www.sciencemag.org/cgi/content/full/science.1194597/DC1

Supporting Online Material for

Lineage Relationship Analysis of RORγt+ Innate Lymphoid Cells

Shinichiro Sawa, Marie Cherrier, Matthias Lochner, Naoko Satoh-Takayama, Hans Jörg Fehling, Francina Langa, James P. Di Santo, Gérard Eberl*

*To whom correspondence should be addressed. E-mail: gerard.eberl@pasteur.fr

Published 23 September 2010 on Science Express

DOI: 10.1126/science.1194597

This PDF file includes:

Materials and Methods Figs. S1 to S16 References

MATERIALS AND METHODS

Mice

Bac-transgenic Rorc(γt)-tTATG mice were generated as described previously (S1). The coding

sequence for tTA, including the stop codon, was inserted into exon 1 of Rorc(γt) in place of

the endogenous ATG translation start codon, on a 200-kb BAC (Invitrogen) carrying at least

70 kb of sequence upstream of the Rorc(γt) translation start site. Rorc(γt)-tTATG mice were

crossed with LC1-Cre mice (S2) and then further crossed with Rosa26-YFP (S3) or Rosa26-

RFP (S4) mice for inducible fate mapping. Mice crossed to Rosa26-RFP reporters were

crossed with BAC-transgenic Rorc(γt)-GfpTG mice described before (S5). CCR6-, Nod1-,

Nod2- and Myd88-deficient mice were described previously (S6, S7). All these mice were

kept in specific pathogen-free conditions. Germfree Rorc(γt)-GfpTG mice were obtained by

aseptic caesarean section followed by the adoption of the caesarean-derived pups by germ-

free foster mothers. Health monitoring tests were performed to verify that the germ-free

colony was also free of parasites and of mouse specific viruses. Colonization of germfree

Rorc(γt)-GfpTG mice with SFB or culturable bacteria was performed as described previously

(S8). All animal experiments were approved by the committee on animal experimentation of

the Institute Pasteur and the French Ministry of Agriculture.

Mouse treatments

Triple transgenic mice were treated with doxycycline (Sigma Aldrich) at 1mg/ml in drinking

water containing 5% Sucrose until analysed. To eradicate the intestinal bacterial flora,

transgenic mice were treated with a cocktail of antibiotics containing 5g/L Streptomycin, 1g/L

Colistin, 1g/L Ampicillin and 2.5% Sucrose (Sigma Aldrich) in the drinking water starting

one day before birth, and until analysis at day 56. To verify the effect of doxycycline itself on

the development of RORγt+ILCs, Rorc(γt)-GfpTG mice were treated with 1mg/ml doxycycline

since one day before birth until analysis at day 42. Antibiotic treatment was renewed every

week. To ablate CD4+ cells, triple transgenic mice were injected i.p. with 50µg of anti-CD4

antibody (GK1.5) or 50µg Rat IgG2b isotype control (both from eBioscience) every two days

between day 7 and day 20 after birth. Mice were analyzed at day 21.

1

Adoptive transfer of cells

Bone marrow cells isolated from 8 weeks old Rorc(γt)-EgfpTG mice were treated with a

combination of lineage depletion kit (the cocktail of antibodies against CD5 B220 CD11b Gr1

Ter-119 (Miltenyi Biotec) and biotin-conjugated anti-mouse NKp46 (R&D systems) and a

total of 1x106 cells was intravenously injected into C57BL/6 mice irradiated at 1000rad.

5x105 of lineage depleted fetal liver cells were also injected into 600rad irradiated d.0

C57BL/6 neonates. Mice were analyzed after 6 weeks.

