Figure 8: TGF-β signalling is involved in Lkb1-implemented T_reg cell suppressor function.

(a) Tgfbr1 and Tgfbr2 mRNA expression in Foxp3^Cre and Foxp3^CreLkb1^f/f T_reg cells by real-time PCR (n=3). (b) Intracellular expression of phosphorylated Smad2/3 in T_reg cells from Foxp3^Cre and Foxp3^CreLkb1^f/f mice, with or without TGF-β stimulation (n=3). (c) Binding capacity of Smad2/3 to recognized DNA sequences in nuclear lysates extracted from T_reg cells from Foxp3^Cre and Foxp3^CreLkb1^f/f mice. (d) CD44 and CD62L expression on CD4⁺Foxp3⁻ T cells from 6-week-old Foxp3^Cre and Foxp3^CreTgfbr2^f/f mice (n=6–11). (e) Foxp3 expression in CD4⁺ T cells from 6-week-old Foxp3^Cre and Foxp3^CreTgfbr2^f/f mice (n=6–11). (f) Suppression of proliferation of T_n cells labelled with CFSE by different ratios of CD4⁺YFP⁺ T_reg cells from Foxp3^Cre and Foxp3^CreTgfbr2^f/f mice. The experiment was repeated three times. (g) Some genes differentially expressed between CD4⁺YFP⁺ T_reg cells from Foxp3^Cre and Foxp3^CreTgfbr2^f/f T_reg cells determined by transcriptional profiling. Fold difference means fold change of gene expression levels in T_reg cells from Foxp3^CreTgfbr2^f/f mice compared with that from Foxp3^Cre mice. (h,i) Expression of indicated proteins on splenic T_reg cells from Foxp3^Cre and Foxp3^CreTgfbr2^f/f mice (n=3). (j) Suppression of proliferation of T_n cells labelled with CFSE by different ratios of CD4⁺YFP⁺ T_reg cells from Foxp3^Cre and Foxp3^CreLkb1^f/f mice that were transduced with retrovirus carrying TGF-βR2 or control vector. (k) Foxp3 expression in CD4⁺YFP⁺ T_reg cells doubly sorted from Foxp3^Cre and Foxp3^CreTgfbr2^f/f mice and transferred together with T_n cells into Rag1^−/− mice for 3 weeks. Two-way analysis of variance (ANOVA) was used for statistical analyses in (a,b,c,d,e,i) (*P<0.05, **P<0.01); error bars represent s.d.; all data are representative of at least two independent experiments.