Type I interferon receptor deficiency in dendritic cells facilitates systemic murine norovirus persistence despite enhanced adaptive immunity

In order for a virus to persist, there must be a balance between viral replication and immune clearance. It is commonly believed that adaptive immunity drives clearance of viral infections and, thus, dysfunction or viral evasion of adaptive immunity is required for a virus to persist. Type I interferons (IFNs) play pleiotropic roles in the antiviral response, including through innate control of viral replication. Murine norovirus (MNoV) replicates in dendritic cells (DCs) and type I IFN signaling in DCs is important for early control of MNoV replication. We show here that the non-persistent MNoV strain CW3 persists systemically when CD11c positive DCs are unable to respond to type I IFN. Persistence in this setting is associated with increased early viral titers, maintenance of DC numbers, increased expression of DC activation markers and an increase in CD8 T cell and antibody responses. Furthermore, CD8 T cell function is maintained during the persistent phase of infection and adaptive immune cells from persistently infected mice are functional when transferred to Rag1-/- recipients. Finally, increased early replication and persistence are also observed in mixed bone marrow chimeras where only half of the CD11c positive DCs are unable to respond to type I IFN. These findings demonstrate that increased early viral replication due to a cell-intrinsic innate immune deficiency is sufficient for persistence and a functional adaptive immune response is not sufficient for viral clearance

Differentiation and Protective Capacity of Virus-Specific CD8

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche. Chronic infections often cause T cell dysfunction, but how noroviruses (NV) evade immunity is unknown. Tomov et al. show that gut-resident T cells against NV remain functional but ignorant of chronic viral replication, suggesting that NV persists in an immune-privileged enteric niche. © 2017 Elsevier Inc

Persistent enteric murine norovirus infection is associated with functionally suboptimal virus-specific CD8 T cell responses

Norovirus (NV) gastroenteritis is a major contributor to global morbidity and mortality, yet little is known about immune mechanisms leading to NV control. Previous studies using the murine norovirus (MNV) model have established a key role for T cells in MNV clearance. Despite these advances, important questions remain regarding the magnitude, location, and dynamics of the MNV-specific T cell response. To address these questions, we identified MNV-specific major histocompatibility complex (MHC) class I immunodominant epitopes using an overlapping peptide screen. One of these epitopes (amino acids 519 to 527 of open reading frame 2 [ORF2(519-527)]) was highly conserved among all NV genogroups. Using MHC class I peptide tetramers, we tracked MNV-specific CD8 T cells in lymphoid and mucosal sites during infection with two MNV strains with distinct biological behaviors, the acutely cleared strain CW3 and the persistent strain CR6. Here, we show that enteric MNV infection elicited robust T cell responses primarily in the intestinal mucosa and that MNV-specific CD8 T cells dynamically regulated the expression of surface molecules associated with activation, differentiation, and homing. Furthermore, compared to MNV-CW3 infection, chronic infection with MNV-CR6 resulted in fewer and less-functional CD8 T cells, and this difference was evident as early as day 8 postinfection. Finally, MNV-specific CD8 T cells were capable of reducing the viral load in persistently infected Rag1(−/−) mice, suggesting that these cells are a crucial component of NV immunity. Collectively, these data provide fundamental new insights into the adaptive immune response to two closely related NV strains with distinct biological behaviors and bring us closer to understanding the correlates of protective antiviral immunity in the intestine

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

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

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