Combined immunostimulation and conditional cytotoxic gene therapy provide long-term survival in a large glioma model

In spite of preclinical efficacy and recent randomized, controlled studies with adenoviral vectors expressing herpes simplex virus-1 thymidine kinase (HSV1-TK) showing statistically significant increases in survival, most clinical trials using single therapies have failed to provide major therapeutic breakthroughs. Because glioma is a disease with dismal prognosis and rapid progression, it is an attractive target for gene therapy. Preclinical models using microscopic brain tumor models (e.g., < or =0.3 mm3) may not reflect the pathophysiology and progression of large human tumors. To overcome some of these limitations, we developed a syngeneic large brain tumor model. In this model, administration of single therapeutic modalities, either conditional cytotoxicity or immunostimulation, fail. However, when various immunostimulatory therapies were delivered in combination with conditional cytotoxicity (HSV1-TK), only the combined delivery of fms-like tyrosine kinase ligand (Flt3L) and HSV1-TK significantly prolonged the survival of large tumor-bearing animals (> or =80%; P < or = 0.005). When either macrophages or CD4+ cells were depleted before administration of viral therapy, TK + Flt3L therapy failed to prolong survival. Meanwhile, depletion of CD8+ cells or natural killer cells did not affect TK + Flt3L efficacy. Spinal cord of animals surviving 6 months after TK + Flt3L were evaluated for the presence of autoimmune lesions. Whereas macrophages were present within the corticospinal tract and low levels of T-cell infiltration were detected, these effects are not indicative of an overt autoimmune disorder. We propose that combined Flt3L and HSV1-TK adenoviral-mediated gene therapy may provide an effective antiglioma treatment with increased efficacy in clinical trials of glioma.     

Immuno-gene therapy with adenoviruses expressing fms-like tyrosine kinase 3 ligand and CD40 ligand for mouse hepatoma cells in vivo

Introduction of genes encoding immuno-stimulatory cytokines into cancer cells is known to enhance antitumor immunity. CD40 ligand (CD40L, CD154) and fms-like tyrosine kinase 3 ligand (Flt3L) are recently identified cytokines, which have been demonstrated to stimulate antitumor immunity in several cancer models. However little is known about antitumor activity of Ftl3L and CD40L against hepatocellular carcinoma (HCC). In the present study, we constructed replication-defective adenoviruses expressing Flt3L and CD40L and examined their therapeutic efficacy on mouse HCC, MH134 cells. Subcutaneous injection of MH134 cells genetically engineered to express Flt3L and/or CD40L developed tumors in all the syngeneic immunocompetent mice, but tumor growth was significantly delayed as compared to control mice. Partial inhibition of this antitumor effect in athymic nude mice suggests that both innate and adaptive immunity appear to play a role. It was shown by immunodepletion of NK cells with anti-asialo-GM1 antibody that the effector cells involved in innate immunity are NK cells. In a therapeutic setting, however, injection of adenovirus expressing Flt3L or CD40L into pre-established MH134 tumors exhibited no efficacy. These data demonstrate that Flt3L and CD40L induce significant, but only weak, antitumor immunity against MH134 cells presumably through both innate and adaptive immunity. Our results suggest that immuno-gene therapy with Flt3L and CD40L may need adjuvant modalities to achieve strong immune response.     

