supplementary information for il-27 confers a ... · γ (xmg1.2) were injected i.p. 7 days after...

Supplementary Information for

IL-27 confers a protumorigenic activity of regulatory T cells via CD39

Young-Jun Park, Heeju Ryu, Garam Choi, Byung-Seok Kim, Eun Sook Hwang, Hun Sik Kim, and

Yeonseok Chung

www.pnas.org/cgi/doi/10.1073/pnas.1810254116

Supplementary Materials and Methods

Cell line and Reagents

B16F10 (ATCC) and EL4 ( Korean Cell Line Bank) were cultured in DMEM or RPMI 1640 supplemented

with 10% FBS and 1% penicillin-streptomycin. In vitro T cell cultures were performed in RPMI 1640

supplemented with 10% FBS, 1% penicillin-streptomycin, and 55 μmol/L 2-mercaptoethanol. ATP,

busulfan, ZM241385, and ARL67165 were obtained from Sigma-Aldrich. The microbeads for

lymphocyte selection were purchased from Miltenyi Biotec (Bergish Galdbach, Germany).

In vivo tumor models

To analyze the characteristics of tumor-infiltrating Tregs (Ti-Tregs), the mice were inoculated

subcutaneously (s.c.) with 2 × 105 B16F10 cells or 5 × 105 EL4 cells. To block IFN-γ, 100 μg of anti-IFN-

γ (XMG1.2) were injected i.p. 7 days after tumor implantation (total 4 times/every other day). For the

Treg transfer tumor model, Foxp3YFP-Cre and Il27ra—/—- × Foxp3YFP-Cre naïve CD4 T cells were

differentiated into iTregs in the presence of 12.5 ng/ml TGF-β for iTreg transfer, and Foxp3YFP-Cre and

Stat1—/— × Foxp3YFP-Cre nTregs were isolated for nTreg transfer. FACS-sorted YFP+ cells were mixed with

CD25-GITR-CD4+ and CD8+ T cells in a ratios of physiological relevance, and transferred into Rag1—/—

mice. B16F10 cells were implanted s.c. 2 days later. To identify the effect of CD39 on tumor growth,

CD39 inhibitor ARL67156 (5 mg/kg) was treated for 5 consecutive days starting from 1 day after tumor

inoculation. In some experiments, the mixture of FACS-sorted YFP+ Tregs (CD45.2+) from Foxp3-YFP

and Il27ra—/— × Foxp3-YFP and B16F10 tumor cells were subcutaneously injected into Rag1—/— mice

reconstituted with naïve CD4+ T and CD8+ T cells (CD45.1+). Tumor size was monitored every 2-3 days

by caliper in 3 dimensions. TILs were obtained and analyzed at 15 days post tumor inoculation unless

otherwise indicated. In some experiments, the amount of IL-27 in tumor lysates was measured by ELISA

(eBioscience), according to the manufacturer's protocol.

In vitro suppression assay

To identify the effect of IL-27-Tregs on T cell functions, naïve CD4 T cells from Foxp3YFP-Cre mice were

differentiated under iTreg–skewing conditions in the presence of IL-2 (20 ng/ml) and TGFβ (5 ng/ml)

with or without IL-27 (10 ng/ml). YFP+ iTregs were sorted and plated with CFSE-labeled naïve CD8 T

cells for 3 days (2 μg/ml anti-CD3/CD28 antibodies, plate-coated). CFSE dilution and cytokine

productions (IFN-γ, TNF-α, and IL-2) were analyzed by FACS. To identify the effect of CD39 and

adenosine on T cell function, CD39 inhibitor ARL67156 (150 μM) and A2A receptor antagonist

ZM241385 (1 μM) were added to Treg-CD8 T cell coculture.

