Fig 1: In Vitro Analysis Identifies Multipotent and ILC3-Restricted ILC Progenitors(A) Schematic of purified bone marrow progenitor populations co-cultured in vitro with OP9 stromal cells to facilitate ILC development.(B) Representative flow-cytometry gating strategy for ILC subsets generated in vitro after co-culture of progenitor cell populations, purified from the bone marrow of the 5x polychromILC mice, with OP9 stromal cells.(C) Flow-cytometry analysis of the proportions of ILC subsets generated in vitro after co-culture of progenitor cell populations IVa, IVb, and IVc, purified from the bone marrow of 5x polychromILC mice, with OP9 stromal cells.(D) Flow-cytometry analysis of the proportions of ILC subsets generated in vitro after co-culture of progenitor cell populations IIIhi, IIIlo, and IIIlo-kat+, purified from the bone marrow of 5x polychromILC mice, with OP9 stromal cells.(E) Characterization of progeny derived from clonal analysis of single IVa, IVb, and IVc progenitor cells, purified from the bone marrow of 5x polychromILC mice, after co-culture with OP9 stromal cells.(F) Characterization of progeny derived from single IIIhi, IIIlo, and IIIlo-kat+ progenitor cells, purified from the bone marrow of 5x polychromILC mice, after co-culture with OP9 stromal cells.(G) Proportion of Eomes+ (NK) and Eomes− (ILC1) cells after co-culture of the indicated progenitor populations, purified from the bone marrow of 5x polychromILC mice, with OP9 stromal cells.(H) Flow-cytometric analysis of cells derived from IVa, IVb, and IVc progenitor populations for the expression of Eomes (co-cultured with OP9 stromal cells).(I) Flow-cytometric analysis of cells derived from IVa, IVb, and IVc progenitor populations for the expression of perforin and IFN-γ (co-cultured with OP9 cells and stimulated for 48 hr with IL-2, IL-15, and IL-18).(J) Flow-cytometric analysis of Bcl11b, Eomes, perforin, and IFN-γ expression in LiveCD45.2+ spleen cells stimulated in vitro with IL-2, IL-15, and IL-18, 6 weeks after transfer of IVa cells into Rag2−/−Il2rgc−/− recipients.(A–D) Data are pooled from 3 independent experiments; mean ± SEM of 5–9 replicate cultures. (E and F) Data are pooled from 3 independent experiments. (G) Data are pooled from 2 independent experiments. (H and I) Data are representative of 3 independent experiments. (J) shows data concatenated from 7 animals taken from 2 independent experiments. Please also see Figure S6.
Fig 2: IL-15 stimulates proliferation of FAPs both in vivo and in vitro. a A schematic showing the experiment in vivo: IL-15 (or combination with Jak inhibitor) was administered from 3dpi to 5 dpi and samples were sectioned on 7 dpi (top). Activated FAPs were detected by staining Ki67 (bottom). Scale bars, 20 μm. b Quantifications of percentage of PDGFRα+Ki67+ FAPs in total FAPs. c The ability of proliferation of FAPs in the presence of IL-15 (or combination with Jak inhibitor) detected by BrdU staining. d Quantifications of percentage of BrdU+ FAPs in total FAPs. e Western blots for activation of Jak-STAT pathways after stimulated by IL-15 with/without inhibitor and (f) quantity analysis. g FITC-Annexin-V/PI assay for apoptosis of FAPs after stimulated by IL-15 for 48 h. Values not sharing a common small letter differ significantly (p < 0.05). Abbreviations: Gly, glycerol; I, Inhibitor
