Mib1-null DCs fail to activate Notch signaling.

<p>A, LPS-stimulated <i>Mib1^f/f</i> or <i>Mx1-Cre;Mib1^f/f</i> DCs were cultured with Notch1-expressing C2C12 cells transfected with WT or MT CBF luciferase constructs, and 24 h after the coculture, luciferase activity was measured. Results are representative of two independent experiments. <i>B</i>, Western blot analysis of Notch1 ICD (NICD) in negatively purified OT-II CD4⁺ T cells before (<i>None</i>) and 6 h after coculture with peptide-pretreated <i>Mib1^f/f</i> or <i>Mx1-Cre;Mib1^f/f</i> DCs. <i>C</i>, Quantitative real-time RT-PCR analysis of Notch target genes, Hes1 and Hes5, in CD4⁺ T cells before (<i>None</i>) and after coculture with <i>Mib1^f/f</i> or <i>Mx1-Cre;Mib1^f/f</i> DCs. Results are representative of three independent experiments. Data represent mean ± SD; *, P<0.05; **, P<0.001.</p

Negative Regulation of Type I IFN Expression by OASL1 Permits Chronic Viral Infection and CD8⁺ T-Cell Exhaustion

<div><p>The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2′–5′ oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected <i>Oasl1</i> knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected <i>Oasl1</i> KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2–3 days post-infection). <i>Oasl1</i> deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.</p></div

Effect of early <i>in vivo</i> blockade of IFN-I receptor signaling on viral clearance and virus-specific CD8⁺ T-cell responses in <i>Oasl1</i> KO mice.

<p>Mice were infected with LCMV CL-13. At 1.5 d p.i., 0.5 mg IFNAR-1 mAb (αIFNAR-1) or its control isotype Ab (Isotype) was administrated i.v. once into <i>Oasl1</i> KO mice. PBS was injected into WT mice as another control. (<b>A</b>) Changes in body weight at the indicated time points. (<b>B</b>) Serum virus titers of mice at indicated time points p.i. Vertical arrowhead indicates the time point of antibody injection and dashed black line represents the virus detection limit. (<b>C</b>) Frequencies of virus (GP33 and GP276)-specific CD8⁺ T cells and their PD-1 expression levels in the spleen at 35 d p.i. Numbers in the plots (left) indicate the percentages of the corresponding cell population among CD8⁺ T cells. Absolute numbers of GP33 tetramer-positive CD8⁺ T cells (top right) and PD-1 MFI values for GP33 tetramer-positive CD8⁺ T cells (bottom right) were also depicted. (<b>D</b>) Cytokine production on CD8⁺ T cells obtained from spleens at 35 d p.i. after <i>in vitro</i> restimulation with GP33 peptide. Percentages of cytokine-producing cells among CD8⁺ T cells (left) are shown. Absolute numbers of CD8⁺ T cells producing IFN-γ (top right) and the frequencies of TNF-α-producing cells among IFN-γ ⁺ CD8⁺ T cells (bottom right) are also depicted. All line graphs and bar graphs show mean ± SD and mean + SD, respectively. Data are representative of two independent experiments (<i>n</i> = 3 per group in each experiment). Statistical significance was determined by comparison between isotype-treated KO mice and each of the other two groups, PBS-treated WT mice or αIFNAR-1-treated KO mice. ns, not significant; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Efficient induction of functional virus-specific T cells and complete viral control in <i>Oasl1</i> KO mice during late phase of LCMV infection.

<p>(<b>A</b>–<b>C</b>) Lymphocytes were isolated from the spleens of LCMV CL-13-infected WT and <i>Oasl1</i> KO mice at 75 d p.i. and restimulated <i>in vitro</i> with GP33 or GP276 peptides for CD8⁺ T cells and GP_66–80 (GP66) peptide for CD4⁺ T cells. (<b>A</b>) Representative data showing the percentages of cytokine-producing cells among CD8⁺ or CD4⁺ T cells. (<b>B</b>) Absolute numbers of T cells producing IFN-γ per spleen. (<b>C</b>) Summary showing the frequency of TNF-α- or IL-2-producing cells among IFN-γ⁺ CD8⁺ or IFN-γ⁺ CD4⁺ T cells. Bar graphs show mean + SD. (<b>D</b>) Virus titers in the kidneys extracted from LCMV CL-13-infected mice at 75 d p.i. (left) and at 130 d p.i. (right). Dashed black line represents the virus detection limit. Undetectable samples were given 20 PFU per gram. The graph includes individual values with their arithmetic mean. All data are representative of at least two independent experiments (<i>n</i> = 3–4 per group in each experiment). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Phenotypic change of virus-specific CD8⁺ T cells in tissues of <i>Oasl1</i> KO mice during late phase of chronic LCMV infection.

