(A–C) Mouse CD4 T cells were polarized into iTreg for 5 days and then treated with the ER stress inducer tunicamycin or tunicamycin/TUDCA in medium supplemented with 1 ng/ml IL-2. FoxP3 expression was analyzed by flow cytometry (n = 6–9 per group). Representative images (A) and statistical analysis of FoxP3 expression (B) and fixable viability dye (C) in Treg are shown. (D and E) CD4⁺FoxP3⁺ Tregs were sorted from the SPL and pLN by YFP expression. The cells then were treated with or without cytokines, including IL-12 (10 ng/ml), IL-4 (10 ng/ml), or IL-6 (50 ng/ml) for 10 hours. The mRNA levels of xbp-1s (D) and chop (E) were evaluated by qPCR analysis after 10 hours of treatment (n is at least 4 biological samples per group). (F) Tregs were cultivated with IL-4 or further with the ER stress inhibitor TD for 2 days; the FoxP3 expression levels were determined. (G) CD4⁺YFP⁺ Tregs were sorted, and Hrd1 mRNA in the Tregs from WT and Hrd1^fl/flFoxP3^Cre mice were measured (n = 5 per group). (H and I) Sorted WT and Hrd1^fl/fl-FoxP3^cre CD4⁺YFP⁺ Tregs were treated with anti-CD3 or anti-CD3 plus IL-12, IL-4, or IL-6 for 10 hours. The mRNA expression level of xbp1 (H) and chop (I) were analyzed by qPCR (n = 3–4 per group). (J and K) Volcano plot comparing the P value versus sorted CD4⁺YFP⁺ Tregs (J) and polarized CD4⁺YFP⁺ iTregs (K) from WT and Hrd1^fl/fl-FoxP3^cre mice. Genes labeled in red are ER stress–associated genes significantly differentially expressed between WT and Hrd1^fl/fl-FoxP3^cre Tregs. (L and M) GSEA was performed using the Gorilla bioinformatics tool for significantly upregulated genes in Hrd1^fl/fl-FoxP3^cre sorted YFP⁺ nTregs and Hrd1^fl/fl-FoxP3^cre polarized iTregs relative to WT. (N) CD4⁺ T cells were polarized into iTregs in vitro for 5 days. Protein expression levels in WT and Hrd1^fl/fl-FoxP3^cre iTregs were then analyzed by immunoblotting. Data shown as mean ± SD in B–E and G–I. *P < 0.05, **P < 0.01, and ***P < 0.005 by 2-tailed Student’s t test.