. Gating strategy is shown in Supplementary Fig. 6. h, i Murine CD4+ T cells were cultured as above with but with Th9-promoting ligands (40 ng/ml IL-4, 20 ng/ml IL-2), and assayed as above after 4 days of culture by staining for intracellular Foxp3 and IL-9. Data are mean ± SEM of three replicates from 1 of 2 independent experiments; data were analysed by multiple t tests which showed p < 0.05 for 1 ng/ml TGF-β v TGM for percentage IL-9+, and p < 0.001 for 10, 50, and 100 ng/ml TGM v TGF-β for percentage Foxp3+. Gating strategy is shown in Supplementary Fig. 6
