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: Immunology

Article Title: Co-immunization with an optimized plasmid-encoded immune stimulatory interleukin, high-mobility group box 1 protein, results in enhanced interferon-? secretion by antigen-specific CD8 T cells

doi: 10.1111/j.1365-2567.2009.03044.x

Figure Lengend Snippet: High-mobility group box 1 (HMGB1) induces strong human immunodeficiency virus (HIV)-specific T-cell responses in mice. Interferon-γ enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). BALB/c mice were immunized three times, each 2 weeks apart, with 25 μg of pVax vector or with pHMGB1 and were killed 1 week later. Splenocytes were harvested and cultured overnight in the presence of R10 (negative control), 10 μg/ml of HIV-1 Gag peptide pools or 10 μg/ml of HIV-1 Env peptide pools against a library of peptides spanning HIV-1 subtype B. (a) Responses to HIV-1 Gag are shown as a stacked group. (b) The total additive response to HIV-1 Env. Spot-forming units (SFU) were quantified using an automated ELISPOT reader, and the raw values were normalized to the number of SFU per million splenocytes. Error bars represent the standard deviation (SD) of ELISPOT results in triplicate wells and the data are representative of three independent experiments.

Article Snippet: 23 , 28 , 29 Briefly, 96-well ELISPOT plates (Millipore, Billerica, MA) were coated with anti-mouse IFN-γ capture antibody and incubated for 24 hr at 4° (R&D Systems).

Techniques: Virus, ELISpot Assay, Enzyme-linked Immunospot, Plasmid Preparation, Cell Culture, Negative Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: CTRP3 inhibits the differentiation of Th17 cells in an autocrine manner. (A) The relative expression of C1qtnf3 mRNA in different Th subsets was determined by real-time PCR, and the expression levels are shown relative to that of Th0 cells. Th0, Th1, Th2 and Th17 cells were prepared as described in the Materials and Methods section. (B) WT or C1qtnf3 –/– (KO) naïve CD4 + T cells were cultured under Th17-polarizing conditions for 4 days (n = 4 each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The numbers in each panel indicates percentage of IL-17 + CD4 + T cells in total CD4 + T cells (2 left panels). The content of IL-17 + CD4 + T cells in total CD4 + T cells (%) (center), and IL-17 concentrations (ng/ml) in culture supernatant determined by ELISA (right). Average and SEM are shown. * p < 0.05, *** p < 0.001. Student’s t -test. (C) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th17-polarizing conditions in the absence or presence of recombinant CTRP3 (50, 100 and 200 ng/ml) for 4 days (n = 4 wells each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates the percentage of IL-17 + CD4 + T cells (left), and the proportion of IL-17 + CD4 + T cells is shown in the center panel. IL-17 concentration in the culture supernatant was determined by ELISA (right). Average and SEM are shown. ** p < 0.01, *** p < 0.001. Student’s t -test. (D) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th1-polarizing conditions for 3 days (n = 4 wells each). Intracellular IFN-γ expression was evaluated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates the percentage of IFN-γ + CD4 + T cells in total CD4 T cells (left). The population of IFN-γ + CD4 + T cells (center). IFN-γ concentration in the culture supernatant determined by ELISA (right). Average and SEM are shown. Student’s t -test. (E) The expression of Adipor1 and Adipor2 mRNA in different Th subsets was determined by real-time PCR and relative expression levels to that in Th0 cells are shown. (F) C1qtnf3 –/– naïve CD4 + T cells were cultured with recombinant CTRP3 (200 ng/ml) under Th17-polarizing conditions in the absence (-) or presence of AdipoR1 blocker (R1 block, 10 μg/ml) or AdipoR2 blocker (R2 block, 10 μg/ml) for 4 days (n = 4 each). Intracellular IL-17 expression was evaluated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates percentage of IL-17 + CD4 + T cells (left). The IL-17 + CD4 + T cell content (center). IL-17 concentration in the culture supernatant determined by ELISA (right). Average and SEM are shown. * p < 0.05, ** p < 0.01. Student’s t -test. (G) The effect of AdipoR1 blocker and AdipoR2 blocker on the regulatory effects of CTRP3 on Th17 cell gene expression was examined. In vitro differentiated C1qtnf3 –/– Th17 cells (1 x 10 5 cells in 200 μl/96 well) were treated with CTRP3 (200 ng/ml) in the presence or absence of AdipoR1 blocker (10 μg/ml, R1 block) or AdipoR2 blocker (10 μg/ml, R2 block), and the expression of Ampka , Ppara , Rorc , Stat3 and Mtor mRNA was determined by real-time PCR. The relative expression levels to that of Gapdh are shown. These data are the average from three independent experiments. Average and SEM are shown. * p < 0.05, ** p < 0.01. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Blocking Assay, Gene Expression, In Vitro

