Efficacy of Dendritic Cells Matured Early with OK-432 (Picibanil?), Prostaglandin E2, and Interferon-α as a Vaccine for a Hormone Refractory Prostate Cancer Cell Line

Dendritic cells (DCs) are potent antigen-presenting cells. OK432 (Picibanil®) was introduced as a potent stimulator of DC maturation in combination with prostaglandin-E₂ and interferon-α. We compared the efficacy of a DC-prostate cancer vaccine using early-mature DCs stimulated with OK432, PGE2 and INF-α (OPA) with that of vaccines using other methods. On days 3 or 7 of DC culture, TNF-α (T), TNF-α and LPS (TL) or OPA were employed as maturation stimulators. DU145 cells subjected to heat stress were hybridized with mature DCs using polyethyleneglycol. T cells were sensitized by the hybrids, and their proliferative and cytokine secretion activities and cytotoxicity were measured. The yields of early-mature DCs were higher, compared to yields at the conventional maturation time (P<0.05). In the early maturation setting, the mean fusion ratios, calculated from the fraction of dual-positive cells, were 13.3%, 18.6%, and 39.9%, respectively (P=0.051) in the T only, TL, and OPA-treated groups. The function of cytotoxic T cells, which were sensitized with the hybrids containing DCs matured early with OPA, was superior to that using other methods. The antitumor effects of DC-DU145 hybrids generated with DCs subjected to early maturation with the OPA may be superior to that of the hybrids using conventional maturation methods.     

Increasing numbers of hepatic dendritic cells promote HMGB1-mediated ischemia-reperfusion injury

Endogenous ligands released from damaged cells, so-called damage-associated molecular pattern molecules (DAMPs), activate innate signaling pathways including the TLRs. We have shown that hepatic, warm ischemia and reperfusion (I/R) injury, generating local, noninfectious DAMPs, promotes inflammation, which is largely TLR4-dependent. Here, we demonstrate that increasing dendritic cell (DC) numbers enhance inflammation and organ injury after hepatic I/R. High-mobility group box 1 (HMGB1), a NF released by necrotic cells or secreted by stimulated cells, is one of a number of ligands promoting TLR4 reactivity. Augmentation of DC numbers in the liver with GM-CSF hydrodynamic transfection significantly increased liver damage after I/R when compared with controls. TLR4 engagement on hepatic DC was required for the I/R-induced injury, as augmentation of DC numbers in TLR4 mutant (C3H/HeJ) mice did not worsen hepatic damage. It is interesting that TLR4 expression was increased in hepatic DC following HMGB1 stimulation in vitro, suggesting a mechanism for the increased liver injury following I/R. It thus appears that functional TLR4 on DC is required for I/R-induced injury. Furthermore, HMGB1 may direct the inflammatory responses mediated by DC, at least in part, by enhancing TLR4 expression and reactivity to it and other DAMPs.     

TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

Purpose

Invariant natural killer T (iNKT) cells may play an important role in the pathogenesis of asthma in mice and humans. Thus, an agent that modulates the function of iNKT cells may have therapeutic potential to control asthma. We hypothesized that lipopolysaccharide (LPS)-, flagellin-, or CpG-induced changes in the cytokine milieu may modify and even inhibit the function of airway iNKT cells in asthma.

Methods

Because increased α-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR) reflects the presence of airway iNKT cells, α-GalCer-induced AHR, as well as inflammatory cells and cytokines in bronchoalveolar lavage (BAL) fluid, were determined 24 hours after in vivo treatment with LPS, flagellin, or CpG in naïve BALB/c mice. Intracellular IL-4 and IFN-γ were measured in spleen iNKT cells after in vitro treatment with LPS, flagellin, or CpG. A role for IL-12 following the treatments was determined.

