DCs have increased activation markers in CD11c-<i>Ifnar1</i>^-/- mice.

<p>DCs (CD3-negative, CD19-negative, CD11c-positive, MHCII-positive) from CD11c-<i>Ifnar1</i>^-/- mice and littermate controls (cre-negative) 3 days after innoculation with CW3 were quantified (<b>A</b>) and stained for surface markers (<b>B</b>). The geometric mean fluorescence intensity (Geo. MFI) is shown for MHC molecules MHCI-K^b and MHCII-IA/IE, and markers of activation: CD40, CD80, and CD86. Geometric MFIs were normalized in each experiment to the average geometric MFI of each marker on DCs from naïve mice (uninfected cre-positive and cre-negative littermates). Representative histograms are shown. Data is combined from two experiments and individual mice are represented by each data point. Statistical significance was determined by 1-way ANOVA. n.s = p>0.05, * = p≤0.05, *** = p≤0.001, **** = p≤0.0001.</p

Type I IFN response in myeloid cells prevents systemic persistence.

<p>CD11c-<i>Ifnar1</i>^-/- mice and littermate controls (<b>A</b>) or <i>IRF3x7</i>^<i>-/-</i> mice (<b>B</b>) were inoculated with CW3 and the indicated tissues were collected on days four, eight, 14, 21, and 35 for viral genome quantification by qPCR. Tissues from <i>IRF3x7</i>^<i>-/-</i> mice (<b>B</b>) were collected on day 21 only. Data is combined from at least two experiments with a total of three to eight mice per group. Statistical significance was determined by 2-way ANOVA (A) or Kruskal-Wallis test (B). n.s = p>0.05, * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001.</p

Dendritic cell-intrinsic IFNAR response is required for clearance of CW3 infection.

<p>Mixed bone marrow chimeras were generated by reconstitution of irradiated CD45.1 recipients with 50% wild-type CD45.2 bone marrow and 50% wild-type CD45.1 bone marrow (labeled control) or 50% CD11c-<i>Ifnar1</i>^-/- (CD45.2) bone marrow and 50% wild-type CD45.1 bone marrow (labeled 50% CD11c-<i>Ifnar1</i>^-/-). (<b>A</b>) Prior to infection, 50% chimerism was confirmed by staining of blood cells with antibodies to CD45.1 and CD45.2. (<b>B</b>) Three days after infection with CW3, viral genomes were quantitated per microliter of blood. (<b>C</b>) Survival curve of mixed bone marrow chimeras. (<b>D</b>) 21 days after infection with CW3, viral genomes were quantitated in spleen, liver, MLN, and colon. Data is combined from two experiments with individual mice shown. Statistical significance was determined by t-test. n.s = p>0.05, **** = p≤0.0001.</p

CW3 persistence in CD11c-<i>Ifnar1</i>^-/- mice is associated with enhanced CD8 T cell expansion.

<p>Cells were collected from spleens, MLNs, or Peyer’s patches of CD11c-<i>Ifnar1</i>^-/- mice and controls eight days after infection with CW3. (<b>A</b>) Total CD8 T cells were analyzed for expression of IFNAR1. (<b>B</b>) The number or percentage of CW3-tetramer positive CD8 T cells was determined by flow cytometry from the indicated tissue. (<b>C</b>) Splenocytes were stimulated <i>ex vivo</i> with CW3 peptide and stained for cell surface CD107a and intracellular IFNγ and TNFα. (<b>D</b>) RNA extracted from spleens was used to quantitate transcripts for <i>Ifng</i>, <i>Tnfa</i>, and <i>Gzmb</i>. (<b>E</b>) The percentage of cells in (C) positive for one, two, or three of the markers of activation is shown. Data in (B) is combined from three experiments and data in (A), (C), (D) and (E) is combined from two experiments with seven mice per data point in (E). Statistical significance was determined by unpaired t test (B, C and D) or 2-way ANOVA (A and C). n.s = p>0.05, * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001.</p

The adaptive immune response generated in persistently infected CD11c-<i>Ifnar1</i>^-/- mice is functional in an <i>Ifnar1</i>-sufficient environment.

<p>(<b>A</b>) CD11c-<i>Ifnar1</i>^-/- mice were infected with CW3 (filled gray histograms) or CW3 engineered to express the SIINFELK peptide (Red-lined histograms). Three days after inoculation, splenocytes were isolated and stained with an antibody that recognizes H2-K^b with SIINFEKL bound in the peptide-binding groove. Overlain histograms from individual mice show fluorescence intensity of staining on T cells (CD3+, CD19-), B cells (CD19+, CD3-), Macrophages (CD3-, CD19-, F4/80+, CD11b^low), or DCs (CD3-, CD19-, CD11c+, MHCII+) as indicated. (<b>B</b>) A portion of the MNoV VP1 gene was amplified by PCR from spleen tissue 21 days after inoculation and sequenced by Sanger sequencing. The shown chromatogram includes the region encoding the immunodominant CD8 T cell epitope and is representative of sequencing results from four mice. (<b>C and D</b>) Splenocytes were collected from CD11c-<i>Ifnar1</i>^-/- and littermate controls 21 days after infection with CW3. 10⁷ splenocytes were injected I.P. into <i>Rag</i>^<i>-/-</i> mice that had been infected with CW3 21 days prior. Spleens and Ileums were collected from <i>Rag</i>^<i>-/-</i> seven days after splenocyte transfer. Viral titers in tissues were determined by qPCR for viral genomes (C) and plaque assay (D). Data in C and D is combined from two experiments with individual mice shown. Statistical significance was determined by one-way ANOVA (A) or Kruskal-Wallis test (B) or. n.s = p>0.05, * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001.</p

