<i>Il10</i>^−/− mice show reduced virus loads and increased body weight loss during acute MCMV infection.

<p>B6 and <i>Il10</i>^−/− mice were infected i.v. with 5×10⁶ PFU <i>Δm157</i> MCMV. A) Virus titers on different days post infection were determined in lungs and salivary glands. Each symbol represents one individual mouse, horizontal line indicates the mean (n = 3–6, dashed line indicates detection limit). Data are representative of 3 independent experiments. B) IFN-γ, TNF-α and IL-12 protein concentrations were determined in the sera of B6 and <i>Il10</i>^−/− mice at day 5.5 post infection by cytometric bead array (BD, Bioscience) and murine IL-12 ELISA Kit (Peprotech). Each symbol represents one individual mouse, horizontal line indicates the mean (n = 3). Data are representative of 2 independent experiments. C, E) Body weight change of B6 and <i>Il10</i>^−/− mice was measured up to 14 or 20 days post infection. Change in percentage of body weight relative to day 0 is shown. One group of <i>Il10</i>^−/− mice was treated with a neutralizing anti-TNF-α antibody (C) or depleted of CD4 T cells (E). Right panel in E indicates the same experiment showing significances for day 6 p.i. Data are representative of 3 independent experiments. Each symbol represents the mean of 3 mice per group, error bars indicate the standard deviation. D) Virus titers in lungs and salivary glands on day 14 post infection of B6 and <i>Il10</i>^−/− mice with or without depletion of CD4 or CD8 T cells. Each symbol represents one individual mouse, horizontal line indicates the mean (n = 5–7, dashed line indicates detection limit). Pooled data from 2 independent experiments are shown. Statistical analysis was performed by 2-tailed unpaired student's t-test (* p<0.05, ** p<0.01, *** p<0.001).</p

NK-like cells are responsible for enhanced MCMV-specific CD4 T cell response in <i>Il10</i>^−/− mice.

<p>B6 and <i>Il10</i>^−/− mice were infected with 5×10⁶ PFU <i>Δm157</i> MCMV. A–D) B6 and <i>Il10</i>^−/− mice were either mock treated or depleted of NK-like cells using αNK1.1 (PK136) antibody. A) Lymphocytes from lungs were isolated at day 5.5. p.i. and <i>ex vivo</i> restimulated with a pool of M14, m18, M25, M112, m139 and m142 peptides (CD4 peptide pool). Percentages of IFN-γ⁺TNF-α⁺ peptide specific CD4 cells are shown (n = 3, error bars indicate standard deviation, data are representative from at least 3 experiments). B) Virus titers were determined in salivary glands at day 14 p.i. Each symbol represents one individual mouse, horizontal line indicates the mean, dashed line indicates detection limit (n = 3, data are representative of 2 independent experiments). C) IFN-γ, TNF-α and IL-12 protein concentrations were determined in the sera of B6 and <i>Il10</i>^−/− mice at day 3.5 p.i. Each symbol represents one individual mouse, horizontal line indicates the mean (n = 3, data are representative of 2 independent experiments). D) Body weight change of B6 and <i>Il10</i>^−/− mice was measured at the indicated time points. Changes in percentage of body weight relative to day 0 are shown. Each symbol represents the mean of 3 mice per group; vertical bars indicate the standard deviation. Data are representative of 3 independent experiments. E) Splenic NK1.1⁺CD3ε⁻ NK cells (NK) were isolated from B6 and <i>Il10</i>^−/− mice at day 3.5 p.i. Total numbers of IFN-γ⁺ NK cells and MFI of IFN-γ⁺ in NK cells are shown (upper panel). Total numbers of TNF-α⁺ NK cells and expression levels of NKG2D and NCR-1 on NK cells are shown (lower panel). Error bars indicate standard deviation; n = 3, data are representative of 3 independent experiments. Statistical analysis was performed by 2-tailed unpaired student's t-test (* p<0.05, ** p<0.01, *** p<0.001).</p

IL-12 contributes to enhanced CD4 T cell priming in <i>Il10</i>^−/− mice.

