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.</p

Dependence of innate lymphoid cell 1 development on NKp46

<div><p>NKp46, a natural killer (NK) cell–activating receptor, is involved in NK cell cytotoxicity against virus-infected cells or tumor cells. However, the role of NKp46 in other NKp46⁺ non-NK innate lymphoid cell (ILC) populations has not yet been characterized. Here, an NKp46 deficiency model of natural cytotoxicity receptor 1 (<i>Ncr1</i>)^gfp/gfp and <i>Ncr1</i>^gfp/+ mice, i.e., homozygous and heterozygous knockout (KO), was used to explore the role of NKp46 in regulating the development of the NKp46⁺ ILCs. Surprisingly, our studies demonstrated that homozygous NKp46 deficiency resulted in a nearly complete depletion of the ILC1 subset (ILC1) of group 1 ILCs, and heterozygote KO decreased the number of cells in the ILC1 subset. Moreover, transplantation studies confirmed that ILC1 development depends on NKp46 and that the dependency is cell intrinsic. Interestingly, however, the cell depletion specifically occurred in the ILC1 subset but not in the other ILCs, including ILC2s, ILC3s, and NK cells. Thus, our studies reveal that NKp46 selectively participates in the regulation of ILC1 development.</p></div

Absence of TRAIL⁺ ILC1s in NKp46-deficient mice.

<p>(A) Percentages of TRAIL⁺ ILC1s were analyzed by flow cytometric analysis in the liver of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates. ILC1s were gated on Lin^─NK1.1⁺NKp46⁺(or GFP⁺ for <i>Ncr1</i>^gfp/gfp mice) TRAIL⁺CD49b^─ among lymphocytes. (B) Quantification of TRAIL⁺ ILC1s in the liver of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates for (A) (<i>n</i> = 5). (C) Quantification of TRAIL⁺ ILC1s in other organs (spleen, <i>n</i> = 5; BM, <i>n</i> = 5; SI, <i>n</i> = 4) of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates. **, <i>p</i> < 0.01; *, <i>p</i> < 0.05. The numerical data for panels B and C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004867#pbio.2004867.s007" target="_blank">S1 Data</a>. Lin^─, CD3^─CD19^─; BM, bone marrow; GFP, green fluorescent protein; ILC1, innate lymphoid cell 1; <i>Ncr1</i>, natural cytotoxicity receptor 1; NK, natural killer; SI, small intestine; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.</p

NKp46 plays a cell-intrinsic role in regulating ILC1 development.

<p>(A) Scheme of BM transplantation using BM cells of CD45.2 <i>Ncr1</i>^gfp/gfp mice or <i>Ncr1</i>^+/+ 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 <i>Ncr1</i>^gfp/gfp mice (<i>n</i> = 5) or <i>Ncr1</i>^+/+ littermates (<i>n</i> = 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 <i>Ncr1</i>^gfp/gfp mice (<i>n</i> = 5) or their <i>Ncr1</i>^+/+ littermates (<i>n</i> = 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 <i>Ncr1</i>^gfp/gfp mice (<i>n</i> = 4) or their <i>Ncr1</i>^+/+ littermates (<i>n</i> = 4). Error bars, standard deviations; ***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; *, <i>p</i> < 0.05. The numerical data for panels B, C, D and E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004867#pbio.2004867.s007" target="_blank">S1 Data</a>. Lin^─, CD3^─CD19^─; BM, bone marrow; ILC, innate lymphoid cells; <i>Ncr1</i>, natural cytotoxicity receptor 1; NK, natural killer; RORγt, retinoic acid receptor (RAR) related orphan receptor gamma t; SI, small intestine.</p

NKp46 is required for ILC1 development.

<p>(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 <i>Ncr1</i> ^gfp/+ or <i>Ncr1</i>^gfp/gfp mice were used. (B) Percentages or quantities of ILC1s were determined by flow cytometric analysis in the liver of <i>Ncr1</i>^gfp/gfp, <i>Ncr1</i>^gfp/+, and <i>Ncr1</i>^+/+ mice. (C) NK cells were gated on Lin^─NK1.1⁺NKp46⁺(or GFP⁺ for <i>Ncr1</i>^gfp/gfp mice)CD49b⁺CD49a^─ among lymphocytes. ILC1s were gated on Lin^─NK1.1⁺NKp46⁺(or GFP⁺ for <i>Ncr1</i>^gfp/gfp mice)CD49b^─CD49a⁺ among lymphocytes. (D) Quantification of ILC1s in different organs or tissues in <i>Ncr1</i>^gfp/gfp mice and <i>Ncr1</i>^+/+ littermates, i.e., summary data for (C). Each line demonstrates percentages of ILC1s for a pair of <i>Ncr1</i>^gfp/gfp and <i>Ncr1</i>^+/+ littermates. (E) Quantities of NK cells or ILC1s in different organs of <i>Ncr1</i>^gfp/gfp mice and their <i>Ncr1</i>^+/+ littermates (<i>n</i> = 4). Error bars, standard deviations; ***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; *, <i>p</i> < 0.05. The numerical data for panels B, D and E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004867#pbio.2004867.s007" target="_blank">S1 Data</a>. 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; <i>Ncr1</i>, natural cytotoxicity receptor 1; SI, small intestine; SSC-A, side scatter area; SSC-H, SSC height; SSC-W, SSC width.</p

IL15 does not prevent loss of NK cell number.

