Fig 2: IL-15 can facilitate regeneration of myofibers in injured muscles. a A shcematic showing the experiment in vivo: IL-15 was administered from 1dpi to 3 dpi and samples were sectioned on 8 dpi. b Immunofluorescence for myofibers 8 days after glycerol injection with administration of IL-15 by staining phalloidine. c Quantifications of average cross-sectional area of myofibers with administration of IL-15. d Immunofluorescence for Laminin α2 and Hoechst in injured muscle 8 dpi with administration of IL-15. e Quantifications of percentages of number of myofibers with centrally located nuclei. f Number of nuclei present in myofibers with administration of IL-15. Abbreviations: Gly, glycerol; Colla 1, Collagen I

Fig 3: 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 4: 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 5: The level of IL-15 is negatively associated with fatty infiltration in chronic muscle injury. After injection of glycerol in tibialis anterior, the pathological changes of myofibers (a) and fatty infiltration (b) were examined by immunofluorescene, an antibody against adipocyte biomarker perilipin were used to detect the adipose formation in injured mice. c The mRNA level of candidate myokines in injured muscle on 0 days and 7 dpi. Were analyzed using real-time PCR. Scale bars, 20 μm (a, b)