www.sciencemag.org/cgi/content/full/science.aac4263/DC1

Supplementary Materials for

The microbiota regulates type 2 immunity through RORγt T cells

Caspar Ohnmacht, Joo-Hong Park, Sascha Cording, James B. Wing, Koji Atarashi, Yuuki Obata, Valérie Gaboriau-Routhiau, Rute Marques, Sophie Dulauroy,

Maria Fedoseeva, Meinrad Busslinger, Nadine Cerf-Bensussan, Ivo G. Boneca, David Voehringer, Koji Hase, Kenya Honda, Shimon Sakaguchi, Gérard Eberl*

*Corresponding author. E-mail: gerard.eberl@pasteur.fr

Published 9 July 2015 on Science Express DOI: 10.1126/science.aac4263

This PDF file includes:

Materials and Methods Figs S1 to S13 References

2

Materials and Methods Mice

Foxp3Rfp mice (43) were crossed to Rorc(γt)Gfp mice (15) and maintained on a C57BL/6

background, as were Cd11cCre x Rosa26Dta (ΔDC) mice (44), IL-2-deficient (45), IL-10-

deficient (46), IL-6-deficient (47), Il23a-deficient (48), Nod1/Nod2- and Myd88/Trif-

double deficient mice, Il10Venus (21) and Gata3GFP mice (49). RORγt-deficient mice (14)

were backcrossed at least 8 generations to a C57BL/6 background. Foxp3Cre mice were

purchased on a NOD/ShiLt background (The Jackson Laboratory) and crossed to

Rorc(γt)Fl/KO mice (The Jackson laboratory) and to Stat3Fl/Fl mice (50) on a C57BL/6

background, as were Rorc(γt)Cre mice (51). OT-II TCR transgenic mice (specific for

OVA323-339 peptide) (52) were crossed with Rag2-deficient or RORγt-deficient mice. Fox-

IRES-cre mice were crossed with CTLA-4-floxed mice (38). Germfree mice were

generated from SPF C57BL/6 mice and maintained at the Gnotobiology Platform of the

Institut Pasteur. In all experiments, littermates were used as controls. All mice were kept

in specific pathogen-free conditions and all animal experiments were approved by the

committee on animal experimentation of the Institut Pasteur and by the French Ministry

of Agriculture.

Mouse treatments

For antibiotic treatment, pregnant females were fed three days before delivery with a

cocktail of the following antibiotics in their drinking water: 1 mg/ml Collistin, 5 mg/ml

Streptomycin, 1 mg/ml Ampicillin and 0.5 mg/ml Vancomycin (Sigma). The antibiotic-

containing drinking water was changed once a week until analysis. For DSS treatment,

mice received three cycles of seven days of 3% DSS (MP Biochemicals) in their drinking

water followed by ten days of normal drinking water. This cycle was repeated twice

before analysis. For oxazolone treatment, mice were presensitized with 200 ul of 2%

(w/v) oxazolone (4-ethoxymethylene-2-phenyl-2-oxazoline-5-one, Fluka) in 100%

ethanol by subcutaneous injection. Five days later, mice were challenged intrarectally

with 150µl of 1% oxazolone in 50% ethanol or only 50% ethanol under

anesthesia. Intrarectal injections were administered with a 3.5F polyurethane catheter.

3

Monocolonizations with SFB, E. coli or Clostridia were performed as described

previously (21, 24). For butyrate treatment, mice were fed a low fiber diet with or without

butyrylated high-amylose maize starches for 4 weeks. For diet study, vitamin A-deficient

and –sufficient diets were purchased from SAFE diets, and administered to pregnant

females from day 15 of gestation until weaning. Offspring were maintained on special

diet for 10 weeks. For inhibition of the retinoic acid receptor, 250 µg of the pan-retinoic

acid receptor inverse agonist BMS493 (R&D) was administered intraperitoneally to

C57BL/6 mice daily for 12 days. For infection with Heligmosomoides polygyrus, 200 L3

larvae were inoculated to each mouse by oral gavage, and from day 14 post-inoculation,

fecal pellets were collected and rehydrated. Following addition of saturated sodium

chloride solution, eggs were counted using a McMaster chamber.

