Nature Immunology: doi: /ni Supplementary Figure 1

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1 Supplementary Figure 1 IL-2R signaling is indispensable for the maintenance and function of T reg cells. (a) The analysis shown in Fig. 1j performed by gating on CD62L hi CD44 lo (upper panels) and CD62L lo CD44 hi (lower panels) YFP + Foxp3 + T reg cell subsets. (b) The expressions of CD62L and CD44 in CD3 + CD4 + Foxp3 + (upper panels) and frequencies of Foxp3 + cells among CD3 + CD4 + cells (lower panels) in the spleen and small intestine lamina propria lymphocytes (SILPL) of the indicated mice (5 wk old). The right graph shows the summary data. (c) The expression of indicated markers by splenic CD3 + CD4 + Foxp3 + cells in the indicated mice (5 wk old). (d) Lifespan of Foxp3 Cre Il2ra fl/fl (solid; n=25) and Foxp3 Cre Il2ra fl/wt (dotted; n=20) mice. (e) Analysis of LN cellularity, Foxp3 expression levels (MFI) and frequencies of Foxp3 + T reg cells among CD3 + CD4 + cells (upper panels) and proinflammatory cytokine production by CD4 + Foxp3 and CD8 + Foxp3 cells (lower panels) in the indicated mice (4 wk old). (f) Histopathology of the indicated organs of 4-wk-old Foxp3 Cre Il2ra fl/fl mice. Scale bar, 100 m. (g) The expression of Foxp3 and CD25 in LN CD4 + T cells in the indicated mice (6 wk old). The lower histogram represents the expression levels of CD25 in Foxp3 + cells shown in the upper panels. (h) The expression of CD62L and CD44 in LN CD3 + CD4 + Foxp3 (upper panels) and CD3 + CD8 + Foxp3 (lower panels) cells. (i, j) Cytokine production by splenic CD4 + Foxp3 (i) and CD8 + Foxp3 (j) cells isolated from indicated mice and in vitro stimulated with anti-cd3/cd28 for 5 hr. (k) Histopathology of the indicated organs of 4-wk-old Foxp3 Cre Stat5a/b fl/fl mice. Scale bar, 100 m. Sex and age matched mice were analyzed. Cells were analyzed by flow cytometry. *, P < 0.05; **, P < 0.01; ***, P < (twotailed unpaired Student s t test). Data are from one experiment representative of three (a) or two (b, c, e) independent experiments with similar results with three or more mice per group in each experiment (a, b, c, e; each dot represents a single mouse; mean ± s.e.m.) or representative data of n=6 (b), n=3 (f), n=8 (g, h, i, j), n=5 (k) mice per group analyzed are shown.

2 Supplementary Figure 2 Restoration of the suppressor activity of IL-2R -deficient T reg cells by expression of an active form of STAT5. (a) The schematic of Rosa26 Stat5bCA allele. CAG, cytomegalovirus early enhancer element with the promoter, the first exon and the first intron of chicken beta-actin gene and the splice acceptor of the rabbit beta-globin gene; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein (b, c) Photographs of 4-wk-old mice. (d) The expression of Foxp3 and CD25 in CD3 + CD4 + cells in the LNs and spleens of the indicated mice (4 wk old). (e) Intracellular phospho-stat5 levels in T reg cells from the indicated mice in vitro stimulated with rmil-2 (1,000 U/ml) for 20 min. (f) The expression of CD62L and CD44 in CD3 + CD4 + Foxp3 and CD3 + CD8 + Foxp3 cells in the LNs of the indicated mice. (g) Cytokine production by splenic CD4 + Foxp3 cells stimulated for 5 hr with anti-cd3/cd28. (h) The numbers of CD3 + CD4 + Foxp3 IFN- + (left graph) and CD3 + CD8 + Foxp3 IFN- + (right graph) cells in the LNs of the indicated mice (4 wk old). (i) The frequencies CD44 hi cells among CD4 + Foxp3 cells (upper left graph) and the frequencies of CD62L lo CD44 hi and CD62L hi CD44 hi cells among CD8 + Foxp3 cells (upper right graph) in the LNs of Foxp3 Cre Il2ra fl/wt, Foxp3 Cre Il2ra fl/wt Rosa26 Stat5bCA, Foxp3 Cre Il2ra fl/fl, and Foxp3 Cre Il2ra fl/fl Rosa26 Stat5bCA mice at the indicated ages. Lower graphs show the numbers of each cell subset in the LNs of indicated mice. (j) Lifespan of Foxp3 Cre Il2ra fl/fl (solid; n=25) and Foxp3 Cre Il2ra fl/fl Rosa26 Stat5bCA (dotted; n=21) mice. ***, P < (two-tailed unpaired Student s t test). Data are from one experiment representative of two (e, i) or three (h) independent experiments with similar results with three or more mice per group in each experiment (h, i; mean ± s.e.m.) or representative data of more than six mice per group analyzed are shown (d, f, g).

