Supplementary Figure 1. Ex vivo IFNγ production by Tregs. Nature Medicine doi: /nm % CD127. Empty SSC 98.79% CD25 CD45RA.

Transcription

1 SSC CD25 1.8% CD127 Empty 98.79% FSC CD45RA CD45RA Foxp3 %IFNγ + cells IL-12 P =.3 IFNγ p=.9 %IL-4+ cells IL-4 P =.4 c %IL-1 + cells IFNγ Control Foxp3 IL-1 P = MS IL-12 %IL-17+ cells %IFNγ+ s 2 IL P =.5 P =.1 P =.5 P =.7 +IL-12 +IL-12 Control MS d Foxp3 MFI IL-12 +IL-12 Supplementary Figure 1. Ex vivo IFNγ production y s Nature Medicine doi:1.138/nm.2389

2 FOXP3 1 5 P =.7 2 TBET P =.2 RORC2 mrna (1 3 relative expression) P =.3 CXCR3 P =.3 TGFβ Control MS Control 4 CCR IL MS Control P =.2 CTLA4 P =.1 IFNγ MS Supplementary Figure 2. Characterization of ex vivo MS s Nature Medicine doi:1.138/nm.2389

3 FOXP3 mrna (1 3 relative expression) Relative cell numer 15 + IL Day 4 Day 1 + IL-12 Isotype c Foxp3 CpG position Foxp3 + IFNγ + T reg cells Foxp3 + IFNγ - T reg cells Foxp3 - IFNγ + T mem cells Day 4 Foxp3 + IFNγ + T reg cells Foxp3 + IFNγ - T reg cells Foxp3 - IFNγ + T mem cells Day 1 1 Methylation % Supplementary Figure 3. Foxp3 staility after IL-12 treatment Nature Medicine doi:1.138/nm.2389

4 IFNγ IL-1 Foxp3 mrna (1 3 relative expression) 2 IFNγ IL-12 (ng/ml) mrna (1 3 relative expression) 2 IL IL-12 (ng/ml) Supplementary Figure 4. IL-12 dose-response Nature Medicine doi:1.138/nm.2389

5 + IL-12 3 Il Ifnγ mrna (1 3 relative expression) 1 3 Il Il Supplementary Figure 5. Cytokine gene expression in ex vivo s Nature Medicine doi:1.138/nm.2389

6 mrna (1 3 relative expression) 3 IL IFNγ + IL IL IFNγ Foxp3 Supplementary Figure 6. Effect of IL-12 family of cytokines on IFNγ production Nature Medicine doi:1.138/nm.2389

7 IFNγ IL IL IL Foxp IFNγ %IFN γ + cells IFNγ P =.1 %IL-1+ cells IL-1 P =.4 +IL-12 +IL-12 s +IL-12 +IL-12 Supplementary Figure 7. IFNγ and IL-1 production y clones. Nature Medicine doi:1.138/nm.2389

8 5 4 IFNγ IL-1 + IL-12 pg/ml P = Foxp3 MFI IL-12 +IL-12 Supplementary Figure 8. IFNγ and IL-1 secretion y clones Nature Medicine doi:1.138/nm.2389

9 4 RORC a Time (d) 5 CMAF IL Time (d) 4 IRF1 3 * Days +IL-12 Merged Relative cell numer T reg cells T naïve cells %Suppression c T-et - Anti-IL-1 Anti-IFNγ Anti-IL-1 + Anti-IFNγ P =.2 P =.4 - Anti-IL-1 Anti-IFNγ Anti-IL-1 + Anti-IFNγ treated T reg cells + IL-12 treatedt reg cells Supplementary Figure 9. Transcription factor expression and suppression upon IL-12 treatment Nature Medicine doi:1.138/nm.2389

10 15 FOXP3 + IL-12 2 TBET 1 1 mrna (1 3 relative expression) GATA3 P = RORC Supplementary Figure 1. Transcription factors gene expression. Nature Medicine doi:1.138/nm.2389

