Supplementary Figures 09-11-17
A) Ear 21.2
ILC2s 23.7
98.1
Lineage
B)
iCD3!
15.0% 26.0% 4.1% 46.5% 8.5%
ILC1s ILC2s ILC3s TrN Ungated
Total=4617.94
ILC3s 6.72
ROR"t
GATA-3
GATA-3
IL-7R⍺
IL-7R⍺
ILC1s 19.5 TrN 39.5
T-bet
Lineage Gate: B220, CD11b, CD11c, CD5, CD3, CD19, Ter119, CD123, Gr1, F4/80, Fc!R1 and CD49b
Supplementary Fig1 Supplementary Fig1
G)
CD3!i
Lineage
60
Ear auLN
IL-7R⍺
IL-7R⍺
Id2+ 1.7
CD3!i
Lineage
Id2-GFP
C) auLN Id2-GFP
IL-7R⍺
IL-7R⍺
IL-7R⍺
Id2+ 89.7
CD3!i
Lineage
Id2-GFP
Text
Id2+ 1.6
IL-7R⍺
IL-7R⍺
WT
IL-7R⍺
D)
80
40
Id2-GFP
WT
IL-7R⍺
B)
IL-7R⍺
IL-7R⍺
IL-7R⍺
Id2+ 89.9
100
% of Id2GFP+ ILCs
A) Ear Id2-GFP
CD3!i
Lineage
Id2-GFP
E) NK control Id2-GFP
CD45
NK1.1
IL-7R⍺
Id2+ 92.5
NKp46
CD3!
Id2-GFP
F)
Id2+ 2.6
CD45
NK1.1
IL-7R⍺
WT
CD3!
NKp46
Id2-GFP
Supplementary Fig2 Supplementary Fig2
A)
Ear – Ear Digestion Protocol (EDP)
CD45
Lineage
iCD3"
ILC3s 3.2
GATA-3
GATA-3
9.5
v IL-7R⍺
IL-7R⍺
Live/Dead
9.5
ILC2s 7.1 TrN 60.1 ILC1s 17.3
T-bet
ROR#t
B) Lung – Lung Digestion Protocol (LDP) ILC2s 44.0
CD45
C)
Lineage
iCD3"
ILC3s 3.8
TrN 34.0
GATA-3
GATA-3
IL-7R⍺
58.6
IL-7R⍺
Live/Dead
1.1
ILC1s 9.6
T-bet
ROR#t
Lung – Ear Digestion Protocol (EDP) ILC2s 20.5
CD45
Lineage
Lung
ILC3s 3.9
ILC1s 1.7
T-bet
ROR#t
Lung
TrN 66.5
GATA-3
GATA-3 iCD3"
Lung
Lung
Lung
Number of ILCs
% of ILCs
E) Number of LiveCD45+ cells
D) % of LiveCD45+ cells
IL-7R⍺
76.0
IL-7R⍺
Live/Dead
1.4
Lung
Number of ILCs
% of ILCs
F)
Supplementary Fig3
Previously Gated: Live CD45+ cells Ear -EDP
A) 61.7
Lung -LDP 28.7
Lung -EDP
Lung
B) 50
25.3
IL-7R⍺
% CD3+
40 30 20 10 0
CD3! 32.4
27.6
IL-7R⍺
0.8
IL-7R⍺
LDP EDP
3 2 1 0
11.2
50
9.5
LDP EDP
40
% F4/80+
IL-7R⍺
10
4
% CD49b+
0.7
9.5
30 20 10 0
F4/80 32.5
20
5
CD49b 18.8
30
0
CD5 21.7
LDP EDP
40
% CD5+
35.2
50
4.5
30
3.8
LDP EDP
IL-7R⍺
% Fc!RI+
20
Fc!RI
10
0 LDP EDP
Supplementary Fig4
Previously Gated: Live CD45+ cells Ear -EDP
A)
Lung -EDP
1.8
1.5
13.9
4.6
1.4
0.4
11.1
0.3
9.1
0.02
Lung
B)
IL-7R⍺
% ROR!t+
35.7
Lung -LDP
ROR!t
IL-7R⍺
% Tbet+
18.8
Tbet
IL-7R⍺
% GATA3+
38.1
GATA-3
IL-7R⍺
% CD4+
0.6
CD4
IL-7R⍺
% CD8+
0.06
CD8
Supplementary Fig5