Isolation of cells and FACS

To isolate mononuclear cells from the small intestinal lamina propria (SI-LP), intestines from

adult mice were cut open and first incubated in PBS (Ca/Mg free) containing 30mM EDTA

for 30 min at 4oC. Tissues were then washed with PBS (Ca/Mg free) by vigorous shaking

three times. Tissues were subsequently cut into 1mm pieces and incubated at 37oC for 60 min

in DMEM (Gibco) containing 1mg/ml collagenase D (Roche) and 1U/ml DNase 1

(Invitrogen). Every 10 minutes, tissues were washed with warm DMEM and re-incubated

with fresh Collagenase and DNase containing medium. Supernatants obtained in each step

were collected. Fetal or neonatal total intestine were isolated and cut into 1mm pieces, and

then incubated at 37oC for 30 min in DMEM containing 1mg/ml collagenase D and 1U/ml

DNase 1. Remaining intestinal fragments were collected and pressed through a 100-µm mesh,

mixed with the collected supernatants, and resuspended in a 40% Percoll solution (GE

Healthcare). Mononuclear cells were collected from the interphase between 80% and 40%

Percoll solutions after spin at 2,700 rpm for 20 min. To obtain single cells from the lymph

node, the spleen and the liver, organs cut into 5mm pieces were incubated at 37oC for 30 min

in DMEM containing 1mg/ml collagenase D and 1U/ml DNase 1 and then pressed through a

100-µm mesh. To obtain mononuclear cells from the liver, 40-80% percoll cell separation was

performed. Fetal liver and adult bone marrow cells were treated with a lineage depletion kit

(Miltenyi Biotec) and lin+ cells were further gated out during analysis. All cells were first

preincubated with mAb 2.4G2 to block Fcγ receptors, and then washed and incubated with

the indicated mAb conjugates for 40 min in a total volume of 100 μl PBS containing 2mM

EDTA and 2% bovine serum. Cells were analysed on FACSCanto I or FACSCantoⅡ(BD

Biosciences) and Flowjo software (Tristar). Cells were sorted with a FACS Aria (BD

Biosciences). The purity of sorted RORγt+ILCs was 95-98%.

2

Antibodies

Purified polyclonal anti-GFP, Alexa Fluor 488 conjugated anti-rabbit IgG (H+L) and

Alexa647-conjugated anti-Syrian Hamster were purchased from Invitrogen. Pacific blue-

conjugated anti-CD3 (500A2); PE-conjugated anti-CD117 (2B8), anti-RORγt (AFKJS-9),

anti-LAMP-1 (Integrin α4β7) (DATK32), anti-IL-22 (1H8PWSR) and RatIgG1 isotype control

(eBRG1); PerCP Cy5.5-conjugated anti-CD4 (RM4-5); PE-Cy7-conjugated anti-

CD8α (53−6.7) and Streptavidin; APC-conjugated anti-CD4 (RM4-5), anti-CD127 (A7R34)

and anti-CD11c (N418); Alexa 647 conjugated Streptavidin and anti-IL-17A (eBio17B7);

APC-Alexa Fluor®780 conjugated anti-CD3ε (17A2), anti-CD117 (2B8), and anti-CD45.2

(104) were purchased from e-Bioscience. Biotin-conjugated anti-NKp46 and PE-conjugated

anti-CCR6 (140706) was from R&D systems. Purified anti-CD3 (500A2) was from BD

Biosciences. To eliminate lineage positive cells from total bone marrow cells, the mouse

Lineage Cell Depletion kit (a cocktail of antibodies against CD5, B220, CD11b, Gr1 and Ter-

119) was used together with biotin-conjugated anti-mouse NKp46, followed by Auto-MACS

(Miltenyi Biotec).

Cell culture.

GFP+ or YFP+ ILCs were FACS-sorted from the SI-LP of either Rorc(γt)-EgfpTG or triple

transgenic E.14-16 fetuses, day 0 neonates or 4 weeks-old adult mice. Sorted (1000) cells

were suspended in culture medium (OPTI MEM (GIBCO) containing 10% FCS (LONZA

Verviers Belgium lot#9SB001), β2-mercaptoethanol, ampicilin and streptomycin) and seeded

into ftat bottom 96-well plates coated with OP9 or OP9DL1 stroma cells in the presence of

20ng/ml mouse rIL-7 and 20ng/ml mouse rSCF (PeproTec). In some experiments, mouse rIL-

2 (eBioscience) was added at the concentration of 10ng/ml. After 6 days of culture, ILCs and

stroma cells were dissociated with Cell Dissociation Buffer (GIBCO) and 100-300 ILCs per

well were recovered. CD45.2+ hematopoietic cells were analysed by FACS Canto. E.16

Rorc(γt)-EgfpTG fetal liver cells were treated with a lineage depletion kit (Miltenyi) and FACS

sorted. Sorted (200) GFP+ fetal liver cells were cultured in the same conditions as SI-LP

RORγt+ILCs and 500-800 GFP+ cells were recovered.