B7-H3, a potential therapeutic target, is expressed in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a brain cancer with a median survival of only 1 year. Lack of molecular characterization of this tumor impedes the development of novel therapies. Membrane protein B7-H3, aka CD276, involved in interactions with host defenses in certain cancers, has been shown to be over-expressed in the majority of malignant neuroectodermal tumors including adult high-grade glioma. Targeting B7-H3 with a monoclonal antibody has demonstrated safety and efficacy in the salvage treatment of stage IV childhood neuroblastoma, another neuroectodermal tumor. It thus stands to reason that B7-H3 might serve as a therapeutic target in DIPG. B7-H3 immunoreactivity was determined in DIPG and non-diffuse brainstem glioma specimens with immunohistochemistry. In addition, B7-H3 mRNA expression was evaluated with microarrays in another set of specimens. All of the nine (100 %) DIPG specimens were shown to be B7-H3 immunoreactive. In the non-diffuse brainstem glioma group, none of the eight WHO grade I specimens showed B7-H3 immunoreactivity and nine of the 24 WHO grade II specimens (37.5 %) showed B7-H3 immunoreactivity. The association between histological grade and B7-H3 immunoreactivity was statistically highly significant. B7-H3 mRNA expression was also significantly higher in DIPG samples than in normal brain and juvenile pilocytic astrocytoma (WHO grade I) specimens. In summary, B7-H3 is over-expressed in DIPG. Given the need for novel treatment in this disease, antibody-based immunotherapy against B7-H3 in DIPG warrants further investigation.     

Induction of antitumor immunity using intercellular adhesion molecule 1 (ICAM-1) transfection in mouse glioma cells.

We investigated the role of intercellular adhesion molecule 1 (ICAM-1) expression in tumorigenicity of gliomas and the antitumor effects of glioma cells genetically engineered to express ICAM- 1. Mouse glioma cells transfected with ICAM-1 grew in T-cell deficient mice but not in syngeneic mice. Vaccination with ICAM-1-transfected tumor cells markedly inhibited the growth of subcutaneously inoculated gliomas but not gliomas located in the brain. In vivo antibody ablation study revealed that CD4+ T, CD8+ T and NK cells were all required to produce the antitumor effect of SR/ICAM- 1. In this study, we demonstrated the therapeutic potential of vaccination with ICAM-1-overexpressing tumor cells for the control of the tumor growth.     

Magnetic resonance imaging determination of tumor grade and early response to temozolomide in a genetically engineered mouse model of glioma.

PURPOSE: The median survival for patients diagnosed with glioblastoma multiforme, the most common type of brain tumor, is less than 1 year. Animal glioma models that are more predictive of therapeutic response in human patients than traditional models and that are genetically and histologically accurate are an unmet need. The nestin tv-a (Ntv-a) genetically engineered mouse spontaneously develops glioma when infected with ALV-A expressing platelet-derived growth factor, resulting in autocrine platelet-derived growth factor signaling. EXPERIMENTAL DESIGN: In the Ntv-a genetically engineered mouse model, T2-weighted and T1-weighted, contrast-enhanced magnetic resonance images were correlated with histology, glioma grade (high or low), and survival. Magnetic resonance imaging (MRI) was therefore used to enroll mice with high-grade gliomas into a second study that tested efficacy of the current standard of care for glioma, temozolomide (100 mg/kg qdx5 i.p., n=13). RESULTS: The Ntv-a model generated a heterogeneous group of gliomas, some with high-grade growth rate and histologic characteristics and others with characteristics of lower-grade gliomas. We showed that MRI could be used to predict tumor grade and survival. Temozolomide treatment of high-grade tv-a gliomas provided a 14-day growth delay compared with vehicle controls. Diffusion MRI measurement of the apparent diffusion coefficient showed an early decrease in cellularity with temozolomide, similar to that observed in humans. CONCLUSIONS: The use of MRI in the Ntv-a model allows determination of glioma grade and survival prediction, distribution of mice with specific tumor types into preclinical trials, and efficacy determination both by tumor growth and early apparent diffusion coefficient response.     

