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1.
FIGURE 3

FIGURE 3. From: p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza A virus.

Expression of antiviral genes in the bone marrow. A-B, Quantitative real time PCR for the indicated antiviral genes. RNA samples were obtained from bone marrow flushed from femurs and tibiae of infected mice at the indicated time points, and used as a template for qRT-PCR. Results represent the average gene induction in three independent bone marrow samples. Results are shown as Mean ± SEM.

César Muñoz-Fontela, et al. J Immunol. ;187(12):6428-6436.
2.
FIGURE 1

FIGURE 1. From: p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza A virus.

Expression of antiviral genes and pro-inflammatory cytokines in IAV-infected wt (black bars) and p53 mice (white bars). A, Quantitative real time PCR for the indicated antiviral genes. RNA samples were obtained from lung tissue of infected mice at the indicated time points and used as a template for qRT-PCR. Results represent the average gene induction in three independent lung samples and are representative of two independent experiments. Results are shown as Mean ± SEM. B, qRT-PCR data for the indicated chemokines, as described above. Asterisks represent statistical significance (p<0.05), as assessed by student’s T test. C, Evaluation of IAV nucleoprotein (NP) and non-structural protein 1 (NS1) gene expression in lung samples of wt (black symbols) and p53−/− mice (white symbols) infected with IAV. At least 5 mice were used for each of the indicated time points. RNA samples were obtained from lung tissue and used as template for qRT-PCR analysis. Asterisks denote statistical significance (p<0.05), as assessed by Mann-Whitney non-parametric test.

César Muñoz-Fontela, et al. J Immunol. ;187(12):6428-6436.
3.
FIGURE 4

FIGURE 4. From: p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza A virus.

Monocyte infiltration and dendritic cell kinetics in lungs and mLNs. A, Flow cytometry analysis of monocyte infiltration in lungs of wt and p53−/− mice infected with IAV for 6 days (left panel) or in a time course (right panel). Monocytes were gated as CD11c MHCII F4/80+ CD11b+ Ly6C+ cells in the low side scatter (SSC). Single cell suspensions were obtained from lungs of wt (full line) and p53−/− mice (dotted line) and subjected to FACS analysis as described on the Methods section. Lungs were pooled from three different mice for each time point. In the right panel, results are represented as absolute number of monocytes per 2×105 events in lungs of infected mice at the indicated time points. B, Flow cytometry analysis of the kinetics of myeloid DCs in the lungs of wt (unbroken line) and p53−/− mice (dotted line) upon IAV infection. DCs were gated as CD11c+ MHC II+ cells in the low SSC, and were further defined as CD103+ CD11b B220 (CD103+) and CD103 CD11b+ B220 (CD11b+). In the right panel, numbers on the x axis represent days post-infection and are represented as Mean ± SEM of two independent experiments. C, Flow cytometry analysis of DC migration from lungs to draining mLNs. Tissue DCs in the mLNs were defined as CD11c+ MHC IImed cells expressing either CD11b or CD103. Each point (black for wt and white for p53−/−) represents the average DC numbers in mLNs pooled from three different mice.

César Muñoz-Fontela, et al. J Immunol. ;187(12):6428-6436.
4.
FIGURE 6

FIGURE 6. From: p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza A virus.