Cell stimulation and intracellular staining

To assess intracellular IL-17A expression, cells were stimulated for 3hr in DMEM containing

50 ng/ml Phorbol 12-Myristate 13-Acetate (PMA) and 500ng/ml ionomycin (Sigma-Aldrich).

3

For the last 1.5 hr, 10µg brefeldin A (Sigma-Aldrich) was added to the culture medium. For

intracellular IL-22 staining, cells were stimulated with 40 ng/ml mouse IL-23 (R&D systems)

for 3 hr and in the presence of brefeldin A for the last 1.5 hr. After surface staining of c-kit

(CD117), CD4, NKp46, IL-7Rα(CD127) and CD3ε, cells were fixed with 4% PFA (Sigma-

Aldrich) and permeabilized by 1% saponin (Sigma-Aldrich) and then intracellular anti-IL-

17A or Il-22 were performed as described previously (S5). To detect intracellular RORγt, the

Foxp3-staining buffer set (e-Bioscience) was used for fixation and permeabilization of the

cells.

Chemokine receptor staining

For the surface CCR6 staining, intestinal lamina propria cells were first incubated with 10%

FCS containing DMEM at 37oC for 30 min in the presence of anti-CCR6 antibody, followed

by staining at 4oC against other surface molecules such as c-kit (CD117), CD4, NKp46, IL-

7Rα (CD127) and CD3ε.

Cell proliferation

To measure DNA synthesis during cell proliferation in vivo, 50µL of 2mM EdU (Invitrogen)

solution was injected i.p. into day 3 dox-treated TTG-YFP mice daily between day 7 and day

13 after birth. Incorporation into cells of 5-ethynyl-2'-deoxyuridine (EdU) was detected at day

14 with the Click-iTTM EdU Flow Cytometry Assay Kits-Pacific BlueTM (Invitrogen). To

enhance YFP expression of the fixed cell, anti-GFP and Alexa Fluor 488 anti-Rabbit

(Invitrogen) were used.

RNA isolation and qPCR

To perform gene expression analysis, epithelium-containing cells pellets obtained by 30mM

EDTA incubation of small intestine was immediately frozen in liquid nitrogen. Cell pellets

were treated with TRIZOL regent and total RNA was purified according to the manufacture’s

protocol (Invitrogen). RNA was subjected to DNase I digestion and additional purification

using RNeasy Mini kit (Quiagen). RNA was transcribed into cDNA using Superscipt III

reverse transcriptase (Invitrogen) according to the manufacture’s protocol. Quantitative real

time PCR was performed using RT2 qPCR Primer sets and RT2 SYBR-Green master mix

(SABiosciences) on a PTC-200 thermocycler equipped with a Chromo4 detector (Bio-Rad

Laboratories). Data was analysed using Opticon Monitor software (Bio-Rad Laboratories).

4

CT values were normalized to the mean CT values obtained for the two house keeping genes

Hsp90 and Gapdh. All the primers used in qRT-PCR were purchased from SABiosciences;

Hsp90 (NM_008302), Gapdh (NM_008084), Reg3b (NM_011036) and Reg3g

(NM_011260).

5

1γttTA

3γ/γtpRorc(gt)RORγt-tTATG

LC-1 (TRE)7-pminhCMV CreLuc

X

X

R26R-YFP YfpSTOPpRosa26 YfppRosa26

DoxtTA

Cre

Triple transgenic mice

Fig. S1. Strategy for inducible genetic fate mapping.Rorc(γt)-tTATG mice express tTA under control of the Rorc(γt) locus on a BAC transgene,LC-1 transgenic mice are induced to express Cre by tTA, and R26R-YFP reporter miceexpress EYFP upon removal of a floxed Stop sequence by Cre. This activation cascade isblocked by doxycycline (dox).

