Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Monophosphoryl lipid A plus IFNγ maturation of dendritic cells induces antigen-specific CD8 + cytotoxic T cells with high cytolytic potential

doi: 10.1007/s00262-010-0843-z

Figure Lengend Snippet: The superior induction of CTLs by MPLA/IFNγ DCs compared to CC-DCs is dependent on IL-12 and not on IL-6 or IL-23. a Blocking antibodies specific for IL-12p40 and IL-6, and an irrelevant antibody were added to MPLA/IFNγ DC-CTL cocultures. 7 days after the second stimulation of the CTLs with DCs (day 17), the CTLs were stained with the MART-1 tetramer (TM) and CD8-FITC. A graph showing the % MART-1 TM cells for all separate cultures of one experiment is shown. The condition treated with anti-IL-12 significantly differed (one-way ANOVA, P < 0.05) from untreated or isotype control treated conditions. A representative experiment out of four is depicted. b Blocking antibodies specific for IL-12p35 and IL-23p19, and an irrelevant antibody were added to MPLA/IFNγ DC-CTL cocultures. 7 days after the third stimulation of the CTLs, the CTLs were stained with the MART-1 TM and CD8-FITC. A graph showing the % MART-1 TM cells for all separate cultures of one experiment is shown. The condition treated with anti-IL-12p35 significantly differed (one-way ANOVA, P < 0.01) from isotype control treated condition

Article Snippet: The following neutralizing antibodies were used; anti-IL-12p40 (C8.6), anti-IL-12p35 (B-T21, Diaclone), anti-IL-23p19 (B-Z23, Diaclone), anti-IL-6 (IL6.8 [ 26 ]).

Techniques: Blocking Assay, Staining, Control

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, Naïve CD4+T cells from WT mice were activated with anti-CD3 and anti-CD28 and cultured with IL-4 and anti-IFN-γ (Th2 conditions) or with IL-4, TGF-β and anti-IFN-γ (Th9 conditions). Each day during differentiation, cells were stained for intracellular-phospho-STAT3 and phospho-STAT6. The bar graphs represent percentages of cells (top) and mean fluorescent intensity (MFI, bottom). Data are average ± S.E.M of 6 mice from 3 experiments.B, C. Naïve CD4+T cells from WT and Stat6−/− (top), WT and Stat3CD4−/− (middle), and WT and Parp14−/− mice (bottom) were cultured under Th9 conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular staining for cytokines IL-9 and IL-4 was performed (B) or stimulated with anti-CD3 and 24h later cell-free supernatant was collected and IL-9 production was assessed by ELISA (C). Data are representative of 3 experiments with similar results.*, p<0.05. ***, p<0.001.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A-C. WT and Il4−/− naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or GATA3-expressing retroviruses (A), control or c-Maf expressing retroviruses (B), or control or JunB-expressing retroviruses (C). After 5d in culture cells were stimulated with PMA and Ionomycin for intracellular staining of IL-9 and IL-4. Data are representative of 2-3 independent experiments with similar results.*, p<0.05. **, p<0.01.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Transduction, Expressing, Staining

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, Naïve CD4+T cells from WT mice were analyzed directly ex vivo (naïve) or cultured under Th1, Th2, Th17, Th9, and Treg cell conditions for 5d. After RNA isolation, quantitative PCR was performed for the indicated genes. Data are representative of 2 experiments with similar results. B, Total cell lysates were prepared from Th1, Th2, and Th9 cells and immunoblot was performed for Runx3, β-actin was used as loading control. C, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or Runx3-expressing retroviruses and control or T-bet expressing retroviruses. Cells were harvested after 5d in culture and stimulated with PMA and ionomycin before intracellular IL-9 and IL-4 staining. Data are average ± S.D of 2 experiments with 4 mice. D, Naïve CD4+T cells from WT and Tbx21−/− (bottom left) or WT and Runx3fl/fl-CD4-Cre (bottom right) were cultured in Th9 cell conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular IL-9 and IL-4 staining. Data are representative of 2-3 experiments with similar results. E, WT and Stat6−/−naïve CD4+T cells were cultured under Th9 cell conditions for 5d. Cells were harvested and quantitative PCR was performed for Runx3and Tbx21.*, p<0.05. **, p<0.01.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Ex Vivo, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Expressing, Staining

