Supporting Information - PNAS · Supporting Information Miyara et al. 10.1073/pnas.1508224112 SI...
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Supporting Information Miyara et al. 10.1073/pnas.1508224112 SI Methods Diagnosis of Human Diseases. Diagnosis for active sarcoidosis, active SLE, Sjögren syndrome, systemic sclerosis, mycosis fun- goides, or myasthenia gravis were made according to previously described criteria (20, 33–36). Cytometry. Human peripheral blood mononuclear cells (PBMCs) and human thymocytes were prepared by Ficoll gradient cen- trifugation and stained with anti-hCD3, anti-hCD8, anti–hCD4- PerCP-Cy5.5 or –APC, anti–hCD25-PE, anti–hCD45RA-PE-Cy7, anti–ICOS-, anti–HLA-DR-PE (from BD Biosciences), anti- CD31 (-APC from eBioscience), anti-hCD127 (-Pacific blue). In- tracellular detection of FOXP3 with anti-hFOXP3 (PE or Alexa Fluor 647, clone 259D/A7, BD Biosciences) and of Ki-67 antigen with Ki-67 antibody (FITC or PE from BD Biosciences) was performed on fixed and permeabilized cells using Intracellular Fixation and Permeabilization Buffer Set (eBioscience). Most mAbs used for the study were obtained from the Lyoplate system (BD Biosciences). All mAbs for the cell surface marker screening were unconjugated and secondary stained. Clones and species for mAbs are described in Dataset S1. For subsequent cytometry analysis, Alexa Fluor 647-conjugated anti-CD15s mAbs (BD) were used. For the analysis of cytokine production, PMBCs were stimulated for 5 h with PMA and ionomycin. Data ac- quired by LSR-Fortessa or FACSCanto-II were analyzed with FlowJo software. Treg Suppression Assays. The 1 × 10 4 CFSE (1 μM, Invitrogen)- labeled responder CD25 − CD45RA + CD4 + T cells were cocul- tured with 1 × 10 4 unlabeled cells assessed for their suppressive capacity together with 1 × 10 5 irradiated autologous accessory cells containing B cells and monocytes. Cells were stimulated with 0.5 μg/mL plate-bound anti-CD3 (OKT3 mAb) in 96-well round-bottom plate in RPMI medium supplemented with 100 mL/L FBS (Bio West), 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino acid MEM, 100 units/mL peni- cillin, 100 μg/mL streptomycin and amphotericin B (all from Gibco). Proliferation of CFSE-labeled cells was assessed by flow cytometry after 84–90 h of culture. In Vitro Sensitization of NY-ESO-1–Specific CD4 + T Cells. CD8 + T cells were depleted from PBMCs with CD8 Microbeads (Miltenyi Biotec). The remaining cells were subjected to negative selection of CD4 + T cells with CD4 + T Cell Isolation Kit (Miltenyi Biotec). CD4 + T cells were treated with biotin-anti-CD15s mAb for 15 min at 4 °C. Subsequently, anti-Biotin MicroBeads (Miltenyi Biotec) were added as described in the manufacturer’s protocol, then washed using PBS containing 20 mL/L FCS. CD15s − cells were separated on autoMACS Pro Separator (Miltenyi Biotec). CD4 − CD8 − cells were used as antigen-pre- senting cells (APCs) after pulsing with pooled peptides (10 