Wim Maes

Dendritic Cell Therapy of High-Grade Gliomas

The prognosis of patients with malignant glioma is poor in spite of multimodal treatment approaches consisting of neurosurgery, radiochemotherapy and maintenance chemotherapy. Among innovative treatment strategies like targeted therapy, antiangiogenesis and gene therapy approaches, immunotherapy emerges as a meaningful and feasible treatment approach for inducing long‐term survival in at least a subpopulation of these patients. Setting up immunotherapy for an inherent immunosuppressive tumor located in an immune‐privileged environment requires integration of a lot of scientific input and knowledge of both tumor immunology and neuro‐oncology. The field of immunotherapy is moving into the direction of active specific immunotherapy using autologous dendritic cells (DCs) as vehicle for immunization. In the translational research program of the authors, the whole cascade from bench to bed to bench of active specific immunotherapy for malignant glioma is covered, including proof of principle experiments to demonstrate immunogenicity of patient‐derived mature DCs loaded with autologous tumor lysate, preclinical in vivo experiments in a murine orthotopic glioma model, early phase I/II clinical trials for relapsing patients, a phase II trial for patients with newly diagnosed glioblastoma (GBM) for whom immunotherapy is integrated in the current multimodal treatment, and laboratory analyses of patient samples. The strategies and results of this program are discussed in the light of the internationally available scientific literature in this fast‐moving field of basic science and translational clinical research.

DC vaccination with anti-CD25 treatment leads to long-term immunity against experimental glioma

We studied the feasibility, efficacy, and mechanisms of dendritic cell (DC) immunotherapy against murine malignant glioma in the experimental GL261 intracranial (IC) tumor model. When administered prophylactically, mature DCs (DCm) ex vivo loaded with GL261 RNA (DCm-GL261-RNA) protected half of the vaccinated mice against IC glioma, whereas treatment with mock-loaded DCm or DCm loaded with irrelevant antigens did not result in tumor protection. In DCm-GL261-RNA-vaccinated mice, a tumor-specific cellular immune response was observed ex vivo in the spleen and tumordraining lymph node cells. Specificity was also shown in vivo on the level of tumor challenge. Depletion of CD8(+) T-cells by anti-CD8 treatment at the time of tumor challenge demonstrated their essential role in vaccine-mediated antitumor immunity. Depletion of CD25(+) regulatory T-cells (Tregs) by anti-CD25 (aCD25) treatment strongly enhanced the efficacy of DC vaccination and was itself also protective, independently of DC vaccination. However, DC vaccination was essential to protect the animals from IC tumor rechallenge. No long-term protection was observed in animals that initially received aCD25 treatment only. In mice that received DC and/or aCD25 treatment, we retrieved tumor-specific brain-infiltrating cytotoxic T-lymphocytes. These data clearly demonstrate the effectiveness of DC vaccination for the induction of long-lasting immunological protection against IC glioma. They also show the beneficial effect of Treg depletion in this kind of glioma immunotherapy, even combined with DC vaccination.

Experimental immunotherapy for malignant glioma: lessons from two decades of research in the GL261 model

Nearly twenty years of experimental immunotherapy for malignant glioma yielded important insights in the mechanisms governing glioma immunology. Still considered promising, it is clear that immunotherapy does not on its own represent the magic bullet in glioma therapy. In this review, we summarize the major immunotherapeutic achievements in the mouse GL261 glioma model, which has emerged as the gold standard syngeneic model for experimental glioma therapy. Gene therapy, monoclonal antibody treatment, cytokine therapy, cell transfer strategies and dendritic cell therapy were hereby considered. Apart from the considerable progress made in understanding glioma immunology in this model, we also addressed its most pertinent issues and shortcomings. Despite these, the GL261 model will remain indispensable in glioma research since it is a fast, highly reproducible and easy-to-establish model system.

Glioma-derived galectin-1 regulates innate and adaptive antitumor immunity.

Galectin‐1 is a glycan‐binding protein, which is involved in the aggressiveness of glioblastoma (GBM) in part by stimulating angiogenesis. In different cancer models, galectin‐1 has also been demonstrated to play a pivotal role in tumor‐mediated immune evasion especially by modulating cells of the adaptive immune system. It is yet unknown whether the absence or presence of galectin‐1 within the glioma microenvironment also causes qualitative or quantitative differences in innate and/or adaptive antitumor immune responses. All experiments were performed in the orthotopic GL261 mouse high‐grade glioma model. Stable galectin‐1 knockdown was achieved via transduction of parental GL261 tumor cells with a lentiviral vector encoding a galectin‐1‐targeting miRNA. We demonstrated that the absence of tumor‐derived but not of host‐derived galectin‐1 significantly prolonged the survival of glioma‐bearing mice as such and in combination with dendritic cell (DC)‐based immunotherapy. Both flow cytometric and pathological analysis revealed that the silencing of glioma‐derived galectin‐1 significantly decreased the amount of brain‐infiltrating macrophages and myeloid‐derived suppressor cells (MDSC) in tumor‐bearing mice. Additionally, we revealed a pro‐angiogenic role for galectin‐1 within the glioma microenvironment. The data provided in this study reveal a pivotal role for glioma‐derived galectin‐1 in the regulation of myeloid cell accumulation within the glioma microenvironment, the most abundant immune cell population in high‐grade gliomas. Furthermore, the prolonged survival observed in untreated and DC‐vaccinated glioma‐bearing mice upon the silencing of tumor‐derived galectin‐1 strongly suggest that the in vivo targeting of tumor‐derived galectin‐1 might offer a promising and realistic adjuvant treatment modality in patients diagnosed with GBM.