Results

Intranasal administration of LPS, flagellin, or CpG reduced development of α-GalCer-induced AHR, eosinophilic airway inflammation, and Th1 and Th2 cytokine responses in BAL fluid, while producing IL-12 in BAL fluid. Intraperitoneal administration of IL-12 mAb blocked the suppressive effect of LPS, flagellin, or CpG. In vitro treatment with LPS, flagellin, or CpG reduced production of IL-4 and IFN-γ from α-GalCer-stimulated spleen iNKT cells; these effects were ameliorated by addition of anti-IL-12 mAb.

Conclusions

TLR4, 5, and 9 agonists may suppress the function of airway and spleen iNKT cells via IL-12-dependent mechanisms. Anergy of iNKT cells by IL-12 might play a role in suppression by these TLR agonists.     

Toll‐like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar Typhimurium infection,but not for the initiation of bacterial clearance

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF^−/− mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4^−/−, MyD88^−/− and TRIF^−/− mice in vitro. Pro-inflammatory responses from Mal^−/− macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF^−/− macrophages were partially restored by the addition of interferon-γ (IFN-γ), and TRIF^−/− mice produced markedly enhanced IFN-γ levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4^−/−, MyD88^−/−, TRIF^−/− and Mal^−/− mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.     

Antigen‐triggered interferon‐γ and interleukin‐10 pattern in cured mucosal leishmaniasis patients is shaped during the active phase of disease

An exacerbated type 1 response to leishmanial antigens is the basis of tissue destruction observed in mucosal leishmaniasis (ML). After therapy, a persistent production of high levels of inflammatory cytokines can confer a poor prognosis. Herein we investigated whether the clinical conditions defined during the active phase of ML affect the magnitude of long-term anti-Leishmania immune response. Twenty clinically cured ML cases were studied. Peripheral blood mononuclear cells (PBMC) were cultured with L. braziliensis antigens (Lb-Ag), Toxoplasma gondii antigens (Tg-Ag), concanavalin-A (Con-A) or medium alone, and the lymphocyte proliferative response and cytokine secretion were quantified. Medical records were reviewed for Montenegro skin test (MST) during diagnosis, duration of ML disease or time elapsed after clinical cure. The duration of disease was correlated positively with MST (r = 0·61). Lb-Ag induced interferon (IFN)-γ was correlated positively with duration of illness (r = 0·69) as well as the frequency of secreting cells [enzyme-linked immunospot (ELISPOT)] assay. No association was observed for Tg-Ag or Con-A. Disease duration was correlated negatively with interleukin (IL)-10 production (r = −0·76). Moreover, a negative correlation between length of time after clinical cure and TNF levels (r = −0·94) or the IFN-γ : IL-10 ratio (r = −0·89) were also seen. We suggest that the magnitude of the IFN-γ inflammatory response triggered by ML can be driven by the time of leishmanial antigens exposition during the active phase of the disease. This pattern could persist even long-term after cure. However, despite IFN-γ levels, the decrease of the TNF and IFN-γ : IL-10 ratio reflects the control of proinflammatory responses achieved by cure of ML, possibly preventing disease relapses.     

The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13

Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T. encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.     

