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, Ian Lang, James Bridges, Ian Lewkowich, Yan Xu, Shawn Ahlfeld, William Zacharias, Theresa Alenghat, and Hitesh Deshmukh
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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