Supplementary Information The induction and function of the anti-inflammatory fate of TH17 cells
Xu, H., Agalioti, T. et al., 2020
Supplementary Figure 1: Evaluation of TH17-specific IL-10 depletion mouse model
Supplementary Figure 2: No immune abnormality observed in the periphery
Supplementary Figure 3: TH17 cells express higher levels of IL-10R compared to TH1 cells
Supplementary Figure 4: TGF-β promotes IL-10 production in mature TH17 cells
Supplementary Figure 5: Tgfbr2 is required for TH17 cells to produce IL-10
Supplementary Figure 6: Tgfbr2 is required for TH17 cells to produce IL-10 during intestinal inflammation
Supplementary Figure 7: TIF1γ is dispensable for IL-10 production in TH17 cells
Supplementary Figure 8: Smad4 is required for TH17 cells to produce IL-10
Supplementary Figure 9: Smad3 is important for IL-10 production in TH17 cells
Supplementary Figure 10: Human intestinal TH17 cells produce IL-10; and TGF-β further promotes IL-10 expression
Supplementary Figure 1. Evaluation of TH17-specific IL-10 depletion mouse model. A. Mouse strains employed to deplete IL-10 production in TH17 cells. B. Flow cytometric analysis of intestinal lymphocytes isolated from Il17aCre Il10Wt/Wt as well as Il17aCre Il10Flox/Flox mice after anti-CD3 treatment. Intracellular staining for IL-10 was then performed. A pre-gate on TH17 cells (CD4+, TCRβ+, IL-17A+) was applied. C. Statistical analysis of frequencies of IL-10+ cells among TH17 cells. Each dot represents one mouse (nwild type=3, nKO=3). Mean ± S.D.; **P < 0.01 by Welch’s t-test. D. ELISA measurement of IL-10 after in vitro culture. Naive CD4+ T cells were isolated from the spleens of Il17aCre Il10Wt/Wt as well as Il17aCre Il10Flox/Flox mice and were cultured under non-inflammatory (IL-6 + TGF-β) or inflammatory (IL-6 + IL-23 + IL-1β) TH17 conditions. IL-10 in the supernatant was measured by ELISA on day 5. Each dot represents one replicate (n=3). Mean ± S.D.; ns, not significant; *P < 0.05 by Mann-Whitney U test. E. Flow cytometric analysis of in vitro cultured TH17 cells. Naive CD4+ T cells were isolated from the spleens of Il17aCre Il10Wt/Wt as well as Il17aCre Il10Flox/Flox mice and were cultured under non-inflammatory (IL-6 + TGF-β) TH17 conditions. Cells were re-stimulated and intracellularly stained for IL-10 on Day 5. On the right the statistics of IL-10+ cells within YFP+ cells are reported. Each dot represents one separate TH17 cell culture (nwild type=4, nKO=8) obtained from pooled naïve T cells coming from two mice. Mean ± S.D.; Mann-Whitney U-test. F. Representative histological pictures of H&E stained small intestines under steady states. One representative experiment of two is shown. Each dot represents one mouse (nwild type=4, nKO=3). Mean ± S.D.; ns, not significant by Welch’s t-test. G. Frequencies of different IL-10-producing lymphocyte populations that are derived from IL-17A producers. Intestinal lymphocytes were harvested from steady state Fate+ mice (Gagliani et al., Nature 523, 224-224, 2015). Different IL-10-producing immune cell populations that have activated IL-17ACRE (indicated by YFP expression) were analyzed by flow cytometry.
