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

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

Delayed-type hypersensitivity responses in ponies following allergen injection in the skin.

<p>Immune reactions of 6 healthy (light bars) and 10 IBH-sensitive animals (dark bars) to allergen injection in winter was determined at the site of injection at different time points after injection. <b>A</b>) relative skin swelling in animals, 30 min following <i>C</i>. <i>obsoletus</i> WBE compared to histamine injection; <b>B</b>) total histological score of the skin; <b>C</b>) number of mast cells; <b>D</b>) total CD3 mRNA expression; <b>E</b>) total MIP1α mRNA expression. Bars represent average +SEM; dots represent individual animals.</p

Increased frequencies of allergen-specific Th1 cells in healthy compared to IBH-affected ponies.

<p>Allergen-specific CD4 T-cells were <i>in vitro</i> expanded in summer from PBMC of 11 healthy and 8 IBH-affected animals, by stimulation with WBE. Cytokine expression of expanded CD4 T-cells was then determined by exposure to monocyte-derived DCs in the presence or absence of allergen. <b>A</b>) representative FACS blot; <b>B</b>) IFNγ- and <b>C</b>) IL4 CD4 T-cells; <b>D</b>) ratio IFNγ/ IL4 CD4 T-cells. Bars represent average +SEM; dots represent individual animals.</p

Type-1 immune response skewing in the skin of healthy ponies following allergen injection.

<p>Cytokine expression at the site of injection, 24 hrs after allergen injection, was determined by qRT-PCR. <b>A</b>) IFNγ and <b>B</b>) IL4 mRNA expression as ratio to 18S; <b>C</b>) ratio IFNγ / IL4 expression. Bars represent average +SEM; n = 6 and n = 10, for healthy and IBH-sensitive animals, respectively; dots represent individual animals.</p

Immunoproteasome-deficient mice have responsive peripheral NK cells.

<p>(A–C) Splenocytes of RAG1^−/− and RAG1^−/−β2i/MECL-1^−/−β5i/LMP7^−/− (RAG1^−/−IS^−/−) mice were analyzed by flow cytometry (A) Frequencies of NK cells (DX5⁺NKp46⁺) as percentage of total lymphocytes. (B,C) Expression of CD11b and CD27 on DX5⁺NKp46⁺ NK cells. (B) Representative FACS plots and (C) graph showing percentages of subsets for individual mice for the regions indicated in (B). (D–H) Splenocytes of RAG1^−/− and RAG1^−/−IS^−/− mice were incubated on plates coated with anti-NKG2D and anti-NKp46, in the presence or absence of IL-2, or left unstimulated. Frequencies of IFNγ⁺, LAMP-1⁺ NK cells and CD69 mean fluorescent intensity (MFI) on NK cells were determined by flow cytometry. (D) Representative FACS plots showing IFNγ⁺ and LAMP-1⁺ NK cells. (E–H) Bars showing the percentages of IFNγ⁺LAMP-1⁺ NK cells (E), IFNγ⁺ NK cells (F), LAMP-1⁺ NK cells (G), and CD69 MFIs on NK cells (H). Results are shown as mean ± S.E.M. Data are representative for 2 independent experiments with 6 mice per group. Statistical analysis was performed using Mann-Whitney U test. *, P<0.05.</p

Rejection of immunosubunit-deficient cells in influenza virus infected mice.

<p>Splenocytes of congenic CD45.1 (<i>wt</i>) and CD45.2 β2i/MECL-1^−/−β5i/LMP7^−/− (IS^−/−) mice were 1∶1 mixed based on total cell numbers, and transferred <i>i.v.</i> into untreated or anti-asialo GM1 treated CD45.1.2 recipients, that were subsequently infected <i>i.n</i>. with influenza virus or left uninfected. Recipient mice were sacrificed 8 days later and spleens were analyzed. (A) Representative FACS plots showing gating strategies and percentages of recovered CD45.1⁺ (<i>wt</i>) and CD45.2⁺ (IS^−/−) cells of CD19⁺ cells in the different groups: uninf – NK (uninfected, anti-asialo GM1 treated), uninf (uninfected, untreated), inf – NK (influenza virus infected, anti-asialo GM1 treated), inf (influenza virus infected, untreated). (B) Representative FACS plots gated on TCRβ⁻ cells showing staining for DX5 and NKp46 on splenocytes from untreated and anti-asialo GM1 treated mice. Cells in the gate are DX5⁺NKp46⁺ NK cells. (C) Ratio of CD45.2 (IS^−/−)/CD45.1 (<i>wt</i>) calculated by dividing absolute numbers of CD19⁺ CD45.2⁺ (IS^−/−) cells by absolute numbers of CD19⁺ CD45.1⁺ (<i>wt</i>) cells. Results are representative for 2 independent experiments with 5–6 mice per group. Statistical analysis was performed using a Mann-Whitney <i>U</i> test. **, P<0.01.</p

Effect of immunoproteasome-deficiency on constitutive expression and activation-induced upregulation of MHC class I molecules.

<p>Splenocytes of untreated or poly(I:C) treated RAG1^−/− and RAG1^−/−β2i/MECL-1^−/−β5i/LMP7^−/− (RAG1^−/−IS^−/−) mice were analyzed for the expression of H2-K^b on DCs (CD11c^hiCD11b^int). (A) Representative histogram of untreated mice showing H2-K^b expression on DCs. (B) H2-K^b mean fluorescent intensity (MFI) on DCs of individual mice. Data are representative of three independent experiments, n = 4–6 mice per group. Statistical analysis was performed using a Mann-Whitney <i>U</i> test. *, P<0.05.</p

Adoptively transferred splenocytes of poly(I:C)-treated immunoproteasome-deficient mice are tolerated by NK cells in naïve wt recipients.

<p>CFSE-labeled splenocytes of untreated β2i/MECL-1−/−β5i/LMP7−/− (IS−/−; CFSElow) and of poly(I:C) treated IS−/− (CFSEhigh) mice were mixed 1∶1 and transferred i.v. into untreated or anti-asialo GM1 treated wt recipients. Recipient mice were sacrificed 17 h later and spleens were analyzed. (A) Representative FACS plots showing gating strategies. (B) Ratios of poly(I:C) treated (CFSEhigh) and untreated (CFSElow) IS−/− cells calculated by dividing absolute numbers of B220+CFSEhigh cells by absolute numbers of B220+CFSElow cells. Results represent one experiment with 6 mice per group.</p