Widespread and efficient deletion of IFNαβR in vivo following tamoxifen activation of Cre-ERT2.

<p>(A) LCMV immune UBC-Cre-ERT2⁺IFNαβR^fl/fl and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were injected with tamoxifen, and the indicated analyses were carried out. (B) The efficiency of deletion at the DNA level was assessed by PCR analysis of genomic DNA extracted from the indicated tissues; M = markers; Splns = splenocytes; LN = lymph nodes; x = empty lane. 1144bp = floxed allele, 309bp = deleted allele. (C & D) The efficiency of Cre activation in T cells of LCMV-immune mice was determined. Cre⁺ or Cre^- IFNαβR^f/fzsGreen^+/wt mice were injected with tamoxifen and, two weeks later, splenocytes were harvested and incubated with each of the four indicated peptides, and virus-specific T cells were identified using standard intracellular cytokine staining for IFNγ. zsGreen expression also was evaluated. The red numbers indicate zsGreen⁺ cells as a percentage of all CD8⁺ T cells (No Peptide groups) or of virus-specific (i.e, IFNγ⁺) cells (peptide-stimulated groups). (C) Gated on CD8⁺ T cells and (D) gated on CD4⁺ T cells. (E) The efficiency with which Cre activation resulted in ablation of IFNαβR biological function was determined, by measuring Stat 1 phosphorylation after <i>in vitro</i> incubation with IFNβ. ZsGreen+CD8⁺ T cells from Cre⁺ mice were compared to CD8⁺ T cells from Cre^- animals. Grey histograms = isotype control antibody. Mouse numbers: C & D, n = 6–7; E, n = 4.</p

Reporter-negative (IFNαβR-intact) CD8+ or CD4+ memory T cells do not preferentially expand during the recall response.

<p>As described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005861#ppat.1005861.g003" target="_blank">Fig 3A</a>, LCMV-immune Cre reporter (zsGreen) mice were generated, treated with tamoxifen, and subjected to secondary LCMV infection. Mice were sacrificed, and virus-specific CD8⁺ and CD4⁺ T cells were enumerated using intracellular cytokine staining, and evaluated for zsGreen expression, days 0, 2, 5 & 23 following the secondary infection. Representative epitope-specific responses in individual mice are shown. Cells were harvested at day 23 following secondary infection, and are gated on (A) CD8⁺ T cells or (B) CD4⁺ T cells. Numbers represent the proportion of the gated cells in each quadrant. (C-F) Cumulative data showing the proportion of zsGreen-positive cells at the indicated time points over the course of the recall response, in each of the four epitope-specific T cell populations. (G) At the indicated time points, splenocytes from Cre⁺ mice were exposed in vitro to IFNβ, and levels of pSTAT expression were evaluated. Representative plots are shown, gated on CD8 and zsGreen. The rightmost panel is a positive control, showing the responsiveness to IFNβ of day 23 post-secondary CD8⁺ T cells from Cre^- animals. Grey histograms = isotype control antibody.</p

Functional type I and II interferons are expressed in response to secondary viral infection.

<p>LCMV-immune mice were rechallenged with 2x10⁶ PFU LCMV-Arm and (A) IFNα and (B) IFNγ levels in the plasma were assessed via ELISA or multiplex, respectively (n = 5 per time point). (C) Type I and type II ISG expression was quantified by PCR array in the spleens of LCMV-immune mice at 12 hours p.i., and the data were normalized to equivalent analyses of sham infected mice (sham n = 6, 12 hrs n = 4).</p

IFNαβR deletion prior to secondary viral infection does not limit LCMV-specific T cell attrition or the expression of MHC Class I & Qa-1 in vivo.

<p>(A) LCMV immune UBC-CreERT2⁺ and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were tamoxifen injected and rechallenged with LCMV or given a sham injection. LCMV-specific (B) CD8⁺ and (C) CD4⁺ T cells were quantified via standard intracellular cytokine staining at the indicated times p.i. (D & E) Qa-1 and (F & G) MHC Class I expression was measured upon D^bGP_33-41⁺ (left column) and D^bNP_396-404⁺ (right column). Shown in panels D & F are representative histograms (gated on D^bGP_33-41⁺ CD8⁺ T cells). Data in B, C, E, & G are summations of 2 independent experiments (n = 4–7). Significance was determined via two-way ANOVA with Sidak correction (**** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05).</p

Secondary expansion, long term maintenance, and qualitative changes of memory CD8+ and CD4+ T cells are not reliant upon T1IFN signaling.

<p>LCMV-immune UBC-Cre-ERT2+ and UBC-Cre-ERT2-IFNαβR^fl/fl mice were injected with tamoxifen and later rechallenged with LCMV. (A-C) CD8⁺ and (D) CD4⁺ T cells specific for the indicated LCMV immunodominant epitope were identified and quantified within the spleen at the indicated times post infection, using standard intracellular cytokine staining. Data are a summation of five independent experiments; each dot represents an individual mouse (at each time point, n = 4–11 per group). (E & F) The ability of CD8⁺ (E) and CD4⁺ (F) memory T cells to produce IFNγ, TNF, and IL-2 was examined using a standard ICCS assay at days 2, 5, 14, and 23 following secondary challenge. The loss of multifunctionality in memory cells soon after secondary infection, and its gradual restoration, are shown by the relative proportion, at each time point, of cells that are capable of producing one, two or three cytokines (black, grey and white segments, respectively).</p

Reduced T1IFN-inducible gene expression does not limit viral control within the spleen or liver during secondary viral infection.