μM) overnight at 37 °C as previously described (17). After irradiation (35 Gy), 3∼5 × 10 5 APCs were added to cultures containing 1∼3 × 10 5 CD4 + T cells, and were fed with IL-2 (10 units/mL; Roche Diagnostics) and IL-7 (20 ng/mL; R&D Systems) in round- bottom 96-well plates (Thermo Fisher Scientic). Subsequently, one- half of the medium was replaced by fresh medium containing IL-2 (20 units/mL) and IL-7 (40 ng/mL) twice per week. Synthetic Peptides of NY-ESO-1. Peptides 1–20 (MQAEGRGTGG- STGDADGPGG), NY-ESO-1 11–30 (STGDADGPGGPGIPD- GPGGN), NY-ESO-1 21–40 (PGIPDGPGGNAGGPGEAGAT), NY-ESO-1 31–50 (AGGPGEAGATGGRGPRGAGA), NY-ESO- 1 41– 60 (GGRGPRGAGAARASGPGGGA), NY-ESO-1 51– 70 (ARASGPGGGAPRGPHGGAAS), NY-ESO-1 61–80 (PRGPHG- GAASGLNGCCRCGA), NY-ESO-1 71–90 (GLNGCCRCGARG- PESRLLEF), NY-ESO-1 81–100 (RGPESRLLEFYLAMPFATPM), NY-ESO-1 91–110 (YLAMPFATPMEAELARRSLA), NY-ESO- 1 101–120 (EAELARRSLAQDAPPLPVPG), NY-ESO-1 111–130 (QDAPPLPVPGVLLKEFTVSG), NY-ESO-1 119–143 (PGVLLKE- FTVSGNILTIRLTAADHR), NY-ESO-1 131–150 (NILTIRLTAA- DHRQLQLSIS), NY-ESO-1 139–160 (AADHRQLQLSISSCLQQL- SLLM), NY-ESO-1 151–170 (SCLQQLSLLMWITQCFLPVF), and NY-ESO-1 161–180 (WITQCFLPVFLAQPPSGQRR) were obtained from Invitrogen. In Vitro Sensitization of CMV-Specific CD8 + T Cells. For in vitro sensitization of CMV-specific CD8 + T cells, 0.5∼1 × 10 6 PBMCs were cultured with CMV peptides (CMV 495–503 for HLA- A*0201 restricted, 10 μM) in a round-bottom 96-well plate. After 8 h, one-half of the medium was replaced by fresh medium containing IL-2 (20 units/mL) and IL-7 (40 ng/mL) and repeated twice per week. Presensitized CD8 + T cells were stained after 7 d culture with PE-labeled HLA-A*0201/ tetramer for 10 min at 37 °C before additional staining with cell surface markers for 15 min at 4 °C. Enzyme-Linked Immunospot Assay. Flat-bottomed, 96-well nitro- cellulose plates (MAHAS4510; Millipore) were coated with anti– IFN-γ mAb (4 μg/mL, 1-D1K; MABTECH) and incubated overnight at 4 °C and washed and blocked with RPMI with 100 mL/L AB serum. Presensitized 2∼5 × 10 4 CD4 + T cells and 5 × 10 4 target cells (peptide-pulsed autologous activated T-cell APCs) were added to each well and incubated for 20–22 h at 37 °C. Spots were developed using biotinylated anti–IFN-γ mAb (0.2 μg/mL, 7-B6-1-biotin; MABTECH), alkaline phosphatase conjugated streptavidin (Roche Diagnostics), and 5-bromo-4- chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma) and counted with a CTL ImmunoSpot S5 Micro Analyzer (Cellular Technologies). Miyara et al. www.pnas.org/cgi/content/short/1508224112 1 of 15
Transcript of Supporting Information - PNAS · Supporting Information Miyara et al. 10.1073/pnas.1508224112 SI...
Supporting InformationMiyara et al. 10.1073/pnas.1508224112SI MethodsDiagnosis of Human Diseases. Diagnosis for active sarcoidosis,active SLE, Sjögren syndrome, systemic sclerosis, mycosis fun-goides, or myasthenia gravis were made according to previouslydescribed criteria (20, 33–36).