0 10 4 10 5
0
-10 3
10 3
10 4
10 5
0 10 4 10 5
0
-10 3
10 3
10 4
10 5
Com
p-BV
421-A
A
Il17a Il10Cre Wt/Wt
Il17a Il10Cre Flox/Flox
CD4 T Cells (92.39%) CD8 T Cells (5.85%)gdT Cells (< 1%)ILCs (< 1%)Others (< 1%)
IL-10+, YFP+ Cell Population
B
IL-10+
(14.4%)
CD4
IL-1
0
Il17a Il10Cre Wt/Wt
Il17a Il10Cre Flox/Flox
IL-10+
(4.69%)
C
Il17a
Cre Il10W
t/Wt
Il17a
Cre Il10Flox
/Flox
0
5
10
15
20
Freq
uenc
y of
IL-1
0+ ce
lls (%
)
**
G
YFP
IL-1
0B
V4
11
EIl17a Il10
Cre Wt/WtIl17a Il10
Cre Flox/Flox
0.0
0.5
1.0
1.5
2.0
2.5
% o
f IL
-10
with
in t
he Y
FP
+ c
ells
p=0.004
Non-in
flammato
ry
Inflam
matory
0
2000
4000
6000
8000
IL-1
0 (n
g/m
l)
Il17aCre Il10Wt/Wt
Il17aCre Il10Flox/Flox*
ns
D
-0.5
0.0
0.5
1.0
1.5
2.0
Score
ns
Histopathological
Score
F
100um
100um
Il17a Il10Cre Wt/Wt
Il17a Il10Cre Flox/Flox
Supplementary Figure 2. No immune abnormality observed in the periphery. Frequencies and cell numbers of different T cell subsets in different immune organs. Lymphocytes from thymus, spleen and peripheral lymph nodes (pLNs) were harvested from steady state Il17aCre Il10Flox/Flox mice as well as their littermate wild-type control mice. Different T cell subsets were then analyzed by flow cytometry. Each dot represents one mouse (nwild type=4, nKO=4). Mean ± S.D.; ns, not significant by Mean ± S.D.; ns, not significant by Mann-Whitney U test.
CD4+ in Thymus
0
1!106
2!106
3!106
4!106
5!106
6!106
Cel
l num
ber
ns
CD4+ in Thymus
0
2
4
6
8
10
Freq
uenc
y (%
)
ns
CD8+ in Thymus
0.0
4.0!105
8.0!105
1.2!106
1.6!106
2.0!106
Cel
l num
ber
ns
CD8+ in Thymus
0
1
2
3
4
Freq
uenc
y (%
)
ns
CD4+, CD8+ in Thymus
0
2!107
4!107
6!107
8!107
Cel
l num
ber
ns
CD4+, CD8+ in Thymus
70
75
80
85
90
Freq
uenc
y (%
)
ns
Il17a Il10Cre Wt/Wt
Il17a Il10Cre Flox/Flox
CD4+ in Spleen
0.0
4.0!106
8.0!106
1.2!107
1.6!107
2.0!107
Cel
l num
ber
ns
CD4+ in Spleen
0
10
20
30
Freq
uenc
y (%
)
ns
CD8+ in Spleen
0.00
2.50!106
5.00!106
7.50!106
1.00!107
1.25!107
Cel
l num
ber
ns
CD8+ in Spleen
0
5
10
15
20
Freq
uenc
y (%
)
ns
Naive CD4+ in Spleen
0
2!106
4!106
6!106
8!106
1!107
Cel
l num
ber
ns
Naive CD4+ in Spleen
40
50
60
70
80
90
100
Freq
uenc
y (%
)
ns
CD4+ in pLNs
0
1!105
2!105
3!105
4!105
Cel
l num
ber
ns
CD4+ in pLNs
10
20
30
40
50
Freq
uenc
y (%
)
ns
CD8+ in pLNs
0.0
4.0!103
8.0!103
1.2!104
1.6!104
2.0!104
Cel
l num
ber
ns
CD8+ in pLNs
0
10
20
30
40
50
Freq
uenc
y (%
)
ns
Naive CD4+ in pLNs
0.0
5.0!104
1.0!105
1.5!105
2.0!105
2.5!105C
ell n
umbe
rns
Naive CD4+ in pLNs
20
40
60
80
100
Freq
uenc
y (%
)
ns
Supplementary Fig. 3 TH17 cells express higher levels of IL-10R compared to TH1 cells. A. Histograms
showing the expression of IL-10R for TH1 cells (defined as CD4+ CD44+ IFN-γKatushka+ IL-17aeGFP-), TH17 cells
(defined as CD4+ CD44+ IFN-γ Katushka- IL-17a eGFP+), TH1/TH17 cells (defined as CD4+ CD44+ IFN-γ Katushka+ IL-
17a eGFP+) and Tregs (defined as CD4+ Foxp3RFP+ IFN-γKatushka- IL-17aeGFP-) isolated from the intestinal lamina
propria of a representative Foxp3RFP IL-17aeGFP IFN-γKatushka reporter mouse injected with anti-CD3 mAb. B.