Plasmacytoid Dendritic Cells in the Tumor Microenvironment: Immune Targets for Glioma Therapeutics

Adenovirus-mediated delivery of the immune-stimulatory cytokine Flt3L and the conditionally cytotoxic thymidine kinase (TK) induces tumor regression and long-term survival in preclinical glioma (glioblastoma multiforme [GBM]) models. Flt3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. Although pDCs can present antigen and produce powerful inflammatory cytokines, that is, interferon α (IFN-α), their role in tumor immunology remains debated. Thus, we studied the role of pDCs and IFN-α in Ad.TK/GCV+ Ad.Flt3L-mediated anti-GBM therapeutic efficacy. Our data indicate that the combined gene therapy induced recruitment of plasmacytoid DCs (pDCs) into the tumor mass; which were capable of in vivo phagocytosis, IFN-α release, and T-cell priming. Thus, we next used either pDCs or an Ad vector encoding IFN-α delivered within the tumor microenvironment. When rats were treated with Ad.TK/GCV in combination with pDCs or Ad-IFN-α, they exhibited 35% and 50% survival, respectively. However, whereas intracranial administration of Ad.TK/GCV + Ad.Flt3L exhibited a high safety profile, Ad-IFN-α led to severe local inflammation, with neurologic and systemic adverse effects. To elucidate whether the efficacy of the immunotherapy was dependent on IFN-α-secreting pDCs, we administered an Ad vector encoding B18R, an IFN-α antagonist, which abrogated the antitumoral effect of Ad.TK/GCV + Ad.Flt3L. Our data suggest that IFN-α release by activated pDCs plays a critical role in the antitumor effect mediated by Ad.TK/GCV + Ad.Flt3L. In summary, taken together, our results demonstrate that pDCs mediate anti-GBM therapeutic efficacy through the production of IFN-α, thus manipulation of pDCs constitutes an attractive new therapeutic target for the treatment of GBM.     

Targeting Wee1 for the treatment of pediatric high-grade gliomas

Background

We investigated the efficacy of the Wee1 inhibitor MK-1775 in combination with radiation for the treatment of pediatric high-grade gliomas (HGGs), including diffuse intrinsic pontine gliomas (DIPGs).

Methods

Gene expression analysis was performed for 38 primary pediatric gliomas (3 grade I, 10 grade II, 11 grade III, 14 grade IV) and 8 normal brain samples using the Agilent 4 × 44 K array. Clonogenic survival assays were carried out in pediatric and adult HGG cell lines (n = 6) to assess radiosensitizing effects of MK-1775. DNA repair capacity was evaluated by measuring protein levels of γ-H2AX, a marker of double strand DNA breaks. In vivo activity of MK-1775 with radiation was assessed in 2 distinct orthotopic engraftment models of pediatric HGG, including 1 derived from a genetically engineered mouse carrying a BRAF^V600E mutation, and 1 xenograft model in which tumor cells were derived from a patient''s DIPG.

Results

Wee1 is overexpressed in pediatric HGGs, with increasing expression positively correlated with malignancy (P = .007 for grade III + IV vs I + II) and markedly high expression in DIPG. Combination treatment of MK-1775 and radiation reduced clonogenic survival and increased expression of γ-H2AX to a greater extent than achieved by radiation alone. Finally, combined MK-1775 and radiation conferred greater survival benefit to mice bearing engrafted, orthotopic HGG and DIPG tumors, compared with treatment with radiation alone (BRAF^V600E model P = .0061 and DIPG brainstem model P = .0163).

Conclusion

Our results highlight MK-1775 as a promising new therapeutic agent for use in combination with radiation for the treatment of pediatric HGGs, including DIPG.     

An experimental xenograft mouse model of diffuse pontine glioma designed for therapeutic testing

The prognosis for diffuse infiltrating pontine gliomas (DIPG) remains extremely poor, with the majority of patients surviving less than 2 years. Here, we have adapted standard xenograft techniques to study glioma growth in the mouse brainstem, and have utilized the mouse model for studying a relevant therapeutic for treating DIPGs. bioluminescence imaging monitoring revealed a progressive increase in signal following the injection of either of two tumor cell types into the brainstem. Mice with orthotopic GS2 tumors, and receiving a single 100 mg/kg dose of temozolomide showed a lengthy period of decreased tumor luminescence, with substantially increased survival relative to untreated mice (P < 0.001). A small molecule inhibitor that targets cdk4/6 was used to test AM-38 brainstem xenograft response to treatment. Drug treatment resulted in delayed tumor growth, and significantly extended survival. Our results demonstrate the feasibility of using an orthotopic brainstem tumor model in athymic mice, and for application to testing therapeutic agents in treating DIPG.     