Cytometry.Human peripheral blood mononuclear cells (PBMCs)and human thymocytes were prepared by Ficoll gradient cen-trifugation and stained with anti-hCD3, anti-hCD8, anti–hCD4-PerCP-Cy5.5 or –APC, anti–hCD25-PE, anti–hCD45RA-PE-Cy7,anti–ICOS-, anti–HLA-DR-PE (from BD Biosciences), anti-CD31 (-APC from eBioscience), anti-hCD127 (-Pacific blue). In-tracellular detection of FOXP3 with anti-hFOXP3 (PE or AlexaFluor 647, clone 259D/A7, BD Biosciences) and of Ki-67 antigenwith Ki-67 antibody (FITC or PE from BD Biosciences) wasperformed on fixed and permeabilized cells using IntracellularFixation and Permeabilization Buffer Set (eBioscience). MostmAbs used for the study were obtained from the Lyoplate system(BD Biosciences). All mAbs for the cell surface marker screeningwere unconjugated and secondary stained. Clones and speciesfor mAbs are described in Dataset S1. For subsequent cytometryanalysis, Alexa Fluor 647-conjugated anti-CD15s mAbs (BD)were used. For the analysis of cytokine production, PMBCswere stimulated for 5 h with PMA and ionomycin. Data ac-quired by LSR-Fortessa or FACSCanto-II were analyzed withFlowJo software.
Treg Suppression Assays. The 1 × 104 CFSE (1 μM, Invitrogen)-labeled responder CD25−CD45RA+CD4+ T cells were cocul-tured with 1 × 104 unlabeled cells assessed for their suppressivecapacity together with 1 × 105 irradiated autologous accessorycells containing B cells and monocytes. Cells were stimulatedwith 0.5 μg/mL plate-bound anti-CD3 (OKT3 mAb) in 96-wellround-bottom plate in RPMI medium supplemented with100 mL/L FBS (Bio West), 2 mM L-glutamine, 1 mM sodiumpyruvate, 1% nonessential amino acid MEM, 100 units/mL peni-cillin, 100 μg/mL streptomycin and amphotericin B (all fromGibco). Proliferation of CFSE-labeled cells was assessed byflow cytometry after 84–90 h of culture.
In Vitro Sensitization of NY-ESO-1–Specific CD4+ T Cells. CD8+
T cells were depleted from PBMCs with CD8 Microbeads(Miltenyi Biotec). The remaining cells were subjected to negativeselection of CD4+ T cells with CD4+ T Cell Isolation Kit (MiltenyiBiotec). CD4+ T cells were treated with biotin-anti-CD15s mAbfor 15 min at 4 °C. Subsequently, anti-Biotin MicroBeads(Miltenyi Biotec) were added as described in the manufacturer’sprotocol, then washed using PBS containing 20 mL/L FCS.CD15s− cells were separated on autoMACS Pro Separator(Miltenyi Biotec). CD4−CD8− cells were used as antigen-pre-
senting cells (APCs) after pulsing with pooled peptides (10 μM)overnight at 37 °C as previously described (17). After irradiation(35 Gy), 3∼5 × 105 APCs were added to cultures containing1∼3 × 105 CD4+ T cells, and were fed with IL-2 (10 units/mL;Roche Diagnostics) and IL-7 (20 ng/mL; R&D Systems) in round-bottom 96-well plates (Thermo Fisher Scientic). Subsequently, one-half of the medium was replaced by fresh medium containing IL-2(20 units/mL) and IL-7 (40 ng/mL) twice per week.
