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12
Figure legends
Fig. 1. Lymphoid cell-specific inhibition of RAR activity depletes ILC1s but not NK cells.
(A–G) Flow cytometric (FCM) analysis of liver and mLN lymphocytes in Rosa26RARa403/+ mice, Il7r-CreWT mice, or Il7r-CreRARa403 mice. Representative FCM profiles in
the liver (A) and mLNs (B), the percentages (upper) and the cell numbers (lower) of T cells
(C), B cells (D), NK cells (E), ILC1s (F), and NKT cells (G) in indicated tissues are shown.
Data represent two to three independent experiments (n = 4–6). Data are presented as mean ±
SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.
Fig. 2. RA signaling is required for development of ILCPs.
(A–D) FCM analysis of CLPs, ILCPs, ILC3s, and ILC2s in Il7r-CreWT (control) or Il7rCreRARa403 mice. Representative FCM profiles in BM (A) and the percentages (upper) and the
cell numbers (lower) of CLPs and ILCPs (B), ILC3s (C), and ILC2s (D) in indicated tissues
are shown. Data represent two to three independent experiments (n = 3–6). Data are presented
as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.
Fig. 3. Cell-intrinsic RAR activity is required for maintenance of peripheral ILC1s.
(A–D) FCM analysis of liver and mLN lymphocytes in Ncr1-CreWT or Ncr1-CreRARa403 mice.
Representative FCM profiles in the liver (A), the percentages of T cells and NKT cells (B),
and the percentages (upper) and the cell numbers (lower) of NK cells (C) and ILC1s (D) in
the liver and mLNs are shown. Data represent two to three independent experiments (n = 3–5).
Data are presented as mean ± SEM. *p < 0.05.
Fig. 4. RA signaling supports the proliferative and functional statuses in ILC1s.
(A) Number of genes significantly upregulated (blue; up-DEGs) and downregulated (red;
down-DEGs) in each cell population of Ncr1-CreRARa403 mice compared to Ncr1-CreWT mice
are shown. (B and C) Dot plots showing the enriched pathways on down-DEGs of liver (B)
and spleen (C) ILC1s. Genes count indicates the number of DEGs included in the pathway.
Gene ratio is the ratio of genes count to the total gene number in the pathway. (D) Heatmap
representing normalized expression levels of the genes related to cell cycle in liver ILC1s
from Ncr1-CreWT (control) or Ncr1-CreRARa403 mice followed by range scaling. (E)
Normalized read counts of Mki67 (padj = 0.254) expressed in liver ILC1s from control or
Ncr1-CreRARa403 mice. (F–H) FCM analysis of proliferation and survival marker expression in
liver NK cells and ILC1s of Ncr1-CreWT or Ncr1-CreRARa403 mice. The percentages of Ki-67+
cells (F), MFI levels of Bcl-2 (G), and the percentages of Annexin V+ cells (H) are shown. (I)
Normalized read counts of Ccl3 (padj = 0.013), Ccl4 (padj = 0.203), and Xcl1 (padj = 0.078)
expressed on liver ILC1s from control or Ncr1-CreRARa403 mice. (J) Venn diagram showing
the overlap between down-DEGs of liver and spleen ILC1s. Data represent two independent
experiments (F–H; n = 4–5) or are from RNA-seq experiments with three biological replicates
(A–E, I, and J). Data are presented as mean ± SEM. *p < 0.05.
13
Supplementary figure legends
Supplementary Figure S1. Transcriptome analysis of G1-ILCs in Ncr1-CreRARa403 mice
(A) IL-7R expression of liver (upper) and spleen (lower) CD49a+CD49b− G1-ILCs in Ncr1CreWT or Ncr1-CreRARa403 mice. (B) Volcano plots showing gene expression of each G1-ILC
population in Ncr1-CreWT (control) mice relative to that in Ncr1-CreRARa403 mice. Genes
significantly downregulated (red; down-DEGs) or upregulated (blue; up-DEGs) in each cell
population of Ncr1-CreRARa403 mice are highlighted. padj, adjusted p value. FC, fold change.
Data represent three (liver) and one (spleen) experiments (A) or are from RNA-seq
experiments with three biological replicates (B).
14
Figure 1
Liver PI–
live cells
R26RAR 403
NKT
CD49a
NK1.1
7.25
NKp46
97.1
22.2
ILC1
mLN CD3–
NK1.1+NKp46+
G1-ILC
NK
4.35
97.3
5.32
97.3
CD49a
25.4
Il7r-Cre WT
403
CD3
20
20
n.s.
15
****
0.04
**
0.02
0.5
0.01
**
0.8
0.6
0.4
0.2
10
0.0
n.s.
n.s.
mLN
0.4
n.s.
0.3
0.2
0.1
0.0
15
n.s.
n.s.