6

RFP

NKp46+

LTi4

0 102 103 104 1050

10

20

30 40

0 102 103 104 1050

20

40

60

80

100

8

A

RORgt-tTATG

LC-1(TRE)7-pminhCMV CreLuc

X

X

R26R-tdRFPRfppRosa26

1γttTA

3γ/γtpRorc(gt)

X

RORgt-gfpTG

1γtgfp

3γ/γtpRorc(gt)

Rorc(γt)-EgfpTG

Triple transgenic mice

4 weeks old

RORγt (GFP)

RFP

RFP+ LTi4

0 102 103 104 105

0

102

103

104

105 0 100

00

RFP- LTi4

0 102 103 104 105

0

102

103

104

105 0 43

570

RORγt+ RFP-

OP9 stroma cell

+ IL-7 & SCF

RORγt+ RFP+ ILCs

or

B

Fig. S2. Distinct labelling efficiencies in fate mapped subsets of RORγt+ ILCs.(A) Cells were isolated from 4 weeks old Rorc(γt)-tTATG mice crossed to LC-1 and R26R-Redfluorescent protein (RFP) mice, as well as to the Rorc(γt)-EgfpTG reporter mice. Histograms showthe RFP labelling efficiencies in LTi4 cells and NKp46+ ILCs through the fate mapping cascade.Data shown are representative of three individual experiments. (B) To assess whether RFPlabelling is stochastic or determined by putative subsets within LTi4 cells, RFP labelled and non-labelled LTi4 cells were flow-sorted from tetra-transgenic mice and cultured on OP9 cells in thepresence of IL-7 and SCF, and analyzed after 6 days. The proportion of cells RFP+ cellsgenerated in vitro from RFP- LTi4 cells was 43% and similar to the proportion of RFP+ cellsamong LTi4 cells in vivo, indicating that labelling is stochastic. Given the 5x lower RFP labellingefficiency in NKp46+ ILCs as compared to LTi4 cells, it is therefore unlikely that NKp46+ ILCsderive from LTi4 cells or a putative subset of LTi4 cells endowed with low RFP labellingefficiency.

7

0 100 1000 10000 1x105

0

100

1000

10000

1x105

3.5

YFP+ CD3ε-

IL-7

Rα

c-kit

CD

3ε

YFP

E.15

0 100 1000 10000 1x105

0

100

1000

10000

1x105

911.4

c-kithiIL-7Rαhi

CD4

LTi4LTi0

0 100 1000 10000 1x1050

1

2

3

76

0 102 103 104 1050

0.5

1

1.5

2

2.5

1.6

NKp46

Fig. S3. Phenotype of fetal RORγt+ ILCsMononuclear cells isolated from the lamina propria of the small intestine (SI-LP cells) of E.15Rorc(γt)-tTATG mice were analyzed by first gating on YFP+CD3- cells and then for expressionof CD4 and NKp46 in c-kithi IL-7Rαhi cells. The numbers indicate percentages of the gatedcells. The data shown are representative of 5 individual fetuses.

8

0 102 103 104 105

0

102

103

104

105

8.5

SI-LP

0 102 103 104 105

0

102

103

104

105

0.2

0 102 103 104 105

0

102

103

104

105

0.1

0 102 103 104 105

0

102

103

104

105

0.04

mLN ingLN SpleenC

D3ε

RORγt (GFP)0 102 103 104 105

0

102

103

104

105

0.05

0 102 103 104 105

0

102

103

104

105

0.2

SI-IEL Liver

0 102 103 104 105

0

102

103

104

105

7.04e-3

Bone Marrow

Fig. S4. RORγt+ ILCs in adult organs.The frequency of RORγt+ ILCs was analyzed in 4 weeks old Rorc(γt)-EgfpTG mice: in small intestinal lamina propria (SI-LP), mesentericlymph node (mLN), inguinal lymph node (ingLN), spleen, liver, bonemarrow and small intestinal epithelium (SI-IE). The numbers indicatepercentages of gated cells. The data shown are representative of threeindividual mice.

