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*

Background: IDO functions as a crucial mediator of tumor-mediated immune tolerance. Results: Here, we found that GSK-3β-dependent IDO expression in the dendritic cell plays a role in anti-tumor activity via the regulation of CD8+ T-cell proliferation and CTL activity. Conclusion: GSK-3β activity functions in the immune enhancement-mediated anti-tumor response via IDO regulation. Significance: We proved the effectiveness of the GSK-3β-dependent IDO regulatory mechanism in DC-based cancer vaccination. 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.

Dendritic cells (DCs) 4 are known to help coordinate the immune system because they play a crucial role in both the induction of immunity and the triggering of T-cell tolerance (1). The DC is an antigen-presenting cell and is deeply involved in immunity via processes that include the capture, processing, and presentation of antigens to T-cells (2). Regarding tolerance, a DC phenotype that is tolerogenic plays a role in immune tolerance. The immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO), which is an essential enzyme that degrades the amino acid tryptophan via the kynurenine pathway, contributes to that tolerance by causing T-cell suppression via tryptophan depletion (1,3). In a tumor environment, an IDO that is highly expressed by tumor cells has been reported to function as a crucial mediator of tumor-mediated immune tolerance (4 -6). Although DCs are crucial for initiating a primary T-cell response (7), IDO-positive DCs are thought to be important in the generation and maintenance of peripheral tolerance via the induction of regulatory T-cell responses (8).
Interferon-␥ (IFN-␥) is known as an inducer of IDO in many cells, including DCs, macrophages, and various cancer cells (9). Transcriptional induction of the IDO gene is mediated by Janus kinase 1 (JAK1) and Stat1 (10). Stat1 acts both directly and indirectly. It works directly by binding to the IFN-␥-activated sites within the IDO promoter. Also, it acts indirectly by induc-ing IFN regulatory factor-1 (IRF-1), which binds to the IDO promoter at two IFN-stimulated response element sites (11). In a previous study, we noted that IFN-␥-induced IDO expression is regulated by both the JAK1/2-Stat1 pathway and the protein kinase C (PKC) pathway (12).
Glycogen synthase kinase-3␤ (GSK-3␤), a multifunctional serine/threonine kinase found in all eukaryotes, was initially identified as a key regulator of insulin-dependent glycogen synthesis (13). In addition, GSK-3␤ is known to be involved in diverse cellular processes, including proliferation, differentiation, motility, and survival (14). Furthermore, dysregulation of GSK-3␤ has also been implicated in tumorigenesis and cancer progression (14). In recent studies, the role of GSK-3 as a regulator of immune responses, including activation and differentiation of DCs and endotoxemia, has been reported (15)(16)(17). Also, GSK-3-mediated regulation of Stat3 in primary astrocytes of the cerebral cortex was demonstrated (18).
Here, we defined the role and regulatory mechanism of GSK-3␤ in Stat-mediated IDO expression. Using a DC-based tumor vaccination murine model, we examined the substantial role of GSK-3␤ involved in IDO expression via the JAK1/2-Stat signaling cascade in DCs, representative cells of initiating the immune response and mediating T-cell proliferation and CTL responses against EG7 thymoma.

EXPERIMENTAL PROCEDURES
Mice-Eight-to 10-week-old male C57BL/6 (H-2K b and I-A b ) mice were purchased from the Korean Institute of Chemistry Technology (Daejeon, Korea). C57BL/6 OT-I T-cell receptor (TCR) transgenic and IDO Ϫ/Ϫ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The animals were housed in a specific pathogen-free environment within our animal facility and used in accordance with the institutional guidelines for animal care. All experiments were done in accordance with the guidelines of the committee of Ethics of Animal Experiments, Konkuk University.
