Supplementary Information for nature05543: Foxp3-dependent programme of regulatory T-cell differentiation

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1 Supplementary Information for nature5543: Foxp3-dependent programme of regulatory T-cell differentiation Supplementary Methods Mice. The Foxp3 gfpko targeting vector was generated by inserting the stop codon/frameshift oligos shown in Fig. 1a into our previously described Foxp3 gfp fusion protein targeting construct 1. Double stranded oligos were inserted into the Avr II site downstream of and the Bam HI site in exon 5. Positive ES cell clones were identified by PCR and Southern blot and mice were generated as described 1. Foxp3 gfpko mice were back-crossed five times onto B6 background. Mice were treated with IL-2 as described 2. Flow cytometry and cell sorting. Conjugated antibodies were purchased from both BD Pharmingen (San Jose, CA) and ebioscience (San Diego, CA) and labeled cells were analyzed with a FACS Canto flow cytometer (BD). The following conjugates were commonly used: CD4-PECy7, CD8-PerCP, HSA-PE, -APC and CD44-APCCy7. Cells were sorted using a FACS Aria cell sorter (BD) (Supplementary Fig. 4). For Ki-67 staining, lymph node cells from Foxp3 gfp/gfp or Foxp3 gfpko/wt mice were fixed for 3 min with 2% paraformaldehyde to help retain soluble. Cells were further fixed, permeabilized and stained for Ki-67 (phycoerytherin) and cell surface markers according to manufacturer s instructions (BD Biosciences, Cytofix/Cytoperm). For PDE3B staining, cells were fixed in 1% paraformaldehyde/pbs for 1 min on ice, washed twice in ice cold Foxp3 Permeabilization Buffer (PB) (ebiosciences) and blocked with PB, 5% donkey serum (Jackson) for 15 min at room temperature. Anti-PDE3B rabbit IgG, raised against rat PDE3B N-terminal peptide 2-18, was added to 1 μg/ml (or no primary antibody as a control) for an additional 3 min incubation, after which cells were washed 3x in PB. Cells were incubated with donkey anti-rabbit IgG-APC (Jackson ImmunoResearch, West Grove, PA), 5% donkey serum and fluorescently labeled cell surface marker antibodies in PB for 3 min at room temperature, washed 3x in PB and resuspended in PBS for analysis. In vitro assays. For suppression assays, indicated T cell populations (2 x 1 4 each) and 6 x 1 4 irradiated (2 rad) T cell-depleted splenocytes were cultured in the presence of.7 μg/ml ConA for 72 hrs as described elsewhere 1. To test for in vitro responsiveness, 8 x 1 3 T cells were stimulated with the indicated concentrations antibodies and 3.2 x 1 4 irradiated T cell depleted splenocytes for 72 hrs. 3 H-TdR was added for the last 7 hrs of culture. For cytokine analysis, cells were stimulated with 1 ng/ml anti-cd3 and 1 ng/ml anti-cd28 in goat-anti-hamster IgG (Jackson ImmunoResearch) coated 96-well plates and supernatants were analyzed by cytokine array (Allied Biotech, Inc., Ijamsville, MD) and IL-17 was detected by ELISA (R&D Systems, Minneapolis, MN). For mixed lymphocyte reaction, CD4 + cells (1.5 x 1 5 ) were stimulated with allogeneic (Balb/c, H-2 d ) or syngeneic (B6, H-2 b ) irradiated (2 rad) T-depleted APC (1 6 ), or a 1:2 mixture of the two. Cells were pulsed with 3 H-TdR overnight on day 3 and harvested on day 4 of culture. Gene expression analysis. RNA was converted to biotinylated crna using commercially available reagent kits (Affymetrix, Inc, Santa Clara, CA; or Ambion, Austin, TX) and biological duplicates were hybridized to Affymetrix mouse microarrays. For thymic and peripheral data sets, after normalization (GCMRA, Bioconductor) genes with log 2 expression values > 4 and absolute log 2 fold-change values > 2 (thymus) or > 1.5 (periphery) for at least one of 6 possible pair-wise cell type comparisons ( /, T FN /, /, /T FN, /T FN, / ) were sorted into 16 by Pearson Squared K- Means Clustering with MultiExperiment Viewer (TIGR). For the IL-2 treatment experiment, an absolute log 2 foldchange > 1 for each IL-2 treated vs. untreated cell type was used. Similar were combined and outlying genes were sorted by hand. PDE3B expression. + CD4 + from B6 mice or CD4 + cells from B6.SJL mice (Ly5.1 + ), isolated by magnetic bead sorting as described 3, were stimulated with 1 ng/ml anti-cd3 and 1 ng/ml anti-cd28 in goat-antihamster IgG (Jackson ImmunoResearch) coated 12-well plates (1 6 cells/well). IL-2 (1 U/ml) was added to + CD4 + cells on day and to CD4 + cells on day 1 of culture. On days 1 and 2 following stimulation, cells were spinfected (9 min, 25 rpm, 37 C) with retroviral supernatants, supplemented with 5 μg/ml polybrene, 1 mm HEPES and IL-2, from transfected ΦNX-E cells transfected with MigR1, MigR1-PDE3B or MigR1- PDE3B-H81A. On day 5 of culture, aliquots of cells were stained for Foxp3. Cultures of + CD4 + cells routinely contained >7% Foxp3 + cells, >55% of which were transduced + (P ), while cultures of CD4 + cells contained >35% transduced cells. CD4 + cells were spinfected only on day 1 because retroviral transduction of these cells was more efficient than for. Cells transduced with the same vectors were mixed and transferred into TCRα -/- recipients (5 x 1 5 Foxp3 + cells and 3 x 1 5 Ly5.1 + cells per recipient). Three weeks after transfer cells were analyzed by flow cytometry and isolated by FACS sorting for gene expression analysis. 1. Fontenot, J. D. et al. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, (25). 2. Fontenot, J. D., Rasmussen, J. P., Gavin, M. A. & Rudensky, A. Y. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol 6, (25). 3. Gavin, M. A., Clarke, S. R., Negrou, E., Gallegos, A. & Rudensky, A. Homeostasis and anergy of CD4(+)(+) suppressor T cells in vivo. Nat Immunol 3, (22).