9

Adult SI-LP RORγt+ ILCs

OP9 stroma cell

+ IL-7 & SCF

0 102 103 104 1050

1

2

3

4

5 81

0 102 103 104 1050

1

2

3 74

0 102 103 104 1050

0.5

1

1.5

2

2.5

4.0

0 102 103 104 1050

2

4

6 9.8

0 102 103 104 105

0

102

103

104

105 0 0.5

99.500 102 103 104 105

0

102

103

104

105 0.9 0

1.897.20 102 103 104 105

0

102

103

104

105 5.6 0

3.291.30 102 103 104 105

0

102

103

104

105 92 1.0

07.0

NKp46+LTi4 LTi0 c-kitL

c-kit

CD4

NK

p46

Fig. S5. Stable phenotype of adult RORγt+ ILCs.Small intestinal lamina propria leukocytes were isolated from 4 weeks old Rorc(γt)-EgfpTG

reporter mice. Each RORγt+ILC subset (LTi4, LTi0, ckitL and NKp46+) was sorted by flowcytometry and seeded on flat bottom 96 well plates coated with OP9 stroma cells in thepresence of IL-7 and SCF. After 6 days of culture, the phenotype of each subset was re-analyzed.

10

ControlAnti-CD4

NKp46+0

CD4+T CD8+T

**

5

10

15

20

25

ckitL0

2

4

6

8

10

LPL

Nbs

(x10

3 )

*

LTi0LTi4

d21

dox

d7d3

Anti-CD4 Ab

n.s n.s

n.s

n.s

Fig. S6. In vivo ablation of CD4+ cells affects only CD4+ ILCs.

Triple transgenic Rorc(γt)-tTATG x LC-1 x R26R-YFP mice weretreated with dox since day 3 after birth, as well as with depleting anti-CD4 (GK1.5) or control (Rat IgG) antibody until intestinal laminapropria lymphocytes (LPLs) were analyzed on day 21. Results areaverage of three independent experiments. Error bars, s.d.; *P < 0.05,**P < 0.005, n.s, statistically not significant, unpaired t-test.

11

RORγt+ Fetal Liver cell

OP9 or OP9DL1Stroma cell

+ IL-7 & SCF

OP9 OP9DL1

GFP+

Integr.α4β7+GFP+

Integr.α4β7-

OP9

100 101 102 103 104100

101

102

103

104

0.1 0.5

981.4100 101 102 103 104

100

101

102

103

104

0.04 0.5

6930CD

3ε

RORγt (GFP)100 101 102 103 104

100

101

102

103

104

0.08 0.1

4555

100 101 102 103 104100

101

102

103

104

0.18 0.01

6.693100 101 102 103 104

100

101

102

103

104

0.17 0.33

1387100 101 102 103 104

100

101

102

103

104

0.1 0.4

5049

NKp46

CD

11cRORγt+

CD3-

A

B

Integr.α4β7+IL-7Rα+GFP-

Fetal Liver cell

OP9 stroma cell

+ IL-7 & SCF + IL-2

NKp46

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 1050

20

40

60

80

100 GFP+ NK1.1-

GFP- NK1.1+

+ IL-2no

0 102 103 104 105

0

102

103

104

105 40 3.7

479.60 102 103 104 105

0

102

103

104

105 69 0.9

237.8

RORγt (GFP)

NK

1.1

Fig. S7. The progeny of fetal liver RORγt+ cells.(A) Fetal liver RORγt+ cells do not acquire CD3 or CD11c in vitro. Integrin α4β7+ or α4β7-

RORγt+ cells were sorted from E.14 fetal liver of Rorc(γt)-EgfpTG reporter mice and seededonto OP9 or OP9DL1 stroma cells in the presence of IL-7 and SCF. After 6 days of culture,expression of CD3ε and CD11c was assessed. (B) IL-2 favours the generation of conventionalNK cells but not of RORγt+ NKp46+ cells from integrin α4β7

+ fetal liver cells. Integrin α4β7+

RORγt- cells were sorted from E.14 fetal liver of Rorc(γt)-EgfpTG mice and cultured on OP9cells in the presence of IL-7, SCF and rIL-2 (10ng/ml). After 6 days of culture, NK1.1 andNKp46 expression on the progeny of the fetal liver cells was analyzed by FACS.