FACS antibodies and recombinant proteins

The following antibodies were used for cell surface and intracellular staining: APC/Cy7-labeled anti-

CD45.2 (104), PerCP/Cy5.5, PE/Cy7 or PacificBlue-labeled anti-CD45.1 (A20), APC-labeled anti-CD44

(IM7), Alexa 488-labeled anti-CD62L (MEL-14), PE-labeled anti-CD25 (PC61), Brilliant Violet 510,

PE/Cy7 or PerCP/Cy5.5-labeled anti-CD4 (GK1.5), PE/Cy7-labeled anti-NK1.1 (PK136), PacificBlue-

labled anti-TCRβ (H57-597), Alexa 488-labeled anti-PD-1 (RMP1-14), APC-labeled anti-CD73

(TY/11.8), PE-labeled anti-CD39 (24DMS1), PE-labeled anti-TIM-3 (RMT3-23), APC-labeled anti-

CTLA4 (UC10-4B9), PE/Cy7 or PerCPCy5.5-labeled anti-ICOS (C398.4A), PacificBlue or PE/Cy7-

labeled anti-granzyme B (GB11), PE/Cy7 or PerCP/Cy5.5-labeled anti-CD8 (53-6.7), APC or eFluor 450-

labeled anti-TNF-α (MP6-XT22), Alexa 488 or eFluor 450-labeled anti-Foxp3 (FJK-16s), Alexa 488 or

PerCP/Cy5.5 or APC-labeled anti-IFN-γ (XMG1.2), PE-labeled anti-IL-2 (JES6-5H4), PerCP/Cy5.5-

labeled anti-CXCR3 (CXCR3-173), PE/Cy7-labeled anti-CCR6 (29-2L17), PE-labeled anti-CCR4

(2G12), biotin-labeled anti-CXCR5 (L138D7), APC-labeled anti-RORγt (AFKJS-9), PE-labeled anti-

BCL6 (IG191E/A8), BV421-labeled anti-GATA3 (L50-823), PE/Cy7-labeled anti-T-bet (eBio4B10),

PacificBlue-labeled anti-pSTAT3 (4/P-STAT3), APC-labeled anti-pSTAT1 (4a), PE-labeled anti-Ki67

(16A8), APC-labeled Annexin V, and strepavidin-labeled APC from BD Biosciences, Biolegend, or

eBioscience. Recombinant mouse IL-2, TGF-β, IL-27, IFN-γ, and IL-35-Fc fusion proteins were obtained

from PeproTech, eBioscience, R&D Systems, and Chimerigen Laboratories.

Antibody staining and flow cytometry analysis

For intracellular cytokine analysis, cells were stimulated with phorbol 12-myristate 13-acetate (100 ng/mL;

Sigma-Aldrich) and ionomycin (1 μmol/L; Sigma-Aldrich) plus Brefeldin A and monensin (both from

eBioscience) before staining. IC fixation buffer and permeabilization buffer from eBioscience were used

for intracellular cytokine staining, and the Foxp3 staining kit (eBioscience) was used for transcription

factor staining, according to the manufacturer's instructions. For phospho-STAT staining, cells were fixed

with IC fixation buffer and permeabilized with ice-cold methanol for 30 min. Samples were analyzed with

the FACSVerse and LSR Fortessa X-20 flow cytometer (BD Biosciences), and acquired data were

analyzed with FlowJo software (TreeStar, Ashland, Ore).

Analysis of tumor-infiltrating lymphocytes

To separate tumor-infiltrating lymphocytes (TILs), tumors were collected and single-cell suspensions were

prepared by using the gentle MACS dissociator (Miltenyi Biotec). Then, tumor cells were incubated at

37 °C for 30 min in RPMI-1640 containing 1 mg/mL collagenase IV (Roche), 50 μg/mL DNase I (Sigma).

The cells were filtered through a 100-μm nylon mesh, and TILs were isolated by density gradient

separation. In some experiments, macrophages, Tregs, non-Tregs T cells in TILs were FACS-sorted, and

the relative expression of Il27 and Ebi3 was determined as described previously23.

Isolation and stimulation of Tregs

Single cell suspension of CD4+ cells from Foxp3YFP-Cre mice was positively selected with CD4 microbeads

(L3T4; Miltenyi Biotec). Subsequently, Foxp3+YFP+ Tregs were sorted with the FACSAria III cell sorter

(BD Biosciences) and stimulated with plate-coated anti-CD3 and anti-CD28 (145-2C11 and 37.51

respectively, 2 μg/mL: Bio X Cell) in the presence of IL-2 (10 ng/ml) ± IL-27 (10 ng/ml) for 3 days. For

iTreg differentiation, naïve CD4+ T cells (CD62LhighCD44low) were isolated from Foxp3YFP-Cre or C57BL/6

mice. The cells were stimulated with plate-coated anti-CD3 and anti-CD28 (1 μg/ml) in the presence of

IL-2 (20 ng/ml) and TGF-β (5 ng/ml or 12.5 ng/ml) ± IL-27, IFN-γ, or IL-35-Fc (10 ng/ml) for 3 days.