11 T resp cells T resp + T reg cells T reg Anti-CD3 + Anti-CD2 + Anti-CD28 eads treatment - anti-il-1 anti-ifnγ anti-il-1 + anti-ifnγ Relative cell numer Unstimulated IL-12 Stimulated CFSE 1 8 treated cells + IL-12 treated cells P =.9 %Suppression Anti-IL-1 Anti-IFNγ Anti-IL-1 + Anti-IFNγ - Anti-IL-1 Anti-IFNγ Anti-IL-1 + Anti-IFNγ Supplementary Figure 11. Suppression assay with IL-12 treated s in APC-free assays. Nature Medicine doi:1.138/nm.2389

12 Before IL-12 treatment Unstimulated Stimulated clones (1-16) Relative cell numer CFSE After IL-12 treatment Unstimulated Stimulated clones (1-16) IL-12 Relative cell numer CFSE IL-12 Supplementary Figure 12. Suppression assay with clones Nature Medicine doi:1.138/nm.2389

13 Day P =.1 75 IFNγ 5.7 IL-1 5. %Suppression IL-12 + IL-12 Foxp3 c IFNγ Day IL IL-12 d %Suppression P =.1 P =.11 (d4) + (d7) Foxp3 IL-12 (d4) + IL-12(d7) IL-12 (d4) + (d7) Supplementary Figure 13. Th1- phenotype is reversile Nature Medicine doi:1.138/nm.2389

14 mrna (1 3 relative expression) CCR5 P =.8 15 P =.8 CXCR IL-12 8 AHR P =.1 CTLA GITR P =.1 2 mrna (1 3 relative expression) 4 ICOS PD LAG3 P = PDL P =.4 TGFβ 15 1 OX Supplementary Figure 14. Characterization of IL-12 driven Th1-s Nature Medicine doi:1.138/nm.2389

15 mrna (1 3 relative expression) Ctla4 * + IL-12 * * Time (d) +IL-12 Merged Relative cell numer Intracellular Extracellular CTLA-4 c Intracellular CTLA-4 + IL-12 Extracellular CTLA P =.2 CTLA-4 MFI %CTLA-4+ cells 4 2 Supplementary Figure 15. CTLA-4 expression Nature Medicine doi:1.138/nm.2389

16 Supplementary Methods Cell culture reagents and antiodies Cells were cultured in RPMI 164 media supplemented with 2 nm L-glutamine, 5 mm HEPES, and 1 U/µg/ml penicillin/streptomycin (Biowhittaker),.5 mm sodium pyruvate,.5 mm nonessential amino acids (Life Technologies), and 5% human AB serum (Gemini Bio-Products). The antiodies used for stimulation were anti-human CD3 (clone UCHT1) and anti-human CD28 (clone 28.2) (BD Biosciences) at 1µg/ml. Inspector Beads (Miltenyi Biotec) were used in suppression assays. IL-12, 3 and 7 (R&D Systems) were used at 1 ng/ml, 2 ng/ml and 5 ng/ml, respectively. was otained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) and was used at 25 U/ml. Study sujects Peripheral lood was drawn from healthy individuals and 25 MS sujects after informed consent and approval y the Institutional Review Board at the Brigham and Women s Hospital. The patients were of ages of 47.1±1.5 years, and all had relapsingremitting disease with Kurtzke Expanded Disaility Status Scale scores etween and 2.5. Untreated patients (n=13) were not treated with any immunomodulatory drugs. Treated patients (n=12) had een receiving IFNβ for at least 12 months.healthy donors were age- and sex-matched with the MS patients, with no history of autoimmune disease. Cell isolation and FACS sorting of T cell populations Peripheral lood mononuclear cells (PBMCs) were isolated from healthy donors after informed consent was otained in accordance with the Declaration of Helsinki, y Ficoll Hypaque gradient centrifugation. Total CD4+ T cells were isolated y negative selection using the CD4+ T cell isolation kit II (Miltenyi Biotec) and stained for fluorescence-activated cell sorting (FACS) with the following antiodies: anti-cd62l (clone Dreg56), CD45RA (clone Hl1), CD25 (clone M-A251) (all from BD Biosciences) and CD127 (clone ebiordr5 from ebioscience). The (CD4 + CD45RA - CD25 hi CD127 low/neg ), Nature Medicine doi:1.138/nm