12

Dox [d3]Dox [d14]Dox [d28]

LTi0LTi4

% o

f YFP

+ ce

lls 100

0 14 28 42 56 70 84

50

10

t1/2=25.5±7.6d

0 14 28 42 56 70 84

t1/2=21.7±8d

c-kitL NKp46+

% o

f YFP

+ ce

lls 100

0 14 28 42 56 70 84

50

10

0 14 28 42 56 70 84Days after birthDays after birth

t1/2=22.3±7.9d t1/2=25.8±6.4d

d3 14 28 56 84

dox

dox

dox

A B

YFPRORγtCD3ε

d3 84dox

x200

Fig. S8. The half-life of RORγt+ ILCs(A) The t1/2 of the different subsets of small intestinal lamina propria RORγt+ ILCs wasdetermined by measuring the proportion of fate mapped YFP+ cells among total RORγt+ ILCs.Triple transgenic mice were treated with dox from day 3, 14 or 28 after birth and analyzed atdifferent time points thereafter. Percentages were calculated as the proportion of YFP+ cellsamong a particular subset of RORγt+ILCs, and normalized to the maximal proportion of YFP+

cells in that subset determined in triple transgenic mice not treated with dox. Each data pointrepresents the mean percentage plus standard deviation from at least 3 individual mice. (B) Thepresence of long-lived progeny of perinatal RORγt+ ILCs. Rorc(γt)-tTATG mice fed continuouslywith dox from day 3 after birth were examined at day 84 for the presence of YFP+ cells in thesmall intestinal lamina propria. White arrows indicate the presence of YFP+ cells in ILFs of theterminal ileum. YFP- RORγt+ ILCs are newly generated RORγt+ ILCs.

13

% o

f YFP

+ ce

lls

Dox [d3]Dox [d7]Dox [d28]

84d3 28 56

dox

dox

dox

7

mLN

-14 0 14 28 42 56 70 84

Days after birth

0

1

10

10050

Spleen

0

1

10

100

-14 0 14 28 42 56 70 84

50

Days after birth

t1/2=25.4±9.2d t1/2=24.8±14.3d

LTi4

Fig. S9. The half-life of CD4+ LTi cells in mLN and spleen.The t1/2 of CD4+ LTi cells in the mesenteric lymph node (mLN) and the spleen was determinedby measuring the proportion of fate mapped YFP+ cells among total CD4+ LTi cells. Tripletransgenic mice were treated with dox from E.15, day 3, 7 or 28 after birth and analyzed atdifferent time points thereafter. Percentages were calculated as the proportion of YFP+ cellsamong CD4+ LTi cells, and normalized to the maximal proportion of YFP+ cells in that subsetas determined in triple transgenic mice not treated with dox. Each data point represents themean percentage plus standard deviation from at least 3 individual mice.

14

0 102 103 104 105

0102

103

104

105

0.4

0 102 103 104 105

0102

103

104

105

4250

RORγt+ CD3ε-C

D3ε

RORγt (GFP)IL

-7Rα

c-kit

c-kithiIL-7Rαhi c-kitlowIL-7Rαlow

0 102 103 104 1050

1

2

3

4

5

23

0 102 103 104 1050

2

4

6

8

80

CD4 NKp46

6 weeks After BMT

Fig. S10. Bone marrow cells generate RORγt+ ILCs1x106 Bone marrow cells from Rorc(γt)-EgfpTG mice were purified witha lineage+ cell depletion kit (Miltenyi Biotec) and adoptively transferredinto irradiated C57BL/6 hosts. Donor-derived RORγt+ ILCs (GFP+) inthe small intestinal lamina propria were analyzed 6 weeks later. Data arerepresentative of three independent mice.