ATP and adenosine quantification assay

To measure enzymatic activity of CD39 induced by IL-27 stimulation, Foxp3+YFP+ nTregs or iTregs,

incubated as described above, were cultured with 100 μM ATP for 6 hrs. The remaining ATP was

quantified by the luminescence ATP detection assay kit (ATPlite, Perkin-Elmer) according to the

manufacturer’s instructions. Adenosine was also measured by colorimetric assay (BioVision) using

adenosine deaminase to convert adenosine to xanthine. For ATP hydrolysis inhibition, iTregs or IL-27-

iTregs were preincubated for 3 hrs in the presence or absence of 200 μM ARL67156. ATP (50 μM) was

added in the last 1 hr. The supernatant was analyzed for ATP and adenosine with the detection kit above

mentioned.

Statistics

Data were analyzed with Prism 6.0 software (GraphPad Software). Statistical significance between 2

groups was determined by using the unpaired 2-tailed Student t-test. For comparison of multiple

conditions within a group or between more than 2 groups, 1-way or 2-way ANOVA with Bonferroni post-

tests was used, respectively. P values of less than 0.05 were considered statistically significant.

Supplementary Figure Legends

Fig. S1. Non-Tregs CD4+ T cells marginally acquire inhibitory phenotype in tumor tissue compared

to Ti-Tregs.

Naïve and B16F10 s.c. tumor-bearing mice were analyzed for the phenotype of CD4+Foxp3— and

CD4+Foxp3+ T cells 15 days after tumor inoculation. The geometric MFI of indicated proteins expression

in tumor-distal, tumor-draining lymph node, tumor tissue, and lymph node from naïve mice was shown.

The graphs show means ± SEM. ***, p<0.001. The data is representative of two independent experiments

(n=6-7).

Fig. S2. Ti-Tregs preferentially express T-bet and CXCR3 in EL4 s.c. tumor model.

EL4 s.c. tumor-bearing mice were analyzed for the phenotype of CD4+Foxp3+ T cells in tumor-draining

lymph node and tumor tissue at 15 days after tumor inoculation. The geometric MFI of (A) indicated

transcription factors and (B) CXCR3 was shown. The representative plots and graphs are shown. The

graphs show means ± SEM. *, p<0.05; ***, p<0.001.

Fig. S3. Immune cell accumulation in tumor tissues of IL-27R-deficient mice.

WT, Il27ra—/— mice (A and B), or WT:Il27ra—/— mixed BM chimeric mice(C) were inoculated s.c. with

B16F10 tumor cells. The frequencies and numbers of Foxp3+ and Foxp3—CD4+T cells (A and C) as well

as the number of total TILs were determined 15 days post tumor inoculation. The graphs show means ±

SEM.

Fig. S4. A role of IL-27 signal on tumor-infiltrating NK and NKT cells.

WT or Il27ra—/— mice were inoculated s.c. with B16F10 and were analyzed 15 days post inoculation. (A-

C) The frequencies and numbers of NK (NK1.1+TCRβ—) and NKT (NK1.1+αGC tetramer+) cells in tumor

tissue were determined. (D-F) The frequency and number of IFN-γ-producing NK and NKT cells were

determined. The graphs show means ± SEM. *, p<0.05; **, p<0.01 (n=3-5).

Fig. S5. Growth of subcutaneous B16F10 tumor Ebi3—/— and Il27ra—/— mice.

(A, B) WT, Ebi3—/—, and Il27ra—/— mice were inoculated s.c. with B16F10, and tumor growth was

monitored. The graphs show means ± SEM. ***, p<0.001. The data are representative of three independent

experiments (n=4-5).

Fig. S6. Expression of IL-27 by tumor-infiltrating cells.