Geometric Mean (GM) values of IL-10R from different CD4 T cell subsets. Each dot represents one mouse
(n=3). Mean ± S.D.
0
500
1000
1500
2000
GM
of IL-1
0R
TH1 TH17 TH1 T
H17 / Tregs
Isotype
TH1
TH17
TH1 T
H17 /
Tregs
A
IL-10RPE
Co
un
tB
Supplementary Fig. 4 TGF-β promotes IL-10 production in mature TH17 cells. A, C. TH17 cells generated
in vitro under either TGF-β condition (IL-6 + IL-23 + TGF-β) (A) or IL-1β condition (IL-6 + IL-23 + IL-1β) (C)
were purified and then re-activated with IL-6, IL-23 as well as different amounts of TGF-β. Cells in the above
dot plots are pre-gated on viable CD4+ T cells. B, D. Statistical analysis of the frequencies of IL-10+ cells in
A, C respectively across different TGF-β conditions. **P < 0.01, by ordinary one-way ANOVA.
A
IL-17AKatushka
IL-10eGFP
0.5ng/ml TGF-β 10ng/ml TGF-β
TH17
(42.2%)
TH17
(42.9%)
B
IL-10+
(2.56%)
IL-10+
(22.7%)
IL-17AKatushka
IL-10eGFP
0.5ng/ml TGF-β 10ng/ml TGF-β
TH17
(57.5%)
TH17
(45.7%)
IL-10+
(1.08%)
IL-10+
(14.2%)
C D
0.5 1 2 5 100
5
10
15
IL-6 + IL-23
Freq
uenc
y of
IL-1
0+ ce
lls (%
) **
TGF-! (ng/ml)
0.5 1 2 5 100
5
10
15
20
25
Freq
uenc
y of
IL-1
0+ ce
lls (%
)
IL-6 + IL-23
**
TGF-! (ng/ml)
Supplementary Fig. 5 Tgfbr2 is required for TH17 cells to produce IL-10. A. Constructs contained in Tgfbr2fl/fl Fate+ mouse line. B. Flow cytometry analysis of TGF-βRII expression in intestinal CD4+ T cells expressing YFP (indicating CRE activation). One representative of two independent experiments is shown. C. Flow cytometry analysis of naive CD4+ T cells polarized under TH17 condition in vitro. Cells are pre-gated on viable CD4+ T cells. D. Frequency of TH17 cells shown in C. One representative of two independent experiments is shown. Mean ± S.D.; ns, not significant by Welch’s t-test. E. Flow cytometry analysis of small intestinal CD4+ T cells under steady state. Top panel is pre-gated on CD4+ Foxp3- T cells and bottom panel on CD4+ Foxp3- YFP+ T cells. The cell populations indicated on the FACS dot plots are identified by different combination of reporter expression (TR1exTH17: IL-10eGFP+ YFP+ IL17aKata-; IL-10+ TH17: IL-10eGFP+ YFP+ IL17a Kata+; TH17: IL-10eGFP- YFP+ IL17aKata+). F, G. Frequencies (F) and numbers (G) of TR1exTH17, IL-10+ TH17 and TH17 cells. Data are pooled from three independent experiments. Each dot represents one mouse (nwild
type=7, nKO=7). Mean ± S.D.; ns, not significant; *P < 0.05, ***P < 0.001 by Mann-Whitney U test. H. Left, Flow cytometric analysis of the Foxp3 expression level among the intestinal YFP+ CD4+ T cells under steady state conditions. Foxp3 expression was determined by RFP reporter fluorescence. A pre-gate on YFP+ CD4+ T cells is applied. Right panel shows the statistical analysis of Foxp3 Mean Fluorescence Intensity (MFI) among YFP+ CD4+ T cells. I. Numbers of YFP+ Foxp3+ Treg cells in the small intestine under steady state conditions in the two indicated mouse lines. Each dot represents one mouse (nwild type=7, nKO=7). Mean ± S.D.; ns, not significant by Mann-Whitney U test.
