Myeloid-derived suppressor cell exosomes have

Supplement

Myeloid-derived suppressor cell exosomes have significant immunoregulatory effects in autoimmune disease as exemplified in mouse alopecia areata 1

1

1

1

2

3

Margot Zöller , Kun Zhao , Natalia Kutlu , Natalie Bauer , Jan Provaznik , Thilo Hackert , Martina 4 Schnölzer 1 2 Tumor Cell Biology, University Hospital of Surgery, Heidelberg, Gene Core Unit, EMBL Heidelberg, 3 4 Pancreas Section, University Hospital of Surgery, Heidelberg, Functional Proteome Analysis, German Cancer Research Center, Heidelberg, Germany

Table S1 Table S2 Table S3 S3A S3B S3C Table S4 S4A S4B S4C Table S5 S5A S5B Table S6 Table S7 S7A S7B

Antibodies and reagents Immunogenic Ha1 and K6irs keratinocyte peptides Comparison between in vitro versus ex vivo generated MDSC Top score recovered peptides Higher peptide recovery in in vitro than ex vivo MDSC Higher peptide recovery in ex vivo than in vitro MDSC Protein profile of MDSC-Exo from in vitro / ex vivo MDSC Top score recovered peptides Higher peptide recovery in Exosomes from in vitro MDSC Higher peptide recovery in Exosomes from ex vivo MDSC Differences in MDSC versus MDCS-Exo protein profile Preferential peptide recovery in MDSC-Exo Higher peptide recovery in MDSC than MDSC-Exo MDSC-Exo peptides recovered in pulldown from Sepharose-coupled lymph node cell membranes In vivo impact of MDSC-Exo on AA spleen cells Increased mRNA recovery in spleen cells of MDSC-Exo treated AA mice Reduced mRNA recovery in spleen cells of MDSC-Exo treated AA mice

Figure S1 Figure S2 Figure S3 Figure S4

Proteome analysis of ex vivo and in vitro generated MDCS and MDSC-exosomes Proteome analysis comparing ex vivo and in vitro generated MDCS with MDSC-exosomes MDSC-exosome membrane attached proteins Examples of hair regrowth in MDSC exosome-treated mice with AA partialis and incipient

1

Table S1 Antibodies and reagents Antibody specificity Akt (p)Akt Alix BAD pBAD BclXl Caspase 3 activ Caspase 8 Caspase9-clv. CCR3 (CD193) CCR4 (CD194) CCR5 (CD195) CCR6 (CD196) CCR7 (CD197) CCR8 (CDw198) CCR9 (CDw199) CD3 CD4 CD8 CD9 CD11b (ITM) (YBM6.6.10) CD11c (ItgX) CD14 CD16 (FcRIIIb) CD16/32 (NK) CD19 CD25 (PC61) CD28 CD32 (2.4G2) CD40 CD47 (IAP) CD54 (ICAM1) CD63 (Tspan30) CD69 CD80 CD81 (Tspan28) CD86 CD103 (ITE) CD115 (M-CSFR) CD117 (c-KIT) CD135 (FLT3) CD152 CD154 CD314 (NKG2D) CXCR3 (CD183) CXCR4 (CD184) Erk1/2 ERK1/2-p F4/80 FoxP3 GM-CSF Gr1 HIF1 IFN IB IB-p IgM IL1 IL4 IL6 IL10 IL12 JAK2 JAK2-p JAK3 JAK3-p JNK pJNK Jun Jun-p Ly6C Ly6G

donor mouse mouse rabbit hamster rabbit rabbit rabbit rabbit rabbit rat goat rat rat rat goat rat rabbit rat rat rat rat hamster rat rabbit rat rat rat monocl.hamster rat rat rat monocl.hamster rabbit monocl.hamster rat monocl.hamster rat rat rabbit rat rat monocl.hamster monocl.hamster rat rat/rabbit rat mouse mouse rat rat rat rat mouse rat rabbit mouse rabbit rat rat rat rat rat rabbit rabbit mouse goat rabbit mouse rabbit rabbit rat rat

supplier Becton Dickinson, HD, G Becton Dickinson, HD, G Santa Cruz, HD, G Becton Dickinson, HD, G Cell Signaling, Frankfurt, G Cell Signaling, Frankfurt, G Becton Dickinson, HD, G Becton Dickinson, HD, G Cell Signaling, Frankfurt, G R&D Systems, G abcam, Cambridge, UK Becton Dickinson, HD, G R&D Systems, G R&D Systems, G abcam, Cambridge, UK R&D Systems, G Abcam, Cambridge, UK EACCb EACCb Becton Dickinson, HD, G EACCb Becton Dickinson, HD, G Becton Dickinson, HD, G abcam, Cambridge, UK Becton Dickinson, HD, G ImmunoTools, Friesoythe, G EACCb Becton Dickinson, HD, G ATCCc Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Santa Cruz, HD, G ImmunoTools, Friesoythe, G Becton Dickinson, HD, G ImmunoTools, Friesoythe, G Becton Dickinson, HD, G Becton Dickinson, HD, G abcam, Cambridge, UK Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Santa Cruz, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G ImmunoTools, Friesoythe, G Novus Biologicals, Littletown, USA Becton Dickinson, HD, G Santa Cruz, HD, G Becton Dickinson, HD, G Dianova, Hamburg, G BioTrend, Cologne, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G SantaCruz, HD, G SantaCruz, HD, G Becton Dickinson, HD, G SantaCruz, HD, G SantaCruz, HD, G Becton Dickinson, HD, G SantaCruz, HD, G SantaCruz, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G

2

Table S1 continued Antibody specificity MHCI (K7-65) MHCII (K25-137) Myc-p MyD88 NFB NK (PK136) NOS2 P38 P38-p PI3K pPI3k Rab5 SCA1 (Ly6A) SCF (KIT ligand) Shc Shc-p Src Src-p STAT3-p STAT4-p STAT6-p Survivin Ter119 TGF1 TLR2 (CD282) TLR3 TLR4 (CD284) TNF TSG101 ZAP70 ZAP70-p dye or biotin labeled secondary antibodies /Streptavidin

donor mouse mouse rabbit rabbit rabbit rat rabbit rabbit rabbit rabbit rabbit mouse rat rabbit rabbit rabbit rabbit rabbit rabbit mouse rabbit rabbit rat rat rat rabbit rat rat rabbit rabbit rabbit

supplier Ref 1d Ref 1d Santa Cruz, HD, G Santa Cruz, HD, G Cell Signaling, Franfurt, G ATCC SantaCruz, HD, G SantaCruz, HD, G SantaCruz, HD, G Cell Signaling, Frankfurt, G Cell Signaling, Frankfurt, G Becton Dickinson, HD, G Becton Dickinson, HD, G abcam, Cambridge, UK Cell Signaling, Franfurt, G Cell Signaling, Franfurt, G Cell Signaling, Franfurt, G Cell Signaling, Franfurt, G Cell Signaling, Franfurt, G Becton Dickinson, HD, G SantaCruz, HD, G SantaCruz, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G SantaCruz, HD, G Becton Dickinson, HD, G Becton Dickinson, HD, G SantaCruz, HD, G Cell Signaling, Frankfurt, G Cell Signaling, Frankfurt, G

Reagent AnnV-FITC / APC CFSE (carboxyfluoresceinsuccinimidylester) GM-CSF IL4 IL6 PGE2 PI Sulfobiotin-X-NHS

Dose 0.2µl-0.5µl

Supplier Becton Dickinson, HD, G

5µM (10-20ng/ml 2ng/ml 5ng/ml 1nM 0.08µg 1mg/ml

Invitrogen, Darmstadt, G ImmunoTools, Friesoythe, G ImmunoTools, Friesoythe, G ImmunoTools, Friesoythe, G BioTrend, Cologne, G Becton Dickinson, HD, G Calbiochem, Darmstadt, G

Dianova, Becton Dickinson, Amersham

a

all rat and mouse antibodies are monoclonal, monoclonal hamster antibodies are marked EACC: European Collection of Animal Cell Cultures ATCC: American Type Cell Culture Collection d Reference 1. Koch N, Hämmerling GJ, Tada N, Kimura S, Hämmerling U. Cross-blocking studies with monoclonal antibodies against IA molecules of haplotypes b, d and k. Eur J Immunol (1982) 12: 909-14. b c

3

Table S2 Immunogenic Ha1 and K6irs keratinocyte peptides MHCI

MHCII

Name-Position

Sequenz

Name-Position

Sequenz

Ha1(K31)-147

TELGLRQLV

Ha1-204

QLGDRLNVEVDAAPT

Ha1(K31)-211

VEVDAAPTV

Ha1-223

RVLNETRCQYEAMVE

Ha1-157

SDINGLRRI

Ha1-249

TQTEELNKQVVSSSE

Ha1-251

TEELNKQVV

Ha1-277

RTVNALEIELQAQHC

K6irs(K71)-205

SELRNVRDV

K6irs-296

LDLDSIIDEVRAQYE

K6irs-178

LEPILEGHI

K6irs-118

VELDPEIQKVRAQER

K6irs-193

LETLSGDRV

K6irs-382

QRGDSALKDARAKLD

K6irs-242

VDAAYANKV

K6irs-289

SMDNNRNLDLDSIID

4

Table S3 Comparison between in vitro versus ex vivo generated MDSC Table S3A Highest score recovered proteins in ex vivo and in vitro generated MDSC MDSC in vitro

MDSC ex vivo

Scorea

Prot.M.

Pept.M.

Scorea

Aconitate hydratase, mitochondrial

1252

78

20

1315

119

32

Actb

Actin, cytoplasmic 1

1972

1165

47

1995

1063

48

Actn1

actinin alpha 1

2631

141

35

2453

116

31

Actn4

actinin alpha 4

2575

128

36

2151

96

29

Aldh2

aldehyde dehydrogenase 2 family

1551

262

29

1555

245

33

Aldoa

aldolase, fructose-bisphosphate A

1401

182

18

1206

131

14

Anxa1

Annexin A1

2043

134

24

1963

134

24

Anxa2

Annexin A2

1679

180

21

1359

160

17

Anxa3

Annexin A3

1474

43

18

869

24

14

Anxa4

Annexin A4

1464

50

17

1018

29

12

Anxa5

Annexin A5

1574

44

20

963

31

13

Anxa6

Annexin A6

1727

80

18

1215

38

15

Atp1a1

ATPase Na+/K+ transporting subunit alpha 1

1058

49

11

1314

105

18

Atp2a2

ATPase Ca2+ transporting 2

1234

72

15

2257

155

26

Atp5a1

ATP synthase, H+ transporting, complex alpha 1

2090

331

25

2025

321

25

Atp5b

ATP synthase, H+ transporting complex, beta 5

2222

296

25

2205

301

25

Atp6v1a

ATPase H+ transporting V1 subunit A

1115

52

16

1807

104

24

Calr

Calreticulin

1533

159

22

1460

143

21

Canx

Calnexin

1538

87

19

1581

121

22

Cap1

Adenylyl cyclase-associated protein 1

1317

170

17

1179

76

16

Capn1

Calpain-1

1186

29

18

759

21

11

Cat

Catalase

1101

30

15

1347

58

21

CD11b

Integrin alpha-M

2995

244

38

2569

268

33

CD13

Alanyl aminopeptidase, membrane

1972

65

24

1874

67

23

CD177

CD177 antigen Cd177

1019

39

13

1283

43

18

CD18

Integrin beta-2

3098

178

39

3099

259

37

CD206

mannose receptor C-type 1

2224

48

27

1580

41

20

Ckap4

Cytoskeleton-associated protein 4

1713

54

20

1668

60

21

Cltc

Clathrin heavy chain 1

3779

99

49

3425

107

42

Cndp2

CNDP dipeptidase 2

1218

77

16

857

40

13

Copa

coatomer protein complex subunit alpha

1159

23

15

1033

34

15

Ctsb

Cathepsin B

1139

66

14

746

25

10

Cybb

Cytochrome b-245 beta chain

787

50

13

1053

130

14

Dync1h1

dynein cytoplasmic 1 heavy chain 1

2332

56

36

3537

89

53

Eef2

Elongation factor 2

2630

269

35

1538

167

21

Ehd4

EH domain-containing 4

884

45

12

1276

57

18

Eif4a1

Eukaryotic initiation factor 4A1

1254

135

15

1141

117

14

Eno1

enolase 1

2380

373

27

2238

280

25

Esyt1

Extended synaptotagmin-1

2753

217

37

3012

261

44

Ezr

Ezrin

1565

123

21

1370

91

17

Fasn

Fatty acid synthase

1018

25

15

1200

22

16

Fermt3

Fermitin family member 3

1519

111

21

1160

126

16

Flna

Filamin-A

5389

391

71

4448

314

54

Ganab

glucosidase II alpha subunit

1962

113

26

2094

115

29

Gapdh

Glyceraldehyde-3-phosphate dehydrogenase

1357

401

13

1158

243

11

Gdi2

GDP dissociation inhibitor 2

1335

92

15

1147

65

16

Glg1

Golgi apparatus protein 1

920

32

10

1423

50

18

Glud1

Glutamate dehydrogenase 1

1589

77

21

1793

131

26

Gnai2

G protein alpha inhibiting 2

1310

140

15

1340

221

17

Got2

Aspartate aminotransferase, mitochondrial

1177

93

19

1064

80

18

Gpi

Glucose-6-phosphate isomerase

1495

113

21

1125

67

15

Synonym

Protein Description

Aco2

5

Prot.M. Pept.M.

Table S3A cont MDSC in vitro Synonym

Protein Description

MDSC ex vivo

Scorea

Prot.M.

Pept.M.

Scorea

48

15

1044

50

12

Prot.M. Pept.M.