Fig 3: IL-15 inhibits the adipogenesis of FAPs both in vivo and in vitro. a Immunofluorescence for fatty infiltration after administration of IL-15 in injured muscles 7 dpi. Scale bars, 20 μm. b Quantifications of adipose occupied area (shown in percentage) in injured muscles 7 dpi with glycerol injection. c Real-time PCR for adipogenic biomarkers (c/ebpα, pparγ and FABP4) in whole injured muscles 7 dpi with glycerol injection. d The effect of IL-15 on adipogenesis of FAPs was detected by Oil Red O staining in vitro. Scale bars, 100 μm (top), 40 μm (bottom). e Quantifications of Oil Red O occupied area (shown in percentage). f qPCR analysis of DHH and TIMP3 in purified FAPs from injured muscles 7 dpi
Fig 4: NIX Is Critical for Formation of Effector Memory in Ova-Specific CD8+ T CellsSpleens from OT-I mice (A–D) or wild-type (WT) and T/NIX-/- mice (E–K) were collected at designated time points.(A) Kinetics of Nix expression in Ova-specific CD8+ T cells (Ova-CD8+) after VSV-Ova immunization.(B) Gene expression of Nix in Ova-CD8+ 24 h after addition of IL-15. CD8+ T cells from naive OT-I mice were activated with anti-CD3 and anti-CD28 for 72 h, followed by IL-15 addition.(C) Kinetics of Nix expression in Ova-CD8+ after CD3-stimulation, followed by IL-15 addition.(D) Kinetics of Il15ra expression in Ova-CD8+ after VSV-Ova immunization. Ova-CD8+ from mice within the same experimental group in (A)–(D) were pooled before analysis.(E) Representative dot plot showing percentage of Ova-EM in WT or T/NIX-/- spleens on day 30 p.i. with 104 plaque-forming units (PFU) of VSV-Ova.(F) Mean frequencies of Ova-EM from (E).(G) Experimental model for adoptive transfer experiment performed in (H).(H) Left: representative plot showing percentages of CD45.1+ WT and CD45.2+ T/NIX-/- Ova-EM in CD45.2+ T/NIX-/- mice 30 days after VSV-Ova immunization. Right: mean frequencies of CD45.1+ WT and CD45.2+ T/NIX-/- Ova-EM from experiment performed in the left panel.(I) Kinetics of effector memory formation in Ova-CD8+ in vivo in WT or T/NIX-/- mice after VSV-Ova immunization.(J) Gene expression of Nix in day 0 naive, day 6 Ova-activated, day 10 Ova-CD8+ MPECs, day 30 Ova-EM, and day 30 Ova-CM in WT mice after VSV-Ova immunization.(K and L) Gene expression of Foxo1 (left panel) and Tcf7 (right panel) in day 10 Ova-CD8+ MPECs (K) or day 30 Ova-EM (L) harvested from WT or T/NIX-/- spleens after VSV-Ova immunization.(M) In vitro differentiation of Ova-EM. Left: representative plot for percentage of WT or T/NIX-/- Ova-EM on day 8. Right: mean frequency of Ova-EM from left panel.In (E) and (M), CD8+Ova_tetramer+ population (Ova-EM) was gated on CD3+CD8+CD43-CD62L-CD44+ population. Data are representative of two or more independent experiments (n = 3–10). Data were analyzed using one-way ANOVA with Bonferroni’s posttest (mean ± SEM) in (A)–(D); two-tailed Student’s t test (mean ± SEM) in (F), (H, right), (J)–(L), and (M, right); and two-way ANOVA with Bonferroni’s posttests (mean ± SEM) in (I). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figures S1 and S2.
Fig 5: The expression of IL-15 is positively correlated with number of FAPs and collagen deposition in subjects with rotator cuff tear. a Immunofluorescence for PDGFRα and Laminin in muscles from subjects with RCT. b Quantification of number of FAPs in muscles from subjects with RCT. c Immunofluorescence for Collagen Iin muscles from subjects with RCT. d Quantification of percentage of collagen deposition area in muscles from subjects with RCT. e qPCT analysis of mRNA expression of IL-15 in samples from patients with RCT. f Pearson’s correlation analysis for mRNA level of IL-15 and number of FAPs in samples from patients with RCT. g Pearson’s correlation analysis for mRNA level of IL-15 and percentage of area of collagen deposition in samples from patients with RCT
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