Silencing of GSK-3␤ by siRNA-To knock down GSK-3␤, BMDCs were transfected with the four sequence pools (ON-TARGET plus SMART pool, LU-001080-00-0020, mouse GSK-3␤, NM_019827 (Dharmacon Lafayette, CO)) using the DharmaFECT 1 transfection reagent according to the manufacturer's protocol. Briefly, BMDCs were seeded in 12-well plates at 80 -90% confluence; the medium was replaced with complete RPMI 1640 medium without antibiotics before transfection. DharmaFECT 1 and siRNA products were incubated separately in serum-free RPMI 1640 medium at room temperature for 5 min. Mixtures were combined, incubated for another 20 min, and added to the cells at the final concentration of 2 l/ml DharmaFECT 1 and 25 nM siRNAs. For IFN-␥ stimulation, siRNA and the transfection reagent were removed 24 h posttransfection, and the complete culture medium was added.
Generation and Culture of DCs-Briefly, bone marrow was flushed from the tibiae and femurs of 6 -8-week-old male C57BL/6 mice and depleted of red blood cells (RBCs) using a red blood cell lysing buffer (Sigma). The cells were then plated in 6-well culture plates (1 ϫ 10 6 cells/ml; 2 ml per well) in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, 20 ng/ml rmGM-CSF, and 10 ng/ml rmIL-4 at 37°C in 5% CO 2 . On days 3 and 5, the floating cells were gently removed from the cultures, and fresh medium was added. On day 6 of culture, nonadherent cells and loosely adherent proliferating dendritic cell aggregates were harvested and re-plated in 60-mm dishes (1 ϫ 10 6 cells/ml; 5 ml/dish) for stimulation and analysis. On day 7, 80% or more of the nonadherent cells expressed CD11c. In certain experiments, the DCs were labeled with bead-conjugated anti-CD11c mAb (Miltenyi Biotec, Gladbach, Germany) and then subjected to positive selection through paramagnetic columns (LS columns; Miltenyi Biotec), according to the manufacturer's instructions, to obtain highly purified populations for subsequent analysis. The purity of the selected cell fraction was Ͼ90%.
Immunofluorescence Assay-Cells were fixed with 4% paraformaldehyde for 10 min and were incubated with IDO antibody purchased from Alexis. Subsequently, the cells were incubated with Alexa 488-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. All images were captured with a fluorescence microscope (AX10; Carl Zeiss, Germany). The results are representative of three independent experiments.
Immunoblot Analysis-In brief, cell lysates were subjected to SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were then blocked with 5% nonfat milk in a washing buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and incubated with the indicated antibodies for 1 h at room temperature. The membranes were washed and incubated for 1 h at room temperature with the appropriate secondary antibodies conjugated with horseradish peroxidase (Amersham Biosciences). Protein bands were visualized using an enhanced chemiluminescence system (Amersham Biosciences).
Coimmunoprecipitation Analysis-In brief, cells were lysed with lysis buffer (20), and the lysates were subjected to 10 min of microcentrifugation at 12,000 ϫ g. The soluble fraction was then incubated for 2 h at 4°C with the appropriate antibodies, after which the mixture was incubated for an additional 1 h at 4°C with protein G-coupled Sepharose beads (Amersham Biosciences). The resultant precipitates were subjected to SDS-PAGE and analyzed via immunoblotting.
Mixed Leukocyte Reaction-Transgenic OVA-specific CD8 ϩ T-cells or CD4 ϩ T-cells were purified from bulk splenocytes via negative selection by using CD4 ϩ T cell isolation kit and CD8 ϩ T cell isolation kit (Miltenyi Biotec, Auburn, CA). The purity of the obtained cell population was assessed to be Ͼ93% by flow cytometry after staining with a Cy5-conjugated anti-CD8 Ab.
