SUPPLEMENTARY INFORMATION
RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating
negative signals from the symbiotic microbiota
Shinichiro Sawa1,2, Matthias Lochner1,2,3, Naoko Satoh-Takayama4,5, Sophie Dulauroy1,2,
Marion Bérard 6, Melanie Kleinschek7, Daniel Cua7, James P. Di Santo4,5 and Gérard Eberl1,2
1
Institut Pasteur, Lymphoid Tissue Development Unit, 75724 Paris, France
2
CNRS, URA1961, 75724 Paris, France
3
Institute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical
Infection Research; a joint venture between the Medical School Hannover (MHH) and the
Helmholtz Centre for Infection Research (HZI), Hannover, Germany
4
Institut Pasteur, Innate Immunity Unit, 75724 Paris, France
5
Inserm, U668, 75724 Paris, France
6
Institut Pasteur, Animalerie Centrale, 25 rue du Dr Roux, 75724 Paris, France
7
Merck Research Laboratories, DNAX Discovery Research, 94304 Palo Alto, USA
1
Nature Immunology doi:10.1038/ni.2002
a
8 weeks
SI-LPL
SI-IEL
mLN
Spleen
105
104
2.6
CD3ε
103
8.0e-3
0.12
0.03
102
0
0
102
103
104
105
RORγt (GFP)
b
% of Max events
B220+
30
CD11c+
CD11b+
0
0
0
20
10
0
0 102
103
104
105
RORγt (GFP)
Supplementary Figure 1
ROR t+ ILCs in IELs and secondary lymphoid tissues. (a ) ROR t+ ILCs in the SI-LPL, intraepithelial
lymphocytes (IEL), mesenteric lymph nodes (mLN) and spleen of 4 weeks old Rorc( t)-GfpTG mice. Data are
representative of n = 4 mice. (b) ROR t (GFP) expressions in SI-LPL B220+, CD11c+ or CD11b+ cells. Numbers
indicate percent positive cells. Data are representative of n = 3 mice.
2
Nature Immunology doi:10.1038/ni.2002
a
0.8
22
2.9
0.2
53
25
76
21
2.8
12
3.5
15
2 weeks
105
105
4
4
10
3
103
10
8 weeks
102
0
67
0
19
102
103
104
CD3ε
IL-22
10
102
0
69
0
105
12
102
103
104
105
CD4
RORγt
(GFP)
b
LTi4
10
26
25
39
24
15 22
E.16
fetus
105
1
RORγt+ CD4+ T
1
10
RORγt+ γδ+ T
1
Isotype
3
105
104
104
103
103
102
0
41
0
20 76
102
103
104
1
35
54
57
39
RatIgG1
IL-22
4 weeks
NKp46+
0
0
102
0
99
0
105
IL-17A
0.8
102
103
104
105
RatIgG1
Trance expression (relative)
c
102
*
Supplementary Figure 2
The production of pro-inflammatory cytokines by R OR t+
ILCs. (a) Small intestinal lamina propria leukocytes (SILPL) from 2 weeks and 8 weeks old Rorc( t)-GfpTG mice
were analyzed by flow cytometry. The production of IL-22
1
was assessed by intracellular cytokine staining after 3 hours
of stimulation with PMA/ionomycine e x vivo, and IL-22+
ROR t+ cells were further analyzed for the expression of
E15 2 weeks
8 weeks
CD3 and CD4. Numbers indicate percent cells per
quadrant. The data are representative of three independent
experiments.
(b) ROR t+ ILCs from E16 mice, and ROR t+ ILCs, ROR t+ CD4+ T cells and ROR t+ T cells from 4 weeks
old mice were analyzed for intracellular expression of IL-17 and IL-22 after 3 hours stimulation ex vivo with
PMA/ionomycin. Data are representative of 3 independent experiments. (c) SI-LPL from E15, 2 w eeks and 8
weeks old Rorc( t)-GfpTG mice were analyzed by real-time qPCR for the expression of transcripts for TRANCE.
Data are mean of three independent experiments. *P < 0.05, unpaired t-test.
