Figure 3. Effect of costimulation on LY49H⁺ NK-cell expansion in liver during acute MCMV infection.

Wild type C57BL/6, B7^-/- or CD28^-/- knockout mice infected with Smith MCMV (5 × 10⁴ pfu) had liver tissues removed 2 or 6 days after infection. Three hours prior to organ removal, each mouse received BrdU. Hepatic MNCs were prepared and NK-cells were enumerated and had division estimated by four color flow cytometry. The MNCs were stained with fluorescent dye-conjugated mAbs to CD3 (PerCP-Cy5.5), NK1.1 (PE) and Ly49H (APC) for cell surface markers, and intracellular staining with mAb to BrdU (FITC) as described in Methods. Percentages of each subset (CD3^- NK1.1⁺Ly49H⁺, CD3^-NK1.1⁺Ly49H^-, CD3^-NK1.1⁺Ly49H⁺BrdU⁺ or CD3^- NK1.1⁺Ly49H^-BrdU⁺) were determined and then multiplied by numbers of bulk MNCs. A. Representative flow cytometric plots at day 2 pi from CD3^-NK1.1⁺ gated cells. B. Mean cell numbers of CD3^-NK1.1⁺ gated Ly49H⁺ and Ly49H^- NK-cells and BrdU incorporation in hepatic MNC over time. Each data point represents mean ± standard error for n=3 mice. This experiment was performed twice with comparable results.