Gsn

Gelsolin

1257

Hbb-b1

hemoglobin, beta adult major chain

1295

503

14

960

184

12

Hbb-b2

hemoglobin, beta adult minor chain

1130

299

14

854

117

12

Hnrnpa2b1

Heterogeneous nuclear ribonucleoproteins A2/B1

1286

82

17

479

10

5

Hsd17b4

hydroxysteroid 17-beta dehydrogenase 4

464

9

5

1082

44

11

Hsp90aa1

Heat shock protein HSP 90-alpha

1900

255

27

1289

173

18

Hsp90ab1

Heat shock protein HSP 90-beta

2507

419

33

1973

309

27

Hsp90b1

heat shock protein 90, beta (Grp94), member 1

2758

457

35

3265

525

42

Hspa5

heat shock protein 5

3362

544

38

3586

610

38

Hspa8

Heat shock cognate 71 kDa protein

3021

436

38

2745

330

35

Hspd1

heat shock protein family D (Hsp60) member 1

1943

69

24

861

44

10

Hyou1

Hypoxia up-regulated protein 1

2463

169

30

2426

161

31

Idh2

Isocitrate dehydrogenase [NADP] 2

920

63

14

1081

77

18

Immt

inner membrane mitochondrial protein

435

7

5

1078

44

14

Iqgap1

IQ motif containing GTPase activating protein 1

3437

233

43

2981

239

36

Lcp1

lymphocyte cytosolic protein 1

3082

486

38

2690

400

37

LDHA

L-lactate dehydrogenase A chain

1329

147

16

1064

129

16

Lmna

Lamin-A/C Lmna

1994

102

25

Lmnb1

Lamin-B1 Lmnb1

693

21

10

2297

131

27

Lnpep

Leucyl-cystinyl aminopeptidase

1122

37

15

1132

34

15

Ltf

Lactotransferrin

3279

223

41

2903

192

37

Mdh2

Malate dehydrogenase 2

1365

114

16

1281

88

16

Mpo

Myeloperoxidase

1761

149

21

1271

68

17

Msn

Moesin

2728

221

36

2573

158

34

Mvp

Major vault protein

1750

45

24

1791

55

25

Myh9

myosin heavy chain 9

5150

207

69

263

3

2

Myof

Myoferlin

191

4

3

3636

186

52

Napa

NSF attachment protein alpha

655

16

8

1066

31

14

Ncl

Nucleolin

1729

87

23

816

51

12

Oat

Ornithine aminotransferase

711

16

9

1119

48

16

Ogdh

oxoglutarate dehydrogenase

1079

38

15

1201

68

20

P4hb

prolyl 4-hydroxylase subunit beta

2412

286

31

2351

312

31

Pdia3

Protein disulfide-isomerase A3

2747

263

37

2655

335

39

Pdia4


2505

182

32

2519

215

35

Pdia6


1286

113

15

1358

155

14

Pgk1

Phosphoglycerate kinase 1

1289

107

13

960

69

13

Pgls

6-phosphogluconolactonase

1191

156

15

1018

113

13

Pkm

Pyruvate kinase PKM

2580

403

31

2108

298

27

Plek

Pleckstrin

352

14

5

1959

53

25

Por

cytochrome P450 oxidoreductase

1104

67

14

1248

92

15

Ppib

Peptidyl-prolylisomerase B

1043

55

13

1130

60

14

Ptpn6

protein tyrosine phosphatase, non-receptor type 6

1348

110

17

1182

106

15

Ptprc

protein tyrosine phosphatase, receptor type C

1229

85

17

1247

113

20

Pygl

Glycogen phosphorylase L

1534

106

22

838

41

13

Rab7a

RAB7A, member RAS oncogene family

1229

149

15

1009

88

13

Rack1

Receptor of activated protein C kinase 1

1145

81

16

762

53

12

Rdx

Radixin

1183

108

17

1075

80

14

Rnh1

ribonuclease/angiogenin inhibitor 1

1332

52

14

1035

50

12

Rpn1

ribophorin I

1331

169

21

1783

250

23

Rpn2

ribophorin II

1212

95

15

1371

150

17

Rrbp1

Ribosome-binding protein 1

1444

46

19

1724

54

24

Sdha

succinate dehydrogenase complex subunit A

497

19

6

1089

66

13

Serpinb1a

Leukocyte elastase inhibitor A

1377

156

19

913

64

12

6

Table S3A cont. MDSC in vitro Synonym

Scorea

Protein Description

Prot.M.

MDSC ex vivo

Pept.M.

Scorea

89

17

Prot.M. Pept.M.

634

40

10

1297

solute carrier family 25 member 4

868

109

13

1169

171

16


1193

204

15

1328

280

18

Snd1

Staphylococcal nuclease and tudor domain-1

1459

51

21

483

17

5

Sun2

SUN domain-containing protein 2

1682

125

20

1539

107

19

Thbs1

Thrombospondin-1

1206

64

16

268

10

3

Tkt

Transketolase

1676

163

20

1625

113

20

Tln1

Talin-1

4912

297

57

5199

342

61

Tuba1b

Tubulin alpha-1B

1440

238

19

1360

157

18

Tuba1c

Tubulin alpha-1C

1357

222

18

1295

137

17

Tuba4a

Tubulin alpha-4A

1352

189

18

1189

122

16

Tubb2a

Tubulin beta-2A

1173

166

15

Tubb4b

Tubulin beta-4B

1454

203

17

1332

160

17

Tubb5

Tubulin beta-5

1628

247

20

1459

179

19

Uba1

Ubiquitin-like modifier-activating enzyme 1

2006

101

23

1243

67

14

Uggt1

UDP-glucose glycoprotein glucosyltransferase 1

3039

116

39

2906

124

38

Vcp

valosin containing protein

2350

132

29

1970

173

24

Vdac1

Voltage-dependent anion channel 1

1431

64

15

1545

73

16

Vim

Vimentin

3056

371

42

2495

153

37

Slc25a12


Slc25a4 Slc25a5

a

Score ≥1000: bold, ≥2-fold difference in significant peptide hits: italics

7

Table S3B Peptides recovered at a significantly higher frequency in in vitro than ex vivo MDSC

Synonym Ap1b1 Ap1m1 Ap2b1 Arg1 Arsb Ca1 Ca2 Cbr2 CD41 CD61 Celf2 Chp1 Cmpk2 Cpd Ddx39a Ddx3x Elavl1 Ethe1 Fam3c Glo1 Gmfg Grb2 Gstm1 Gsto1 Hadh Hck Hdac1 Hnrnpa1 Hnrnpa2b1 Hnrnph1 Hnrnpl Hprt1 Hsd17b10 Ifi44 Ifit1

MDSC in vitro MDSC ex vivo Prot.M. Pept.M. Prot.M. Pept.M. 33 12 17 2 5 4 23 6 35 10 5 3 6 3 21 7 6 2 5 4 23 12 14 6 10 5 5 3 37 12 6 3 80 14 4 4 17 7 13 6 6 3 10 4 12 6 1 1 13 6 2 1 13 7 1 1 26 12 1 1 13 6 7 4 12 4 9 6 95 15 2 1 82 17 10 5 67 13 10 4 31 11 5 3 9 5 6 3 27 9 40 12

Protein Description AP-1 complex subunit beta-1 AP-1 complex subunit mu-1 AP-2 complex subunit beta Arginase-1 Arylsulfatase B Carbonic anhydrase 1 Carbonic anhydrase 2 Carbonyl reductase 2 Integrin alpha-IIb Integrin beta-3 CUGBP Elav-like family member 2 calcineurin like EF-hand protein 1 cytidine/uridine monophosph kinase 2 Carboxypeptidase D DEAD-box helicase 39A DEAD-box helicase 3, X-linked ELAV like RNA binding protein 1 ETHE1, persulfide dioxygenase family with similarity 3 member C glyoxalase I Glia maturation factor gamma Growth factor receptor-bound prot. 2 Glutathione S-transferase Mu 1 Glutathione S-transferase omega-1 hydroxyacyl-CoA dehydrogenase HCK protooncogene, Src family TK Histone deacetylase 1 Heterogeneous ribonucleoprotein A1 Heterogeneous ribonucleoprot. A2/B1 Heterogeneous ribonucleoprotein H1 Heterogeneous ribonucleoprotein L Hypoxanthine phosphoribosyltransf. 1 hydroxysteroid 17- dehydrogenase 10 IFN-induced protein 44 IFN-induc. prot. w tetratricopep.rep.1

Protein Family membrane traffic. ECM receptor membrane traffic., prot. transporter cysteine protease Hydrolase dehyratase dehyratase dehydrogenase Adhesion Adhesion nucleic acid & RNA binding phosphatase nucleotide kinase metalloprotease RNA helicase GTPase activity protein & RNA & nucleic acid binding hydrolase lyase signaling non-receptor tyrosine kinase transferase cytoskel., transferase, elong. factor dehydrogenase tyrosine kinase reductase RNA-, nucleic acid binding RNA-, miRNA binding protein & RNA & nucleic acid binding protein, mRNA binding glycoyltransferase dehydrogenase, reductase protein & RNA binding

8

Reactome pathway protein transporter & binding vesicle biogenesis & transport vesicle med. transport, endocyt., signaling scavenger receptor metabolism metabolism reductase, protein binding adhesion and signaling adhesion and signaling metabolism, hyaluronan degradation vesicle biogenesis mRNA processing protein-, RNA-, DNA-binding splicing ion binding platelet activation & signaling citric acid cycle, electron transport Arp2/3 complex binding multiple signaling pathways metabolism metabolism protein kinase, transferase protein binding, transcription factor activity signaling splicing and signaling splicing metabolism metabolism molecular function

Table S3B cont. Synonym Ifit2 Ifit3 Ilf2 Iqgap2 Irgm1 Isg15 Kpnb1 Mcm4 Mcm7 Mcpt8 Mrps34 Msra Mta2 Mybbp1a Myh9 Myl12b Nipsnap3b Npc2 Nsf Nudt21 Ostf1 Park7 Pcmt1 Pddc1 Pds5a Pf4 Pfdn2 Ppil1 Ppp1ca Ppp1cb Ppp1cc Ppp2ca Ppp2r1a Prdx6 Psma2 Psma3 Psma5

MDSC in vitro MDSC ex vivo Prot.M. Pept.M. Prot.M. Pept.M. 19 6 20 4 5 3 4 3 7 3 52 4 55 13 20 4 7 4 13 3 11 4 5 4 5 3 10 6 29 12 2 1 207 69 3 2 27 8 8 4 7 4 0 0 7 4 13 7 9 6 9 4 5 3 8 3 6 3 4 3 6 4 1 1 4 3 36 13 7 4 19 9 25 11 12 7 1 1 15 7 3 2 30 13 14 8 1 1 14 6 1 1 19 8

Protein Description IFN-induc. prot. w tetratricopep. rep. 2 IFN-induc. prot. W tetratricopep. rep. 3 Interleukin enhancer-binding factor 2 IQ motif contain. GTPase active. prot.2 Immunity-relat. GTPase family M prot.1 ISG15 ubiquitin-like modifier karyopherin subunit beta 1 minichromosome mainten. complex 4 minichromosome mainten. complex 7 Mast cell protease 8 Mcpt8 mitochondrial ribosomal protein S34 Methionine sulfoxide reductase 1 metastasis assoc. 1 family member 2 Myb-binding protein 1A myosin heavy chain 9 myosin light chain 12B NipSnap homolog 3B NPC intracell. cholesterol transporter 2 Vesicle fusing ATPase nudix hydrolase 21 Osteoclast-stimulating factor 1 Parkinsonism assoc. deglycase, DJ-1 protein-L-isoaspartate methyltransfer. Parkinson disease 7 domain-cont. 1 PDS5 cohesin associated factor A Platelet factor 4 Prefoldin subunit 2 peptidylprolyl isomerase like 1 protein phosphatase 1 subunit alpha protein phosphatase 1 subunit beta protein phosphatase 1 subunit gamma protein phosphatase 2 subunit alpha Protein phosphatase 2A subunit A Peroxiredoxin-6 Proteasome subunit alpha 2 Proteasome subunit alpha 3 Proteasome subunit alpha 5

Protein Family

kinase activator G-protein modulator hydrolase protein binding transfer / carrier protein DNA helicase, hydrolase hydrolase, DNA helicase serine protease reductase chromatin binding transcription factor G-protein modul., actin binding binding cholesterol binding hydrolase hydrolase SH3 domain binding protease, transcription methyltransferase RNA binding, transcription factor chromatin binding chemokine chaperone isomerase phosphatase phosphatase phosphatase phosphatase phosphatase peroxidase endopeptidase peptidase, hydrolase peptidase, hydrolase

9

Reactome pathway RNA binding protein binding Rho GTPase signaling cytokine signaling apoptosis execution cell cycle cell cycle proteolysis protein synthesis, ribosome constituent metabolism DNA & protein binding protein & RNA-binding protein binding Rho GTPase signaling vesicular trafficking cholesterol transport COPI-,COPII-mediated vesicle transport mRNA splicing bone resorption scaffold protein binding

cell cycle CXCR3 signaling protein folding splicing GPCR signailng Rho GTPase signaling, cell cycle protein binding, phosphatase activity TLR4 cascade, MAPK signaling, TLR & multiple signaling peroxidase, hydrolase, binding multiple signaling pathways signaling signaling

Table S3B cont. Synonym Psma6 Psma7 Psmb10 Psmb3 Psmb4 Psmb8 Psmb9 Psme1 Ptbp3 S100a13 Sept7 Sept9 Set Sf3a1 Sf3b3 Siglec1 Skap2 Smarca5 Snd1 Snrpa Snrpa1 Snx5 Sptan1 Srsf10 Ssb Ssr4 Stip1 Tfrc Tgm2 Thbs1 Tln2 Tmed4 Tpr Tpt1 Trex1 Tubb2a U2af2

MDSC in vitro MDSC ex vivo Prot.M. Pept.M. Prot.M. Pept.M. 18 8 3 2 17 10 6 3 11 5 20 7 14 6 15 5 3 1 38 11 4 3 19 8 9 3 5 3 15 3 9 4 16 4 6 4 1 1 32 9 6 5 0 0 5 3 12 5 51 21 17 5 8 3 10 5 13 4 1 1 7 5 4 3 14 7 2 2 29 5 13 6 30 9 21 6 64 16 10 3 26 6 12 5 2 1 14 9 30 4 14 5 166 15 9 5

Protein Description Proteasome subunit alpha 6 Proteasome subunit alpha 7 Proteasome subunit beta 10 Proteasome subunit beta 3 Proteasome subunit beta 4 Proteasome subunit beta 8 Proteasome subunit beta 9 proteasome activator subunit 1 Polypyrimidine tract-binding protein 3 S100 calcium binding protein A13 Septin-7 Septin-9 SET nuclear proto-oncogene Splicing factor 3A subunit 1 Splicing factor 3B subunit 3 Sialoadhesin Src kinase-assoc. phosphoprotein 2 regulator of chromatin, subfamily a5 Staphyloc. nuclease & tudor dom.1 small nuclear ribonucleoprotein A small nuclear ribonucleoprotein A' Sorting nexin-5 Spectrin alpha, non-erythrocytic 1 Serine/arginine-rich splicing factor 10 Sjogren syndrome antigen B Stomatostatin receptor type 4 Stress-induced-phosphoprotein 1 Transferrin receptor transglutaminase 2 Thrombospondin-1 Talin-2 transmembrane p24 trafficking prot. 4 translocated promoter tumor protein, translation.-control. 1 Three-prime repair exonuclease 1 Tubulin beta-2A U2 snRnP auxiliary factor 2

Protein Family peptidase, hydrolase endopeptidase endopeptidase peptidase peptidase endopeptidase endopeptidase endopeptidase NA & RNA binding calmodulin cytoskeleton cytoskeleton chaperon, phosphatase inhibitor RNA & protein binding RNA & protein binding Ig superfam. adhesion, signaling protein binding DNA helicase transcription cofactor RNA binding RNA bindingr trafficking regulator non-motor actin binding RNA & nucleic acid binding tRNA / RNA binding GPCR chaperone receptor acyltransferase integrin-, fibrinogen-, collagen V-bind. actin family (cytoskeleton) transfer protein, vesicle coat RNA-, protein-binding microtubule binding nuclease, hydrolase, protein binding GTP binding RNA & protein binding

10

Reactome pathway signaling multiple signaling pathways multiple signaling pathways

GPCR signaling multiple signailng pathways regulator of differentiation binding & signaling binding binding cell cycle cell cycle splicing factor protein & carbohydrate binding signal regulator biological processes protein, NA, RNA binding splicing factor splicing factor vesicle biogenesis, -mediated transport multiple signaling splicing factor splicing factor signal transduction axon guidance clathrin-mediated endocytosis apoptosis platelet aggreg., aniogenesis, inflamm., matrix adhesion actin filament assembly signal transduction SLC-mediated transmembrane transport regulator of growth and differentiation mismatch repair membrane trafficking, vesicle-mediated transport splicing factor

Table S3B cont. Synonym Trex1 Tubb2a U2af2 U2surp Vps29 Xpo2

MDSC in vitro MDSC ex vivo Prot.M. Pept.M. Prot.M. Pept.M. 14 5 166 15 9 5 4 3 4 3 9 4

Protein Description Three-prime repair exonuclease 1 Tubulin beta-2A U2 snRnP auxiliary factor 2 U2 snRNP-associated SURP motif vacuolar protein sorting 29 homolog Exportin-2

Protein Family GTP binding splicing factor mRNA processing vesicle coat GTPase, transporter

Reactome pathway nuclease, hydrolase, protein binding membrane trafficking, vesicle-mediated transport protein-, RNA-binding splicing protein & ion binding protein binding, Ran GTPase binding