LTi4
NKp46+
LTi4
NKp46+
LTi4
10
3
Nature Immunology doi:10.1038/ni.2002
a
CD3- RORγt+
105
105
104
104
CD4- NKp46105
104
CD3ε
102
103
102
0
0
102
103
104
105
ckitL
102
0
RORγt
(GFP)
b
103
IL-7Rα
21
103
NKp46
2 weeks
0
102
103
104
0
105
0
CD4
*
LTi0
c-kitL
LTi0
1
8 weeks
8 weeks
% of Max events
10
2 weeks
100
105
ckitL
63±3.7
43±4.4
28±2.7
22±2.3
60
40
20
0
0 102
103
104
105
LTi0
ckitL
102
33±4.2
16±2.8
4.3±1.1
10±1.8
2 weeks
0 102
8 weeks
Ifng expression (relative)
102
*
10
104
105
102
10
ND
ND ND
1
LTi0
c-kitL
LTi0
LTi0
c-kitL
1
2 weeks
103
IL-17A
8 weeks
2 weeks
Supplementary Figure 3
LTi0
2 weeks
c-kitL
LTi0
c-kitL
LTi0
8 weeks
c-kitL
ND ND
1
% of Max events
10
c-kitL
Il17a expression (relative)
104
80
IL-22
Lta expression (relative)
103
2 weeks
102
c-kitL
Il22 expression (relative)
*
102
c-kit
LTi0
103
30 LTi
0
53
66
8 weeks
The production of pro -inflammatory cytokines by CD4- NKp46- ROR t+ ILCs. (a) SI-LPL from 2 weeks old
Rorc( t)-GfpTG mice were analyzed by flow cytometry. CD4-NKp46- ROR t+ILCs were subdivided into c -kithi
(LTi0) and c-kitlo (c-kitL) cells. Data are representative of at least n = 8 mice. (b) Expression of transcripts and
proteins for IL-22, IL-17, LT and IFN by LTi0 and c-kitL cells from 2 weeks and 8 weeks old Rorc( t)-GfpTG
mice. Intracellular IL-22 and IL-17 protein expression was measured after 3 hours stimulation ex vivo with rIL23 or PMA/ionomycin, respectively. The data are representative of five independent experiments. In histograms,
numbers are mean of n = 10 mice ± S.E.M percentages of IL-22+ or IL-17+ cells. *P < 0.05, unpaired t-test, ND,
non-detected.
4
Nature Immunology doi:10.1038/ni.2002
b
c
NS
NS
NS
25
20
103
15
102
10
5
SPF GF
PBS IL-25
i.p
WT IL-25
KO
PBS IL-25
i.p
d
LTi4
1
0
1
10
LTi4
NKp46+
10
NS
104
Il23r expression (relative)
NS
*
IL-23 (pg/ml)
Il23 expression (relative)
102
NKp46+
a
+PBS
+IL-25
e
PMA+ Iono
rIL-23
15±4.2
PMA+ Iono
23±7.7
19±4.6
PBS
WT
3.0
13±8.5
16±3.9
21±6.1
Anti-IL-23R
p19-KO
p19-KO
+IL-25
104
CD3ε
103
2.3
102
0
100
100
4.4±2.5
3.0±1.2
80
Anti-IL-23R
+IL-25
60
40
20
0
0 102
103
104
105
RORγt
0 102
103
104
105
% of Max events
105
% of Max events
2.3
4.4±0.8
80
60
40
20
0
0
IL-22
102
103
104
105
IL-22
Supplementary Figure 4
No role for IL -23 in th e IL-25-mediated regulation of IL-22 production by R OR t+ ILCs. (a) Expression of
transcripts for IL-23p19 (Il23a) in the terminal ileum of 8 weeks-old SPF, GF, wild type and IL-25-deficient
mice, and 6 weeks-old wild-type mice injected with rIL-25 or PBS. (b) IL-23a protein expression in the terminal
ileum of wild-type mice injected with rIL-25 or PBS. (c) IL-23 receptor (Il23r) expression by ROR t+ ILCs from
6 weeks-old RAG2-deficient mice treated with rIL-25 or PBS. *P < 0.05, NS, statistically not significant,
unpaired t-test. (d) Expression of IL-22 by ROR t+ ILCs in the small intestine of adult p19-deficient mice treated
with rIL-25 or PBS. Cells were stimulated for 3 hours ex vivo with PMA/ionomycin (middle columns) or rIL-23
(right columns). Data are mean ± S.E.M of n = 3 mice per group. (d) Expression of IL-22 by ROR t+ ILCs in 8
weeks old Rorc( t)-GfpTG mice treated with neutralizing anti-IL23R antibody and rIL -25 or P BS. Cells were
stimulated for 3 hours ex vivo with PMA/ionomycin. Data are mean ± S.E.M of n = 3 mice per g r o u p .
5
Nature Immunology doi:10.1038/ni.2002
105
NS
*
*
RORγt+
CD4+ T cells
% of Max events
DSS
DSS+IL-25
100
5.6±1.6
80
2.0±0.8
Cells
104
PBS
*
IL-25
103
60
40
20
0
0
102
103
104
105
102
Total
IL-22
IL-22+
RORγt+ CD4+ T
Supplementary Figure 5
Total
IL-22+
RORγt+ ILCs
IL-25 represses IL-22 production by ROR t+ T cells during colitis. (Left) Expression of IL-22 by small intestinal
lamina propria CD4+ ROR t+ T cells in 9 weeks old C57/BL6 wild type mice treated with DSS. Data are mean ±
S.E.M of n = 3 mice per group. Cells were stimulated for 3 hours ex vivo with PMA/ionomycin. (Right)
Absolute numbers of small intestinal lamina propria cells in DSS-treated 9 weeks old C57/BL6 wild type mice.
Data are mean ± S.E.M of n = 3 mice per group. *P < 0.05, unpaired t-test.
6
Nature Immunology doi:10.1038/ni.2002