Cell preparation and cultures

Small intestinal and colonic lamina propria cells were isolated as described previously

(53). Briefly, Peyer’s Patches were removed, and whole small intestine and colon were

opened longitudinally, cut into pieces and incubated for 30 minutes in 30mM EDTA,

washed extensively, and incubated for several rounds in 0.7mg/ml collagenase D. These

preparations, as well as thymus, spleen and lymph nodes, were then grinded through a

100μm filter to generate single cell suspensions. Cells were separated by a 40/80% (w/v)

Percoll (GE Healthcare) density gradient and washed prior to staining for flow cytometry

analysis. For intracellular cytokine staining, cells were restimulated with 50 ng/ml PMA

and 500 ng/ml ionomycin for 4 hours. Brefeldin A was added for the final 2 hours. For

analysis of cytokine production, cells were cultured for 1 day in round-bottomed 96-well

plates coated with anti-CD3 (10 µg/ml) in RPMI 1640 (Invitrogen) containing soluble

anti-CD28 (1 μg/ml) and 10% FCS, 25 mM HEPES, 0.05 mM 2-mercaptoethanol, and

100 U/ml of penicillin and streptomycin. Mesenteric lymph node cells isolated from H.

polygyrus-infected mice were incubated as above for 3 days. Cytokine levels in culture

supernatants were analysed by ELISA or LEGENDplexTM (Biolegend).

4

Flow cytometry

Cells were preincubated with Fc-Block for 5 minutes and stained for 20 minutes with the

following antibodies: FITC-conjugated anti-CD3 (clone 145-2C11), eFluor450-

conjugated anti-CD3 (clone 17A2), APC-eFluor780-conjugated CD3 (clone 17A2),

PerCP-Cy-5.5-conjugated anti-CD3 (clone 17A2) or Brilliant Violett 785-conjugated

anti-CD3 (clone 17A2; BioLegend), eFluor450-conjugated anti-CD4 (clone GK1.5) or

Horizon V500-conjugated anti-CD4 (clone RM4-5; BD), biotin-conjugated anti-CD8

(clone 53-6.7), eFluor450-conjugated anti-CD25 (clone PC61.5), eFluor660-conjugated

anti-CD39 (clone 24DMS1), APC-conjugated anti-CD44 (clone IM7), FITC-conjugated

anti-CD45.2 (clone 104; BD), APC-conjugated anti-CD45R (clone RA3-6B2) or A700-

conjugated anti-CD45R (clone RA3-6B2), Brilliant Violet 650-conjugated anti-CD19

(clone 6D5, Biolegend), eFluor450-conjugated anti-MHC II (clone M5/114.15.2),

eFluor660-conjugated anti-CD11c (clone N418), PE-Cy7-conjugated anti-CD80 (clone

16-10A1), FITC-conjugated anti-CD86 (clone GL1), PE-conjugated anti-CD49b (clone

DX5), biotin-conjugated anti-CD62L (MEL-14), APC-conjugated PE-Cy7-conjugated

anti-CD73 (clone Ty/11.8), biotin-conjugated anti-CD103 (clone 2E7), PE-conjugated

CD196 (clone 140706; R&D), APC-conjugated anti-CD278 (clone C398.4A), APC-

conjugated ployclonal anti-Neuropilin 1 (R&D), PE-conjugated anti-TCR Vβ5.1&5.2

(clone MR9-4; BD), PE-Cy7-conjugated anti-TCR Vα2 (clone B20.1, BD), eFluor660-

conjugated anti-IRF4 (clone 3E4, eBioscience). PE-, PerCP-Cy-5.5- (both eBioscience)

or AlexaFluor647- (Invitrogen) conjugated streptavidin was used for detection of

biotinylated antibodies. For intracellular stainings, cells were fixed and permeabilized

with a commercially available fixation/permeabilization buffer (eBioscience). After

restimulation, LIVE/DEAD fixable violet dead stain kit (Invitrogen) was used prior to

fixation. Intracellular staining was performed with the following antibodies: Pacific Blue-

conjugated anti-Helios (clone 22F6; BioLegend), PE- or APC-conjugated anti-RORγt

(clone AFKJS-9), PerCP-Cy5.5-conjugated Foxp3 (clone FJK-16s), APC-conjugated

anti-CD152 (UC10-4B9), eFluor660-conjugated anti-Gata3 (clone TWAJ), APC-

conjugated anti-IL-10 (clone JES5-16E3), APC- or PE-conjugated anti-IL-17A (clone

TC11-18H10; BD). All antibodies were from eBioscience unless stated otherwise.