Table S3C

Proteins recovered at a significantly higher level in ex vivo than in vitro MDSC Synonym Atp6ap2 Atp6v0a1 Capzb Casp6 Chmp2a Cpt2 D1Pas1 Dnajc10 Emc1 Erlin2 Fundc2 Galnt7 Gas7 Glrx Igf2r Lmna Lmnb2 Mmp12 Mrpl41 Mrpl49 Mtch2 Myof Nono Nup155 Nup210 Olfm4

MDSC in vitro MDSC ex vivo Prot.M. Pept.M. Prot.M. Pept.M. 9 5 23 8 17 4 6 4 5 3 1 1 10 7 8 4 2 1 13 5 16 8 15 6 7 4 1 1 7 6 6 5 8 4 5 3 102 25 17 3 1 1 18 6 7 4 3 3 16 5 4 3 186 52 6 3 8 5 3 1 45 12 8 5

Protein Description ATPase H+ transporting 2, Renin receptor ATPase, H+ transporting, V0 subunit A1 capping protein muscle Z-line, beta Caspase-6 Charged multivesicular body protein 2a Carnitine O-palmitoyltransferase 2 DNA segment, Chr 1, Pasteur Institute 1 DnaJ Hsp family (Hsp40) member C10 ER membrane protein complex subunit 1 ER lipid raft associated 2 FUN14 domain-containing 2 N-acetylgalactosaminyltransferase 7 Growth arrest-specific 7 Glutaredoxin insulin-like growth factor 2 receptor Lamin-A/C Lmna Lamin-B2 matrix metallopeptidase 12 mitochondrial ribosomal protein L41 mitochondrial ribosomal protein L49 Mitochondrial carrier 2 Myoferlin Non-POU domain-con. octamer-binding nucleoporin 155 nucleoporin 210 Olfactomedin-4

Protein Family ATP synthase, hydrolase non-motor actin binding protease, protease inhibitor . transferase RNA helicase . . . . transferase actin family oxidoreductase receptor intermediate filament intermediate filament . ribosomal protein ribosomal protein transfer / carrier protein membrane traffic mRNA splicing . membrane traffic protein receptor, structural protein

11

Reactome pathway angiotensin metabolism InsR signal., ROS, transmembr. & ion transp. b-tubulin-, actin-, protein-binding apoptotic execution phase vesicle biogenesis, ECSRT, traffic, budding lipid / lipidprotein metabolism ATP-, RNA-, DNA-binding chaperone binding, Hsp70 binding molecular function transmembrane transport molecular function glycosylation membrane trafficking regulator electron carrier, nucleotide metabolism clathrin endocytos., memb. traffic, vesicle biogen. apoptotic execution, cell cycle structural molecule ECM organization, ECM degradation mitochondrial biogenesis and maintenance mitochondrial biogenesis and maintenance molecular function phospholipid binding protein-, RNA-, DNA-binding heat stress regulation, SLC-mediated transport heat stress response, transmembrane transport protein binding

Table S3C cont. MDSC in vitro MDSC ex vivo Synonym Prot.M. Pept.M. Prot.M. Pept.M. Parp1 4 3 Pdha1 13 6 Pfkp 2 1 13 6 Ptbp1 18 5 Samm50 6 3 Sel1l 7 4 23 13 Slc25a13 47 8 Snap29 5 3 Snx12 4 3 Ssr2 0 0 46 4 Timm50 6 4 Tomm40 14 5 Trpv2 2 1 18 5 Uqcrc1 7 2 46 7

Protein Description poly (ADP-ribose) polymerase family 1 pyruvate dehydrogenase alpha 1 phosphofructokinase, platelet Polypyrimidine tract-binding protein 1 SAMM50 sorting and assembly machinery SEL1L ERAD E3 ligase adaptor subunit solute carrier family 25 member 13 Synaptosomal-associated protein 29 Sorting nexin-12 signal sequence receptor subunit 2 translocase of inner mitoch. membrane 50 translocase of outer mitoch. membrane 40 Transient receptor cation channel V 2 ubiquinol-cytochrome c reductase I

Protein Family DNA ligase dehydrogenase carbohydrate kinase . protein binding and sorting enzyme modulator AA transporter, protein transfer SNARE protein phosphatidyl & lipid bindingr G-protein-coupled receptor phosphatase membrane trafficking ion channel metalloprotease

12

Reactome pathway TGFbR complex signaling RA signaling, electron transport, sign. transduct. glucose metabolism mRNA splicing, FGFR signaling protein assembly transmembrane transport, NOTCH signaling glucose metabolism membrane traffic, vesicle-mediated transport membrane traffick regulato GPCR signaling IL2R binding protein channel, transmembrane transport transmembrane transport electron transport

Table S4 Protein profile of MDSC-Exosomes from in vitro / ex vivo MDSC Table S4A Top score peptides of MDSC-Exosomes from in vitro / ex vivo MDSCa MDSC-Exo in vitro

MDSC-Exo ex vivo

Synonym

Protein Description

Prot.M.

Pept.M.

Prot.M.

Pept.M.

Actb

Actin, cytoplasmic 1

477

42

589

38

Actr3

Actin-related protein 3

34

12

47

13

Ahcy

Adenosylhomocysteinase

21

7

53

10

Anxa1

Annexin A1

88

18

23

11

Anxa11

Annexin A11

53

10

29

6

Anxa2

Annexin A2

108

18

34

15

Anxa4

Annexin A4

56

14

23

10

Anxa5

Annexin A5

43

17

34

13

Anxa6

Annexin A6

64

19

10

3

Ap2m1

AP-2 complex subunit mu

21

10

2

1

Arpc2

Actin-related protein 2/3 complex subunit 2

24

12

C3

Complement C3

119

16

152

15

Cap1

Adenylyl cyclase-associated protein 1

19

11

38

10

Ccdc47

Coiled-coil domain-containing 47

33

14

Cct2

chaperonin containing TCP1 subunit 2

39

12

18

9

Cct3


32

10

4

4

Cct4


32

10

7

4

CD11b

Integrin alpha-M

83

21

39

16

CD13

Alanyl aminopeptidase, membrane

36

16

CD18

Integrin beta-2

78

27

32

17

Clic1

Chloride intracellular channel 1

26

10

26

7

Cltc

Clathrin heavy chain 1

208

50

14

9

Coro1a

Coronin-1A

41

10

24

8

Ctsb

Cathepsin B

9

5

47

10

Ctsd

Cathepsin D

25

6

52

10

Dera

Deoxyribose-phosphate aldolase

11

6

20

12

Eea1

Early endosome antigen 1

113

13

91

12

Eif3a

Eukaryotic translation initiation factor 3 A

127

23

Eif3b

Eukaryotic translation initiation factor 3 B

42

17

Eif3c

Eukaryotic translation initiation factor 3 C

128

19

Eif3e

Eukaryotic translation initiation factor 3 E

49

18

13

8

Eif3l

Eukaryotic translation initiation factor 3 L

32

10

Eif4a1

Eukaryotic initiation factor 4A1

46

15

26

12

Emilin2

elastin microfibril interfacer 2

20

10

Eno1

enolase 1

194

20

152

23

Ezr

Ezrin

33

12

Fasn

Fatty acid synthase

27

16

2

1

Fermt3

Fermitin family member 3

33

14

65

18

Flna

Filamin-A

27

16

23

10

Fn1

Fibronectin 1

580

71

71

25

Fth1

Ferritin heavy chain

171

15

132

12

Ftl1

Ferritin light chain 1

226

14

271

13

Gapdh

Glyceraldehyde-3-phosphate dehydrogenase

210

13

215

13

Gdi2

GDP dissociation inhibitor 2

21

12

13

8

Gsn

Gelsolin

86

11

H2-Ab1

histocompatibility 2, class II antigen A, beta 1

53

10

37

9

Hbb-b1

hemoglobin, beta adult major chain

205

10

507

13

Hbb-b2

hemoglobin, beta adult minor chain

122

10

306

13

Hmgb1

High mobility group box 1

95

11

16

4

Hmgb2

High mobility group box 2

52

10

26

7

Hsp90ab1

Heat shock protein HSP 90-beta

133

17

102

18

13

.

Table S4A cont. MDSC-Exo in vitro

MDSC-Exo ex vivo

Synonym

Protein Description

Prot.M.

Pept.M.

Prot.M.

Pept.M.

Hspa5

heat shock protein 5

43

11

22

4

Hspa8

Heat shock cognate 71 kDa protein

217

25

208

25

Ighm

Ig mu chain C region

9

5

86

14

Iqgap1

IQ motif containing GTPase activating protein 1

46

15

2

1

Klhdc4

Kelch domain-containing 4

27

11

Krt73

Keratin 73

31

4

67

13

LDHA

L-lactate dehydrogenase A chain

49

9

38

10

Lgals3bp

Galectin-3-binding protein Lgals3bp

149

21

116

19

Lpl

Lipoprotein lipase

174

12

59

9

Ltf

Lactotransferrin

98

29

89

26

Mfge8

Lactadherin

120

18

50

14

Mmp12

matrix metallopeptidase 12

324

22

229

20

Mmp19

matrix metallopeptidase 19

56

14

39

10

Mpo

Myeloperoxidase

15

9

56

16

Msn

Moesin

52

19

22

8

Mvp

Major vault protein

26

14

Myh1


54

13

Myh4


.

63

15

Myh9


235

60

69

20

Ncl

Nucleolin

302

30

13

7

Pdcd6ip

Programmed cell death 6-interacting protein

118

27

12

6

Pgam1

Phosphoglycerate mutase 1

19

9

19

10

Pkm

Pyruvate kinase PKM

186

24

Ppib

Peptidyl-prolylisomerase B

39

12

26

9

Prdx1

Peroxiredoxin-1

79

10

16

6

Prkcd

protein kinase C delta

222

25

Psma1

Proteasome subunit alpha 1

34

9

38

11

Psma5


29

8

33

10

Psma6


34

10

39

10

Psma7


30

11

43

10

Psmb8

Proteasome subunit beta 8

34

8

49

11

Psmd11

proteasome 26S subunit, non-ATPase 11

37

13

7

4

Psmd2


32

19

3

3

Psmd3


24

13

2

1

Psmd6


35

16

19

10

Rab11b

RAB11B, member RAS oncogene family

35

11

30

9

Rack1

Receptor of activated protein C kinase 1

33

12

50

14

Rplp0

ribosomal protein P0

36

7

38

10

Ruvbl2

RuvB like AAA ATPase 2

20

10

4

3

Ssrp1

structure specific recognition protein 1

48

10

Tcp1

T-complex 1

42

14

2

1

Thbs1

Thrombospondin-1

60

17

104

23

Tln1

Talin-1

24

14

Tuba1b

Tubulin alpha-1B

127

18

210

36

Tuba1c

Tubulin alpha-1C

115

17

145

17

Tuba4a

Tubulin alpha-4A

91

16

144

18

Tubb4b

Tubulin beta-4B

107

19

120

19

Tubb5

Tubulin beta-5

115

20

124

19

Uba1

Ubiquitin-like modifier-activating enzyme 1

33

10

12

5

Vcp


226

41

37

14

a

.

.

Only proteins are shown with ≥10 significant peptides by at least one MDSC-Exo population

14

Table S4B Higher recovery in Exosomes from in vitro MDSC Synonym Actn1 Adam10 Anp32b Anxa6 Ap2a2 Ap2b1 Ap2m1 Arrb2 Ass1 Bin2 Brox Capg Capns1 Cbx3 Ccdc47 Ccl9 Cct6a Cct7 Cct8 CD13 CD316 CD36 Cltc Copb2 Csnk2a1 Cyp4f3 Ddost Dek Dhx9 Dip2b Dnaja2 Dync1h1 Eftud2 Eef1d Eif3a Eif3b

Exo - in vitro Prot.M. Pept.M. 8 4 9 5 5 3 64 19 10 4 28 6 21 10 13 6 16 8 17 4 4 3 7 4 9 3 11 5 33 14 13 3 20 7 5 3 13 7 36 16 24 8 14 7 208 50 10 5 6 4 17 6 6 3 38 9 5 4 4 3 6 3 32 21 14 8 3 3 127 23 42 17

Exo - ex vivo Prot.M. Pept.M. Protein Description actinin alpha 1 Disintegrin and metalloproteinase 10 Acidic nuclear phosphoprotein 32B 10 3 Annexin A6 AP-2 complex subunit alpha-2 AP-2 complex subunit beta 2 1 AP-2 complex subunit mu arrestin beta 2 Argininosuccinate synthase 1 Bridging integrator 2 BRO1 domain and CAAX motif capping actin protein, gelsolin like Calpain small subunit 1 Chromobox 3 Coiled-coil domain-containing 47 C-C motif chemokine 9 7 2 chaperonin TCP1 subunit 6A chaperonin containing TCP1 subunit 7 2 1 chaperonin TCP1 subunit 8 Alanyl aminopeptidase 13 3 2 Immunoglobulin superfamily 8 1 1 CD36, platelet glycoprotein 4 14 9 Clathrin heavy chain 1 coatomer complex beta 2 2 1 Casein kinase II alpha 1 3 1 cytochrome P450 family 4F3 DDoligosaccharide-glycosyltransferase DEK proto-oncogene DEAH-box helicase 9 Disco-interacting protein 2 homolog B DnaJ heat shock protein member A2 3 2 dynein cytoplasmic 1 heavy chain 1 elongation factor Tu GTP binding 2 Elongation factor 1-delta Eukaryotic translation initiation 3 A Eukaryotic translation initiation 3 B

Molecular function integrin, actin, vinculin binding metallopeptidase RNA & histone & protein binding protein complex & cholesterol binding lipid & protein binding transporter binding, signaling enzyme modulator binding, ligase phospholipid binding,kinase protein binding, molecular function actin binding, protein complex binding endopeptidase transcription factor ion & RNA binding cytokine & chemokine activity chaperonin ATP & protein binding chaperonin metalloprotease protein binding lipoprotein binding vesicle coat vesicle coat, structural molecule PTK, phosphatase regulator oxygenase DNA & histone binding hydrolase, protein binding molecular function, catalytic activity glycoprotein & HSP binding microtubule activity hydrolase, translation initiat & elong. translation elongation translation initiation translation initiation

15

Reactome pathway cell junction organization, cell-cell communication NOTCH, Ephrin, EGFR signaling apoptosis, differentiation contraction clathrin endocytosis, lipid transport, vesicle transport lipid & vesicle transport, clathrin endocytosis, signaling lipid transport, Gap junction traffic, clathrin endoc. clathrin endocyt., vesic.-med.transport, GPCR signaling AA & polyamine metabolism brassinosteroid signaling pathway actin filament capping epigenetic regul. of gene expression ER organization, osteopblast differention GPCR signaling protein folding chaperon- & protein folding protein folding angiogenesis, signaling, proteolysis CD81 partner, cell component movement TLR cascade, vesicle-mediated transport lipid transport, vesicle transport & biog., clathrin endoc. transport, vesicle-med.trasport,, membrane traffic. signaling, cell cycle Lipid metabolism T cell activ., neutrophil degranulation, cytokine response regulation of gene expression & AP-2 transcription MyD88, TLR, type I IFN signaling cascade cochaperone activity, protein refolding Vesicle & vesicle-med. transport, organelle biogenesis splicing translation, protein metabolism

Table S4B cont. Exo - in vitro Synonym Prot.M. Pept.M. Eif3c 128 19 Eif3i 11 6 Eif3l 32 10 Emilin2 20 10 Esyt1 22 9 Ezr 33 12 Fasn 27 16 Fgl2 11 5 Gca 4 3 Glg1 5 3 Gm2a 6 3 Gpi 5 3 Gsn 86 11 Hnrnpk 18 5 Iqgap1 46 15 Klhdc4 27 11 Lamp2 5 3 Lcp1 20 9 M6pr 11 4 Man2a1 12 8 Mvp 26 14 Myadm 10 3 Mybbp1a 10 3 Myh9 235 60 Ncl 302 30 Nrdc 10 7 Opn 20 3 Pabpc1 15 8 Pcbp1 5 3 Pdcd6ip 118 27 Pfkp 4 3 Pfn1 82 7 Prkcd 222 25 Psma2 21 8 Psmb5 7 3 Psmc3 7 3 Psmd1 12 7

