Supporting Information

Miyara et al. 10.1073/pnas.1508224112 SI Methods senting cells (APCs) after pulsing with pooled peptides (10 μM) Diagnosis of Human Diseases. Diagnosis for active sarcoidosis, overnight at 37 °C as previously described (17). After irradiation 5 active SLE, Sjögren syndrome, systemic sclerosis, mycosis fun- (35 Gy), 3∼5 × 10 APCs were added to cultures containing 5 + goides, or myasthenia gravis were made according to previously 1∼3 × 10 CD4 T cells, and were fed with IL-2 (10 units/mL; described criteria (20, 33–36). Roche Diagnostics) and IL-7 (20 ng/mL; R&D Systems) in round- bottom 96-well plates (Thermo Fisher Scientic). Subsequently, one- Cytometry. Human peripheral blood mononuclear cells (PBMCs) half of the medium was replaced by fresh medium containing IL-2 and human thymocytes were prepared by Ficoll gradient cen- (20 units/mL) and IL-7 (40 ng/mL) twice per week. trifugation and stained with anti-hCD3, anti-hCD8, anti–hCD4- PerCP-Cy5.5 or –APC, anti–hCD25-PE, anti–hCD45RA-PE-Cy7, Synthetic Peptides of NY-ESO-1. Peptides 1–20 (MQAEGRGTGG- anti–ICOS-, anti–HLA-DR-PE (from BD Biosciences), anti- STGDADGPGG), NY-ESO-111–30 (STGDADGPGGPGIPD- CD31 (-APC from eBioscience), anti-hCD127 (-Pacific blue). In- GPGGN), NY-ESO-121–40 (PGIPDGPGGNAGGPGEAGAT), tracellular detection of FOXP3 with anti-hFOXP3 (PE or Alexa NY-ESO-131–50 (AGGPGEAGATGGRGPRGAGA), NY-ESO- Fluor 647, clone 259D/A7, BD Biosciences) and of Ki-67 antigen 141–60 (GGRGPRGAGAARASGPGGGA), NY-ESO-151–70 with Ki-67 antibody (FITC or PE from BD Biosciences) was (ARASGPGGGAPRGPHGGAAS), NY-ESO-161–80 (PRGPHG- performed on fixed and permeabilized cells using Intracellular GAASGLNGCCRCGA), NY-ESO-171–90 (GLNGCCRCGARG- Fixation and Permeabilization Buffer Set (eBioscience). Most PESRLLEF), NY-ESO-181–100 (RGPESRLLEFYLAMPFATPM), mAbs used for the study were obtained from the Lyoplate system NY-ESO-191–110 (YLAMPFATPMEAELARRSLA), NY-ESO- (BD Biosciences). All mAbs for the cell surface marker screening 1101–120 (EAELARRSLAQDAPPLPVPG), NY-ESO-1111–130 were unconjugated and secondary stained. Clones and species (QDAPPLPVPGVLLKEFTVSG), NY-ESO-1119–143 (PGVLLKE- for mAbs are described in Dataset S1. For subsequent cytometry FTVSGNILTIRLTAADHR), NY-ESO-1131–150 (NILTIRLTAA- analysis, Alexa Fluor 647-conjugated anti-CD15s mAbs (BD) DHRQLQLSIS), NY-ESO-1139–160 (AADHRQLQLSISSCLQQL- were used. For the analysis of cytokine production, PMBCs SLLM), NY-ESO-1151–170 (SCLQQLSLLMWITQCFLPVF), and were stimulated for 5 h with PMA and ionomycin. Data ac- NY-ESO-1161–180 (WITQCFLPVFLAQPPSGQRR) were obtained quired by LSR-Fortessa or FACSCanto-II were analyzed