Fig 2: Rora-Teal Expression Distinguishes ILCs from NK Cells(A) Flow-cytometry gating strategy for ILC1 or ex-ILC3 in the siLP of 5x polychromILC mice.(B) Flow-cytometry analysis of Rora-Teal expression in splenic NK cells (CD3-CD19-NKp46+NK1.1+) and siLP ILC1or exILC3 (gated as shown in A) from 5x polychromILC mice.(C) Flow-cytometry analysis of the indicated cell surface markers in Lin-NKp46+NK1.1+ cells from the spleen, liver, and siLP of Rora+/Teal mice.(D) Flow-cytometry analysis of CD11c, Eomes, and T-bet expression in NK or ILC1 cells from the spleen of Rora+/Teal mice.(E) Flow-cytometry analysis of CD11c, Eomes, and T-bet expression in NK or ILC1 cells from the liver of Rora+/Teal mice.(F) Flow-cytometry analysis of CD11c, Eomes, and T-bet expression in NK or ILC1 cells from the siLP of Rora+/Teal mice.Data are representative of 2 independent experiments.

Fig 3: FACS in mouse lung cells on PND7 (n = 6–8). a FACS gating strategies for CD4+ T cells, CD8+ T cells, neutrophils (CD45+Ly6GhiCD11bhiCD11ChiF4/80–) and natural killer cells (CD45+NKp46+CD3–). b FACS gating strategies for CD103+CD11c+ dendritic cells (CD45+CD103hiCD11ChiLy6C-F4/80loMHCIIhiCD11bloSiglec-F– cells) and interstitial macrophages (CD45+CD103loCD11CloLy6CloF4/80intMHCIIhiCD11bloSiglec-F–). c, d There were no differences in CD4+ and CD8+ populations across groups. e There was no difference in the percentage of neutrophils between groups. f–h Percentage of CD103+CD11c+ dendritic cells (f) and natural killer cells (g) increased in the injured group, but was only significantly returned to control levels by early treatment. h Percentage of interstitial macrophages significantly increased in the injured animals, but was brought back to control levels by early treatment. Data expressed as mean ± standard error of mean (SEM). Statistical significance determined with one-way ANOVA accompanied by the Bonferroni post-hoc test. **p < 0.01, ****p < 0.0001. FSC forward scatter, SSC side scatter, NK natural killer, DC dendritic cell, hAEC human amnion epithelial cell

Fig 4: Murine ILCPs yield group 1, 2, and 3 ILCs after co-culture with SIOs(A) Confocal image of SIOs with apical ZO-1 and Lyzozyme-1+ Paneth cells (scale bar: 50 µm).(B) Diagram of co-culture system and representative confocal image of SIO-ILCP co-culture (scale bar: 50 µm).(C) Schematic of experimental design.(D) Representative flow plot of DAPI-Lineage- (hematopoeitic stem cell [HSC] cocktail [CD3, CD45R, CD11b, TER-119] Ly-G6, CD5, CD19, and NK1.1), CD127+a4ß7+Flt3-, PD-1+ ILCP, and CD25+ ILC2P, gated based on PD-1 fluorescence minus one (FMO) controls (overlaid in red).(E) Quantification of ILCP count yielded per animal (N = 15 animals, five experiments).(F) Representative flow plots of DAPI-EpCAM+CD45- IECs and EpCAM-CD45+ ILCs from 7-day cultures of PD-1+ ILCPs only, ILCP + SIO, and SIO-only cultures. Arrows indicate corresponding lack of Lineage markers.(G) Fold change of CD45+Lineage- cells yielded after culture relative to the number of seeded precursors (N = 15 animals, five independent experiments).(H) Contour plot overlay representing expression of NKp46, ROR?t, NKp46, NK1.1, Klrg1, and Sca-1 in Lineage- populations derived from (F) in populations pregated as putative group 1 (magenta; Live, EpCAM-, CD45+, Lin-, ROR?t-, Klrg1-, NK1.1+, NKp46+), group 2 (green; Live, EpCAM-, CD45+, Lin-, ROR?t-, NKp46-, Klrg1+, Sca-1+), NKp46+ group 3 (lavender; Live, EpCAM-, CD45+, Lin-, Klrg1-, ROR?t+, NKp46+), NKp46- group 3 (blue; Live, EpCAM-, CD45+, Lin-, Klrg1-, ROR?t+, NKp46-), and a putative immature “other” population (gray; Live, EpCAM-, CD45+, Lin-, ROR?t-, Klrg1-, NK1.1-, NKp46-) in cultures derived from ILCPs only or ILCP co-culture with SIOs.(I) Quantification of pooled ILC groups depicted in (H) (ILCPs from N = 4 animals).Error bars represent SEM; p values are from unpaired Student’s t tests.

Fig 5: mTORC2 and mTORC1 promote NK cell maturation by controlling the expression of Tbx21 and Eomes in a cooperative and nonredundant manner. a Intracellular flow cytometric analysis and cumulative results for the phosphorylation (p-) of S6 at ser235/236 (p-S6ser235/236) (top) and Akt at Ser473 (p-AktSer473) (bottom) in splenic NK cells (CD3-CD19-NK1.1+) from mice of the indicated genotype. b Cumulative ratio and enumeration of NK cells (CD3-CD19-NK1.1+) in the BM, spleen, and peripheral lymph nodes (pLNs) from mice of the indicated genotype. c Flow cytometric analysis and cumulative frequencies of subpopulations of NK cells (CD3-CD19-NK1.1+NKp46+) in the BM, spleen, and pLNs (left) and the calculated ratio of CD27- versus CD27+ cells among CD11b+ NK cells (right). d The cumulative frequencies depicting the CD43+KLRG1+ subset of NK cells (CD3-CD19-NK1.1+NKp46+) in the BM, spleen, and pLNs from mice of the indicated genotype. e, f Tbx21 and Eomes mRNA and protein expression in control versus Rictor?/? mice (e) and control versus Raptor?NK mice (f), as assessed by quantitative RT-PCR and flow cytometry, respectively. Purified splenic NK cells were used for quantitative RT-PCR (left). Cumulative data for Tbx21 and Eomes protein expression in total splenic NK cells (CD3-CD19-NK1.1+NKp46+) and the indicated subpopulations thereof were analyzed by flow cytometry (right). g Cumulative data for Tbx21 and Eomes protein expression in total splenic NK cells (CD3-CD19-NK1.1+NKp46+) and the indicated subpopulations thereof from control, Rictor?NK and Rictor?NKRaptorfl/+ mice. The control represents Rictorfl/+/Ncr1-Cre+ mice; Rictor?NK represents Rictorfl/fl/Ncr1-Cre+ mice; and Rictor?NKRaptorfl/+ represents Rictorfl/fl/Rptorfl/+/Ncr1-Cre+ mice. The dashed line indicates the control group, and the solid line indicates the gene knockout group. The MFI was calculated relative to total NK cells or to the CD27SP subset of NK cells from control littermates. The subpopulations of NK cells are distinguished by surface expression of CD27 and CD11b, and CD27SP, DP, and CD11bSP represent the CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK cell subsets, respectively. Each dot represents one mouse, and all experiments were replicated 3 (a, e left, f, g), 4 (e right), or 5 (b–d) times; error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; one-way ANOVA (a–d, g), unpaired two-tailed Student’s t test (e, f)