Briefly, the cells were resuspended in 5 M carboxyfluroscein diacetate succinimidyl ester (CFSE) in phosphate-buffered saline (PBS) and shaken for 10 min at room temperature. Next, the cells were washed once in pure FBS and twice in PBS with 10% FBS. Immature DCs, OVA-pulsed DCs, OVA-pulsed and IFN-␥-treated DCs, or OVA-pulsed IFN-␥ and GSK-3␤ inhibitor-treated DCs were cultured with CFSE-labeled splenocytes of OT-I T-cell receptor transgenic mice (1 ϫ 10 6 per well) for 96 h. After 4 days, the cells were harvested and stained with Cy5-labeled anti-CD8 Ab (to gate OT-I T-cells) and analyzed by flow cytometry.
Cytokine Analysis-The levels of IFN-␥ were measured, using an ELISA kit from R&D Systems (Minneapolis, MN), according to the manufacturer's instructions.
In Vitro Cytotoxicity Assays-To determine the effect of IFN-␥ and GSK-3␤ on OVA-specific CTLs, cytotoxicity assays were performed. In mixed cultures, immature DCs, OVApulsed DCs, OVA-pulsed and IFN-␥-treated DCs, or OVApulsed IFN-␥ and GSK-3␤ inhibitor-treated DCs were first cul- Cell lysates were directly subjected to immunoblot analysis with the indicated antibodies. C, BMDCs were pretreated with or without a GSK-3␤ inhibitor for 30 min and then incubated with IFN-␥ (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. Preventive Tumor Challenge Experiments-Mice were intraperitoneally injected once a week for 3 weeks with immature DCs, OVA-pulsed DCs, OVA-pulsed and IFN-␥-treated DCs, or OVA-pulsed IFN-␥ and GSK-3␤ inhibitor-treated DCs, followed by subcutaneous injection of EG7 thymoma cells (1 ϫ 10 6 ) into the right lower back. The tumor size was measured every 4 days, and the tumor mass was calculated as follows: V ϭ (2A ϫ B)/2, where A is the length of the short axis, and B is the length of the long axis.

GSK-3␤-mediated IDO Regulation in Dendritic Cell
Statistical Analysis-All experiments were repeated at least three times, and consistent results were obtained. Unless otherwise stated, data are expressed as the mean Ϯ S.E. Analysis of variance was used to compare experimental groups with control values, whereas comparisons between multiple groups were made using Tukey's multiple comparison tests (Prism 3.0 GraphPad software). p values of less than 0.05 were considered statistically significant.

GSK-3␤ Activity Is Crucial for the Expression and Activity of IDO via the JAK1/2-Stat Signaling
Cascade-In a previous study, it was revealed that a GSK-3 inhibitor disturbs the activation of Stat3 by blocking the interaction between IFN-␥ and Stat3 in primary astrocytes (18). However, the physiological meaning of a GSK-3 inhibitor-mediated reduction of Stat activity in IFN-␥-stimulated conditions was not defined. Here, we illuminate the precise regulatory mechanism of GSK-3␤ by examining the influence of a GSK-3␤ inhibitor on the JAK1/2-Stat signaling axis and PKC␦ on the IFN-␥-induced expression of IDO, an immunoregulatory enzyme in DCs. Moreover, by using DC-mediated immune enhancement via T-cell proliferation and a DC-vaccinated murine EG7 thymoma model system, we investigated the physiological Consistent with a previous study (18), IFN-␥ provokes the activation of GSK-3␤ in BMDCs (Fig. 1A). In addition, in IFN-␥-treated conditions, GSK-3␤ inhibition blocked the activation of Stat3 (Fig. 1B). However, the activity of JAK1 and JAK2, upstream kinases of Stat3, did not change (Fig. 1B, left panel). Therefore, we inferred that a GSK-3␤ inhibitor (SB415286) prohibits the JAK1/2-Stat signaling cascade by inhibiting Stat3. In addition, the IFN-␥-mediated expressions of IFN-regulatory factor-1 (IRF-1), a downstream transcription factor of Stat3, and IDO were also inhibited (Fig. 1B, right panel). As shown in Fig. 1C, using an immunofluorescence assay, we confirmed that IFN-␥-induced expression of IDO is reduced by adding a GSK-3␤ inhibitor. Those results indicate that GSK-3␤ modulates the expression of IDO, a crucial immunoregulatory protein, by regulating Stat3 in the IFN-␥ signaling cascade.