Foxp3 E13 IRES mRFP polyA5’ 3’
Foxp3
Il10 E5 IRES eGFP polyA5’ 3’
IL-10
Il17a E3 IRES Katushka polyA5’ 3’
IL-17A
E1 polyA5’ 3’
IL-17A
iCRE polyA E3E2
polyA5’ 3’
Rosa26
neo tpA eYFP
loxP loxP
polyA5’ 3’
TGFβRII
Tgfbr2 E4
loxP loxP
Exons
FIR
TIGER
IL-17AKatushka
IL-17ACRE
eYFP
TGFβRIIFlox/Flox
A B
TGFβRII
Count
Isotype
Tgfbr2Wt/WtTgfbr2Flox/Flox
CD4+, YFP+
E FTgfbr2
Wt/Wt
YFP
IL-1
7A
Katu
shka
Fate+ Tgfbr2Flox/Flox
Fate+
YFP+
(3.35%)
YFP+
(7.12%)
IL-17AKatushka
IL-1
0eG
FP IL-10+ T
H17
(1.02%)
IL-10+ TH17
(11.7%)
TH17
(19.1%)
TH17
(4.19%)
TR1exT
H17
(0.96%)
TR1exT
H17
(0.31%)IL-10+ TH17
0
1!103
2!103
3!103
4!103
Cel
l Num
ber
***
IL-10+ TH17
0
5
10
15
Freq
uenc
y (%
)
***
TH17
0.0
5.0!103
1.0!104
1.5!104
Cel
l Num
ber
*
TH17
0
10
20
30
40
Freq
uenc
y (%
)
***
TR1exTH17
0
1!102
2!102
3!102
4!102
5!102
6!102
Cel
l Num
ber
ns
TR1exTH17
0.0
0.5
1.0
1.5
2.0
2.5
Freq
uenc
y (%
)
*
G
H
C
CD4
IL-1
7A
Ka
tush
ka T
H17
(13.1%)
CD4
IL-1
7A
Ka
tush
ka T
H17
(11.9%)
TH17
0
5
10
15
20
25
Freq
uenc
y (%
)
ns
Tgfbr2Wt/Wt
Fate+ Tgfbr2Flox/Flox
Fate+
D
Foxp3RFP
Count
CD4+, YFP+
Tgfbr2Flox/Flox
Tgfbr2Wt/WtFoxp3+ Treg, YFP+
0
1000
2000
3000
4000
Cel
l Num
ber
ns
Tgfbr2Wt/Wt
Fate+
Tgfbr2Flox/Flox
Fate+
0
100
200
300
400
MF
IofF
oxp3
RF
P
ns
I
Supplementary Fig. 6 Tgfbr2 is required for TH17 cells to produce IL-10 during intestinal inflammation. A. Percentages of initial body weight of mice after anti-CD3 mAb treatment. The arrows indicate the injection of anti-CD3 mAb. Data are cumulative of three independent experiments. Mean ± SEM.; *P < 0.05, by 2-way ANOVA with Bonferroni’s post-test. B. Representative histological pictures of H&E stained small intestines after anti-CD3 treatment. Statistical analysis is representative of two experiments. Each dot represents one mouse (nwild type=4, nKO=5). Mean ± SEM, *P < 0.05 by Mann-Whitney U test. C. Left, flow cytometric analysis of Foxp3 expression among YFP+ CD4+ T cells in the small intestine during anti-CD3 mAb-induced intestinal inflammation. Foxp3 expression was determined by RFP reporter fluorescence. A pre-gate on YFP+ CD4+ T cells is applied. Right panel shows the statistical analysis of Foxp3 Mean Fluorescence Intensity (MFI) among YFP+ CD4+ T cells between the two indicated mouse lines. Each dot represents one mouse (nwild type=5, nKO=5). D. Numbers of YFP+ Foxp3+ Treg cells in the small intestine in the two indicated mouse lines. Each dot represents one mouse (nwild type=5, nKO=5). Mean ± S.D.; ns, not significant by Mann-Whitney U test. E. As part of Fig.4G, here it is reported the statistical analysis of YFP+ and TH17 cells after CD4+ T cells transfer. CD4+ T cells were isolated form the indicated mouse lines. Each dot represents one mouse (nwild type=3, nKO=3). Mean ± S.D.; **P < 0.01 by Welch’s t-test.
ATgfbr2
Wt/Wt
Fate+Tgfbr2Flox/FloxFate+
0 2 4 680
85
90
95
100
Days
Per
cent
age
of In
itial
Bod
y W
eigh
t (%
)
0
2
4
6
8
Score
Histopathological
Score
B
*
*
*
ETH17
0
5
10
15
20
25
30
35
Freq
uenc
y (%
)
**
YFP+
0
20
40
60
Freq
uenc
y (%
)
**
(n=8)
(n=10)
C
D
Foxp3RFP
Co
un
t
CD4+, YFP+
Tgfbr2Flox/Flox
Tgfbr2Wt/Wt
Foxp3+ Treg, YFP+
0
2000
4000
6000
8000
Cel
l Num
ber
ns
Tgfbr2Wt/Wt Fate+
Tgfbr2Flox/Flox Fate+
0
200
400
600
800
MF
IofF
oxp3
RF
P
ns
Tgfbr2Wt/Wt Fate+
Tgfbr2Flox/Flox Fate+
Tgfbr2Wt/Wt Fate+
Tgfbr2Flox/Flox Fate+
Supplementary Fig. 7 TIF1γ is dispensable for IL-10 production in TH17 cells. A. Constructs contained
in Tif1gfl/fl Fate+ mice. B. Flow cytometry analysis of small intestinal CD4+ T cells after anti-CD3-induced
intestinal inflammation. Top panel, pre-gated on Foxp3- CD4+ T cells; bottom panel, pre-gated on YFP+ Foxp3-
CD4+ T cells. The populations indicated on the FACS dot plots are identified by different combination of
reporter expression (TR1exTH17: IL-10eGFP+ YFP+ IL17aKata-; IL-10+TH17: IL-10eGFP+ YFP+ IL17aKata+; TH17: IL-
10eGFP- YFP+ IL17aKata+). C, D. Frequencies (C) and numbers (D) of TR1exTH17, IL-10+ TH17 and TH17 cells.
One representative of three independent experiments is shown. Each dot represents one mouse (nwild type=4,
nKO=4). Mean ± S.D.; ns, not significant by Mann-Whitney U test.
