Figure 4.

JunB does not influence Treg stability but is continuously required for select Treg functions. (A) Treg cell stability, determined as frequency of Foxp3⁻ cells among labeled TCRβ⁺CD4⁺ZsGreen⁺ cells 14 days after tamoxifen-induced Junb deletion. Data are aggregated from at least 14 mice from seven independent experiments. Statistical significance determined by Mann-Whitney U-test with Holm-Bonferroni correction. (B) Representative flow cytometry plots (left) and summary statistics (right) showing frequency of Foxp3⁺ZsGreen⁺ Treg cells among TCRβ⁺CD4⁺ cells following tamoxifen-induced deletion of Junb. Data are aggregated from at least 17 mice from eight independent experiments. Statistical testing by two-way ANOVA identified a significant organ:genotype interaction and was followed by Welch's t-test with Holm-Bonferroni correction. (C) Frequency of TCRβ⁺CD4⁺ZsGreen⁻ cells expressing the indicated cytokines 14 days after tamoxifen administration. Data are aggregated from at least seven mice per group from four independent experiments. Significance determined by Mann-Whitney U-test with Holm-Bonferroni correction. (D) Frequency of PD-1^hiBcl-6⁺ Tfr cells among TCRβ⁺CD4⁺Foxp3⁺ZsGreen⁺ cells. Data are aggregated from at least 13 mice per group from six independent experiments. Statistical significance determined by Mann-Whitney U-test with Holm-Bonferroni correction. (E) Relative expression of CD25 (left) and frequency of CD25⁺ cells (right) among TCRβ⁺CD4⁺Foxp3⁺ZsGreen⁺ Treg cells. Data are aggregated from at least 11 mice per group from five separate experiments. Statistical significance determined by Mann-Whitney U-test with Holm-Bonferroni correction. (F) Frequency of Foxp3⁺ZsGreen⁺ Treg cells among TCRβ⁺CD4⁺ cells in the spleen and VAT of 14–16 week old male mice. Data are aggregated from at least eight mice per group from three independent experiments. Statistical significance determined by Mann-Whitney U-test with Holm-Bonferroni correction. (G) Representative contour plots (left) and summary statistics (right) showing the frequency of TCRβ⁺CD4⁺Foxp3⁺ZsGreen⁺ displaying the characteristic VAT Treg phenotype. Data are aggregated from at least eight mice per group from three independent experiments. (H) Frequency of TCRβ⁺CD4⁺ZsGreen⁺ cells expressing IL-10. Data are aggregated from seven mice per group from two independent experiments. Statistical significance determined by Mann-Whitney U-test with Holm-Bonferroni correction. (I) Representative histogram showing CTLA-4 expression in Tregs from iHET and iKO mice compared to splenic CD4⁺Foxp3⁻ cells in gray (left) and summary data (right) showing expression of CTLA-4 as determined by flow cytometry, normalized to the gMFI of splenic Treg cells from iHET mice for each experiment. Data are aggregated from at least six mice per group from three independent experiments. Statistical significance determined by Mann-Whitney U-test with Holm-Bonferroni correction. All summary data represent mean with a bootstrapped 95% confidence interval. *p < 0.05; **p < 0.01; ***p < 0.001.