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4⁺ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155^−/−) mice. Infection was controlled significantly quicker in the miR155^−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155^−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ⁺CD8⁺ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155^−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155^−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155^−/− T cells was significantly enhanced in L. major−infected miR155^−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Leishmania are obligate intracellular protozoans that infect phagocytes and cause a spectrum of clinical diseases such as cutaneous leishmaniasis (CL) and visceral leishmaniasis. Common in the tropical and subtropical regions, leishmaniasis affects over 1 billion people worldwide, with an incidence of up to 1 million cases per year.¹ CL is the most common type of Leishmania infection, manifesting as localized skin lesions that can become chronic, leading to significant tissue destruction and disfigurement.^2,3 It is well documented that the induction of a Th1 response and interferon (IFN)-γ are indispensable in the resolution of CL caused by Leishmania major,⁴ whereas disease progression is associated with the induction of a Th2 response and the production of cytokines such as IL-4 and IL-10.⁵ Establishing a disease-resolving response in the host is largely dependent on the ability to mount an appropriate Th1 immune response.⁴ Crucial in this response is the stimulation and activation of DCs that direct T-cell proliferation and differentiation toward IFN-γ–producing Th1 cells.^6,7 In addition to activating of phagocytic cells, IFN-γ induces the production of reactive nitrogen species, specifically nitric oxide (NO), leading to enhanced parasite clearance.⁴miR155 is a recognized regulator of immune cell function and immune response. miR155 enhances macrophage and DC activation and induces inflammatory response,^8,9 and up-regulation of miR155 in CD4⁺ T cells promotes preferential Th1 differentiation and IFN-γ production¹⁰ by suppressing the expression of suppressor of cytokine signaling (SOCS)-1.11, 12, 13, 14 Conversely, miR155 gene–deficient mice exhibit diminished levels of Th1/Th17 cells, macrophages, and DCs.¹⁵ miR155 has also been shown to play a role in regulating effector Th2 response.16, 17, 18 Collectively, these findings suggest that miR155 regulates both Th1 and Th2 responses, which control the outcome of CL caused by L. major. Therefore, the role of miR155 in immunity to L. major using miR155^−/− mice was investigated in the present study. The findings show that miR155 is not required for the induction of a Th1 response and IFN-γ in L. major infection. Rather, miR155 plays a disease-exacerbating role in CL by attenuating DC function and Th1 response and promoting Th2 response.     

EBV LMP2A-specific T Cell Immune Responses Elicited by Dendritic Cells Loaded with LMP2A Protein

Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an ideal target for immunotherapy. In previous studies we have demonstrated that using LMP2A protein loaded dendritic cells, the most powerful antigen processing cells in the body can elicit specific and robust anti-tumor cellular immune response in vitro. In this paper, we further investigated the T cell profile of the anti-tumor immune response. We found that LMP2A specific CD4＋ and CD8＋ T cells could be stimulated by LMP2A protein loaded dendritic cells （DCs）. The Thl type immune response is dominant in the immune response mediated by LMP2A specific CD4^＋ T cells. The CD8^＋ cytotoxic T cells can lyse LMP2A bearing cells effectively and specifically. The CD8^＋ cytotoxic T cells can also secrete high level of intracellular IFN-γ, which indicates these cells are EBV-LMP2A specific cytotoxic T cells. Altogether, our studies proved that LMP2A protein loaded DCs can elicit anti-tumor cellular immune responses efficiently. This study provides a rationale for the DC-based immunotherapy against EBV-LMP2A expressing malignancies.     

Regulation of inflammasome activity

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTg)-sensitized splenocytes activated with MTg and interleukin (IL)-12. Our previous studies showed that, when used as donors and recipients, interferon (IFN)-γ^−/− and wild-type (WT) DBA/1 mice both develop severe G-EAT. Thyroid lesions in IFN-γ^−/− mice have many eosinophils and few neutrophils, while those in WT mice have extensive neutrophil infiltration and few eosinophils. Thyroid lesions in IFN-γ^−/− mice consistently resolve by day 40–50, whereas those in WT mice have ongoing inflammation and fibrosis persisting for more than 60 days. To determine if the extensive infiltration of eosinophils in thyroids of IFN-γ^−/− mice contributes to thyroid damage and/or early resolution of G-EAT, anti-IL-5 was used to inhibit migration of eosinophils to thyroids. G-EAT severity was compared at day 20 and day 40–50 in IFN-γ^−/− recipients given anti-IL-5 or control immunoglobulin G (IgG). Thyroids of anti-IL-5-treated IFN-γ^−/− mice had few eosinophils and more neutrophils at day 20, but G-EAT severity scores were comparable to those of control IgG-treated mice at both day 20 and day 40–50. Expression of chemokine (C-X-C motif) ligand 1 (CXCL1) mRNA was higher and that of chemokine (C-C motif) ligand 11 (CCL11) mRNA was lower in thyroids of anti-IL-5-treated IFN-γ^−/− mice. IL-5 neutralization did not influence mRNA expression of most cytokines in IFN-γ^−/− mice. Thus, inhibiting eosinophil migration to thyroids did not affect G-EAT severity or resolution in IFN-γ^−/− mice, suggesting that eosinophil infiltration of thyroids occurs as a consequence of IFN-γ deficiency, but these cells have no apparent pathogenic role in G-EAT.     