with from Invitrogen. FlowJo software. In Vitro Sensitization of CMV-Specific CD8+ T Cells. For in vitro + 6 Treg Suppression Assays. The 1 × 104 CFSE (1 μM, Invitrogen)- sensitization of CMV-specific CD8 T cells, 0.5∼1 × 10 PBMCs − + + labeled responder CD25 CD45RA CD4 T cells were cocul- were cultured with CMV peptides (CMV 495–503 for HLA- tured with 1 × 104 unlabeled cells assessed for their suppressive A*0201 restricted, 10 μM) in a round-bottom 96-well plate. After capacity together with 1 × 105 irradiated autologous accessory 8 h, one-half of the medium was replaced by fresh medium cells containing B cells and monocytes. Cells were stimulated containing IL-2 (20 units/mL) and IL-7 (40 ng/mL) and repeated + with 0.5 μg/mL plate-bound anti-CD3 (OKT3 mAb) in 96-well twice per week. Presensitized CD8 T cells were stained after round-bottom plate in RPMI medium supplemented with 7 d culture with PE-labeled HLA-A*0201/ tetramer for 10 min at 100 mL/L FBS (Bio West), 2 mM L-glutamine, 1 mM sodium 37 °C before additional staining with cell surface markers for pyruvate, 1% nonessential amino acid MEM, 100 units/mL peni- 15 min at 4 °C. cillin, 100 μg/mL streptomycin and amphotericin B (all from Gibco). Proliferation of CFSE-labeled cells was assessed by Enzyme-Linked Immunospot Assay. Flat-bottomed, 96-well nitro- flow cytometry after 84–90 h of culture. cellulose plates (MAHAS4510; Millipore) were coated with anti– IFN-γ mAb (4 μg/mL, 1-D1K; MABTECH) and incubated + + In Vitro Sensitization of NY-ESO-1–Specific CD4 T Cells. CD8 overnight at 4 °C and washed and blocked with RPMI with + T cells were depleted from PBMCs with CD8 Microbeads 100 mL/L AB serum. Presensitized 2∼5 × 104 CD4 T cells (Miltenyi Biotec). The remaining cells were subjected to negative and 5 × 104 target cells (peptide-pulsed autologous activated + + selection of CD4 T cells with CD4 T Cell Isolation Kit (Miltenyi T-cell APCs) were added to each well and incubated for 20–22 h + Biotec). CD4 T cells were treated with biotin-anti-CD15s mAb at 37 °C. Spots were developed using biotinylated anti–IFN-γ mAb for 15 min at 4 °C. Subsequently, anti-Biotin MicroBeads (0.2 μg/mL, 7-B6-1-biotin; MABTECH), alkaline phosphatase (Miltenyi Biotec) were added as described in the manufacturer’s conjugated streptavidin (Roche Diagnostics), and 5-bromo-4- protocol, then washed using PBS containing 20 mL/L FCS. chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma) and − CD15s cells were separated on autoMACS Pro Separator counted with a CTL ImmunoSpot S5 Micro Analyzer (Cellular − − (Miltenyi Biotec). CD4 CD8 cells were used as antigen-pre- Technologies).