2 a TCR with an increased affinity for self antigen that facilitates sustained Foxp3 expression and lineage commitment. Supplementary Fig. 1 Foxp3 wt pre- Foxp3 + high IL-7R low CTLA-4 high GITR high Suppressor mechanisms. Il2 repression. b self antigen 1 Apoptosis. Foxp3 gfpko pre- 2 Pro-inflammatory effector mechanisms. Cell cycle. IL-2 expression. Foxp3 gfpko mrna and expression. self antigen 3 Partial acquisition of phenotype resulting from unique TCR signals. Foxp3 gfpko mrna and expression. c TCR that promotes conventional CD4 + T cell development. Foxp3 gfpko or Foxp3 wt naïve CD4 T cell Pro-inflammatory effector mechanisms. Cell cycle. Low and transient Foxp3 expression but no development High affinity foreign antigen Supplementary Fig. 1. Potential fates of T cells expressing the Foxp3 gfpko allele in Foxp3 gfpko/wt female mice. a, Under normal circumstances, precursors interact with their cognate self-peptide-mhc ligand which induces sustained high level Foxp3 expression and consequential development. b, In female heterozygous Foxp3 gfpko/wt mice, three potential cell fates can be envisioned for + T cells transcribing the Foxp3 gene yet lacking Foxp3 protein expression (termed T FN for Foxp3 null -expressing T cell): 1) The cells may undergo apoptosis, indicating that Foxp3 rescues precursors from thymic or peripheral deletional mechanisms of tolerance. However, analyses of TCR repertoires in Foxp3 null mice argues strongly against this scenario (Hsieh et al., 26. Nat Immunol 7:41). 2) The cells may become highly activated and produce proinflammatory cytokines, similar to Foxp3 + CD4 + cells. Observations of induction of T cell anergy and inhibition of IL-2 and Th1 cytokine expression upon forced Foxp3 expression in conventional peripheral T cells are predictive of this outcome. 3) The signals that induce Foxp3 transcription also induce some properties characteristic of mature cells. Here, Foxp3 gfpko precursors would develop into a unique cell type exhibiting expression and some, but not all, properties of. Comparison of this cell to would allow for the distinction of Foxp3-dependent properties from those that are Foxp3-independent. c, Recent reports have described transient low level Foxp3 expression without development (Kretschmer, et al. 25. Nat Immunol 6:1219). In this experimental model, TCR transgenic T cells were stimulated in vivo with a high dose of cognate antigen or with potent co-stimulatory signals, resulting in proliferation and transient Foxp3 expression. Thus, one scenario that may lead to appearance of + cells in Foxp3 gfpko/wt mice is responses of conventional CD4 + T cells to newly encountered high-affinity peripheral self antigens or infectious agents. However, according to the reported observations, such cells should appear highly activated and cycling while T FN are not. Furthermore, such T cells are undetectable in the course of the immune response in vivo in mice displaying polyclonal TCR repertoire (Fontenot et al. 25. Immunity 22:329). Thus, even if such + T cells appear in Foxp3 gfpko/wt mice their numbers and contribution to the entire + population should be negligible.