Foxp3YFP-Cre mice were injected s.c. with B16F10. Tumor-bearing mice were sacrificed 12 and 15 days

after tumor inoculation. Tregs (Foxp3-YFP+), Foxp3—CD4+ T cells (TCRβ+Foxp3-YFP—), and

macrophages (F4/80+CD11c+/—) cells were sorted from tumor and spleen, and the relative gene expression

of Il27 (A) and Ebi3 (B) were examined by qPCR. The tumor growth (C), IL-27 level in tumor lysates (D),

Foxp3+ frequency among CD4+ T cells (E), CD39 geometric MFI on Ti-Tregs (F), and CD11c+ frequency

among CD11b+F4/80+ macrophages (G) were analyzed at indicated time points. The graphs show means

± SEM. *, p<0.05 (n=3-5).

Fig. S7. IL-27-STAT1 signal induces CD39 upregulation on Tregs in steady-state.

(A) Schematic outline of mixed BM chimeric mouse experiments. (B) CD4+Foxp3+ T cells in the thymus

and the spleen from indicated BM chimeric mice were analyzed for CD39 expression. Representative

plots and geometric MFI of CD39 are shown. The graphs show means ± SEM. *, p<0.05; **, p<0.01 (n=3-

4).

Fig. S8. CD39 expression on Tregs stimulated with IL-35.

Naïve CD4+ T cells were differentiated under iTreg-skewing condition in the presence or absence of IL-

27 or IL-35-Fc protein. The geometric MFI of CD39 expression on CD4+Foxp3+ T cells was determined.

The graphs show means ± SEM. ***, p<0.001.

Fig. S9. Putative STAT1 binding sites in Entpd1 locus.

(A) The Entpd1 conservation alignment plot for mouse was depicted by using ECR Browser. The vertical

position corresponds to the level of nucleotide identity in this alignment. Corresponding Entpd1 loci were

depicted below (B) Five highly conserved regions (Red squares, 1-5) were selected to predict STAT1,

Smad3, and Foxp3 binding affinity. % Dissimilarity presented by PROMO was shown. nd, no binding

detected.

Fig. S10. A role for IFN-γ on CD39 expression on Tregs.

(A) WT and Il27ra—/— mice were inoculated s.c. with B16F10 tumor cells. The mice were treated with

either IFN-γ neutralizing or isotype-matched control antibodies starting from 7 days after tumor

inoculation (n=3-4). The geometric MFI of CD39 expression on Ti-Foxp3+ cells was analyzed. (B) Naïve

CD4+ T cells from WT or Stat1—/— mice were differentiated under iTreg-skewing condition in the presence

or absence of IL-27 or IFN-γ. The geometric MFI of CD39 expression on Foxp3+ cells was analyzed. The

graphs show means ± SEM. **, p<0.01; ***, p<0.001.

Fig. S11. Adenosine-A2A adenosine receptor interaction is crucial for IL-27-stimulated Treg-

mediated suppression.

(A) CFSE-labeled naïve CD8+ T cells were incubated with iTregs or IL-27-iTregs for 3 days. %

suppression on T cell proliferation is shown. (B) The effect of ARL67156, CD39 blockade, on adenosine

production, and (C) the effect of ZM241385, A2A receptor blockade, on IL-2-producing CD8+ T cells. The

graphs show means ± SEM. **, p<0.01; ***, p<0.001.

Fig. S12. IL-27 signal on Tregs is required for its protumorigenic activity in tumor.

(A-C) WT and Il27ra—/— Tregs (CD45.2+) were mixed with B16f10 tumor cells, and subcutaneously

injected into Rag1—/— mice reconstituted with naïve CD4+ T and CD8+ T cells (CD45.1+). The tumor

growth was monitored (A). The frequency of indicated cytokine-producing CD8+ T cells (B) and NK cells

(C) were analyzed 15 days after tumor inoculation. The graphs show means ± SEM. *, p<0.05 (n=3-5).

(D-G) Separately, WT and Il27ra—/— Tregs or (H-K) WT and Stat1—/— Tregs (CD45.2+) were mixed with

B16F10 tumor cells, and subcutaneously injected into Rag1—/— mice reconstituted with T cells as indicated

above. Some mice were treated i.p. with ARL67156 (D-G). Tumor weight, TIL number (D and H), Treg

frequency/number (E and I), Annexin V (F and J), and Ki67 levels (G and K) were analyzed 12 days after

tumor inoculation. The graphs show means ± SEM. *, p<0.05 (n=4-5).

