Figure 7. IκB-ζ is dispensable for the stability of Foxp3 gene expression.

(A) Relative IκB-ζ expression in immune cells (B cells, CD4⁺FOXP3⁻ cells, CD4⁺FOXP3⁺ cells, CD8⁺ T cells, and CD11b⁺ cells) from Nfkbiz^f/+ Foxp3^YFP-Cre and Nfkbiz^f/f Foxp3^YFP-Cre mice. Data shown are the means ± sd of duplicate samples and are representative of 2 independent experiments. (B and C) Helios staining (B) or the activation status of CD4⁺ cells (C) in the spleens of 6- to 9-months-old male Nfkbiz^flox/+Foxp3^YFP-cre and Nfkbiz^flox/floxFoxp3^YFP-cre mice. Data shown are from 1 experiment that was representative of 3 independent experiments. (D) Costimulatory molecule expression in T_regs isolated from the spleens of 6- to 9-month-old Nfkbiz^flox/+Foxp3^YFP-cre and Nfkbiz^flox/floxFoxp3^YFP-cre mice. Relative values were obtained by comparing the mean fluorescence intensities ± sem; n = 3. Red, Nfkbiz^flox/+Foxp3^YFP-cre; blue line, Nfkbiz^flox/floxFoxp3^YFP-cre. (E) Cytokine production from CD4⁺ T cells in spleens of 6- to 9-month-old male Nfkbiz^flox/+Foxp3^YFP-cre (open circles) and Nfkbiz^flox/floxFoxp3^YFP-cre (filled circles) mice. Horizontal bars represent the calculated means. (F) Bisulfite sequencing of genomic DNA from YFP-positive T_regs isolated from the spleens of 8-week-old male Nfkbiz^flox/+Foxp3^YFP-cre or Nfkbiz^flox/floxFoxp3^YFP-cre mice. CpG methylation status in the CNS2 region of the Foxp3 gene was analyzed and is shown in sector graphs; 21–25 cDNA clones were sequenced from each subset. (G and H) Flow cytometry analysis of 6- to 8-month-old female Nfkbiz^flox/+Foxp3^YFP-cre/+ and Nfkbiz^flox/floxFoxp3^YFP-cre/+ mice. The percentages of FOXP3⁺ T_regs in the spleens of the indicated mice are shown.