Fig. 6. Cis-deficiency impairs Treg cell function in an IL-4 signaling-dependent manner.

(A) RT-qPCR of gene expression in CD4⁺Foxp3-YFP⁺CD25⁺ Treg cells sorted from Cis^fl/fl,Cre and Cis^fl/+,Cre mice. Ebi3 and p35 encode IL-35. Tbx21 encodes T-bet. Actb was used as a loading control. (B) ELISA of effector cytokine expression in purified Treg cells from above mice following stimulation with plate-bound α-CD3 and α-CD28. (C) Intracellular stain of Foxp3 in Cis^fl/fl,Cre and Cis^fl/+,Cre iTreg cells cultured on α-CD3 and α-CD28 coated plate for 8 h in the presence of α-IL-4, α-IL-6 + IL-21R, α-IFNγ or a control antibody (Ctr). The numbers are the percentages of Foxp3^lo cells (Cis^fl/+,Cre/Cis^fl/fl,Cre). (D) ³H-thymidine incorporation of effector CD4⁺ T cells in the presence or absence of purified Foxp3-GFP⁺ WT and Cis^−/− iTreg cells differentiated from naïve cells isolated from Cis^+/+,GFP and Cis^−/−,GFP mice. Data shown are a representative of 2 experiments (3 biological replicates per group) (A, B), 2 (C) independent experiments, or a combination of 2 experiments (4-6 biological replicates per group) (D). Values (A, B, D) are means and S.D. (B, D). Student t test, * p< 0.05, ** p< 0.005.