Fig. S13. Schematic model of the mechanisms by which IL-27 tunes Tregs to inhibit effector CD8+

T cells.

Upon encounter of IL-27 with its hetero-dimeric receptor (gp130-IL-27Rα) on Tregs in tumor tissue, the

phosphorylation of STAT1 induces the expression of ectonucleotidase CD39 by interplay with unknown

molecular partners. The increased CD39 on Tregs vigorously hydrolyzes ATP, leading to an increase in

adenosine which, in turn, suppresses effector function of tumor-infiltrating CD8+ T cells. Blockade of IL-

27 signaling thus prevents Tregs from STAT1 phosphorylation and CD39 upregulation, which relives

CD8+ T cells from Treg-mediated suppression in tumor tissue.

010002000300040005000 ***

Naive

Distal

LNTdLN TIL TIL

0

500

1000

1500 ***

050

100150200250 ***

Naive

Distal

LNTdLN TIL TIL

0

500

1000

1500 ***

0

500

1000

1500

2000 ***

0

2000

4000

6000 ***

TIM

3 M

FI

PD

1 M

FI

CTL

A4

MFI

CD

39 M

FIC

D73

MFI

ICO

S M

FI

Supplementary Figure 1

T-bet

TILTdLN

CD4+Foxp3+

0

500

1000

1500

2000

0

500

1000

1500 * TdLNTIL

0500

1000150020002500

0

2000

4000

6000

8000T-

bet M

FIB

CL6

MFI

GA

TA3

MFI

RO

Rγt

MFI

0

500

1000

1500 ***

CXC

R3

MFI

A

GATA3

RORγt

BCL6

CXCR3

B CD4+Foxp3+


CD4

Foxp3

WT

Il27ra─/─

Den

sity

-sep

arat

edce

ll N

o. (x

105 )

D15A B

0

20

40

60

80

WTIl27ra-/-

CD4

Foxp

3

C

Cel

l %

in F

oxp3

+ce

lls

TIL

WT C

D45.1

Il27ra

-/- C

D45.2

0

20

40

60

80 Foxp3+ gated

WT C

D45.1

Il27ra

-/- C

D45.2

0

20

40

60

80Foxp3-CD4+ gated

Cel

l %

in F

oxp3

- CD

4+ce

lls

0

20

40

60

WTIl27ra/

0

20

40

60

Foxp

3+ %

CD

4+Fo

xp3-

%

0

20

40

60

0

20

40

60Fo

xp3+

No.

(x10

4 )

CD

4+Fo

xp3-

No.

(x10

4 )


0

5

10

15

20 *

0.0

0.5

1.0

1.5

NK1.1

CD1dTetramer

TCRβ

020406080

100*

0

20

40

60 **

NK1.1

IFN-γ

WT Il27ra─/─

NK

NKT

WT Il27ra─/─

0

20

40

60

80 WTIl27ra-/-

0

10

20

30

NK

No.

(x10

4 )N

KT

No.

(x10

3 )

NK

%N

KT

%IF

N-γ

+ %

IFN

-γ+

%

01020304050

0

5

10

15 *

IFN

-γ+

No.

(x10

4 )IF

N-γ

+ N

o.(x

103 )

A B C

D E F


0 5 10 150

100

200

300WTIl27ra-/-

0 5 10 150

100

200

300

400

500 WTEbi3-/-

***

Tum

or g

row

th (m

m3 )

Day after tumor inoculation

Tum

or g

row

th (m

m3 )


A B


D12 D150

1000

2000

3000

4000

D12 D150

20

40

60 *

CD

39 M

FIC

D11

c+ M

. %

F4/80

CD11c

CD39

D12 D150

100200300400500 *

Tum

or g

row

th (m

m3 )

Ti Treg Ti T

Ti CD11

c+ M

Ti CD11

c- M

SPL Treg

SPL T

SPL M0

5

10

15

Ti Treg Ti T

Ti CD11

c+ M

Ti CD11

c- M

SPL Treg

SPL T

SPL M0

1020304050

Rel

. exp

ress

ion

D12 D150

5

10

15

Tum

or IL

-27

pg/m

g pr

otei

n

Il27 Ebi3

D12 D15

Rel

. exp

ress

ion

Ti-Treg

Mac.

