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Immunity, Volume 52
Supplemental Information
Insulin-like Growth Factor 1 Supports
a Pulmonary Niche that Promotes Type 3 Innate
Lymphoid Cell Development in Newborn Lungs
Katherine Oherle, Elizabeth Acker, Madeline Bonfield, Timothy Wang, Jerilyn Gray, IanLang, James Bridges, Ian Lewkowich, Yan Xu, Shawn Ahlfeld, WilliamZacharias, Theresa Alenghat, and Hitesh Deshmukh
Supplementary Figure Legends: Supplementary Figure 1 (Refers to Fig. 1 and 2): IL-22 producing pulmonary ILC3 are essential
for host defense against respiratory pathogens in the newborn.
A) Numbers of ILCs (defined as live CD45+Lin
-CD127
+ cells) or ILC3s (defined as live CD45
+Lin
-
CD127+RORγt
+GATA3
- cells) in the lungs of newborn (PN7) B6 mice were quantified by flow cytometry.
B) Representative bivariate density plots of ILC3s (defined as CD45+Lin
-CD127
+GATA3
-RORγt(GFP)
+
cells) in the lungs of newborn RorcGFP/+ mice. Numbers represent the frequency of different ILC3 subsets.
Lineage panel includes CD3ε, CD5, F4/80, CD11b, CD19 and Ly6G.
C) Newborn B6 mice were inoculated with either S. pneumoniae or sham via intratracheal (i.t.) route on
PN3. Frequency of interleukin (IL)-22 in the pulmonary ILC3s (defined as CD45+Lin
-CD127
+GATA3
-
RORγt+ cells) was quantified by FACS. Numbers represent frequency of IL-22
+ ILC3.
D) Mean fluorescent intensity (MFI) of IL-22 in the pulmonary ILC3s or small intestinal (SI) ILC3s in
newborn B6 mice inoculated with either S. pneumoniae or sham via intratracheal route on PN3.
E) Newborn B6 mice were inoculated with either S. pneumoniae or sham via i.t. route on PN3. Frequency
of IL-22+ ILC3s in SI was quantified by FACS.
F) Frequency of IL-22+ ILC3s in the lungs or the G) SI of newborn (PN3) RorcCre
or RorcΔIl22 or Il22fl/fl
mice was quantified by flow cytometry.
H) Frequency of different ILC3 subsets in lungs of newborn (PN3) RorcCre or RorcΔIl22
or Il22fl/fl mice
mice was quantified by flow cytometry.
I) Frequency of common lymphoid progenitors (CLP) in bone marrow of age-defined newborn (PN1, 3,
7, 14 and 21) or adult mice (PN28) quantified by flow cytometry. CLPs were identified as CD45+Lin
-
CD127+CD25
-ICOS
-CD117
+Sca-1
-α4β7
-CD135
+RORγt
-ZΒΤΒ16
- cells.
J) BM of the newborn (PN3) Zbtb16EGFPCre/+ mice were examined for ZBTB16
+ ILC precursors by flow
cytometry. Lineage panel includes CD3ε,CD5,F4/80,CD11b,CD19 and Ly6G. GFP gate is indicated in
black. The cells were pooled from six PN3 newborn mice.
K) Expression of eGFP in indicated cell types isolated from the lungs of newborn (PN3) Zbtb16EGFPCre/+
or WT mice. NK cells are defined as live CD45+CD3ε
-NK1.1
+ cells. T cells are defined as
CD45+CD4
+CD3ε
+ cells. γδ T cells are defined as CD45
+CD3ε
+TCRγδ
+ cells. Frequency of indicated cells
co-expressing GFP are indicated in red. Data represents cells pooled from six PN3 newborn mice. Data is
representative of three independent experiments.
L) Expression of ZBTB16 in indicated cell types isolated from the lungs of newborn (PN3) B6 mice was
quantified by flowcytometry. NK cells are defined as live CD45+CD3ε
-NK1.1
+ cells. T cells are defined
as CD45+CD4
+CD3ε
+ cells. γδ T cells are defined as CD45
+CD3ε
+TCRγδ
+ cells. Frequency of indicated
cells co-expressing ZBTB16 are indicated in red. Data represents cells pooled from six PN3 newborn
mice. Data is representative of three independent experiments.
Data is representative of three independent experiments. Results are shown as the means ± s.e.m (ANOVA
[Fig. S1A,S1B, S1E, S1F, S1G, S1H, S1I], *P ≤ 0.05; **P ≤ 0.01 and number of individual animals [n]
are indicated in the figures).