3 a c Foxp3 wt ko gfpko thymus CD4SP HSA low 7 day old gfpko d gfpko lymph node CD4 + 17d gfpko b % + of CD4 + LN or CD4SP HSA low thymocytes d gfpko gfpko LN Thy age (days) f cytokine (pg/ml) IL-2 Foxp3 CD4 subset genotype gfpko gfpko + gfpko IL N.D IL-5 IL-1 GM-CSF N.D. N.D Supplementary Fig. 2 IFN-γ TNF-α g + CD4 + gfp/gfp gfpko/wt 7d gfpko 17d gfpko 21d gfpko e d gfp/gfp lymph node CD4 + gfpko/wt 7d gfpko 17d gfpko 21d gfpko d gfpko thymus CD4SP HSA low gfpko/wt day old gfpko CD d gfpko CD4 + + CD4 + CD IL forward scatter IL-1 forward scatter CD45RB forward scatter CD45RB IL-4 CD62L GITR CD62L GITR h CD4 + donor population from male gfpko mice: CD44 CD44 + CD total +.35% 11.5% 5.1% 8.4% ICOS ICOS cell size (forward scatter) (LN CD4 + cells in recipient TCRβδ -/- mice are shown)

4 Supplementary Fig. 2. T FN in autoimmune male Foxp3 gfpko mice. In male Foxp3 gfpko mice, Foxp3 protein was not detected by flow cytometric (a) or western blot analyses (data not shown) of P+ CD4 + cells. Staining for Foxp3 was performed with rabbit-anti-foxp3 polyclonal IgG as described 1. b-e, In male Foxp3 gfpko mice, T FN were more abundant than in Foxp3 gfpko/wt mice. At 7 days of age, prior to the onset of thymic involution due to systemic inflammation, a mean of 4% of mature CD4 + thymocytes expressed (b). This frequency is approximately half that of among mature thymocytes from 7 day old Foxp3 gfp/gfp mice 2, consistent with our observations in Foxp3 gfpko/wt mice that the persistence of precursors during development is less efficient without Foxp3. In the periphery of 7 day old males, T FN constituted 8-11% of CD4 + cells, and with the progression of autoimmunity, T FN accumulated to nearly 2% of CD4 + cells in 21 day old mice (b,c). While thymic T FN in young male mice were mostly CD44 high, two populations of P+ cells could be distinguished in the periphery: a CD44 high subset with a highly activated phenotype and a CD44 intermdiate subset of small cells bearing a more naïve T cell phenotype (d). Data for female Foxp3 gfpko/wt mice are shown for comparison (d). Values represent gated cell percentages for the gates of the corresponding colour (d). With the exception of, the CD44 intermdiate cells expressed activation markers characteristic of naïve T cells. In contrast, the CD44 high + cells were more similar to CD44 high cells in their expression of several T cell activation markers (e). Histogram overlays show, for 7 and 17 day old Foxp3 gfpko mice, comparative staining of the 4 gated CD4 + T cell subsets as shown with the indicated PE-labeled CD44 low (blue), + CD44 high (purple), CD44 high (green) (e). Thus, in the absence of functional, T FN accumulate and become highly activated. antibodies. CD44 low (gray), + P fixation and staining with antibodies as described (Cytofix/Cytoperm staining kit, BD). h, The stability of expression in T FN from male mice was examined in cell transfer experiments. Four CD4 + T cell subsets, based on, CD44 and expression, from 3 week old Foxp3 gfpko mice were transferred into TCRβδ / recipients. Following 11 days in recipient animals, T cells were analyzed by flow cytometry (h). Most T FN lost expression, with only ~8% of donor T FN retaining. A similar percentage of activated-phenotype donor CD44 + and CD cells induced expression in this experimental system, while only ~.4% of naïve-phenotype donor CD44 induced low levels of. The unimodal distribution of expression among P± CD44 + CD4 + T cells in male Foxp3 gfpko mice (d) suggests that Foxp3 transcription is also transient for T FN in autoimmune Foxp3 null mice following acquisition of an activated phenotype. Furthermore, these data suggest that in older Foxp3 null mice only activated cells bear TCRs capable of promoting Foxp3 transcription. 1. Gavin, M. A. et al. Single-cell analysis of normal and FOXP3- mutant human T cells: FOXP3 expression without regulatory T cell development. Proc Natl Acad Sci U S A 13, (26). 2. Fontenot, J. D., Dooley, J. L., Farr, A. G. & Rudensky, A. Y. Developmental regulation of Foxp3 expression during ontogeny. J Exp Med 22, 91-6 (25). 3. Wildin, R. S., Smyk-Pearson, S. & Filipovich, A. H. Clinical and molecular features of the immunodysregulation, polyendocrinopathy, enteropathy, X linked (IPEX) syndrome. J Med Genet 39, (22). f-g, Cytokine production by CD4 + T cell subsets from male Foxp3 gfpko mice was analyzed. Cells (5 x 1 4 ) sorted from 2 day old Foxp3 gfpko based on and expression mice were stimulated with PMA (2 ng/ml) and ionomycin (5 ng/ml) for 16 hrs and supernatants were assayed for cytokines. T FN efficiently generated IL-2 and both T H 1 and T H 2 cytokines (f). In similar experiments, flow cytometric analysis revealed that up to half of T FN produced IL-4, and cells co-expressing IL-17 and IL-1 were readily detectable (g). High IL-4 production might explain the hyper-ige syndrome observed in human IPEX patients harboring Foxp3 loss-of-function mutations 3. For intracellular cytokine staining, total lymph node cell suspensions were activated with PMA and ionomycin in the presence of GolgiPlug (BD Biosciences) for 4.5 hours. Cells were fixed with 2% paraformaldehyde/pbs for 3 min on ice prior to

5 Supplementary Fig. 3 a T FN CD44 CD13 ICOS b mrna relative to Tbp 1e-4 1e-2 1e+ 1e+2 Fos Jun Rorc Il17 Il1 Supplementary Fig. 3. Phenotypic characterization of T FN. a, Histogram overlays show comparative staining of CD4 + lymph node cells with the indicated PE-labeled antibodies. b, cdna encoding the indicated genes was quantified by real-time PCR relative to TATA-binding protein (Tbp) using primer pairs corresponding to 3 UTR sequences. High Il1 mrna in indicated that their reduced IL-1 production following stimulation with plate-bound antibody was not due to transcriptional repression (Fig. 1g). Indeed, efficient IL-1 production by was observed following anti-cd3/28 stimulation in the presence of antigen presenting cells (data not shown).

6 Supplementary Fig 4. Foxp3 gfpko/wt Foxp3 gfp/gfp Supplementary Fig. 4. Representative post sort analysis of peripheral and T FN. Post sort analysis indicated that sorted T FN cells were not contaminated with + cells (i.e. ) from Foxp3 gfpko/wt mice.

