Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 1. Antitumor effect of intratumoral IFN-α gene transfer. (a) Growth of tumors injected with Ad-mIFN. Tumor volumes were measured at indicated days following the intratumoral injection of Ad-mIFN (n = 6) or Ad-AP (n = 8). Relative tumor volumes compared with those at day10 were presented. Data are shown as means ± standard deviation (s.d.). (b) ELISpot assay of IFN-γ-producing cells in response to stimulation of CT26 cells. Twenty-two days after tumor inoculation, splenocytes were isolated from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3), and were cultured with CT26 or syngeneic lymphocytes. Data are presented as means ± s.d. (c) Intracellular cytokine staining of IFN-γ-producing cells in response to CT26 cells. The splenocytes from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3) at day 22 were incubated with CT26 cells and stained by anti-mouse IFN-γ antibody. The activated cell fractions were analyzed by staining with anti-mouse CD8 antibody. Representative FACS plots (right panel) are shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Standard Deviation, Enzyme-linked Immunospot, Isolation, Cell Culture, Staining, Incubation

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 2. Intratumoral IFN-α gene transfer reduced the frequency of Tregs in tumors. (a) Frequency of CD4+Foxp3+ Tregs per CD4+ T cells in tumors. Tumors injected with viruses were harvested at days 16, 22 and 28, and processed into single-cell suspension. The percentage of CD4+

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Suspension

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 3. Intratumoral IL-6 concentration was significantly increased by IFN-α gene transfer. (a) IL-6 concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at days 16, 22 and 28, and IL-6 concentration was measured by ELISA. (b) Relationship between IL-6 and IFN-α concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at day 16, and the concentrations of IL-6 and IFN-α were compared by ELISA. (c) IL-6 production from tumor CD11c+ cells. The CD11c+ and CD11c −cells were isolated from tumors injected with Ad-mIFN (n = 2) or Ad-AP (n = 2) at day 16, and 5 × 104 cells were plated in 96-well plates. After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA. (d) IL-6 production from splenic CD11c+ cells in response to a recombinant IFN-α protein. The CD11c+ and CD11c−cells isolated from naïve splenocytes, and 5 × 104 of CT26 cells were cultured in 96-well plates with depicted concentration of recombinant mouse IFN-α (Miltenyi Biotech). After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA (n = 2 for each group).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Concentration Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 4. IL-6 receptor blockade suppressed IFN-α-mediated Treg reduction in tumors. (a) Schema of experiment. The 1000 μg of the monoclonal anti-IL-6 receptor antibody was intraperitoneally injected into the mice at days 7, 14 and 21 after tumor inoculation. Ad-mIFN or Ad-AP was injected once at day 10 after inoculation. (b) Frequency of CD4+Foxp3+ cells per CD4+ T cells in tumors treated with IL-6R ab. Tumors were harvested at day 22, and CD4+ T cells and CD4+Foxp3+ Tregs were analyzed by flow cytometry (n = 5 for the group of Ad-AP i.t. +IL-6R ab i.p., n = 4 for the other groups). (c) Ratio of CD8+ T cells to CD4+Foxp3+ Tregs in tumors. Frequency of CD8+ T cells within whole tumor cells (left panel). Frequency of CD4+Foxp3+ Tregs within whole tumor cells (middle panel). The number of CD8+ T cells was compared with that of CD4+Foxp3+ Tregs in tumors at day 16 (right panel) (n = 4 for the group of Ad-mIFN i.t.+IL-6R ab i.p., n = 6 for the other groups). IL-6R ab, anti-IL-6 receptor antibody; i.t., intratumoral injection; i.p., intraperitoneal administration; TDLNs, tumor-draining lymph nodes.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Cytometry