17 (CD4 + CD45RA - CD25 low/neg CD62L + ) and Tnaïve (CD4 + CD45RA + CD25 low/neg CD62L + ) populations were sorted on a FACS Aria (BD Biosciences). T cell activation and intracellular staining T cell populations were stimulated with 1 µg/ml plate-ound anti-cd3, 1 µg/ml solule anti-cd28 and 25 U/ml in the presence or asence of IL-12. At day 4, cells were stimulated with 3 nm phorol-12-myristate-13-acetate (PMA) and 2 nm ionomycin for 4 hours in the presence of GolgiStop (BD Biosciences) and intracellular staining of cytokines (IFNγ, IL-1, IL-17, IL-4), Foxp3 and T-et was performed with Foxp3 staining uffers (ebioscience) per manufacturer s recommendations and the following antiodies: IFNγ (clone 4S.B3), IL-17 (clone BL168) and T-et (clone 4B1) from Biolegend, IL-1 (clone JES3-19F1) and IL-4 (clone ), from BD Biosciences and Foxp3 (clone PCH11) from ebioscience. Foxp3 locus methylation analysis FACS-sorted s from healthy controls were stimulated for 4 days in the presence and asence of IL-12 and ex vivo FACS sorted s from healthy controls and RRMS patients were stimulated for four hours with PMA and ionomycin. Samples were intracellularly stained for Foxp3 and IFNγ. Fixed IFNγ + Foxp3 + and IFNγ - Foxp3 + s were resorted and sujected to genomic DNA isolation as previously descried {Hansmann #111}. Sodium isulfate treatment was performed on purified genomic DNA using the EpiTect Bisulfite kit (QIAGEN), following manufacturer s recommendations. PCR and purification of gel ands were performed as previously descried {Floess, 27 #112} and sequenced directly. Trace files were interpreted using 4Peaks software (Mekentosj). Enzyme-linked immunosorent assay ELISA measurement of IFNγ and IL-1 from stimulated clone supernatants was performed according to manufacturer s recommendations (BD Biosciences). Antiodies used were human IFNγ MA (clone 2G1) and human IFNγ MA iotin-laeled Nature Medicine doi:1.138/nm

18 (Thermo Scientific, Rockford, IL) and rat anti-human and viral IL-1 and iotin anti-human and viral IL-1 (BD Biosciences). Quantification of mrna expression levels y RT-PCR RNA was isolated using QIAGEN RNeasy Micro Kit (QIAGEN), following manufacturer s guidelines and converted to cdna y reverse transcription (RT) with random hexamers and Multiscrie RT (TQMN, Reverse Transcription Reagents; Applied Biosystems). For mrna gene expression assays, proes were purchased from Applied Biosystems (Tale 1) and the reactions were set up following manufacturer s guidelines and run on a 75 Fast Real-Time PCR System (Applied Biosystems). Values are represented as the difference in Ct values normalized to β2-microgloulin for each sample as per the following formula: Relative RNA expression = (2 dct ) x 1. Single-cell clones and cells were sorted at one cell per well in XVIVO15 medium containing 5% human serum and stimulated with solule CD3 and CD28 (oth at 1 µg/ml), irradiated T cell-depleted APCs (1 5 /well) and 5 U/ml. Half of the medium was replaced with fresh medium containing (5 U/m) starting at day 1 and every 3 to 4 days thereafter. After 4 weeks of expansion, each clone was tested for Foxp3 expression, IL-17, IFNγ, IL-1 and IL-4 production y intracellular staining. Suppression assays s previously treated with IL-12 (and ) or untreated (and cultured with alone) were co-cultured with CFSE-laelled responder CD4+CD25- T cells at a 1:2 :Tresp ratio (125 s and 25 Tresp). The stimuli used were plate-ound antihuman CD3 at.3µg/ml and 15x1 3 irradiated CD2-depleted APC or Inspector Beads (Miltenyi) at manufacturer s recommended concentration. At day 3, co-cultures were stained for viaility with the LIVE/DEAD stain kit (Invitrogen) and fixed using Foxp3 staining uffer (ebioscience). Proliferation of viale responder T cells was analyzed on a Nature Medicine doi:1.138/nm