GSK-3␤ Regulates the Expression of IDO in Both a PKC␦-dependent and -independent
Manner-In a previous report, we revealed that PKC␦-dependent Stat regulation was critical for IDO expression in IFN-␥-treated BMDCs (12). Thus, we examined whether or not GSK-3␤ inhibition-mediated regulation of Stat3 and IDO was via PKC␦. As shown in Fig. 2A, IFN-␥-induced activation of PKC␦ was blocked by a GSK-3␤ inhibitor in BMDCs. Using GSK-3␤ Ϫ/Ϫ MEFs, we confirmed that GSK-3␤ is crucial for PKC␦ activity (Fig, 2A, right). Next, we investigated whether or not GSK-3␤ inhibition-mediated suppression of Stat3 and IDO is independent on PKC␦ using rottlerin (PKC␦ inhibitor). Here, we found that a GSK-3␤ inhibitor partially inhibits Stat3 activity and IDO expression in the presence of rottlerin (Fig. 2B).
Previously, we found that Stat1 activity is modulated by inhibiting protein kinase C␦ activation in BMDCs (12); therefore, we examined whether GSK-3␤ activity influences Stat1 activity in IFN-␥-stimulated conditions. Here, we found that GSK-3␤ inhibition suppressed the activity of Stat1 in a PKC␦dependent manner (Fig. 2C). Thus, we concluded that GSK-3␤ inhibition-mediated regulation of Stat and IDO occurs via PKC␦dependent or -independent pathways in IFN-␥-treated BMDCs.
In GSK-3␤ Ϫ/Ϫ MEFs and GSK-3␤ Knockdown Dendritic Cells, the Expressions of IDO and IRF1 Were Blocked Due to Impaired Stat Activity-Because of the fact that we found that GSK-3␤ regulates the expressions of IDO and IRF1 by modulating Stat in IFN-␥-treated BMDCs, we confirmed the phenomenon using GSK-3␤ Ϫ/Ϫ MEFs. Consistent with data in dendritic cells, GSK-3␤ abolition did not influence the activity of JAK1 and JAK2,

GSK-3␤-mediated IDO Regulation in Dendritic Cell
upstream kinases of Stat in an IFN-␥-stimulated condition. Instead, it blocked the activation of Stat1 and Stat3 (Fig. 4A). In addition, IFN-␥-mediated expression of IRF-1, a downstream transcription factor of Stat, and IDO were also inhibited (Fig. 4B). Furthermore, we confirmed that the activation of Stat1 and Stat3 and the expression of IDO were also inhibited by the silencing of GSK-3␤ expression (Fig. 4C). From that data, we confirmed that GSK-3␤ modulates the expression of IDO by regulating Stat in IFN-␥-stimulated conditions.

Interferon (IFN)-␥-induced IDO Expression via GSK-3␤ Activity Plays a Role in CD8 ϩ T Cell Proliferation-To determine whether or not IFN-␥-induced IDO expression via
GSK-3␤ activity in DCs affects T-cell proliferation, we performed a mixed lymphocyte reaction (MLR) assay using CD8 ϩ T-cells of OT-I TCR transgenic mice, which express a TCR specific for the MHC class I-restricted OVA peptide 257-264 antigen (OVA(257-264)) in DCs (21). The proliferation of CFSElabeled OVA-specific CD8 ϩ T-cells co-cultured with DCs pulsed with OVA(257-264) was significantly higher than that of T-cells co-cultured with unpulsed DCs (Fig. 5A, upper panel). In addition, the enhanced CD8 ϩ T-cell proliferation by DCs pulsed with OVA peptide was dramatically inhibited by treatment with IFN-␥ (Fig. 5A, upper panel). Furthermore, IFN-␥-mediated suppressed CD8 ϩ T-cell proliferation was restored by the treatment of a GSK-3␤ inhibitor (Fig. 5A, upper panel).
