NKp46 staining allows the in situ visualization of mouse NK cells. For C57BL/6 mouse spleen (A) or inguinal lymph node (B), 7-μm serial frozen sections were fixed with acetone and stained with anti-NKp46 antiserum (green), CD19 mAb (red), and either CD3, CD11c, or CD11b mAb (blue). Samples were analyzed by confocal microscopy. A representative picture for each group is shown in the same anatomical region for each staining. WP, white pulp; RP, red pulp; T, T cell zone. (Original magnification, ×16.)

NKP46 genomic sequence can be used to genetically tag mouse NK cells. (A) Schematic representation of the human genomic fragment used for transgenesis. NKP46 exons are shown as black bars. (B) Flow cytometric measurement of human NKp46 expression in various gated subsets from huNKp46 transgenic mice. The indicated cell types were identified as described in Methods. Granulo., granulocytes; DC, dendritic cells. Human NKp46 expression (open histogram, thick line) or isotype control (gray histogram, thin line) is shown in the indicated subsets in B and in subsequent panels. (C) Flow cytometric measurement of human NKp46 expression on NK precursors (NKp), immature and mature NK cells, identified as described in Fig. 1. (D) Flow cytometric measurement of human NKp46 expression in gated NK1.1⁺ CD3⁻ cells from huNKp46 Tg mouse lymph nodes (inguinal), lung, liver and peripheral blood. (E) Redirected lysis assay of LAK cells derived from B6 (C57BL/6) or huNKp46 Tg (Tg) spleen cells against Daudi cells incubated with the indicated antibodies. The cytolytic function of LAK cells prepared from B6 and huNKp46 Tg mice were comparable. Results are representative of three experiments.

Expression of the activating NKp46 cell surface receptor on mouse NK cells. (A) Flow cytometric measurement of NKp46 expression in gated NK1.1⁺ spleen cells from RAG^−/− and RAG^−/−CD3ζ^−/−FcRγ^−/− mice (C57BL/6 background). NKp46 expression (open histogram, thick line) or isotype control (gray histogram, thin line) is shown in the indicated subsets in A and in subsequent panels. (B) Indicated cell types were identified from C57BL/6 splenocytes; Granulo., granulocytes; DC, dendritic cells. (C) Flow cytometric measurement of NKp46 expression on C57BL/6 NK precursors (NKp), immature and mature NK cells, identified as described (47) as CD122⁺lin⁻NK1.1⁻DX5⁻, CD122⁺NK1.1⁺DX5⁻, and CD122⁺NK1.1⁺DX5⁺, respectively. (D) Flow cytometric measurement of NKp46 expression in gated NK1.1⁺ CD3⁻ cells from C57BL/6 mouse lymph nodes (inguinal), lung, liver, and peripheral blood. (E) Comparison of the percentage of CD3⁺ cells within the NKp46⁺ (black bars), NK1.1⁺ (gray bars), and DX5⁺ subsets in various organs of C57BL/6 mice. (F) Flow cytometric measurement of NKp46 expression in gated splenic DX5⁺CD3⁻ cells from the indicated strains. (G) Flow cytometric measurement of NKp46/DX5 expression in gated NK1.1⁺CD3⁻ cells from the spleen or the liver (27), obtained from 5-week-old C57BL/6 mice. (Left) Isotype staining. Results in A–C and D–G show one representative set of data of three independent experiments. Results in E are presented as the mean ± SD of at least three independent experiments. Similar staining results were obtained by using either affinity purified goat anti-mouse NKp46 polyclonal antibodies and the rat anti-NKp46 29A1.4 mAb (SI Fig. 7).

Mouse NK cells can be activated via NKp46. C57BL/6 spleen cell suspensions were stimulated for 4 h in wells coated with the indicated antibodies, in the presence of soluble anti-CD107a antibodies. Control antibodies included mouse and goat IgG. Cells were then stained for surface DX5/CD3 and intracellular IFN-γ. Results show the expression of CD107a and IFN-γ in gated DX5⁺CD3⁻ cells, and are representative of five independent experiments.

Dissection of NKP46 regulatory sequences and generation of NKDTR/EGFP mice. (A) The putative 400-bp human NKP46 promoter was used in a dual luciferase assay in K562 or NKL cell lines. The assay was also performed with promoterless or SV40 promoter vectors for negative and positive control, respectively. (B) Schematic representation of the transgenic construct used to make NKDTR/EGFP transgenic mice. For details, see Methods. (C) Flow cytometric measurement of EGFP expression in immune cell types of NKDTR/EGFP or control mice, identified as in Fig. 1. Granulo, granulocytes. Results are representative of at least three experiments. (D) NKDTR/EGFP transgenic or control littermates were injected i.p. twice with DT within a 24-h interval. The percentage of NKp46⁺CD3⁻ or CD3⁺ cells in the blood was measured over time after DT injections. Results are representative of at least three experiments. (E) NKDTR/EGFP (open histograms) and wild-type littermates (filled histograms) were injected with DT. Twenty-four hours later, the number of NK cells was measured in the indicated organs. Results show the mean ± SD number of NK cells in DT-treated wild-type or NKDTR/EGFP mice, expressed as the percentage of the controls (NK cell numbers in untreated wild-type mice); four mice per group. (F) NKDTR/EGFP transgenic or control mice were injected i.p. twice with DT within a 24-h interval. Forty-eight hours after DT injections, the percentage of NK cells, γδ T cells and CD1d-restricted NKT cells was measured in the peripheral blood. Representative results of three experiments are expressed as the percentage of indicated cell subsets as compared with control mice. (G) Spleen cells from mice in E were cultured o/n with IL-2 and used as effector cells in a standard 4-h Cr⁵¹ cytotoxicity assay against YAC-1 cells. Results are expressed as the mean ± SD percentage of specific lysis of YAC-1 cells with four mice in each group.