3 Supplementary Figure 3 Effects of in vivo neutralization of IL-2 on the activation of CD4 + or CD8 + cells. (a) Representative flow cytometric analyses for the experiment shown in Fig. 2e. (b) LN cells of Foxp3 Cre (upper 6 panels) and Foxp3 Cre Il2rb fl/fl (lower 8 panels) mice were unstimulated (US) or stimulated with rmil-2 (1,000 or 10 U/ml) for 20 min, and intracellular levels of phospho-stat5 in T reg (CD4 + YFP + CD25 hi ), T naive (YFP CD44 lo CD25 lo ; CD4 + and CD8 + ), and T eff (YFP CD44 hi ; CD25 lo and CD25 hi ; CD4 + and CD8 + ) cells were analyzed by flow cytometry. Data are representative of two independent experiments with three biological replicates in each experiment.

4 Supplementary Figure 4 Characterization of mice with T reg cells expressing an active form of STAT5. (a) The frequencies of STAT5b-CA-expressing CD4 + Foxp3 + cells among total CD4 + Foxp3 + cells in the spleen of Foxp3 Cre- ERT2 Rosa26 Stat5bCA mice after a single tamoxifen gavage on day 0. (b) The frequencies of STAT5b-CA-expressing CD4 + Foxp3 + cells among total CD4 + Foxp3 + cells in the indicated organs of Foxp3 Cre-ERT2 Rosa26 Stat5bCA mice three months after a single tamoxifen gavage. (c) TCR V usage of the T reg cells in various tissues 2 months after tamoxifen gavage. MLNs, mesenteric lymph nodes; PPs, Peyer s patches. (d) Changes in body weights after tamoxifen gavage. 4-month-old Foxp3 Cre-ERT2 (black, n=7) and Foxp3 Cre- ERT2 Rosa26 Stat5bCA (blue, n=7) mice were gavaged with tamoxifen and body weights were monitored the following 4 months. (e) Serum chemistry profiles 4.5 months after tamoxifen gavage. (f h) General characterization of T reg cells of Foxp3 Cre-ERT2 (black) and Foxp3 Cre- ERT2 Rosa26 Stat5bCA (blue) mice three months after a single tamoxifen gavage. (f) The expression levels of indicated molecules on CD4 + Foxp3 + cells in the indicated organs. (g) The frequencies of Foxp3 + cells among CD3 + CD4 + cells (upper graph) and the expression levels of Foxp3 in the CD3 + CD4 + Foxp3 + cells (lower graph) in the indicated organs. (h) The frequencies of Foxp3 + cells among CD3 + CD8 + cells in the indicated organs. (i) In vitro suppressor activity of T reg cells. T reg cells were isolated from Foxp3 Cre-ERT2 (control) and Foxp3 Cre-ERT2 Rosa26 Stat5bCA (STAT5b-CA) mice and co-cultured with T naive cells (responder cells). The proliferative activities of T reg and responder cells were determined by flow cytometry based on the dilution of CellTrace Violet (CTV) fluorescence intensity. Typical dye dilution patterns of T naive cells at a 4:1 responder vs. T reg cell ratio are shown in the left two panels. Summary graphs showing the proliferation of co-cultured responder T cells (Foxp3 ) and T reg cells (Foxp3 + ) are shown in the right two panels. CTV MFI of cells inversely correlates with cell division. Cells were analyzed by flow cytometry (a c, f i). *, P < 0.05; **, P < 0.01; ***, P < (two-tailed unpaired Student s t test). Data are from one experiment representative of two (c, d, i) or three or more (a, b, f h) independent experiments with similar results with three or more biological replicates per group in each experiment. Each dot represents a single mouse (b, e h). mean ± s.e.m. (a, b, d i).