Next, to examine whether or not IFN-␥-mediated suppression of CD8 ϩ T-cell proliferation is due to IDO regulated by GSK-3␤, we performed an MLR assay using IDO Ϫ/Ϫ dendritic cells. In that experiment, OVA-pulsed DC-induced CD8 ϩ T-cell proliferation was not suppressed by IFN-␥ in the absence of IDO (Fig. 5A, lower panel). We then investigated IFN-␥ production in the abovementioned MLR conditions. A higher level of IFN-␥ was produced by syngeneic T-cells primed with OVA peptide-pulsed DC reduced by IFN-␥ (IDO inducer)-treated DC (Fig. 5B). By using GSK-3␤-inhibited DC, IFN-␥-treated DC-mediated attenuation of OVA-pulsed DC-mediated IFN-␥ production in T-cells was restored (Fig. 5B). To examine whether the attenuation of OVA-pulsed DC-mediated IFN-␥ production in T-cells by IFN-␥-treated DC is due to IDO, we checked the level of IFN-␥ in an MLR assay using IDO Ϫ/Ϫ dendritic cells. Here, the reduced level of OVA-pulsed DC-mediated IFN-␥ production in T-cells by IFN-␥-treated DC was not restored (Fig. 5B). From these results, we inferred that IFN-␥induced IDO expression via GSK-3␤ activity in DCs modulates CD8 ϩ T-cell proliferation.

GSK-3␤ Activity Involving IFN-␥-induced IDO Expression
Regulates Cytotoxic T Cell Responses-Next, we investigated the cytotoxic T-cell responses of CD8 ϩ T-cells generated by OVA-pulsed DCs. OVA-pulsed DCs reduced the population of CSFE-stained OVA-expressing EG7 tumor cells compared with nonpulsed DCs, and the reduced OVA-expressing EG7 tumor cells were restored by IFN-␥-treated and OVA-pulsed DCs (Fig.  6, upper panel). Furthermore, the restored CTL activity by IFN-␥ was abolished by GSK-3␤ inhibition (Fig. 6, upper panel). However, DC-mediated modulation of CTL responses did not affect non-OVA-expressing EL4 tumor cells (Fig. 6, upper  panel). In addition, by IFN-␥-treated and OVA-pulsed DCs or IFN-␥ ϩ SB415286-treated and OVA-pulsed DCs, the modulation of OVA-pulsed DC-mediated CTL activity against OVAexpressing EG7 tumor cells was not observed in the absence of IDO (Fig. 6, lower panel). From that data, we concluded that OVA-specific CTL activity was consistent with the previous CD8 ϩ T-cell proliferation data.

IFN-␥-induced IDO Expression via GSK-3␤ Is Effective for OVA-pulsed DC Vaccinations against EG7 Thymoma-Next,
we assessed that IFN-␥-induced IDO expression via GSK-3␤ in DCs is crucial for cell-based anti-tumor activity using an EG7 lymphoma model. To determine the anti-tumor potential of immunization with OVA-treated DCs, mice were intraperitoneally injected once a week for 3 weeks with immature DCs, OVA-pulsed DCs, OVA-pulsed IFN-␥-treated DCs, or OVApulsed IFN-␥ and GSK-3␤ inhibitor-treated DCs (1 ϫ 10 5 ), followed by subcutaneous injection of EG7 thymoma cells (1 ϫ 10 6 ) into the right lower back. Injection of DCs pulsed with OVA(257-264) significantly reduced the size of the OVA-expressing EG7 tumors compared with tumors in mice receiving DCs that had not been pulsed with OVA (Fig. 7A). Interestingly, EG7 tumors in mice that received IFN-␥-treated DCs pulsed with OVA were larger than those in mice that received DCs pulsed with OVA (Fig. 7A). In the group of IFN-␥ and GSK-3␤ inhibitor-treated DCs pulsed with OVA, the attenuated anti- tumor activity was restored (Fig. 7A). Consistent with that finding, the group of mice injected with OVA-pulsed DCs showed prolongation of survival compared with mice injected with OVA-pulsed IFN-␥-treated DCs (Fig. 7B). The group of mice injected with IFN-␥ and GSK-3␤ inhibitor-treated DCs pulsed with OVA had an enhanced survival score compared with mice injected with IFN-␥-treated DCs pulsed with OVA (Fig. 7B). From those results, we concluded that IFN-␥-mediated IDO regulation via GSK-3␤ activity plays a role in dendritic cellbased vaccination against EG7 thymoma tumor.