5

Adoptive cell transfer

Single cell suspensions of spleens and mesenteric lymph nodes from OT-II Rag-/- mice

(or from OT-II RORγt-/- and OT-II RORγt+/- mice) were stained, and naïve

CD4+TCRβ+CD25-CD62LhiCD44lo T cells (CD4+TCRVβ5.1/5.2+Vα2+CD25-

CD62LhiCD44lo for OT-II RORγt-/- and OT-II RORγt+/- mice) isolated by FACS. 1.5-2 x

106 naïve T cells were injected i.v. into CD45.1 congenic mice, and recipients received

1.5% chicken ovalbumin (Sigma) in the drinking water for seven consecutive days prior

to analysis.

Cultures of colon explants

Small pieces of colon were isolated, weighed and cultured overnight in 24-well tissue

culture plates in 500μl of complete RPMI 1640 at 37°C. Supernatants were analysed for

IL-33 using LEGENDplex capture bead (Biolegend), and were normalized to the weight

of the colon explant.

Quantitative PCR

To isolate mRNA, cells were directly FACS-sorted into RLT buffer (Qiagen)

supplemented with β2-mercaptoethanol. Total RNA was prepared using RNeasy micro

kit (Qiagen) according to the manufacturer’s instructions. Concentration and integrity

was assessed using the Bioanalyzer (Agilent). Linear amplification of mRNA was

performed using the MessageBooster cDNA kit (Epicentre). cDNA was synthesized by

reverse transcription using Superscript III (Invitrogen). Detection of specific gene

expression was performed using primers from Superarray Biosciences and SYBR green

(Qiagen). Gene expression was normalized to Gapdh, Hsp90 and Hprt in each sample.

Statistical analysis

P-values were calculated with unpaired, two-tailed t test or Mann-Whitney test using

GraphPad PRISM software (version 5).

6

Fig. S1. Phenotype of RORγγt+ Tregs. (A) Frequency of RORγt+ Tregs in the colon lamina propria of wild type mice at the indicated age. n = 4. (B) Expression of RORγt and Helios by thymic Tregs in control (left) or RORγt-deficient mice (right). (C) Expression of the indicated markers by RORγt+ Tregs (red) and RORγt- Tregs (blue). Data are representative of at least three independent experiments.

A 9 weeks 2 weeks 1 year

RORγt

Cel

l num

ber

0 102 103 104 1050

5

10

15

20

25

3.74

0 102 103 104 1050

3

6

9

12

# C

ells

47

0 102 103 104 1050

10

20

30

40

63.3

0 102 103 104 1050

5

10

15

20

90.4

5 weeks

RORγt- Tregs RORγt+ Tregs

CCR6

CD62L ICOS CTLA-4 ic

CD103

CD39

CD73 Nlrp1

CD44

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 105

PE A CD44

0

20

40

60

80

100

%

Helios

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 105

APC A CCR6

0

20

40

60

80

100

%

0 102 103 104 1050

20

40

60

80

100

C

2 5 9 52 (weeks)

B

0 102 103 104 105

0

102

103

104

105 1.0 1.5

69.628.00 102 103 104 105

0

102

103

104

105 1.3 2.3

79.916.7

Control RORγt-/-

RO

Rγt

Helios

Thymic Tregs

%R

ORγt

+ of

Tre

gs

7

Fig. S2. Gata3+ Tregs and RORγγt+ Tregs are distinct populations. (A) Expression of Gata3, RORγt and Helios in small intestine (upper) and colon Tregs (lower). (B) Relative expression, measured by qPCR, of the indicated genes in FACS-purified Gata3+ and Gata3- Tregs isolated from small intestine and colon of Gata3GFP mice. Error bars, s.d.