Flt3L and TK gene therapy eradicate multifocal glioma in a syngeneic glioblastoma model

The disseminated characteristics of human glioblastoma multiforme (GBM) make it a particularly difficult tumor to treat with long-term efficacy. Most preclinical models of GBM involve treatment of a single tumor mass. For therapeutic outcomes to translate from the preclinical to the clinical setting, induction of an antitumor response capable of eliminating multifocal disease is essential. We tested the hypothesis that expression of Flt3L (human soluble FMS-like tyrosine kinase 3 ligand) and TK (herpes simplex virus type 1-thymidine kinase) within brain gliomas would mediate regression of the primary, treated tumor mass and a secondary, untreated tumor growing at a distant site from the primary tumor and the site of therapeutic vector injection. In both the single-GBM and multifocal-GBM models used, all saline-treated control animals succumbed to tumors by day 22. Around 70% of the animals bearing a single GBM mass treated with an adenovirus expressing Flt3L (AdFlt3L) and an adenovirus expressing TK (AdTK + GCV) survived long term. Approximately 50% of animals bearing a large primary GBM that were implanted with a second GBM in the contralateral hemisphere at the same time the primary tumors were being treated with AdFlt3L and AdTK also survived long term. A second multifocal GBM model, in which bilateral GBMs were implanted simultaneously and only the right tumor mass was treated with AdFlt3L and AdTK, also demonstrated long-term survival. While no significant difference in survival was found between unifocal and multifocal GBM-bearing animals treated with AdFlt3L and AdTK, both treatments were statistically different from the saline-treated control group (p < 0.05). Our results demonstrate that combination therapy with AdFlt3L and AdTK can eradicate multifocal brain tumor disease in a syngeneic, intracranial GBM model.     

The use of antihuman glioma monoclonal antibodies for targeting chemotherapy of brain gliomas

Athymic nude mice bearing subcutaneous and intracerebral human glioma xenografts were used to assess the therapeutic efficacy of monoclonal antibody-adriamycin immunoconjugates against malignant gliomasin vivo. Immunoconjugates showed a significantly stronger antitumor effect with a T/C (treated/control tumor volume) of 30% as compared with free drug (T/C of 84%). The targeting treatment with immunoconjugates significantly prolonged 54% of median survival time of nude mice. Side effects of immunoconjugates on the normal bone marrow and small intestines were much slighter than those of the free drug. The results of this study indicate that the use of monoclonal antibodies as carriers of antitumor agents may have many therapeutic advantages and potential for the treatment of brain gliomas.     

Diagnostics and treatment of diffuse intrinsic pontine glioma: where do we stand?

Journal of Neuro-Oncology - Diffuse intrinsic pontine glioma (DIPG) is a rare clinically, neuro-radiologically, and molecularly defined malignancy of the brainstem with a median overall survival of...     

Therapeutic efficacy and safety of TRAIL-producing human adipose tissue-derived mesenchymal stem cells against experimental brainstem glioma

Mesenchymal stem cells (MSCs) have an extensive migratory capacity for gliomas, which is comparable to that of neural stem cells. Among the various types of MSCs, human adipose tissue-derived MSCs (hAT-MSC) emerge as one of the most attractive vehicles for gene therapy because of their high throughput, lack of ethical concerns, and availability and ease of isolation. We evaluated the therapeutic potential and safety of genetically engineered hAT-MSCs encoding the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against brainstem gliomas. Human AT-MSCs were isolated from human fat tissue, characterized, and transfected with TRAIL using nucleofector. The therapeutic potential of TRAIL-producing hAT-MSCs (hAT-MSC.TRAIL) was confirmed using in vitro and in vivo studies. The final fate of injected hAT-MSCs was traced in long-survival animals. The characterization of hAT-MSCs revealed the expression of MSC-specific cell-type markers and their differentiation potential into mesenchymal lineage. Short-term outcomes included a 56.3% reduction of tumor volume (P < .001) with increased apoptosis (3.03-fold, P < .05) in animals treated with hAT-MSC.TRAIL compared with the control groups. Long-term outcomes included a significant survival benefit in the hAT-MSC.TRAIL-treated group (26 days of median survival in the control group vs 84 days in the hAT-MSC.TRAIL-treated group, P < .0001), without any evidence of mesenchymal differentiation in vivo. Our study demonstrated the therapeutic efficacy and safety of nonvirally engineered hAT-MSCs against brainstem gliomas and showed the possibility of stem-cell-based targeted gene therapy for clinical application.     

