MCP-1 is expressed in the airways of influenza virus infected mice.

<p>(A) C57BL/6 mice were infected with HKx31 and the presence of MCP-1 in the BALF was determined by ELISA at the indicated days post-infection. Data shown are means+S.E.M. from one experiment with 5 mice per time point. Similar results were found in an independent experiment at day 2 and 5 post-infection.</p

NK cells migrate CCR2-dependent to the BAL during influenza virus infection.

<p>Mixed BM chimeric mice were constructed by injecting a mix of BM from CCR2^−/− (CD45.2) and C57BL/6.SJL (CD45.1) into lethally irradiated CD45.1.2. recipients. After 6–8 weeks, mice were infected with influenza virus or left uninfected. (A) Representative FACS plots showing CD45.1 and CD45.2 staining of NK cells (TCRβ⁻NK1.1⁺) recovered from the indicated organs 5 days p.i. (B-D) Ratio of CD45.2 (CCR2^−/−)/CD45.1 (<i>wt</i>) NK cells calculated by dividing absolute numbers of CD45.2⁺ NK cells by absolute numbers of CD45.1⁺ (<i>wt</i>) NK cells. Ratio of CD45.2/CD45.1 NK cells recovered from the indicated organs shown as average ± S.E.M. at the indicated days (B) and shown as individual mice at day 5 (C), and the ratio in the spleen and BM of individual mice are connected by a line at day 5 (D) after influenza virus infection. Representative results of two independent experiments are shown with 4–6 mice per group. Statistical analysis was performed using a Mann-Whitney U test. *, P<0.05.</p

A subset of NK cells expresses the CCR2 receptor.

<p>(A) Gating strategy (upper panel) for NK cells (TCRβ⁻NK1.1⁺DX5⁺) and representative histograms (lower panel) showing CCR2 expression on NK cells recovered from the indicated organs (blood, lung, BM and spleen) of naïve C57BL/6 (<i>wt</i>) or CCR2^−/− mice. (B,C) Expression of CD27 and CD11b (C) or KLRG1 on electronically gated NK cells (TCRβ⁻NK1.1⁺) or CCR2⁺ NK cells. The depicted FACS plots are representatives of 9 mice that were analyzed in two independent experiments. Statistical analysis was performed using a Mann-Whitney U test (C) and KLRG-1-expression on CCR2⁺ or CCR2⁻ NK cells is not significantly different (Mann-Whitney U test, *, P<0.05).</p

Epitope prediction for MHC B4, B15, B19 and B21 based on anchor residues.

<p>Predicted epitopes and their localization based on anchor residues that have been described for B4, B15, B19 and B21 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Kaufman1" target="_blank">[32]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Koch1" target="_blank">[34]</a>. X represents any amino acid. Anchor residues specific for the different MHC types are indicated in bold. A variable number of amino acids between the anchor residues is indicted with (X).</p

Identification of MHC B4-, B15-, B19- and B21-restricted CD8+ T-cell epitopes using peptide pools.

<p>Lung cells isolated at 10 dpi were stimulated with B4 restricted peptide pools (A) or, B15 (B), B21 (C) and B19-restricted peptide pools (D–F) and IFNγ producing cells were determined by IFNγ Elispot analysis. Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p

Identification of MHC B12-restricted CD8+ T-cell epitopes using peptide pools.

<p>Lung cells were stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis. Results for three individuals birds are shown at 5 dpi (A–C), 7 dpi (D–F), 10 dpi (G–I) and 14 dpi (J–L). Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p

CD8+ T-cell frequencies in tissues of LPAIV infected chickens.

<p>Percentages of CD8αα+CD3+ T cells (A–C) and CD8βα+CD3+ T cells (D–F) were analysed by flowcytometry in lung, spleen and PBMC at several days post infection. Absolute numbers were calculated by multiplying the percentage of CD8αα+CD3+ T or CD8βα+CD3+ T cells with the total number of cells isolated from lung (G, I) and spleen (H, J). Mean plus SEM is shown. In white: uninfected controls (UNINF, n = 12), in grey infected birds (n = 3 per time point).</p

Novel AIV-specific CD8+ T cell epitopes within the LPAI H7N1 virus.

<p>Epitope mapping resulted in 11 novel CD8+ T-cell epitopes in the nucleoprotein (A) and 5 epitopes in the matrix 1 protein (B) of the LPAI H7N1 virus. In black, B12-restricted epitopes, in grey, B4-restricted epitopes (A) or B19-restricted epitopes (B).</p

Epitope prediction for MHC B12 based on anchor residues.

<p>Predicted epitopes and their localization based on anchor residues that have been described for B12 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Kaufman1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Wallny1" target="_blank">[33]</a>. X represents any amino acid. Anchor residues specific for the different MHC types are indicated in bold. A variable number of amino acids between the anchor residues is indicted with (X).</p

Screening of MHC B12-restricted CD8+ T-cell epitopes using individual peptides.

<p>Lung cells were isolated, <i>in vitro</i> re-stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis at 7 dpi (A) and 10 dpi (B). Mean plus SEM is shown, n = 4 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p