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 Fig. S1. Miyara et al. (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 CD77 CD66b CD58 CD49d CD45RB CD39 CD30 CD20 CD11b CD4 FOXP3 CD79b CD66f CD59 CD49e CD45RO CD40 CD31 CD21 CD11c CD5 2of15 Fig. S1. Miyara et al. (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 FOXP3 CD244 CD209 CD183 CD164 CD151 CD138 CD124 CD116 CD103 CD91 CD255 CD220 CD184 CD165 CD152 CD140a CD126 CD117 CD105 CD94 3of15 Miyara et al. Fig. S1. (Continued) www.pnas.org/cgi/content/short/1508224112 Ms IgG3 Control cell NKB1 HPC CXCR4 CD326 CD212 CDw327 BUFFER BUFFER 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 CXCR7 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 IgMControl 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 FOXP3 Ms IgG2b BUFFER RAT MIC A/B TCR CMRF-56 CD321 CD210 2-microglobulin BUFFER BUFFER 4of15 flow cytometry of PBMCs gated on CD4 anti-CD4 mAb. dye-conjugated anti-CD4 mAb waswith dye-conjugated of the antibodies same (CD3, clone CD8, (RPA-T4), CD4, CD45RA, the CD25, dye-conjugated HLA-DR, anti-CD4 ICOS, mAb CD31, failed FOXP3, to Ki-67, stain and after Helios). In incubating CD4 with panels, the because unconjugated unconjugated and Fig. S1. Miyara et al. Cell surface marker expression by FOXP3-expressing CD4 www.pnas.org/cgi/content/short/1508224112 IL15R CD357 CD298 CD265 CD240CE CD178 CD143 CD79a CD52 CCR9 Bambi + T cells. Data are representative of 6 healthy donors. PBMCs were first incubated with unconjugated antibodies,Integrin then 5 c-Met CD300e CD266 CD247 CD179b CD144 CD82 CD60 CCR10 B7-H4 Notch 1 CMKLR1 CD307 CD270 CD254 CD182 CD148 CD101 CD65s CDCP1 CCR1 SLP-76 CRACC CD317 CD271 CD255 CD202b CD155 CD111 CD66ce CD218a CCR2 + T cells. Expression of intracellular FOXP3 and each indicated surface marker assessed by TSLPR CXCR3 CD318 CD276 CD256 CD203c CD167a CD115 CD66e CD1c CCR3 TWEAKr DR3 CD319 CD277 CD258 CD222 CD169 CD116 CD68 CD42c CCR4 BUFFER CD334 CD282 CD261 CD235B CD172a CD120a CD68Bis CD42d CCR5 Markers FOXP3 FceR1a CD344 CD286 CD262 CD236 CD172g CD129 CD75s CD44 CCR6 GITRL CD349 CD289 CD263 CD239 CD173 CD139 CD75 CD52Bis CCR7 5of15 Fig. S2. Miyara et al. (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 CD77 CD66b CD58 CD49d CD45RB CD39 CD30 CD20 CD11b CD4 Ki-67 CD79b CD66f CD59 CD49e CD45RO CD40 CD31 CD21 CD11c CD5 6of15 Fig.