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 5. IL-6 receptor blockade partially attenuated IFN-α-mediated tumor growth suppression. (a) Growth of tumors treated with IL-6R ab. Tumor volumes in mice treated with the viruses and/or IL-6R ab were measured at the indicated days (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups). Relative tumor volumes compared with those at day 10 were presented. (b) ELISpot assay of IFN-γ- producing cells in mice treated with IL-6R ab. The splenocytes were isolated from mice as shown in Figure 4a at day 28, and the cells were cultured with CT26 or syngeneic splenocytes (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunospot, Isolation, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 6. Intratumoral IFN-α expression increased the number of Th17 cells in tumors. (a) Expression of RORγt and Foxp3 genes in tumors. The tumors injected with viruses were harvested at day 16 and were subjected to real-time PCR analysis (Ad-mIFN: n = 4, Ad-AP: n = 3). (b) Expression of IL-17 in tumors. The tumors were harvested at day 28, and subjected to RT-PCR for IL-17 expression (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 2 for the group of Ad-mIFN i.t.+PBS i.p., n = 3 for the group of Ad-mIFN i.t.+IL-6R ab i.p.). (c) Intracellular cytokine staining of IL-17A in CD4+ T cells. The tumors and tumor-draining lymph nodes were harvested at day 22, and IL-17A expressions were analyzed by flow cytometry (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 4 for the group of Ad-mIFN i.t.+PBS i.p., n = 4 for the group of Ad-AmIFN i.t.+IL-6R ab i.p.) (upper panel). Representative FACS plots of tumors (lower left panel) and tumor-draining lymph nodes (lower right panel) were shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining, Cytometry

Journal: Mucosal immunology

Article Title: Type I IFN ineffectively activates neonatal dendritic cells limiting respiratory antiviral T cell responses

doi: 10.1038/s41385-019-0234-5

Figure Lengend Snippet: a K b -OVA + cDC1s from the dLN of RSV-ova infected adult and neonatal mice were enriched by sorting, and analyzed using RNA-seq to identify DEG ( red and green shaded genes ). Heat map showing the log fold change (Log FC ) of adult compared to neonatal interferon signaling genes. The top five genes, indicated in grey, were identified by IPA to define a difference in the interferon signaling pathway between neonates and adults. Additional genes are shown to provide further context for the different IFN signaling genes. b, c Quantification of b IFNα or c IFNβ by ELISA from neonatal and adult lungs at indicated hours PI. d, e Effect of intranasal IFNα (10 4 U at 8- and 24-hours PI) on the d expression of CD86 on neonatal cDC1s from the dLN 2 DPI and e neonatal RSV-ova-specific CD8 + T cells 7 DPI from the lung. f, g Effect of intranasal IFNβ (10 3 U at 8- and 24-hours PI) on f expression of CD86 on neonatal cDC1s from the dLN 2 DPI and g neonatal RSV-ova-specific CD8 + T cells 7 DPI from the lung. b, c, d, f Data are representative of three independent experiments showing similar results, 4–6 samples per group, 2–8 mice pooled per sample. e, g one mouse per sample, 4–5 samples per group. Mean ± SD are depicted (* p < 0.05, ** p < 0.001, *** p < 0.0001, **** p < 0.00001). b, c Mann-Whitney U test. d-g One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: IFN-I was quantified using mouse IFNα ELISA (limit of detection 125ng/ml) (PBL Assay Science, Piscataway, NJ) or mouse/human IFNβ ELISA (limit of detection 12.5pg/ml) (R&D).

Techniques: Infection, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY, Comparison

Journal: Cell reports

Article Title: Direct type I interferon signaling in hepatocytes controls malaria

doi: 10.1016/j.celrep.2022.111098

Figure Lengend Snippet: (A) Scatterplots showing relative liver-parasite burdens in B6 or Ifnar1 knockout (KO) mice inoculated with 3 × 10 4 P. berghei sporozoites (spzs) at 36-h post infection (p.i.). (B) Scatterplots showing relative liver-parasite burdens in B6 mice treated with vehicle control, STING agonist DMXAA, or TLR3 agonist polyI:C (−1 day p.i.), and inoculated with 3 × 10 4 P. berghei spzs at 36-h p.i. (A and B) Data points represent individual mice, presented as mean ± SEM and analyzed using two-tailed t test (A) or one-way ANOVA with Tukey’s correction (B), and are combined from three separate experiments with ≥3 mice/group. (C) Frequency of P. falciparum -infected primary human hepatocyte in cultures treated with the indicated concentrations of IFNα/β at 4 days p.i. Data are combined from three separate experiments with ≥3 technical replicates. (D) Parasite loads indicated by luciferase activity in primary mouse hepatocyte cultures infected with firefly luciferase expressing P. berghei (Pb-luc) and treated with the indicated concentrations of IFNα/β at 36-h p.i. A.U., arbitrary units. (C and D) Data are presented as mean ± SEM and analyzed using ANOVA comparing the indicated groups with the group treated with 0 U/mL IFNα/β. Data are normalized for background signal and combined from three separate experiments with ≥3 technical replicates. (E) Representative (>10 fields) pseudo-colored confocal images depicting IRF-9 translocation into host cell nuclei, indicating type I IFN signaling in P. berghei -infected B6 mice liver (cryosection) at 24-h p.i. The arrows indicate P. berghei exoerythrocytic forms (EEFs) stained for Hep17 protein in the parasitophorous vacuolar membrane (PVM) in the infected hepatocytes. (F) Principal-component analysis representing gross transcriptional differences between P. berghei ( Pb-GFP )-infected primary murine hepatocytes sorted from infected cultures (36-h p.i.) and naive B6 hepatocytes from parallel uninfected in vitro cultures. (G) Transcriptional perturbations in the interferon regulated genes of P. berghei -infected or uninfected hepatocytes isolated from P. berghei -infected or naive primary murine (B6) hepatocyte cultures at 36-h p.i. (F) and (G) represent three replicate infections. *p ≤ 0.05, **p < 0.01, n.s. p > 0.05.