A

0 102 103 104 105

0

102

103

104

105 24.2 1.0

56.618.20 102 103 104 105

0

102

103

104

105 4.5 21.2

12.961.40 102 103 104 105

0

102

103

104

105 57.3 0.8

33.68.4

0 102 103 104 105

0

102

103

104

105 63.3 1.1

23.012.70 102 103 104 105

0

102

103

104

105 3.3 6.1

17.972.8AP

CA

GAT

A3

0 102 103 104 105

0

102

103

104

105 8.4 1.4

63.226.9

Helios

RO

Rγt

Gat

a3

Gat

a3

RORγt

SI-LPL

Co-LPL

B

Rel

ativ

e ex

pres

sion

IL10IL17

aCcl2

0Rorc

IL23R

FoxP3

STAT3

Il1rl1

Ffar2

Ffar3

10-3

10-2

10-1

100

SI GATA3+TregsSI GATA3-TregsCo GATA3+TregsCo GATA3-Tregs

8

Fig. S3. Gata3+ Tregs are not induced by microbiota. (A) Expression of RORγt and Gata3 by Tregs in the colon of SPF or germfree (GF) mice, and ratio of Gata3+ Tregs versus RORγt+ Tregs in SPF and germfree mice, n ≥ 5. (B) Expression of Gata3 and Helios by Tregs, and frequencies and total cell numbers of Gata3+ Tregs, in the small intestine and the colon of germfree mice or germfree mice that have been recolonized for 2 weeks with SPF microbiota (ex-germfree), n = 4. Error bars, s.d.; **P<0.01, as calculated by Student’s t-test.

0

2

4

6

8

10

0 102 103 104 105

0

102

103

104

105 4.9 11.9

42.241.10 102 103 104 105

0

102

103

104

105 3.0 6.8

14.975.3

0 102 103 104 105

0

102

103

104

105 7.7 40.1

38.314.00 102 103 104 105

0

102

103

104

105 7.7 36.7

16.439.1

Germfree ex-Germfree

Helios

Gat

a3

SI-LPL

Co-LPL S

I-LP

L

Co-

LPL

Abs

olut

e nu

mbe

r

SI-L

PL

Co-

LPL

% o

f Tre

gs

A

B

Rat

io in

Tre

g

Gata3 R

ORγt

SPF GF Gata3+/RORγt+ Tregs

SPF GF

Gata3+ Tregs Gata3+ Tregs

**

9

Fig. S4. Induction of RORγγt+ Tregs by SPF microbiota. (A) Expression of RORγt and Helios by Tregs in the colon of germfree mice or germfree mice that have been recolonized for 2 weeks with SPF microbiota (ex-germfree). (B) Absolute numbers of RORγt+ Tregs and (RORγt-) Helios+ Tregs in the organs of SPF, germfree and ex-germfree mice. n = 4. Error bars, s.d.; ns, not significant, **P<0.01, ***P<0.001, as calculated by Student’s t-test.

Germfree ex-Germfree

Helios R

ORγt

0 102 103 104 105

0

102

103

104

105

3.83

26.4

66

0 102 103 104 105

0

102

103

104

105

g 67

9.05

21.1

A

B

** ***

*** ***

*** ***

ns ns

SP

mLN

SI-L

PL

Co-LP

L

RORγt+ Tregs

Helios+ Tregs

Abs

olut

e nu

mbe

r

10

Fig. S5. Butyrate induce RORγγt+ Tregs. Expression of Foxp3 and RORγt by CD4+ T cells, and frequencies of RORγt+ Tregs and RORγt- Tregs in the colon of mice fed for 4 weeks with a control diet or a diet containing butyrylated high-amylose maize starches. n≥ 8. Error bars, s.d.; ***P<0.001, as calculated by Student’s t-test.

Butyrate Control

Control Butyrate

***

Foxp

3

RORγt

14.2 10.5

3.26720

105

104

g

103

102

1051041031020

12.6 6.24

5.4375.80

105

104

g

103

102

1051041031020

% o

f Tre

gs

0

20

40

60

80 *** RORγt+ Tregs RORγt- Tregs

11

Fig. S6. TLRs and NLRs are not required for the generation of RORγγt+ Tregs. Expression of RORγt and Helios by Tregs in the colon of wild-type control and double-mutant Myd88-/-Triff-/- or Nod1-/-Nod2-/- mice.