5 Supplementary Figure 5

6 Systemic reduction in the T eff cell population in the presence of STAT5b-CA + T reg cells. (a d) Analysis of Foxp3 Cre-ERT2 (black dots) and Foxp3 Cre-ERT2 Rosa26 Stat5bCA (blue dots) mice three months after a single tamoxifen treatment. (a, b) The frequencies of Ki-67 + (upper graphs), CD62L hi CD44 lo (middle; % T naive), and CD62L lo CD44 hi (lower; % T eff ) cells among CD4 + Foxp3 (a) and CD8 + Foxp3 (b) cells of the indicated organs. (c) Cytokine production by CD4 + Foxp3 cells in the spleen and mesenteric LN cells stimulated with anti-cd3/cd28 for 5 hr. (d) Serum Ig levels determined by ELISA. (e) The numbers and sizes of polyps in the distal small intestines of 5-month-old Foxp3 Cre-ERT2 Apc Min/+ and Foxp3 Cre-ERT2 Rosa26 Stat5bCA Apc Min/+ mice treated with tamoxifen at 4 wk of age. Cells were analyzed by flow cytometry (a c). *, P < 0.05; **, P < 0.01; ***, P < (two-tailed unpaired Student s t test). Data are from one experiment representative of three or more independent experiments with similar results with three or more mice per group in each experiment (a c) or pooled samples with n=10 or more were analyzed (d, e). Each dot represents a single mouse (a e). mean ± s.e.m. (a e).

7 Supplementary Figure 6 RNA-seq analysis (supplementary information for Fig. 5) (a) Plot of gene expression (as log 2 normalized read count) in control T naive versus STAT5b-CA T naive cells (i.e., naive CD4 + T cells from Foxp3 Cre-ERT2 Rosa26 Stat5bCA mice). The diagonal lines indicate fold change of at least 1.5 or 0.67 fold. Significantly up- and down-regulated genes (defined as genes with at least 1.5 or 0.67 fold change, adjusted P-value 0.05, and expression above a minimal threshold based on the distribution of all genes) are colored red or blue, respectively, and their numbers are shown. (b) Volcano plot showing log 10 FDR-adjusted P-values versus log 2 fold change between STAT5b-CA and control T reg cells. Genes that fall outside of the x- or y-axis range of this plot are shown on the axes as empty triangles. The vertical and horizontal gray lines indicate 1.5 or 0.67 fold change (±log = ±0.58) and P = 0.05 ( log = 1.3), respectively. (c) Network analysis of GO term enrichment among significantly downregulated genes in STAT5b-CA expressing vs. control T reg cells. Downregulated genes were analyzed for over-represented GO terms using BiNGO in Cytoscape, and the resulting network was calculated and visualized using

8 EnrichmentMap. Groups of similar GO terms were manually circled. Edge thickness and color are proportional to the similarity coefficient between connected gene sets. Node color is proportional to the FDR-adjusted P-value of the enrichment. Node size is proportional to gene set size.

9 Supplementary Figure 7 Strategies for targeting Il2rb fl and Rosa26 Stat5bCA alleles. (a) Generation of a conditional Il2rb allele and Il2rb targeting strategy. The targeting vector was constructed such that upon Cremediated deletion, the promoter region and exon 2 which comprises the first ATG of Il2rb were deleted with simultaneous activation of egfp expression. Shown from top to bottom i) the Il2rb locus with the promoter region, exons and translational start site in exon 2 (E2); ii) the targeting vector comprising an egfp, a triple SV40 polya site (tpa), a PGK neopa cassette, a PGK promoter (Pr.) downstream of exon 2, a TK gene, and loxp and frt sites; arrows denote the orientation; iii) the targeted Il2rb locus. Restriction sites, probes used for detection and the expected fragments detected by Southern blot analysis are indicated. Correctly targeted embryonic stem (ES) cell lines were identified by Southern blot analysis of XbaI-digested DNA that displayed the 4.0 kb band of the integrated transgene along with the 14.0 kb wild-type band. Co-integration of the 3 loxp site was verified by PCR analysis using primers that hybridize in a unique region spanning the PGK promoter and the 3 frt site (forward primer) and in a region upstream of intron 3 of Il2rb (reverse primer). (b) The schematic of, and targeting strategy for Rosa26 Stat5bCA allele. The targeting vector was constructed such that CAG promoter driven STAT5b-CA is expressed upon Cre-mediated deletion of a STOP cassette. Correctly targeted ES cell lines were identified by Southern blot analysis of EcoRI-digested DNA that displayed the 5.9 kb (probe A; 5 side) and 11.6 kb (probe F; 3 side) bands of the integrated transgene along with the 15.6 kb wild-type band (probe A and F; both sides).