7 Supplementary Fig. 5. Gene expression in selected. Genes in P3 and P4 that are also present in and are marked. For larger, ~1 of the most differentially expressed genes are shown, except for P3, where more genes are included. Color scales are adjusted for each cluster with maximum and minimum log 2 fold change values shown F4Rik 11159G2Rik 137C21Rik 1718L22Rik 23116F22Rik O19Rik 3111A13Rik J19Rik J6Rik G5Rik A3313N21Rik A6377B13Rik Adss Ahcy Ahnak AI79276 Ankrd17 Arf1 Atp1d BB BC87945 BM Bmpr2 Btg2 C338J1Rik Capg Card4 Cd38 Cd44 Cd48 Ctla4 Cyfip1 D14Ertd29e Drg1 Endod1 Foxp3 Gltscr2 Glud1 Gpr177 Gpx4 Gucy1a3 Gvin1 Hint1 Hipk1 Hrasls3 Il1r2 Il2ra Il2rb Inpp5f Irf4 Itga6 Itgae Itgav Itgb1 Itgb8 Kars Klra16 Klra18 Lrig1 Maf1 Mthfs Myc Niban Nrip1 Nrp1 Nt5e Nucb2 Odc1 P2rx7 Penk1 Plekhb2 Plscr1 Ppm1l Psmb3 Psmd4 Psmd8 Rab6ip1 Rnh1 Rora Rpn2 Rragd Sc5d Selk Sema4a Sh3bgrl Sh3bp5 Slc3a7 Smpdl3a Smu1 Snag1 Snx14 Socs2 Stat4 Stx11 Syngr2 Syt11 Tcrb-J Tcrb-V13 Tiam1 Timm1 Tmem65 Tnfrsf9 Xbp1 Ywhaq Zfp622 Zfpn1a2 Cluster L1Rik 2738C9Rik 2942A17Rik 321K1Rik 59343O7Rik A1314H13Rik AA47659 Akr1b3 Alg5 Amd1 Anxa6 Atp5b Atp5g2 Bach2 BB Bcl2l11 BM Bmyc Cast Ccdc22 Cct2 Cd2 Cd24a Cd47 Cdc37 Cep11 Chchd2 Cyba DHXS9928E D1Wsu52e Dctn3 Ddx54 E432D4Rik Ech1 Eif3s3 Eif3s6ip Eif3s9 Eno1 Fis1 Ftl1 Ga17 Gdi2 Gmps Gpr83 Gpr97 Gsta4 Guk1 Hcls1 Hnrpf Ik Itm2b Jak1 Jak2 Kcnn4 Klhdc2 Lgals1 Lig1 LOC Lrpap1 Marcks Mcm6 Mdh1 Mkrn1 Mrpl46 Mtf2 Mthfd1 Mtmr9 Myl6 Ndufa9 Nfkb2 Nkg7 Nrgn Oaz1 Pdrg1 Pnp Ppm1m Prps1 Prss15 Psmb8 Pstpip2 Ptp4a2 Qars Rac2 Rap1gds1 Reep5 Rpl1 Rpl18 Rpl32 Rps7 Rps9 Ruvbl1 Samsn1 Sar1a Smo Snrpb Specc1 Stambpl1 Stat1 Strap Strn3 Supt4h1 Tcrb-V13 Timm13 Tpr Uck2 Ugp2 Vim Xpo4 Cluster T F5Rik 1117C2Rik 123I7Rik 1791H14Rik 1819M1Rik 2519E7Rik 261E1Rik 2615L7Rik 278G24Rik C13Rik J5Rik O4Rik E19Rik E19Rik A5321J7 Abcb1a AI Aim1l Air Bambi-ps1 BB52693 BC165 BC27249 BC29169 BC55324 Bcl2 BE Bicc1 Blr1/ CXCR5 Bmp7 Casp4 Ccl2 Ccl5 Cd2r4 Cd22 Cd7 Ches1 Cpe Cpm Ctse Cxcr3 Deadc1 Dsp Eps15 Fcgr3 Gaa Galns Gemin5 Gja1 Gm288 Gstt2 Gucy1b3 H28 Ifi22b Igfbp7 Il17a Iqcc Itga4 Kif23 Klk1b22,1b9 Lck Ldhd Lime1 Lincr LOC Ly6k Mbnl3 Mtmr9 Myo6 Nckap1l Oprs1 Otud4 Padi2 Pcm1 Peli1 Pex3 Prickle1 Pstpip2 Ptger3 Rab6b Rgs11 Sec61a2 Serpinb1a Siat7a Slc2a8 Slfn5 Snf1lk Ss18 St6galnac1 Tbx21 Tm6sf1 Tmem38b Tnfsf8 Txnl4 Vdr Cluster P F1Rik 1215M12Rik 137C21Rik 23114G6Rik 23115A1Rik 26119F3Rik 28155G22Rik 31145G13Rik 31152M2Rik G5Rik L24Rik 57359K17Rik I19Rik L14Rik 99321J17Rik A134M12Rik A3349M8Rik A934K21Rik Abca2 Acoxl Acpp Actg2 Actr3b AI Akap7 Aldoc Alox8 Arhgef12 Arl5a Atp6va1 Atp6vd2 Baiap3 BB22611 BC28789 Bcdo2 BE Bicd1 Bik C1qtnf6 Camk2b Camk2n1 Casp3 Cbx7 Ccr8 Ccrl2 Cd2r1 Cd244 Cd47 Cd74 Cd8 Cdcp1 Crem Ctla2a Ctla2a,b