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Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression - PubMed

  • ️Thu Jan 01 2015

Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression

Kyung Tae Noh et al. J Biol Chem. 2015.

Abstract

Indoleamine 2,3-dioxygenase (IDO) functions as a crucial mediator of tumor-mediated immune tolerance by causing T-cell suppression via tryptophan starvation in a tumor environment. Glycogen synthase kinase-3β (GSK-3β) is also involved in immune and anti-tumor responses. However, the relativity of these proteins has not been as well defined. Here, we found that GSK-3β-dependent IDO expression in the dendritic cell (DC) plays a role in anti-tumor activity via the regulation of CD8(+) T-cell polarization and cytotoxic T lymphocyte activity. By the inhibition of GSK-3β, attenuated IDO expression and impaired JAK1/2-Stat signaling crucial for IDO expression were observed. Protein kinase Cδ (PKCδ) activity and the interaction between JAK1/2 and Stat3, which are important for IDO expression, were also reduced by GSK-3β inhibition. CD8(+) T-cell proliferation mediated by OVA-pulsed DC was blocked by interferon (IFN)-γ-induced IDO expression via GSK-3β activity. Specific cytotoxic T lymphocyte activity mediated by OVA-pulsed DC against OVA-expressing EG7 thymoma cells but not OVA-nonexpressing EL4 thymoma cells was also attenuated by the expressed IDO via IFN-γ-induced activation of GSK-3β. Furthermore, tumor growth that was suppressed with OVA-pulsed DC vaccination was restored by IDO-expressing DC via IFN-γ-induced activation of GSK-3β in an OVA-expressing murine EG7 thymoma model. Taken together, DC-based immune response mediated by interferon-γ-induced IDO expression via GSK-3β activity not only regulates CD8(+) T-cell proliferation and cytotoxic T lymphocyte activity but also modulates OVA-pulsed DC vaccination against EG7 thymoma.

Keywords: cancer biology; cancer vaccine; dendritic cell; glycogen synthase kinase 3 (GSK-3); indoleamine-pyrrole 2,3-dioxygenase (IDO1); interferon.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.
FIGURE 1.

GSK-3β activity is crucial for the expression and activity of IDO via the JAK1/2-Stat signaling cascade. A, BMDCs were treated with or without IFN-γ (100 units/ml) for 30 min and harvested. Cell lysates were directly subjected to immunoblot (IB) analysis with the indicated antibodies. B, right panel, BMDCs were pretreated with or without a GSK-3β inhibitor (SB415286) for 30 min and then harvested after incubating with IFN-γ (100 units/ml) for 30 min. Cell lysates were directly subjected to immunoblot analysis with the indicated antibodies. Left panel, BMDCs were pretreated with or without a GSK-3β inhibitor for 30 min and then harvested after incubating with IFN-γ (100 units/ml) for 24 h. Cell lysates were directly subjected to immunoblot analysis with the indicated antibodies. C, BMDCs were pretreated with or without a GSK-3β inhibitor for 30 min and then incubated with IFN-γ (100 units/ml) for 18 h. Cells were fixed with 4% paraformaldehyde for 10 min, stained with rabbit anti-IDO antibodies overnight at 4 °C, and then stained with Alexa 488-conjugated anti-rabbit antibodies for 1 h at room temperature. Fluorescence intensity was analyzed using the Zeiss AX10 fluorescence microscope. The results are representative of three independent experiments.

FIGURE 2.
FIGURE 2.

GSK-3β regulates the expression of IDO in a PKCδ-dependent and -independent manner. A, left panel, BMDCs were pretreated with or without a GSK-3β inhibitor for 30 min and then harvested after incubating with IFN-γ (100 units/ml) for 30 min. Cell lysates were directly subjected to immunoblot (IB) analysis with the indicated antibodies. Right panel, GSK-3β +/+ and GSK-3β −/− MEFs were harvested and lysed. Cell lysates were directly subjected to immunoblot analysis with the indicated antibodies. B, left panel, BMDCs were pretreated with or without a GSK-3β inhibitor or PKCδ inhibitor (rottlerin) and then harvested after incubating with IFN-γ (100 units/ml) for 30 min. Cell lysates were directly subjected to immunoblot analysis with indicated antibodies. Right panel, BMDCs were pretreated with or without a GSK-3β inhibitor or PKCδ inhibitor and then harvested after incubating with IFN-γ (100 units/ml) for 18 h. Cell lysates were directly subjected to immunoblot analysis with the indicated antibodies. C, BMDCs were pretreated with or without a GSK-3β inhibitor or PKCδ inhibitor incubated with IFN-γ (100 units/ml) for 30 min and then harvested. Cell lysates were directly subjected to an immunoblot analysis with the indicated antibodies. The results are representative of three independent experiments.