19 LSRII flow cytometer (BD Biosciences). For suppression assays with ex vivo FACS sorted s and clones, Inspector Beads were used. Statistics A standard two-tailed t test was used for statistical analysis with P values of.5 or less considered significant. Supplementary Figure 1. IL-12 induced IFNγ secretion y healthy control and MS s. A. Sorting strategy and Foxp3 staining for purity. B. Bars diagrams show percentage of cytokine-secreting cells as mean±sem from 5 independent experiments performed. Memory CD4+ T cells () were assayed in parallel as controls. C. Representative example of intracellular staining of healthy control and MS s stimulated in the presence/asence of IL-12 for 4 days (left) and statistical analysis (right) n=3. D. Foxp3 mean fluorescence intensity (MFI) in healthy control s after 4 days in the presence/asence of IL-12 (n=12). Supplementary Figure 2. Characterization of ex vivo s from RRMS patients. A. FACS-sorted s from healthy controls and 1 untreated RRMS patients were stimulated directly ex vivo with PMA and ionomycin for 4 hours and RNA was isolated to measure gene expression. Supplementary Figure 3. Foxp3 staility after IL-12 treatment. A. FOXP3 mrna relative expression 24 hours after activation (n=5). B. Histograms show Foxp3 expression in untreated s (light) or IL-12-treated (old) after 4 and 1 days in culture. Light grey histograms correspond to isotype control. C. Methylation analysis of the Foxp3 locus from IFNγ + Foxp3 - and IFNγ - Foxp3 + at days 4 and 1 after IL-12 treatment. Memory IFNγ + Foxp3 - CD4 + T cells used as negative control. Nature Medicine doi:1.138/nm

20 Supplementary Figure 4. IL-12 dose-response. A. IFNγ and IL-1 secretion y s after 4 days stimulation in the presence of different concentrations of IL-12. B. mrna gene expression at day 1 after stimulation in the presence of various doses of IL-12. Supplementary Figure 5. Cytokine gene expression in ex vivo s. mrna gene expression of IL17, IFNγ, IL1 and IL4 on s activated with (lack ars) and without (white ars) IL-12 at 24 hours. Supplementary Figure 6. Effects of IL-12 family of cytokines in IFNγ production. A. IFNγ and IL1 relative gene expression on s activated in the presence of IL-12, 3 or 7 at day 1. B. Intracellular staining showing Foxp3 versus IFNγ at day 4. Supplementary Figure 7. IFNγ and IL-1 production y clones. A. Representative example of a clone treated with IL-12 (lower row) or alone (upper row) for 1 days. Dots plots show IFNγ (left) and IL-1 (middle) production versus Foxp3 expression, and IFNγ versus IL-1 production (right column). B. Percentage of IFNγ and IL-1-producing cells from 16 and clones (mean±sem). Supplementary Figure 8. IFNγ and IL-1 secretion in clones. A. ELISA of 16 and clones stimulated for 1 days with and without IL-12. Supernatant was collected after a 4 hour stimulation with PMA and ionomycin. B. Foxp3 MFI of the same clones after stimulation with PMA and ionomycin (Mean±SEM). Supplementary Figure 9. Transcription factor gene expression and suppression in IL-12-treated s. A. s were stimulated in the presence Nature Medicine doi:1.138/nm

