Fig 1.
NKp46 is required for ILC1 development.
(A) Gating strategy for ILC1s. ILC1s were gated on lymphocytes and then were further defined as Lin−NK1.1+NKp46+CD49b─CD49a+, with the exception that GFP+ was used to replace NKp46+ when Ncr1 gfp/+ or Ncr1gfp/gfp mice were used. (B) Percentages or quantities of ILC1s were determined by flow cytometric analysis in the liver of Ncr1gfp/gfp, Ncr1gfp/+, and Ncr1+/+ mice. (C) NK cells were gated on Lin─NK1.1+NKp46+(or GFP+ for Ncr1gfp/gfp mice)CD49b+CD49a─ among lymphocytes. ILC1s were gated on Lin─NK1.1+NKp46+(or GFP+ for Ncr1gfp/gfp mice)CD49b─CD49a+ among lymphocytes. (D) Quantification of ILC1s in different organs or tissues in Ncr1gfp/gfp mice and Ncr1+/+ littermates, i.e., summary data for (C). Each line demonstrates percentages of ILC1s for a pair of Ncr1gfp/gfp and Ncr1+/+ littermates. (E) Quantities of NK cells or ILC1s in different organs of Ncr1gfp/gfp mice and their Ncr1+/+ littermates (n = 4). Error bars, standard deviations; ***, p < 0.001; **, p < 0.01; *, p < 0.05. The numerical data for panels B, D and E can be found in S1 Data. Lin─, CD3─CD19─; BM, bone marrow; FSC-A, forward scatter area; FSC-H, FSC height; FSC-W, FSC width; GFP, green fluorescent protein; ILC1, innate lymphoid cell 1; NK, natural killer; Ncr1, natural cytotoxicity receptor 1; SI, small intestine; SSC-A, side scatter area; SSC-H, SSC height; SSC-W, SSC width.
Fig 2.
Absence of TRAIL+ ILC1s in NKp46-deficient mice.
(A) Percentages of TRAIL+ ILC1s were analyzed by flow cytometric analysis in the liver of Ncr1gfp/gfp mice and their Ncr1+/+ littermates. ILC1s were gated on Lin─NK1.1+NKp46+(or GFP+ for Ncr1gfp/gfp mice) TRAIL+CD49b─ among lymphocytes. (B) Quantification of TRAIL+ ILC1s in the liver of Ncr1gfp/gfp mice and their Ncr1+/+ littermates for (A) (n = 5). (C) Quantification of TRAIL+ ILC1s in other organs (spleen, n = 5; BM, n = 5; SI, n = 4) of Ncr1gfp/gfp mice and their Ncr1+/+ littermates. **, p < 0.01; *, p < 0.05. The numerical data for panels B and C can be found in S1 Data. Lin─, CD3─CD19─; BM, bone marrow; GFP, green fluorescent protein; ILC1, innate lymphoid cell 1; Ncr1, natural cytotoxicity receptor 1; NK, natural killer; SI, small intestine; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
Fig 3.
NKp46 deficiency does not affect ILC2s and ILC3s.
(A) Gating strategy for ILC2s and ILC3s. ILC2s were gated on CD45+Lin─CD127+Gata3+, and ILC3s were gated on CD45+Lin─CD127+RORγt+. (B) Percentages of ILC2s or ILC3s were analyzed by flow cytometry in SI in Ncr1gfp/gfp mice and Ncr1+/+ littermates. ILC2s were gated on CD45+Lin─CD127+Gata3+RORγt─ lymphocytes. ILC3s were gated on CD45+Lin─CD127+RORγt+Gata3─ lymphocytes. (C) Quantities of ILC2s or ILC3s were determined in SI of Ncr1gfp/gfp mice and their Ncr1+/+ littermates (n = 4). (D) Lin─(or CD3─CD19─)NK1.1+NKp46+(or GFP+ for Ncr1gfp/gfp mice)CD49b+ NK cells were sorted from the spleen of Ncr1gfp/gfp mice or Ncr1+/+ littermates and were co-stimulated with IL-12 (10 ng/ml) and IL-18 (10 ng/ml) for 16 h, followed by the measurement of IFN-γ production by intracellular flow cytometric analysis (D, left panel, n = 3) or ELISA assays (D, right panel, n = 3). Golgi Plug was added at a 1:1,000 dilution to the culture 4 h prior to cell harvesting. (E) Homogenized SI cells isolated from Ncr1gfp/gfp mice or Ncr1+/+ littermates were stimulated with IL-23 (10 ng/ml) for 4 h, followed by the measurement of IL-22 production by flow cytometric analysis after gating ILC3s on CD45+Lin─CD127+RORγt+. Golgi Plug was added at a 1:1,000 dilution to the culture 3 h prior to cell harvesting. Error bars, standard deviations. The numerical data for panel C and D can be found in S1 Data. ELISA, Enzyme-linked immunosorbent assay; FSC-A, forward scatter area; FSC-H, FSC height; IFN, interferon; IL, interleukin; ILC, innate lymphoid cell; Ncr1, natural cytotoxicity receptor 1; NK, natural killer; NS, no significance; RORγt, retinoic acid receptor (RAR) related orphan receptor gamma t; SI, small intestine; SSC-A, side scatter area; SSC-H, SSC height; SSC-W, SSC width.
Fig 4.
NKp46 plays a cell-intrinsic role in regulating ILC1 development.
(A) Scheme of BM transplantation using BM cells of CD45.2 Ncr1gfp/gfp mice or Ncr1+/+ littermate controls as donor cells to inject into CD45.1 recipients via tail vein. Development of ILC subsets was analyzed 2 weeks after transplantation. (B) Percentages of CD45.2+ NK cells or CD45.2+ ILC1s were analyzed by flow cytometric analysis in the liver of CD45.1 recipients, which were engrafted with BM cells of Ncr1gfp/gfp mice (n = 5) or Ncr1+/+ littermates (n = 4). (C) Percentages of CD45.2+ NK cells or CD45.2+ ILC1s were analyzed in the spleen or BM of CD45.1 recipient mice, which were engrafted with BM cells of Ncr1gfp/gfp mice (n = 5) or their Ncr1+/+ littermates (n = 4). (D and E) Data shown are representative dot plots of flow cytometric analysis (left panel) and summary data (right panel) of CD45.2+ILC2 (D) or CD45.2+ ILC3 (E) in SI in CD45.1 recipients, which were engrafted with BM cells of Ncr1gfp/gfp mice (n = 4) or their Ncr1+/+ littermates (n = 4). Error bars, standard deviations; ***, p < 0.001; **, p < 0.01; *, p < 0.05. The numerical data for panels B, C, D and E can be found in S1 Data. Lin─, CD3─CD19─; BM, bone marrow; ILC, innate lymphoid cells; Ncr1, natural cytotoxicity receptor 1; NK, natural killer; RORγt, retinoic acid receptor (RAR) related orphan receptor gamma t; SI, small intestine.