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and were cultured with increasing doses of TGF-β in the presence (Th9) or absence (Treg) of IL-4 for 5d. Cells were then stained for intracellular Foxp3 (top) and IL-9 (middle). Differentiated Th9 cells were stimulated with anti-CD3 and 24h later IL-9 production was assessed by ELISA (bottom). B, Naïve CD4+T cells were cultured under Th9 and Treg cell conditions as in A (left) or cultured with 2 ng/ml TGF-β and increasing doses of IL-4 (Th9, right) for 5d. After differentiation, RNA was isolated and Sfpi1 expression was measured by quantitative PCR. Data are representative of 2 experiments with similar results. C, WT and Stat6−/− naïve CD4+T cells were cultured under Th9 cell conditions for 5d and Foxp3 expression was measured using qPCR. Data are average ± S.D of 4 mice from 2 experiments. D, Naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured in Th9 cell conditions for 48h before being transduced with control or Foxp3-expressing retroviruses. After 5d in culture cells were stimulated with PMA and Ionomycin and intracellularly stained for IL-9 and IL-17A. Data are representative of 2 experiments with similar results.E, Schematic showing a summary of a transcription factor network in Th9 cells.*, p<0.05.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Transduction

Journal: The Journal of Biological Chemistry

Article Title: Wingless-type Mammary Tumor Virus Integration Site Family, Member 5A (Wnt5a) Regulates Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Glycoprotein 120 (gp120)-induced Expression of Pro-Inflammatory Cytokines via the Ca 2+ /Calmodulin-dependent Protein Kinase II (CaMKII) and c-Jun N-terminal Kinase (JNK) Signaling Pathways *

doi: 10.1074/jbc.M112.381046

Figure Lengend Snippet: Wnt5a is critical for gp120 to induce IL-1β, IL-6, and TNF-α in the SDH. A–C, time courses of IL-1β (A), IL-6 (B), and TNF-α (C) protein levels after i.t. gp120 (100 ng) administration. D–F, inhibition of Wnt5a by Box5 impaired gp120-induced increase of IL-1β (D), IL-6 (E), and TNF-α (F) protein in the SDH at 12 h after gp120 injection. G–I, sFRP3 attenuated gp120-induced increase of IL-1β (G), IL-6 (H), and TNF-α (I) protein levels in the SDH at 12 h after gp120 injection. Box5 (10 μg), sFRP3 (2 μg) or vehicle was intrathecally administered 30 min prior to 100 ng of gp120 or vehicle injection. In summary graphs, the levels of target proteins were normalized against β-actin and expressed as relative units to the control (0 h or vehicle). Data presented in graphs are means ± S.E. from at least 3 mice per group from three experiments. *, p < 0.05; **, p < 0.01 versus control (one way ANOVA).

Article Snippet: The primary antibodies used included: rabbit anti-Wnt5a (0.5 μg/ml; cat. 72583; abcam), rabbit anti-Wnt5a (1 μg/ml; cat. 2530; CST), goat anti-IL-1β (0.2 μg/ml; cat. AF-401-NA; R&D systems), rat-anti-IL-6 (0.5 μg/ml; cat. 554400; BD Biosciences), goat-anti-TNF-α (0.1 μg/ml; cat. AF-410-NA; R&D systems), rabbit anti-p-αCaMKII (Thr-286) (1:1000; cat. 3361; Cell Signaling Technology (CST)), rabbit anti-CaMKII (1:1000; cat. 3362; CST), rabbit anti-p-JNK (Thr-183/Tyr-185) (1:1000; cat. 9251; CST), rabbit anti-JNK (1:1000; cat. 9252; CST), rabbit anti-p-PKC (pan) (Thr-514) (1:1000; cat. 9379; CST), and rabbit anti-β-actin (1:1000; cat. 4967; CST) antibodies.

