Research ArticleCancer Immunology

Bypassing STAT3-mediated inhibition of the transcriptional regulator ID2 improves the antitumor efficacy of dendritic cells

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Science Signaling  27 Sep 2016:
Vol. 9, Issue 447, pp. ra94
DOI: 10.1126/scisignal.aaf3957
  • Fig. 1 ID2 expression enhances the antitumor efficacy of GM-DCs.

    (A) Purified GM-DCs derived from bone marrow samples from Id2+/GFP mice (CD45.2+) were injected intratumorally into established B16 melanoma tumors in congenic CD45.1+ tumor-bearing mice. GFP expression in CD45.2+ GM-DCs was analyzed before (0 hours) and 24 hours after intratumoral delivery by flow cytometry. GM-DCs from wild-type (WT) (CD45.2+ Id2+/+) mice served as a control. (B) Id2+/GFP mice were injected subcutaneously with 5 × 105 B16 melanoma cells. After 7 days, mononuclear cells were isolated from tumors or the adjacent skin region (outside the tumor margin). Flow cytometry was used to detect GFP+ cells within the CD11c+ CD103+ CD24+ DC population. CD11c+ CD103+ CD24+ DCs from WT (Id2+/+) mice were analyzed as a control. Data in (A) and (B) are representative flow plots and mean median fluorescence intensity (MFI) ± SEM from three independent assays. ****P < 0.0001, by Student’s t test. (C and D) Purified ID2–GM-DCs and RV–GM-DCs were stimulated with LPS (0.1 μg/ml) for 6 hours. After thorough washing with sterile phosphate-buffered saline (PBS) three times, ID2–GM-DCs and RV–GM-DCs were delivered intratumorally (i.t.) into B16 melanoma tumors in C57BL/6 mice (7 days after tumor establishment), as indicated. Tumor size was assessed every 2 to 3 days (C), and survival of tumor-bearing animals was analyzed (D). Mice were euthanized upon tumors reaching 20 mm in any direction or at the completion of the experiment. Data represent four independent experiments. n = 5 to 6 mice per vaccination condition per experiment. **P < 0.01, by analysis of variance (ANOVA) followed by Bonferroni’s post test (C); indicated P value (D) determined by Kaplan-Meier log-rank test.

  • Fig. 2 STAT3-dependent regulation of Id2 by tumor-associated cytokines.

    (A) Purified GM-DCs derived from Id2+/GFP or control Id2+/+ bone marrow samples were untreated (NT) or treated ex vivo with IL-6, IL-10, VEGF, or all three for 24 hours. GFP expression was analyzed by flow cytometry. A representative flow plot and mean MFI ± SEM from three independent assays are shown. **P < 0.01, by Student’s t test. (B) Immunoblotting for total and Tyr-phosphorylated STAT3 (p-STAT3) in whole-cell lysates derived from GM-CSF–differentiated bone marrow cells (7 days) treated with IL-6, IL-10, or VEGF (10 ng/ml) for up to 6 hours. Blots are representative of three independent assays. (C) Id2 expression analyzed by quantitative polymerase chain reaction (qPCR) in GM-CSF–differentiated bone marrow cells (7 days) from CD11c Cre Stat3f/f (left panel) or CD11c Cre+ Stat3f/f (right panel) mice, treated ex vivo for 6 hours with IL-6, IL-10, VEGF (10 ng/ml), or B16-conditioned medium (B16-CM) in the absence (−) or presence of blocking antibodies to IL-6 (αIL-6), IL-10 (αIL-10), VEGF (αVEGF), all three (All), or control immunoglobulin G (IgG) (10 μg/ml). Data are means ± SEM from four independent assays. **P < 0.01, ***P < 0.001, compared to controls (NT, −), by Student’s t test. (D) CD11c Cre+ Stat3f/f Id2+/GFP mice and CD11c Cre Stat3f/f Id2+/GFP littermate controls were injected subcutaneously with 5 × 105 B16 melanoma cells. GFP expression was measured in tumor-infiltrating or skin-resident CD103+ DCs by flow cytometry 14 days after tumor establishment. Representative flow plot and mean MFI ± SEM from three independent assays are shown. (E) Chromatin immunoprecipitation (ChIP) assays with STAT3 antibody or IgG control and the Id2 promoter in D2SC/1 cells either untreated or treated with the indicated cytokine for 1 hour. Data are means ± SEM from four independent assays. (F) Id2 promoter activity was measured by luciferase assay in D2SC/1 cells transfected with the pGL3/Id2 reporter construct in the presence of STAT3 WT or ​STAT3–transactivation domain mutant (TAD) plasmids and stimulated with IL-6, IL-10, VEGF, or all three (10 ng/ml for 2 hours). Data are mean fold change of promoter activity upon cytokine treatment relative to untreated controls from four independent assays. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Student’s t test.