Increased type I IFN stimulated genes in in CD11c-<i>Ifnar1</i>^-/- mice.

<p>CD11c-<i>Ifnar1</i>^-/- mice and C57BL/6 controls were inoculated with CW3 and spleens were collected on days three for quantification of the indicated interferon stimulated genes by qPCR. Data is combined from two experiments. Statistical significance was determined by unpaired t test. n.s = p>0.05, * = p≤0.05, ** = p≤0.01.</p

Type I IFN response in myeloid cells prevents viral persistence.

<p><i>Ifih1</i>^<i>-/-</i>, <i>Mavs</i>^<i>-/-</i>, <i>Myd88</i>^<i>-/-</i>, <i>Ticam1</i>^<i>-/-</i>, <i>IRF3x7</i>^<i>-/-</i>, and <i>Ifnar1</i>^<i>-/-</i> mice (<b>A</b>) or Cre-positive and Cre-negative littermates from <i>Ifnar1</i>^{<i>flox/flox</i>} x LysM-cre, <i>Ifnar1</i>^{<i>flox/flox</i>} x CD11c-cre, and <i>Ifnar1</i>^{<i>flox/flox</i>} x villin-cre crosses (<b>B</b>) were tested for the ability to clear CW3 infection. Mice were inoculated with CW3, and viral genomes were quantified in the MLN on day 21. Data is combined from at least two experiments with a total of 5–11 mice per group. Statistical significance determined by Kruskal-Wallis test. n.s = p>0.05, * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001.</p

Model for the relationship between IFNAR signaling, viral replication, the adaptive immune response, and persistence.

<p>The contribution of the adaptive immune response depends on the level of type I IFN deficiency. When the type I IFN response is intact, innate immunity is relatively effective at control of MNoV and a small magnitude of adaptive immune response is sufficient to clear the infection. When a susceptible cell type (i.e. DCs) lacks the ability to respond to type I IFN, MNoV replication is increased, a larger magnitude of adaptive immune response is elicited, and infection is controlled but not cleared. When type I IFN signaling is completely absent, MNoV replication is at a maximum and the host succumbs to infection prior to generation of a protective adaptive immune response.</p

CW3-specific CD8 T cells are functional during persistent infection.

<p>Cells were collected from spleens of CD11c-<i>Ifnar1</i>^-/- mice and littermate controls 21 days after infection with CW3. The number and percentage of CD8 T cells that were tetramer positive (<b>A</b>) or responded to <i>ex vivo</i> peptide stimulation (<b>B</b>) were determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005684#ppat.1005684.g004" target="_blank">Fig 4</a>. (<b>C</b>) RNA extracted from spleens was used to quantitate transcripts for <i>Ifng</i>, <i>Tnfa</i>, and <i>Gzmb</i>. (<b>D</b>) The percentage of cells in (B) positive for one, two, or three of the markers of activation is shown. (<b>E</b>) Tetramer positive CD8 T cells in the spleens of CD11c-<i>Ifnar1</i>^-/- (red lines) and littermate controls (blue lines) were analyzed for cell surface expression of Ly6C, CD103, and PD-1. Grey lines are histograms of total CD8⁺ T cells. Dot plots show the geometric MFI for each marker. Data in (E) is representative of two independent experiments. Data in (A-D) is combined from two (C) or three (A, B, D) experiments with individual mice shown in (A-C) and 13 mice per data point in (D). Statistical significance was determined by unpaired t test (A, B, C) or 2-way ANOVA (D). n.s = p>0.05, * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001.</p

Autophagy is induced by wild-type, Δ<i>actA</i>, Δ<i>plcAB</i> but not by Δ<i>hly</i> or heat-killed <i>L. monocytogenes</i>.

<p>(<b>A</b>) Western blot of LC3I and LC3II in wild-type and <i>atg5^−/−</i> BMDMs infected with wild-type <i>L. monocytogenes</i>. All images are from a single gel. (<b>B</b>) Western blot of LC3I and LC3II in wild-type BMDMs at 60 minutes post-infection. BMDMs were infected with wild-type, Δ<i>hly</i>, Δ<i>actA</i>, Δ<i>plcAB</i> mutant <i>L. monocytogenes</i> as well as heat-killed <i>L. monocytogenes</i>. Images taken from a single gel, cropped and combined. Data shown are representative of results obtained in three independent experiments.</p