Supplementary Figure 2 (Refers to Fig. 3): Intratracheal delivery of diphtheria toxin in newborn
mice.
A) Equipment used for intratracheal delivery of diphtheria toxin. 50 μl of air was aspirated into a 100 μl
gas-tight Hamilton syringe, followed by 50 μl of trypan blue by advancing the Teflon plunger from 50 µl
mark to the 100 µl mark on the syringe body. The syringe was then fitted with 24 or 26-gauge, 19 mm
long intravascular catheter.
B) The anesthetized newborn mouse was secured on intubation platform with Velcro strips and the
platform was inclined to 45°.
C) An operating otoscope fitted with 2 mm speculum was gently introduced into oropharynx using the
nondominant hand while maintaining tongue retraction.
D) The gas-tight Hamilton syringe preloaded with trypan blue and fitted with 24-gauge intravascular
catheter was then gently introduced through the speculum with the dominant hand.
E) The 24-gauge intravascular catheter was advanced through the vocal cords under direct visualization
using the side of the speculum as a guide.
F) The liquid/air bolus was injected into the trachea in one fluid motion by depressing the plunger with
dominant hand and immediately withdrew the catheter and the speculum.
G and H) Fifteen minutes post injection, the animals were euthanized and presence or absence of trypan
blue in stomach and the lung was confirmed by direct visualization.
Supplementary Figure 3 (Refers to Fig. 3): Pulmonary ZBTB16+ ILC precursors contribute to the
homeostatic pool of mature ILC3 in the newborn lung.
A) Single cells from lungs pooled from 8-10 (PN1) newborn RorcGFP/+ mice (on CD45.1 background)
were stained with 7-AAD Viability staining solution and lineage panel (CD3ε, CD4, CD8, TCRβ, CD11b,
CD11c, CD19, Β220 and LY6G antibody) anti–mouse CD127 antibody, anti-mouse KLRG1 and anti-
mouse NK1.1. ILC2 were sorted as Live CD45+
Lineage (CD3ε, CD5, CD11b, CD11c, FcεR1 and B220)
CD127+(RORγt) GFP
- KLRG
+ NK1.1
- cells. ILC3 were sorted as Live CD45
+Lineage (CD3ε, CD5,
CD11b, CD11c, FcεR1 and B220) CD127+(RORγt) GFP
+ KLRG
- NK1.1
- cells. Sorted cells were then
stained with anti–mouse RORγt antibody and anti-mouse GATA3 antibody. Representative histograms
from 3 independent experiments.
B) Newborn Zbtb16iDTR or Rosa26iDTR
mice treated with intratracheal (i.t.) diphtheria toxin (DT) on PN3,
received adoptive transfer of ILC3s (sorted as CD45+Lin
-CD127
+(RORγt)GFP
+KLRG
-NK1.1
- cells
isolated from lungs of age-matched RORCGFP/+
mice [on CD45.1 background]) or sham on PN5. Two
days later, the newborn mice were examined for susceptibility after i.t. inoculation with S. pneumoniae.
C) Newborn Zbtb16iDTR or Rosa26iDTR
mice treated with i.t. DT on PN3 were examined for the presence
of ILC2 (defined as CD45+Lin
−CD127
+GATA3
+RORγt
- cells) in lungs of newborn or adult (PN28) mice
by flow cytometry.
D) Newborn Zbtb16iDTR or Rosa26iDTR
mice treated with i.t. DT on PN3, received adoptive transfer of
ILC2s (sorted as CD45+Lin
-CD127
+(RORγt) GFP
-KLRG
+NK1.1
-cells from lungs of age-matched
RORCGFP/+
mice [on CD45.1 background]) or sham on PN5. The relative proportion of ILC2s carrying
respective congenic markers were examined by FACS one- or two-weeks post transfer.
E) Newborn Zbtb16iDTR or Rosa26iDTR
mice treated with i.t. DT on PN3, received adoptive transfer of
ILC2s or sham from age-matched donor mouse on PN3. Two days later, the newborn mice were examined
for susceptibility after intratracheal inoculation with S. pneumoniae.
F) Newborn Zbtb16iDTR or Rosa26iDTR
mice treated with i.t. DT on PN3 were examined for the presence
of NK cells (defined as live CD45+CD3ε
-NK1.1
+ cells) in the lungs two days later by flow cytometry.