Antiviral responses in mixed bone marrow chimeric mice. A, Generation of mixed chimeras. Bone marrow cells from WT and p53−/− mice (expressing CD45.1 and CD45.2, respectively; 2 × 106 cells from each) were mixed in a 1:1 ratio and transplanted into lethally irradiated congenic WT or p53−/− recipients (expressing CD45.2). Engraftment was evaluated 4 weeks after transplant by flow cytometry analysis of CD45.1 and CD45.2 cells in blood. Right panel shows a representative dot plot indicating the contribution of WT and p53−/− cells to hematopoietic cells in the mLNs. B, Analysis of IAV-specific DCs in the mLNs of wt and p53−/− recipients. Dot plot shows representative data of IAV-specific DCs both in the CD45.1 (wt) and CD45.2 (p53−/−) background. DCs were gated as CD11c+ MHC IImed expressing either CD45.1 (red) or CD45.2 (blue). Right panel indicates absolute numbers of IAV-specific DCs per 2×105 events. C, OT-I T cell proliferation assay. Wt and p53−/− recipient mice were infected with PR8-OT-I virus and adoptively transferred with CFSE-labeled T cells isolated from Rag2/OT-I mice. At day 4 post-infection, mice were sacrificed and mLNs extracted and prepared for flow cytometry. Left panel indicates T cell proliferation as assessed by CFSE dilution. The red line shows the histogram profiles and the blue lines the peaks of proliferation detected using FlowJo’s proliferation platform. The panels on the right indicate % of cells divided from the parent population of CD8+ T cells.

César Muñoz-Fontela, et al. J Immunol. ;187(12):6428-6436.
5.
FIGURE 2

FIGURE 2. From: p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza A virus.

Viral replication and pathogenesis. A, Quantification of viral titers by plaque assay followed by immunostaining of plaques using anti-influenza NP antibodies. Lungs from mice infected at the indicated time points were harvested and mechanically homogenized. Lung supernatants were disposed in ten-fold dilutions in 6-well plates seeded with 100% confluent Madin-Darby canine kidney (MDCK) cells for 60 min at room temperature and covered with an overlay of 2% agar for 48 hours. Cells were fixed with 4% paraformaldehide and permeabilized with 0.2% of Triton X-100. Immunostaining of viral plaques was performed as described in experimental procedures. B, Analysis of weight loss in wt (unbroken line) and p53−/− mice (dotted line). The numbers in the x axis represent days post-infection. 10 mice of each genotype were used for the assay and results are represented as % of weight loss relative to initial weight. Results are represented as Mean ± SEM. Asterisks represent statistical significance (p<0.05) as assessed by student’s T test. C, Survival in response to A/Puerto Rico/8/1934 (H1N1) infection. Mice were infected intranasally with 1000 pfu of PR8 virus under isoflurane anesthesia. Weight loss and body score were assessed daily, and mice were euthanized if their body weight dropped more than 25% from the initial weight, as per institutional guidelines.

César Muñoz-Fontela, et al. J Immunol. ;187(12):6428-6436.
6.
FIGURE 5

FIGURE 5. From: p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza A virus.

T cell responses in wt and p53−/− mice infected with IAV. A, CTL in vivo assay. Wt and p53−/− mice mock-infected with PBS or infected with PR8-OT-I virus for 8 days were adoptively transferred with splenocytes isolated from wt donor mice and pulsed with SIINFEKL peptide or irrelevant control peptide for 60 min. SIINFEKL positive cells and control peptide positive cells were labeled with high vs low CFSE respectively and analyzed by flow cytometry. Analysis of specific killing was done as described in Materials and Methods. B, OT-I T cell proliferation. Mice were infected with PR8-OT-I virus and adoptively transferred with CFSE-labeled T cells isolated from Rag2/OT-I mice. At day 4 post-infection, mice were sacrificed and mLNs extracted and prepared for flow cytometry. Left panel represents T cell proliferation as assessed by CFSE dilution. The red line represents the histogram profiles and the blue lines the peaks of proliferation detected using FlowJo’s proliferation platform. The panels on the right represent % of cells divided from the parent population of CD8+ T cells, and the proliferation index (average of divisions of each population). These parameters were determined using FlowJo’s proliferation platform. C, ELISA analysis of IAV specific IgG isotypes in serum 10 days after IAV infection. At least 10 mice were used for the analysis. Serum samples were collected as described in the Methods section and subjected to evaluation in X-31 activated ELISA plates. Results are representative of two independent experiments.

César Muñoz-Fontela, et al. J Immunol. ;187(12):6428-6436.

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