Cxcr6 Cybb Cyp4f16 D7335F11Rik Dgat2 Dgkd Dhx37 Dnajb13 Dzip1 E432D4Rik E434N4Rik Ehd3 Ela1 Entpd1 Ephx1 Ercc8 Evi2a F13a1 F7331O2Rik Faim3 Fgl2 Gabarapl1 Galm Ggt1 Gjb2 Gnaq Gpd2 Gpr16 Gpr34 Grb7 Hdac9 Id2 Igfbp4 Igh-1a Igh-6 Il1ra Il18 Il18r1 Il21 Irf5 Cluster P3 also in / Itch Itfg3 Itgae Itgb5 Itgb8 Itsn1 Jak1 Kctd12b Klrg1 continued Ky Lamc1 Lgmn LOC Lyl1 Malat1 Matk Matn2 Mbp MGC3332 Mgea5 Ms4a4b Ms4a4c Mtm1 Myo1e Myo1f Myst2 Naprt1 Ndg1 Neo1 Pde2a Pde3b Pde7a Pglyrp1 Phlpp Phxr4 Pkp4 Plxdc1 Pnkd Pou2af1 Pou2f2 Ppm1l Prdm1 Prdx6 Prkch Psen2 Ptpn13 Ptprj Ptprv Rabgap1l Ralgds Rapgef5 Rhobtb1 Rin2 Rnf144 Rnf166 Rsn S1a4 S1a6 Samsn1 Sdc4 Sema4a Serinc3 Sfi1 Sfrs1 Slc15a2 Slc22a5 Slc2a3 Slc35d1 Slfn1 Sorcs2 Sox4 Sp4 Spnb2 Spock2 St14 St8sia1 Sytl2 Tacstd1 Tcf7 Tgfbr3 Tgif Tmbim1 Tspan3 Uap1 Ugt1a Usp18 Usp48 Zfp also in / H23Rik 155K14Rik 21111I1Rik 2311M24Rik 23116F22Rik 23132F3Rik 2926A2Rik O11Rik J19Rik J6Rik J16Rik H17Rik 9348N13Rik A5352I6Rik A631O12Rik A7395J18Rik Acpl2 Acsbg1 Adh1 Afp AI79276 Alcam Als2cl Anxa4 Arhgap29 Atp8b4 AV71699 Bach2 BC3324 Capg Capn3 Cd79b Cd81 Cd83 Cdkn2c Chd7 Cnga1 Ctla4 Cttn E434N4Rik Enc1 Epas1 Fah Fasl Foxp3 Frmd4b Gadd45b Gbp4 Ggtla1 Gm489 Gpld1 Gpr15 Gpr83 Gprc5b Gsto1 Hod Hspa1b Il1r2 Il1rl1 Il1rl2 Il2ra Il2rb Itgae Lycat Map3k8 Mbnl3 Mmd Mxd1 Myo1 Myo1c Ncf1 Neb Nfil3 Niban Nrp1 Osbpl3 Pdk1 Penk1 Pim1 Plagl1 Pmaip1 Ppm1l Prc1 Prkar1b Ramp3 Rapgef4 Rragd Sema4a Sema4f Sh3bgrl2 Slc16a5 Soat1 St3gal6 Stx11 Swap7 Syt11 Tanc1 Tiam1 Tmie Tnfrsf18 Tnfrsf1b Tnfrsf4 Tnfrsf9 Ttc7b Twsg1 Vav2 Zfpn1a2 Cluster P also in / 28142A17Rik H13Rik B12Rik 57357H5Rik 63343K7Rik A5321J7 Abcb1a Abhd4 Acot7 Alad Ar Aspm Atf6 Aurka Aurkb BC23488 Birc5 Bmp7 Bub1 Bub1b Ccna2 Ccnb1 Ccnb1-rs1 Ccnb2 Ccr2 Cdc2 Cdc2a Cdca1 Cdca3 Cdca5 Cdca8 Cenpe Cenpf Cep55 Chdh Cks1b Clspn Cst7 Cysltr2 D17H6S56E-5 D4Ertd89e Dctd Ebi3 F7347E7Rik Fbxw8 Fignl1 Gzmb Hist1h1c Hist2h3c2 Hmmr Icam1 Ifi44 Igf2bp3 Il12rb1 Il17rb Irf8 Kif2a Kif2c Ksr1 Lgals3 Luzp5 Mcm1 Mtmr7 Myo1f Nedd4 Nek2 Nrn1 Nusap1 Odc1 Pbk Pdcd1lg2 Plk1 Rad51 Rad54l Rasgrp2 Rpl17 Rrm2 Serpina3g Slc39a4 Snx9 Solt Spag5 Spbc24 Spbc25 St8sia6 Stard1 Tgfbr2 Tk1 Tktl1 Tnfrsf9 Tpi1 Tpx2 Tyki Tyms Unc119 Vars2 Zcchc18 Cluster P H23Rik 23131A18Rik 23137P21Rik 2811G2Rik O19Rik C17Rik P3Rik L22Rik 64357G G5Rik Abcb1b Actn1 Adamts6 Afp Agpat4 AI Amigo2 Anxa1 Apob48r App Arhgap2 BB Bmpr2 Cyfip1 Cyp2s1 D18Ertd653e D6339A3Rik Dip3b Dnahc12 Dst Ecm1 Emp1 Enc1 Eno3 Enpp1 Evi2a Fasl Fgd6 Fos Galm Gata1 Gbp1 Glipr2 Gm16 Gpr177 Gramd3 Gsta4 Gucy1a3 Gzmk Hectd2 Hipk2 Hs3st3b1 Ifitm3 Ift8 Igf2r Il1 Itih5 Jun Kcnk6 Klrd1 LOC Lrrc8d Lyst Mbnl3 Msra Mtap6 Mtmr3 Mxd1 Myo1e Ncoa7 Nt5e Ntrk3 Pard6g Pdlim4 Ppic Ppp1r3e Prf1 Prg4 Prnp Qpct Rab6ip1 Rabgap1l Rnf32 Selp She Snn Socs2 Sostdc1 Specc1 St3gal6 Tlr7 Tmem64 Tmsb1 Tnfrsf13b Trub1 Usp27x Xkrx Zfpn1a4 Cluster P