C D

A B

G

E

D12 D150

20

40

60

80

Foxp

3+ %

CD4

Foxp3

F


Thymus

Spleen

CD39

CD45.1:

CD45.2:

WT

Il27ra─/─

WT

Stat1─/─

WT

Stat3CD4

CD

39 M

FI

in T

reg

CD

39 M

FI

in T

reg

WT KO WT KO WT KO0

200400600800

1000** **

050

100150200250 *

Il27ra─/─ Stat1─/─ Stat3CD4

ACD45.1+

(WT)CD45.2+

(Il27ra─/ ─, Stat1─/ ─, Stat3CD4)

Bone marrow cells (1:1)

Tcrb─/ ─

recipients

B16F10 s.c.

Ti-Treg cell analysis

B


-IL-

27

Con-Fc

IL-35

-Fc

0

500

1000

1500***

CD

39 M

FI

CD39


1 2 3 4 5

STAT1 5.8 5.8 2.9 5.8 5.8

Foxp3 3.08 4.26 0 0 3.08

Smad3 3.2 1.94 0.94 0 nd

1 2 3 4 5

B

A

Mouse

Conservation plot for Entpd1

Dissimilarity %

95.72-95.74 95.74-95.76 95.78-95.81 95.81-95.83 95.86-95.87Entpd1

correspondingloci (Mb)


010002000300040005000

***** WT+iso AbWT+αIFNγIl27ra-/-+iso AbIl27ra-/-+αIFNγ**

CD39C

D39

MFI

CD

39 M

FI

010002000300040005000

iTregIL-27-iTregIFN-γ-iTregIL-27-iTregIFN-γ-iTreg

WT

Stat1-/-

******

***

CD39

In vitro

In vivo

B

A


B C

1:2 1:4 1:80

20

40

60

% s

uppr

essi

on

Treg : Teff

A

ATP

iTreg

IL-27-iTreg

ARL.

+

+

-

-

+

-

+

-

+

-

+

+

0.0

0.5

1.0

1.5

2.0 *** ***

0

10

20

30 ***** **

Rel

. ade

nosi

nepr

oduc

tion

Teff

iTreg

IL-27-iTreg

ZM.

+

+

-

-

+

-

+

-

+

-

+

+

IL-2

+ %


0

20

40

60

80 *

IFN

-γ+G

ZB+

%IF

N-γ

+ %

NK1.1

IFN-γ

CD8

NK

IFN-γ

GZB

TregWT TregIl27ra─/─

0 5 10 150

100

200

300

400

500 TregWT

TregIl27ra─/─ **

*Tum

or g

row

th (m

m3 )


A B

C

0

20

40

60 *


0.000.050.100.150.200.25

Tum

or w

eigh

t (g)

0

20

40

60

Tota

l TIL

No.

(x10

4 )

0

1

2

3

4

*

CD

4+Fo

xp3+

%

Treg

No.

(x10

3 )

02468

10

D E F

01020304050 TregWT

TregIl27ra─/─

TregWT + ARL.

Ki67

+%

0

5

10

15

Anne

xin

V+%

G

0

1

2

3

0

20

40

60 TregWT

TregStat1─/ ─

0

10

20

30

0.00

0.05

0.10

0.15

*

0

20

40

60

0

2

4

6

8

Tum

or w

eigh

t (g)

Tota

l TIL

No.

(x10

4 )

CD

4+Fo

xp3+

%

Treg

No.

(x10

3 )

H I J

Ki67

+%

Anne

xin

V+%

K

Graphical summary

IL-27

pSTAT1 up

?

CD39

ATPAdenosine

Treg

CD8+ T cell

IL-27Rα

Tumor tissue

gp130

CD73

IFN-γ

GZB

IL-27

pSTAT1 down

?

CD39

ATP Adenosine

Il27ra─/ ─ Treg

CD8+ T cell

gp130

CD73

IFN-γ

GZB

Anti-tumor immunity

Treg suppression


supplementary information for il-27 confers a ... · γ (xmg1.2) were injected i.p. 7 days after...

Documents