(A) Schematic representation of FoxP3, GFP-FoxP3, and FoxP3-I-GFP. Protein convertase cleavage sites (RXXR), proline rich regions (Pxxp1 and Pxxp2), leucine zipper (Leu-zip) and forkhead (FKH) domains are represented. (B) Arthritis clinical score and ankle measurements of 8 week-old K/BxN.Foxp3^fgfp males, K/BxN.Foxp3^fgfp/wt heterozygote females and WT littermate control mice. (C) Anti-GPI IgG reactivity in sera of 8 week-old K/BxN.Foxp3^fgfp males, K/BxN.Foxp3^fgfp/wt females and WT littermate control mice (mean +/− SD, n=6–8). (D) Arthritis clinical score, ankle thickness, and anti-GPI antibodies in 8 week-old male and female K/BxN.Foxp3^igfp and WT controls. (E) Diabetes incidence curves of NOD.Foxp3^fgfp males NOD.Foxp3^fgfp/wt, females and littermate controls (10 mice/group). Two different cohorts of NOD.Foxp3^fgfp/wt heterozygous females were followed, in two different animal facilities.

(A and B) Percentage of FoxP3 positive cells within CD4⁺ T cells. (A) Spleens and draining lymph nodes (popliteal) of 8 week-old WT and K/BxN.Foxp3^fgfp males (each point is an individual mouse). (B) Spleen and pancreas of 9 week-old NOD.Foxp3^fgfp males and controls. (C) Proportion of FoxP3⁺ Treg cells among LN and visceral adipose tissue of 40 week-old B6 males. (D) Representative dot plots showing the percent FoxP3⁺ Helios⁻ Treg cells in colon lamina propria and spleen of 6 week-old B6.Foxp3^fgfp and B6.Foxp3^igfp males. Splenocytes from K/BxN.Foxp3^fgfp/wt heterozygote females were stained with anti-CD3, -CD4, and –FoxP3 antibodies. (E) Gates and values represent the fraction of GFP positive and negative cells among FoxP3⁺ population. Summary of data in adjacent plot shows fraction of GFP⁺ cells among FoxP3⁺ cells in thymus, spleen and draining lymph nodes of K/BxN.Foxp3^fgfp/wt females; the dashed line denotes the 50/50 ratio expected from random X-inactivation (F) Linear regression analysis of serum anti-GPI levels vs the fraction of GFP⁺ FoxP3⁺ Treg cells in K/BxN.Foxp3^fgfp/wt heterozygote females.

(A) IL17 producing cells in spleen and small intestine lamina propria from 4–5 week old K/BxN and K/BxN.Foxp3^fgfp male mice. Gates and values represent fraction of IL17 positive cells in CD4 population. Data is summarized in adjacent graph. Data is representative of 3 independent experiments. (B) In vitro Th17/Treg differentiation of congenically marked naïve cells from WT (CD45.1) and Foxp3^fgfp B6 mice. Data is representative of 2 independent experiments.

(A) Anti-FoxP3 staining profiles of CD3⁺CD4⁺ from different organs of K/BxN.Foxp3^fgfp male or WT littermates (shaded area is profile from isotype control). Data representative of 3 independent experiments. (B) FoxP3 mean fluorescence intensity (MFI) of splenic Treg cells on 3 different backgrounds. Each dot represents an individual mouse, and values are normalized to the mean FoxP3 MFI of WT mice in each experiment. (C) FoxP3 MFI for LN and visceral fat from 40 week-old B6.Foxp3^fgfp mice. (D) Foxp3 mRNA levels in CD4⁺GFP⁺ splenic Treg cells from Foxp3^fgfp or WT male mice on 2 different backgrounds.

(A) In vitro suppressive ability of CD4⁺CD25⁺ Treg cells from B6.Foxp3^fgfp or WT littermates, titrated at different ratios in co-cultures with CD4⁺ Tconv responder cells and APCs. Data represent mean proliferation +/− SD of 3 independent experiments. (B) Th17 differentiation suppressed by GFP-FoxP3 (FGFP) and FoxP3-ires-GFP (IGFP) Tregs. Ifng, Il17a, Il4 mRNA expression level assessed by rt-PCR.

Gene expression profiles were generated by microarray of Treg and Tconv cells from B6.Foxp3^fgfp males, with parallel profiling from B6.Foxp3^igfp as a reference. (A) Comparison of expression values in Treg versus Tconv splenocytes from Foxp3^fgfp and Foxp3^igfp B6 males. The canonical Treg signature is highlighted in red (Treg up-regulated transcripts) and blue (Treg down-regulated transcripts). (B) Comparison of expression values in Foxp3^fgfp and control Foxp3^igfp Treg splenocytes, on B6 and NOD backgrounds. Genes concordantly over-expressed in Foxp3^fgfp relative to Foxp3^igfp in both backgrounds are highlighted in red. Transcription factor genes are represented in green. (C) Heatmap representation of transcripts over-represented in Foxp3^fgfp on both backgrounds. (D) Flow cytometric confirmation of CTLA-4, CD103, and KLRG1 over-expression in CD4⁺FoxP3⁺ Treg cells from spleens of Foxp3^fgfp heterozygote females, on the K/BxN and NOD backgrounds. The values shown are the percent CD103 or KLRG1 positive cells within the GFP-positive or -negative fraction of Treg cells (mean +/− SD of 8 mice). (E) Over-representation of the Irf4 Treg signature: The ratio of expression in Foxp3^fgfp vs Foxp3^igfp of Irf4-responsive transcripts is shown (log2 scale) for both B6 and NOD datasets. The p-value is calculated with a χ² test for departure from a null hypothesis of random distribution.

(A) Association of FoxP3 with FoxP1 determined by co-immunoprecipitation. Anti-FoxP3 was used to immunoprecipitate FoxP3 from nuclear lysates of CD4⁺CD25⁻ Tconv or CD4⁺CD25⁺ Treg cells from B6.Foxp3^fgfp or WT mice, and immunoblots were probed for FoxP1 (anti-FoxP3 as control). (B) Nuclear lysates of B6.Foxp3^fgfp and B6.Foxp3^igfp CD25⁺ Treg cells were immunoprecipitated with anti-FoxP3 or control IgG, and probed with anti-Hif1a (anti-FoxP3 and anti-β–actin as controls). (C) Anti-Irf4 was used to immunoprecipitate Irf4 from nuclear lysates CD4⁺CD25⁺ Treg cells from B6.Foxp3^fgfp or WT mice, and immunobloted for FoxP3. (D) Western blot analysis of Irf4 from nuclear lysates of CD4⁺CD25⁺ Treg cells from B6.Foxp3^fgfp or WT mice, two independent experiments shown.