Synthetic Peptides of NY-ESO-1. Peptides 1–20 (MQAEGRGTGG-STGDADGPGG), NY-ESO-111–30 (STGDADGPGGPGIPD-GPGGN), NY-ESO-121–40 (PGIPDGPGGNAGGPGEAGAT),NY-ESO-131–50 (AGGPGEAGATGGRGPRGAGA), NY-ESO-141–60 (GGRGPRGAGAARASGPGGGA), NY-ESO-151–70(ARASGPGGGAPRGPHGGAAS), NY-ESO-161–80 (PRGPHG-GAASGLNGCCRCGA), NY-ESO-171–90 (GLNGCCRCGARG-PESRLLEF), NY-ESO-181–100 (RGPESRLLEFYLAMPFATPM),NY-ESO-191–110 (YLAMPFATPMEAELARRSLA), NY-ESO-1101–120 (EAELARRSLAQDAPPLPVPG), NY-ESO-1111–130(QDAPPLPVPGVLLKEFTVSG), NY-ESO-1119–143 (PGVLLKE-FTVSGNILTIRLTAADHR), NY-ESO-1131–150 (NILTIRLTAA-DHRQLQLSIS), NY-ESO-1139–160 (AADHRQLQLSISSCLQQL-SLLM), NY-ESO-1151–170 (SCLQQLSLLMWITQCFLPVF), andNY-ESO-1161–180 (WITQCFLPVFLAQPPSGQRR) were obtainedfrom Invitrogen.
In Vitro Sensitization of CMV-Specific CD8+ T Cells. For in vitrosensitization of CMV-specific CD8+ T cells, 0.5∼1 × 106 PBMCswere cultured with CMV peptides (CMV 495–503 for HLA-A*0201 restricted, 10 μM) in a round-bottom 96-well plate. After8 h, one-half of the medium was replaced by fresh mediumcontaining IL-2 (20 units/mL) and IL-7 (40 ng/mL) and repeatedtwice per week. Presensitized CD8+ T cells were stained after7 d culture with PE-labeled HLA-A*0201/ tetramer for 10 min at37 °C before additional staining with cell surface markers for15 min at 4 °C.
Enzyme-Linked Immunospot Assay. Flat-bottomed, 96-well nitro-cellulose plates (MAHAS4510; Millipore) were coated with anti–IFN-γ mAb (4 μg/mL, 1-D1K; MABTECH) and incubatedovernight at 4 °C and washed and blocked with RPMI with100 mL/L AB serum. Presensitized 2∼5 × 104 CD4+ T cellsand 5 × 104 target cells (peptide-pulsed autologous activatedT-cell APCs) were added to each well and incubated for 20–22 hat 37 °C. Spots were developed using biotinylated anti–IFN-γmAb(0.2 μg/mL, 7-B6-1-biotin; MABTECH), alkaline phosphataseconjugated streptavidin (Roche Diagnostics), and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma) andcounted with a CTL ImmunoSpot S5 Micro Analyzer (CellularTechnologies).
Miyara et al. www.pnas.org/cgi/content/short/1508224112 1 of 15
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Fig. S1. Cell surface marker expression by FOXP3-expressing CD4+ T cells. Expression of intracellular FOXP3 and each indicated surface marker assessed byflow cytometry of PBMCs gated on CD4+ T cells. Data are representative of 6 healthy donors. PBMCs were first incubated with unconjugated antibodies, thenwith dye-conjugated antibodies (CD3, CD8, CD4, CD45RA, CD25, HLA-DR, ICOS, CD31, FOXP3, Ki-67, and Helios). In CD4 panels, because unconjugated anddye-conjugated anti-CD4 mAb was of the same clone (RPA-T4), the dye-conjugated anti-CD4 mAb failed to stain after incubating with the unconjugatedanti-CD4 mAb.