Exo - ex vivo Prot.M. Pept.M. Protein Description Eukaryotic translation initiation 3 C 1 1 Eukaryotic translation initiation 3 I Eukaryotic translation initiation 3 L elastin microfibril interfacer 2 1 1 Extended synaptotagmin-1 Ezrin 2 1 Fatty acid synthase Fibroleukin Grancalcin Golgi apparatus protein 1 GM2 ganglioside activator Glucose-6-phosphate isomerase Gelsolin 4 1 Heterogeneous ribonucleoprotein K 2 1 IQ motif GTPase activating protein 1 Kelch domain-containing 4 Lysosome-associated glycoprotein 2 3 2 lymphocyte cytosolic protein 1 1 1 mannose-6-phosphate receptor mannosidase 2, alpha 1 Major vault protein Myeloid-assoc. differentiation marker Myb-binding protein 1A 69 20 myosin heavy chain 9 13 7 Nucleolin nardilysin convertase secreted phosphoprotein 1,Osteopontin poly(A) binding protein 1 Poly(rC)-binding protein 1 12 6 Programmed cell death 6-interact phosphofructokinase, platelet Profilin-1 protein kinase C delta Proteasome subunit alpha 2 Proteasome subunit beta 5 proteasome 26S subunit, ATPase 3 2 1 proteasome 26S subunit 1

Molecular function translation initiation translation initiation, protein binding translation initiation ECM protein ion & lipid binding cytoskeletal protein binding hyrolysae, transferase, ligase peptidase, signaling dimerization, metal ion binding fibroblast growth factor binding enzyme activator, hydrolase growth factor activity actin & myosin binding enzyme modulator G-protein modulator molecular function protein domain specific binding actin & protein binding GPCR activity hydrolase, binding phosphatase & kinase binding transcription corepressor, prot.binding actin & protein & RNA binding RNA & protein binding metalloprotease ECM binding, cytokine activity RNA & protein binding DNA & protein binding protein binding transferase and kinase activity binding non-receptor kinase endopeptidase endopeptidase hydrolase enzyme regulator

16

Reactome pathway

cell adhesion lipid metabolism Netrin1 signaling lipid metabolism signaling membrane fusion, neutrophil degranulation hemostasis lipid metabolism carbohydrate metabolism apoptosis, programmed cell death splicing Rho GTPase effector signaling platelet activation & signaling T cell activation, IL12 & PKA signaling clathrin endocytosis, vesicle transport protein transport & modification InsR signaling, transport, endocytosis raft organization, migration epigenetic regulation membrane traffic, Rho GTPase signaling LIF response, angiogenesis migration, proliferation ECM organization, signal transduction splicing, RNA silencing, translation splicing protein binidng Glucose metabolism Rho GTPase signaling IFN & cytokine & multiple signaling pathways ABC transporter, multiple signaling pathways multiple signaling pathways multiple signaling pathways multiple signaling cascades

Table S4B cont. Exo - in vitro Synonym Prot.M. Pept.M. Psmd11 37 13 Psmd13 19 7 Psmd2 32 19 Psmd3 24 13 Pygl 14 7 Rab10 26 3 Rap2b 4 3 Rdx 29 10 Rnh1 6 4 Rpl3 9 4 Rtn4 28 8 Ruvbl1 17 8 Ruvbl2 20 10 Scamp3 8 4 Sf3b3 12 6 Slc25a5 9 4 Smarca5 15 5 Snrpb 10 3 Ssrp1 48 10 Stk24 17 6 Tcp1 42 14 Tln1 24 14 Tm9sf2 15 9 Tm9sf3 8 3 Tmed4 7 4 Tmed9 11 5 Tra2b 36 4 U2surp 16 6 Vat1 10 5 Vcp 226 41 Vim 20 7 Vrk1 7 4 Wdr5 10 5 Ybx1 16 5 Ypel5 7 4

Exo - ex vivo Prot.M. Pept.M. 7 4 1 1 3 3 2 1

6 4 4

2 2 3

1

1

2

1

2 1

1 1

37

14

2 1 1

1 1 1

Protein Description proteasome 26S subunit 11 proteasome 26S subunit 13 proteasome 26S subunit 2 proteasome 26S subunit 3 Glycogen phosphorylase L RAB10, member RAS oncogene family RAP2B, member of RAS oncogene fam. Radixin ribonuclease/angiogenin inhibitor 1 ribosomal protein L3 Reticulon-4 RuvB like AAA ATPase 1 RuvB like AAA ATPase 2 secretory carrier membrane protein 3 Splicing factor 3B subunit 3 solute carrier family 25 member 5 Regulator of chromatin a5 Small nuclear ribonucleoprotein B & B1 structure specific recognition protein 1 Serine/threonine-protein kinase 24 T-complex 1 Talin-1 Transmembrane 9 member 2 Transmembrane 9 superfamily 3 transmembrane p24 trafficking protein 4 transmembrane p24 protein 9 Transformer-2 beta homolog U2 snRNP-associated SURP vesicle amine transport 1 valosin containing protein Vimentin vaccinia related kinase 1 WD repeat domain 5 Y-box binding protein 1 yippee-like 5

Molecular function protein binding, structural molecule endopeptidase. structural molecule enzyme regulator enzyme regulator phosphorylase GTPase, myosinV binding GTPase actin binding nuclease, hydrolase RNA binding protein binding protein binding protein binding ubiquitin ligase binding RNA & protein binding transporter hydrolase, protein binding protein & RNA binding transcription factor, signaling protein kinase peptidase, chaperonin cytoskeleton organization, binding transporter, molecular function transporter, molecular function signal transducer transfer / carrier, vesicle coat nucleic acid & RNA & protein binding mRNA processing reductase, AA transporter hydrolase, protein binding structural protein protein kinase, ATP binding esterase, kinase inhibitor, splicing transcription activator metal ion binding

17

Reactome pathway multiple signaling cascades APC transport, multiple signaling pathways multiple signaling pathways multiple signaling pathways carbohydrate metabolism membrane traffic, vesicle transport signal transduction, platelet activation RAP & PKA signal., junction organiz., endosome traffic. RNA processing signal transduction protein metabolism, signal transduction DNA repair, transcription vesicle-mediated transport splicing energy metabolism epigenetic gene regulation splicing transcriptional regulation Apoptosis, programmed cell death chaperonin & protein folding, protein metabolism integrin signaling

transmembrane transport transport & vesicle-med. transport splicing splicing transmembrane transporter APC & small molecule transporter, stress response apoptotic cleavage, programmed cell death cell cycle HOX gene activation transcription, splicing neutrophil degranulation

Table S4C Higher recovery in Exosomes from ex vivo MDSC Synonym Adh5 Alad Apoe Aprt Arf4 Arpc2 C1qc Ca2 CD147 CD177 CD41 Cfp Ctsg Hgd Hspa1a Hspa1l Ilk Itih2 Itm2b Jup Myh1 Myh4 Ngp Pkm Prdx2 Prdx6 Psap Rab5a Rpl7 Rps17 Tfrc Ube2n Vcl Ywhaq

Exo - in vitro Prot.M. Pept.M. . . 10 2 . . . 3 1 2 1 . 3 2 2 1 . 1 1 . . . . . . . . . 1 1 . 1 1 . 1 1 . 7 1 . . . . .

Exo - ex vivo Prot.M. Pept.M. 5 3 13 7 22 8 8 3 22 4 24 12 5 4 8 4 4 3 14 9 6 5 143 7 13 4 3 3 38 6 41 5 7 6 100 7 5 4 12 7 54 13 63 15 19 4 186 24 9 4 10 6 8 6 17 5 18 5 8 4 20 5 4 3 5 3 18 3

Protein Description Alcohol dehydrogenase class-3 aminolevulinate dehydratase Apolipoprotein E Adenine phosphoribosyltransferase ADP-ribosylation factor 4 Actin-related protein 2/3 subunit 2 complement C1q C chain Carbonic anhydrase 2 Basigin CD177 antigen Cd177 Integrin alpha-IIb complement factor properdin Cathepsin G Homogentisate 1,2-dioxygenase Heat shock 70 kDa protein 1A Heat shock 70 kDa protein 1-like Integrin-linked kinase Inter-alpha-trypsin inhibitor H2 Integral membrane protein 2B Junction plakoglobin myosin heavy chain 1 myosin heavy chain 4 Neutrophilic granule protein Pyruvate kinase PKM Peroxiredoxin-2 Peroxiredoxin-6 Prosaposin RAB5A, RAS oncogene family ribosomal protein L7 ribosomal protein S17 Transferrin receptor Ubiquitin-conjugating enzyme E2 N Vinculin 14-3-3 protein theta

Molecular function oxidoreductase, ion & fatty acid binding lyase, ion & protein binding protein & lipid binding transferase, AMP binding GTP & protein binding cytoskeleton, protein complex binding endopeptidase dehydrase, lyase, metal ion binding carbohydrate binding molecular function protein binding ECM component serine protease oxidoreductase protein binding protein folding, GPCR binding non-receptor tyrosine kinase protease inhibitor protein binding protein binding G-protein modulator G protein modulator peptidase inhibitor catalytic activity, protein binding protein binding, oxidase binding, catalytic & hydrolase activity immune defense, surfactant GTPase DNA & RNA binding structural ribosome constituent receptor activity, protein binding transferase, protein binding Rho GTPase, protein & actin binding chaperone, protein binding

18

Reactome pathway metabolism, oxidation Porphyrin & heme metabolsim vesicle & lipid transport, signaling purine metabolism organelle biogenesis, trafficking & transport Rho GTPase & Ephrin signal., endocyt., ves.transport complement cascade, granuloc. & M differentiation O2/CO2 metabolism ECM organization, membrane transport fibrin clot formation integrin signaling, ECM organization complement activation ECM organization, MMP activation, IGF transport AA metabolism stress response ECM & cell junction organization hyaluronan & protein metabolism protein metabolism, amyloid precursor regulation junction organization, VEGF signaling membrane & vesicle med. trafficking defense, angigoenesis regulation carbohydrate metabolism stress response stress response TLR signaling clathrin endocyt., membr. & ves. transport, ves. biogen translation initiation translation, mRNA catabolism clathrin endocyt., membr. & ves. transport, ves. biogen. IFN-, cytokine-,TLR signaling platelet activation & signaling Rho GTPase sign., BAD activ., vesicle transport

Table S5 Differences in MDSC versus MDSC-Exo protein profile Table S5A Preferential peptide recovery in MDSC-Exo Synonym. Ap2m1 Ap2S1 Arrb2 Atp6ap2 C1qtnf3 C3 Casp6 Ccdc47 Ccl9 Cct4 CD316 CD81 Col1a1 CSN3 CSN4 CSN6 CSN8 Cyp4f3 Dek Dera Eea1 Eftud2 Eif3a Eif3b Eif3c Eif3e Eif3f Eif3g Eif3l Eif3m Emilin2 Fgl2 Fn1 H2afy

MDSC Prot.M. Pept.M. 5 2 . 2 1 . . 19 5 3 . 9 . . . . . . . 3 4 . 5 . 9 21 3 2 8 1 1 2 . . . .

2 3

2 2 4 4 4 2 2 1 1 1 1

MDSC-Exo Prot.M. Pept.M. 21 10 11 5 13 6 9 6 7 2 119 16 5 4 33 14 13 4 32 10 24 8 14 5 4 4 3 2 4 4 4 4 3 2 17 6 38 9 11 6 113 13 14 8 121 21 42 17 128 19 49 18 42 9 20 5 32 10 27 7 20 10 11 5 580 71 82 14

Protein Description AP-2 complex mu AP-2 complex sigma arrestin beta 2 ATPase H+ transporting 2 C1q and TNF related protein 3 Complement C3 Caspase-6 Coiled-coil domain- 47 C-C chemokine 9 chaperonin TCP1 subunit 4 Immunoglobulin superfamily 8 CD81 antigen collagen type I alpha 1 COP9 signalosome subunit 3 COP9 signalosome subunit 4 COP9 signalosome subunit 6 COP9 signalosome subunit 8 cytochrome P450 family 4F3 DEK proto-oncogene Deoxyribose-phosphate aldolase Early endosome antigen 1 elongation factor Tu 2 Eukaryotic initiation factor 3 A Eukaryotic initiation factor 3 B Eukaryotic initiation factor 3 C Eukaryotic initiation factor 3 E Eukaryotic initiation factor 3 F Eukaryotic factor 3 G Eukaryotic initiation factor 3 L Eukaryotic initiation factor 3 M elastin microfibril interfacer 2 fibroleukin Fibronectin 1 Core histone H2A.1

19

Function protein binding, receptor vesicle coat protein enzyme modulator enzyme binding, receptor protein binding cytokine, protease inhibitor peptidase calcium & RNA binding chemokine chaperonin protein binding adhesion, signaling protein binding, ECM molecular function protein binding, protease transcription, peptidase protein binding, signaling oxygenase DNA / RNA binding lyase ion/protein binding G protein, hydrolase translation protein/RNA binding, transcript. translation binding, translation peptidase, translation binding, translation translation translation ECM protein signaling, peptidase binding, signaling histone, binding

Reactome pathway trafficking, transmission, endocytosis, signaling lipid & vesicel transport, endocytosis, cargo recognition, signaling multiple signaling pathways (HH, WNT5A, FZD4, GPCR), metabolism (angiotensin) complement cascade GPCR signaling, complement activation apoptosis execution Ca++ homeostasis, ER organization, osteoblast differentiation GPCR signaling protein folding & metabolism proliferation, cell component movement immune regulation ECM organization endocytosis, cargo recogn., membrane traffic, vesicle transport excision repair, endocytosis, vesicle transport transcription, endocytosis, membrane traffic, vesicle transport endocytosis, trafficking, cargo recognition, vesicle transport lipid metabolism regulation of gene expressio carbohydrate metabolism TLR cascades splicing translation initiation translation, metabolism translation initiation translation initiation translation, protein metabolism translation initiation translation initiation translation, protein metabolism cell adhesion neutrophil degranulation matrix formation, multiple sign. cell cycle, rRNA regulation

Table S5A cont. Synonym. Hist1h3a Hist1h3b Hist2h2aa1 Ighm Klhdc4 Lpl Mfge8 Mmp12 Mmp19 Nrdc Opn,Spp1 Paics Pdcd6ip Plscr3 Polr2c Prkcd Psmd1 Psmd13 Psmd2 Psmd3 Psmd6 Ruvbl1 Ruvbl2 Sbno1 Scimp Sdcbp Sec13 Serpinc1 Srsf1 Ssrp1 Stk24 Sub1 Supt16h Tnfsf13 Tspan14 Vps28 Wdr5

MDSC Prot.M. Pept.M. . . . . . . 19 5 1 1 . . . . 6 3 . . 6 4 1 1 1 1 7 6 5 3 1 1 2 1 4 3 . . 4 1 . . 6 2 . . 1 1 1 1 . 1 1 . 1 1

MDSC-Exo Prot.M. Pept.M. 64 5 69 5 107 7 9 5 27 11 174 12 120 18 324 22 56 14 10 7 20 4 8 5 118 27 5 3 10 6 222 25 12 7 19 7 32 19 24 13 35 16 17 8 20 10 2 2 6 2 68 6 4 2 41 6 67 8 48 10 17 6 58 8 7 5 4 2 9 5 11 5 10 5