Miyara et al. www.pnas.org/cgi/content/short/1508224112 1of15 CD5 CD4 CD2 CD3 CD1a CD1b CD1d CD4v4 BUFFER CD6 CD7 CD9 CD8a CD8b CD10 CD11c CD11a CD11b CD16 CD13 CD14 CD20 CD21 CD15 CD18 CD19 CD15s CD24 CD25 CD31 CD26 CD30 CD27 CD29 CD23 CD28 CD32 CD39 CD40 CD38 CD35 CD33 CD34 CD36 CD37 CD43 CD44 CD41a CD42b CD41b CD42A CD45RB CD45RA CD45RO CD45 CD48 CD47 CD46 CD49c CD49d CD49a CD49b CD49e CD50 CD58 CD59 CD54 CD56 CD57 CD51 CD53 CD55 CD61 CD63 CD64 CD66f CD66b CD62L CD62E CD62P CD66(a,c,d,e) CD77 CD74 CD75 CD69 CD70 CD71 CD72 CD73 CD79b CD80 CD81 CD83 CD84 CD85 CD22 Markers FOXP3

Fig. S1. (Continued)

Miyara et al. www.pnas.org/cgi/content/short/1508224112 2of15 CD89 CD91 CD94 CD86 CD87 CD88 CD90 CDw93 BUFFER CD95 CD99 CD97 CD98 CD100 CD103 CD105 CD102 CD99R CD112 CD114 CD116 CD117 CD108 CD106 CD109 CD107a CD107b CD119 CD118 CD122 CD123 CD124 CD126 CD120a CD121a CD121b CD130 CD127 CD134 CD137 CD138 CD135 CD140a CD137L CD128b (CD182) CD146 CD147 CD151 CD152 CD142 CD144 CD150 CD140b CD141 CD161 CD164 CD165 CD154 CD162 CD163 CD153 CD158a CD158b CD180 CD166 CD171 CD177 CD178 CD181 CD183 CD184 CD172b CD193 CD195 CD196 CD197 CD200 CD205 CD206 CD209 CD220 CD231 CD244 CD255 CD226 CD227 CD229 CD243 CD221 CD235a CD273 CD275 CD271 CD274 CD278 CD268 Markers FOXP3

Fig. S1. (Continued)

Miyara et al. www.pnas.org/cgi/content/short/1508224112 3of15 BLTR-1 BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER RAT BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER BUFFER 2-microglobulin CD49f CD104 CD201 CD132 CD210 CD120b CDw327 CDw328 CDw329 CD267 CD294 CD212 CD279 CD282 CD305 CD309 CD314 CD321 CLIP CD336 CD337 CD340 CD326 CD335 CD338 CMRF-56 CMRF-44 CLA DGD2 EGFR fMLPR TCR CXCR4 CXCR5 CXCR6 CXCR7 HPC HLA-A2 MIC A/B HLA-DQ HLA-A,B,C Integrine 7 Invariant NK T Invariant HLA-DR-DP-DQ V 8 NKB1 V 23 SSEA4 SSEA-1 SSEA-3 TRA-1-81 TRA-1-60 BUFFER RAT BUFFER Ms IgG1 Ms Ms IgG2b Ms Ms IgG2a Ms Control cell Control cell Control cell Control cell Control cell Control Control cell Control Rt IgM HLA-DR Ms IgM Ms Rt IgG1 Ms IgG3 Rt IgG2a Rt IgG2b

Markers FOXP3

Fig. S1. (Continued)

Miyara et al. www.pnas.org/cgi/content/short/1508224112 4of15 CCR3 CCR4 CCR5 CCR6 CCR7 CCR1 CCR2 Bambi B7-H4 CD1c CD44 CCR9 CD42c CD42d CCR10 CDCP1 CD218a CD52Bis CD68 CD52 CD60 CD75 CD75s CD65s CD66e CD66ce CD68Bis CD82 CD111 CD116 CD115 CD139 CD79a CD101 CD129 CD120a CD143 CD144 CD148 CD169 CD173 CD155 CD167a CD172a CD172g CD178 CD182 CD239 CD222 CD236 CD203c CD179b CD202b CD235B CD247 CD258 CD261 CD254 CD255 CD256 CD262 CD263 CD240CE CD265 CD266 CD270 CD271 CD277 CD282 CD289 CD286 CD276 CD334 CD298 CD317 CD319 CD349 CD307 CD318 CD344 CD300e DR3 c-Met GITRL CD357 CXCR3 FceR1a CRACC BUFFER CMKLR1 IL15R TSLPR SLP-76 Notch 1 TWEAKr Integrin 5 Markers FOXP3

Fig. S1. Cell surface marker expression by FOXP3-expressing CD4+ T cells. Expression of intracellular FOXP3 and each indicated surface marker assessed by + flow cytometry of PBMCs gated on CD4 T cells. Data are representative of 6 healthy donors. PBMCs were first incubated with unconjugated antibodies, then with dye-conjugated antibodies (CD3, CD8, CD4, CD45RA, CD25, HLA-DR, ICOS, CD31, FOXP3, Ki-67, and Helios). In CD4 panels, because unconjugated and dye-conjugated anti-CD4 mAb was of the same clone (RPA-T4), the dye-conjugated anti-CD4 mAb failed to stain after incubating with the unconjugated anti-CD4 mAb.