21 nd asence of IL-12 for 5 days and RNA was isolated every 24 hours. Representative example of 3 performed with similar results., p<.5. B. T-et staining in s (upper row) and naïve CD4+ T cells as controls (lower row) activated in the asence (left column) or presence (middle row) of IL-12. Open histograms in the first two columns correspond to isotype control. Representative example of 4 performed. C. CFSE-laeled Tresp were cocultured with s previously treated with IL-12 or alone at a Tresp: ratio of 2:1 and anti-cd3 and T-depleted APC as stimulation. At day 3, cells were collected, stained for viaility and fixed. Bars diagram showing the percentage of suppression as per the following formula: %Suppression = [1-(Tresp in coculture/tresp alone)]x1. Statistical analysis of three independent experiments performed with two donors per experiment (mean±sem). Supplementary Figure 1. Transcription factors gene expression in clones. Transcription factor relative gene expression of 16 and clones stimulated for 1 days with and without IL-12. RNA was collected after a 4 hour stimulation with PMA and ionomycin (Mean±SEM). Supplementary Figure 11. Suppression assay with IL-12 treated s. Suppression assay performed as in Figure 4, ut with Inspector Beads (Miltenyi Biotec) as stimulation. Supplementary Figure 12. Clones suppression assays. clones were tested for suppressive function efore (A) and after restimulation in the presence/asence of IL-12 for 1 days (B) in co-culture assays (1:2 ratio :Tresp) with CFSE-laelled responder T cells. Histograms represent CFSE dilution of laelled responder cells for each co-culture assay. Nature Medicine doi:1.138/nm

22 Supplementary Figure 13. The acquisition of a Th1-like phenotype y s is reversile. A. s were activated in the presence and asence of IL-12 and stained intracellularly for Foxp3, IFNγ and IL-1 at day 4. B. Suppression after 4 days in culture. C. Cells treated with IL-12 were divided into two wells, one of them was washed and resuspended in fresh media without IL-12 (lower row) and the other was left with IL-12 for 3 days (upper row). Dot plots represent the intracellular staining at day 7 of IFNγ and IL-1. Suppression assays were performed as descried at day 4 (B) and day 7 (D). Bars diagrams show percentage of suppression as mean±sem of three independent experiments. Supplementary Figure 14. Phenotype of IL-12-reprogrammed s. s were stimulated in the presence or asence of IL-12 and RNA was collected at 36 hours. Relative gene expression of different Th1-related (A) and -related cell surface markers and cytokines (B) n=4. Supplementary Figure 15. CTLA-4 expression. A. Kinetics of CTLA4 gene expression on IL-12-treated and untreated s. Representative example of 3 experiments performed with similar results. B. Intracellular (upper row) and extracellular (lower row) CTLA-4 expression at day 4 on IL-12-treated (middle column) and untreated (left column) s. C. Bars diagram showing CTLA-4 MFI (left) for intracellular staining and percentage of CTLA-4 positive s (right) for extracellular staining, as mean±sem of three independent experiments performed. Nature Medicine doi:1.138/nm

23 Supplementary Tale 1. TaqMan proes used in this work. Proe Catalog Numer Il17A Hs936345_m1 Il4 Hs929862_m1 Ifnγ Hs989291_m1 Il1 Hs961622_m1 Foxp3 Hs185834_m1 Tet Hs23436_m1 Rorc2 Hs176112_m1 Gata3 Hs231122_m1 Cmaf Hs193519_m1 Irf1 Hs97196_m1 Gitr Hs188346_m1 Ox4 Hs533968_m1 Tgfβ Hs998133_m1 Ctla4 Hs111591_m1 Icos Hs _m1 Pd1 Hs169472_m1 Pdl1 Hs24257_m1 Ahr Hs169233_m1 Cxcr3 Hs17141_m1 Lag3 Hs158563_m1 Ccr5 Hs152917_m1 β2m E Nature Medicine doi:1.138/nm