8 b a # of genes in thymic cluster # of genes shared between corresponding % 35% T1 T2 T5 T6 T7 T differentially expressed in: thymus alone both thymus and periphery periphery alone P1 P2 P3 35% P4 61% P5 P6 P7 P # of genes in peripheral cluster red = upgregulated genes blue = downregulated genes black = both upregulated and downregulated 1 GO: Biological Process 2 GO: Cellular Component 3 GO: Molecular Function 4 KEGG Pathways 5 Interpro Supplementary Fig. 6 * p < 5e-2 ** p < 5e-3 *** p < 5e-4 **** p < 5e-5 THYMUS T1 T2 T5 T6 T7 T8 Also in periphery Unique to thymus All genes in Ubiquitin cycle 1 (21)** cluster ABC transporter 3 (1)*** Transcription 1 (32)* Nucleus 2 (57)** Extracellular space 2 (29)**** Integral to membrane 2 (37)**** Metabolism 1 (19)* Protein/RNA/Sphingolipid metabolism 1 (92/16/5)* Insulin signaling pathway 4 (9)** Receptor activity 3 (12)*** Organ morphogenesis 1 (6)* Intracellular 2 (94)** ATP binding 3 (27)* Unfolded protein binding 3 (7)*** c PERIPHERY P1 P2 P3 P4 P5 P6 P7 P8 Also in thymus Unique to periphery All genes in cluster C-type lectin5 (6)*** Immune response 1 (13)* Extracellular space 2 (18)* Integral to membrane 2 (23)* Cell communication 1 (53)* Electron transport 1 (9)* Intracellular signaling 1 (25)** Immune response 1 (7)** Receptor activity 3 (14)* Receptor activity 3 (15)* Cell communication 1 (19)* Cell cycle 1 (2)**** Intracellular 2 (37)*** Glycolysis 1 (3)* Supplementary Fig. 6. Overlap of thymic and peripheral differential gene expression and enriched functional gene designation. a, Intersection of thymic and peripheral data sets. The number of genes in each cluster is plotted along with the subset of each cluster that contained genes differentially expressed among both thymic and peripheral cells (dark blue bars). The membership of these shared genes in thymic and peripheral was enumerated in the displayed 8x8 heat map in which the intensity of blue represents the number of genes at each intersection. b-c, Functional designations for gene, or subsets of gene, are shown. Subsets were derived based on whether genes were upregulated (red font), downregulated (blue font), shared between thymic and peripheral data sets (top row), unique to thymic or peripheral genes (middle row) or represented within the full cluster (bottom row). Gene subsets were compared to all other genes in the thymic or peripheral gene set to elucidate significant enrichment of functional terms using Babelomics FatiGO-Plus (Al- Shahrour F., et al. 26. Nucleic Acids Res 34: W472), and the number of genes and p value for each term is shown. GO, Gene Ontology.