<p>(<b>A</b>–<b>D</b>) Lymphocytes were isolated from the spleen (SP), lung (LG), and liver (LV) of LCMV CL-13-infected WT and <i>Oasl1</i> KO mice at 75 d p.i. and analyzed by flow cytometry. (<b>A</b>) Representative data showing the frequency of GP33 and GP_276–286 (GP276) tetramer-positive cells among CD8⁺ T cells. (<b>B</b>) Absolute numbers of the tetramer-positive CD8⁺ T cells in the indicated tissues. (<b>C</b>, <b>D</b>) PD-1 and CD127 expression levels on virus-specific CD8⁺ T cells in the spleen of WT and KO mice. Numbers in the plots indicate PD-1 MFI value (<b>C</b>) on GP33 and GP276 tetramer-positive CD8⁺ T cells and CD127⁺ cell percentage (<b>D</b>) among the tetramer-positive cells. Blue vertical lines in the plots of (<b>D</b>) divide CD127⁺ and CD127⁻ tetramer-positive CD8⁺ T cells. The PD-1 MFI values and CD127⁺ cell frequencies in the indicated tissues are also summarized in the graphs. (<b>E</b>) Co-expression pattern of CD127 and CD62L on virus-specific CD8⁺ T cells <i>ex vivo</i>. Splenocytes isolated from LCMV CL-13-infected WT and KO mice at 75 d p.i. (top) and 130 d p.i. (bottom) were analyzed by flow cytometry. GP33 and GP276 tetramer-positive CD8⁺ T cells were plotted: the numbers in the plots indicate percentages of CD127⁺CD62L⁺ (central memory) and CD127⁺CD62L⁻ (effector memory). All bar graphs show mean + SD. Data are representative of at least two independent experiments (<i>n</i> = 3–4 per group in each experiment). ns, not significant; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Production of sustained IFN-I and higher IRF7 protein in <i>Oasl1</i> KO mice early after LCMV CL-13 infection and the cellular sources of the IFN.

<p>WT and <i>Oasl1</i> KO mice were infected with LCMV CL-13 and their sera (<b>A</b>), spleens (<b>B–C</b>), and sorted splenic cells (<b>D</b>) were analyzed. (<b>A</b>) The levels of IFN-α and IFN-β in the sera were measured by ELISA. Line graphs are shown as mean ± SD. The data are representative of at least four independent experiments (<i>n</i>>5 per group in each experiment). (<b>B</b>) mRNAs prepared from the spleens were analyzed by quantitative RT-PCR at the indicated days: mRNA expression levels (<i>n</i> = 3 per group) normalized to <i>Gapdh</i> was recalculated by dividing each expression value with the least mRNA expression value among the samples and shown as a relative mRNA level. (<b>C</b>) The protein levels for OASL1, IRF7, IRF3, and β-actin (loading control) were measured by immunoblot. (<b>D</b>) mRNA expression levels of IFN-I and OASL1 for the sorted cell populations following the sorting strategy shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003478#ppat.1003478.s009" target="_blank">Figure S9</a>. The mRNA expression level (<i>n</i> = 3 per group) of <i>Ifna2</i>, <i>Ifna</i>5<i>/6/13</i>, <i>Ifnb1</i>, or <i>Oasl1</i> normalized to <i>Gapdh</i> was shown as a relative mRNA level. All bar graphs show mean + SD. Data of (<b>B</b>–<b>D</b>) are representative of two independent experiments. ns, not significant; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Expression of ligands and E3 ligases for Notch signaling in DCs.

<p><i>A</i> and <i>B</i>, Semi-quantitative RT-PCR analyses of Notch ligands (<i>A</i>) and E3 ubiquitin ligases (<i>B</i>) in BM-derived DCs prior to LPS stimulation. Serial 2-fold dilutions of PCR products were electrophoresed in agarose gel <i>C</i>, Quantitative real-time RT-PCR analysis of ligands and E3 ligases in DCs before (<i>None</i>) and after 24 h of LPS stimulation (<i>Sti</i>). The results are representative of more than five independent experiments. <i>D</i>, Flow cytometry analysis for Notch ligands in DCs. IgG, Armenian hamster IgG isotype control; None, DCs before LPS stimulation; Sti, DCs after LPS stimulation. Results are representative of three independent experiments. <i>E</i>, Western blot analysis of Mib1 in BM culture with rGM-CSF on days 5, 7, and 9, and 24 h after LPS treatment on day9 (<i>Sti</i>). <i>F</i>, Quantitative real-time RT-PCR analysis of Mib1 in BM culture with rGM-CSF on days 5, 7, and 9. <i>G</i>, Quantitative real-time RT-PCR analysis of Mib1 in the BM precursors of classical spleen DC. MP, myeloid progenitor; MDP, macrophage and DC precursor; CDP, common DC precursor; pre-cDC, committed precursors of classical spleen DC. Results are representative of three independent experiments. Data represent mean ± SD; *, P<0.05.</p

