Functional defect in the NK-cell activity of IK^L/L Rag^−/− mice after TLR9 stimulation. (A) CD69 expression on splenic NK cells (gated on NK1.1⁺ DX5⁺) from C57BL/6, IK^+/+ Rag^−/−, or IK^L/L Rag^−/− mice 18 hours after intravenous injection of PBS, CpG, CpG-Dotap, or PIC. Numbers indicate mean fluorescence intensity for each histogram. (B-C) C57BL/6, IK^+/+ Rag^−/−, or IK^L/L Rag^−/− mice were injected with PBS, CpG, CpG-Dotap, or PIC. Some IK^L/L Rag^−/− mice were also reconstituted with 1 to 2 × 10⁶ purified pDCs 1 day before CpG-Dotap injection (CpG-Dotap + pDC). After 4 hours (B) or 18 hours (C), spleen cells were labeled, and IFN-γ (B) or GrzB (C) production by NK cells (NK1.1⁺ DX5⁺ cells) was determined by intracellular staining. Results are expressed as the mean of the percentage of IFN-γ⁺ (B) or GrzB⁺ (C) NK cells ± SD from 5 mice. Data are from 3 experiments. *P < .05. **P < .01. ***P < .001. (D) Top panel: the morphology and enumeration of GrzB⁺ spots in NKp46⁺ DX5⁺ NK1.1⁺ cells from C57BL/6 mice 18 hours after injection of CpG-Dotap by multispectral imaging. Representative images show NK cells (NKp46⁺ DX5⁺ NK1.1⁺) with 1 (first row), 3 (second row), and 10 (third row) GrzB⁺ spots. Bottom panel: the indicated mouse strains were injected with PBS alone, CpG, CpG-Dotap, or PIC. After 18 hours, the numbers of GrzB⁺ spots in NK cells were quantified. Bars represent the distribution of GrzB⁺ spot counts on NKp46⁺ DX5⁺ NK1.1⁺ cells, and numbers indicate the mean of GrzB⁺ spots per NK cell. One representative experiment of 4 is shown. (E) C57BL/6, IK^+/+ Rag^−/−, and IK^L/L Rag^−/− mice were injected with PBS, CpG, CpG-Dotap, or PIC. Some IK^L/L Rag^−/− mice were also reconstituted with 1 to 2 × 10⁶ purified pDC 1 day before CpG-Dotap injection (CpG-Dotap + pDC). After 18 hours, NK-cell cytotoxic activity was determined with a 4-hour in vivo killing assay using β2m knockout splenocytes as target cells. Data shown are from 4 experiments. *P < .05.