10
NKT
20
**
15
mLN
0.3
60
40
Il7r-Cre RAR
0.1
80
Il7r-Cre WT
0.2
10
0.0
NK
Liver
0.00
0.0
20
mLN
0.03
1.0
30
CD49b
Liver
n.s.
ILC1
Liver
Frequency (%)
n.s.
10
1.5
40
Frequency (%)
50
Cell number (×10⁴)
n.s.
40
mLN
Frequency (%)
80
60
40
Cell number (×10⁶)
mLN
Cell number (×10⁴)
Frequency (%)
Liver
CD49b
Cell number (×10⁴)
n.s.
CD3
Cell number (×106)
60
0.67
0.69
Frequency (%)
Il7r-Cre RAR
8.45
20
n.s.
403
Figure 2
0.33
4 7
CD25
IL-7R
BM PI–
live cells
ILC2 18.0
ILCP 11.2
CLP
55.1
Il7r-Cre WT
24.0
0.37
Il7r-Cre RAR
8.91
2.71
53.3
403
33.5
Lin
BM
0.4
0.2
0.010
0.3
0.2
0.005
0.1
0.000
0.0
Cell number (×10⁴)
0.015
p = 0.091
0.25
p = 0.085
0.15
0.10
0.05
0.00
Il7r-Cre WT
0.1
Il7r-Cre RAR
403
BM
mLN
ILC2
ILC2
****
0.06
0.15
**
0.10
0.04
0.05
0.02
0.00
0.20
0.08
n.s.
Frequency (%)
p = 0.064
ILC3
Cell number (×10⁴)
Frequency (%)
0.5
ILCP
Frequency (%)
CLP
mLN
PD-1
Cell number (×10⁴)
FLT3
1.5
0.00
**
1.0
0.5
0.0
Figure 3
Liver PI–
live cells
Ncr1-Cre WT
93.6
Liver
21.0
Frequency (%)
2.56
CD49a
4.60
21.7
3.14
Ncr1-Cre RAR
NKp46
NK1.1
7.73
93.5
40
65
30
60
20
55
10
50
45
NKT
3.16
403
mLN
Liver
mLN
CD3
n.s.
0.25
0.15
0.10
0.05
0.00
15
10
n.s.
Liver
n.s.
0.20
n.s.
CD49b
ILC1
mLN
Frequency (%)
Frequency (%)
Liver
Cell number (×10⁴)
NK
Cell number (×10⁴)
CD3
1.0
mLN
0.05
0.8
0.04
0.6
0.03
0.4
0.02
0.2
0.01
0.0
0.00
0.8
0.6
0.4
0.2
0.0
Frequency (%)
19.3
25
20
15
10
n.s.
0.6
0.4
0.2
0.0
n.s.
Ncr1-Cre WT
Ncr1-Cre RAR
n.s.
403
Figure 4
Liver NK
30
60
20
40
10
20
Ncr1-Cre WT
Ncr1-Cre RAR
n.s.
n.s.
MFI (×103)
NK
n.s.
40
20
20
cytokine-mediated
signaling pathway
2.6
3.0
3.00 3.25 3.50 3.75 4.00
Genes count
Gene ratio
Ccl4
Ccl3
n.s.
2.8
-(LogP)
ILC1
60
Spleen ILC1
neg. reg. of T cell
activation
Annexin V
Genes count
Gene ratio
403
reg. of leukocyte
proliferation
20
5.0 7.5 10.0 12.5
Bcl-2
-(LogP)
neg. reg. of cell projection
organization
ILC1
20
Normalized
read count (×102)
Frequency (%)
NK
10
ILC1
15
10
Mki67
n.s.
40
30
neg. reg. of cytokine
production
lymphocyte differentiation
NK
60
40
meiotic cell cycle
neg. reg. of protein
modification process
Ki-67
25
pos. reg. of interleukin-1
beta production
–1
80
cytokinesis
Down-DEG
20
Cdc20
Ncaph
Kif4
Cdk1
Cep55
Spc25
Ube2c
Nusap1
Stmn1
Bub1b
100
Reg. of APC/C activators
between G1/S and
early anaphase
reg. of cytokine production
Up-DEG
40
Control RAR 403
Liver ILC1
mitotic cell cycle process
60
Normalized
read count (×102)
80
Spleen ILC1
Liver ILC1
40
Frequency (%)
Number of DEGs
1.5
0.5
Xcl1
Egr1
Gimap7
Filip1l
Gimap6
Gimap4
51
37
Liver ILC1
Spleen ILC1
Supplementary Figure S1
CD49a + CD49b – G1-ILC
Ncr1-Cre WT
403
NK1.1
Liver
Ncr1-Cre RAR
Spleen
58.9
41.1
55.9
44.1
18.0
82.0
25.6
74.4
IL-7R
–log (padj)
Liver NK
Liver ILC1
Spleen ILC1
15
15
15
10
10
10
–10
–5
RAR 403
10
–10
–5
log (FC)
10
–10
–5
10
Control
...