Miyara et al. www.pnas.org/cgi/content/short/1508224112 5of15 CD4 CD2 CD3 CD5 CD1a CD1b CD1d CD4v4 BUFFER CD9 CD6 CD7 CD10 CD8a CD8b CD11c CD11b CD11a CD13 CD14 CD16 CD20 CD15 CD18 CD19 CD21 CD15s CD27 CD30 CD23 CD24 CD25 CD26 CD28 CD29 CD31 CD39 CD36 CD32 CD34 CD33 CD35 CD37 CD38 CD40 CD44 CD43 CD41a CD41b CD42b CD42A CD45RB CD45RA CD45RO CD45 CD46 CD47 CD48 CD49c CD49d CD49a CD49b CD49e CD53 CD55 CD58 CD50 CD51 CD54 CD56 CD57 CD59 CD63 CD61 CD64 CD66f CD62L CD66b CD62E CD62P CD66(a,c,d,e) CD77 CD73 CD71 CD69 CD70 CD72 CD75 CD74 CD79b CD80 CD81 CD83 CD84 CD85 CD22 Markers Ki-67

Fig. S2. (Continued)

Miyara et al. www.pnas.org/cgi/content/short/1508224112 6of15 i.S2. Fig. iaae al. et Miyara (Continued) www.pnas.org/cgi/content/short/1508224112

CD268 CD221 CD193 CD166 CD153 CD140b CD127 CD118 CD106 CD95 BUFFER

CD271 CD226 CD195 CD171 CD154 CD141 CD128b (CD182) CD119 CD107a CD97 CDw93

CD273 CD227 CD196 CD172b CD158a CD142 CD130 CD120a CD107b CD98 CD86

CD274 CD229 CD197 CD177 CD158b CD144 CD134 CD121a CD108 CD99R CD87

CD275 CD231 CD200 CD178 CD161 CD146 CD135 CD121b CD109 CD99 CD88

CD278 CD235a CD205 CD180 CD162 CD147 CD137L CD122 CD112 CD100 CD89

CD243 CD206 CD181 CD163 CD150 CD137 CD123 CD114 CD102 CD90

Markers

Ki-67 CD244 CD209 CD183 CD164 CD151 CD138 CD124 CD116 CD103 CD91

CD255 CD220 CD184 CD165 CD152 CD140a CD126 CD117 CD105 CD94 7of15 iaae al. et Miyara S2. Fig. (Continued) www.pnas.org/cgi/content/short/1508224112

Ms IgG3 Control cell NKB1 HPC CXCR4 CD326 CD212 CDw327 BUFFER BUFFER TCR

Ms IgM Control cell SSEA-1 HLA-A,B,C CXCR5 CD335 CD267 CDw328 BUFFER BUFFER BLTR-1

Rt IgG1 Control cell SSEA4 HLA-A2 CXCR6 CD336 CD279 CDw329 BUFFER BUFFER BUFFER RAT

Rt IgG2a Control cell SSEA-3 HLA-DQ CD158b CD337 CD282 CD49f BUFFER BUFFER BUFFER

Rt IgG2b Control cell TRA-1-60 HLA-DR-DP-DQ CLA CD338 CD294 CD104 BUFFER BUFFER BUFFER

Rt IgM Control cell TRA-1-81 HLA-DR DGD2 CD340 CD305 CD120b BUFFER BUFFER BUFFER

Ms IgG1 V 8 Invariant NK T EGFR CLIP CD309 CD132 BUFFER BUFFER BUFFER

Markers Ms IgG2a V 23 Integrine 7 fMLPR CMRF-44 CD314 CD201 BUFFER BUFFER BUFFER Ki-67