Protein Description Histone H3.1 Histone H3.2 Histone H2A type 2A Ig mu chain C region Kelch domain-containing 4 Lipoprotein lipase Lactadherin matrix metallopeptidase 12 matrix metallopeptidase 19 nardilysin convertase Osteopontin p-ribosylamin lase/synthase Programmed cell death 6-interact. Phospholipid scramblase 3 RNA polymerase II subunit C protein kinase C delta proteasome 26S, non-ATPase 1 proteasome 26S, non-ATPase 13 proteasome 26S, non-ATPase 2 proteasome 26S, non-ATPase 3 proteasome 26S, non-ATPase 6 RuvB like AAA ATPase 1 RuvB like AAA ATPase 2 strawberry notch homolog 1 SLP adapter & CSK-interact. p. syndecan binding protein SEC13, COPII coat complex serpin family C member 1 Serine/arginine-rich splicing F1 1 structure recognition protein 1 Serine/threonine-protein kinase 24 SUB1 transcriptional regulator SPT16 chromatin remodeling TNF superfamily member 13 Tetraspanin-14 VPS28, ESCRT-I subunit WD repeat domain 5

20

Function histone histone, binding histone binding molecular function esterase, lipase binding, protease, transporter peptidase metallopeptidase metalloprotease cytokine ligase, lyase, binding binding transfer/carrier protein DNA & protein binding transferase, kinase, carrier enzyme modulator enzyme modulator binding, enzyme modulator enzyme modulator regulator binding, hydrolase protein & ATP binding molecular function protein binding binding, traffic regul. traffic. reulator protease inhibitor RNA & protein binding HMG box TF, signaling kinase, transferase transcription coactiv. RNA binding, transcript. cofactor cytokine adhesion, signaling binding esterase, protein binding

Reactome pathway multiple signaling pathways, stress response cytokine/interleukin signaling, stress response metalloprotease, cell cycle regulation BCR signaling retinoid/lipid metabolsim/transport signaling, transporter ECM organization/degradation ECM degradation migration, proliferation ECM organization, signaling metabolism TJ & MVB & Exo assembly, protein transport apoptosis, cholesterol & glucose homeostasis splicing, transcription, signaling multiple signaling pathways multiple sign.pathways multiple signaling pathways multiple signaling pathways multiple signaling pathways multiple signaling pathways, protein metabolism, ß-cat/TCF activation DNA repair, transcription activation, protein folding transcription regulation immune synapse formation Ephrin signaling Rho signaling, cell cycle, vesicle transport, membrane traffic. clotting cascade splicing, mRNA transport transcription apoptosis execution transcription regulation, SMAD signaling transcription cytokine signaling (TNFR) plasma membrane organization, NOTCH sign. neutrophil degranulation ESCRT, vesicle transport kinase inhibitor

Table S5B Higher peptide recoveryn in MDSC than MDSC-Exo Synonym. Acaa1a Acad Aco2 Acsl1 Actn1 Actn4 Adpgk Agps Ak2 Akr1a1 Aldh2 Alox5 Ankfy1 Anp32b Ap1b1 Api5 Apmap Arf4 Arg1 Arhgap1 Arl8a Arl8b Arpc2 Asah1 Atp1a1 Atp2a2 Atp2a3 Atp5a1 Atp5b Atp5c1 Atp5f1 Atp5h Atp5i Atp5o Atp6v1a Atp6v1b2 B4galnt1 Bax Bcap31 Blvrb Bzw1 Ca2 Calr Canx Capn1 Cat CD166 CD177 CD180 CD206 CD29 CD41 CD61 CD74 Celf2 Chil3 Ckap4 Cmpk2 Cndp2

Protein Description acetyl-CoA acyltransferase 1A Acyl-CoA dehydrogenase Aconitate hydratase, mitochondrial Acyl-CoA synthetase long-chain family member 1 actinin alpha 1 actinin alpha 4 ADP-dependent glucokinase alkylglycerone phosphate synthase Adenylate kinase 2 aldo-keto reductase family 1 member A1 aldehyde dehydrogenase 2 family Arachidonate 5-lipoxygenase Ankyrin repeat and FYVE domain containing 1 Acidic nuclear phosphoprotein 32 B AP-1 complex subunit beta-1 Apoptosis inhibitor 5 Adipocyte plasma membrane-associated protein ADP-ribosylation factor 4 Arginase-1 Rho GTPase-activating protein 1 ADP-ribosylation factor-like GTPase 8A ADP-ribosylation factor-like GTPase 8B Actin-related protein 2/3 complex subunit 2 N-acylsphingosine amidohydrolase 1 ATPase Na+/K+ transporting subunit alpha 1 ATPase Ca2+ transporting 2 ATPase Ca2+ transporting 3 ATP synthase, H+ transporting F1 alpha subunit 1 ATP synthase, H+ transporting F1 beta subunit ATP synthase, H+ transporting F1 gamma subunit ATP synthase, H+ transporting, F0 subunit B1 ATP synthase, H+ transporting, F0 subunit D ATP synthase, H+ transporting, F0 subunit E ATP synthase, H+ transporting, F1 subunit O ATPase H+ transporting V1 subunit A ATPase H+ transporting V1 subunit B2 Beta-1,4 N-acetylgalactosaminyltransferase 1 BCL2 associated X, apoptosis regulator B-cell receptor-associated protein 31 biliverdin reductase B Basic leucine zipper and W2 domain 1 Carbonic anhydrase 2 Calreticulin Calnexin Calpain-1 Catalase CD166 antigen, Alcam CD177 antigen CD180 antigen mannose receptor C-type 1 Integrin beta-1 Integrin alpha-IIb Integrin beta-3 CD74 antigen, invariant chain associated CUGBP Elav-like family member 2 Chitinase-like 3 Cytoskeleton-associated protein 4 cytidine/uridine monophosphate kinase 2 CNDP dipeptidase 2

21

MDSCinvitro Prot.M. Pept.M. 20 7 4 9 78 20 30 9 141 35 128 36 36 11 23 5 30 9 43 11 262 29 49 13 8 5 30 14 33 12 8 5 8 6 38 8 35 10 12 5 23 8 37 11 83 15 12 6 49 11 72 15 29 6 331 25 296 25 46 6 30 11 21 8 12 5 42 9 52 16 20 5 17 12 14 6 10 5 10 5 14 8 21 7 159 22 87 19 29 18 30 15 24 12 39 13 15 8 48 27 27 9 23 12 14 6 23 5 10 5 63 13 54 20 37 12 77 16

MDSC in vitro-Exo Prot.M. Pept.M.

8

4

7

5

5

3

4

3

3

2

8

4

1 1

1 1

2

1

1

1

2 3

1 2

1 6 2

1 3 1

3

1

5

2

6

3

Table S5B cont. Synonym. Colgalt1 Copz1 Cox4i1 Cox5a Cox6c Cs Ctsa Ctsc Ctsg Ctsz Cyb5r3 Cybb Cycs Dars Ddost Ddx39a Ddx39b Ddx3x Ddx5 Dhx9 Diaph1 Dlat Dld Dlst Dnajb11 Dnajc3 Dock8 Dpysl2 Dstn Ear2 Echs1 Eef1b Eef1d Eef1g Eef2 Ehd1 Ehd4 Eif1 Eif4a3 Eif5a Elane Elavl1 Emb Entpd1 Erap1 Ero1a Erp29 Erp44 Esyt1 Etfa Etfb Fabp5 Fam129b Fam162a Fam49b Fh Fis1 Fkbp2 Fkbp3

Protein Description collagen beta(1-O)galactosyltransferase 1 Coatomer protein complex subunit zeta 1 cytochrome c oxidase subunit IV isoform 1 cytochrome c oxidase subunit 5A Cytochrome c oxidase subunit 6C Citrate synthase cathepsin A cathepsin C Cathepsin G Cathepsin Z cytochrome b5 reductase 3 Cytochrome b-245 beta chain Cytochrome c, somatic Aspartate--tRNA synthase dolichyl-diphosphooligosacch glycosyltransfer. DExD-box helicase 39A DExD-box helicase 39B DEAD-box helicase 3, X-linked DEAD-box helicase 5 DEAH-box helicase 9 diaphanous related formin 1 dihydrolipoamide S-acetyltransferase dihydrolipoamide dehydrogenase dihydrolipoamide S-succinyltransferase DnaJ heat shock protein family B11 DnaJ heat shock protein family C3 Dedicator of cytokinesis 8 Dihydropyrimidinase like 2 Destrin eosinophil-associated, ribonuclease A2 Enoyl-CoA hydratase, short chain 1 Elongation factor 1-beta Elongation factor 1-delta Elongation factor 1-gamma Elongation factor 2 EH domain-containing 1 EH domain-containing 4 Eukaryotic translation initiation factor 1 Eukaryotic initiation factor 4A3 Eukaryotic translation initiation factor 5A elastase, neutrophil expressed ELAV like RNA binding protein 1 Embigin Ectonucleos. triphosphate diphosphohydrolase 1 Endoplasmic reticulum aminopeptidase 1 endoplasmic reticulum oxidoreductase 1 alpha Endoplasmic reticulum protein 29 Endoplasmic reticulum protein 44 Extended synaptotagmin-1 Electron transfer flavoprotein alpha subunit Electron transfer flavoprotein beta subunit Fatty acid-binding protein 5 family with sequence similarity 129 member B family with sequence similarity 162 member A family with sequence similarity 49 member B Fumarate hydratase fission, mitochondrial 1 FK506 binding protein 2 FK506 binding protein 3

22



2

2

4 1 2

2 1 1

6 1

3 1

4 4

2 3

3

2

2 4

2 2

1 2 8 29 6 8

1 2 4 8 2 4

4

1

1

1

3

2

22 2

9 1

1

1

3

2

Table S5B cont. Synonym. Flna G3bp1 Ganab Gba Glb1 Glg1 Glo1 Glud1 Gmfg Gnai2 Gnb1 Gnb2 Got2 Gpd2 Gpi Gpx1 Grb2 Gsr Gstm1 Gsto1 H2-D1 H2-Eb1 Hadha Hadhb Hars Hdac1 Hexa Hexb Hk1 Hk2 Hk3 Hmox1 Hmox2 Hnrnpa1 Hnrnpa2b1 Hnrnpa3 Hnrnpab Hnrnpd Hnrnpf Hnrnph1 Hnrnpk Hnrnpl Hnrnpm Hpcal1 Hprt1 Hsd11b1 Hsd17b11 Hsd17b12 Hsd17b4 Hsp90aa1 Hsp90b1 Hspa1a Hspa5 Hspd1 Hspe1 Hyou1 Idh2 Idh3a Ifi44

Protein Description Filamin-A GTPase activating protein binding protein 1 glucosidase II alpha subunit Glucosylceramidase beta galactosidase, beta 1 Golgi apparatus protein 1 glyoxalase I Glutamate dehydrogenase 1 Glia maturation factor gamma guanine nucleotide binding protein alpha inhibit 2 guanine nucleotide binding protein beta 1 guanine nucleotide binding protein beta 2 Aspartate aminotransferase, mitochondrial Glycerol-3-phosphate dehydrogenase 2 Glucose-6-phosphate isomerase Glutathione peroxidase 1 Growth factor receptor-bound protein 2 Glutathione disulfide reductase Glutathione S-transferase Mu 1 Glutathione S-transferase omega-1 histocompatibility 2, D region locus 1 histocompatibility 2, class II antigen E beta hydroxyacyl-CoA dehydrogenase alpha subunit hydroxyacyl-CoA dehydrogenase beta subunit histidyl-tRNA synthetase Histone deacetylase 1 Hexosaminidase subunit alpha Hexosaminidase subunit beta Hexokinase-1 Hexokinase-2 Hexokinase-3 Heme oxygenase 1 Heme oxygenase 2 Heterogeneous ribonucleoprotein A1 Heterogeneous ribonucleoproteins A2/B1 Heterogeneous ribonucleoprotein A3 Heterogeneous ribonucleoprotein A/B Heterogeneous ribonucleoprotein D0 Heterogeneous ribonucleoprotein F Heterogeneous ribonucleoprotein H1 Heterogeneous ribonucleoprotein K Heterogeneous ribonucleoprotein L Heterogeneous ribonucleoprotein M Hippocalcin-like 1 Hypoxanthine phosphoribosyltransferase 1 hydroxysteroid 11-beta dehydrogenase 1 hydroxysteroid 17-beta dehydrogenase 11 hydroxysteroid 17-beta dehydrogenase 12 hydroxysteroid 17-beta dehydrogenase 4 Heat shock protein HSP 90-alpha heat shock protein 90, beta 1 Heat shock 70 kDa protein 1A heat shock protein 5 heat shock protein family D (Hsp60) member 1 heat shock protein family E (Hsp10) member 1 Hypoxia up-regulated protein 1 Isocitrate dehydrogenase [NADP] 2 Isocitrate dehydrogenase [NAD] alpha Interferon-induced protein 44

23


MDSC in vitro-Exo Prot.M. Pept.M. 27 16

5

3

19

5

3

2

5

3

2

2

1

1

7

4

2 2

1 1

1 2 4

1 1 2

14 1

3 1

2

1

20

4

43

11

Table S5B cont. Synonym. Ifit1 Ifit2 Immt Kctd12 Kpnb1 Lap3 Lbr Lcp1 Lgals1 Lgals9 Lmnb1 Lnpep Lpcat2 Lrpap1 Lrpprc Lrrc59 Lta4h Man2b1 Mapre1 Mcm2 Mdh1 Mdh2 Me2 Mogs Mta2 Mthfd1l Mybbp1a Naga Naa50 Napa Nceh1 Ncf1 Ncf2 Ncf4 Ncstn Ndufa13 Ndufa8 Ndufb10 Ndufs3 Nme1 NOMO1 Nucb1 Nudt21 Oas1a Oat Ogdh Osbpl8 Ostf1 Oxct1 P4hb Pai2 Pcbp2 Pdcd10 Pdia3 Pdia4 Pdia6 Phb Phb2 Pitpna

Protein Description Interferon-induced protein 1 Interferon-induced protein 2 inner membrane mitochondrial protein potassium channel 12 karyopherin subunit beta 1 leucine aminopeptidase 3 Lamin-B receptor lymphocyte cytosolic protein 1 Galectin-1 Galectin-9 Lamin-B1 Lmnb1 Leucyl-cystinyl aminopeptidase Lysophosphatidylcholine acyltransferase 2 LDL receptor related protein associated protein 1 leucine rich pentatricopeptide repeat containing Leucine-rich repeat-containing protein 59 Leukotriene A-4 hydrolase mannosidase alpha class 2B member 1 Microtubule-associated protein RP/EB member 1 minichromosome maintenance complex 2 Malate dehydrogenase 1 Malate dehydrogenase 2 Malic enzyme 2 Mannosyl-oligosaccharide glucosidase metastasis associated 1 family member 2 methylenetetrahydrofolate dehydrogenase 1-like Myb-binding protein 1A Alpha-N-acetylgalactosaminidase N-alpha-acetyltransferase 50 NSF attachment protein alpha Neutral cholesterol ester hydrolase 1 Neutrophil cytosol factor 1 Neutrophil cytosol factor 2 Neutrophil cytosol factor 4 Nicastrin NADH:ubiquinone oxidoreductase A13 NADH:ubiquinone oxidoreductase A8 NADH:ubiquinone oxidoreductase B10 NADH:ubiquinone oxidoreductase S3 NME/NM23 nucleoside diphosphate kinase 1 Nodal modulator 1 Nucleobindin-1 nudix hydrolase 21 2'-5'-oligoadenylate synthase 1A Ornithine aminotransferase oxoglutarate dehydrogenase Oxysterol-binding protein like 8 Osteoclast-stimulating factor 1 3-oxoacid CoA-transferase 1 prolyl 4-hydroxylase subunit beta Plasminogen activator inhibitor 2 Poly(rC)-binding protein 2 Programmed cell death 10 Protein disulfide-isomerase A3 Protein disulfide-isomerase A4 Protein disulfide-isomerase A6 Prohibitin Prohibitin-2 Phosphatidylinositol transfer protein alpha