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Fig. S2. (Continued)
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Fig. S2. (Continued)
Miyara et al. www.pnas.org/cgi/content/short/1508224112 7 of 15
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R
CD
w32
7
CD
w32
8
CD
w32
9
CD
49f
CD
104
CD
120b
CD
132
CD
201
CD
210
CD
212
CD
267
CD
279
CD
282
CD
294
CD
305
CD
309
CD
314
CD
321
CD
326
CD
335
CD
336
CD
337
CD
338
CD
340
CLI
P
CM
RF-
44
CM
RF-
56
CXC
R4
CXC
R5
CXC
R6
CD
158b
CLA
DG
D2
EG
FR
fMLP
R
TCR
HP
C
HLA
-A,B
,C
HLA
-A2
HLA
-DQ
HLA
-DR
Inte
grin
e 7
MIC
A/B
HLA
-DR
-DP
-DQ
Inva
riant
NK
T
BU
FFE
R R
AT
2-m
icro
glob
ulin
NK
B1
SS
EA
-1
SS
EA
4
SS
EA
-3
TRA
-1-6
0
TRA
-1-8
1
V 8
V 2
3
Con
trol c
ell
Con
trol c
ell
Con
trol c
ell
Con
trol c
ell
Con
trol c
ell
Con
trol c
ell
Ms
IgG
1
Ms
IgG
2a
Ms
IgG
2b
Ms
IgG
3
Ms
IgM
Rt I
gG1
Rt I
gG2a
Rt I
gG2b
Rt I
gM
BU
FFE
R R
AT
Ki-67Mar
kers
Fig. S2. (Continued)
Miyara et al. www.pnas.org/cgi/content/short/1508224112 8 of 15
Bam
bi
B7-
H4
CC
R1
CC
R2
CC
R3
CC
R4
CC
R5
CC
R6
CC
R7
CC
R9
CC
R10
CD
CP
1
CD
218a
CD
1c
CD
42c
CD
42d
CD
44
CD
52B
is
CD
52
CD
60
CD
65s
CD
66ce
CD
66e
CD
68
CD
68B
is
CD
75s
CD
75
CD
79a
CD
82
CD
101
CD
111
CD
115
CD
116
CD
120a
CD
129
CD
139
CD
143
CD
144
CD
148
CD
155
CD
167a
CD
169
CD
172a
CD
172g
CD
173
CD
178
CD
179b
CD
182
CD
202b
CD
203c
CD
222
CD
235B
CD
236
CD
239
CD
240C
E
CD
247
CD
254
CD
255
CD
256
CD
258
CD
261
CD
262
CD
263
CD
265
CD
266
CD
270
CD
271
CD
276
CD
277
CD
282
CD
286
CD
289
CD
298
CD
300e
CD
307
CD
317
CD
318
CD
319
CD
334
CD
344
CD
349
CD
357
c-M
et
CM
KLR
1
CR
AC
C
CXC
R3
DR
3
BU
FFE
R
FceR
1a
GIT
RL
IL15
R
Inte
grin
5
Not
ch 1
SLP
-76
TSLP
R
TWEA
Kr
Ki-67Mar
kers
Fig. S2. Cell surface marker expression by Ki-67–expressing CD4+ T cells. Expression of intracellular Ki-67 and each indicated surface marker assessed byflow 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 unconjugatedand 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 un-conjugated anti-CD4 mAb.
Miyara et al. www.pnas.org/cgi/content/short/1508224112 9 of 15
BU
FFE
R
CD
1a
CD
1b
CD
1d
CD
2
CD
3
CD
4v4
CD
4
CD
5
CD
6
CD
7
CD
8a
CD
8b
CD
9
CD
10
CD
11a
CD
11b
CD
11c
CD
13
CD
14
CD
15s
CD
15
CD
16
CD
18
CD
19
CD
20
CD
21
CD
23
CD
24
CD
25
CD
26
CD
27
CD
28
CD
29
CD
30
CD
31
CD
32
CD
33
CD
34
CD
35
CD
36
CD
37
CD
38
CD
39
CD
40
CD
41a
CD
41b
CD
42A
CD
42b
CD
43
CD
44
CD
45R
A
CD
45R
B
CD
45R
O
CD
45
CD
46
CD
47
CD
48
CD
49a
CD
49b
CD
49c
CD
49d
CD
49e
CD
61
CD
62E
CD
62L
CD
62P
CD
63
CD
64
CD
66b
CD
66f
CD
69
CD
70
CD
71
CD
72
CD
73
CD
74
CD
75
CD
77
CD
79b
CD
80
CD
81
CD
83
CD
84
CD
85
CD
22
CD
66(a
,c,d
,e)
HeliosMar
kers
CD
50
CD
51
CD
53
CD
54
CD
55
CD
56
CD
57
CD
58
CD
59
Fig. S3. (Continued)
Miyara et al. www.pnas.org/cgi/content/short/1508224112 10 of 15
BU
FFE
R
CD
w93
CD
86
CD
87
CD
88
CD
89
CD
90
CD
91
CD
94
CD
95
CD
97
CD
98
CD
99R
CD
