Supplementary Figure S1 Wang et. al.

1. Supplementary Figure S1 Wang et. al. Pre-sort Post-sort a 105 4.12 0.07 104 WT 103 102 0 105 5.47 0.16 104 Bim/ 103 102 CD25 0 0 102 103 104 105 0 102 103 104 105 Foxp3 b Bim/ WT 105 104 103 102 Foxp3 0 102 103 104 105 0 102 103 104 105 0 WT Tconv Bim/ Tconv BrdU c 25 80 NS NS 20 60 15 #CD4+Foxp3+BrdU+ (x103) 40 #CD4+Foxp3BrdU+ (x105) 10 NS NS 20 5 0 0 Supplementary Figure S1: (a) Purity of transferred Tconv. Tconv (CD4+CD45RBhiCD25) were sorted from the spleen and lymph nodes of C57BL/6 (WT) or Bim/ mice, stained with anti-Foxp3/anti-CD25 and analyzed by flow cytometry. (b) Representative dot plots of BrdU staining. Cells from Rag1/ recipients were analyzed as in Figure 1 and stained with antibodies against CD4, Foxp3 and BrdU. (c) Total number of BrdU positive CD4+Foxp3 and CD4+Foxp3+ cells in the spleen and MLN. Data are the mean ± s.e. of 2 independent experiments with 4 mice per group and statistical analysis performed using standard unpaired t test.

2. Supplemental Figure S2 Wang et. al. a b R1 150 100 #Cells R1 R2 R2 50 0 Foxp3 eFluor 670 c d e NS NS *** WT Tconv Bim/ Tconv NS %IL-2+ (CD4+CD25) WT Bim/ Bim/ WT Supplemental Figure S2: Correlation of T cell proliferation and iTreg induction. (a) Tconv were isolated from Foxp3GFP reporter mice and induced to become iTregs. Cells were then labeled with the cell proliferation tracker eFluor 670, activated and proliferation measured by flow cytometry. (b) Tconv from Foxp3GFP mice were activated for 3 days with various concentrations of plate bound anti-CD3 with soluble anti-CD28 with or without TGF-β (5 ng/ml). The percentage of Foxp3+ cells (iTregs) was analyzed by flow cytometry. (c) Percentage of iTreg induction of WT or Bim/ Tconv. Data is mean ± s.e. of 4 independent experiments and statistical analysis performed using standard unpaired t test (***p<0.005). (d) Tconv were sorted by FACS from WT or Bim/ mice and activated for 12 h with anti-CD3/CD28 coated beads. Supernatants were analyzed for IL-2 expression by ELISA (e) CD4+CD25 cells were isolated 4 weeks after Tconv transfer and stimulated 12 h. IL-2 frequency was determined by intracellular staining. Data are mean ± s.e. of 3 independent experiments with 2-3 individual mice per group in each experiment .

3. Supplementary Figure S3 Wang et. al. pre post * * * NS * ** Foxp3 Foxp3+ Foxp3 Foxp3+ Foxp3 Foxp3+ Supplementary Figure S3: iTregs and activated Tconv were prepared as in Figure 2. mRNA was isolated from either freshly sorted iTregs (Foxp3+) and activated Tconv (Foxp3) before (pre) or after an additional 48 h of culture in the absence of IL-2 (post). Expression levels of genes indicated was assessed by qPCR. Data shown is the mean ± s.e. and is representative of 1 (4 samples) of 3 (9 total samples) independent experiments with statistical analysis performed using standard paired t test (*p<0.05, **p<0.01). 

4. Supplementary Figure S4 Wang et. al. *** ** ** Supplementary Figure S4: Correlation of decreased Bim/Bcl-2 ratio in iTregs and their survival following IL-2 treatment. Tconv were isolated by FACS from WT Foxp3GFP reporter mice and activated with anti-CD3/CD28 coated beads in the presence of TGFβ (5 ng/ml) and IL-2 (100U/ml) for 3 days to induce iTreg differentiation. mRNA was prepared from iTregs cultured in the presence or absence of IL-2 for 48 h and qPCR performed. Data is mean of  3 independent experiments (**p<0.01, ***p<0.005). NT= not treated.