Previously Gated: Live CD45+ cells 0.6
Lung -LDP 1.9
Lung -EDP
Lung
0.1
1.9
IL-7R⍺
B)
% CD25+
Ear -EDP
A)
CD25 2.0
5.1
13.9
9.1
43.1
45.2
1.0
1.9
IL-7R⍺
% ICOS+
13.6
IL-7R⍺
% KLRG1+
ICOS Ki67 21.8
KLRG1
IL-7R⍺
% MCHII+
80.1
MHCII
IL-7R⍺
% ST2+
5.0
ST2
Supplementary Fig6
A
WT Ear
WT SI
9.5 2.4
WT Lung
WT mLN
WT SI Lineage+ IL-7R⍺-
69.0
96.4
83.9
B
CD25
KLRG-1
IL-7R⍺ IL-7R⍺ MHCII
99.0
IL-7R⍺ ST2
KLRG-1
35.4
MHCII
26.2
IL-7R⍺ ST2
IL-7R⍺
IL-7R⍺ IL-7R⍺
IL-7R⍺
IL-7R⍺ IL-7R⍺
19.4
KLRG-1
0.5
MHCII
ICOS
70.0
KLRG-1
3.9
MHCII
ICOS
81.1
KLRG-1
3.9 9.5
70.3
ICOS
95.0
IL-7R⍺
9.5 8.7
IL-7R⍺
IL-7R⍺ ICOS
CD25
IL-7R⍺
ICOS
CD25
97.3
99.3
IL-7R⍺
IL-7R⍺
71.8 9.5
CD25
MHCII
80.1
IL-7R⍺
CD25
IL-7R⍺
IL-7R⍺
IL-7R⍺
GATA-3+ ILC2s
ST2
ST2
ST2
Supplementary Fig7
WT
60
**
40 20 0
ICOS
WT
ICOS
ILC1
9,378 7,664
104
103
WT
ICOS
% of ILC subsets
mLN
WT
ICOS
ILC2
WT
ICOS
WT
ILC3
80
105
C)
*
80
ICOS
105
***
202,975 172,408 102,446 25,893 70,660 24,285 9.411 10,202
104 103 102 WT
ICOS
ILC1
104
*
40
106
TrN
***
60
Number of ILC subsets
105
B)
Number of ILC subsets
145,012 127,878
% of ILC subsets
106
Number of ILCs
SI LP
Number of ILCs
A)
WT
ICOS
ILC2
WT
ICOS
WT
ILC3
ICOS TrN
*** 4,790 1,863 2,403
1,454
2,457 1,767
923 611
103
20
102
0 WT
ICOS
ILC1
WT
ICOS
ILC2
WT
ICOS
ILC3
WT
ICOS TrN
WT
ICOS
ILC1
WT
ICOS
ILC2
WT
ICOS
ILC3
WT
ICOS TrN
Supplementary Fig8 Supplementary Fig5
ILC2s 23.7
98.1
Lineage
B)
iCD3!
15.0% 26.0% 4.1% 46.5% 8.5%
ILC1s ILC2s ILC3s TrN Ungated
Total=4617.94
ILC3s 6.72
ROR"t
GATA-3
GATA-3
IL-7R⍺
IL-7R⍺
ILC1s 19.5 TrN 39.5
T-bet
Lineage Gate: B220, CD11b, CD11c, CD5, CD3, CD19, Ter119, CD123, Gr1, F4/80, Fc!R1 and CD49b
Supplementary Fig1 Supplementary Fig1
G)
CD3!i
Lineage
60
Ear auLN
IL-7R⍺
IL-7R⍺
Id2+ 1.7
CD3!i
Lineage
Id2-GFP
C) auLN Id2-GFP
IL-7R⍺
IL-7R⍺
IL-7R⍺
Id2+ 89.7
CD3!i
Lineage
Id2-GFP
Text
Id2+ 1.6
IL-7R⍺
IL-7R⍺
WT
IL-7R⍺
D)
80
40
Id2-GFP
WT
IL-7R⍺
B)
IL-7R⍺
IL-7R⍺
IL-7R⍺
Id2+ 89.9
100
% of Id2GFP+ ILCs
A) Ear Id2-GFP
CD3!i
Lineage
Id2-GFP
E) NK control Id2-GFP
CD45
NK1.1
IL-7R⍺
Id2+ 92.5
NKp46
CD3!
Id2-GFP
F)
Id2+ 2.6
CD45
NK1.1
IL-7R⍺
WT
CD3!