G) Single cells from lungs pooled from 12 (P1) newborn Zbtb16CreGFP/+ mice were stained with 7-AAD
Viability staining solution and lineage panel (CD3ε, CD4, CD8, TCRβ, CD11b, CD11c, CD19, Β220 and
LY6G antibody), anti-mouse NKp46 antibody, anti-mouse NK1.1 antibody, anti-mouse α4β7 antibody,
anti-mouse KLRG1, anti-mouse CCR6 antibody, anti–mouse CD127 antibody and anti-mouse CD25
antibody. ZBTB16+ ILC precursors were sorted as Live CD45
+Lineage (CD3ε, CD4, CD8, TCRβ,
CD11b, CD11c, CD19, B220 and Ly6G), CD127+α4β7
+NKp46
-NK1.1
-KLRG1
-CCR6
- CD25
- ZBTB16
(GFP)+
cells.
H) Sorted cells were then stained with anti–mouse RORγt antibody, anti-mouse T-bet antibody, anti-
mouse GATA3 antibody, anti-mouse ID2 antibody and anti-mouse PD1 antibody. Representative
histograms from 3 independent experiments. Isotype control antibody indicated in grey. Respective gates
indicated in red and numbers represent the frequency of cells expressing the respective protein.
Data is representative of three independent experiments. Results are shown as the means ± s.e.m (ANOVA
[Fig. S4C, S4D, S4F] or Kaplan-Meier log-rank test [Fig. S4B, S4E] , *P ≤ 0.05; **P ≤ 0.01 and number
of individual animals [n] are indicated in the figures).
Supplementary Figure 4 (Refers to Fig. 4) : Differential requirements for development and
maturation of pulmonary ZBTB16+ ILC precursors
A) Global transcriptomic analysis of ZBTB16+ ILC precursors (sorted as CD45
+Lineage [CD3ε, CD4,
CD8, TCRβ, CD11b, CD11c, CD19, B220 and Ly6G]-, CD25
-CD127
+α4β7
+NKp46
-Nk1.1
-KLRG1
-
CCR6-ZBTB16(GFP)
+ cells) isolated from lungs or bone marrow (BM) of newborn Zbtb16EGFPCre
mice
(PN3).
B) Differentially abundant transcripts (indicated by red closed circles) in pulmonary ZBTB16+ ILC
precursors compared to their BM counterparts.
C) Gene Ontology terms overrepresented in differentially abundant transcripts.
D) Clustering of differentially abundant genes. Expression results are presented as log2 RPKM values
('reads per kilobase of transcript per million reads'; mean and maximum value for each gene).
E) ZBTB16+ ILC precursors isolated from lungs or bone marrow of newborn Zbtb16EGFPCre
mice were
examined for expression of cell surface proteins and transcription factors. Representative histograms of 3
separate experiments. Isotype antibody staining is indicated in grey. Gates indicated in red.
Supplementary Figure 5 (Refers to Fig. 5): Figure 5: Insulin-like growth factor signaling is critical
for the proliferation of ILC3 precursors in the pulmonary niche
A) Pulmonary ZBTB16+ ILC precursors from newborn mice were co-incubated with three-dimensional
aggregate of alveolar epithelial cells (CD45−CD131
+CD146
-CD310
-) and alveolar fibroblast
(CD45−CD131
-CD146
+CD310
-) from age-matched newborn mice in the presence of or absence of
cytokines, interleukin (IL)-33 or IL-23 or sham.
B) Frequency of different ILC subsets, in the indicated experimental groups was quantified by flow
cytometry.
C) Pulmonary ZBTB16+ ILC precursors sorted as CD45
+Lineage [CD3ε, CD4, CD8, TCRβ, CD11b,
CD11c, CD19, B220 and Ly6G]-, CD25
-CD127
+α4β7
+NKp46
-Nk1.1
-KLRG1
-CCR6
-ZBTB16(GFP)
+
cells) from newborn Zbtb16EGFPCre (PN3) mice were co-incubated with three-dimensional aggregate of
alveolar epithelial cells and alveolar fibroblast from either sham or tamoxifen treated age-matched
newborn Gli1CreER x Igf1fl/fl
or Igf1fl/fl mice in the presence of BrdU.
D) Frequency of BrdU+ ZBT1B6
+ ILC precursors and
E) fold increase in numbers of ILC3, in the indicated experimental groups was quantified by flow
cytometry.