9 Supplementary Fig. 7. T FN are hyperresponsive to IL-2 in vivo and associated changes in gene expression suggest an IL-2 dependent component of transcription. If Foxp3-dependent genes predominantly effect signal transduction, then the reduced numbers, lack of division and small size of T FN should be due to a deficiency in T FN responsiveness to their environment. One likely explanation for these differences is that expression on T FN is too low to make them competitive for paracrine IL-2. While expression on cells is similar to that of T FN, their ability to generate IL-2 is likely to promote the proliferative activity they share with. To determine if increased IL-2 signaling restores T FN proliferation, we investigated changes in cellular phenotype and gene expression in, T FN and following high dose IL-2 treatment in vivo. Indeed, we found that T FN were transcriptionally hyperresponsive to high-dose IL-2 exposure in vivo. Gene expression analysis revealed that the set of cell cycle genes shared by peripheral and cells (cluster P6) was upregulated in, T FN and, with slightly higher increases occurring in T FN cells, indicating that T FN proliferation can also be induced by IL-2 in vivo as observed in in vitro assays (b,c, cluster L7; data not shown). However, IL-2 responses in T FN were more dramatic in that approximately 8 additional genes, significantly enriched for mitochondrial genes and genes that promote biosynthesis, altered expression in T FN alone (c). These data suggest that the reduced frequency of T FN results at least partially from cytokine starvation. This notion is in agreement with a 5% reduction in thymic and peripheral in the absence of IL-2 signaling and a 7-1-fold reduction of Il2ra -/- relative to wt in mixed bone marrow chimeras (Fontenot et al., 25. Nat Immunol 6: 1142). Thus, acute IL-2 sensitivity of T FN cells is likely to result from coincidental IL-7R downregulation, block in IL-2 production and intermediate levels that prevent them from competing with Foxp3 + for paracrine IL-2. a PBS IL-2 Supplementary Fig. 7 b differentially expressed in: both periphery and IL-2 treatment IL-2 treatment alone c T FN IL-2 treated vs. untreated for each cell subset (log 2 ratio) Forward Scatter L1 L2 L Also in periphery Unique to IL-2 d St14 Tnfrsf4 Foxp3 Mcm7 AI Sdh1 Il1ra Kif3b Il2rb Gpr83 Elovl O8Rik D18Rik Rragd 9348N13Rik BC6478 Hipk2 Il2ra Sypl D1337M23Rik Map3k8 All genes in cluster Receptor activity 1 (17)*** Extracellular space 2 (18)* Integral to membrane 2 (3)** Biosynthesis 1 (27)*** Mitochondrion 2 (24)**** Cytoplasm 2 (56)**** Apoptosis 4 (5)** Transcription 1 (39)* Transport 1 (68)** T FN + IL-2 T FN + IL-2 + IL-2 BB23511 BF C1Rik AI66844 Ian6 BB391 Kif1b BE6867 Tmie BB22318 D7335F11Rik # of genes shared between corresponding P1 P2 P3 P4 P5 P6 P7 P8 28 L L2 29 L3 L4 L5 L6 7 L7 L4 L5 L6 L # of genes in IL-2 treatment cluster Cell cycle 1 (23)**** Intracellular organelle 2 (38)* Cell cycle 1 (14)*** Intracellular organelle 2 (29)* Methods a, Foxp3 gfp/gfp and Foxp3 gfpko/wt mice were injected with IL-2 or vehicle (PBS) four times within 24 hrs. 3 min following the last treatment, lymph node cells were analyzed by flow cytometry. Both and T FN responded in a similar fashion by increasing expression and cell volume, with increased cell size being slightly more pronounced among T FN. b-c, The, and T FN subsets were sorted from pooled lymph node and spleen cells for gene expression profiling. Genes were clustered based on fold change values between IL-2 treated and PBS treated cells for each of the three subsets. The number of genes in each cluster and overlap with peripheral P1-P8 (b) and the mean and range of fold change values (c) are shown. Overrepresented functional terms (c) were determined as described for Supplementary Fig 6. d, In addition to Foxp3, Il2ra and Il2rb, IL-2 exposure of T FN changed expression of ~3 other genes to levels similar to those observed in cells revealing aspects of the Foxp3 transcriptional program depending exclusively on increased IL-2 signaling. Genes whose expression in IL-2-treated T FN approaches that of are shown. For each gene, the 6 heat map panels represent log 2 gene expression fold-changes in the indicated T cell type with or without IL-2 treatment relative to cells.

10 Supplementary Fig. 8 a 5' LTR Ly5.1 + allelic marker + Ψ PDE3B PDE3B-H81A (metal binding domain: HDYDH) or empty vector Transduce, mix and transfer. IRES TCRα 3' LTR 3 weeks post-transfer: Flow cytometry and cell sorting for gene expression profiling. b PDE3B Vector Transcription, DNA-dependent 1 (55)** Negative regulation of apoptosis 1 (6)* (Birc6 Cfdp1 Dapk1 Hdh Pdcd6ip Snai2) Nucleus 2 (119)** Biosynthesis 1 (65)**** Protein biosynthesis 1 (3)** Amino acid metabolism 1 (18)** Cytoplasm 2 (161)** Mitochondiron 2 (49)* Golgi stack 2 (27)* c d P1 P2 P3 P4 P5 P6 P7 P8 N.A./2 23/27 44/58 1/11 8/12 3/31 N.A./13 N.A./21 T FN T PDE3B ( R Vector) % 85% 78% 82% 75% 9% 85% 62% -3 H81A PDE3B Vector Vector e Ctla4 Cybb Ebi3 Entpd1 F13a1 Fgl2 Gzmb Hmox2 Il1 Il18 Il2ra Lamc1 Ncf1 Ncf4 Nt5e Sypl Syt11 Sytl1 Sytl2 Tfpi Tgfb1 Tnfsf1 T FN T PDE3B ( R Vector) + f -Foxp3 g vector PDE3B H81A empty 465 PDE3B PDE3B (MFI) H81A pmol camp/min/mg protein

