Fig. 1: Characterization of CD4⁺Foxp3⁺CD69⁺ Tregs.

a Density plots show CD69 expression in gated CD4⁺Foxp3⁺ cells from freshly isolated Spl, PLN and MLN, PPs, IEL, and colonic LPL in Foxp3^GFP knock-in mice. b Real-time PCR was performed to assess mRNA expression of T-bet, GATA3, RORγt and Foxp3 genes in CD69⁺ Treg and CD69⁻Tregs. β-actin mRNA was used for the normalization. c 1 × 10⁶/ml naïve CD4⁺ T, CD69⁺ Treg or CD69⁻ Treg cells were cultured under Th1-cell-differentiation conditions for four days. The CD4⁺ naïve T-cells treatment without IL-12 were regarded as negative control. Each group of T-cells were stimulated with the cell stimulation cocktail for 6 h and then stained with anti-mouse CD4 and IFN-γ antibodies, followed by flow cytometry (left). The levels of IFN-γ in the supernatants of cultured T-cells were detected by ELISA (right). d BMDCs co-cultured respectively with 1 × 10⁶/ml naïve CD4⁺ T, CD69⁺ Treg, or CD69⁻Treg cells at a ratio of 1:5 under Th17-differentiation conditions for four days. The CD4+ naïve T-cells treatment without IL-6 and TGF-β1 were regarded as negative control. Each group of T-cells were stimulated with the cell stimulation cocktail for 6 h and then stained with anti-mouse CD4 and IL-17 antibodies, followed by flow cytometry (left). The levels of IL-17 in the supernatants of cultured T-cells were detected by ELISA (right). e The expression levels of immunosuppressive markers in CD69⁺ Treg and CD69⁻Tregs were analyzed using flow cytometry with indicated antibodies. Data are representative images or expressed as the mean ± SD of three independent experiments (n = 5). *P < 0.05, **P < 0.01, ****P < 0.0001, ns not significant, analyzed by ANOVA or Student’s t-test