Ad-CD40L mobilizes CD4 T cells for the treatment of brainstem tumors

BackgroundDiffuse midline glioma, formerly DIPG (diffuse intrinsic pontine glioma), is the deadliest pediatric brainstem tumor with median survival of less than one year. Here, we investigated (i) whether direct delivery of adenovirus-expressing cluster of differentiation (CD)40 ligand (Ad-CD40L) to brainstem tumors would induce immune-mediated tumor clearance and (ii) if so, whether therapy would be associated with a manageable toxicity due to immune-mediated inflammation in the brainstem.MethodsSyngeneic gliomas in the brainstems of immunocompetent mice were treated with Ad-CD40L and survival, toxicity, and immune profiles determined. A clinically translatable vector, whose replication would be tightly restricted to tumor cells, rAd-Δ24-CD40L, was tested in human patient–derived diffuse midline gliomas and immunocompetent models.ResultsExpression of Ad-CD40L restricted to brainstem gliomas by pre-infection induced complete rejection, associated with immune cell infiltration, of which CD4+ T cells were critical for therapy. Direct intratumoral injection of Ad-CD40L into established brainstem tumors improved survival and induced some complete cures but with some acute toxicity. RNA-sequencing analysis showed that Ad-CD40L therapy induced neuroinflammatory immune responses associated with interleukin (IL)-6, IL-1β, and tumor necrosis factor α. Therefore, to generate a vector whose replication, and transgene expression, would be tightly restricted to tumor cells, we constructed rAd-Δ24-CD40L, the backbone of which has already entered clinical trials for diffuse midline gliomas. Direct intratumoral injection of rAd-Δ24-CD40L, with systemic blockade of IL-6 and IL-1β, generated significant numbers of cures with readily manageable toxicity.ConclusionsVirus-mediated delivery of CD40L has the potential to be effective in treating diffuse midline gliomas without obligatory neuroinflammation-associated toxicity.     

Antitumor effect of secreted Flt3-ligand can act at distant tumor sites in a murine model of head and neck cancer

The Flt3 ligand (Flt3-L) manifests antitumor activity, presumably due to its capacity to recruit dendritic cells and cause their proliferation. To assess whether local production of Flt3-L can mediate a "distant bystander" effect, murine B4B8 squamous cell carcinoma cells were transfected with a plasmid encoding a secretory form of Flt3-L to produce B4B8FL cells. Similarly, B4B8FL and B4B8 cells were transfected with herpes simplex virus thymidine kinase (HSVTK) to produce B4B8TK and B4B8FL/TK cells, which should be sensitive to ganciclovir (GCV), to know whether the effects of Flt3-L and HSVTK/GCV would be synergistic. To test for a distant bystander effect in vivo, B4B8FL, B4B8TK, and B4B8FL/TK cells were injected subcutaneously into the left flank of syngeneic Balb/c mice, and na?ve B4B8 cells were injected into the right flank. The formation of tumors derived from B4B8FL and B4B8FL/TK cells was significantly delayed in both flanks compared with na?ve B4B8 and B4B8TK cells. Growth of B4B8TK tumors in the ipsilateral flank was retarded following GCV treatment, but in contrast to B4B8FL and B4B8FL/TK cells, no distant bystander effect in the contralateral flank was observed. Immunohistochemistry showed lymphocytic infiltrates in both flanks of the B4B8FL and B4B8FL/TK groups. The data indicate that in these cells, local secretion of Flt3-L is sufficient to evoke a distant bystander effect but that expression of HSVTK, even after GCV administration, is not. Furthermore, the combination of local Flt3-L and HSVTK production, together with GCV administration, does not enhance the distant bystander effect produced by Flt3-L alone.     