<p>B6 and <i>Il10</i>^−/− mice were infected with 5×10⁶ PFU <i>Δm157</i> MCMV and treated <i>in vivo</i> with αIL-12 antibody at days 0, 3, 4 p.i. A) Splenic NK1.1⁺CD3ε⁻ NK cells (NK) were isolated from B6 and <i>Il10</i>^−/− mice at day 3.5 p.i. Total numbers of IFN-γ⁺ NK and TNF-α⁺ cells are shown (error bars indicate standard deviation; n = 3, data are representative of 2 independent experiments). B) Lymphocytes were isolated from lungs at day 5.5 p.i. and <i>ex vivo</i> restimulated with the CD4 peptide pool. Fold increase in IFN-γ⁺ TNF-α⁺c peptide specific CD4 T cells between <i>Il10</i>^−/− and B6 is shown (n = 3, error bars indicates standard deviation, data are representative from 3 independent experiments). Statistical analysis was performed by 2-tailed unpaired student's t-test (** p<0.01, *** p<0.001, n.d. = not detected).</p

IL-10 differentially affects MCMV-specific CD4 vs CD8 T cell responses.

<p>B6 or <i>Il10</i>^−/− mice were infected i.v. with 5×10⁶ PFU <i>Δm157</i> MCMV. Lymphocytes from spleen, lungs, liver and salivary gland (A) or lung (B) were isolated at day 14 post infection and <i>ex vivo</i> restimulated with appropriate peptides. A, B) CD4 T cells were restimulated with a pool of M14, m18, M25, M112, m139 and m142 peptides (CD4 peptide pool, A, B), or with M25 and m142 peptide alone (B). Fold increase in percentage of IFN-γ⁺ TNF-α⁺ peptide-specific CD4 T cells between <i>Il10</i>^−/− and B6 mice (A, right panel, (n = 3), data are representative for at least 3 experiments, error bars indicate the standard deviation). (B) Total numbers (lower row) and percentages (upper row) of IFN-γ ⁺ TNF-α⁺ peptide-specific CD4 T cells from B6 and <i>Il10</i>^−/− mice. C) Lung lymphocytes were isolated from infected mice and M45- or M38-specific CD8 T cells were quantified by tetramer staining (n = 3, data are representative from at least 3 experiments, error bars indicate the standard deviation). Statistical analysis was performed by 2-tailed unpaired student's t-test (* p<0.05, ** p<0.01, *** p<0.001).</p

IL-10 is produced early upon MCMV infection.

<p>B6 or <i>Il10</i> GFP reporter (Tiger) mice were infected with 5×10⁶ PFU <i>Δm157</i> MCMV. A) IL-10 protein concentration was determined in the sera of infected B6 mice by IL-10 ELISA Set (BD, Biosciences) at indicated time points (n = 3, data are representative of 2 independent experiments, error bars indicate the standard deviation). B) Lung and spleen lymphocytes were isolated from infected <i>Il10</i> GFP reporter (Tiger) mice and control littermates at day 5.5 p.i. Percentages of GFP⁺ cells (after substraction of background fluorescence from littermate controls) are shown for the indicated cell populations: CD4⁺ cells, CD8⁺ cells, B220⁺ cells (B cells), NK1.1⁺CD3ε⁻ cells (NK cells), NK1.1⁺CD3ε⁺ cells (NK T cells), CD11b⁺CD11c⁻LyG/C⁻ cells (monocytes/macrophages) and splenic CD11c⁺CD11b⁺MHCII⁺B220⁻ cells (myeloid DCs). (n = 3, data are representative of 2 independent experiments, error bars indicate the standard deviation).</p

Unleashed NK/DC crosstalk promotes CD4 T cell priming in <i>Il10</i>^−/− mice.