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11 Supplementary Table 2 Antibody list Antibody Clone Fluorescein, Dilution Manufacturer B220 RA3-6B2 PE-TexasRed, 1:500 BD Biosciences CD103 2E7 PE, 1:500, ebioscience CD11b M1/70 APC-eFluor780, 1:300 Tonbo Biosciences CD11c N418 PE-Cy7, 1:500 ebioscience CD122 5H4 PE, 1:200 ebioscience CD127 A7R34 PE, 1:200; PerCP-Cy5.5, 1:200 ebioscience CD132 TUGm2 PE, 1:200; APC, 1:200 BD Biosciences CD25 PC61 PE, 1:400; PE-Cy7, 1:500; APC, 1:300 ebioscience CD3 17A2 efluor450, 1:200; PerCP-Cy5.5, 1:300 ebioscience CD4 RM4-5 PE-eFluor610, 1:500 ebioscience CD44 IM7 PE, 1:500; efluor450 1:300 ebioscience CD45 30-F11 Alexa700, 1:400 ebioscience CD62L MEL-14 APC, 1:300 ebioscience CD69 H1.2F3 APC, 1:300 ebioscience CD8 5H10 BV605, 1:300 BioLegend CD A1 APC, 1:300; PE, 1:500 ebioscience CD86 GL1 APC, 1:300; PE, 1:500 ebioscience CTLA-4 UC10-4B9 PE, 1:300; APC, 1:300 ebioscience Foxp3 FJK-16s efluor450, 1:200, FITC, 1:300; PE-Cy7, 1:500; APC, 1:300 ebioscience GITR DTA-1 efluor450, 1:300; APC, 1:500 ebioscience Gr-1 RB6-8C5 PE, 1:400 BD Biosciences ICOS 7E.17G9 efluor450, 1:200; PE, 1:400 ebioscience IFN-γ XMG1.2 PE, 1:500 Tonbo Biosciences IL-13 ebio13a PE, 1:300 ebioscience IL-17 ebio17b7 APC, 1:300 ebioscience IL-4 11B11 PE, 1:300; APC, 1:300 ebioscience Ki-67 B56 PE, 1:400 BioLegend KLRG1 2F1 APC, 1:300 ebioscience MHC Class II (I-A/I-E) M5/ efluor450, 1:1000 ebioscience py-stat5 47/Stat5(pY694) Alexa647, 1:100 BD Biosciences TCRβ H PerCP-Cy5.5, 1:200 BioLegend TNFα MP6-XT22 PE-Cy7, 1:500; PE, 1:500 BD Biosciences Vβ10b B21.5 FITC, 1:50 BD Biosciences Vβ11 RR3-15 FITC, 1:50 BD Biosciences Vβ12 MR11-1 FITC, 1:50 BD Biosciences Vβ13 MR12-3 FITC, 1:50 BD Biosciences Vβ FITC, 1:50 BD Biosciences Vβ2 B20.6 FITC, 1:50 BD Biosciences Vβ3 KJ25 FITC, 1:50 BD Biosciences Vβ4 KT4 FITC, 1:50 BD Biosciences Vβ5.1, 5.2 MR9-4 FITC, 1:50 BD Biosciences Vβ6 RR4-7 FITC, 1:50 BD Biosciences Vβ7 TR310 FITC, 1:50 BD Biosciences Vβ8.1, 8.2 MR5-2 FITC, 1:50 BD Biosciences Vβ8.3 1B3.3 FITC, 1:50 BD Biosciences Vβ9 MR10-2 FITC, 1:50 BD Biosciences