24



4 1

2 1

20 1

9 1

1

1

5 11

3 5

10

3

3

2

3

2

3

2

6

2

3

2

3

2

Table S5B cont. Synonym. Pkm Plbd1 Pld4 Plec Plek Pnkp Pnp Por Ppa1 Ppif Ppp1ca Ppp1cb Ppp1cc Ppp2ca Ppp2r1a Prdx2 Prdx3 Prdx4 Prdx5 Prdx6 Prtn3 Psma4 Psme1 Ptbp3 Ptgr1 Ptgs1 Ptpn1 Ptprc Pycard Pygl Rab18 Rab21 Rab27a Rab2a Rab31 Rab5a Ralb Rap2c Rbbp4 Rbbp7 Rbmxl1 Rnf213 Rnh1 Rpl13 Rpl13a Rpl15 Rpl17 Rpl18a Rpl4 Rpl7 Rpn1 Rpn2 Rps14 Rps16 Rps17 Rps9 Rrbp1 Samhd1 Sars

Protein Description Pyruvate kinase PKM phospholipase B domain containing 1 Phospholipase D4 Plectin Pleckstrin polynucleotide kinase 3'-phosphatase Purine nucleoside phosphorylase cytochrome P450 oxidoreductase pyrophosphatase 1 Peptidyl-prolylisomerase F protein phosphatase 1 catalytic subunit alpha protein phosphatase 1 catalytic subunit beta protein phosphatase 1 catalytic subunit gamma protein phosphatase 2 alpha Protein phosphatase 2A subunit A Peroxiredoxin-2 Peroxiredoxin-3 Peroxiredoxin-4 Peroxiredoxin-5 Peroxiredoxin-6 proteinase 3 Proteasome subunit alpha 4 proteasome activator subunit 1 Polypyrimidine tract-binding protein 3 Prostaglandin reductase 1 prostaglandin-endoperoxide synthase 1 protein tyrosine phosphatase, non-receptor type 1 protein tyrosine phosphatase, receptor type C PYD and CARD domain containing Glycogen phosphorylase L RAB18, member RAS oncogene family RAB21, member RAS oncogene family RAB27A, member RAS oncogene family RAB2A, member RAS oncogene family RAB31, member RAS oncogene family RAB5A, member RAS oncogene family RAS like proto-oncogene B RAP2C, member of RAS oncogene family RB binding protein 4 RB binding protein 7 RNA binding motif protein, X-linked-like-1 ring finger protein 213 ribonuclease/angiogenin inhibitor 1 ribosomal protein L13 ribosomal protein L13a ribosomal protein L15 ribosomal protein L17 ribosomal protein L18a ribosomal protein L4 ribosomal protein L7 ribophorin I ribophorin II ribosomal protein S14 ribosomal protein S16 ribosomal protein S17 ribosomal protein S9 Ribosome-binding protein 1 SAM and HD domain cont. triphosphohydrolase 1 seryl-tRNA synthetase

25


MDSC in vitro-Exo Prot.M. Pept.M. 2

1

1

1

2

2

5

2

1

1

4

3

2 1

1 1

14

7

5

2

1 2

1 1

6 8

4 2

2 2

1 1

5 7

2 1

14

2

1

1

Table S5B cont. Synonym. Scp2 Sdf2l1 Sdha Sec11c Sec22b Sec61a1 Sept11 Serpinb1a Sfpq Sfxn1 Sgpl1 Sh3bgrl Siglec1 Slc25a11 Slc25a12 Slc25a3 Slc25a4 Slc25a5 Slc3a2 Snd1 Snx2 Sod2 Spcs2 Sptan1 Ssr4 Stat1 Stfa1 Stfa3 Stip1 Stt3a Stx7 Sun2 Surf4 Syncrip Tagln2 Taldo1 Tap1 Tap2 Tapbp Tfrc Tgm2 Tkt Tln1 Tln2 Tmem43 Tmpo Tor1aip1 Tpi1 Tpr Trap1 Trex1 Tubb2a Txn Txndc5 U2af2 Ube2n Ube2v1 Uggt1 Ugt1a7c

Protein Description sterol carrier protein 2 Stromal cell-derived factor 2-like 1 succinate dehydrogenase complex flavoprotein A SEC11 homolog C, signal peptidase complex SEC22 homolog B, vesicle trafficking protein Sec61 translocon alpha 1 Septin-11 Leukocyte elastase inhibitor A Splicing factor, proline- and glutamine-rich Sideroflexin-1 Sphingosine-1-phosphate lyase 1 SH3 domain binding glutamate rich protein like sialic acid binding Ig-like lectin 1, sialoadhesin solute carrier family 25 member 11 solute carrier family 25 member 12 solute carrier family 25 member 3 solute carrier family 25 member 4 solute carrier family 25 member 5 solute carrier family 3 member 2 Staphylococcal nuclease & tudor domain 1 Sorting nexin-2 Superoxide dismutase 2 Signal peptidase complex subunit 2 Spectrin alpha, non-erythrocytic 1 signal sequence receptor subunit 4 Signal transducer and activator of transcription 1 Stefin A1 Stefin A3 Stress-induced-phosphoprotein 1 STT3A, oligosaccharyltransferase complex Syntaxin-7 SUN domain-containing protein 2 Surfeit 4 synaptotagmin binding protein Transgelin-2 Transaldolase 1 transporter 1, ATP binding cassette subfamily B transporter 2, ATP binding cassette subfamily B TAP binding protein Transferrin receptor transglutaminase 2 Transketolase Talin-1 Talin-2 Transmembrane protein 43 thymopoietin Torsin-1A-interacting protein 1 Triosephosphate isomerase translocated promoter region TNF receptor associated protein 1 Three-prime repair exonuclease 1 Tubulin beta-2A Thioredoxin Thioredoxin domain containing 5 U2 small nuclear RNA auxiliary factor 2 Ubiquitin-conjugating enzyme E2 N Ubiquitin-conjugating enzyme E2 variant 1 UDP-glucose glycoprotein glucosyltransferase 1 UDP glucuronosyltransferase 1 polypeptide A7C

26


MDSC in vitro-Exo Prot.M. Pept.M. 1

1

1

1

3

2

2

1

9 11 1

4 5 1

2

1

1

1

4

2

4 8

2 3

4

1

1 24

1 14

1

1

6

3

1

1

Table S5B cont. Synonym. Uqcrc2 Uso1 Vamp8 Vapa Vars Vcl Vdac1 Vdac2 Vdac3 Vim Vps35 Vwa5a Wdr1 Xpo1 Ywhaq

Protein Description ubiquinol-cytochrome c reductase core protein II USO1 vesicle transport factor Vesicle-associated membrane protein 8 VAMP associated protein A valyl-tRNA synthetase Vinculin Voltage-dependent anion channel 1 Voltage-dependent anion channel 2 Voltage-dependent anion channel 3 Vimentin vacuolar protein sorting 35 homolog von Willebrand factor A domain 5A WD repeat domain 1 Exportin-1 14-3-3 protein theta

27

MDSCinvitro Prot.M. Pept.M. 22 11 17 6 12 5 21 5 15 7 27 14 64 15 63 11 40 7 371 42 49 13 24 12 57 14 11 5 48 6


2

1

2

2

20 6

7 2

1

1

Table S6 a MDSC-Exo peptides recovered in pulldown from Sepharose-coupled lymph node cell membranes MDSC-Exo over LNC membrane Prot.M.

cellular localization

Synonym

Protein Description

Acaa2

acetyl-CoA acyltransferase 2

14

9

mitochondrion

Acadvl

acyl-CoA dehydrogenase, very long chain

21

12

mitochondrial inner membr.

Aco2

Aconitate hydratase, mitochondrial

8

5

mitochondrion, myelin sheet

Acta2

actin, alpha 2, smooth muscle, aorta

68

20

cytoskeleton, cytoplasm

Aldh2

aldehyde dehydrogenase 2 family

15

9

mitochondrion

Ank1

Ankyrin-1

6

5

cytoskel., plasma membrane

Anxa11

Annexin A11

9

5

cytoskel., membrane

Anxa4

Annexin A4

8

5

plasma membrane

Arhgdib

Rho GDP-dissociation inhibitor beta

7

6

cytoplasm, membrane

Arhgef1

Rho guanine nucleotide exchange factor 1

6

5

cytoplasm, plasma membrane

Atp1a1

ATPase Na+/K+ transporting subunit alpha 1

22

11

ER & plasma membrane, raft

Atp2a1

ATPase ER Ca2+ transporting 1

27

13

ER membrane

Atp2a3

ATPase ER Ca2+ transporting 3

14

8

ER membrane

Cap1

CAP, adenylate cyclase-associated protein 1

17

8

cytoskeleton, raft

Capg

capping actin protein, gelsolin like

9

5

nucleus, cytoplasm

CD11b

Integrin alpha-M

7

5

plasma membrane

Copg1

coatomer protein complex subunit gamma 1

6

5

cytoplasmic vesicle coat

Cyc1

Cytochrome c1

10

5

nucleus, mitoch. inner membr.

Ddx39b

DExD-box helicase 39B

9

5

nucleus, cytopl.,spliceosome

Ddx5

DEAD-box helicase 5

11

7

nucleus, cytopl.,spliceosome

Dync1h1

dynein cytoplasmic 1 heavy chain 1

8

6

cytopl., cytoskel. (complex)

Eftud2

elongation factor Tu GTP binding domain containing 2

28

15

nucleus, spliceosome compl.

Eif3b

eukaryotic translation initiation factor 3 subunit B

9

5

cytoplasm (complex)

Eif4a2

Eukaryotic initiation factor 4A-II Eif4a2

13

6

nucleolus, cytoplasm

Eno1

enolase 1

17

8

raft, synaptic membrane

Etfa

electron transfer flavoprotein alpha subunit

10

6

mitochondrion (complex)

Etfdh

electron transfer flavoprotein dehydrogenase

8

5

mitochondrial membrane

Fgg

Fibrinogen gamma chain

7

5

cytopl,plasm.membr. (extern.)

Flna

Filamin-A

17

9

nucleus, cytosol, pl.membrane

Gimap4

GTPase IMAP family member 4

9

6

cytoplasm

Gpd2

glycerol phosphate dehydrogenase 2, mitochondrial

20

11

membrane (mitochondr.)

H2afx

H2A histone family member X

20

6

nucleus

H2afy

H2A histone family member Y

15

7

nucleus

H2-D1

histocompatibility 2, D region locus 1

18

8

ER, plasma membr., raft

Hadha

hydroxyacyl-CoA dehydrogenase, alpha subunit

43

20


Hadhb

hydroxyacyl-CoA dehydrogenase, beta subunit

12

7


Hist1h1c

histone cluster 1 H1 family member c

13

6

nucleus

Hist1h1e

histone cluster 1 H1 family member e

11

5

nucleosome

Hist1h2bb

histone cluster 1 H2B family member b

84

5

nucleus, cytoplasm

Hnrnpk

heterogeneous nuclear ribonucleoprotein K

11

5

nucleus, spliceosome compl.

Hnrnpu

heterogeneous nuclear ribonucleoprotein U

15

7

mitotic spindle, spliceosome

Hsp90ab1

heat shock protein 90 alpha, class B member 1

30

16

plasma & vesicle membrane

Idh3a

isocitrate dehydrogenase 3 (NAD+) alpha

17

7

mitochondrion, myelin sheet

Krt8

Keratin 8

10

6


Kpnb1

karyopherin (importin) beta 1

11

6

nuclear membr., ER network

Lman1

lectin, mannose binding 1

8

5

organelles (membrane bound)

Lmnb1

Lamin-B1 Lmnb1

10

6

nucl. membr., intermed.filam.

Msn

Moesin

7

5

plasma membrane (cytopl)

Mtco2

Cytochrome c oxidase subunit 2 Mtco2

16

4

mitochondrial inner membr.

Myh14

Myosin-14

14

7

stress fibers (complex)

Myh4

Myosin-4

156

72

cytoplasm, myosin filament

28

Pept.M

Table S6 cont. MDSC-Exo over LNC membrane

cellular localization

Synonym

Protein Description

Prot.M.

Pept.M

Myh9

Myosin-9

161

70

pl.membr.,signalos.,imm.synapse

Myl1

Myosin light chain 1/3, skeletal muscle isoform

21

8

myosin complex (fibres)

Mylpf

Myosin regulatory light chain 2, skeletal muscle isoform

10

6

lysosome complex

Ndufs1

NADH-ubiquinone oxidoreductase 75 kDa subunit

8

5

mitoch.inner membrane

Ogdh

oxoglutarate dehydrogenase

8

5


Pcbp1

Poly(rC)-binding protein 1

14

7

nucleus, ribosome complex

Pcbp2

Poly(rC)-binding protein 2

9

5

nucleus, cytopl., membrane

Pdia6

protein disulfide isomerase family A member 6

6

5

ER (complex), pl.membr.

Pfn1

Profilin-1

5

3

cytoskeleton, membrane

Phgdh

phosphoglycerate dehydrogenase

7

5

myelin sheath

Pkm

Pyruvate kinase, muscle

19

10

cytopl.,mitochondr. (complex)

Pml

promyelocytic leukemia

8

5

nucleus, cytosol, ER membr.

Psmd1


9

6

proteasome complex

Psmd13


14

7

proteasome complex

Psmd2


9

6

proteasome complex

Psmd6


17

9

proteasome complex

Psme1

Proteasome activator subunit 1

9

5

proteasome complex

Ptprc

protein tyrosine phosphatase, receptor type C

6

5

plasma membr. (outer side)

Rack1

receptor for activated C kinase 1

14

7

cytopl., ribosome, pl.membr.

Rpl12

ribosomal protein L12

9

5

ribosome complex

Rpn1

ribophorin I

9

6

ER membrane

Sdha

succinate dehydrogenase complex flavoprotein subunit A

16

8

mitoch.inner membr.(complex)

Sf3b3

Splicing factor 3B subunit 3

15

8

spliceosome complex

Slc25a11


4

3

mitochondrial inner membrane

Slc25a12


18

11

mitochondr.inner membrane

Slc25a4


15

8

mitoch.membr., cytoskel., raft

Sptb

spectrin beta, erythrocytic

71

32

cytoskel.,pl.membr., (complex)

Stat1

Signal transducer and activator of transcription 1

12

7

nucleus, cytoplasm (complex)

Tap2

transporter 2, ATP binding cassette subfamily B member

7

5

ER membrane (complex)

Tfrc

Transferrin receptor

15

9

endosome & plasma membr.

Tgtp1

T cell specific GTPase 1

10

4

ER membrane

Tln1

Talin-1

38

19

cytoskel.,plasma membrane

Tmed10

transmembrane p24 trafficking protein 10

9

5

organelle (membr., compl.)

Ucp1

uncoupling protein 1 (mitochondrial, proton carrier)

10

7

mitochondr.inner membrane

Vcp


13

9

proteas.compl., ER membr.