Article Snippet: Recombinant mouse IFNα , PBL Assay Science , Cat# 12100-1.

Techniques: Knock-Out, Infection, Control, Two Tailed Test, Luciferase, Activity Assay, Expressing, Translocation Assay, Staining, Membrane, In Vitro, Isolation

Journal: Cell reports

Article Title: Direct type I interferon signaling in hepatocytes controls malaria

doi: 10.1016/j.celrep.2022.111098

Figure Lengend Snippet: (A) Histograms representing the expression levels of IFNAR in Pb-Cre -infected Ifnarl fl hepatocytes at 36-h p.i. Pb-Ova -infected Ifnar1 fl hepatocytes, Pb-Cre -in-fected Ifnar1 fl -AlbCre, or uninfected Ifnar1 fl -AlbCre hepatocytes served as controls. The numbers indicate mean fluorescence intensities. Spzs were stained using CellTrace Violet (CTV) to identify the Plasmodium -infected hepatocytes from the infected cultures. Representative data are shown from one of three separate experiments. (B) Frequencies of infected B6 or Ifnar1 fl primary hepatocytes in culture co-incubated with Pb-Cre or control Pb-Ova parasites at 36-h p.i. Combined data are presented as mean ± SEM from three experiments and are analyzed using one-way ANOVA with Tukey’s correction. (C) Scatterplots showing relative liver-parasite burdens at 36-h p.i. in the various groups of mice inoculated with 3 × 10 4 Pb-Cre or Pb-Ova spzs as indicated and treated with or without DMXAA at 24-h p.i. Combined data are presented as mean ± SEM from three experiments and are analyzed using one-way ANOVA with Tukey’s correction. Dots represent individual mice. Dotted line indicates the mean background signal derived from two naive mice. (D) Kinetics of parasitemia in the indicated groups of mice inoculated with 200 Pb-Cre spzs and treated with IFNα/β or control PBS at 12- and 24-h p.i. Combined data from three experiments are presented as mean ± SEM and are analyzed using two-way ANOVA with Tukey’s correction. (E) Representative (>10 fields) pseudo-colored confocal image indicating tdTomato expression, Cre localization, or Pb-Cre in primary Ai14 mouse hepatocytes in culture at 36-h p.i.; green arrows indicate Pb-Cre infection in hepatocytes, red arrows indicate tdTomato-expressing hepatocytes, and yellow arrow shows tdTomato-expressing cells that have detectable Pb-Cre (Hep17 + ) parasites in them. Picture inset shows magnified image of a single Pb-Cre -infected hepatocyte with detectable Pb-Cre in it. See for individual channels. (F) Scatterplot indicating the frequencies of primary Ai14 mouse hepatocytes exhibiting tdTomato expression or detectable infection when co-incubated with Pb-Cre spzs for 36 h. Combined data are presented as mean ± SEM and are analyzed using two-tailed t tests, with each dot representing data from a replicate experiment. *p ≤ 0.05, **p < 0.01, n.s. p > 0.05.

Article Snippet: Recombinant mouse IFNα , PBL Assay Science , Cat# 12100-1.

Techniques: Expressing, Infection, Fluorescence, Staining, Incubation, Control, Derivative Assay, Two Tailed Test

Journal: Cell reports

Article Title: Direct type I interferon signaling in hepatocytes controls malaria

doi: 10.1016/j.celrep.2022.111098

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant mouse IFNα , PBL Assay Science , Cat# 12100-1.

