Figure 2. CD11c^high DC but not macrophages are required for NK cell priming.

(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.