Vdac1

voltage-dependent anion channel 1

20

9

mitochonr. & plasma membrane

Vdac2


12

7

mitochonr. & plasma membr., raft

Vdac3


11

5


Vim

Vimentin

12

6


Xpo1

Exportin-1

10

5

cytopl., organelle membr.

a

proteins with ≥3 peptide hits are shown

29

Table S7 In vivo impact of MDSC-Exo on AA spleen cells Table S7A Increased mRNA recovery in spleen cells of MDSC-Exo treated AA mice AA ExoSynonym SC treated Protein name Abca1 1595 10791 ATP-binding cassette sub-family A member 1 Abcg1 1370 5488 ATP-binding cassette sub-family G member 1 Adgre1 1664 7722 Adhesion G protein-coupled receptor E1 Agap1 316 1005 Arf-GAP with GTPase,ANK repeat & PH domain prot.1 Aldh1l2 238 1042 Mitochondrial 10-formyltetrahydrofolate dehydrogenase Als2cl 3427 12879 ALS2 C-terminal-like protein Angptl2 301 1478 Angiopoietin-related protein 2 Ankrd55 270 1993 Ankyrin repeat domain-containing protein 55 Arg1 14325 57553 Arginase-1 Atf3 1102 3326 Cyclic AMP-dependent transcription factor ATF-3 Atf5 2046 6599 Cyclic AMP-dendent transcription factor ATF-5 Atn1 3185 9859 Atrophin-1 Atp6v0d2 1684 6401 V-type proton ATPase subunit d2 C3ar1 1476 8124 C3a anaphylatoxin chemotactic receptor Cacna1d 329 1108 Voltage-dependent L-type calcium channel alpha-1D Cacnb1 284 1131 Voltage-dependent L-type calcium channel beta-1 Ccl2 1445 8996 C-C motif chemokine 2 Ccl7 262 2216 C-C motif chemokine 7 Ccnd1 299 1254 G1/S-specific cyclin D1 Ccr9 420 1790 C-C chemokine receptor type 9 Cd200 820 3180 OX-2 membrane protein Cd302 473 1423 CD302 antigen Cd36 2234 11144 Platelet glycoprotein 4 Cd83 2025 11032 CD83 antigen Cd86 656 2229 T-lymphocyte activation antigen Cdk18 422 1483 Cyclin-dependent kinase 18 Cdkn2a 279 1003 Tumor suppressor ARF Ch25h 123 1868 Cholesterol 25-hydroxylase Ckb 295 1416 Creatine kinase B-type Clec4a1 239 1181 C-type lectin domain family4 member a1 Clec4a2 361 1233 C-type lectin domain family4 member a2 Clec4d 1784 6243 C-type lectin domain family4 member d Clmp 308 1901 CXADR-like membrane protein Colec12 213 1060 Collectin-12 Csf1r 3625 10806 Macrophage colony stimulating factor 1 receptor Ctsk 509 8274 Catheppsin K Ctsl 29630 225943 Catheppsin L1 Cxcl1 265 1346 Growth-regulated alpha protein Cxcl16 1035 3221 C-X-C motif chemokine 16 Cxcl2 1539 5103 C-X-C motif chemokine 2

function transport, lipoprotein biosynthesis, LPS response transport, lipoprotein biosynthesis signal transduction, GPCR signaling ARP3 complex regulation oxidation-reduction GTPase endothelial growth factor protein-protein interaction hydrolase, ion binding transcription, gluconeogenesis chromatin binding, transcription factor transcription corepressor ion transport C3a binding, GPCR activ. ion chanel, ankyrin binding calcium transport cytokine activity, CCR2 binding, chemotaxis cytokine activity, CCR2 binding, chemotaxis transcription chemotaxis, chemokine / GPCR signaling protein binding, adhesion C-type lectin transmembrane receptor cytokine secretion humoral IR TLR & PI3K & BCR & FGFR signaling transferase cell cycle regulation oxidation-reduction kinase activity, IL6, IL12, TNF production , dendritic cell inhibitory receptor, inflamm. monocytes carbohydrate binding Ig receptor binding cell-cell adhesion pattern recognition receptor RTK, cytokine binding, migration peptidase, proteolysis peptidase, histone binding chemokine activ, CXCR binding chemokine & cytokine activity, CXCR binding chemokine & cytokine activity, CXCR binding

30

biological processesa lipoprotein metab, endosomal transport, phagocytosis lipoprotein metabolism, ABC family transport IR regulation membrane traffic, cytoskeleton organization vitamin metabolism endosome organization organism development suggested involvement in autoimmune diseases AA metabolism, T proliferation, IFN signaling TRAIL-activation, apoptotic signaling cell cycle regulation, proliferation, apoptosis CNS development, neuron apoptosis ROS/RNS production, endocytosis, transport chemotaxis, inflamm.IR, C' cascade, GPCR signaling transmembrane transport transmembrane transport, synaptic transmission IL1, IFN, TNF, TGFß, GPCR signaling GPCR signaling, IL1, IFN, TNF response cell cycle regul., Wnt signaling, DNA damage response IR regulation IR (lymphoid-nonlymphoid) regulation myeloid DC migration, phagocytosis antigen cross-present.,vesicl.-med.transport, TLR sign. CD4 different., IL2 & IL10 production CD28-dependent T cell stimulation phosphorylation cell growth (T & B cells) , NFB signaling lipid & lipoprotein metabolism, B cell chemotaxis innate IR, Myd88/TLR, NFB, IL1 signaling  innate & adaptive IR FcR signaling, IR regulation phagocytosis, TLR signaling RTK, STAT, ERK1/2 signaling, innate IR, hemopoiesis TLR cascades ECM organiz, MHCII antig.present., hair follicle morph.. chemotaxis regulation, inflamm. IR, GPCR signaling T cell chemotaxis, TNF, IFN, GPCR signaling chemotaxis, inflammatory IR, GPCR signaling

Table S7A cont. AA ExoSynonym SC treated Protein name Cxcl3 1929 10842 C-X-C motif chemokine 3 Cxcl5 247 2461 C-X-C motif chemokine 5 Dab2 1152 3841 Disabled homolog 2 Dtx1 476 1729 E3 ubiquitin-prootein ligase DTX1 Dusp10 1832 8026 Dual specificity protein phosphatase 10 Ebf1 342 1128 Transcription factor COE1 Ephx1 783 2807 Epoxide hydrolase 1 Fam19a3 1563 5337 Protein FAM19A3 Fam20c 943 3954 Extracellular serine/threonine protein kinase FAM20C Flrt2 617 3655 Leucine-rich repeat transmembrane protein FLRT2 Foxp3 544 2328 Forkhead box protein P3 Fpr1 809 2453 fMet-Leu-Phe receptor Gja1 307 1080 Gap junction alpha-1 protein Gm15056 80 1496 Protein Gm15056 Gpnmb 22678 105624 Transmembrane glycoprotein nMB Hdac9 209 1557 Histone deacetylase 9 Hmox1 9634 49122 Heme oxygenase 1 Ifngr2 1310 4129 Ifngr2 protein Igf1 194 4505 Insulin-like growth factor 1 Igsf23 332 1321 Igsf23 protein Il1r1 301 1675 Interleukin-1 receptor type 1 Il6st 3286 10126 Interleukin6 receptor subunit beta Inadl 1109 4672 InaD-like protein Inhba 814 2779 Inhibin beta A chain Irak3 1023 3230 Interleukin-1 receptor-associated kinase 3 Jag1 289 1143 Protein jagged-1 Kcna2 544 2961 Potassium voltage-gated channel subfamily A 2 Lgmn 3430 10461 Legumain Lhfpl2 1198 4169 Lipoma HMGIC fusion partner-like 2 protein Lpl 1797 11860 Lipoprotein lipase Lrp1 2537 10001 Prolow-density lipoprotein receptor-related protein 1 Lrpap1 5607 18250 Alpha-2-macroglobulin receptor-assoc. protein Lrrc25 505 1584 Leucine-ricj repeat-cont. protein 25 Maf 902 2842 Transcription factor Maf Marcks 726 2765 Myristoylated alanine-rich C-kinase Marco 66 1218 Macrophage receptor Marco Mfge8 2382 22112 Lactadherin Mmp12 33388 179224 Matrix metalloproteinase 12 Mmp13 2876 32434 Matrix metalloproteinase 13 Mmp14 982 3534 Matrix metalloproteinase 14 Mmp9 1637 13706 Matrix metalloproteinase 9 Mrc1 930 5855 Macrophage mannose receptor 1 Msr1 3838 16063 Macrophage scavenger receptor types i and II

function chemokine & cytokine activity, CXCR binding chemokine activity, CXCR binding, chemotaxis AP2 complex binding, clathrin adaptive activity ion binding, transferase MAPK, JNK cascade transcription hydrolase, catalytic activity C-C chemokine family protein serine/threonine kinase protein kinase inhibitor transcription factor, IL2, IL5, IL10, IFN production chemotaxis, leukocyte migration channel activity, signaling beta defensin gene family receptor-ligand activity, TNF production transcription factor binding oxidation-reduction IL10 receptor activity receptor binding & signaling IR gene family IL-1 signaling signaling protein binding cytokine activ, receptor binding, cell cycle kinase activity, neg.regul.IL6, IL12, TNF product.,. regulation of proliferation ion channel activity peptidase, proteolysis tetraspanin family, platelet degranulation lipid metabolism, chemokine secretion endocytosis, transcytosis lipase binding binding, monocytes and plamacytoid dendtritc cells transcription regulation actin filament assembly endocytosis, apoptotic clearance adhesion, phagocytosis binding, proteolysis, virus IR regulation proteolysis peptidase proteolysis, leukocyte migration,collagen metabolism endocytosis, LPS response endocytosis, phagocytosis, cholesterol transport

31

biological processes chemotaxis, inflammatory IR, GPCR signaling cytokine production, inflamm.IR, GPCR, JAK/Stat sign. myeloid cell diff., endocytosis, vesicle-med. transport T cell differentiation, NOTCH signaling Treg differentiation, innate IR regulation adipocyte differentiation response to toxic substances microglia cell activation muscle development adhesion, migration, FGFR1 signaling T cell prolif.,chronic inflammation, Treg differentiation GPCR signaling Junction assembly, membr.trafficking, vesicle- transport T cell activation , ERK1/2 signaling transcript.regulation, inflamm. IR, B cell activation hypoxia resp., intrinsic apoptotic sign., IB-NFB sign. IR regulation, IFN signaling regulation IGF1R signaling, hemostasis, development IR regulation IL6, oncostatinM, NOTCH, IL27 signaling, T cell prolif. multimeric complex organization, TJ, signaling extrinsic apoptosis, SMAD phosphoryl., hair follicle  innate IR, Myd88/TLR, NFB sign. , IL1 signaling NOTCH signaling ion transmembrane transport TLR cascade, MHCII antigen processing hemostasis lipid & lipoprotein metabolism actin organiz., GPCR signaling, lysosomal catabolism regulation endocytosis & cell death cell development, cytokine production energy metabolisms GPCR signaling, vesicle-mediated transport angiogenesis, phagocytosis, apoptotic cell clearance ECM organization, regulation IFN/ß signaling ECM disassembly regulation cell migration, proteolysis, ECM organization axon guidance, EPH-Ephrin signaling IL4 & IFN response Ligand uptake, vesicle-mediated transport

Table S7A cont. AA ExoSynonym SC treated Protein name Nrp2 2457 8104 Neuropilin Pacsin1 563 2113 PKC and casein kinase substrate in neurons protein1 Pcdh7 325 1321 Prootein Pcdh7 Pdlim4 156 1026 PDZ and LIM domain protein 4 Pdpn 1397 6282 Podoplanin Pid1 295 2137 PTB-containing, cubilin and LRP1-interacting protein Plau 1864 6029 Urokinase-type plasminogen activator Pltp 650 2297 Phospholipid transfer protein Pmepa1 746 2509 Protein TMEPAI Pou2f2 834 2612 POU domain, class 2, transcription factor 2 Ppap2b 536 1947 Phospholipid phosphatase 3 Pros1 1325 4996 Vitamin K-dependent protein S Ptges 330 1688 Prostagalandin E synthase Qsox1 5343 21041 Sulfhydryl oxidase 1 Rgl1 987 3411 Ral guanine nucleotide dissociation stimulator-like 1 Rin2 462 1406 Ras and Rab interactor 2 Rragd 208 1273 Ras-related GTP-binding protein D Rxra 657 2037 Retinoic acid receptor RXR-alpha Saa3 2181 13528 Serum amyloid A-3 protein Sash1 434 1884 SAM and SH3 domain containing protein 1 Sbf2 473 1538 Protein Sbf2 Sdc4 1157 5059 Syndecan 4 Serpinb8 241 1042 Serpin B8 Siglec1 184 1303 Sialoadhesin Slc15a3 1830 5825 Solute carroer family 15 member 3 Slc6a8 370 1314 Sodium- nd chloride-dependent creatine transporter 1 Slc7a2 6214 29655 Cationic amino acid transporter 2 Slfn5 2137 7068 Schlafen family member 5 Snx29 272 1148 Sortin nexin-29 Spib 1227 4146 Transcription factor Spi-B Spsb1 269 1736 SPRY domain-containing SOCS box Stab1 387 1270 Stabilin-1 Stac2 323 1025 SH3 and cystein-rich domain-containing protein2 Tgfbi 2818 12397 Transforming growth factor-beta-induced protein ig-h3 Timp2 638 2240 Metalloproteinase inhibitor 2 Tlr8 294 1240 Toll-like recptor 8 Tmie 278 1580 Transmembrane inner ear expressed proteii Tnfrsf4 889 2817 Tumor necrosis factor receptor superfamily member 4 Tnfsf8 870 3307 Tumor necrosis factor ligand superfamily member 8 Trat1 238 2001 T-cell receptor-associated transmembrane adaptor 1 Trem2 602 3204 Triggering receptor expressed on myeloid cells 2 Vcan 1087 5151 Versican core protein Wfdc17 1545 5877 Activated macrophage/microglia WAP domain protein

function protein binding, migration, chemotaxis phospholipid & cytoskeleton binding protein binding actin cytoskeleton organization binding response to IL6 & TNF peptidase lipid transporter R-SMAD binding regulation of transcription cell-cell adhesion, regul.phosphorylation phagocytosis, negative regulation coagulation lipid metabolism oxidation-reduction GTPase signal transduction GTPase activator, GTPase regulation of transcription chemotaxis, response to IL-1 protein complex scaffold activity phosphatase regulator fibronectin binding, thrombospondin receptor activity peptidase inhibitor endocytosis, extrinsic apoptosis signaling peptide, protein transport ion transport AA transport, NO production, Mf activation ATP binding phosphatidylinositol binding transcription ubiquitin transferase scavenger receptor ion binding, intracellular signaling adhesion cytokine response, proteolysis  IL1ß, IL6, IL8, IFN/ß regulation/secretion, inflamm.IR regulation IR & AICD CD8 T cell differentiation, response to bacterium calcium-mediated signaling transmembrane signaling receptor, adhesion endopeptidase inhibitor

32

biological processes LIF response, VEGFR signaling endocytosis, actin filament organization platelet activation & signaling bone marrow development cell-cell adhesion, morphogenesis, platelet activation regulation of ROS response regulation proliferation, migration, hemostasis lipid metabolism & transport SMAD & TGFßR signaling  B cell maturation lipid & lipoprotein metabolism, Wnt signaling clotting cascade prostaglandin synthesis hemostasis regulation of GTPase activity & lipid metabolism endocytosis, signaling PI3K & TOR signaling impact on organ development TLR signaling LPS & JUN & NFB & p38MAPK signaling membrane organization, myelination ECM organiz., focal adhesion, Exo assembly & secret. cell-cell adhesion, platelet activation T cell apoptotic process transmembrane transport amino acid metabolism transmembrane transport, inflammatory IR regulation cell differentiation M differentiation MHCI antigen process./present., adaptive IS response inflammatory IR, vesicle-mediated transport ion channel inactivation ECM organization, angiogenesis ECM organization,regul. Ras, cAMP, MAPK signaling MyD88-TLR, IB-NFB, sign., MAPK, TLR7/8,9 activ. sensory hair development inner ear cytokine signaling, TNFR2-NFB signaling IR receptor recycling, T cell receptor signaling IR (lymph.-nonlymph.), CCR7, ERK1/2, CD40 signaling GAG synthesis, ECM organization .

Table S7B Reduced mRNA recovery in spleen cells of MDSC-Exo treated AA mice AA SC Synonym Adamts14 Add2 Alox12 Angpt1 Aqp1 Arsb Atp1b2 B4galnt2 Bzrap1 Camp Capn5 Car1 Car2 Casp12 Ccnb2 Cd160 Cd177 Cd244 Cd34 Ces2g Chil1 Clca3a1 Clec12a Clnk Clstn3 Cpa3 Ctla2a Ctla2b Dixdc1 Dsc2 Elane Epx Ermap Esco2 Fads2 Fam64a Fcrl6 Fdps Gfi1b Gp1ba Gp5 Gp9

2537 2158 3067 1447 4415 5461 3492 2486 1884 1519 3836 9846 10370 2002 3305 1718 3385 3150 4550 2691 1095 1379 1465 1719 1407 43212 13192 1894 1248 2697 14228 5245 1811 1810 1463 1393 3671 1967 1565 2834 1396 1090

Exotreated 356 44 445 217 183 969 106 298 295 43 702 404 2065 204 620 240 99 515 739 327 193 226 260 321 185 6709 2223 221 205 57 162 186 49 336 136 278 45 289 251 520 253 126

Protein name Adamts14 Beta-adducin Arachidonate 12-lipoxygenase 12S Angiopoietin-1 Aquaporin-1 Arylsulfatase B Sodium/potassium-transport ATPase subunit ß2 Beta-1,4 N-acetylgalactosaminyltransferase 2 Peripheral-type benzodiazepine rec.-assoc. protein1 Cathelicidin antimicrob. Peptide Calpain 5 Carbonic anhydrase 1 Carbonic anhydrase 2 Caspase 12 G2/mitotic-specific cyclin-B2 CD160 antigen CD177 NK receptor 2B4 Hematopoieitc progenitor cell antigen CD34 Carboxylic ester hydrolase Chitinase-3-like protein-1 Calcium-sens. chloride conductane protein1 C-type lectin domain fam.12memberA Cytokine-dep. hematopoietic cell linker Calsystenin-3 Mast cell carboxypeptidase A Protein CTLA-2a CTLA-2b Dixin Desmocollin-2 Neutrophil elastase Eosinophil peroxidase Erythroid membr.-assoc. protein N-acetyltransferase Fatty acid desaturase 2 Protein FAM64A Fc receptor-like protein6 Farnesyl pyrophosphate synthase Zinc finger protein Gfi-1b Platelet glycoprotein Ib- Platelet glycoprotein V Platelet glycoprotein IX

function peptidase actin & protein kinase binding oxidoreductase activity angiogenesis transporter hydrolase ATPase activity glycosylation protein binding antimicrobial peptide protease inhibition carbon metabolism carbon metabolism protease cyclin-dependent serine/threonine kinase activity MHCI receptor activity fibrin clot formation NK & mDC activation binding oxidation glycosidase peptidase, ion channel signaling transmembrane receptor ion binding proteolysis peptidase peptidase inhibitor enzyme modulator, signaling ion binding inflammatory response, phagocytosis, proteolysis defense to nematode erythroid membrane receptor DNA replication oxidorectuctase controls metaphase to anaphase transition MHCII ligand acyltransferase regul.transcription coagulation collagen binding adhesion

33

biological processes ECM organization transmembrane transport, leukocyte migration lipid & lipoprotein metabolism multiple signaling pathways transmembrane transport autophagy ion transport negative regulation adhesion synapse formation innate IR, chemotaxis proteolysis O2/CO2 exchange (erythrocytes) chloride transport intrinsic apoptosis signaling cell cycle, T cell homeostasis IR (bacterium) clotting cascade hemostasis hemopoiesis, TGFß&IL10 production, leukoc. migr. Inflamm. IR, apoptosis, PKB & NFB&ERK1/2 activ. ion transport immune response regulation tyrosine kinase signaling, IR synapse assembly and transmission peptide hormone metabolism regulation Treg differentiation Treg conversion microtubule organization, JNK cascade cell adhesion ECM organization & degradation oxidative stress response cell cycle lipid & lipoprotein metabolism cell cycle CTL & NK receptor, upregulated in chronic imm.stim. lipid & lipoprotein metabolism hemopoiesis clotting cascade clotting cascade clotting cascade

Table S7B cont. AA SC ExoSynonym treated Protein name Gucy1a3 1838 321 Guanylate cyclase soluble subunit 3 Gucy1b3 1844 341 Guanylate cyclase soluble subunit 1 Gzma 91251 5036 Granzyme A Gzmb 149194 12697 Granzyme B Gzmc 43167 1693 Granzyme C Gzmd 75163 898 Granzyme D Gzme 28887 345 Granzyme E Gzmf 26081 359 Granzyme F Gzmg 96614 1092 Granzyme G Havcr2 8662 649 Hepatatis A virus receptor 2 Hmgcs1 10861 1759 Hydroxymeth.-CoA synthase Ifitm1 52676 10125 IFN-induced transmembrane protein Ifng 1637 255 Interferon  Itga2b 23212 4422 Integrin -Ib Kalrn 1263 171 Kalirin Klrg1 1073 57 Killer cell lectin-like receptor G1 Lipg 4652 620 Endothelial lipase Ltbp1 1839 206 Latent transforming growth factor ß-binding protein 1 Ltf 4922 181 Lactotransferrin Ly6c1 6129 768 Lymphocyte antigen 6C1 Ly6c2 9867 1265 Lymphocyte antigen 6C2 Maob 1138 149 Amine oxidase B Mcpt8 18692 2308 Mast cell protease Mfsd2b 3503 511 Major facilitator superfam.dom.-contain.protein2B Mns1 1283 140 Meiosis-specific nuclear struct.protein 1 Mpl 1661 229 Thrombopoietic receptor Mpo 23160 789 Myeloperoxidase Mrvi1 5207 768 Mrvi1 Ms4a2 7186 1234 High affinity Igepsilon R subunit ß Ms4a3 2383 80 Membr.-spanning 4 domain subfamily A3 Myb 5422 809 Transcriptional activator Myb Mycn 1669 268 N-myc proto-oncogene protein Myl10 1050 180 Myosin regulatory light chain 10 Mylk 3936 642 Myosin light chain kinase Ngp 31130 394 Neutrophilic granule protein Nkg7 17727 2786 Protein NKG7 Nlrp6 5308 912 NACHT,LRR & PYD dom. containing prot.6 Nqo1 1689 276 NAD(P)H dehydrogenase 1 Nrgn 1471 206 Neurogranin Pkd2l1 1615 298 Polycystic kidney disease 2-like 1 Podxl 5715 920 Podocalyxin Prf1 39787 2235 Perforin Prg2 3593 335 Bone marrow proteoglycan

function cGMP biosynthesis cGMP biosynthesis, Hsp90 binding peptidase peptidase peptidase peptidase peptidase peptidase peptidase adaptive and innate IR metabolism response to virus regulation of cytokine production leukocyte migration, platelet aggregation guanyl exchange factor innate IR phospholipase TGFß binding protease, ROS/RNS production monocyte marker monocyte marker oxidation-reduction proteolysis enzyme activity protein binding cytokine receptor activity oxidation-reduction process regulation of smooth muscle contraction FcRI signaling CD20L,cell cycle regulation transcription factor transcription cytoskeletal protein actin binding peptidase inhibition cytokeletal protein transcription cofactor regulation of oxidate and apoptotic processes synaptic potentiol ion transport protein binding wide pore formation component of eosinophil granule

34

biological processes hemostasis NO-mediated signaling proteolysis, cytolysis cytolysis, T cell cytotoxicity proteolysis, cytolysis proteolysis, cytolysis proteolysis, cytolysis proteolysis, cytolysis proteolysis cytokine signaling lipid & lipoprotein metabolism immune regulation (nonlymphoid-lymphoid) cytokine signaling in the IS hemostasis, multiple signaling pathways MAPK & Rho & GPCR signaling, death signaling Immunoregulation (lymphoid-nonlymphoid) lipid & lipoprotein metabolism ECM organization, TFGßR signaling regulation cytokine production, innate IR inflammatory process orchestration inflammatory process orchestration biological oxidation inflammatory respone in the skin transport cilium organization regulation of hemopoiesis and homeostasis azurophilic granules, innate IR cGMP signaling MAPK & NFB activation, inflammatory IR IS signal transduction, neutrophil degranulation TH & B & myeloid cell differentiation regulation cell death regulation focal adhesion, leukocyte migration regul. migration & transport, RhoGTPase signaling regulation defense response adhesion, transporter IR , TLR&MAPK&IB/NFB signaling  oxidation-reduction process intracellular signaling channel activity regulation leukocyte adhesion & migration channel activity, apoptosis, cytolysis IR to nematode & bacterium

Table S7B cont. AA SC Synonym Prss34 6940 Prtn3 5128 Rab27b 3767 Rab38 4129 Rhag 1136 S100a4 9322 S100a6 14291 S100a8 37682 S100a9 57146 Samd14 1645 Scin 14700 Sdpr 1753 Serpinb9b 3296 Sh2d5 1812 Slc6a4 1510 Spns2 2092 Spta1 1842 Sytl2 3530 Tfr2 1478 Timp3 6914 Tjp1 1380 Tmem119 4546 Tmem40 1096 Treml1 1496 Tubb1 1651 Vash1 3055 Vcam1 1055 Vwf 13563 Zbtb32 5045 a

Exotreated 934 168 698 787 110 692 2415 4415 4653 304 2765 268 376 123 259 317 66 462 240 995 211 467 104 255 154 345 39 2121 816

Protein name Mast cell protease 11 Myeloblastin Ras-related protein Rab-27B Ras-relat. protein Rab-38 Ammonium transporter Rh type A Protein S100-A4 Protein S100-A6 Protein S100-A8 Protein S100-A9 Sterile alpha motic dom.cont. protein 14 Adseverin Serum-depriv. response protein Serpinb9b SH2 domain-contain.protein 5 Sodium-depend. serotonin transporter Protein spinster homolog 2 Spectrin  chain,erythrocytic-1 Synaptogamin-like protein2 Transferrin receptor protein2 Metalloproteinase inhibitor 3 TJ protein ZO1 Transmembrane protein 119 Transmembrane protein 40 Trem-like transcript 1 protein Tubulin 1 chain Vasohibin1 Vascular cell adhesion protein1 vonWillebrand factor Zinc finger and BTB domain-cont. protein 32

function peptide crosslinking & proteolysis protease inhibition GTPase activity GTPase activity iron homeostasis actin binding calcium and protein binding neutrophil chemotaxis, apoptosis chemotaxis, inflamm.response, apoptosis aberrant promoter methylation actin binding PKC substrate, phosphatidyl binding cytolysis protein binding transporter lipid transport actin organization membrane traffic regulator regulation iron ion homeostasis protease inhibition GAP junction formation CD11b+ cells in microglia Ig receptor GTPase protein binding integrin binding blood coagulation transcription corepressor

biological processes basophil mediated tissue damage proteolysis, phagocytosis Exo sorting & exocytosis organelle organization & transport transmembrane transport IB/NFB signaling ion transport, plasma membrane tubulation inflammatory response TLR cascade actin nucleation, apoptosis regulation plasma membrane tabulation protection from cell-mediated cytotoxicity synapse transmission B & T cell homeostasis, lymphocyte migration regul.hemopoiesis, T cell prolif., multiple signaling vesicle docking & exocytosis regulation of endocytosis regulation proteolysis, TRAIL signaling pathway membrane & vesicle-mediated transport osteoblast differentiation, spermatogenesis biological process lymphoid-nonlymph. interaction, neg.regul. signaling organelle biog., membr.traff., vesicle-med. transport angiogenesis regulation, cellular sensecence IR regulation (lymphoid-nonlymphoid) clotting cascade, beta3 signaling osteoblast different.,hematopoietic prog. proliferation

AA: amino acid, AICD: activation-induced cell death, DC: dendritic cells, ECM: extracellular matrix, IS: immune system, IR: immune response, TJ: tight junction, : reduced recovery / activity, : pronounced recovery / activity, red: engagement in hair follicle organization

35

36

Figure S1 Proteome analysis of ex vivo and in vitro generated MDCS and MDSC-exosomes. (A) MDSC were either isolated from SC by magnetic bead sorting for CD11b+Gr1+ cells or were generated in vitro from BMC cultured in the presence of GMCSF, IL6 and PGE2. After SDS-Page and digestion, proteins were identified by mass spectrometry. Proteins preferentially recovered in ex vivo or in vitro generated MDSC were clustered using Reactome according to their engagement in immune response, cell cycle, transport and cytoskeleton organization, proteolysis / apoptosis and signaling. Proteins engaged in two clusters are identified by a distinct frame color. (B,C) Exo were collected from MDSC as described above after 2d culture in medium with 3% Exo-depleted FCS. After purification, SDS-Page and digestion, proteins were identified by mass spectrometry. (B) Comparison of abundantly recovered proteins in MDSC-Exo derived from ex vivo versus in vitro generated MDSC including proteins that are selectively recovered from the two distinct MDSC preparations and (C) MDSC-Exo preferentially recovered from ex vivo or in vitro generated MDSC were clustered according to major functional activities as described for MDSC. A proteome analysis confirmed no significant differences in ex vivo versus in vitro generated MDSC according to immune system-related functions. The abundance of distinctly in in vitro generated MDSC relying mostly on pronounced cell division and associated metabolic processes due to the culture condition. This also accounts for the differential recovery in Exo from ex vivo or in vitro MDSC, where the significant increase in proteins engaged in cell division and metabolic processes is also reflected in Exo from culture-derived MDSC.

37

Figure S2 Proteome analysis comparing ex vivo and in vitro generated MDCS with MDSC-exosomes. MDSC were generated in vitro from BMC. After maturation, MDCS were cultured for 2d in medium with 3% FCS that was Exo-depleted. MDSC and MDSC-Exo proteins were identified by mass spectrometry. (A) Panther Gene Analysis of molecular functions of in vitro and ex vivo MDSC versus MDSC-Exo; (B) Panther Gene Analysis of molecular functions of proteins that are distinctly recovered in in vitro and ex vivo MDSC versus MDSC-Exo. (C) Reactome analysis of distinctly recovered proteins in BMC-derived MDSC versus MDSC-Exo, which are engaged in immune response regulation including activation of signaling cascades. Three proteins engaged in MDSC activity, Arg1, Sod2 and Bax were recovered at a lower level in MDSC-Exo. On the other hand, tetraspanins, C’ and proteasome subunits were more abundant in MDSC-Exo than MDSC. Hypothesizing that pro and contra of function-relevant proteins in MDSC versus MDCS-Exo could possibly be balanced, we proceeded comparing their effectivity in immune response regulation.

38

Figure S3 MDSC-exosome membrane attached proteins. A lymphocyte membrane preparation was coupled to Sepharose. MDSC-Exo were lysed (mild conditions, Lubrol) and were passed over the lymphocyte-membrane Sepharose column. After washing, Exo lysates were eluted, dialyzed, concentrated and subjected to SDS-PAGE for proteome analysis. (A) Pulled-down MDSC-Exo proteins were clustered according to molecular functions (Panther Gene Analysis) and (B) using Reactome according to proteins engaged in Exo biogenesis, transport, cytoskeleton organization, engagement in innate and adaptive immune responses as well as lipid metabolism. Proteins engaged in signal transduction are framed in red. The pulldown of MDSC-Exo indicated that invaginated membrane domains remain intact during Exo biogenesis including attached cytoskeletal and signaling molecules. The latter may well contribute to immune target cell modulation.

39

40

Figure S4 Examples of hair regrowth in MDSC exosome-treated mice with AA partialis and incipient. (A) Hair loss progression in untreated mice with AA partialis and partial hair growth recovery in MDSC-Exo treated mice is demonstrated after 2wk, 6wk and 10wk (3 mice / group); (B) Hair loss progression in untreated mice with AA incipient 16wk after initiating MDSC-Exo application; hair growth in MDSC-Exo treated mice is shown after 2wk, 6wk, 10wk and 16wk. MDSC-Exo treatment is curative when started at a very early stage of hair loss. At an intermediate state it prevents progression. Partial hair regrowth proceeds from remaining nonaffected areas.

41