(A) IL-10 protein concentrations in lung and spleen homogenates from naïve C57BL/6 (wt) mice (closed bars) and wt mice infected for 4 days with MCMV (open bars) were measured by cytometric bead array. Results represent the mean +/− SEM of 4-6 mice per group and 2 experiments are shown. Naive mice are combined from 2 separate experiments. (B and C) IL-10 expression by pulmonary (B) and splenic (C) leukocytes in naïve mice (closed bars) and mice infected for 4 days with MCMV (open bars). Expression was analysed following ex vivo stimulation for 5 hours with PMA/ionomycin (CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells and NK1.1⁺CD3⁻ cells), LPS (inflammatory macrophages, 7/4^hiF4/80^intLy6G⁻CD11c^low/intCD11b^hi/int; alveolar macrophages, CD11c^hiCD11b^lo/int; dendritic cells, CD11c^hiMHCII^hi) or PMA/ionomycin and LPS (B cells, CD3⁻CD19^hi). Results represent the mean +/− SEM of 7 mice per group, and represent three independent experiments.

MCMV-infected mice were treated with IgG or anti-IL-10R on day 0 and cellular accumulation assessed 2, 4 and 7 days later. (A and B) Representative bivariate flow cytometry plots showing NK1.1+CD3− cell accumulation in lungs (A) and spleens (B) of IgG (left panel) and anti-IL-10R (right panel) treated mice, 4 days post infection. (C and D) Numbers of NK1.1+CD3− cells in the lungs (C) and spleens (D) of IgG (closed circles) and anti-IL-10R (open circles) treated mice analyzed over 7 days. Results are expressed as the mean +/− SEM of 6-10 mice per group and are representative of 5 independent experiments.

Mice were infected with MCMV and treated with IgG or anti-IL-10R. (A and B) Apoptosis of NK cells was assessed by Annexin V binding. Representative overlay histograms of Annexin V binding of NK cells in the lungs (A) and spleens (B) of mice following treatment with anti-IL-10R (solid line) or IgG (dashed line) 4 days post infection (shaded histogram = fluorescence minus one (FMO) control). Annexin V binding by NK cells in the lungs (C) and spleens (D) of mice treated with IgG (closed circles) or anti-IL-10R (open circles) shown as % of NK+CD3− cells over a 7-day time course. Results are expressed as the mean +/− SEM of 4-6 mice per group and are representative of 2-3 independent experiments. (E) Intracellular expression of active caspase-3 by NK+CD3− cells in the lungs and spleens of mice 4 days post infection, treated with IgG (closed bars) or anti-IL-10R (open bars) directly ex vivo. Results are expressed as the mean +/− SEM of 10 mice per group and are representative of 3 independent experiments.

(A and B) MCMV-infected mice were treated with IgG (closed symbols) or anti-IL-10R (open symbols) and on days 2, 4 and 7, MCMV glycoprotein B (gB) was detected by quantitative PCR and normalized to β-actin. Data is expressed as genome copy number per 100ng genomic DNA. Median of 4-6 mice/group +/− interquartile range is shown. Data represents 2 (day 2), 5 (day 4) or 3 (day 7) experiments in total. (C and D) Mice were infected with MCMV, treated with combinations of IgG or anti-IL-10R +/− anti-NK1.1, and at 4 days post-infection genomic DNA was measured in the lungs (C) and spleen (D). Horizontal dashed lines depict the lower limit of detection. Circles: IgG; diamonds: anti-IL-10R; triangles: IgG and NK depletion; inverted triangles: anti-IL-10R and NK depletion. Data from 2-3 experiments is shown.

MCMV-infected mice were treated with IgG (closed bars) or anti-IL-10R (open bars) on day 0. (A and B) Numbers of NK+CD3− cells in the lungs (A) and spleens (B) expressing surface CD107a after ex vivo incubation with monensin. (C-F) IFNγ production by NK cells was assessed ex vivo. (C and D) Representative zebra flow cytometry plots showing direct ex vivo IFNγ production by NK+CD3− cells in lungs (C) and spleens (D) of IgG (left panel) and anti-IL-10R (right panel) treated mice, 4 days post infection. % and MFI are shown and are representative of over 18 mice/group from at least 3 experiments (E and F) Total numbers of IFNγ producing NK+CD3− cells in the lungs (E) and spleens (F) of IgG (closed bars) and anti-IL-10R (open bars) treated mice analysed over 7 days. Results are expressed as the mean +/− SEM of 6 mice per group and are representative of 2-3 independent experiments. (G and H) Cytokine protein concentrations in lung (G) and spleen (H) homogenates from mice infected for 4 days with MCMV and treated with IgG (closed bars) or anti-IL-10R (open bars) were measured by either cytometric beads (IL-1α, IL-6, IL-10, IFNγ and TNF) or ELISA (IL-12). Results represent the mean +/− SEM of 6 mice per group.

MCMV-infected mice were treated with IgG (closed bars) or anti-IL-10R (open bars) and cellular analysis performed 4 days later. NK cell subsets in the lung (A) and spleen (B) were defined based on CD11b and CD27 co-expression, and Annexin V binding measured. Expression of CD25 (C) and CD69 (D) by Annexin V− (closed bars) and Annexin V+ (open bars) NK cells in the lungs and spleens was assessed. (E) Representative histograms of CD25 (left) and CD69 (right) expression by pulmonary (top) and splenic (bottom) NK cells from IgG (dashed line) and anti-IL-10R (solid line) treated mice. All results are representative of 2 independent experiments, each constituting of 8 mice/group. (F and G) Mean fluorescent intensity (MFI) of CD25 and CD69 expression by pulmonary (F) and splenic (G) NK cells from IgG (closed bars) and anti-IL-10R (open bars) treated mice 4 days after infection. (H and I) Percentages of NK cells in the lung (H) and spleen (I) co-expressing intracellular active caspase-3 and CD25 or CD69. Results are expressed as mean +/− SEM of 8 mice per group from 3 independent experiments.