Techniques: Inhibition, Injection

Journal: The Journal of Biological Chemistry

Article Title: Wingless-type Mammary Tumor Virus Integration Site Family, Member 5A (Wnt5a) Regulates Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Glycoprotein 120 (gp120)-induced Expression of Pro-Inflammatory Cytokines via the Ca 2+ /Calmodulin-dependent Protein Kinase II (CaMKII) and c-Jun N-terminal Kinase (JNK) Signaling Pathways *

doi: 10.1074/jbc.M112.381046

Figure Lengend Snippet: Exogenous Wnt5a evokes the expression of IL-1β, IL-6, and TNF-α in the SDH. The time courses of IL-1β (A), IL-6 (B), and TNF-α (C) protein dynamics after i.t. Wnt5a (20 ng). In summary graphs, the levels of target proteins were normalized against β-actin and presented as relative units to 0 h. Data are expressed as means ± S.E. from at least three independent experiments with at least three animals. *, p < 0.05; **, p < 0.01 versus 0 h (one way ANOVA).

Article Snippet: The primary antibodies used included: rabbit anti-Wnt5a (0.5 μg/ml; cat. 72583; abcam), rabbit anti-Wnt5a (1 μg/ml; cat. 2530; CST), goat anti-IL-1β (0.2 μg/ml; cat. AF-401-NA; R&D systems), rat-anti-IL-6 (0.5 μg/ml; cat. 554400; BD Biosciences), goat-anti-TNF-α (0.1 μg/ml; cat. AF-410-NA; R&D systems), rabbit anti-p-αCaMKII (Thr-286) (1:1000; cat. 3361; Cell Signaling Technology (CST)), rabbit anti-CaMKII (1:1000; cat. 3362; CST), rabbit anti-p-JNK (Thr-183/Tyr-185) (1:1000; cat. 9251; CST), rabbit anti-JNK (1:1000; cat. 9252; CST), rabbit anti-p-PKC (pan) (Thr-514) (1:1000; cat. 9379; CST), and rabbit anti-β-actin (1:1000; cat. 4967; CST) antibodies.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Wingless-type Mammary Tumor Virus Integration Site Family, Member 5A (Wnt5a) Regulates Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Glycoprotein 120 (gp120)-induced Expression of Pro-Inflammatory Cytokines via the Ca 2+ /Calmodulin-dependent Protein Kinase II (CaMKII) and c-Jun N-terminal Kinase (JNK) Signaling Pathways *

doi: 10.1074/jbc.M112.381046

Figure Lengend Snippet: Wnt5a/CaMKII and Wnt5a/JNK pathways differentially regulate the expression of IL-1β, IL-6, and TNF-α in the SDH. A and B, effects of KN-93 (KN) and SP600125 (SP) on i.t. Wnt5a-evoked pT286-αCaMKII (A) and pT183/Y185-JNK (B) in the SDH. C–E, effects of KN-93, SP600125, and stearoylcarnitine (ST) on Wnt5a-induced IL-1β (C), IL-6 (D), and TNF-α (E) in the SDH. F–H, effects of KN-93, SP600125, and stearoylcarnitine (ST) on gp120-induced IL-1β (F), IL-6 (G), and TNF-α (H) in the SDH. KN-93 (25 μg), SP600125 (10 μg), and ST (20 μg) were intrathecally administered 30 min prior to i.t. Wnt5a (20 ng) or gp120 (100 ng). At 12 h after Wnt5a, gp120 or vehicle injection, the SDH was collected. In summary graphs, the levels of target proteins were normalized with the β-actin loading control and expressed as relative units to the vehicle. Data in the graphs are presented as means ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (one way ANOVA).

Article Snippet: The primary antibodies used included: rabbit anti-Wnt5a (0.5 μg/ml; cat. 72583; abcam), rabbit anti-Wnt5a (1 μg/ml; cat. 2530; CST), goat anti-IL-1β (0.2 μg/ml; cat. AF-401-NA; R&D systems), rat-anti-IL-6 (0.5 μg/ml; cat. 554400; BD Biosciences), goat-anti-TNF-α (0.1 μg/ml; cat. AF-410-NA; R&D systems), rabbit anti-p-αCaMKII (Thr-286) (1:1000; cat. 3361; Cell Signaling Technology (CST)), rabbit anti-CaMKII (1:1000; cat. 3362; CST), rabbit anti-p-JNK (Thr-183/Tyr-185) (1:1000; cat. 9251; CST), rabbit anti-JNK (1:1000; cat. 9252; CST), rabbit anti-p-PKC (pan) (Thr-514) (1:1000; cat. 9379; CST), and rabbit anti-β-actin (1:1000; cat. 4967; CST) antibodies.

Techniques: Expressing, Injection