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+

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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