A
Foxp3 E13 IRES mRFP polyA5’ 3’
Foxp3
Il10 E5 IRES eGFP polyA5’ 3’
IL-10
Il17a E3 IRES Katushka polyA5’ 3’
IL-17A
E1 polyA5’ 3’
IL-17A
iCRE polyA E3E2
polyA5’ 3’
Rosa26
neo tpA eYFP
loxP loxP
polyA5’ 3’
Smad4
Tif1g E?
loxP loxP
Exons
FIR
TIGER
IL-17AKatushka
IL-17ACRE
eYFP
TIF1γFlox/Flox
B
Tif1gWt/Wt
YFP
IL-1
7A
Ka
tush
ka
Fate+ Tif1gFlox/Flox
Fate+
YFP+
(8.40%)
YFP+
(6.93%)
IL-17AKatushka
IL-1
0e
GF
P IL-10+ TH17
(14.7%)
IL-10+ TH17
(14.3%)
TH17
(69.4%)
TH17
(65.0%)
TR1exT
H17
(1.85%)
TR1exT
H17
(2.04%)
IL-10+ TH17
0.0
5.0!103
1.0!104
1.5!104
2.0!104
Cel
l Num
ber
ns
IL-10+ TH17
0
5
10
15
20
25
Freq
uenc
y (%
)
ns
TH17
0
2!104
4!104
6!104
8!104
Cel
l Num
ber
ns
TH17
0204050
60
70
80
Freq
uenc
y (%
)
ns
TR1exTH17
0.0
5.0!102
1.0!103
1.5!103
2.0!103
Cel
l Num
ber
ns
TR1exTH17
0
1
2
3
4
Freq
uenc
y (%
)
ns
DC
Supplementary Fig. 8 Smad4 is required for TH17 cells to produce IL-10. A. Constructs contained in
Smad4fl/fl Fate+ mice. B. Flow cytometric analysis of in vitro cultured naive CD4+ T cells. Cells were polarized
under TH17 condition for 5 days. Top panel, pre-gated on Foxp3- CD4+ T cells; bottom panel, pre-gated on
YFP+ Foxp3- CD4+ T cells. The populations indicated on the FACS dot plots are identified by different
combination of reporter expression (TR1exTH17: IL-10eGFP+ YFP+ IL17aKata-; IL-10+TH17: IL-10eGFP+ YFP+
IL17aKata+; TH17: IL-10eGFP- YFP+ IL17aKata+). B. Statistical analysis of TR1exTH17, IL-10+ TH17 and TH17 cells.
Data are representative of one out of three experiments. Each dot represents one mouse (nwild type=3, nKO=3).
Mean ± S.D.; *P < 0.05, ***P < 0.001 by Welch’s t-test.
Foxp3 E13 IRES mRFP polyA5’ 3’
Foxp3
Il10 E5 IRES eGFP polyA5’ 3’
IL-10
Il17a E3 IRES Katushka polyA5’ 3’
IL-17A
E1 polyA5’ 3’
IL-17A
iCRE polyA E3E2
polyA5’ 3’
Rosa26
neo tpA eYFP
loxP loxP
polyA5’ 3’
Smad4
Smad4 E1
loxP loxP
Exons
FIR
TIGER
IL-17AKatushka
IL-17ACRE
eYFP
SMAD4Flox/Flox
Smad4Wt/Wt
YFP
IL-1
7A
Ka
tush
ka
Fate+ Smad4Flox/Flox
Fate+
YFP+
(9.62%)
YFP+
(9.94%)
IL-17AKatushka
IL-1
0e
GF
P IL-10+ TH17
(4.30%)
IL-10+ TH17
(6.41%)
TH17
(67.3%)
TH17
(73.5%)
TR1exT
H17
(3.83%)
TR1exT
H17
(2.33%)
A
B C
TR1exTH17
0
2
4
6
8
10
Freq
uenc
y (%
)
*
IL-10+ TH17
0
3
6
9
12
15
Perc
enta
ge (%
) ***
Smad4Wt/Wt
Fate+Smad4 Flox/FloxFate+
Supplementary Fig. 9 Smad3 is important for IL-10 production in TH17 cells. In vitro differentiated TH17
cells were purified and cultured in the presence 2ng/ml TGF-β together with DMSO or different amount of
SIS3. A. Representative flow cytometry analysis of DMSO and 2uM SIS3 conditions. Cells were pre-gated
on viable CD4+ T cells. Expression of IL-10eGFP was detected by reporter fluorescence. B. Statistical analysis
of the frequencies of IL-10+ cells across different conditions. Each dot represents one replicate (n=3). Mean
± S.D.; **P < 0.01 by Kruskal-Wallis test.