<p>(A) Type I and II ISG expression at 1 day post-secondary LCMV infection was assessed via PCR array, in LCMV-immune tamoxifen-treated Cre⁺ and Cre^- IFNαβR^f/f mice (Cre⁺ n = 5, Cre^- n = 6). Relative expression (Cre^- /Cre⁺) is shown for each ISG, together with statistical significance. (B & C) LCMV RNA was quantified by qPCR in the spleens (B) and livers (C) of Cre+ and Cre-IFNαβR^fl/fl mice at the indicated times following secondary challenge. Data are summations of two (A & C) and five (B) independent experiments. Each dot in panel B & C represents an individual mouse.</p

Tamoxifen-treated naïve Cre⁺IFNαβR^f/f mount primary responses that are similar to those reported for conventional IFNαβR knockout animals.

<p>(A) LCMV naive UBC-Cre-ERT2⁺IFNαβR^fl/fl and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were injected with tamoxifen and ~9 weeks later were given a primary infection with LCMV. Seven days p.i., virus specific CD8⁺ (B & C) and CD4⁺ (D & E) T cells were identified and quantified via standard intracellular staining. For each cell type, representative contour plots from individual mice are shown (B & D), followed by cumulative data from all animals (C & E). (F) LCMV vRNA was quantified within the spleens of Cre⁺ and Cre^- mice (n = 2).</p

The IFIT locus limits early virus replication, contributes to subsequent mortality, and is required for a successful response to IFNβ.

(A-G; black = B6; green = IFITKO mice) B6 and IFITKO mice were infected with CVB3 (104 pfu/mouse, i.p.). (A) Body weight changes were monitored over a 7-day period. Body weights at the time of infection of each individual are set as 100% (n = 6, Means ± SEM). (B) Survival of both B6 (n = 12) and KO (n = 9) mice was assessed over a 12-day period. (C) Mice were sacrificed at 12 days p.i. and images of internal organs and extracted pancreata and livers were taken. (D-F) Mice were sacrificed at day 1 (n = 7 each), day 7 (B6, n = 7; KO, n = 6) and days 11–12 (B6, n = 3; KO, n = 7) p.i. Virus titers in the pancreas (D), liver (E) and heart (F) are represented as PFU/gram. The lower limit of virus detection was 10 PFU per gram of tissue sample (shown as dashed line). Each symbol represents an individual value. (G) Fecal samples were collected and virus titers were determined. (H & I: open circles = PBS-treated; blue circles = IFNβ treated). B6 and IFITKO mice were infected with CVB3 (104 pfu/mouse, i.p.). 24 hours later, mice received either PBS or recombinant IFNβ. (H) Body weight changes were monitored over a 12-day period. Body weights at the time of infection of each individual were set as 100%. (I) The mice were sacrificed at 12 days p.i., and viral titers were determined in the heart (above) and in pancreata and livers (S6 Fig). The XY plots show, for the two mouse strains, the relationships among titer, body weight loss, and treatment status (PBS or IFNβ).</p

Ifit1, 2, 3 and 3b are highly induced in cardiomyocytes during CVB3 infection.

Type I IFN-related gene expression was quantified by PCR array in the hearts of CVB3-infected (500 pfu i.p.) mice at 2 days p.i. (A) Schematic representation of the four different data sources subjected to PCR array analysis. (B and C) Only genes that are induced at statistically significant levels (P - and CMMCM mice are shown (n = 4–5, means ± SEM). (B) Heat map showing the fold-change of gene expression (compared to uninfected mice) after CVB3 infection in Cre- mice (left column) and CMMCM mice (right column). (C) Cardiomyocyte-specific changes in gene expression (left column of heatmap divided by right column of heatmap; IFIT genes are arrowed). (D and E) Primary cardiomyocytes were isolated from C57BL/6 (B6) mice and infected with CVB3 at MOI of 1. At indicated hours p.i., supernatants and cells were collected. (D) Infectious virus titers in the supernatant were determined by plaque assays and represented as PFU/ml. Data are representative of two independent experiments (n = 2, geometric means ± SD). (E) Real-time RT-PCR analysis of each IFIT family gene expression in primary cardiomyocytes. Each value was normalized to the values of Gapdh and divided by the values of uninfected controls (n = 1). (F) B6 mice were infected with 104 PFU of CVB3 i.p., and IFIT mRNA expression was analyzed 48 hours later by real-time RT-PCR. Each value was normalized to the value of Gapdh, and divided by the values of uninfected controls (n = 3, means + SEM).</p

The IFIT locus is required for preventing CVB3-induced myocarditis.

C57BL/6 (B6) and IFITKO (KO) mice were infected with CVB3 (104 pfu/mouse, i.p.). (A) Representative histological sections of hearts stained with Masson’s trichrome (12 days p.i.) are shown. (B) Vibratome sections of the above hearts were stained as indicated, and were imaged by confocal microscopy. Iba-1 (macrophages) (Red), F-actin (Green), and nuclei (Blue). (C) Representative hematoxylin and eosin (H&E) -stained sections of hearts stained taken at 7 days p.i. are shown. Yellow arrowheads indicate sites of cellular infiltration. (D) The extent of this infiltration, and its cellular composition, was assessed using RT-PCR for the four indicated RNAs: Cd4, Cd8b1, Emr1 (F4/80), Ly6G (Gr-1). (B6, n = 6; KO, n = 8, Means + SEM). Each symbol represents an individual value.</p