Stefaan Van Gool

CD80, CD86 and CD40 provide accessory signals in a multiple-step T-cell activation model

In this review, a sequential multiple-step model for T-cell activation is proposed. In a series of in vitro studies, highly purified freshly isolated human peripheral blood T lymphocytes were stimulated through the CD28 receptor, with mAb or with natural ligands B7-1 or B7-2, along with TCR stimulation, in the absence of other costimulatory interactions. Ligation of the CD28 receptor, along with stimulation of the TCR, was found to up-regulate pleiotropic in vitro activities, including the secretion of both Th1 and Th2-type cytokines, B-cell help, and the development of cytotoxic activity. This costimulatory action involves CD4+ and CD8+ as well as naive and memory T-cell subsets. The expression of B7-1 and B7-2 on professional APC in situ in both normal and pathological tissues, and its up-regulation on monocytes by GM-CSF and IFN-gamma is consistent with this role. Additional studies have addressed the contribution of interactions between CD28 and B7-1 and B7-2 in T-cell activation initiated by normal un-engineered APC, such as stimulation with recall antigens and primary MLR. Blockade of the interaction between CD28 and B7-1/B7-2 under these conditions failed to completely inhibit T-cell responses or to induce anergy. Complete inhibition and anergy were, however, induced with a combination of CsA, targeting downstream TCR-triggered signalling, as well as anti-B7-1- and anti-B7-2-directed reagents. Interestingly, and in contrast to anti-LFA-1 mAb, the addition of anti-B7-1 or anti-B7-2 reagents could be delayed until at least 48 h after the initiation of T-cell stimulation, indicating a requirement for a late interaction between CD28 and its counter-receptors. Interactions between CD40L on activated T cells and CD40 on APC may serve to sustain, enhance or prolong the presentation of B7-1 or B7-2 on the APC, and thus to prevent anergy induction, or ineffective or abortive T-cell stimulation. Based on these data a sequential multiple-step T-cell activation model is proposed, and novel strategies for immuno-intervention can be designed.

Integration of autologous dendritic cell-based immunotherapy in the primary treatment for patients with newly diagnosed glioblastoma multiforme: a pilot study

Despite resection, radiochemotherapy, and maintenance temozolomide chemotherapy (TMZm), the prognosis of patients with glioblastoma multiforme (GBM) remains poor. We integrated immunotherapy in the primary standard treatment for eight pilot adult patients (median age 50xa0years) with GBM, to assess clinical and immunological feasibility and toxicity in preparation of a phase I/II protocol HGG-2006. After maximum, safe resection, leukapheresis was performed before radiochemotherapy, and four weekly vaccinations with autologous GBM lysate-loaded monocyte-derived dendritic cells were given after radiochemotherapy. Boost vaccines with lysates were given during TMZm. During the course of vaccination, immunophenotyping showed a relative increase in CD8+CD25+ cells in six of the seven patients, complying with the prerequisites for implementation of immunotherapy in addition to postoperative radiochemotherapy. In five patients, a more than twofold increase in tumor antigen-reacting IFN-γ-producing T cells on Elispot was seen at the fourth vaccination compared with before vaccination. In three of these five patients this more than twofold increase persisted after three cycles of TMZm. Quality of life during vaccination remained excellent. Progression-free survival at six months was 75%. Median overall survival for all patients was 24xa0months (range: 13–44xa0months). The only serious adverse event was an ischemic stroke eight months postoperatively. We conclude that tumor vaccination, fully integrated within the standard primary postoperative treatment for patients with newly diagnosed GBM, is feasible and well tolerated. The survival data were used to power a currently running phase I/II trial.

Integration of autologous dendritic cell-based immunotherapy in the standard of care treatment for patients with newly diagnosed glioblastoma: results of the HGG-2006 phase I/II trial.