A
IL-17AKatushka
IL-1
0e
GF
P
DMSO 2uM SIS3
IL-10+ Cells
(11.7%)
IL-10+ Cells
(4.62%)
B
Perc
enta
ge o
f IL
-10
eG
FP
+ (
%)
DM
SO
SIS
3 (1uM
)
SIS
3 (2uM
)
0
5
10
15**
Supplementary Fig. 10 Human intestinal TH17 cells produce IL-10 and TGF-β further promotes IL-10 expression. A. The dot plot shows the purity of the sorted CD4+ IL17A+ cells corresponding to blood donor
1 shown in Fig. 7. B. Gating strategy of human TH17 cells isolated from PBMCs of one representative healthy
human donor. This strategy applies to the data shown here and in Figure 7B. C. Dot plots of PBMC-sorted
TH17 cells from the human blood, stimulated and cultured under increasing TGF-β1 concentrations four 5
days and stained for IL-17A and IFN-γ. The graphs below show the percentages of IL-17A+, IL-17A+/IFN-γ+,
and IFN-γ+ cells from four different blood donors. Each colored line represents one donor (n=4). D. Absolute
total numbers of living CD4+ T cells corresponding to (C). E. Foxp3 expression (MFI) among the in vitro
cultured human TH17 cells shown in Fig. 7, in the presence of increasing TGF-β concentrations.
AIL
-17
A
-PE
CD4-FITC
95.3%
B
16% 9.48% 13.5% 13.6%35.1% 28.8% 37.4% 30%
20.3% 20.8% 13.9% 15.4%
BV786
IL-1
7A
BV
711
0 ng/mL 0.15 ng/mL 0.5 ng/mL 1.5 ng/mL
0
5
10
15
20
25
0
10
20
30
40
Fre
qu
en
cy o
f IL
-17
A+
- ce
lls (
%)
0
20
40
60
80 Donor 1
Donor 2
Donor 3
Donor 4
Fre
qu
en
cy o
f IL
-17
A+
+ c
ells (
%)
Fre
qu
en
cy o
f IL
-17
A-
+ c
ells (
%)
C
0 0 0
SS
C
FSC CD4FITC
FOXP3PacBlue
IL-10PE
L-D
Am
Cyan
IL-1
7AB
V7
11
IL-1
7AB
V7
11
1 x 105
Num
bers
of C
D4
+ c
ells
8 x 104
6 x 104
4 x 104
2 x 104
0
0
Donor 1
Donor 2
Donor 3
Donor 4
D
0
5000
10000
15000
Ge
om
etr
ic m
ea
n
E
0
Supplementary Table 1: Table of Antibodies Name Clone Company Cat. Number Use Anti-Smad3 (phosphoS423+S425)
EP823Y Abcam ab52903 WB, PLA
Anti-Smad3 - Abcam ab52903 ChIP Smad4 Monoclonal Antibody
4G1C6 ThermoFischer Scientific
MA5-15682 WB, PLA, ChIP
Anti-Smad4 EP618Y Abcam ab40759 ChIP Anti-acetyl Histone H4 - Merck 06-866 WB Pac Blue anti-mouse CD4
GK1.5 Biolegend 100428 FACS
Alexa Fluor® 700 anti-mouse CD4
RM4-5 Biolegend 100536 FACS
PE.Cy5 anti-mouse CD8
53-6.7 Biolegend 100722 FACS