F) Frequency of common lymphoid precursors (CLP) (defined as CD45+Lin
-CD25
-ICOS
-CD117
+Sca-1
-
CD127+α4β7
-CD135
+Id2
-PD1
-RORγt
-GATA3
- cells) in the bone marrow of newborn (PN3) Zbtb16EGFPCre
or Zbtb16ΔIgf1r mice was quantified by flow cytometry.
G) Expression of IGF1R in indicated cells isolated from lungs of newborn (PN5) Zbtb16EGFPCre (blue) or
Zbtb16ΔIgf1r (red)
or
Igf1rfl/fl (green) mice. NK cells are defined as live CD45
+CD3ε
-NK1.1
+ cells. T cells
are defined as CD45+CD4
+CD3ε
+, γδ T cells are defined as CD45
+CD3ε
+TCRγδ
+.
H Expression of IGF1R in indicated cells isolated from small intestine of newborn (PN3) Zbtb16EGFPCre
(blue) or Zbtb16ΔIgf1r (red)
or
Igf1rfl/fl (green) mice.
I) Frequency of ILC3 (defined as CD45+Lin
-CD127
+RORγt
+GATA3
- cells) in the small intestine of
newborn (PN3) Zbtb16EGFPCre or Zbtb16ΔIgf1r
mice was quantified by flow cytometry.
J) Breeding strategy to generate Rorc ΔIgf1r mice.
K) Frequency of common lymphoid precursors (CLP), (defined as CD45+Lin
-CD25
-ICOS
-CD117
+Sca-1
-
CD127+α4β7
-CD135
+Id2
-PD1
-RORγt
-GATA3
- cells) in the bone marrow of newborn (PN3) RorcCre
(blue) or RorcΔIgf1r (red)
or
Igf1rfl/fl (green) mice was quantified by flow cytometry.
L) Absolute numbers of ILC3 (defined as CD45+ Lin
−CD127
+GATA3
-RORγt
+ cells) or M) frequency of
different ILC subsets or the N) geometric mean fluorescent intensity of interleukin (IL)-22 in the
pulmonary ILC3 in newborn (PN3) RorcCre (blue) or RorcΔIgf1r
(red) or
Igf1rfl/fl (green) mice was quantified
by flow cytometry. Live cells were pooled from six PN3 newborn mice of each genotype.
Data is representative of three independent experiments. Results are shown as the means ± s.e.m (ANOVA
[Fig. S5B, S5D, S5E, S5F, S5I, S5K, S5L, S5M, S5N], *P ≤ 0.05; **P ≤ 0.01 and number of individual
animals [n] are indicated in the figures).
Supplementary Figure 6 (Refers to Fig. 6 and 7) Expansion and maturation of pulmonary ILC
precursors is linked to postnatal lung development.
A) Frequency of common lymphoid precursors (CLP) (defined as CD45+Lin
-CD25
-ICOS
-CD117
+Sca-1
-
CD127+α4β7
-CD135
+Id2
-PD1
-RORγt
-GATA3
- cells) in the bone marrow of newborn (PN3) germ free
(GF) or conventional (CNV) B6 mice was quantified by flow cytometry.
B) Newborn GF or CNV mice (PN3) were injected with BrdU and euthanized two hours later. The relative
proportions of pulmonary ZBTB16+ ILC precursors (defined as CD45
+Lin
-CD25
-ICOS
-
CD127+α4β7
+CD135
-Id2
+PD1
+GATA3
-RORγt
-ZBTB16
+ cells) incorporating BrdU or the C) absolute
numbers of pulmonary ILC precursors in newborn GF or CNV mice (PN3) was quantified by flow
cytometry. Live cells were pooled from six PN3 newborn mice of each genotype.
D) Representative high magnification images showing IGF1+ cells (yellow arrowheads) in newborn GF
or CNV mice (P3). Scale bars represent 20 micro M.
E) Numbers of pulmonary IGF1+ cells in per high power field in newborn GF or CNV mice (PN3).
Data is representative of three independent experiments.
F) Sorted fibroblasts (CD45-CD326
-CD31
-CD144
-) from human newborn, child or adult were examined
for expression of Igf1. Expression results presented as RPKM values ('reads per kilobase of transcript per
million reads'; mean and maximum value for each gene).
G) Bivariate density plots of ILC3 (defined as live CD45+Lin
-CD127
+RORγt
+GATA3
- cells) from
bronchial lavage fluid of human infant with BPD. Numbers represent the frequency of IL-22+ ILC3.
Lineage panel includes CD3,CD5,CD11b, CD11c, CD14, FcεR1. Isotype antibody indicated in red.
