Fig. 7

Effect of TGFβ1 on Garp-deficient Tregs. A Basal SMAD2/3 phosphorylation and expression in freshly isolated Tregs were determined by intracellular flow cytometry. Representative staining for phosphorylated SMAD2 and SMAD3 (pSMAD2/3) and total SMAD3 in control and Garp-deficient Tregs and a summary of the results from one representative experiment out of two independent experiments with five mice per group. Horizontal lines indicate mean values. MFI mean fluorescence intensity. B The pSMAD2/3 level in purified control and Garp-deficient Tregs in response to exogenous TGFβ1 determined by intracellular flow cytometry. The results from one representative experiment and a summary of the results from one representative experiment out of two independent experiments with five mice per group are shown. Horizontal lines indicate mean values. Serum starv. serum starvation, MFI mean fluorescence intensity. C The Hdac9 mRNA level in relation to the Actb mRNA level in purified Tregs stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of TGFβ1 for 20 h analyzed by real-time PCR. The results of five independent experiments are demonstrated. Horizontal lines indicate mean values. D, E CD4⁺ T cells from control and Garp-deficient mice were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of TGFβ1 for 20 h. D Analysis of Foxp3 acetylation. Representative staining results with a FRET antibody pair specific for Foxp3 and AcK in control and Garp-deficient CD4⁺ T cells and a summary of the results of one representative experiment out of six independent FRET assay experiments with cells from two mice pooled per group. Horizontal lines indicate mean values. E Analysis of Treg stability. The frequency of Foxp3⁺ Tregs within CD4⁺ T cells cultured in the absence (left panel) or presence (right panel) of TGFβ1 was assessed by intracellular flow cytometry. CHX was added to T cell cultures after 20 h of incubation and incubated for an additional 20 h. The results of five independent experiments are summarized and shown as the mean ± SD for Treg frequency and as the results of individual experiments with the calculated mean value indicated by the line for the Treg half-life. Linear regression calculated based on normalized frequencies of Foxp3⁺ Tregs cells is shown on the left, and the half-life of Foxp3⁺ Tregs calculated based on linear regression is shown on the right