(A-E) Groups of CD11c DTR tg mice were injected with DT (open symbols). Control mice received PBS injections (solid symbols). One day or 4 days later, mice were injected with the indicated TLR ligand and analyzed 18-24h later (A). Immunophosphatase staining of splenic sections from TLR stimulated mice for the indicated cell surface markers 2 or 5 days after DT injection. Magnification, 200x (B). The percentage of CD11c^high MHC-II⁺ cells in the spleen was evaluated by flow cytometry analysis at the indicated time points after DT injection (C). Cytotoxicity (D) and IFN-γ production (E) of splenic NK cells were determined at the indicated timepoints after DT injection against RMA-S/H60 targets. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=3) and results are shown from two independent experiments, which are representative of three experiments performed.
(F) Dendritic cells but not macrophages rescue NK cell priming. CD11c DTR tg mice were injected i.v. with 3-10×10⁶ BM-derived DC or macrophages one day prior to DC ablation. One day later, mice were injected with TLR3 ligand and cytotoxicity (left) and IFN-γ (right) production of NK cells were assayed against RMA-S/H60 cells 18h after TLR stimulation. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=3-4) and results are representative of five (cytotoxicity data) or three (IFN-γ data) separate experiments. nd, not done.
(G) NK cells do not require cell-autonomous TLR-signaling for priming. Mixed BM chimeric mice were generated by injecting lethally irradiated mice with BM cells derived from the indicated TLR signaling-deficient mouse strains or from B6 control mice expressing the indicated congenic markers. BM chimeric mice were injected 8-12 weeks later with the indicated TLR ligands. IFN-γ production of CD45.1⁺ (solid bars) and CD45.2⁺ NK cells (open bars) was assayed in response to RMA-S/H60 target cells 18h after TLR stimulation. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=3-4) and results are representative of two separate experiments.

(A and B) One day after DT injection, B6 mice (solid symbols) or CD11c DTR tg mice (open symbols) were injected subcutaneously (s.c.) into one footpad with the indicated TLR ligand. NK cells were purified from the indicated organs and IFN-γ production (A) and cytotoxicity (B) was determined in response to RMA-S/H60 cells 18-24h later. Similar results were obtained with YAC-1 and RMA-S target cells (data not shown). Error bars display s.d. (n=3-4) and results are representative of three separate experiments.
(C and D) Blocking entry of NK cells into LN results in dramatically decreased numbers of effector NK cells in peripheral tissues. B6 mice were injected with control Ig (Ctrl. Ig; solid symbols) or an anti-CD62L antibody (open symbols) blocking entry of NK cells into the LN. One day later, mice were injected with the indicated TLR ligands. Cytotoxicity (C) and IFN-γ production (D) of NK cells purified from the indicated tissues in response to RMA-S/H60 cells was determined 18-24h after local TLR stimulation. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=3-4) and results are representative of three separate experiments.
(E and F) Circulating naïve NK cells need to enter the draining LN for priming. B6 mice (CD45.2⁺) were injected i.p. with control Ig (solid symbols) or an anti-CD62L antibody (open symbols). Two hours later, 3×10⁶ purified NK cells (CD45.1⁺) were injected i.v. followed by TLR stimulation 12h later. IFN-γ production (E) and granzyme B expression (F) of transferred CD45.1⁺ NK cells isolated from the indicated tissues in response to RMA-S/H60 cells was determined by intracellular cytokine staining and electronic gating on NK cells 18-24h after local TLR stimulation. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=3-4) and results are representative of two separate experiments. nd, not done.

(A) Highly purified IFNAR^-/- NK cells were either cultured in the presence (solid squares) or absence (open squares) of BM-DC from the indicated mouse strains stimulated as indicated. Lysis of RMA-S/H60 cells by NK cells was determined 12h later. Similar results were obtained with RMA-S and YAC-1 targets (data not shown).
(B and C) BM-DC from B6 mice were stimulated with the indicated TLR ligands, an agonistic anti-CD40 antibody (B) or with IFN-I (C) and co-cultured with NK cells in the presence of control serum (solid squares) or antisera specific for the indicated cytokines (open squares). NK cell cytotoxicity against RMA-S/H60 target cells was determined 12h later. Similar results were obtained with RMA-S and YAC-1 targets (data not shown).
(D) Cytotoxicity of NK cells either co-cultured with IFN-I stimulated DC in the same well (solid squares) or spatially separated (open squares) by a membrane (0.4μm pores) was assayed against RMA-S/H60 cells. Similar results were obtained with RMA-S and YAC-1 targets (data not shown).