PurposeDendritic cell (DC)-based tumor vaccination has rendered promising results in relapsed high-grade glioma patients. In the HGG-2006 trial (EudraCT 2006-002881-20), feasibility, toxicity, and clinical efficacy of the full integration of DC-based tumor vaccination into standard postoperative radiochemotherapy are studied in 77 patients with newly diagnosed glioblastoma.Patients and methodsAutologous DC are generated after leukapheresis, which is performed before the start of radiochemotherapy. Four weekly induction vaccines are administered after the 6-week course of concomitant radiochemotherapy. During maintenance chemotherapy, 4 boost vaccines are given. Feasibility and progression-free survival (PFS) at 6xa0months (6mo-PFS) are the primary end points. Overall survival (OS) and immune profiling, rather than monitoring, as assessed in patients’ blood samples, are the secondary end points. Analysis has been done on intent-to-treat basis.ResultsThe treatment was feasible without major toxicity. The 6mo-PFS was 70.1xa0% from inclusion. Median OS was 18.3xa0months. Outcome improved significantly with lower EORTC RPA classification. Median OS was 39.7, 18.3, and 10.7xa0months for RPA classes III, IV, and V, respectively. Patients with a methylated MGMT promoter had significantly better PFS (pxa0=xa00.0027) and OS (pxa0=xa00.0082) as compared to patients with an unmethylated status. Exploratory “immunological profiles” were built to compare to clinical outcome, but no statistical significant evidence was found for these profiles to predict clinical outcome.ConclusionFull integration of autologous DC-based tumor vaccination into standard postoperative radiochemotherapy for newly diagnosed glioblastoma seems safe and possibly beneficial. These results were used to power the currently running phase IIb randomized clinical trial.

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.

Formulations for Intranasal Delivery of Pharmacological Agents to Combat Brain Disease: A New Opportunity to Tackle GBM?

Despite recent advances in tumor imaging and chemoradiotherapy, the median overall survival of patients diagnosed with glioblastoma multiforme does not exceed 15 months. Infiltration of glioma cells into the brain parenchyma, and the blood-brain barrier are important hurdles to further increase the efficacy of classic therapeutic tools. Local administration methods of therapeutic agents, such as convection enhanced delivery and intracerebral injections, are often associated with adverse events. The intranasal pathway has been proposed as a non-invasive alternative route to deliver therapeutics to the brain. This route will bypass the blood-brain barrier and limit systemic side effects. Upon presentation at the nasal cavity, pharmacological agents reach the brain via the olfactory and trigeminal nerves. Recently, formulations have been developed to further enhance this nose-to-brain transport, mainly with the use of nanoparticles. In this review, the focus will be on formulations of pharmacological agents, which increase the nasal permeation of hydrophilic agents to the brain, improve delivery at a constant and slow release rate, protect therapeutics from degradation along the pathway, increase mucoadhesion, and facilitate overall nasal transport. A mounting body of evidence is accumulating that the underexplored intranasal delivery route might represent a major breakthrough to combat glioblastoma.

Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohns disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

Peripheral blood lymphocyte subset shifts in patients with untreated hematological tumors: Evidence for systemic activation of the T cell compartment

Flow cytometry immunophenotyping of peripheral blood lymphocyte subsets and multivariate data-analytical techniques revealed that among untreated hemato-oncological patients (n = 48) with lymphomas, acute and chronic myeloid and lymphocytic leukemias, monoclonal gammopathy of undetermined significance, and multiple myeloma, 42% had (nonmalignant) lymphocyte profiles clearly distinct from healthy donors. Notably, a similar pattern of increased CD3+ CD57+, CD3+ HLA-DR+, CD3+ CD(16 + 56)+, CD4- CD8+, CD8+ CD57+, CD8+ CD28-, and CD8+ CD62L- subsets was detected. More extensive three-color immunophenotyping on an additional group of 49 untreated patients revealed that both CD4+ and CD8+ T cells displayed significant increases of activation markers: CD69, CD(16 + 56), HLA-DR, CD71, and CD57, and a loss of CD62L and CD28, which is also interpreted as a sign of activation. Consistent with the phenotypical signs of in vivo immune activation, polyclonal cytolytic activity, measured ex vivo in an anti-CD3-redirected assay, was detected within immunomagnetically purified CD4+ T cells of three out of six B-CLL patients investigated, but not within purified CD4+ T cells of five healthy donors. The purified CD8+ T cells of patients (n = 28) and donors (n = 5) on the other hand displayed similar polyclonal cytotoxic activities at the various effector:target ratios investigated. Tumor-directed cytotoxic activity of purified CD4+ (n = 6) and/or CD8+ T cells (n = 15) against freshly isolated autologous tumor cells was not detected in any of the experiments. Collectively, our results demonstrate systemic T cell activation as a common feature in hematological neoplasia, and a markedly enhanced cytolytic activity of the CD4- subset in CLL patients. The reason(s) for this expansion of activated T cells and its pathophysiologic significance, however, remain unclear.