<p>B6 and <i>Il10</i>^−/− mice were infected with 5×10⁶ PFU <i>Δm157</i> MCMV. A–C,F) DX5⁺CD3⁻ (NK) cells were isolated from B6 and <i>Il10</i>^−/− mice by MACS at day 3.5 p.i. Splenic DCs were isolated from naive B6 (A–C) mice or naive <i>Il10r</i>^−/− mice (F) by enrichment for CD11c⁺ cells. MCMV-specific CD4 T cells were isolated by MACS from spleens of naive M25-II mice and labeled with CFSE. CD11c⁺ cells were loaded with M25 peptide and co-cultured with CFSE-labeled M25-II cells without (no NK) or with addition of DX5⁺CD3⁻ (NK) cells isolated from B6 (A, B, F) or <i>Il10</i>^−/− (A, C) mice. As indicated, blocking antibodies for IFN-γ, TNF-α, NKG2D and NCR-1 were added to cultures. The frequencies of CFSE^low M25 II cells are shown. (n = 3, data are representative of 3 independent experiments). Dotted lines indicate the mean level of M25-II CFSE dilution in cultures without NK cells (no NK). D, E) B6 and <i>Il10</i>^−/− mice were treated <i>in vivo</i> with αIFN-γ antibodies at days 3 and 4 p.i. (D); with αTNF-α and αNKG2D antibodies at days 0, 3, 4 p.i. (E). Lymphocytes from lungs of infected mice were isolated at day 5.5 p.i. and <i>ex vivo</i> restimulated with a pool of M14, m18, M25, M112, m139 and m142 peptides (CD4 peptide pool). Fold increase (D, E) of IFN-γ⁺ TNF-α⁺ peptide specific CD4 cells between <i>Il10</i>^−/− and B6 mice is shown (n = 3, error bars indicates standard deviation, data are representative for 3 experiments). G) DC- <i>Il10r</i>^flox/flox, <i>Il10r</i>^flox/flox and <i>Il10r</i>^−/− mice were infected with 5×10⁶ PFU <i>Δm157</i> MCMV. Lymphocytes from lungs were restimulated with the CD4 peptide pool. Total numbers of IFN-γ⁺ TNF-α⁺ peptide-specific CD4 cells are shown. (n = 3, data are pooled from 2 independent experiments). Statistical analysis was performed by 2-tailed unpaired student's t-test (* p<0.05, ** p<0.01, *** p<0.001).</p

IL-10 alters the phenotype and function of DCs.

<p>B6 and <i>Il10</i>^−/− mice were infected with 5×10⁶ PFU <i>Δm157</i> MCMV. A) Splenocytes from infected mice were isolated at day 5.5 p.i. Representative FACS plots showing CD11c and I-A/I-E stainings of total spleen leukocytes (upper row). Total numbers of CD11c⁺CD8α⁻CD11b⁺MHCII⁺B220⁻ DCs and CD11c⁺CD8α⁺MHCII⁺B220⁻ DCs from infected B6 and <i>Il10</i>^−/− mice and naive <i>Il10</i>^−/− mice are shown (lower row, n = 3 except for naive <i>Il10</i>^−/− mice n = 1; data are representative for at least 3 experiments, error bars indicate the standard deviation). B) Expression levels of I-A/I-E and costimulatory molecules CD80, CD86 and CD40. Plots are gated on CD11c⁺CD8α⁻MHCII⁺B220⁻DC population at day 5.5 p.i. are shown (n = 3, error bars indicates standard deviation, data are representative from at least 3 experiments). Representative FACS pots (left column) and summary of MFI data (right column). C) Lung lymphocytes were isolated at day 5.5. p.i. and CD4 T cells were restimulated with a pool of M14, m18, M25, M112, m139 and m142 peptides (CD4 peptide pool) and CD8 T cells were restimulated with M45 peptide. Total numbers of IFN-γ⁺ TNF-α⁺ peptide specific CD4 and CD8 T cells are shown (n = 3, error bars indicate standard deviation, data are representative from at least 3 experiments). D) Splenic CD11c⁺ cells isolated at day 3.5 p.i. from B6 and <i>Il10</i>^−/− mice were enriched by MACS, loaded with M25 peptide and incubated with the naive MACS purified CFSE labeled TCR transgenic CD4 T cells specific for the M25 protein (M25-II cells) for 3 days. The frequencies of CFSE^low M25 II cells are indicated in the representative FACS plot. (n = 3 triplicates of respective cell cultures, data are representative of 3 independent experiments). Statistical analysis was performed by 2-tailed unpaired student's t-test (* p<0.05, ** p<0.01, *** p<0.001).</p