(A-D) CD11c DTR tg mice or RAG1^-/- CD11c DTR tg were injected with diphtheria toxin (DT) to ablate all CD11c^high DC (open symbols). Control mice received PBS injections (solid symbols). One day after DT injection, mice were injected i.p. with the indicated TLR ligands or an agonistic anti-CD40 antibody or with control injections of PBS or control Ig (not shown). NK cell effector functions were determined 18h after stimulation. IFN-γ production by highly purified splenic NK cells in response to immobilized anti-NKR-P1C/NK1.1, anti-Ly49D and anti-NKG2D antibodies (A). Cytotoxicity (B) and IFN-γ production (C) of NK cells against RMA-S/H60 targets. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). The percentage of NK1.1⁺CD3⁻ cells in the lymphocyte populations was determined prior to the cytotoxicity assay and lymphocyte numbers were adjusted to contain the same number of NK cells. Thus, an NK cell:target ratio of 6:1 is equivalent to a 100-200:1 splenocyte:target ratio. Granzyme B (D) expression by NK cells. IFN-γ production and granzyme B expression were determined by intracellular cytokine staining and electronic gating on NK cells. Error bars display s.d. (n=3) and results are representative of at least three separate experiments.
(E) Groups of CD11c DTR tg mice were injected with DT (open squares). Control mice received PBS injections (solid squares). Some mice received two injections of anti-asialo GM1 antiserum to deplete all NK cells (open circles) and control mice received equal amounts of normal rabbit serum (NRS). One day later, mice were injected with TLR3 ligand or PBS. Antibody-dependent cytotoxicity (ADCC) of NK cells was evaluated against RMA cells pre-incubated with anti-Thy1.2 or control antibodies 18h after TLR stimulation.

(A and B) DC-ablated (open symbols) and control mice (solid symbols) were infected with the indicated pathogens 12h after DT injection. NK cell activation was analyzed 24h (Listeria) or 48h (LCMV and Vaccinia) later by determining cytotoxicity (A) and IFN-γ production (B) in response to RMA-S/H60 target cells. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=3-4) and results are representative of three separate experiments.
(C) NK cell-mediated control of local Listeria infection requires the presence of DC. Mice were depleted of NK cells by injection anti-NK1.1 antibody two days prior to and at the day of infection. Control mice received the same amount of mouse Ig. DC were ablated by DT injections one day before infection. Mice were infected subcutaneously with Listeria and bacterial titers from the draining LN and spleen were determined one day later. Each symbol represents results from an individual mouse and bars represent the arithmetic mean.

(A and B) IL-15 is sufficient to prime NK cells in vitro. Highly purified NK cells were incubated overnight with the indicated cytokines. Cytotoxic activity of NK cells (A) and IFN-γ production (B) was determined in response to RMA-S/H60 target cells. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). The numbers in the histograms (B) represent the percentage of IFN-γ⁺ NK cells as determined by intracellular cytokine staining.
(C-E) NK cell priming requires IFN-I signaling. B6 (solid symbols) or IFNAR^-/- mice (open symbols) were injected with the indicated TLR ligands or pathogens and cytotoxicity (C) or IFN-γ production (D) in response to RMA-S/H60 target cells and granzyme B expression (E) by NK cells were assayed 18h later. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=4) and results are representative of five separate experiments.
(F and G) NK cell priming does not require NK cell-autonomous IFN-I signaling. Purified CD45.2⁺ B6 (solid bars) or CD45.2⁺ IFNAR^-/- NK cells (open bars) were transferred i.v. into CD45.1⁺ B6 recipients. One day later, mice received i.p. injections of PBS or the indicated TLR ligands and IFN-γ production (F) of CD45.2⁺ NK cells in response to RMA-S/H60 cells and granzyme B expression (G) by donor NK cells was assayed 18-24h after TLR stimulation. Similar results were obtained with RMA-S and YAC-1 targets (data not shown). Error bars display s.d. (n=3-4) and results are representative of three separate experiments.

(A and B) IL-15Rα expression (shaded histograms) by splenic DC 24h after TLR or anti-CD40 stimulation in vivo (A) or by BM-DC 24h after in vitro stimulation (B). Open histograms represent staining with control Ig. The numbers indicate the percentage of IL-15Rα⁺ CD11c^high MHC-II⁺ cells. Data are representative of at least three independent experiments.
(C) Highly purified CD45.1⁺ NK cells were injected into either CD45.2⁺ B6 or CD45.2⁺ IL-15^-/- mice. The percentage of annexin V^- CD45.1⁺ NK cells (in the lymphocyte gate) recovered from the spleens of B6 or IL15^-/- mice 6h after transfer are indicated. Data are representative of five independent experiments.
(D-F) Highly purified CD45.1⁺ NK cells were injected i.v. into either CD45.2⁺ B6 (solid bars) or CD45.2⁺ IL-15^-/- mice (open bars). Mice were injected 2h later with the indicated TLR ligands or anti-CD40. Control mice received injections of PBS or control Ig (Ctrl. Ig), respectively. IFN-γ production of gated CD45.1⁺ NK cells against RMA-S/H60 target cells was determined 4h after TLR stimulation by intracellular cytokine staining and electronic gating on donor NK cells. Some IL-15^-/- mice received i.p. injections of 4μg IL-15 at the time of TLR stimulation (IL-15 i.p.). In some experiments, 10ng/ml IL-15 was added during the in vitro restimulation of donor NK cells with RMA-S/H60 target cells required for the determination of intracellular cytokine expression (IL-15 in vitro). Similar results were obtained with RMA-S or YAC-1 target cells (data not shown). Error bars display s.d. (n=3) and data are representative of at least three independent experiments. nd, not done.