CX3CR1 expression defines 2 functionally distinct KLRG1⁺ NK-cell subsets. (A) Freshly isolated cells from BM and spleen of CX3CR1^+/GFP mice were incubated for 18 hours, at 37°C, 5% CO₂ in the presence of IL-2 (500U) and IL-12 (100 ng/mL). Brefeldin A (10 μg/mL) was added during the last 6 hours. At the end of treatment, cells were stained with anti-NK1.1–, anti-KLRG1–, and anti-CD3ϵ–specific mAbs. Permeabilized cells were stained with anti–IFN-γ–specific mAb, and analyzed by FACS. Dot plots show a representative experiment of 3 performed. Numbers in dot plots indicate the percentage of IFN-γ–producing NK cells relative to CX3CR1-GFP expression on KLRG1⁺ NK-cell population. Histograms show the mean values ± SD of the percentage of IFN-γ⁺ NK cells in the 2 different KLRG1⁺ populations from BM and spleen of a total of 8 animals analyzed in independent experiments. *P < .05. □ indicates KLRG1⁺CX3CR1-GFP⁻; indicates KLRG1⁺/CX3CR1-GFP⁺. (B) Sorted KLRG1⁺/CX3CR1-GFP⁻ and KLRG1⁺/CX3CR1-GFP⁺; NK-cell subsets were treated as described in panel A and intracellular IFN-γ expression was analyzed by FACS. Numbers in the dot plots indicate the mean percentage ± SD of IFN-γ⁺ cells within each NK-cell subset from the spleen of a total of 5 animals analyzed in independent expriments. (C) Sorted KLRG1⁺/CX3CR1-GFP⁻ and KLRG1⁺/CX3CR1-GFP⁺ splenic NK-cell subsets were cultured 24 hours in the presence of IL-15 (100 ng/mL), and used as effector cells in a 4 hour ⁵¹Cr release assay against Yac-1 target cells at the indicated effector:target ratios. Data shown are representative of 1 of at least 3 experiments performed.