  • Fig. 3 STAT3 deficiency in DCs enhances antitumor function.

    (A to C) CD11c Cre+ Stat3f/f mice and CD11c Cre Stat3f/f littermate controls were injected subcutaneously with 5 × 105 B16 melanoma cells. Tumor growth (A) and mouse survival (B) were analyzed as described in Fig. 1 (C and D, respectively). TILs were analyzed for intracellular cytokine amounts by flow cytometry 14 days after tumor establishment; data are presented as number of cells in each subset per 1 g of tumor (C). (D to F) C57BL/6 mice were injected subcutaneously with 5 × 105 B16 melanoma cells. One week later, purified GM-DCs derived from CD11c Cre+ Stat3f/f (Stat3Δ/Δ), CD11c Cre Stat3f/f (Stat3+/+), or CD11c Cre+ Stat3f/f Id2GFP/GFP (Stat3Δ/Δ Id2GFP/GFP) bone marrow samples were stimulated with LPS for 4 to 6 hours, washed extensively, and injected into B16 melanoma tumors in mice. As in (A) to (C), tumor growth (D), mouse survival (E), and TIL profiles [at 13 days (6 days after DC vaccine)] (F) were analyzed. (A to F) *P < 0.05, **P < 0.01, determined by ANOVA followed by Bonferroni’s post test (A and D); P values determined by Kaplan-Meier log-rank test (B and E) or Student’s t tests (C and F). Results represent at least three independent experiments. n = 5 to 6 mice per vaccination condition per experiment.

  • Fig. 4 Constitutive ID2 expression suppresses GM-DC cytokine production and NF-κB activity.

    (A) Expression of cytokine-encoding mRNAs in purified ID2–GM-DCs or RV–GM-DCs stimulated with LPS [0.1 μg/ml for up to 6 hours], analyzed by qPCR. Data are means ± SEM from five independent assays. *P < 0.05, **P < 0.01, by Student’s t tests. (B) Cytokine production in LPS-treated (6 hours) or untreated ID2–GM-DCs and RV–GM-DCs, evaluated by intracellular staining and flow cytometry. Representative flow plots (left) and mean MFI ± SEM from three independent assays (right) are shown. *P < 0.05, **P < 0.01, ***P < 0.001, by Student’s t tests. (C) NF-κB activity in nuclear extracts of purified ID2–GM-DCs and RV–GM-DCs untreated or stimulated with LPS (0.1 μg/ml for the indicated time), assessed by electrophoretic mobility shift assay (EMSA). Data are presented as densitometry values relative to controls (densitometry of NF-Y EMSA) from three independent experiments; means ± SEM are indicated. (D) Autoradiography of kinase assay (KA) detecting phosphorylated GST-IκBα(154) in solution with IKK isolated by NEMO pulldown from untreated or LPS-stimulated RV–GM-DCs or ID2–GM-DCs. IKKβ immunoblot (IKK IB) was performed as a loading control. Blots are representative of two independent experiments. (E) Immunoprecipitation (IP) with Flag or hemagglutinin (HA) antibodies in whole-cell lysates from 293T cells transfected with plasmids encoding Flag-tagged ID2 and HA-tagged IKKα, IKKβ, NEMO, IκBα, or a control vector (−), followed by Western blotting as indicated. Blots represent three independent assays.

  • Fig. 5 Effect of constitutive ID2 expression in GM-DCs on CD4+ T cell responses.

