Viggo Van Tendeloo

Induction of complete and molecular remissions in acute myeloid leukemia by Wilms’ tumor 1 antigen-targeted dendritic cell vaccination

Active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far, most dendritic cell trials have been performed in end-stage cancer patients with high tumor loads. Here, in a phase I/II trial, we investigated the effect of autologous dendritic cell vaccination in 10 patients with acute myeloid leukemia (AML). The Wilms’ tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two patients in partial remission after chemotherapy were brought into complete remission after intradermal administration of full-length WT1 mRNA-electroporated dendritic cells. In these two patients and three other patients who were in complete remission, the AML-associated tumor marker returned to normal after dendritic cell vaccination, compatible with the induction of molecular remission. Clinical responses were correlated with vaccine-associated increases in WT1-specific CD8+ T cell frequencies, as detected by peptide/HLA-A*0201 tetramer staining, and elevated levels of activated natural killer cells postvaccination. Furthermore, vaccinated patients showed increased levels of WT1-specific IFN-γ–producing CD8+ T cells and features of general immune activation. These data support the further development of vaccination with WT1 mRNA-loaded dendritic cells as a postremission treatment to prevent full relapse in AML patients.

Balancing between immunity and tolerance: an interplay between dendritic cells, regulatory T cells, and effector T cells

Dendritic cells (DC), professional antigen‐presenting cells of the immune system, exert important functions both in induction of T cell immunity, as well as tolerance. It is well established that the main function of immature DC (iDC) in their in vivo steady‐state condition is to maintain peripheral tolerance to self‐antigens and that these iDC mature upon encounter of so‐called danger signals and subsequently promote T cell immunity. Previously, it was believed that T cell unresponsiveness induced after stimulation with iDC is caused by the absence of inflammatory signals in steady‐state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. Moreover, several reports indicate that traditional DC maturation can no longer be used to distinguish tolerogenic and immunogenic properties of DC. This review will focus on the complementary role of dendritic cells in inducing both tolerance and immunity, and we will discuss the clinical implications for dendritic cell‐based therapies.

Clinical use of dendritic cells for cancer therapy

Since the mid-1990s, dendritic cells have been used in clinical trials as cellular mediators for therapeutic vaccination of patients with cancer. Dendritic cell-based immunotherapy is safe and can induce antitumour immunity, even in patients with advanced disease. However, clinical responses have been disappointing, with classic objective tumour response rates rarely exceeding 15%. Paradoxically, findings from emerging research indicate that dendritic cell-based vaccination might improve survival, advocating implementation of alternative endpoints to assess the true clinical potency of dendritic cell-based vaccination. We review the clinical effectiveness of dendritic cell-based vaccine therapy in melanoma, prostate cancer, malignant glioma, and renal cell carcinoma, and summarise the most important lessons from almost two decades of clinical studies of dendritic cell-based immunotherapy in these malignant disorders. We also address how the specialty is evolving, and which new therapeutic concepts are being translated into clinical trials to leverage the clinical effectiveness of dendritic cell-based cancer immunotherapy. Specifically, we discuss two main trends: the implementation of the next-generation dendritic cell vaccines that have improved immunogenicity, and the emerging paradigm of combination of dendritic cell vaccination with other cancer therapies.

Regulatory T Cells and Human Disease

The main function of our immune system is to protect us from invading pathogens and microorganisms by destroying infected cells, while minimizing collateral damage to tissues. In order to maintain this balance between immunity and tolerance, current understanding of the immune system attributes a major role to regulatory T cells (Tregs) in controlling both immunity and tolerance. Various subsets of Tregs have been identified based on their expression of cell surface markers, production of cytokines, and mechanisms of action. In brief, naturally occurring thymic-derived CD4+CD25+ Tregs are characterized by constitutive expression of the transcription factor FOXP3, while antigen-induced or adaptive Tregs are mainly identified by expression of immunosuppressive cytokines (interleukin-10 (IL-10) and/or transforming growth factor-β (TGF-β)). While Tregs in normal conditions regulate ongoing immune responses and prevent autoimmunity, imbalanced function or number of these Tregs, either enhanced or decreased, might lead, respectively, to decreased immunity (e.g., with tumor development or infections) or autoimmunity (e.g., multiple sclerosis). This review will discuss recent research towards a better understanding of the biology of Tregs, their interaction with other immune effector cells, such as dendritic cells, and possible interventions in human disease.

The Use of TLR7 and TLR8 Ligands for the Enhancement of Cancer Immunotherapy

The importance of Toll-like receptors (TLRs) in stimulating innate and adaptive immunity is now well established. In view of this, TLR ligands have become interesting targets to use as stand-alone immunotherapeutics or vaccine adjuvants for cancer treatment. TLR7 and TLR8 were found to be closely related, sharing their intracellular endosomal location, as well as their ligands. In this review, we describe the agonists of TLR7 and TLR8 that are known so far, as well as their contribution to antitumor responses by affecting immune cells, tumor cells, and the tumor microenvironment. The major benefit of TLR7/8 agonists as immune response enhancers is their simultaneous stimulation of several cell types, resulting in a mix of activated immune cells, cytokines and chemokines at the tumor site. We discuss the studies that used TLR7/8 agonists as stand-alone immunotherapeutics or cancer vaccine adjuvants, as well as the potential of TLR7/8 ligands to enhance antitumor responses in passive immunotherapy approaches.

Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice

BackgroundCell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.ResultsIn this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.ConclusionWe here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.

Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.

INTRODUCTION Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. MATERIALS AND METHODS Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1,550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 degrees C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 degrees C for 7 and 28 days. A final aliquot was further centrifuged at 10,000g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 microm. RESULTS High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p=0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b=0.611, p=0.025). Storage at 4 degrees C did not affect EMP quantification. However, freezing at -80 degrees C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p<0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance >0.487; p<0.05). CONCLUSION The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols.

A maximal exercise bout increases the number of circulating CD34+/KDR+ endothelial progenitor cells in healthy subjects. Relation with lipid profile

Mobilization of bone marrow-derived endothelial progenitor cells (EPC) might explain exercise-induced improvement of endothelial function. We assessed whether a maximal exercise bout could alter the number of circulating EPC in healthy subjects and whether this effect is related to their cardiovascular risk profile. Additionally, we investigated possible mediators of this effect, namely nitric oxide (NO) bioavailability and vascular endothelial growth factor (VEGF) release. Healthy subjects (group 1, n = 11; group 2, n = 14) performed a symptom-limited cardiopulmonary exercise test on a bicycle ergometer. Numbers of CD34+/kinase insert domain receptor (KDR)+ cells were determined by flow-cytometric analysis, either after magnetic separation of CD34+ cells (group 1) or starting from whole blood (group 2). Serum concentrations of VEGF and NO metabolites were measured by using ELISA. Following exercise, EPC increased by 76% (15.4 +/- 10.7 cells/ml vs. 27.2 +/- 13.7 cells/ml; P = 0.01) in group 1 and by 69% in group 2 (30.9 +/- 14.6 cells/ml vs. 52.5 +/- 42.6 cells/ml; P = 0.03). The increase in EPC correlated positively with LDL and total cholesterol/HDL ratio and negatively with peak oxygen consumption and oxygen consumption at anaerobic threshold. VEGF levels increased with exercise, with a strong trend toward significance (P = 0.055). NO levels remained unchanged. The present study demonstrates that a maximal bout of exercise induces a significant shift in CD34+ cells toward CD34+/KDR+ cells. This response was larger in subjects with a less favorable lipid profile.

Cancer immunotherapy using RNA-loaded dendritic cells

Dendritic cells (DC) are the most professional antigen‐presenting cells of the immune system and are capable of initiating immune responses in vitro and in vivo. One of the great challenges in immunotherapy protocols is to introduce relevant antigens into DC for stimulation of major histocompatibility complex (MHC) class I‐ and class II‐restricted anti‐tumour or anti‐viral immunity. This review will focus on the development of mRNA‐loaded DC‐based immunotherapy vaccines. First, several published results concerning mRNA transfection efficiency in DC are compared. Next, an overview is given for several published studies describing CD8+ and CD4+ T‐cell clone activation using RNA‐loaded DC. These data show that RNA‐loaded DC efficiently process and present antigenic epitopes. Next, published data from in vitro T‐cell activation studies using RNA‐loaded DC are summarized and provide evidence that RNA‐loaded DC can efficiently stimulate in vitro primary and secondary immune responses. Finally, the summarized data provide evidence that RNA‐loaded DC are a promising strategy for the development of future cancer vaccination strategies.

Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: antagonistic effects of IL-4 and IFN-gamma and selective involvement of TNF-alpha receptor-1.

We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two‐stage culture system in which, besides granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and tumour necrosis factor‐α (TNF‐α), stem‐cell factor (SCF) was added during the first 5 days, while interleukin‐4 (IL‐4) and/or interferon‐γ (IFN‐γ) were added during the secondary culture period of 9 days. Addition of IL‐4 favoured the outgrowth of CD1a+, HLA‐DR+, CD4+, CD40+, CD80+ but CD14− cells with dendritic morphology and strong antigen‐presenting capacity. Addition of IFN‐γ selectively induced HLA‐DR and CD86 but did not up‐regulate CD1a expression or antigen‐presenting capacity of the differentiated cells. An antagonism between IL‐4 and IFN‐γ could further be confirmed in that, as compared with IL‐4 alone, the simultaneous addition of IL‐4 and IFN‐γ to GM‐CSF plus TNF‐α during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor‐specific TNF‐α mutants, we investigated the relative involvement of TNF‐α receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA‐DR, as well as the increase in allostimulatory capacity were dependent on TNF‐R1 triggering, whereas triggering through TNF‐R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two‐stage culture assay using SCF, GM‐CSF, TNF‐α and IL‐4; second, that the effect of TNF‐α in DC generation involves signalling via the TNF‐R1 receptor; and third, that IFN‐γ counteracts some of the effects of IL‐4 in DC generation.