RO

Rγt

Control Myd88-/-/Trif-/- Nod1-/-/Nod2-/-

0 102 103 104 105

0

102

103

104

105 68.3 1.71

16.713.30 102 103 104 105

0

102

103

104

105 49.6 0.758

29.919.70 102 103 104 105

0

102

103

104

105

g

41.1 1.28

45.811.8

Helios

12

Fig. S7. Gata3+ Tregs are generated independently of IL-6 and IL-23. (A) Frequencies of Gata3+ Tregs, Gata3+ non-Treg cells and ILC2s (right) in the colon of Il6-

/- (open circles) or littermate control mice (filled circles). n = 4. (B) Frequencies of Gata3+ Tregs, Gata3+ non-Treg cells and ILC2s in the colon of Il23a-/- (open circles) or control mice (filled circles). n ≥ 3. Error bars, s.d.; ns, not significant, *P<0.05, **P<0.01, as calculated by Student’s t-test.

0

1

2

3

4

5

0

2

4

6

8

0

2

4

6

8

10

0

10

20

30

A

B

*

ns

ns

ns

% o

f Th

% o

f ILC

s

0102030405060

% o

f Tre

gs

**

0

10

20

30

40

50

% o

f Tre

gs

**

GATA3+ Tregs Th2 ILC2

GATA3+ Tregs Th2 ILC2

% o

f Th

% o

f ILC

s

Control Il6-/-

Control Il23a-/-

13

Fig. S8. Retinoic acid promotes the generation of RORγγt+ Tregs over Th17 cells. Frequencies of RORγt+ Tregs, Th17 cells, Helios+ Tregs, and ratio of Th17 cells versus RORγt+ Tregs in the colon of mice treated either with DMSO or with an RAR-inhibitor for 12 days, n=3. Error bars, s.d.; ns, not significant, **P<0.01, ***P<0.001, as calculated by Student’s t-test.

Control RARi

ns **

*** %

of T

regs

% o

f Tre

gs

% o

f Th

0.0

0.1

0.2

0.3

0.4

0.5

Rat

io in

CD

4

ns RORγt+ Tregs Helios+ Tregs Th17 Th17/RORγt+ Tregs

14

Fig. S9. RORγγt+ cells control type 2 immune responses. (A) Production of type 2 cytokines by T cells isolated from colons of oxazolone-treated control mice (filled symbols) or Foxp3Cre x Rorc(γt)Fl/Fl mice (open symbols). n = 5. (B) Expression of Gata3 and FoxP3 by CD4+ T cells from the small intestine of littermate control or RORγt-deficient mice. Histograms show frequency of Gata3+ Tregs (left) and Th2 cells (right) in control (filled symbols) and RORγt-deficient (open symbols) mice, n = 5. (C) Serum levels of IgE (left), mean fluorescence intensity of IgE staining on B cells (middle) and basophils (right) in control (filled symbols) and RORγt-deficient (open symbols) mice, n≥ 4. Error bars, s.d.; *P<0.05, ***P<0.001, as calculated by Student’s t-test.

2.67 79.1

6.84 11.2

0102030405060

*** *** * C

* ns

B RORγt+/- RORγt-/-

Foxp

3

Gata3 RORγt+/-

RORγt-/-

RORγt+/-

RORγt-/-

Ser

um Ig

E (n

g/m

l)

MFI

IgE

on

B22

0+ c

ells

MFI

IgE

on

baso

phils

%

of T

regs

% o

f Th

Th2 Gata3+ Tregs

24.4 62.2

6.6 6.7

A

0

50

100

150

200

250

300 * C

ytok

ine

(pg/

ml)

0

200

400

600

800

1000

0

100

200

300

400

500* ns

IL-4 IL-5 IL-13

Control Foxp3Cre x RORγtFL/FL

15

Fig. S10. RORγγt+ Tregs do not regulate type 1 or type 3 immunity. Adult mice received 3 cycles of DSS and were analysed on day 10 after the last DSS cycle. Left, frequency of total Tregs isolated from colon of littermate control (filled circles) or Foxp3Cre x Rorc(γt)Fl/Fl mice (open circles). Middle and right, frequency of IL-17+ or IFN-γ+ CD4 T cells, respectively. Data are pooled from three independent experiments, n= 10. Error bars, s.d.; ns, not significant, *P<0.05, as calculated by Student’s t-test.