    (A) B16 melanoma–bearing C57BL/6 mice were vaccinated with purified RV–GM-DCs, ID2–GM-DCs, or PBS, as described in Fig. 1. Six days after DC vaccine, TIL profiles were analyzed by flow cytometry. Data are means ± SEM from three independent experiments. n = 5 mice per vaccination condition per experiment. (B) Representative flow cytometry (left) for intracellular amounts of IFN-γ and FOXP3 in CD4+ T cells isolated from OT-II mice, after coculture of naïve CD4+ T cells with purified, LPS-stimulated RV–GM-DCs or ID2–GM-DCs and OVA323–339 peptide (10 μg/ml) for 4 days in the presence of recombinant TNF-α (10 ng/ml), TNF-α antibody (10 μg/ml), or IgG control (−) as indicated. Relative ratio of IFN-γ+ versus FOXP3+ T cells (right) determined from mean percent positive ± SEM of three independent experiments. (C) Flow cytometry analysis of the proliferation of naïve CD4+ T cells labeled with eFluor670 cell proliferation dye and cocultured with purified, LPS-stimulated RV–GM-DCs or ID2–GM-DCs. Results represent three independent assays. (D) Tumor growth (monitored as described in Fig. 1) in Tcrb−/−/Tcrd−/− mice bearing B16 melanoma tumors (subcutaneously) and vaccinated with the indicated DC vaccine (2 × 106 cells intratumorally per mouse) and naïve CD4+, CD8+ or CD4+ and CD8+ T cells (106 cells intravenously per mouse). (E and F) Tumor growth in (E) and survival of (F) C57BL/6 mice bearing B16 melanoma tumors and vaccinated with the indicated DC vaccine (2 × 106 cells intratumorally per mouse) and either IgG or PD-1 antibody (IgG or α–PD-1; 250 μg intraperitoneally every other day × 3), as indicated. Data in (D) to (F) are means ± SEM from three independent assays. n = 5 mice per vaccination condition per experiment. *P < 0.05, **P < 0.01, ***P < 0.001, by Student’s t test (A, B, and D) or by ANOVA followed by Bonferroni’s post test (E) or Kaplan-Meier log-rank test (F).

  • Fig. 6 Working model for the roles of STAT3 and ID2 in DC-mediated antitumor immunity.

    Tumor-secreted cytokines stimulate STAT3 activity, which inhibits DC-intrinsic expression of ID2 and dampens DC-mediated immunostimulatory function (left). Constitutive ID2 expression or ablation of STAT3 circumvents ID2 inhibition, resulting in ID2-mediated suppression of NF-κB signaling and reduced IL-6 and TNF-α production, promoting antitumor immunity. Teff, effector T cell.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/447/ra94/DC1

    Fig. S1. Characterization and function of RV–GM-DCs and ID2–GM-DCs.

    Fig. S2. Characterization of STAT3 signaling pathways.

    Fig. S3. Regulation of Id2 by STAT3-activating cytokines in D2SC/1 cells.

    Fig. S4. Phenotypic characterization of Stat3-sufficient and Stat3-deficient GM-DCs.

    Fig. S5. RV–GM-DC and ID2–GM-DC cell surface phenotypes.

    Fig. S6. Effects of ID2 in D2SC/1 cells and cytokine production in B16 melanoma tumors.

    Fig. S7. MAPK activation in RV–GM-DCs and ID2–GM-DCs.

    Fig. S8. Mononuclear cell infiltration in B16 melanomas upon GM-DC vaccination.

    Table S1. Oligonucleotide sequences.

  • Supplementary Materials for:

    Bypassing STAT3-mediated inhibition of the transcriptional regulator ID2 improves the antitumor efficacy of dendritic cells

    Haiyan S. Li, Chengwen Liu, Yichuan Xiao, Fuliang Chu, Xiaoxuan Liang, Weiyi Peng, Jianhua Hu, Sattva S. Neelapu, Shao-Cong Sun, Patrick Hwu, Stephanie S. Watowich*

    *Corresponding author. Email: swatowic{at}mdanderson.org

    This PDF file includes:

    • Fig. S1. Characterization and function of RV–GM-DCs and ID2–GM-DCs.
    • Fig. S2. Characterization of STAT3 signaling pathways.
    • Fig. S3. Regulation of Id2 by STAT3-activating cytokines in D2SC/1 cells.
    • Fig. S4. Phenotypic characterization of Stat3-sufficient and Stat3-deficient GM-DCs.
    • Fig. S5. RV–GM-DC and ID2–GM-DC cell surface phenotypes.
    • Fig. S6. Effects of ID2 in D2SC/1 cells and cytokine production in B16 melanoma tumors.
    • Fig. S7. MAPK activation in RV–GM-DCs and ID2–GM-DCs.
    • Fig. S8. Mononuclear cell infiltration in B16 melanomas upon GM-DC vaccination.
    • Table S1. Oligonucleotide sequences.

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    Citation: H. S. Li, C. Liu, Y. Xiao, F. Chu, X. Liang, W. Peng, J. Hu, S. S. Neelapu, S.-C. Sun, P. Hwu, S. S. Watowich, Bypassing STAT3-mediated inhibition of the transcriptional regulator ID2 improves the antitumor efficacy of dendritic cells. Sci. Signal. 9, ra94 (2016).

    © 2016 American Association for the Advancement of Science

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