Fig. 6

Let-7i transfection decreases expression of IGF1R and TGFBR1 on CD4⁺ T cells. a Induction of Treg cells in the presence of IGF1, TGFβ and IL-2. Naive CD4⁺ T cells were cultured in the presence of various combinations of IGF1 (10 ng/mL), TGFβ (1 ng/mL) and IL-2 (50 U/mL) under stimulation with anti-CD3 and anti-CD28 mAbs for 72 h. The frequency of IFN-γ⁻IL-17A⁻Foxp3⁺CD4⁺ Treg cells among CD4⁺ T cells was determined. b T cells were transfected with let-7i and then cultured under stimulation with anti-CD3 and anti-CD28 mAbs for 72 h. Representative histograms of the expression levels of IGF1R and TGFBR1 are shown. The expression levels of IGF1R and TGFBR1 were determined by measuring the frequency of each receptor-positive cells among CD4⁺ T cells and the MFI of each receptor on CD4⁺ T cells. c Naive CD4⁺ T cells were transfected with siRNAs targeting TGFBR1 and IGF1R as indicated, and then differentiated towards Treg cells with TGFβ (1 ng/mL) and IL-2 (50 U/mL) in addition to stimulation with anti-CD3 and anti-CD28 mAbs for 72 h. The frequency of Treg cells was evaluated. Data are representative of two independent experiments. n = 3 a or 4 b and c in each group. An unpaired t test was used in a and b, and a one-way ANOVA with Dunnett’s comparison test was used in c for statistical analysis. Error bars represent the mean ± s.e.m. **p < 0.01, ***p < 0.001. s.e.m. standard error of the mean, A.U. arbitrary unit, MFI mean fluorescence intensity, ANOVA analysis of variance, IGF1R insulin like growth factor 1 receptor, TGFBR1 transforming growth factor beta receptor 1