Foxp3Cre x RORγtFL/FL Control

%IL

17+

of T

h

%Fo

xp3+

of C

D4

%IF

Nγ+

of T

h

* ns ns

Th17 Tregs Th1

16

Fig. S11. Mechanisms of regulation by RORγγt+ Tregs. (A) Naïve CD4+ T cells were isolated from CD45.2+ OT-II wild-type or CD45.2+ OT-IIRORγt -/- mice and adoptively transferred into CD45.1+ congenic mice, subsequently fed for 7 days with 1.5% chicken ovalbumin. Frequency of RORγt+ Tregs and Th17 cells in the small intestine in host (CD45.1) and donor (CD45.2) cells, n = 3. (B) Expression of RORγt and Gata3 by Tregs and effector CD4+ T cells in the colon of littermate control and RORγt Cre x Stat3Fl/Fl mice. (C) Production of IL-33 in cultures of colon explant from SPF and germfree mice, n ≥ 4. Error bars, s.d.; ns, not significant, *P<0.05, ***P<0.001, as calculated by Student’s t-test.

0.0

0.1

0.2

0.3

0.4

0

20

40

60

80

100

A

B C

Gata3

RO

Rγt

RORγtCre x STAT3FL/FL Control

Tregs

Effectors pg

/ml p

er m

g tis

sue

SPF GF

OT-II control OT-II RORγt-/-

Th17 RORγt+ Tregs

% o

f Tre

gs

Host Donor

*** ns

% o

f CD

4 T

0

10

20

30

40

Host Donor

***

ns

*IL-33

17

Fig. S12. IL-10 regulates type 3 immunity. (A) Frequencies of RORγt+ Tregs (left) and RORγt+ Foxp3- T cells in small intestine and colon of IL-10-deficient or control mice. n = 3. (B) Frequencies of Gata3+ Tregs and Gata3+ Foxp3- T cells in small intestine and colon of IL-10-deficient or control mice. n = 3. Error bars, s.d.; ns, not significant, *P<0.05, **P<0.01, ***P<0.001, as calculated by Student’s t-test.

0102030405060

0.0

0.5

1.0

1.5

2.0

2.5

0

10

20

30

40

50

A

B

Control Il10-/-

Control Il10-/-

** *

Co-LPL SI-LPL Co-LPL SI-LPL

** **

* **

Co-LPL SI-LPL Co-LPL SI-LPL

% o

f Th

% o

f Th

% o

f Tre

gs

RORγt+ Tregs Th17

Th2 Gata3+ Tregs

** ***

% o

f Tre

gs

18

Fig. S13. A model for the regulation of type 2 responses by microbiota. Type 2 responses are triggered by tissue injury to perform repair functions, and therefore are a default immune pathway in the absence of microbes. In contrast, microbiota induce type 3 responses that antagonize type 2 responses cell intrinsically through activation of Stat3, as well as cell extrinsically through the regulation of DC functions. In the absence of symbiotic microbiota, RORγt+ T cells are not generated, which leads to an increase intype 2 responses, which improve protection to worm infection but also exacerbate allergic inflammation. Retinoic acid produced from diet-derived vitamin A by several types of cells within the intestine favours the generation of RORγt+ Tregs, or “type 3 Tregs”, over Th17 cells to maintain a balance between pro- and anti-inflammatory responses at steady state. During infection and inflammation, the production of RA may be perturbed, a context that would favour the generation of Th17 cells over RORγt+

Tregs.

Bacterial Flora

Steady state intestine

Naïve Th cell

+ Gata3

TGFβ

Th2 Gata3+ Treg

Injury

Type 2

+ RORγt

RORγt+ Treg

Th17

RA Symbionts

Type 3

STAT3

Cell-intrinsic regulation

CD80 CD86

CTLA4 IRF4

Cell-extrinsic regulation

19

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