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

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

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

Early folding of human amyloid β (Aβ), around S26 and N27.

<p>The figure is a simplification of the model designed by Olofsson <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019110#pone.0019110-Olofsson1" target="_blank">[28]</a>. Residues in red are solvent accessible; residues in blue (and to a lesser extent in gray) are shielded from the solvent. The peptide cyclo[Aβ(22–28)-YNGK′] is a mimic for misfolded Aβ. YNGK′ is a turn-stabilizing sequence and K′ is a side-chain-modified lysyl residue for selective conjugation to a protein carrier. Other YNGK′-containing cyclic peptides prepared (23–28, 24–29, 25–30, 21–27, 22–28, 23–29, 24–30, and 25–31) did not mimic misfolded Aβ.</p

Western blot of a lysate of SW13 adrenal cells transfected with amyloid precursor protein (APP).

<p>The cells were transfected with either human APP⁶⁹⁵wt (wild type) or with APP⁶⁹⁵swe (Swedish mutation). Monoclonal antibody 6E10 is specific for the largely unstructured N-terminus of amyloid β (Aβ) and recognized both APPs (amino acid residues 2–8 in Aβ correspond to residues 598–604 in APP⁶⁹⁵). Anti-cyclo[Aβ (22–28)-YNGK′] antibodies stained neither of the APPs under the denaturing conditions of the assay and thus seem to recognize a conformational epitope in Aβ.</p

Binding of anti-cyclopeptide antibodies to oligomeric amyloid β (Aβ) as determined by ELISA.

<p>Threefold serial dilutions (starting from 1/150) of pooled antisera obtained were tested. Antisera were raised by immunization with oligomeric (black ▴) or fibrillar (black ▪) Aβ(1–42) and TTd-conjugates of cyclo[Aβ(22–28)-YNGK′] (red ▴) or linear K′-Aβ(22–28)-YNG (red ▪). Monoclonal antibody 6E10 (green •) served as control. Antibody binding to fibrillar Aβ(1–42) gave comparable results.</p

IgG response of individual Balb/c mice against Aβ (1–42) as determined by ELISA.

<p>As indicated on the x-axis, groups of eight mice were immunized with cyclo[Aβ (22–28)-YNGK′]-TTd, oligomeric Aβ(1–42), or fibrillar Aβ (1–42). <b>A</b>, coating antigen: oligomeric Aβ (1–42); <b>B</b>, coating antigen: fibrillar Aβ (1–42). A mouse with a serum titer ≤100 is considered to be a non-responder. The two titers out of range in the right panel of <b>A</b> have values of 40<b>×</b>10³ and 50<b>×</b>10³. The figure demonstrates that anti- cyclo[Aβ (22–28)-YNGK′] antibodies recognize oligomeric and fibrillar Aβ (1–42).</p

Cytokines produced by spleen cells after restimulation with FHA.

<p>Spleen cells from C57BL/6, TLR2−/−, TRIF-deficient (TRIFdef) mice (A), or C3H/HeOuJ and C3H/HeJ mice (B) immunized with PBS or whole cell pertussis vaccine were incubated for 4 days with 500 ng/ml FHA Cytokines IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were determined in the supernatant with Luminex. Data are expressed as means of three mice per group (PBS), or six mice per group (whole cell pertussis vaccine), error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Antibody titers of mice after immunization with <i>N. meningitidis</i> P1.5-1,2-2 OMVs.

<p>Mice were immunized with <i>N. meningitidis</i> P1.5-1,2-2 OMVs and antigen-specific titers of IgG, IgG1, IgG2a/c, IgG2b, and IgG3 in sera were determined with ELISA. Also total IgE in sera of mice immunized with PBS or <i>N. meningitidis</i> P1.5-1,2-2 OMVs was measured with ELISA. Results for C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice are shown in panel A, results for C3H/HeOuJ and C3H/HeJ mice are shown in panel B. Levels of serum bactericidal antibodies after immunization with <i>N. meningitidis</i> P1.5-1,2-2 OMVs are shown in panel C. Data are expressed as means of log₁₀ titers for 6 mice per group, except levels of serum bactericidal antibodies in C57BL/6 and TLR2−/− mice (n = 12). Error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p