Figure 7: Expansion and maturation of pulmonary ILC precursors was linked to postnatal lung development.

A) IGF1 mRNA expression in sorted pulmonary fibroblast (CD45⁻CD326⁻CD140⁺CD31⁻) isolated from age-defined B6 mice.

B) Representative high magnification images showing Insulin-like growth factor (IGF)1⁺ cells (yellow arrowheads) in age-defined newborn B6 or adult (PN28) mice. Scale bars represent 20 micro M. C) IGF1⁺ cells per high power field in lungs of age defined newborn B6 or adult mice (mean, interquartile range, minimum and maximum value for defined age is displayed).

D) The relative proportions of pulmonary ZBTB16⁺ ILC precursors (defined as CD45⁺Lin⁻CD25-ICOS⁻CD127⁺α4β7⁺CD135⁻Id2⁺PD1⁺GATA3⁻RORγt⁻ZBTB16 (GFP)⁺ cells) incorporating BrdU were quantified by flow cytometry in age-defined B6 newborn or adult mice.

E) Correlation between number of IGF1⁺ cells, the relative proportion of BrdU incorporating ZBTB16⁺ ILC precursors and absolute number of mature ILC3 (defined as CD45⁺Lin-CD127⁺GATA3⁻RORγt⁺ cells) in lungs of age-defined newborn or adult mice.

F) Pulmonary ZBTB16⁺ ILC precursors from newborn (PN5) or adult (PN28) ZBTB16^GFP mice were co-incubated with three-dimensional aggregate of alveolar epithelial cells (CD45⁻CD326⁺CD140⁻CD31⁻), and alveolar fibroblast (CD45⁻CD326⁺CD140⁻CD31⁻) from either age-matched newborn mice or adult mice as indicated.

G) Numbers represent the frequency of BrdU⁺ ILC precursors. Representative histograms. H) The relative proportion of pulmonary ZBTB16⁺ ILC precursors incorporating BrdU or I) fold increase in mature pulmonary ILC3s quantified by flow cytometry.

J) Equal number of newborn (PN1) or adult (P28) (both CD45.2⁺) common lymphoid precursors (CLP) (sorted as CD45⁺Lin⁻CD25⁻ICOS⁻CD117⁺Sca-1⁻CD127⁺α4β7⁻CD135⁺ cells) were co-transplanted into sublethally irradiated alymphoid newborn (PN1) or adult (PN28) Rag2^−/−;Il2rg^−/− mice. ZBTB16 ILC precursors or mature ILC3s were examined 2 weeks later in the lungs by flow cytometry. Numbers represent the frequency of donor (CD45.2⁺) CLP-derived ZBTB16⁺ ILC precursors (left panel) or ILC3s (right panel).

K) Newborn B6 mice were continuously exposed to hyperoxic environment (hyperoxia) or room air from PN1 for seven days. Representative low or high magnification images showing Insulin-like growth factor (IGF)1⁺ cells (yellow arrowheads) in lungs of newborn B6 mice (PN7) exposed to hyperoxic environment (hyperoxia) or room air. Scale bars represent 100 micro M and 20 micro M in lower and higher magnification images respectively.

L) IGF1⁺ cells per high power field in lungs in lungs of newborn B6 mice (PN7) exposed to hyperoxic environment (hyperoxia) or room air (mean, interquartile range, minimum and maximum value for defined age is displayed).

M) Relative proportions of pulmonary ZBTB16⁺ ILC precursors incorporating BrdU in newborn B6 mice continuously exposed to hyperoxic environment (hyperoxia) or room air from PN1 for seven days or N) absolute number of mature ILC3s in the lung was quantified by flow cytometry.

O) Newborn B6 mice (PN7) exposed to hyperoxic environment (hyperoxia) or room air were examined for susceptibility after intratracheal inoculation with S. pneumoniae on PN8. Some newborn mice in the hyperoxia experimental group received adoptive transfer of WT ILC3s (sorted as Live CD45⁺Lin-CD127⁺(RORγt)GFP⁺ KLRG⁻ NK1.1⁻ cells from RORC^GFP/+ mice) on PN7 before intratracheal inoculation with S. pneumoniae on PN8.

P) Bronchial lavage fluid (BLF) from infants with BPD or no BPD (Control) were examined for IGF1 protein levels by ELISA or

Q) relative abundance of ILC3s (defined as live CD45⁺Lin⁻CD127⁺RORγt⁺GATA3⁻ cells) was quantified by flow cytometry (mean, interquartile range, minimum and maximum value for each group is displayed).

Data is representative of three independent experiments. Results shown as the means ± s.e.m (Student’s t-test [Fig. 7A,C,D, L,M,N,P,Q] or ANOVA [Fig. 7H,I] or Kaplan-Meier log-rank test [7O] *P ≤ 0.05; **P ≤ 0.01 and number of individual animals [n] are indicated in the figures). See also Fig. S6.