Haptoglobin deficiency facilitates the development of autoimmune inflammation

Haptoglobin (HP) is an acute phase protein synthesized by liver cells in response to IL‐6. HP has been demonstrated to modulate the immune response and to have anti‐inflammatory activities. To analyze HPs effect on autoimmune inflammation, we here studied the course of EAE induced by immunization of Hp knockout (Hp−/−) and syngeneic WT mice with myelin oligodendrocyte glycoprotein peptide (MOG35–55). Hp−/−mice suffered from a more severe disease that was associated with increased expression of IL‐17A, IL‐6, and IFN‐γ mRNA in the CNS and with a denser cellular infiltrate in the spinal cord. During the recovery phase, a significantly higher number of myeloid DC, CD8+ cells, IL‐17+ CD4+ and IFN‐γ+ CD4+ cells persisted in the CNS of Hp−/− mice. Absence of HP affected the priming and differentiation of T cells after MOG35–55 immunization, as levels of Th2 cytokines produced in response to MOG stimulation by Hp−/− T cells were reduced. These results suggest that HP plays a modulatory and protective role on autoimmune inflammation of the CNS.

Depletion of Regulatory T Cells in a Mouse Experimental Glioma Model through Anti-CD25 Treatment Results in the Infiltration of Non-Immunosuppressive Myeloid Cells in the Brain

The recruitment and activation of regulatory T cells (Tregs) in the micro-environment of malignant brain tumors has detrimental effects on antitumoral immune responses. Hence, local elimination of Tregs within the tumor micro-environment represents a highly valuable tool from both a fundamental and clinical perspective. In the syngeneic experimental GL261 murine glioma model, Tregs were prophylactically eliminated through treatment with PC61, an anti-CD25 mAb. This resulted in specific elimination of CD4+CD25hiFoxp3+ Treg within brain-infiltrating lymphocytes and complete protection against subsequent orthotopic GL261 tumor challenge. Interestingly, PC61-treated mice also showed a pronounced infiltration of CD11b+ myeloid cells in the brain. Phenotypically, these cells could not be considered as Gr-1+ myeloid-derived suppressor cells (MDSC) but were identified as F4/80+ macrophages and granulocytes.

Foxp3+ regulatory T cells are activated in spite of B7-CD28 and CD40-CD40L blockade

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)‐cell activity. We previously reported that the blockade of the B7‐CD28 and CD40L‐CD40 interaction efficiently suppresses allogeneic T‐cell activation in vivo. This was characterized by an initial rise in Foxp3+ cells, followed by depletion of host‐reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4+ cell activation. When CTLA‐4Ig and anti‐CD40L mAb (MR1) were added to the cultures, T‐cell proliferation and IL‐2 production were strongly reduced. However, Foxp3+ cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4+ cells much more efficiently than did freshly isolated Treg cells. CD4+ cells activated by allogeneic cells in the presence of MR1 and CTLA‐4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4+ cells when Foxp3+ Treg cells were removed. We conclude that natural Treg cells are less dependent on B7‐CD28 or CD40‐CD40L costimulation compared with Foxp3− T cells. Reduced costimulation therefore alters the balance between Teff and Treg‐cell activation in favor of Treg‐cell activity.

Browser protection against cross-site request forgery

As businesses are opening up to the web, securing their web applications becomes paramount. Nevertheless, the number of web application attacks is constantly increasing. Cross-Site Request Forgery (CSRF) is one of the more serious threats to web applications that gained a lot of attention lately. It allows an attacker to perform malicious authorized actions originating in the end-users browser, without his knowledge. This paper presents a client-side policy enforcement framework to transparently protect the end-user against CSRF. To do so, the framework monitors all outgoing web requests within the browser and enforces a configurable cross-domain policy. The default policy is carefully selected to transparently operate in a web 2.0 context. In addition, the paper also proposes an optional server-side policy to improve the accuracy of the client-side policy enforcement. A prototype is implemented as a Firefox extension, and is thoroughly evaluated in a web 2.0 context.

Technical advancement in regulatory T cell isolation and characterization using CD127 expression in patients with malignant glioma treated with autologous dendritic cell vaccination.

We have successfully treated over two hundred high-grade glioma (HGG) patients with immunotherapy consisting of vaccination with autologous dendritic cells (DCs) loaded with autologous tumour lysate. It has been documented that regulatory T cells (Treg) can counteract anti-tumour immune responses. Therefore, monitoring of Treg in these patients is essential. Up till now, Treg have been characterized based on the expression of the transcription factor Foxp3. Here, we validated IL-7 receptor alpha subunit (CD127)dim expression as a marker for human Treg within HGG patients, as a less laborious assay for routine use in tumour vaccination trials. We noted a strong positive correlation between Foxp3 expression and CD127dim expression in CD4+CD25+ and CD4+ cells. The suppressive function of CD4+CD127dim cells was assessed in an allogeneic mixed lymphocyte reaction (MLR). We conclude that CD127 staining is a fast, well-suited and reproducible Treg monitoring tool in HGG patients treated with immunotherapy.

Lens Epithelium-derived Growth Factor/p75 Qualifies as a Target for HIV Gene Therapy in the NSG Mouse Model

Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF(325-530)) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD(4)(+) T-cells were transduced with lentiviral vectors encoding LEDGF(325-530), the interaction-deficient mutant LEDGF(325-530)D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF(325-530) overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD(4)(+) T-cells expressing LEDGF(325-530) in comparison with engraftment with LEDGF(325-530)D366N cells. Four weeks after transplantation with LEDGF(325-530)D366N cells, 70% of the CD(4)(+) cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF(325-530) cells only a 20% decrease in CD(4)(+) cells was measured. Liver and spleen sections of LEDGF(325-530) mice contained less HIV than LEDGF(325-530)D366N mice as measured by p24 antigen detection. LEDGF(325-530) overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD(4)(+) T-cells.