Human T Cell Activation by Costimulatory Signal-Deficient Allogeneic Cells Induces Inducible Costimulator-Expressing Anergic T Cells with Regulatory Cell Activity

Although immunoregulation by several types of regulatory T cells is now clearly established in mice, the demonstration of such regulatory T cells in humans has been proven more difficult. In this study we demonstrate the induction of anergic regulatory T cells during an MLR performed in the presence of blocking mAb to the costimulatory molecules CD40, CD80, and CD86. Despite this costimulation blockade, which totally blocks T cell proliferation and cytokine production, a nonproliferating T cell subpopulation was activated to express inducible costimulator (ICOS). These ICOS+ cells were anergic when restimulated with unmanipulated allogeneic stimulator cells at the level of proliferation and Th1 and Th2 cytokine production, but they did produce IL-10. These ICOS-expressing cells also blocked the capacity of reciprocal ICOS-negative cells to proliferate and to produce cytokines. ICOS+ anergic cells could suppress allogenic responses of either primed or naive T cells through inhibition of IL-2 gene transcription. Suppression was not mediated by IL-10 and did not require ICOS-ICOS ligand interaction, but depended on cell-cell contact. Thus, a subtype of regulatory T cells in human blood can be activated in the absence of costimulatory signals from CD40, CD80, and CD86, and they can be identified by expression of ICOS after activation.

Uptake and presentation of malignant glioma tumor cell lysates by monocyte-derived dendritic cells

Malignant glioma of the CNS is a tumor with a very bad prognosis. Development of adjuvant immunotherapy is hampered by interindividual and intratumoral antigenic heterogeneity of gliomas. To evaluate feasibility of tumor vaccination with (autologous) tumor cells, we have studied uptake of tumor cell lysates by dendritic cells (DCs), and the T-cell stimulatory capacity of the loaded DCs. DCs are professional antigen-presenting cells, which have already been used as natural adjuvants to initiate immune responses in human cancer. An efficacious uptake of tumor cell proteins, followed by processing and presentation of tumor-associated antigens by the DCs, is indeed one of the prerequisites for a potent and specific stimulation of T lymphocytes. Human monocytes were differentiated in vitro to immature DCs, and these were loaded with FITC-labeled tumor cell proteins. Uptake of the tumor cell proteins and presentation of antigens in the context of both MHC class I and II could be demonstrated using FACS analysis and confocal microscopy. After further maturation, the loaded DCs had the capacity to induce specific T-cell cytotoxic activity against tumor cells. We conclude that DCs loaded with crude tumor lysate are efficacious antigen-presenting cells able to initiate a T-cell response against malignant glioma tumor cells.

Identification of an enriched CD4+ CD8α++ Cd8β+ T-cell subset among tumor-infiltrating lymphocytes in human renal cell carcinoma

Three‐color immunofluorescence flow‐cytometric analysis of freshly isolated tumor‐infiltrating lymphocytes (TIL) from patients with primary renal cell carcinoma (RCC) revealed a unique, not previously described TIL subset with a CD3+ CD4+ CD8α++ CD8β+ phenotype. This subset represented at least 5% of CD3+ TIL in 15 of 21 patients with clear cell RCC, whereas it was not or only marginally represented in patients with papillary RCC or sarcomatoid RCC. In one‐third of the patients with clear cell RCC, more than 20% of CD3+ TIL and in one patient more than half of the CD3+ TIL displayed this phenotype. The occurrence of this subset was not associated with pathological stage, tumor diameter, nuclear grade, cytogenetic abnormalities or vascular invasion in this patient cohort. When present, the CD3+ CD8α++ CD8β+ subset was detected in similar proportions in tumor tissue and tumor capsula, and it was also detected in adjacent non‐tumoral renal tissue, albeit in much lower proportions. Despite strong cell surface expression of various activation markers (CD69, CD54 and HLA‐DR), CD3+ CD4+ CD8α++ anti‐CD3‐redirected cytotoxicity assay. In contrast with CD3+ CD4+ CD8− cells from the same tumor sample, they were markedly deficient in IL‐2Rα up‐regulation following anti‐CD3 triggering. The possibility that these cells represent either anergic cells or a highly specialized effector population with a discrete, as yet undescribed function is discussed. Int. J. Cancer 71:178–182, 1997.