15

[E16]

[d3]

[d14][d28]

No dox

14 28 56E16 d3

dox

dox

YFP dox

dox

Age

dox

is s

tart

ed

NKp46+

LTi4

0

20

40

60

80

100

[E16] [d3] Nodox

[d3] [d14] [d28] Nodox

n.s

n.s

% in

YFP

+ CD

3ε-

E16 d3 14 56

n.s

n.s

0

20

40

60

[E16] [d3] Nodox

[d3] [d14] [d28] Nodox

Age of analysis

Age of analysis

Fig. S11. The relative proportions of RORγt+ ILC subsets is determined by mouse ageThe proportions of small intestinal lamina propria LTi4 and NKp46+ ILCs was determined inthe progeny of fetal (E16), perinatal (day 3), pre-weaned (d14), adult (day 28) or total RORγt+ ILCs in triple transgenic mice not treated with dox, and analyzed at E16, day 3, day 14 andday 56. It is concluded that the relative proportions of the RORγt+ ILC subsets is determinedby mouse age, and not by the age of the cells. Error bars, s.d.; n.s, statistically not significantby students’ t-test.

16

0 102 103 104 105

0

102

103

104

105

7.4

0 102 103 104 105

0

102

103

104

105

3057

0 102 103 104 1050

5

10

15

20

25

18

0 102 103 104 1050

30

60

90

120

85

0 102 103 104 1050

200

400

600

4.8

0 102 103 104 1050

50

100

150

200

250

36C

D3ε

CD4

Doxtreated

IL-7

Rα

c-kit

RORγt+CD3ε-

NKp46

Non treated

CD4+ T

Dox treated

RORγt (GFP)

RORγt (GFP)

c-kitlowIL-7Rαlow c-kithiIL-7Rαhi

0

10

20

30

40

50

60

LTi4 LTi0 ckitL NKp46+

% in

RO

Rγt

+ CD

3-

102

103

104

105

106

Total GFP+ Total LTi4 NKp46+

CD4+T RORγt+ILC

nonDox*

Nb

of L

PL

n.s

n.sn.s

n.s

* n.s

n.s

n.s

Fig. S12. Doxycyclin affects the generation of Th17 cells, but not of RORγt+ILCs.Oral treatment of Rorc(γt)-GfpTG mice with 1mg/ml doxycycline from birth until 6 weeks ofage effectively affects the bacterial symbiotic microbiota and thereby prevents the generationof Th17 cells (top panel), but has no impact on the subsets of RORγt+ ILCs (middle and lowerpanels) residing in the small intestinal lamina propria. Numbers of Th17 cells are indicated asGFP (RORγt)+ cells in CD4+ T cells. The data shown in dot plots and histograms arerepresentative or a compilation of results obtained in 4 individual mice. Error bars, s.d.; *P <0.05, n.s, statistically not significant, unpaired t-test.

17

0

10

20

30

40

50

LTi4 LTi0 ckitL NKp46+

n.s

n.s

n.s

n.s

% in

RO

Rγt

+ CD

3-

GF+SFB

102

103

104

105

106

Total GFP+ Total LTi4 LTi0 ckitL NKp46+

CD4+T RORγt+ILCs

** * n.s

n.sn.s

n.s

n.s

Nb

of L

PL+SFB

0 102 103 104 1050

10

20

30

40

2.7

0 102 103 104 1050

50

100

150

200

33

0 102 103 104 1050

10

20

30

40

50

26

+SFBGF SPF

CD4+T

RORγt (GFP)

NKp460 102 103 104 105

0

10

20

30

86

0 102 103 104 105

0

102

103

104

105

6.3

CD

3ε

0 102 103 104 105

0

102

103

104

105

6429

IL-7

Rα

c-kit

RORγt+CD3ε-

0 102 103 104 1050

10

20

30

40

22

CD4RORγt (GFP)

c-kitlowIL-7Rαlow c-kithiIL-7Rαhi

Fig. S13. Re-colonization of Germfree mice with Segmented Filamentous Bacteria (SFB) hasno impact on the development of RORγt+ILCs.Re-colonization of 6 weeks old germfree (GF) Rorc(γt)-GfpTG mice with SFB for 3 weekseffectively induces the generation of Th17 cells (top and lower panels), but has no effect on thesubsets of RORγt+ ILCs (middle and lower panels) residing in the small intestinal lamina propria.The data shown in dot plots and histograms are representative or a compilation of results obtained in3 individual mice. Error bars, s.d.; *P < 0.05, **P < 0.005, n.s, statistically not significant, unpairedt-test.