Reduced Immune Response to Borrelia burgdorferi in the Absence of �æ� T Cells

Little is known regarding the function of γδ T cells, although they accumulate at sites of inflammation in infections and autoimmune disorders. We previously observed that γδ T cells in vitro are activated by Borrelia burgdorferi in a TLR2-dependent manner. We now observe that the activated γδ T cells can in turn stimulate dendritic cells in vitro to produce cytokines and chemokines that are important for the adaptive immune response. This suggested that in vivo γδ T cells may assist in activating the adaptive immune response. We examined this possibility in vivo and observed that γδ T cells are activated and expand in number during Borrelia infection, and this was reduced in the absence of TLR2. Furthermore, in the absence of γδ T cells, there was a significantly blunted response of adaptive immunity, as reflected in reduced expansion of T and B cells and reduced serum levels of anti-Borrelia antibodies, cytokines, and chemokines. This paralleled a greater Borrelia burden in γδ-deficient mice as well as more cardiac inflammation. These findings are consistent with a model of γδ T cells functioning to promote the adaptive immune response during infection.     

The Presence of Alpha Interferon at the Time of Infection Alters the Innate and Adaptive Immune Responses to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating and costly diseases to the swine industry worldwide. Overall, the adaptive immune response to PRRS virus (PRRSV) is weak, which results in delayed elimination of virus from the host and inferior vaccine protection. PRRSV has been shown to induce a meager alpha interferon (IFN-α) response, and we hypothesized that elevated IFN-α levels early in infection would shorten the induction time and increase elements of the adaptive immune response. To test this, we measured both antibody and cell-mediated immunity in pigs after the administration of a nonreplicating human adenovirus type 5 vector expressing porcine IFN-α (Ad5–pIFN-α) at the time of PRRSV infection and compared the results to those for pigs infected with PRRSV alone. Viremia was delayed, and there was a decrease in viral load in the sera of pigs administered the Ad5–pIFN-α. Although seroconversion was slightly delayed in pigs receiving Ad5–pIFN-α, probably due to the early reduction in viral replication, little difference in the overall or neutralizing antibody response was seen. However, there was an increase in the number of virus-specific IFN-γ-secreting cells detected in the pigs receiving Ad5–pIFN-α, as well as an altered cytokine profile in the lung at 14 days postinfection, indicating that the presence of IFN-α at the time of infection can alter innate and adaptive immune responses to PRRSV.     

Death-associated protein kinase 1 is an IRF3/7-interacting protein that is involved in the cellular antiviral immune response

Interferon regulatory factor （IRF） 7 has been demonstrated to be a master regulator of virus-induced type I interferon production （IFN）, and it plays a central role in the innate immune response against viruses. Here, we identified death-associated protein kinase 1 （DAPK1） as an IRF7-interacting protein by tandem affinity purification （TAP）. Viral infection induced DAPKI-IRF7 and DAPKI-IRF3 interactions and overexpression of DAPK1 enhanced virus-induced activation of the interferon-stimulated response element （ISRE） and IFN-p promoters and the expression of the IFNB1 gene. Knockdown of DAPK1 attenuated the induction of IFNB1 and RIG.lexpression triggered by viral infection or I FN-p, and they were enhanced by viral replication. In addition, viral infection or IFN-p treatment induced the expression of DAPK1. IFN-p treatment also activated DAPK1 by decreasing its phosphorylation level at serine 308. Interestingly, the involvement of DAPK1 in virus-induced signaling was independent of its kinase activity. Therefore, our study identified DAPK1 as an important regulator of the cellular antiviral response.     

Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer

A dendritic cell (DC)-based vaccine strategy could reduce the risk of recurrence and improve the survival of breast cancer patients. However, while therapy-induced apoptosis of hepatocellular and colorectal carcinoma cells can enhance maturation and antigen presentation of DCs, whether this effect occurs in breast cancer is currently unknown. In the present study, we investigated the effect of doxorubicin (ADM)-induced apoptotic MCF-7 breast cancer cells on the activation of DCs. ADM-induced apoptotic MCF-7 cells could effectively induce immature DC (iDC) maturation. The mean fluorescence intensity (MFI) of DC maturity marker CD83 was 23.3 in the ADM-induced apoptotic MCF-7 cell group compared with 8.5 in the MCF-7 cell group. The MFI of DC co-stimulatory marker CD86 and HLA-DR were also increased after iDCs were treated with ADM-induced apoptotic MCF-7 cells. Furthermore, the proliferating autologous T-lymphocytes increased from 14.2 to 40.3% after incubated with DCs induced by apoptotic MCF-7 cells. The secretion of interferon-γ by these T-lymphocytes was also increased. In addition, cell-cell interaction between apoptotic MCF-7 cells and iDCs, but not soluble factors released by apoptotic MCF-7 cells, was crucial for the maturation of iDCs. These findings constitute a novel in vitro DC-based vaccine strategy for the treatment of breast cancer by ADM-induced apoptotic MCF-7 cells.     

IFN-γ Mediates the Antitumor Effects of Radiation Therapy in a Murine Colon Tumor

Cancer treatments using ionizing radiation (IR) therapy are thought to act primarily through the induction of tumor cell damage at a molecular level. However, a new concept has recently emerged, suggesting that the immune system is required for effective IR therapy. Our work here has identified interferon gamma (IFN-γ) as an essential cytokine for the efficacy of IR therapy. Local IR (15 Gy) to mice bearing Colon38, a colon adenocarcinoma, decreases tumor burden in wild-type animals. Interestingly, IR therapy had no effect on tumor burden in IFNγKO mice. We further determined that intratumoral levels of IFN-γ increased 2 days following IR, which directly correlated with a decrease in tumor burden that was not a result of direct cytotoxic effects of IFN-γ on tumor cells. T cells from IR-treated tumors exhibited a far greater capacity to lyse tumor cells in a ⁵¹Cr release assay, a process that was dependent on IFN-γ. CD8⁺ T cells were the predominant producers of IFN-γ, as demonstrated by IFN-γ intracellular staining and studies in IFN-γ reporter mice. Elimination of CD8⁺ T cells by antibody treatment reduced the intratumoral levels of IFN-γ by over 90%. More importantly, elimination of CD8⁺ T cells completely abrogated the effects of radiation therapy. Our data suggest that IFN-γ plays a pivotal role in mediating the antitumor effects of IR therapy.Historically, ionizing radiation (IR) therapy was thought to control cancer predominantly by inducing tumor cell death through DNA damage. However, a new paradigm is emerging, strongly suggesting that the immune system mediates many of the antitumor effects of radiotherapy. Our laboratory has previously demonstrated that local radiation therapy in a melanoma mouse model results in tumor cell death, facilitating the release of tumor antigen.¹ Freed antigen can be processed by antigen-presenting cells and used to stimulate effector cells within the draining lymph node. Consequently, these effector cells were able to traffic to the tumor where they could recognize and lyse malignant cells, thereby reducing tumor growth. Additional reports have supported the concept that the immune system is pivotal in determining the effectiveness of radiation therapy.^2–5 Lee et al³ demonstrated that CD8⁺ T cells partially mediated the therapeutic effects of radiation, whereas Apetoh et al² identified Toll-like receptor 4–positive (TLR4⁺) dendritic cells as potent immunostimulatory cells capable of processing and presenting antigen from dying irradiated tumor cells. As a whole, there is increasing evidence of immune involvement in the anticancer effects of radiotherapy; however, the exact mechanisms governing this response are largely unknown and may differ from one malignancy to another.Irradiation of normal tissue, and now more recently described, tumor tissue, induces an inflammatory state often resulting in the secretion of cytokines into the microenvironment.^6–9 This is likely the result of danger signals released by damaged or dying cells in response to DNA strand breaks.⁸ Whereas once largely ignored, it is now appreciated that these cytokines may impact the effectiveness of radiotherapy, either positively or negatively, by contributing to radioresistance or radiosensitivity as well as inducing or suppressing a radiation-mediated immune response. An example of a cytokine with opposing effects on tumors is interferon gamma (IFN-γ). IFN-γ, an inflammatory cytokine that we determined was up-regulated following radiation in a melanoma model,¹⁰ has conventionally been viewed as antagonistic to tumor growth.¹¹ The biological functions of this cytokine include direct cytotoxic/antiproliferative effects on tumor cells as well as stimulation of the adaptive arm of the immune system against tumor antigens.¹⁰ However, recent reports have questioned the efficacy of IFN-γ as an antitumor agent, instead citing situations where IFN-γ appears to promote tumor progression.¹¹ Depending on the dose, IFN-γ was shown to promote metastases of B16 to the lung as well as stimulate the proliferation of NIH-3T3 cells.^12,13 Additionally, under certain circumstances, IFN-γ inhibited the antitumor function of NK cells and stimulated immunosuppression through the development of T regulatory (Treg) and/or myeloid-derived suppressor cells along with the production of inhibitory molecules like indoleamine 2,3-dioxygenase (IDO).^14–17 Therefore, in a unique situation such as tumor radiotherapy in which the efficacy depends on a potent immune response, we addressed whether IFN-γ is detrimental or beneficial with regard to modulating the immune system following radiotherapy.In this report, we determined that IFN-γ was not only beneficial, but was essential in mediating the antitumor effects of radiation in a mouse colon adenocarcinoma tumor.¹⁸ IFN-γ was up-regulated in the tumor microenvironment 2 days following radiation, and CD8⁺ T cells were the chief producers of this cytokine. Elimination of CD8⁺ T cells, not only greatly reduced the intratumoral (i.t.) levels of IFN-γ, but also abrogated any antitumor effect of radiation. Furthermore, we demonstrated that although IFN-γ had no direct effect on tumor cells in promoting tumor control, it enhanced the cytolytic capacity of T cells, possibly in an autocrine manner, resulting in a decrease of tumor burden.     