Generation of Mib1-null DCs.

<p><i>A</i> and <i>B</i>, Quantitative real-time RT-PCR (<i>A</i>) and western blot (<i>B</i>) analyses of Mib1 in DCs from <i>Mib1^f/f</i> or <i>Mx1-Cre;Mib1^f/f</i> mice. <i>C</i>, Flow cytometry analysis for DC marker, CD11c, costimulatory molecules, CD40, CD80 (B7.1), and CD86 (B7.2), and MHC II (I-A^b) in <i>Mib1^f/f</i> or <i>Mx1-Cre;Mib1^f/f</i> DCs before (<i>None, gray-filled histogram</i>) or after LPS stimulation (<i>Sti, black line</i>). Numbers on flow cytometry plots represent the mean percentage of cells ± SD. <i>D</i>, Quantitative real-time RT-PCR analysis of Notch ligands in LPS-stimulated DCs from <i>Mib1^f/f</i> and <i>Mx1-Cre;Mib1^f/f</i> mice. Results are representative of three independent experiments. <i>E</i>, Flow cytometry analysis for Jag1 and Jag2 in <i>Mib1^f/f</i> or <i>Mx1-Cre;Mib1^f/f</i> DCs. Numbers indicate the relative median fluorescence intensities of IgG isotype control (<i>IgG, gray-filled histogram</i>), DCs before LPS stimulation (<i>None, dotted line</i>), and DCs after LPS stimulation (<i>Sti, black line</i>), respectively, in each plots. Data represent mean ± SD.</p

Rapid expansion and maintenance of virus-specific CD8⁺ T cells and accelerated viral control in <i>Oasl1</i> KO mice during chronic LCMV infection.

<p>WT and <i>Oasl1</i> KO mice were infected with LCMV CL-13. (<b>A–E</b>) PBMCs were collected at indicated time points p.i. and analyzed by flow cytometry. (<b>A</b>) Representative data showing CD8⁺ T-cell percentage (green) among lymphocytes and CD44^hi cell percentage (red) among CD8⁺ T cells. (<b>B</b>) Summary showing CD8⁺ T-cell percentage among lymphocytes and CD44^hi cell percentage among CD8⁺ T cells. (<b>C</b>) Representative data showing the frequency of GP_33–41 (GP33) tetramer-positive cells among CD8⁺ T cells. Numbers in plots indicate percentage of the tetramer-positive cells. (<b>D</b>) The numbers of GP33 tetramer-positive CD8⁺ T cells per 10⁶ PBMCs during the course of LCMV CL-13 infection. (<b>E</b>) Summary showing the PD-1 expression level on GP33 tetramer-positive CD8⁺ T cells as represented by mean fluorescence intensity (MFI), and the percentage of CD127⁺ cells among GP33 tetramer-positive CD8⁺ T cells. (<b>F</b>) Serum viral titer in WT and <i>Oasl1</i> KO mice at the indicated time p.i. Dashed black line represents the virus detection limit. All line graphs show mean ± standard deviation (SD). Data are representative of four independent experiments (<i>n</i>>6 per group in each experiment). ns, not significant; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Protein delivery efficiency of dNP2 and TAT in various immune cells.

<p>(A) 5 μM of EGFP, TAT-EGFP, dNP2-EGFP, or PBS were treated to splenocytes from 6-week-old C57BL/6 mice and protein delivery efficiencies were analyzed by flow cytometry after cell staining with cell type specific markers for CD4⁺ CD4 T cells, CD19⁺ B cells, NK1.1⁺ NK cells, CD11c^lowCD11b^highF4/80⁺ macrophages, and MHCII⁺CD11c^high DCs. (B-D) The experiments were independently repeated three times and are presented as the relative mean fluorescence intensity (relative MFI). The values were normalized with the MFI of PBS treated samples. Bar graphs indicate the mean ± s.d. One-way ANOVA was used for statistical analysis and * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.</p