18

0

10

20

30

40

50

60

70

80

LTi4 LTi0 ckitL NKp46+

Nod2 HetNod2KO

% in

RO

Rgt

+ CD

3-

n.s

n.sn.s

n.s

0

10

20

30

40

50

LTi4 LTi0 ckitL NKp46+

WTNod1KOMyd88KO%

in R

OR

gt+ C

D3-

n.s

n.s

n.s

n.s

*

Fig. S14 . Nod1, Nod2 and Myd88 are not involved in the generation of RORγt+ILCs.Small intestinal lamina propria leukocytes were prepared from 3 or 6 months old deficientmice and age-matched controls. The RORγt-expressing cells were identified byintracellular RORγt staining. The data shown in the histograms are compilation of resultsobtained in 3 individual mice. Error bars, s.d.; n.s, statistically not significant, unpaired t-test.

19

WTRAG2KO

% in

RO

Rγt

+ CD

3ε−

0

10

20

30

40

50

LTi4 LTi0 c-kitL NKp46+

n.s

n.sn.s

*

RORγt (GFP)0 100 1000 10000 1x105

0

10

20

30 16

CD4

0 100 1000 10000 1x1050

20

40

60

70

NKp46

GFP+CD3ε-

0 100 1000 10000 1x105

0

100

1000

10000

1x105

51

CD

3ε

0 100 1000 10000 1x105

0

100

1000

10000

1x105

3756

c-kit

IL-7

Rα

RORγt+CD3ε- c-kithiIL-7Rαhi c-kitlowIL-7Rαlow

Fig. S15. RORγt+ ILCs in RAG2-deficient mice.Small intestinal lamina propria RORγt+ ILCs cells from 6 weeks old RAG2-deficient micewere analyzed by FACS. The numbers indicate percentages of gated cells. The data shownin dot plots and histograms are representative or a compilation of results obtained in 5individual mice. Error bars, s.d.; *P < 0.05, n.s, statistically not significant, unpaired t-test.

20

0 102 103 104 1050

1

2

3

78

0 102 103 104 1050

1

2

3

4

45

0 102 103 104 1050

2

4

634

0 102 103 104 1050

2

4

6

8 1.4

0 102 103 104 1050

1

2

3

4 89

0 102 103 104 1050

1

2

3

53

0 102 103 104 1050

3

6

9

12 25

0 102 103 104 1050

2

4

6

8

2.2

LTi4 LTi0 c-kitL NKp46+

CCR6

SPF

GF

Fig. S16. CCR6 expression by RORγt+ILCs and development in CCR6o mice(A) CCR6 expression by RORγt+ILCs is independent of bacterial colonization.Numbers indicate percent of CCR6+ cells in RORγt+ILCs isolated from 8 weeksold SPF and germ free mice. Data are representative of results obtained in threeindividual mice. (B) Small intestinal lamina propria RORγt+ ILCs cells from 6weeks old CCR6-deficient mice were analyzed by FACS. Data are a compilationof results obtained in 5 individual mice. Error bars, s.d.; *P < 0.05, n.s, statisticallynot significant, unpaired t-test

WTCCR6KO

10

20

30

40

50

LTi4 LTi0 ckitL NKp46+0

% in

GFP

+ CD

3-

n.s

n.s

n.s

n.s

A

B

21

SUPPORTING REFERENCES

S1. T. Sparwasser, S. Gong, J. Y. Li, G. Eberl, Genesis 38, 39 (2004).

S2. K. Schonig, F. Schwenk, K. Rajewsky, H. Bujard, Nucleic Acids Res 30, e134 (2002).

S3. S. Srinivas et al., BMC Dev Biol 1, 4 (2001).

S4. H. Luche, O. Weber, T. Nageswara Rao, C. Blum, H. J. Fehling, Eur J Immunol 37, 43

(2007).

S5. M. Lochner et al., J Exp Med 205, 1381 (2008).

S6. R. Varona et al., J Clin Invest 107, 37 (2001).

S7. J. H. Fritz et al., Immunity 26, 445 (2007).

S8. V. Gaboriau-Routhiau et al., Immunity 31, 677 (2009).

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