Techniques: Recombinant, Virus, Microarray, Gene Expression, Luciferase, Expressing, CRISPR, Plasmid Preparation, Software

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation *

doi: 10.1074/mcp.RA117.000447

Figure Lengend Snippet: Increased SUMOylation of PML, STAT1 and UBC9 by IFNα. A, Relative changes in abundances of PML, STAT1 and UBC9 SUMOylation in response to IFNα, as determined by quantitative mass spectrometry. Means and standard deviations of three technical replicates are shown. B, Relative changes in PML, STAT1 and UBC9 protein expression in response to IFNα, as determined by Western blotting. C–E, PML, STAT1 and pSTAT1 SUMOylation as determined by Ni-NTA. C, HEK293-wt and HEK293-SUMO3m cells untreated or treated with the proteasome inhibitor for 4 h, were incubated with IFNα or IFNγ for 0.75 h, immunoblots of Ni-NTA-purified extracts were performed using anti-SUMO2/3 or anti-PML antibodies. D, HEK293-wt and HEK293-SUMO3m cells were untreated or treated with IFNα for 0.75 h. Inputs and immunoblots of Ni-NTA-purified extracts were performed using anti-STAT1 or anti-pSTAT1 antibodies. E, HEK293-wt and HEK293-SUMO3m cells untreated or treated with the proteasome inhibitor for 4 h, were incubated with IFNα for 0.75 h. Inputs (upper panel) and immunoblots of cytoplasmic and nuclear Ni-NTA-purified extracts (lower panel) were analyzed by Western blotting using anti-His, anti-H3 and anti-pSTAT1 antibodies. See also supplemental Fig. S3.

Article Snippet: Recombinant human IFNα2 was from Schering (USA), human IFNγ from Roussel Uclaf (Romainville, France), recombinant murine IFNα and IFNγ from R&D Systems (Minneapolis, MN).

Techniques: Mass Spectrometry, Expressing, Western Blot, Incubation, Purification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation *

doi: 10.1074/mcp.RA117.000447

Figure Lengend Snippet: PML is required for IFN-enhanced SUMOylation. A, B, wt MEFs and PML−/− MEFs were untreated or treated with murine IFNα for short (0.75 and 1 h) (A) or long (16 h) (B) periods and their extracts were analyzed by Western blotting for SUMO2/3, SUMO1, PML or Actin. C, D, PML−/− MEFs were transduced with HIV-1 derived lentiviral vectors expressing each human PML isoform (C) (PMLI, PMLII, PMLV, PMLVI or PMLVII), (D) (PMLIII or PMLIV). Two days post-transduction, PML expressing cells were untreated or treated with murine IFNα for 16 h and their extracts were analyzed by Western blotting for SUMO2/3, PML and Actin.

Article Snippet: Recombinant human IFNα2 was from Schering (USA), human IFNγ from Roussel Uclaf (Romainville, France), recombinant murine IFNα and IFNγ from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Transduction, Derivative Assay, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation *

doi: 10.1074/mcp.RA117.000447

Figure Lengend Snippet: IFN enhances the recruitment of UBC9 to PML NBs. A, wt MEFs and PML−/− MEFs untreated or treated with murine IFNα for 0.75 or 16 h were analyzed by confocal microscopy for UBC9 (green) and PML (red) staining. PML was stained with mouse monoclonal anti-PML antibody (clone 36.1–104, Merck millipore) that recognized murine PML. B, wt MEFs and PML−/− MEFs were untreated or treated with murine IFNα for 16 h. Cytoplasmic (C), RIPA soluble (R) and RIPA insoluble (P) fractions were analyzed by Western blotting with anti-UBC9 and anti-PML antibodies. The Coomassie brilliant blue (CBB)-stained proteins are shown. C, Expression of human PMLIII in PML−/− MEFs can recruit endogenous UBC9. Two days post-transduction with PMLIII, PML−/− MEFs were untreated or treated with murine IFNα for 16 h and analyzed by confocal microscopy for UBC9 (green) and PML (red) staining. PML was stained with mouse monoclonal anti-PML antibody (sc 966, Santa Cruz) that recognized human PML.

Article Snippet: Recombinant human IFNα2 was from Schering (USA), human IFNγ from Roussel Uclaf (Romainville, France), recombinant murine IFNα and IFNγ from R&D Systems (Minneapolis, MN).

Techniques: Confocal Microscopy, Staining, Western Blot, Expressing, Transduction