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: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Viral RNA in the supernatant of Calu-3 cells infected with indicated SARS-CoV-2 variants was quantified by qRT–PCR at 48 h postinfection (MOI 0.05). Cells were treated preinfection with increasing concentrations of indicated IFNs (α2 at 5 and 500 IU/ml, β, and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml). Dots represent the mean of n = 3 + SEM. (B) Percentage of viral RNA in the supernatant compared with the no IFN control (set to 100%) at 500 IU/ml IFN (100 ng/ml for IFN-λ1) as indicated. Bars represent the mean of n = 3 ± SEM. (C) Infectious SARS-CoV-2 particles determined by the TCID50 assay in the supernatant of Calu-3 cells infected with indicated SARS-CoV-2 variants (MOI 0.05, 48 h postinfection). Cells were left untreated or were pretreated with 500 IU/ml IFN-β or IFN-γ. Bars represent the mean of n = 3 ± SEM. d.l., detection limit. (B, C, D) Correlation of infectious titer of viral particles ((C), TCID50) with viral RNA in the supernatant ((B), qRT–PCR), r, Pearson’s correlation. Statistical significance was calculated using t tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR, Control, TCID50 Assay

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Metabolic activity of Calu-3 cells as assessed by MTT assay after treatment with IFNs (α2, β, and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml) as in . Dots represent the mean of n = 3 ± SEM. (B) Mean of viral RNA in the supernatant of Calu-3 cells infected with indicated SARS-CoV-2 variants quantified by qRT–PCR at 6 h (wash CTRL) and 48 h postinfection (MOI 0.05). Bars represent the mean of n = 3 ± SEM (undetermined values in the 6 h sample were excluded from the calculation). (C) Representative immunofluorescence images of Calu-3 cells infected with Omicron BA.1 (MOI 0.05) or uninfected. Cells were stained for SARS-CoV-2 nucleocapsid (red), SARS-CoV-2 spike (green), and nuclei (blue) at 24 or 48 h postinfection. Scale bar = 50 μm. (D) Metabolic activity of iAT2 cells as assessed by MTT assay after treatment with IFNs (α2, β, and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml) as in . Dots represent the mean of n = 3 ± SEM.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Activity Assay, MTT Assay, Infection, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Viral RNA in the supernatant of iAT2 cells infected with indicated SARS-CoV-2 variants was quantified by qRT–PCR at 48 h postinfection (MOI 0.5). The cells were treated 24 h preinfection with increasing concentrations of indicated IFNs (α2 at 5 and 500 IU/ml, β and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml). Dots represent the mean of n = 3–4 + SEM. (B) Percentage of viral RNA in the supernatant compared with the no IFN control (set to 100%) at 500 IU/ml IFN (100 ng/ml for IFN-λ1) as indicated. Bars represent the mean of n = 3–4 ± SEM. Statistical significance was calculated using t tests. * P < 0.05.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR, Control

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Viral RNA levels harvested from the apical surfaces of ALI cultures infected with indicated SARS-CoV-2 variants 1 h post-treatment with IFNs (α2, β, and γ at 0.5, 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml) quantified by qRT–PCR at 5 d postinfection (MOI 0.5). Individual donors are indicated. (B) Viral RNA levels harvested from the apical surfaces of ALI cultures infected with SARS-CoV-2 omicron BA.1 (MOI 0.5) quantified by qRT–PCR at 2 h (wash CTRL), and 3- and 5-d postinfection. Individual donors are indicated.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Normalized viral RNA of ALI cultures from three donors (Donors A, B, and C) infected with indicated SARS-CoV-2 variants (MOI 0.5) quantified by qRT–PCR at 5 d postinfection. RNA was harvested from the apical surfaces (top panel) or intracellularly (bottom panel). ALI cultures were treated 1 h preinfection with increasing concentrations of indicated IFNs (α2, β and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml). All values are normalized to the no IFN control (set to 100%) and dots represent the mean of n = 3 (δ and ο BA.1) or n = 2 (NL-02-2020) individual donors + SEM. (B) Percentage of viral RNA from the apical surfaces of ALI cultures compared with the no IFN control (set to 100%) at 500 IU/ml IFN (100 ng/ml for IFN-λ1) as indicated. Bars represent the mean of n = 3 (δ and ο BA.1) or n = 2 (NL-02-2020), individual donors are represented by different symbols and connected with lines. Statistical significance was calculated using ratio paired t tests compared with NL-02-2020. * P < 0.05; ** P < 0.01.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR, Control

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: (A) Spleen cells from cotton rats were stimulated with Concanavalin A for 72 hours and lymphoblasts purified by centrifugation on a ficoll-gradient. Subsequently, cells were stained with antisera specific for the human interferon receptor to test for crossreactive binding to cotton rat cells and analyzed by flow cytometry. Antibody specific for human interferon alpha receptor-1 (IFNAR-1) did not react with cotton rat cells (left) whereas antibody specific for human interferon alpha receptor-2 (IFNAR-2) did (right). IFNR specific antibody is shown as black line, controls as gray areas. B. IFNAR-2 antibody was added to cotton rat spleen cells. One hour later different concentrations of a combination of ODN2216 and poly I:C was added. 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments. Significant differences were seen between cells treated with the combination of ODN2216 and poly I:C alone, or the combination of ODN2216 and poly I:C with antibody. IFNAR-1 antibody (not reactive with cotton rat cells) had no effect. C. Cotton rat spleen cells were stimulated with ODN 2216 or poly I:C, with or without the addition of recombinant cotton rat interferon alpha. (One unit equals one picogram of type I interferon.) 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments, and significance was compared to cells without type I interferon treatment. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Purification, Centrifugation, Staining, Binding Assay, Flow Cytometry, Recombinant

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: A) The number of MeV-specific B cells was measured from bone marrow cells of MeV-immune cotton rats. The addition of ODN 2216 and poly I:C individually and in combination increased B cell numbers whereas the addition of sera neutralizing cotton rat interferon alpha and IL-6 reduced this stimulation. Each bar graph represents the mean ± SD of triplicate wells. B) Cotton rats were immunized intranasally (upper panel) or subcutaneously (lower panel) with MeV, or MeV with ODN 2216 and/or pI:C in the presence or absence of human MeV-specific IgG (neutralization titer of 100) which had been injected intraperitoneally one day before immunization. Sera were collected at seven weeks post vaccination and the titer of neutralizing antibody was determined by neutralization assay. Each bar graph represents the average titer of four animals ± SD. The experiment is representative of three experiments. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Neutralization, Injection