H) Model of proposed pulmonary ILC3 niche.
Results are shown as the means ± s.e.m (unpaired two-tailed Student’s t-test [Fig. S6A, S6B, S6C, S6D,
S6F, S6G and S6H], number of individual animals [n] are indicated.
Supplementary Table 1 (Refers to Fig. 4, Supplementary Fig. 4)
Gene name Protein score MW [kDa] Fold change p value
STC2 170.6 33.25 2.01 ↓ 2.8087716
ANP32B 231.5 33.49 2.01 ↓ 4.0241944
ATP6AP1 199.6 52.03 2.01 ↓ 4.1004673
IGFBP7 185.5 29.1 2.02 ↓ 4.1980876
PSMA5 205 26.41 2.02 ↓ 4.1980876
PRDX1 131.3 22.11 2.01 ↓ 3.6041037
NCL 153.9 76.613 2.1 ↓ 4.7878587
LMNB1 151.5 66.408 2.12 ↓ 2.7377348
PLEC 136.1 531.78 2.21 ↓ 3.1719937
FGF17 187.1 25.04 2.21 ↓ 3.092076
DAG1 189.6 97.44 2.29 ↓ 2.9452447
ENO2 193.3 47.17 2.28 ↓ 3.3560245
TGFB1 151.9 26.1 2.23 ↓ 3.0757002
PSMA3 190.4 28.433 2.35 ↓ 3.0757002
FGF19 190.6 21.85 2.43 ↓ 2.6543129
LGALS3 129.1 26.15 2.69 ↓ 2.7781957
HSPA8 178.5 70.9 2.71 ↓ 3.2241534
HNRNPA1 131.2 38.845 2.73 ↓ 2.6786392
LMAN2 181.6 40.23 2.79 ↓ 4.0848337
EEF1B2 158.6 24.763 3.13 ↓ 2.6631968
RPLP0 179.7 34.27 3.14 ↓ 2.8862974
ANP32B 194 34.882 3.17 ↓ 2.7471141
NACA 261.5 94.68 3.37 ↓ 2.9429403
TPI1 182.7 30.8 3.52 ↓ 4.1037517
VIM 108.4 53.65 3.53 ↓ 3.6806068
CCN2 147 37.84 3.54 ↓ 3.6806068
FGF2 168.9 18.25 3.63 ↓ 4.6145609
PA2G4 131.9 45.15 3.63 ↓ 4.6145609
GAPDH 181.7 36.05 3.8 ↓ 4.6145609
EF1-alpha 135.7 50.14 3.82 ↓ 2.8796085
IGFBP2 166.7 35.14 4.56 ↓ 3.2904665
PGAM1 213.8 28.8 4.57 ↓ 3.3968646
IGF1 152.9 7.5 5.41 ↓ 2.8839233
IGFBP4 171 26.1 5.54 ↓ 3.8311088
PSAP 149.2 61.9 6.23 ↓ 2.9226432
GOLM1 176.1 46.27 6.3 ↓ 2.663306
ARCN1 183.6 61.63 6.5 ↓ 5.1674347
TWSG1 168.4 25 7.97 ↓ 3.4725435
KITLG 166.2 30.85 7.98 ↓ 3.3042451
TIMP1 181.8 23.17 9.39 ↓ 2.66007
IGFBP6 143.5 22.6 10.72 ↓ 2.7229805
Supplementary Table 2: Biological function analysis of differentially abundant proteins in
the conditioned medium. (Refers to Fig. 4, Supplementary Figure 4)
GO Biological process
Fold
enrichment
Raw P
value FDR
positive regulation of insulin-like growth factor
receptor signaling pathway
92.76 2.42E-04 1.85E-02
positive regulation of cell proliferation 7.14 5.96E-08 1.31E-04
positive regulation of developmental process 3.32 5.68E-06 1.15E-03
regulation of cell differentiation 3.8 4.12E-05 4.70E-03
pathway restricted SMAD protein
phosphorylation 85.62 3.25E-04 2.34E-02
glucose metabolic process 18.35 6.40E-04 3.78E-02
glycolytic process 61.84 7.74E-07 3.62E-04
positive regulation of collagen biosynthetic
process 53.86 3.08E-05 3.92E-03
fibroblast growth factor receptor signaling
pathway 39.75 7.20E-05 7.50E-03
Supplementary Table 3: Patient Demographics (Refers to Fig. 6, Supplementary Figure 6)