Glioma Stem Cell-Targeted Dendritic Cells as a Tumor Vaccine Against Malignant Glioma

Purpose

Cancer stem cells have recently been thought to be closely related to tumor development and reoccurrence. It may be a promising way to cure malignant glioma by using glioma stem cell-targeted dendritic cells as a tumor vaccine. In this study, we explored whether pulsing dendritic cells with antigens of glioma stem cells was a potent way to induce specific cytotoxic T lymphocytes and anti-tumor immunity.

Materials and Methods

Cancer stem cells were cultured from glioma cell line U251. Lysate of glioma stem cells was obtained by the repeated freezing and thawing method. Dendritic cells (DCs) were induced and cultured from the murine bone marrow cells, the biological characteristics were detected by electron microscope and flow cytometry. The DC vaccine was obtained by mixing DCs with lysate of glioma stem cells. The DC vaccine was charactirizated through the mixed lymphocyte responses and cell killing experiment in vitro. Level of interferon-γ (IFN-γ) in the supernatant was checked by ELISA.

Results

After stimulation of lysate of glioma stem cell, expression of surface molecules of DC was up-regulated, including CD80, CD86, CD11C and MHC-II. DCs pulsed with lysate of glioma stem cells were more effective than the control group in stimulating original glioma cells-specific cytotoxic T lymphocytes responses, killing glioma cells and boosting the secretion of IFN-γ in vitro.

Conclusion

The results demonstrated DCs loaded with antigens derived from glioma stem cells can effectively stimulate naive T cells to form specific cytotoxic T cells, kill glioma cells cultured in vitro.