Fig. 5.

Impaired expression of ERβ allows aberrant overexpression of Tsc22d3 (GILZ) in Tregs. (A and B) CD4⁺CD25^highCD127^lo Tregs were isolated from mLNs of SAMP^ΔERβ mice and SAMP^WT littermate controls by FACS sorting. (A) Tregs were analyzed by next-generation RNA-seq for changes to the global transcriptome, and a dendrogram shows the top 50-most differentially expressed genes by interaction-contrast analysis. (B) Gene expression of Tsc22d3 was analyzed among mLN-isolated Tregs from indicated mice by qPCR. (C and D) CD4⁺CD44⁺ naïve T cells were isolated from spleens of SAMP^ΔERβ male (C) or female (D) mice, then cultured ex vivo with α-CD3/CD28, TGF-β, and neutralizing antibodies α-IL-4 and α-IFN-γ for 5 d. Data represents relative gene expression of Tsc22d3, compared to day 0, in cells from indicated mice. (E) Gene expression of TSC22D3 was analyzed among peripheral blood-derived CD4⁺CD25^highCD127^lo Tregs isolated from Ctrl or CD patients. Data represents the mean ± SEM (NS, not significant; **P ≤ 0.01; ***P ≤ 0.001, n = 3 to 8 per group). (F and G) Amnis Imagestream cytometry was used to visualize colocalize ERβ and GILZ in peripheral blood-derived CD4⁺CD25^highCD127^lo Tregs isolated from Ctrl or CD patients. (F) Representative Imagestream data from indicated patients is shown; similarity score indicates correlation between MFI (ERβ) and MFI (GILZ). (G) Correlation analysis showing ΔMFI (GILZ) versus ΔMFI (ERβ) for Tregs isolated from male versus female CD patients.