NKp46
Id2-GFP
Supplementary Fig2 Supplementary Fig2
A)
Ear – Ear Digestion Protocol (EDP)
CD45
Lineage
iCD3"
ILC3s 3.2
GATA-3
GATA-3
9.5
v IL-7R⍺
IL-7R⍺
Live/Dead
9.5
ILC2s 7.1 TrN 60.1 ILC1s 17.3
T-bet
ROR#t
B) Lung – Lung Digestion Protocol (LDP) ILC2s 44.0
CD45
C)
Lineage
iCD3"
ILC3s 3.8
TrN 34.0
GATA-3
GATA-3
IL-7R⍺
58.6
IL-7R⍺
Live/Dead
1.1
ILC1s 9.6
T-bet
ROR#t
Lung – Ear Digestion Protocol (EDP) ILC2s 20.5
CD45
Lineage
Lung
ILC3s 3.9
ILC1s 1.7
T-bet
ROR#t
Lung
TrN 66.5
GATA-3
GATA-3 iCD3"
Lung
Lung
Lung
Number of ILCs
% of ILCs
E) Number of LiveCD45+ cells
D) % of LiveCD45+ cells
IL-7R⍺
76.0
IL-7R⍺
Live/Dead
1.4
Lung
Number of ILCs
% of ILCs
F)
Supplementary Fig3
Previously Gated: Live CD45+ cells Ear -EDP
A) 61.7
Lung -LDP 28.7
Lung -EDP
Lung
B) 50
25.3
IL-7R⍺
% CD3+
40 30 20 10 0
CD3! 32.4
27.6
IL-7R⍺
0.8
IL-7R⍺
LDP EDP
3 2 1 0
11.2
50
9.5
LDP EDP
40
% F4/80+
IL-7R⍺
10
4
% CD49b+
0.7
9.5
30 20 10 0
F4/80 32.5
20
5
CD49b 18.8
30
0
CD5 21.7
LDP EDP
40
% CD5+
35.2
50
4.5
30
3.8
LDP EDP
IL-7R⍺
% Fc!RI+
20
Fc!RI
10
0 LDP EDP
Supplementary Fig4
Previously Gated: Live CD45+ cells Ear -EDP
A)
Lung -EDP
1.8
1.5
13.9
4.6
1.4
0.4
11.1
0.3
9.1
0.02
Lung
B)
IL-7R⍺
% ROR!t+
35.7
Lung -LDP
ROR!t
IL-7R⍺
% Tbet+
18.8
Tbet
IL-7R⍺
% GATA3+
38.1
GATA-3
IL-7R⍺
% CD4+
0.6
CD4
IL-7R⍺
% CD8+
0.06
CD8
Supplementary Fig5
Previously Gated: Live CD45+ cells 0.6
Lung -LDP 1.9
Lung -EDP
Lung
0.1
1.9
IL-7R⍺
B)
% CD25+
Ear -EDP
A)
CD25 2.0
5.1
13.9
9.1
43.1
45.2
1.0
1.9
IL-7R⍺
% ICOS+
13.6
IL-7R⍺
% KLRG1+
ICOS Ki67 21.8
KLRG1
IL-7R⍺
% MCHII+
80.1
MHCII
IL-7R⍺
% ST2+
5.0
ST2
Supplementary Fig6
A
WT Ear
WT SI
9.5 2.4
WT Lung
WT mLN
WT SI Lineage+ IL-7R⍺-
69.0
96.4
83.9
B
CD25
KLRG-1
IL-7R⍺ IL-7R⍺ MHCII
99.0
IL-7R⍺ ST2
KLRG-1
35.4
MHCII
26.2
IL-7R⍺ ST2
IL-7R⍺
IL-7R⍺ IL-7R⍺
IL-7R⍺
IL-7R⍺ IL-7R⍺
19.4
KLRG-1
0.5
MHCII
ICOS
70.0
KLRG-1
3.9
MHCII
ICOS
81.1
KLRG-1
3.9 9.5
70.3
ICOS
95.0
IL-7R⍺
9.5 8.7
IL-7R⍺
IL-7R⍺ ICOS
CD25
IL-7R⍺
ICOS
CD25
97.3
99.3
IL-7R⍺
IL-7R⍺
71.8 9.5
CD25
MHCII
80.1
IL-7R⍺
CD25
IL-7R⍺
IL-7R⍺
IL-7R⍺
GATA-3+ ILC2s
ST2
ST2
ST2
Supplementary Fig7
WT
60
**
40 20 0
ICOS
WT
ICOS
ILC1
9,378 7,664
104
103
WT
ICOS
% of ILC subsets
mLN
WT
ICOS
ILC2
WT
ICOS
WT
ILC3
80
105
C)
*
80
ICOS
105
***
202,975 172,408 102,446 25,893 70,660 24,285 9.411 10,202
104 103 102 WT
ICOS
ILC1
104
*
40
106
TrN
***
60
Number of ILC subsets
105
B)
Number of ILC subsets
145,012 127,878
% of ILC subsets
106
Number of ILCs
SI LP
Number of ILCs
A)
WT
ICOS
ILC2
WT
ICOS
WT
ILC3
ICOS TrN
*** 4,790 1,863 2,403
1,454
2,457 1,767
923 611
103
20
102
0 WT
ICOS
ILC1
WT
ICOS
ILC2
WT
ICOS
ILC3
WT
ICOS TrN
WT
ICOS
ILC1
WT
ICOS
ILC2
WT
ICOS
ILC3
WT
ICOS TrN
Supplementary Fig8 Supplementary Fig5
