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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+
Supplementary Figure 2
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 )
Supplementary Figure 3
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
Supplementary Figure 4
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 )
Day after tumor inoculation
A B
Supplementary Figure 5
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
Supplementary Figure 6
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
Supplementary Figure 7
-IL-
27
Con-Fc
IL-35
-Fc
0
500
1000
1500***
CD
39 M
FI
CD39
Supplementary Figure 8
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)
Supplementary Figure 9
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
Supplementary Figure 10
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
+ %
Supplementary Figure 11
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 )
Day after tumor inoculation
A B
C
0
20
40
60 *
Supplementary Figure 12
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 Figure 13
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