11 Supplementary Fig. 8. Impaired homeostasis of PDE3B transduced and effects of ectopic PDE3B on gene expression. a, + CD4 + cells from B6 mice, or CD4 + cells from B6.SJL mice (Ly5.1 + ) were transduced with MigR1, MigR1- PDE3B or MigR1-PDE3B(H81A). B6 and B6.SJL cells transduced with the same vector were mixed (i.e. a mixture of transduced and non-transduced and ) and transferred into TCRα -/- mice. 3 weeks after transfer, cells were analyzed by flow cytometry and sorted for RNA isolation (see Supplementary Methods). b-e, To examine in greater detail the effects of ectopic PDE3B expression on, P+ Ly5.1 CD4 + GITR + cells (pooled from 3 recipient mice for each expression construct) were sorted for gene profiling. In comparing genes differentially expressed among transduced with PDE3B, PDE3B(H81A) or empty vector, 42% were specifically altered in PDE3B expressing and 47% were expressed similarly in both PDE3B and H81A transduced cells, leaving only 11% affected mostly by H81A. Gene ontology analysis of all genes differentially expressed by PDE3B vs. empty vector-transduced cells revealed that upregulated genes were enriched for transcription factors and other nuclear proteins, while cytoplasmic proteins, particularly mitochondrial proteins and proteins involved in biosynthesis were downregulated (b). This polarization suggested that PDE3B-expressing were metabolically deprived or quiescent. For genes found to be differentially expressed both in this experiment and in our analysis of peripheral T cells (Fig. 3b) the effect of PDE3B expression was juxtaposed to the gene expression pattern observed for, T FN and cells. Expression values were plotted as the sum of the fold change values (log 2 ratio) for (PDE3B/empty vector) + ( / ) (c). A striking trend emerged in which ectopic PDE3B reversed the Foxp3-dependent expression of 83% of shared genes (i.e. genes shared with P2-P6) resulting in an expression profile similar to that of T FN cells (c, dashed boxes). Values represent the number of genes whose expression reverted back towards that of T FN in PDE3B-transduced vs. the total number of shared genes (c). The H81A/empty vector and PDE3B/empty vector values for the same genes were also plotted along with the percentage of genes whose differential expression was greater with PDE3B vs. H81A (d). Here, 79% of all shared genes exhibited reduced differential expression in PDE3B(H81A)-expressing, consistent with the intermediate effect this mutant exerted on homeostasis (d). This intermediate effect may be due to previously unrecognized adaptor-like properties of PDE3B, as has been shown recently for PDE3B-associated PI3Kγ 1. The reversal of the - to a T FN -like gene expression pattern was particularly remarkable for the cell cycle genes shared with cluster P6, whose expression was uniformly reduced by a factor similar to the fold-change between T FN and, and in a manner that was dependent upon, or enhanced by, catalytically active PDE3B. In contrast, putative and known effector genes were not altered by ectopic PDE3B suggesting that Pde3b repression influences homeostasis rather than effector function (e). PDE3B-affected genes in other than P6 were not enriched for any particular functional designation relative to other genes in the cluster. Because PDE3B can integrate both camp-pka and PI3K-AKT signaling 2, 3, highly complex biochemical mechanisms affecting either survival or expansion might be elicited as the result of overriding Foxp3-dependent Pde3b repression in. Importantly, PDE3B is known to be activated by PKA or PKB phosphorylation 2 with the latter pathway likely to be operating in a higher gear in cells due to sustained IL-2R and TCR signals. This suggests that Pde3b repression might be an adaptation unique to the lineage while lack of PDE3B expression might be inconsequential to nonregulatory T cells. Although further in-depth biochemical and genetic analysis is required to understand a role for PDE3B repression in cells, we suggest, based on aforementioned in vitro findings and gene expression analyses, that some PDE3B transduced were rapidly lost to apoptosis following transfer while those surviving elicited new negative feedback inhibition of -specific signaling as a compensatory mechanism. Indeed, while or TCR levels were not altered in PDE3B-transduced, Jak3 and PKCζ were among the most reduced in expression, suggesting that IL-2 and perhaps other proliferation-inducing signals were attenuated. Furthermore, anti-apoptotic genes were found upregulated in surviving PDE3B-transduced, suggesting that survival mechanisms were engaged in these cells under lymphopenic conditions (b). Notably, loss of PDE3B-transduced was observed within the first week after transfer followed by subsequent stabilization (data not shown). Thus, an observed lack of progressive and more profound loss of PDE3B-transduced cells in these experiments may result from an anti-apoptotic component of adaptation to ectopic PDE3B expression. f, In experiments similar to those described in (a), total CD4 + cells from Foxp3 gfp/gfp mice were transduced with retroviral vectors in which Thy1.1 replaced as a marker gene. Upon recovery from recipient mice, cells were stained for PDE3B (goat-antimouse PDE3B; C-2, Santa Cruz Biotechnology) and cell surface markers. Total CD4 + cells are shown. Values represent mean fluorescence intensity for PDE3B in the indicated quadrant g, The H81A mutation abolishes PDE3B catalytic activity. ΦNX-E cells transfected with MigR1, MigR1-PDE3B or MigR1- PDE3B(H81A) (with transfection efficiencies of 48%, 43% and 47%, respectively, as determined by flow cytometry) were homogenized in PBS containing 1% Nonidet (Roche Applied Science, Indianapolis, IN), 1 mm EGTA, 2 mm DTT,.2 mm PMSF, and Sigma protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO). The homogenate was then assayed for cyclic nucleotide phosphodiesterase activity in the presence of 1 mm 3 H-cAMP substrate, 4 mm MOPS (ph 7.5), 15 mm Mgacetate, 2 mm EGTA, and.2 mg/ml BSA as in Thompson, W. J. & Appleman, M. M. (1971) Biochemistry 1: Protein concentration in the cell extract was assayed with the Bradford assay. 1. Patrucco, E. et al. PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and -independent effects. Cell 118, (24). 2. Degerman, E. et al. Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B. Methods 14, (1998). 3. Ahmad, F. et al. Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells. J Immunol 164, (2).