PE.Cy7 anti-mouse NK1.1
PK136 Biolegend 108713 FACS
PE anti-mouse CD19 6D5 Biolegend 115508 FACS PE.Cy7 anti-mouse CD11b
M1/70 Biolegend 101216 FACS
PE.Cy7 anti-mouse CD11c
N418 Biolegend 117318 FACS
PE.Cy7 anti-mouse γδTCR
GL3 Biolegend 118123 FACS
PE anti-mouse TGFβRII
- R&D Systems FAB532P FACS
BV421 anti-mouse IL-17A
TC11-18H10.1
Biolegend 506925 FACS
PE anti-mouse IFN-γ XMG1.2 Biolegend 554412 FACS FITC anti-human CD4 OKT4 Biolegend 317408 FACS Alexa Fluor® 700 anti-human CD45RA
HI100 Biolegend 304120 FACS
PE/Cy7 anti-human CD127
019D5 Biolegend 351320 FACS
Brilliant Violet 650™ anti-human CD25
BC96 Biolegend 302633 FACS
Brilliant Violet 711™ anti-human IL-17A
BL168 Biolegend 512328 FACS
PE anti-human IL-10 JES3-19F1
Biolegend 506804 FACS
Pacific Blue™anti-mouse CD4
RM4-5 Biolegend 100531 FACS
WB: Western Blot
PLA: Proximity Ligation Assay
ChIP: Chromatin Immunoprecipitation
FACS: Fluorescent Activation Cell Sorting
Supplementary Table 2: Table of primers Name Sequence Amplicon
size Use
Il10pr Fw 5’ gttgcttctgctgttggaaacg 3’ 1447bp PCR, CL
Il10pr Rev 5’ gtagacctcctgttcttggtccc 3’ PCR, CL
Il10pr-pGL2 Fw
5’ gcgtcgtagctcgaggttgcttctgctgttggaaacg 3’ 1477bp PCR, CL
Il10pr-pGL2 Rev
5’ cggaatgccaagcttgtagacctcctgttcttggtccc 3’ PCR, CL
Smad3 Fw 5’ atgtcgtccatcctgcccttcac 3’ 1278bp PCR, CL
Smad3 Rev 5’ ctaagacacactggaacagcggatg 3’ PCR, CL
Smad3-pTNT Fw
5’ ctttttgcactcgagatgtcgtccatcctgcccttcac 3’ 1308bp PCR, CL
Smad3-pTNT Rev
5’ cgggtcgactctagactaagacacactggaacagcggatg 3’ PCR, CL
Smad4 Fw 5’ atggacaatatgtctataacaaatacaccaa 3’ 1656bp PCR, CL
Smad4 Rev 5’ tcagtctaaaggctgtgggtccg 3’ PCR, CL
Smad4-pTag2 Fw
5’ cgggctgcaggaattcatggacaatatgtctataacaaatacaccaa 3’
1687bp PCR, CL
Smad4-pTag2 Rev
5’ cgtatcgataagctttcagtctaaaggctgtgggtccg 3’ PCR, CL
Il10A Fw 5’ caggttgagtggaggaaacaa 3’ 138bp ChIP Il10A Rev 5’ ggcagacagctgttctatgt 3’ ChIP Il10B Fw 5’ gcccagggtacagaatgaaa 3’ 173bp ChIP Il10B Rev 5’ agctgttgaaggatggagatg 3’ ChIP Il10C Fw 5’ agcccatttatccacgtcatta 3’ 94bp ChIP
Il10C Rev 5’ actggtcggaatgaacttctg 3’ ChIP Gapdh Fw 5’ accagggagggctgcagtcc 3’ 237bp ChIP Gapdh Rev 5’ tcagttcggagcccacaggc 3’ ChIP
PCR:PolymeraseChainReaction
CL:Cloning
ChIP:ChromatinImmunoprecipitation