<p>IL15Tg and littermate controls were injected with PBS or EL4. After 3 weeks, bone marrow was evaluated for NK cell number. A. FACs profile of NK1.1⁺CD3⁻ cells obtained from WT or IL15Tg mice with or without tumor. B. Bone marrow cellularity and absolute number of NK cells. The absolute number of NK cells was calculated by multiplying the percentage of NK1.1⁺CD3⁻ cells by the number of bone marrow cells. Data shown were means and SEM, involving 4 mice per group.</p

EL4 tumor growth causes a decrease in splenic and bone marrow NK cells.

<p>Mice were injected subcutaneously with 5×10⁶ EL4 cells and compared to mice that received PBS. The number of NK cells was calculated by multiplying the percentage of NK cells by the cellularity of the spleen or bone marrow. A. FACs profile of splenocytes stained with CD3 and NK1.1. NK cells were characterized as NK1.1⁺CD3⁻. The percentage of NK cells is given as the mean±SEM of five animals. B. Kinetics of tumor growth and reduction of NK cells. Spleens from EL4 bearing mice were obtained every six days and compared to control mice that were sacrificed on day 0. EL4 tumor growth was measured using calipers and the average diameter was obtained from the width and length of the tumor. C. Splenic cellularity and NK cell number from control and EL4-bearing mice. Each symbol represents the mean of a separate experiment. A paired T test was used to calculate the p-value. D. FACs profile of the bone marrow. NK cells are characterized as NK1.1⁺CD3⁻. The percentage of NK cells is given as the mean±SEM of five animals. E. Bone marrow cellularity and NK cell number obtained from control and EL4-bearing mice from several experiments. The number of NK cells was calculated by multiplying the percentage of NK cells by the number of bone marrow cells. Each symbol represents the mean of a separate experiment. A paired T test was used to calculate the p-value. Data in C–E were obtained when mice reached early removal criteria, i.e., the tumor reached 2 cm in diameters, usually at 4 weeks after tumor challenge.</p

NK cell progenitors are reduced in EL4 tumor-bearing mice.

<p>B6 mice were injected with PBS or EL4 and sacrificed after 21 days. NK cell progenitors were characterized as CD122⁺NK1.1⁻CD3⁻. A. FACs profile of NK cell progenitors. B. Absolute number of progenitors was calculated by multiplying the percentage of CD122⁺NK1.1⁻CD3⁻ cells by the total number of NK cells. Data shown were means and SEM, involving 4 mice per group.</p

Decrease in CLPs, NKPS and Block in NKP progression.

<p>B6 mice were sublethally irradiated with 500 Rad prior to EL4 or PBS injection. Twenty-four hours later 5FU treated bone marrow from congenic CD45.1 mice was injected into control or EL4-bearing mice. Three weeks later bone marrow was harvested to evaluate NK cell progenitors. Donor NK cells were identified by an anti-CD45.1. A. Diagram of experiments. B. FACs profile of CD45.1⁺ NK1.1⁺ cells. Data shown are from gated CD45.1⁺CD3⁻ cells. The numbers shown in the panels are means and SEM, involving a total of 5 mice per group. C. Absolute number of CD45.1⁺ NK cells. D. Absolute number of donor B cells. E. FACS profile of donor CLP, characterized as CD45.1⁺Lin⁻CD127⁺cKit^int. Data are representative of 5 mice per group. F. Absolute number of CD45.1⁺ and CD45.1⁻ CLP. Data shown are means and SEM, involving a total of 5 mice per group.</p

Apoptosis and NK cell proliferation do not account for NK cell depletion.

<p>Mice were injected subcutaneously with PBS or 5×10⁶ EL4 cells. After 3 weeks, control and EL4-bearing mice were sacrificed and splenocytes were stained. A. The percentage of Annexin V⁺NK1.1⁺CD3⁻ cells was used to determine the level of NK cell apoptosis. B. Three hours prior to euthanasia control and tumor-bearing mice were injected with BrdU. Splenic NK cells were then stained for intracellular BrdU. The splenic NK cell population was gated on CD122⁺CD3⁻ cells. Data shown were means and SEM, involving 5 mice per group.</p