99
CD
100
CD
102
CD
103
CD
105
CD
106
CD
107a
CD
107b
CD
108
CD
109
CD
112
CD
114
CD
116
CD
117
CD
118
CD
119
CD
120a
CD
121a
CD
121b
CD
122
CD
123
CD
124
CD
126
CD
127
CD
130
CD
134
CD
135
CD
137L
CD
137
CD
138
CD
140a
CD
140b
CD
141
CD
142
CD
144
CD
146
CD
147
CD
150
CD
151
CD
152
CD
153
CD
154
CD
158a
CD
158b
CD
161
CD
162
CD
163
CD
164
CD
165
CD
166
CD
171
CD
172b
CD
177
CD
178
CD
180
CD
181
CD
183
CD
184
CD
193
CD
195
CD
196
CD
197
CD
200
CD
205
CD
206
CD
209
CD
220
CD
128b
(CD
182)
CD
221
CD
226
CD
227
CD
229
CD
231
CD
235a
CD
243
CD
244
CD
255
CD
268
CD
271
CD
273
CD
274
CD
275
CD
278
HeliosMar
kers
Fig. S3. (Continued)
Miyara et al. www.pnas.org/cgi/content/short/1508224112 11 of 15
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 byflow cytometry of PBMCs gated on CD4+ T cells. Data are representative of 6 healthy donors. PBMCs were first incubated with unconjugated antibodies, thenwith dye-conjugated antibodies (CD3, CD8, CD4, CD45RA, CD25, HLA-DR, ICOS, CD31, FOXP3, Ki-67, and Helios). In CD4 panels, because unconjugated anddye-conjugated anti-CD4 mAb was of the same clone (RPA-T4), the dye-conjugated anti-CD4 mAb failed to stain after incubating with the unconjugatedanti-CD4 mAb.
CD
127
CD
26
CD
55
CD
100
CD
221
CD
130
CD
305
CD
321
FOXP3Mar
kers
Fig. S4. Cell surface markers down-regulated in FOXP3high eTreg cells. Eight surface markers were down-regulated in FOXP3high eTreg cells detected byanalyzing 323 mAbs as shown in Fig. S1. Expression of intracellular FOXP3 and each indicated surface marker was analyzed by flow cytometry of PBMCs gatedon CD4+ T cells. Data are representative of six healthy donors.
Miyara et al. www.pnas.org/cgi/content/short/1508224112 13 of 15
Ki-67
A B
Helios
CD
71IC
OS
CD
39
ICO
S-L
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 sixhealthy 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
586(42.7%)
36,134(99.0%)
4,690(83.8%)
1,148(48.0%)
Day 4
CD25-CD45RA+
Responderalone
eTreg cells+
Responder cells(1 to 1 ratio)
CFSE (Responder cells)
Day 6
Cel
l num
ber
Fig. S6. Complete suppression of effector T cell proliferation by eTreg cells. Flow cytometric analysis of CFSE dilution by human CFSE-labeled responder cellsafter 4 d (Top) or 6 d (Bottom) coculture with (Right) or without eTregs isolated by the previously described protocol (4) (Left). Number (and percentage) ofcycling 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 thementered the third cycle of division, whereas most responder cells cultured alone had entered the third and fourth cycle. Furthermore, on day 6, most dividingresponder 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 thefew responder cells that have proliferated in the first few days of coculture have arrested their proliferation. Thus, human Treg cells can potently arrest theproliferation 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