Ms IgG2b BUFFER RAT MIC A/B TCR CMRF-56 CD321 CD210 2-microglobulin BUFFER BUFFER 8of15 lwctmtyo BC ae nCD4 on gated PBMCs of cytometry flow n y-ojgtdat-D A a o mAb. was anti-CD4 mAb conjugated HLA-DR, anti-CD4 CD25, CD45RA, dye-conjugated CD4, CD8, and (CD3, antibodies dye-conjugated with then i.S2. Fig. iaae al. et Miyara elsraemre xrsinb Ki-67 by expression marker surface Cell www.pnas.org/cgi/content/short/1508224112

IL15R CD357 CD298 CD265 CD240CE CD178 CD143 CD79a CD52 CCR9 Bambi

+ Integrin 5 c-Met CD300e CD266 CD247 CD179b CD144 CD82 CD60 CCR10 B7-H4 el.Dt r ersnaieo elh oos PBMC donors. healthy 6 of representative are Data cells. T h aecoe(P-4,tedecnuae anti- dye-conjugated the (RPA-T4), clone same the f – xrsigCD4 expressing Notch 1 CMKLR1 CD307 CD270 CD254 CD182 CD148 CD101 CD65s CDCP1 CCR1

SLP-76 CRACC CD317 CD271 CD255 CD202b CD155 CD111 CD66ce CD218a CCR2 + el.Epeso fitaellrK-7an Ki-67 intracellular of Expression cells. T

TSLPR CXCR3 CD318 CD276 CD256 CD203c CD167a CD115 CD66e CD1c CCR3 CS D1 OP,K-7 n eis.I D aes eas unconjugate because panels, CD4 In Helios). and Ki-67, FOXP3, CD31, ICOS,

TWEAKr DR3 CD319 CD277 CD258 CD222 CD169 CD116 CD68 CD42c CCR4

BUFFER CD334 CD282 CD261 CD235B CD172a CD120a CD68Bis CD42d CCR5 D A aldt ti afte stain to failed mAb CD4 eefrticbtdwt nojgtdantibodies, unconjugated with incubated first were s Markers

Ki-67 FceR1a CD344 CD286 CD262 CD236 CD172g CD129 CD75s CD44 CCR6 ahidctdsurfa indicated each d

GITRL CD349 CD289 CD263 CD239 CD173 CD139 CD75 CD52Bis CCR7 nuaigwt h un- the with incubating r emre a marker ce sse by ssessed 9of15 d i.S3. Fig. iaae al. et Miyara (Continued) www.pnas.org/cgi/content/short/1508224112

CD80 CD69 CD61 CD50 CD45 CD41a CD32 CD23 CD13 CD6 BUFFER

CD81 CD70 CD62E CD51 CD46 CD41b CD33 CD24 CD14 CD7 CD1a

CD83 CD71 CD62L CD53 CD47 CD42A CD34 CD25 CD15s CD8a CD1b

CD84 CD72 CD62P CD54 CD48 CD42b CD35 CD26 CD15 CD8b CD1d

CD85 CD73 CD63 CD55 CD49a CD43 CD36 CD27 CD16 CD9 CD2

CD22 CD74 CD64 CD56 CD49b CD44 CD37 CD28 CD18 CD10 CD3

CD75 CD66(a,c,d,e) CD57 CD49c CD45RA CD38 CD29 CD19 CD11a CD4v4

Markers

Helios CD77 CD66b CD58 CD49d CD45RB CD39 CD30 CD20 CD11b CD4

CD79b CD66f CD59 CD49e CD45RO CD40 CD31 CD21 CD11c CD5 0o 15 of 10 i.S3. Fig. iaae al. et Miyara (Continued) www.pnas.org/cgi/content/short/1508224112

CD268 CD221 CD193 CD166 CD153 CD140b CD127 CD118 CD106 CD95 BUFFER

CD271 CD226 CD195 CD171 CD154 CD141 CD128b (CD182) CD119 CD107a CD97 CDw93

CD273 CD227 CD196 CD172b CD158a CD142 CD130 CD120a CD107b CD98 CD86

CD274 CD229 CD197 CD177 CD158b CD144 CD134 CD121a CD108 CD99R CD87

CD275 CD231 CD200 CD178 CD161 CD146 CD135 CD121b CD109 CD99 CD88

CD278 CD235a CD205 CD180 CD162 CD147 CD137L CD122 CD112 CD100 CD89

CD243 CD206 CD181 CD163 CD150 CD137 CD123 CD114 CD102 CD90

Markers CD244 CD209 CD183 CD164 CD151 CD138 CD124 CD116 CD103 CD91 Helios

CD255 CD220 CD184 CD165 CD152 CD140a CD126 CD117 CD105 CD94 1o 15 of 11 Fig. S3. (Continued)