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: AdipoRon suppresses Th17 cell differentiation via AdipoR2. (A) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th17-polarizing conditions in the absence or presence of AdipoRon (0, 1, 2 and 3 μg/ml) for 5 days (n = 4 wells each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates percentage of IL-17 + CD4 + T cells (2 left panels). The population of IL-17 + CD4 + T cells (center). IL-17 concentrations in culture supernatant determined by ELISA (right). Average and SEM are shown. ** p < 0.01 and *** p < 0.001. Student’s t -test. (B) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th17-polarizing conditions in the absence or presence of AdipoRon (1 μg/ml, Ron) for 4 days (n = 3 wells each), and the effect of AdipoR1 (10 μg/ml, R1) or AdipoR2 blocker (10 μg/ml, R2) on the Th17 cell differentiation was examined. Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The Number in each panel indicates percentage of IL-17 + CD4 + T cells (4 left panels). The population of IL-17 + CD4 + T cells (center). IL-17 concentrations in culture supernatant determined by ELISA (right). Average and SEM are shown. * p < 0.05, ** p < 0.01 and *** p < 0.001. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Cell Differentiation, Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: Development of EAE is exacerbated in C1qtnf3 –/– mice associated with an augmented Th17 cell differentiation. (A–D) Incidence of affected mice (A) , onset time (B) , disease score time course (C) and mean maximum disease score (D) are shown after induction of EAE (11 mice for each group). Average and SEM are shown. * p < 0.05 and *** p < 0.001. χ 2 -test. Mann-Whitney U -test. Student’s t -test. (E, F) Tissue sections of the spinal cord were stained with H&E (E) and LFB (F) at day 28 after induction of EAE. (G, H) CD4 + T cells (G) , CD4 + IL-17 + and CD4 + IFN-γ + T cells (H) in the whole spinal cord from WT and C1qtnf3 –/– (KO) mice at day 21 after immunization were determined by flow cytometry after PMA/ionomycin stimulation (WT, KO: n = 5 each). The numbers in each panel indicate the percentage of IL-17 + , IFN-γ + , and IL-17 + IFN-γ + T cells ( H , 2 left panels). The population of IL-17-single positive, IFN-γ-single positive and IL-17-, IFN-γ-double positive T cells (H, 3 right panels). Average and SEM are shown. *** p < 0.001. Student’s t -test. (I, J) T cell recall response against the MOG peptide. Seven days after MOG/CFA-immunization, LN cells were isolated from WT and C1qtnf3 –/– (KO) mice and re-stimulated with MOG 35-55 (0, 25, 50 μg/ml) for 72 h (n = 5 each). Then, the proliferative response was measured by [ 3 H]TdR incorporation (I) . IL-17 ( J , left) and IFN-γ ( J , right) concentrations in the culture supernatant were determined by ELISA. Average and SEM are shown. * p < 0.05. Student’s t -test. (K) C1qtnf3 –/– 2D2 T cells and C1qtnf3 -/- BMDCs were cultured with 5 μg/ml MOG 35-55 in the presence or absence of CTRP3 (0, 1, 10, 100 ng/ml) for 5 days, and Th17 cell content (left), and IL-17 (center) and IFN-γ (right) concentrations in the culture supernatant were determined (n = 4 wells each). Average and SEM are shown. * p < 0.05 and ** p < 0.01. Student’s t -test. (L) WT or C1qtnf3 -/- 2D2 T cells were cultured with WT or C1qtnf3 -/- BMDCs in the presence of 5 μg/ml MOG 35-55 for 5 days (n = 3 each), and IL-17 (left) and IFN-γ (right) concentrations in the culture supernatant were determined. Average and SEM are shown. * p < 0.05, ** p < 0.01 and *** p < 0.001. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Cell Differentiation, MANN-WHITNEY, Staining, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: DC differentiation is normal in C1qtnf3 -/- mice. (A) C1qtnf3 mRNA in immune cells was determined by real-time PCR, and the relative expression levels to that in B cells are shown in left panel. C1qtnf3 mRNA expression in DCs after stimulation with zymosan or LPS was determined by real-time PCR, and the relative expression levels to that in cells without stimulation (-) are shown in right panel. (B, C) Effect of CTRP3 on the differentiation and proliferation of BMDCs. BMDCs were differentiated from WT or C1qtnf3 –/– (KO) BMCs (B) , or from C1qtnf3 –/– BMCs in the presence of indicated concentrations of CTRP3 (C) . Then, viable cell number was counted at day 8 by using Cell Counting Kit-8. 4 wells for each. Average and SEM are shown. Student’s t -test. (D) WT or C1qtnf3 -/- (KO) BMCs were cultured under BMDC-differentiation conditions, and the expression of CD11c, CD40, CD80 and CD88 was determined by flow cytometry at day 8. (E) IL-6 and TNF-α concentration in the culture supernatant of WT or C1qtnf3 –/– (KO) BMDCs were measured by ELISA after stimulated with zymosan (0, 40 and 80 μg/ml). Four wells for each. Average and SEM are shown. Student’s t -test. (F) IL-6 and TNF-α concentration in the culture supernatant of C1qtnf3 –/– BMDCs were measured by ELISA after stimulated with zymosan (0, 40 and 80 μg/ml) with/without CTRP3. Four wells for each. Average and SEM are shown. Student’s t -test. (G) IL-6 and TNF-α concentration in the culture supernatant of WT or C1qtnf3 –/– (KO) BMDCs were measured by ELISA after stimulated with LPS (0, 10 and 20 ng/ml). Four wells for each. Average and SEM are shown. * p < 0.05, *** p < 0.001. Student’s t -test. (H) IL-6 and TNF-α concentration in the culture supernatant of C1qtnf3 –/– BMDCs were measured by ELISA after stimulated with LPS (0, 10 and 20 ng/ml) with/without CTRP3. Four wells for each. Average and SEM are shown. * p < 0.05. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Counting, Cell Culture, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay