20 nm

a

Supplemental figure 1

b

0

20

40

60

80

100

120

140

160

Medium EL4 TS/A CT26

** *

Ace

tylc

ho

line

este

rase

act

ivit

y (a

.u.)

Figure S1

a : Picture from the ultracentrifuged pellet from culture supernatant of EL4.

b: Acetylcholine esterase activity in ultracentrifuged fraction (TDE).

Supplemental figure 2

Figure S2 :Stat3 activation in purified MDSCs

Purified splenic MDSCs from naïve mice were treated with TCS or TDSF or TDE. pStat3

expression was determined by FACS analysis. Data are Mean ± s.d. fluorescence intensity (n=3)

Norm

aliz

ed p

Sta

t3 M

FI

0

50

100

150

200

250

300

350

400

TSA EL4 CT26 TSA EL4 CT26 TSA EL4 CT26

CM Tumor cell supernatant TDSF TDE

* * * * * *

TCS

Supplemental figure 3

0

10

20

30

40

50

60

70

80

90

CM TDSF TDSF + aGM-CSF GM-CSF

% G

r1+

CD

11b c

ells

* *

Figure S3. Expansion of MDSCs by GM-CSF

Bone marrow from naïve mice were cultured 3 days in complete medium alone or with TDSF or TDSF

+ blocking anti GM-CSF or rGM-CSF . The percentage of Gr1+ CD11b+ precursor cells ± s.d. was

determined by flow cytometry.

a GM-CSF

Supplemental figure 4

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 2 4 6

p6

5 O

.D.

Time (h)

0

0,2

0,4

0,6

0,8

1

1,2

1,4

Co TDSF GM-CSF

p6

5 O

.D.

TDE exposure

Figure S4. NFkB p65 transcriptional activition by TDE and TDSF

(Left panel) MDSC clone were stimulated with TDE for 2h, 4h or 6h . Then NFkB p65 transcriptional

activity was determined with TransAm assay.

(Right panel) MDSC clone were stimulated with TDSF or GM-CSF for 2h. Then NFkB p65

transcriptional activity was determined with TransAm assay.

*

*

*

Supplemental figure 5

pStat3

Naive mice Tumor bearing mice

IgLy6Cint Ly6G+Ly6Chi Ly6G-

Figure S5 . pStat3 expression in MDSCs Subpopulations

MDSC from tumor-bearing mice were analysed for Ly6C Ly6G CD11b and pStat3 expression by

cytometry.

Monocytic MDSC were characterized by Ly6ChighLy6G-CD11b+; granulocytic MDSC by Ly6Cintermediate

Ly6G+ CD11b+.

Supplemental figure 6

a

b

0

50

100

150

SCF PGE2 IL-6 IL-10

EL4

TS/A

CT26

Figure S6. Cytokines production by tumor cells and TDE

A. Tumor cell supernatant SCF, PGE2, IL-10 and IL-6 were determined by ELISA.

B. PGE2 levels in TDE were determined by ELISA.

pg

/ml

0

1000

2000

3000

4000

5000

1 2 3TS/A CT26 EL4

5

4

3

2

1

0

ng

/µg

Supplementary figure 7

Il-1b

pg

/ml

0

50

100

150

TS/A EL4 CT26 TS/A EL4 CT26 3T3

CM UC Medium Exosome

TNF-a

pg

/ml

a

b

IL-1

0 p

g/m

l

c

d

VEG

F p

g/m

l

0

50

100

150

0

50

100

150

0

50

100

150

TDSF TDE

Figure S7. Cytokines production by MDSCs.

Purified MDSC from naïve mice were treated 24h with complete conditioned medium (CM) or

soluble fraction (TDSF) or exosome (TDE) obtained from CT26, TS/A, EL4 and 3T3 cell cultures

before and after ultracentrifugation (UC). IL-1b (a), TNF-a (b), IL-10 (c) and VEGF (d)

concentrations were determined by ELISA.