Miyara et al. www.pnas.org/cgi/content/short/1508224112 12 of 15 Fig. S3. Cell surface marker expression by Helios-expressing CD4+ T cells. Expression of intracellular Helios and each indicated surface marker assessed by flow cytometry of PBMCs gated on CD4+ T cells. Data are representative of 6 healthy donors. PBMCs were first incubated with unconjugated antibodies, then with dye-conjugated antibodies (CD3, CD8, CD4, CD45RA, CD25, HLA-DR, ICOS, CD31, FOXP3, Ki-67, and Helios). In CD4 panels, because unconjugated and dye-conjugated anti-CD4 mAb was of the same clone (RPA-T4), the dye-conjugated anti-CD4 mAb failed to stain after incubating with the unconjugated anti-CD4 mAb. CD130 CD221 CD55 CD100 CD26 CD305 CD321 CD127 Markers FOXP3

Fig. S4. Cell surface markers down-regulated in FOXP3high eTreg cells. Eight surface markers were down-regulated in FOXP3high eTreg cells detected by analyzing 323 mAbs as shown in Fig. S1. Expression of intracellular FOXP3 and each indicated surface marker was analyzed by flow cytometry of PBMCs gated on CD4+ T cells. Data are representative of six healthy donors.

Miyara et al. www.pnas.org/cgi/content/short/1508224112 13 of 15 A B CD71 ICOS CD39

Helios ICOS-L

Ki-67

Fig. S5. Surface markers correlated with Ki-67 and Helios expression. (A) Expression of Ki-67 correlated with surface expression of CD71, ICOS, and ICOS-L. + Representative dot plots depicting CD4 T-cell expression of intranuclear Ki-67 and the surface markers CD71, ICOS, and ICOS-L. Data are representative of six + healthy blood donors. (B) Expression of CD39 correlated with intracellular expression of Helios. Representative dot plots depicting the expression by CD4 T cells of intracellular Helios and CD39. The expression of CD39 corresponded to intracellular expression of Helios in some donors (Bottom) but not in others (Top).

Miyara et al. www.pnas.org/cgi/content/short/1508224112 14 of 15 CD25-CD45RA+ eTreg cells Responder + alone Responder cells (1 to 1 ratio) Day 4

4,690 1,148 (83.8%) (48.0%)

Day 6 Cell number 36,134 586 (99.0%) (42.7%)

CFSE (Responder cells)

Fig. S6. Complete suppression of effector T cell proliferation by eTreg cells. Flow cytometric analysis of CFSE dilution by human CFSE-labeled responder cells after 4 d (Top)or6d(Bottom) coculture with (Right) or without eTregs isolated by the previously described protocol (4) (Left). Number (and percentage) of cycling cell is indicated. In coculture of human eTregs with responder cells, proliferating responder cells could be detected on day 4, yet only a minority of them entered the third cycle of division, whereas most responder cells cultured alone had entered the third and fourth cycle. Furthermore, on day 6, most dividing responder cells cultured alone had entered the sixth or the seventh cycle of cell division. Treg-cocultured responder cells still remained halted at the third cycle, whereas decreasing amplitudes are observed in successive peaks of proliferation. This pattern of CFSE dilution in suppressed responder cells indicates that the few responder cells that have proliferated in the first few days of coculture have arrested their proliferation. Thus, human Treg cells can potently arrest the proliferation of responder cells.

Dataset S1. Monoclonal antibodies used in the study

Dataset S1

Miyara et al. www.pnas.org/cgi/content/short/1508224112 15 of 15