FIGURE 3.
FIGURE 3.

GSK-3β activity contributes to the interaction between Stat3 and JAK1/2. A, coimmunoprecipitation between Stat3 and JAK1/2. BMDCs were pretreated with or without a GSK-3 inhibitor and then harvested after incubating with IFN-γ (100 units/ml) for 30 min. Cell lysates were immunoprecipitated (IP) with anti-Stat3 and then were immunoblotted (IB) with an anti-JAK1/2 antibody. B, GSK-3β +/+ and GSK-3β −/− MEFs were harvested after incubating with IFN-γ (100 units/ml) for 30 min. Cell lysates were immunoprecipitated with anti-Stat3 and then were immunoblotted with an anti-JAK1/2 antibody. The results are representative of three independent experiments.

FIGURE 4.
FIGURE 4.

GSK-3β knock-out or knockdown-mediated impairment of Stat activity blocked the expression of IDO. A, GSK-3β+/+ and GSK-3β−/− MEFs were harvested after incubating with IFN-γ (100 units/ml) for 30 min. Cell lysates were directly subjected to immunoblot (IB) analysis with the indicated antibodies. B, GSK-3β+/+ and GSK-3β−/− MEFs were harvested after incubating with IFN-γ (100 units/ml) for 18 h. Cell lysates were directly subjected to immunoblot analysis with the indicated antibodies. C, BMDCs were transfected with GSK-3β siRNA or scrambled siRNA for 24 h. Cells were harvested after incubating with IFN-γ (100 units/ml) for 30 min (upper panel) or 18 h (lower panel). Cell lysates were directly subjected to an immunoblot analysis with the indicated antibodies. The results are representative of three independent experiments.

FIGURE 5.
FIGURE 5.

IFN-γ-induced IDO expression via GSK-3β activity is a determinant in CD8+ T-cell proliferation. Immature, OVA (1 μg/ml)-pulsed, OVA-pulsed IFN-γ-treated, and OVA-pulsed IFN-γ + GSK-3β inhibitor-treated IDO+/+ and IDO−/− DCs were co-cultured with CFSE-labeled splenocytes of OT-I T-cell receptor transgenic mice (1 × 106 per well) for 96 h. A, cells were then harvested and stained with Cy5-labeled anti-CD8 Ab and analyzed by flow cytometry. Histograms show CD8+ T-cell proliferation as assessed by flow cytometry. The results are representative of three independent experiments. **, p < 0.01 compared with No OVA control. B, culture supernatants obtained from the abovementioned condition were harvested after 24 h and were measured, using an ELISA. The mean ± S.E. values represent three independent experiments. **, p < 0.01 (n.s, not significant).

FIGURE 6.
FIGURE 6.

IFN-γ-induced IDO expression via GSK-3β activity plays a role for cytotoxic T-cell responses. Immature, OVA (1 μg/ml)-pulsed, OVA-pulsed IFN-γ-treated, and OVA-pulsed IFN-γ + GSK-3β inhibitor-treated IDO+/+ and IDO−/− DCs were mixed and cultured with OT-I T-cell receptor transgenic mouse splenocytes (1 × 106 per well) for 72 h and then co-cultured with EL4 (1 × 106, 0.5 μ

m

CFSE-stained (CFSElow)) and EG7 (1 × 106, 10 μ

m

CFSE-stained (CSFEhigh)) cells. After 4 h, the mixed lymphocyte tumor cultures were analyzed by flow cytometry. The mean ± S.E. values represent three independent experiments. *, p < 0.05; **, p < 0.01.

FIGURE 7.
FIGURE 7.

IFN-γ-induced IDO expression via GSK-3β is effective for OVA-pulsed DC vaccination against EG7 thymoma. Mice were intraperitoneally injected three times (a week apart) with immature DCs, OVA (1 μg/ml)-pulsed, OVA-pulsed IFN-γ-treated, or OVA-pulsed IFN-γ + GSK-3β inhibitor-treated DCs, followed by subcutaneous injection into the right lower back with EG7 thymoma cells (1 × 106). Tumor size was measured every 4 days, and tumor mass was calculated. For statistical analysis, analysis of variance was used. n = 9 mice per group. *, p < 0.05, and **, p < 0.01. Survival of mice was recorded for up to 100 days.

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