Figure 5. Foxo1 controls nTreg cell homeostasis and development.

(A–I) 8 week old Foxo1^f/f Foxo3^f/f (filled bars, symbols or histograms) and Cd4Cre Foxo1^f/f Foxo3^f/f (open bars, symbols, or histograms) unless otherwise indicated.

(A) Analysis of Foxp3⁺ cells within thymic CD4 SP cells (pooled results from 2 experiments).

(B) Total thymic CD4 SP Foxp3⁺ cells (mean + s.e.m.; n=3 to 7 mice per time point) with age.

(C) Total thymic CD4 SP Foxp3⁺ cells (mean + s.e.m.; n=3 to 7 mice per genotype and time point). Numbers indicates geoMFI or percentages (when bar is present) according to the color code.

(D) LN TCRβ⁺ cells from 8 week old mice (n≥5 mice per genotype analyzed in 2 independent experiments). Enumeration of TCRβ⁺ CD4⁺ Foxp3⁺ cells in peripheral lymphoid organs of 8 to 15 week old mice (open bars) mice (n≥9 mice per genotype analyzed in 3 independent experiments).

(E) Analysis of Ki67 expression assessed by flow cytometry on gated TCRβ⁺ CD4⁺ Foxp3⁺ LN cells (n≥9 mice per genotype analyzed in 3 independent experiments).

(F) Phenotype of TCRβ⁺ CD4⁺ Foxp3⁺ cells in peripheral lymphoid organs (n≥9 mice per genotype analyzed in 3 independent experiments).

(G) CTLA-4 expression on CD4⁺ TCRβ⁺ Foxp3⁺ LN cells.

(H) CTLA-4 expression after three days of stimulation in culture with anti-CD3 and anti-CD28.

(I) Chromatin immunoprecipitation of Foxo1. Fold enrichment over Ig control indicates the difference between the real-time PCR signal using anti-Foxo1 vs. that of the Ig control in the immunoprecipitation step. Primers are indicated by their position relative to the transcription start site (Figure S4). Representative of three experiments.

(J) Hematoxylin and eosin-stained sections of pancreata from 9 month old Foxo1^f/f and Cd4Cre Foxo1^f/f mice. Inflammatory cells were absent (7/10) to minimal (3/10) in the periductal and interstitial areas of the pancreas in wild type mice. In contrast, scattered foci with prominent lymphocytic infiltrates centered on periductal and perivascular connective tissues with extension of inflammatory cell infiltrates into the exocrine pancreas were observed in Cd4Cre Foxo1^f/f mice (11/11). No insulitis or islet involvement was noted other than bystander damage.