(A) Lymph node and spleen CD4⁺ GFP⁺ T cells were FACS purified from Foxp3-GFP-Cre mice and CD4⁺ YFP⁺ T cells sort-purified from Foxp3-GFP-Crex R26-YFPmice. Purified populations were analyzed for CD127, CD25, and GITR and Foxp3 by intracellular staining. (B) As in (A), analysis of FR4, CTLA-4 and CD103 expression and Foxp3 by intracellular staining. Histograms show YFP⁻ T_conv (thick line), YFP⁺ Foxp3⁺ T_reg (thin line), and YFP⁺ Foxp3⁻ (filled histogram). Representative plots from 6–9 week old mice of 3 experiments.

(A)Percent of GFP⁻YFP⁺ exFoxp3 cells among CD4⁺YFP⁺ cells in the pancreas, inguinal (ILN) and pancreatic (PLN) lymph nodes of NOD mice. Data points represent individual mice. (B,C) CD25 (B) orCD127 (C) expression on exFoxp3 cells isolated from each site, expressed as mean fluorescence intensity (MFI). Six 16–18-week old mice were analyzed in 2 independent experiments. (D) Development of T_reg and exFoxp3 cells in BDC2.5 TCR Tg mice. Analysis of GFP and YFP expression in thymocytes, LN cells and splenocytes of 6–9 week-old BDC2.5 TCR Tg⁺ and control TCR Tg⁻ mice. Plots are gated on live thymocytes and enriched CD4⁺ T cells from LN and spleen. Representative of 3 mice. (E) CD4⁺ YFP⁺ T cells purified from pancreatic LN (PLN) of BDC2.5.TCR Tg or non-Tg control mice were analyzed for V_β4 (BDC2.5 TCR chain) and Foxp3 expression. Plots are representative of 3 mice, and each point on the graph represents the percentage of YFP⁺ FoxP3⁻ cells in individual mice.

(A)CD4⁺ T cells from Foxp3-GFP-Cre × R26-YFPBDC2.5 mice were sorted to >95% purity into GFP⁺YFP⁺ and GFP⁻YFP⁺ cells. The percentage of Foxp3⁺ within these sorted populations was determined using intracellular staining (pre-culture). (B) Purified cells were cultured withanti-CD3 and anti-CD28 coated beads + IL-2for 6–8days, and were stained by flow cytometry (post-culture). Representative plots from 3 experiments.(C) 5 × 10⁵ expanded GFP⁻YFP⁻, GFP⁺YFP⁺ or GFP⁻YFP⁺ cells were transferred into NOD Rag2^−/− mice, and blood glucose concentrations were recorded from 8 days later and onwards. Diabetes is considered when blood glucose exceeds 250 mg/dl. Each point represents an individual mouse. (D) Hematoxylin and eosin staining of pancreas sections from NOD Rag2^−/− mice that did not receive cells, or that received GFP⁻YFP⁺ cells 9 days prior as described in (B). Representative of 4 mice.

(A)Thymocytes were isolated, stained for CD4 and CD8, and GFP and YFP expression was analyzed by flow cytometry. Thymocytes were gated as CD4⁺CD8⁺ double positive (DP) and individual CD4 or CD8 single positive (SP) populations. (B) The histograms show CD24 expression on the CD4SP GFP and YFP populations gated as shown in panel A. (C) GFP and YFP expression by gated CD4⁺ T cells from lymph nodes (LN) and spleen. (D) Methylation analysis of the Foxp3 locus. The methylation status of the CpG motifs of the TSDR of sub-cloned from purified T_conv, T_regs and exFoxp3 cells. Data is the average of data generated in3 independent experiments. (E) Graphs show the indicated GFP and YFP populations as a percentage of CD4⁺ T cells in spleen, LN, liver and Peyer’s patch of Foxp3-GFP-CrexR26-YFP mice. Each point represents an individual mouse. Experiments were performed with 6 -9 week old mice and are representative of 4 (A, B) and 20 (C) independent experiments.

Analysis of peripheral lymphoid organ resident CD4⁺ GFP⁺YFP⁺, CD4⁺ GFP YFP⁺, and CD4⁺GFP⁻YFP⁻ cells. (A) Spleen CD4⁺ populations were gated and analyzed for CD62L and CD44 expression. (B) Spleen CD4⁺ YFP⁺ and CD4⁺ YFP⁻ T cells were purified then stimulated with PMA, ionomycin and monensin for 4 h; cells were then stained for intracellular Foxp3 and IFN-γ. (C,D) Graphs show the percent of cells in each population that produce IFN-γ (C) or IL-17 (D). Each point represents an individual mouse. Experiments were performed on 6 – 9 week old mice and are representative of 5 (A, B) independent experiments.

(A)GFP⁺YFP⁺ GITR^high BDC2.5 TCR Tg purified cells (left dot plot) were transferred into NOD Tcra^−/− mice. After four weeks splenic YFP⁺ cells were purified, exposed to PMA, ionomycin and monensin for 3–4 hours and stained for intracellular Foxp3 and IFN-γ (right dot plot). Representative of 4 mice. (B) GFP⁻YFP⁻ cells purified from inguinal LNs (ILN) of BDC2.5 TCR Tg⁺ mice were transferred into NOD Rag2^−/− mice. Seven days after transfer, pancreatic mononuclear cells of acutely diabetic NOD Rag2^−/− mice that received CD4⁺ BDC2.5 TCR Tg⁺ YFP⁻ cells, or of control NOD Rag2^−/− mice that did not receive cells were analyzed by flow cytometry. CD4⁺ V_β4⁺ cells were gated and analyzed for YFP expression. Representative of 3 mice.

CD4⁺ GFP⁻YFP⁻, GFP⁻YFP⁺ and GFP⁺YFP⁺ T cells were sorted from BDC2.5 TCR Tg mice, cDNA was amplified with Vα2-specific primers and the amplicons were subcloned and sequenced. The frequency of each unique CDR3 amino acid sequence in T_conv (un-filled bar), exFoxp3 (grey bar) and T_reg cells (black bar). Data is one mouse representative of 2 analysed.