Inhibiting TGF-beta signaling restores immune surveillance in the SMA-560 glioma model

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.     

Visualizing the dynamics of EGFR activity and antiglioma therapies in vivo

Many altered pathways in cancer cells depend on growth factor receptors. In primary malignant gliomas, the amplification/alteration of the epidermal growth factor receptor (EGFR) has been shown to play a significant role in enhancing glioma burden. In an effort to dissect the role of EGFR expression in glioma progression in vivo and evaluate targeted therapies for gliomas, we have genetically engineered glioma cells to visualize the dynamics of EGFR and targeted therapies in real time in vivo. Using engineered lentiviral vectors bearing fusions between EGFR and its exon 2 to 7 deleted variant (EGFRvIII) with green fluorescent protein (GFP) and Renilla luciferase (Rluc), we show that there is a direct correlation between EGFR expression and glioma cell proliferation in the initial stages of glioma progression. To monitor and evaluate EGFR-targeted therapies, we have engineered (a) short hairpin RNAs (shRNA) and (b) clinically used monoclonal antibody, cetuximab. Using EGFR-GFP-Rluc/firefly luciferase (Fluc)-DsRed2 glioma model, we show that both shRNAs and cetuximab result in a considerable reduction in glioma cell proliferation in culture and glioma burden in vivo that can be monitored in real time at a cellular resolution. This study serves as a template to follow the role of growth factor receptor expression in tumor progression and to image therapeutic efficacy of targeted therapies in cancer.     

Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.     

Induction of T-cell apoptosis in rats by genetically engineered glioma cells expressing granulocyte-macrophage colony-stimulating factor and B7.1.

PURPOSE: To evaluate antitumor effects on intracerebral gliomas of genetically engineered tumor vaccines expressing granulocyte-macrophage colony-timulating factor (GM-CSF), B7.1, or both (combination). EXPERIMENTAL DESIGN: A rat glioma cell line, RT-2, was engineered with a retroviral vector to express GM-CSF, B7.1, or combination. Tumorigenicity of engineered cells and therapeutic effects of s.c. given irradiated or live tumor vaccines on parental intracerebral gliomas were studied. Immune cell infiltration induced at vaccine and tumor sites was examined by histologic and immunohistochemical staining. Apoptosis of T cells from vaccine sites was analyzed with fluorescence-activated cell sorting. RESULTS: Engineered RT-2 cells exhibited reduced s.c. tumorigenicity in rats with reduced tumor growth and prolonged animal survival time compared with control rats. Rats with intracerebral gliomas s.c. treated with irradiated or live GM-CSF-expressing vaccines had 60% and 100% survival rates, respectively, significantly better than the control groups (P < 0.05). In contrast, rats treated with vaccines expressing B7.1 or the combination had no or mild therapeutic effects. Studies revealed less T-cell infiltration at both vaccine and tumor sites in rats treated with vaccines expressing B7.1 or the combination than in rats treated with a vaccine expressing GM-CSF. Cell sorting analyses revealed higher proportions of apoptotic T cells at vaccine sites of rats treated with the combination than those treated with vaccine expressing GM-CSF. CONCLUSIONS: Combination of GM-CSF- and B7.1-expressing tumor vaccines exerted no synergistic, or even worse, therapeutic effects on gliomas compared with single GM-CSF-secreting tumor vaccine. The worse therapeutic effects of the GM-B7.1-expressing tumor vaccine than the GM-CSF-expressing tumor vaccine were related to the reduced T-cell amount and increased T-cell apoptosis in the former.