Here, we illuminate the precise regulatory mechanism of GSK-3␤ by examining the influence of a GSK-3␤ inhibitor on the JAK1/2-Stat signaling axis and PKC␦ on IFN-␥-induced expression of IDO, an immunoregulatory enzyme in DCs. IDO is an essential enzyme that degrades the amino acid tryptophan via the kynurenine pathway (1,3). It has been reported that IDO is functionally expressed in various immune cells such as DCs and macrophages. It has been shown to also play a role in the immune response as a crucial modulator of tumor-mediated immune tolerance by causing T-cell suppression (1, 23, 24).  . IFN-␥-induced IDO expression via GSK-3␤ is effective for OVA-pulsed DC vaccination against EG7 thymoma. Mice were intraperitoneally injected three times (a week apart) with immature DCs, OVA (1 g/ml)-pulsed, OVA-pulsed IFN-␥-treated, or OVA-pulsed IFN-␥ ϩ GSK-3␤ inhibitor-treated DCs, followed by subcutaneous injection into the right lower back with EG7 thymoma cells (1 ϫ 10 6 ). Tumor size was measured every 4 days, and tumor mass was calculated. For statistical analysis, analysis of variance was used. n ϭ 9 mice per group. *, p Ͻ 0.05, and **, p Ͻ 0.01. Survival of mice was recorded for up to 100 days.
Based on the function of IDO as a crucial mediator of immune tolerance, we further examined the idea that the DC-based immune response mediated by IFN-␥-induced IDO expression via GSK-3␤ activity influences CD8 ϩ T-cell proliferation, cytotoxic T lymphocyte activity, and OVA-pulsed DC vaccination against EG7 thymoma.
DCs function as crucial regulatory cells in the innate and adaptive immune response; therefore, DC-based immunotherapy via the enhancement of host immunogenicity could be a crucial strategy to prevent immune rejection, a major problem in cancer therapy (25,26). Because of those advantages, antitumor immune responses via DC-based vaccination have been utilized in tumor therapy, and vaccination has been studied with regard to the above-mentioned DC functions. Tumor peptide-loaded DCs effectively lead a CTL response in the tumor environment, thereby rendering tumor cell lysis by CTLs. In this study, we applied our finding, the regulation of IFN-␥-induced IDO expression via GSK-3␤ activity, to DC-based cancer vaccination. We found that the regulation of IDO levels by GSK-3␤ activity is a determinant for the efficiency of cell-based cancer vaccination using DCs.
Through various experiments, we clarified that GSK-3␤ activity is crucial for IFN-␥-induced IDO expression in various cells, and we defined that the regulation of DC function by GSK-3␤-dependent IDO regulation is important for CD8 ϩ T-cell proliferation and cytotoxic T lymphocyte activity. Thus, we concluded that GSK-3␤ activity plays an important role in the immune enhancement-mediated anti-tumor response via IDO regulation.
Further studies are needed to define the pivotal role of the regulation of IDO via GSK-3␤ in various disease models such as graft-versus-host disease and clinical immune imbalances, including asthma and autoimmune diseases.