Supplementary figure 8

0

200

400

600

800

WT TLR2 TLR4 Myd88 Trif

Untreated

LPS

PolyICIL-6

pg

/ml

a

0

100

200

300

400

500

WT TLR2 TLR4 Myd TRIF

untreated

IL-6

No

rmal

ized

Pst

at3

MFI

pSt

at3

MFI

WT TLR2-/- TLR4-/- MyD88-/- Trif-/-

WT TLR2-/- TLR4-/- MyD88-/- Trif-/-

b

Figure S8. IL-6 production and pStat3 expression by MDSCs from wild type and TLR

deficient mice

a. MDSCs from wild-type or TLR2-, TLR4-, MyD88- or TRIF-deficient tumor-free mice were

cultured for 24h with PBS alone or supplemented with LPS (10 ng/mL) or Poly(I:C) (50μg/mL),

then IL-6 concentrations were determined by ELISA in the supernatant.

b. MDSCs from wild-type or TLR2-, TLR4-, MyD88- or TRIF-deficient tumor-free mice were

cultured for 24h with PBS alone or supplemented with rIL-6 (10ng/mL). pStat3 expression was

determined by FACS analysis on MDSCs gated cells. MFI = mean fluorescence intensity ± s.d.

Supplemental figure 9

tsg101

Grp94

Hsp72

Exo

som

es

Mic

rop

arti

cles

EL4

Figure S9. Expression of Hsp72, Tsg101 and Grp72 in exosome and other microparticules

EL4- exosome were separated from other microparticules by ultracentrifugation on 30% sucrose/D2O.

Both fractions were blotted for tsg101 (exosomal protein), Grp94 (endoplasmic reticulum protein) and

Hsp72.

Supplemental figure 10

0

100

200

300

400

500

600

700

untreated rHsp72 Pam3CSK4 Pam3CSK4+ anti Hsp72

Pg/mL

IL-6

TNF-a

Figure S10. IL-6 and TNF-a production by MDSCs

Naïve MDSCs were stimulated 24h with rHsp72 or PAM3CSK4 or PAM3CSK4 plus a blocking anti

Hsp72. IL-6 and TNF-a in the supernatant were determined by ELISA.

Supplemental figure 11

rHsp72pErk

Erk

pStat3

Stat3rHsp72

0 5 15 30 60Time (min) 0 2 4 6Time (h)

0

0,4

0,8

1,2

1,6

0 2 4 6

p6

5 O

.D.

Time (h)

rHsp72

0

0,2

0,4

0,6

0 2 4 6

pSt

at3

O.D

.

Time (h)

rHsp72

Figure S11. Erk ,Stat3 and NFkB activation by Hsp72 in MDSCs

MDSC clone were stimulated with rHsp72, pERK (a) and pStat3 (b) expression was determined western

blotting.

MDSC clone were stimulated with rHsp72, then NFkB p65 (c) and Stat3 (d) transcriptional activity was

determined with TransAm assay.

a b

c d

Supplemental figure 12

1

0.8

0.6

0.4

0.2

Hsp72

b-actin

Mock H95 H96 H97 H98 H99

Fo

ld d

ecre

ase

0

b-actin

Hsp72

00.20.40.60.8

1

Fold

decre

ase

Mock H96 H97

TDE

Figure S12. Hsp72 expression in CT26 clones transfected with ShRNA targeting Hsp72

Hsp72 expression was determined by western blotting in shRNA Mock CT26, and in 5 different shRNA

Hsp72 CT26 clones. Hsp72 expression was also determined in TDE from shRNA Mock CT26 and shRNA

Hsp72 CT26 clones H96 and H97.

Supplementary figure 13

0

500

1000

1500

2000

2500

0 5 10 15 20

0

500

1000

1500

2000

2500

0 5 10 15 20

Wild-Type NudeTu

mo

r vo

lum

e (m

m3)

Tum

or

volu

me

(mm

3)

TS/A

EL4

CT26

PBS

CTX

0

1000

2000

3000

4000

5000

0 6 9 12 14 17 20

Tum

or

volu

me

(mm

3)

0

1000

2000

3000

4000

5000

0 6 9 12 14 17 20

Tum

or

volu

me

(mm

3)

0

200

400

600

1 6 11

0

200

400

600

1 2 3 4 5 6 7 8 9 10 110 10 20

1500

3000

4500

* * *

** **

*

** * *

0 10 20

Tum

or

volu

me

(mm

3)

Figure S13. Tumor growth in wild type and n udemice after cyclophosphamide treatment.

Wild-type and nude mice were subcutaneously injected with 1.106 TS/A, EL4 or CT26 tumor cells. 10

days later, mice were injected with PBS or cyclophosphamide (CTX) (100 mg/kg). Tumor volume was

determined using a caliper.

4500

1500

3000

Tum

or

volu

me

(mm

3)

H23

b-actin

Hsc73

Hsp72

Supplemental figure 14

Figure S14.

Hsc73 and Hsp72 expression in H23 and H23 TDE was determined by western blotting.