Peter Ponsaerts

Microglia: gatekeepers of central nervous system immunology

Microglia are perhaps the most underestimated cell type of our immune system. Not only were immunologists unaware of their capabilities until recently, but also, some neuroscientists denied their actual existence until the late 20th century. Nowadays, their presence is confirmed extensively, as demonstrated by numerous reports describing their involvement in virtually all neuropathologies. However, despite distinct approaches, their origin remains a point of controversy. Although many agree about their myeloid‐monocytic ancestry, the precise progenitor cells and the differentiation mechanisms, which give rise to microglia in the different developmental stages of the CNS, are not unraveled yet. Mostly, this can be attributed to their versatile phenotype. Indeed, microglia show a high morphological plasticity, which is related to their functional state. This review about microglia aims to introduce the reader extensively into their ontogeny, cell biology, and involvement in different neuropathologies.

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.

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.

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.

Cellular and molecular neuropathology of the cuprizone mouse model: Clinical relevance for multiple sclerosis

The cuprizone mouse model allows the investigation of the complex molecular mechanisms behind nonautoimmune-mediated demyelination and spontaneous remyelination. While it is generally accepted that oligodendrocytes are specifically vulnerable to cuprizone intoxication due to their high metabolic demands, a comprehensive overview of the etiology of cuprizone-induced pathology is still missing to date. In this review we extensively describe the physico-chemical mode of action of cuprizone and discuss the molecular and enzymatic mechanisms by which cuprizone induces metabolic stress, oligodendrocyte apoptosis, myelin degeneration and eventually axonal and neuronal pathology. In addition, we describe the dual effector function of the immune system which tightly controls demyelination by effective induction of oligodendrocyte apoptosis, but in contrast also paves the way for fast and efficient remyelination by the secretion of neurotrophic factors and the clearance of cellular and myelinic debris. Finally, we discuss the many clinical symptoms that can be observed following cuprizone treatment, and how these strengthened the cuprizone model as a useful tool to study human multiple sclerosis, schizophrenia and epilepsy.

mRNA-Mediated Gene Delivery Into Human Progenitor Cells Promotes Highly Efficient Protein Expression

Gene transfer into human CD34+ haematopoietic progenitor cells (HPC) and multi‐potent mesenchymal stromal cells (MSC) is an essential tool for numerous in vitro and in vivo applications including therapeutic strategies, such as tissue engineering and gene therapy. Virus based methods may be efficient, but bear risks like tumorigenesis and activation of immune responses. A safer alternative is non‐viral gene transfer, which is considered to be less efficient and accomplished with high cell toxicity. The truncated low affinity nerve growth factor receptor (ÄLNGFR) is a marker gene approved for human in vivo application. Human CD34+ HPC and human MSC were transfected with in vitro‐transcribed mRNA for ΔLNGFR using the method of nucleofection. Transfection efficiency and cell viability were compared to plasmid‐based nucleofection. Protein expression was assessed using flow cytometry over a time period of 10 days. Nucleofection of CD34+ HPC and MSC with mRNA resulted in significantly higher transfection efficiencies compared to plasmid transfection. Cell differentiation assays were performed after selecting ΔLNGFR positive cells using a fluorescent activating cell sorter. Neither cell differentiation of MSC into chondrocytes, adipocytes and osteoblasts, nor differentiation of HPC into burst forming unit erythroid (BFU‐E) colony forming unit‐granulocyte, erythrocyte, macrophage and megakaryocyte (CFU‐GEMM), and CFU‐granulocyte‐macrophage (GM) was reduced. mRNA based nucleofection is a powerful, highly efficient and non‐toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may be useful to transiently manipulate stem cell characteristics and thus combine principles of gene therapy and tissue engineering.

Immunosuppression induced by immature dendritic cells is mediated by TGF‐β/IL‐10 double‐positive CD4+ regulatory T cells

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (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. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)‐β and interleukin (IL)‐10 double‐positive CD4+ T cells within 1 week of autologous DC/T cell co‐cultures. In iDC/T cell cultures, where antigen‐specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF‐β and IL‐10 secreted by CD4+CD25−FOXP3− T cells. In addition, the suppressive capacity of CD4+ T cells conditioned by iDC was transferable to already primed antigen‐specific CD8+ T cell cultures. In contrast, addition of CD4+ T cells conditioned by mDC to primed antigen‐specific CD8+ T cells resulted in enhanced CD8+ T cell responses, notwithstanding the presence of TGF‐β+/IL‐10+ T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine‐secreting regulatory T cells. We show that iDC‐conditioned CD4+ T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4+ T cells. Furthermore, TGF‐β+/IL‐10+ T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.

mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation

Genetically modified dendritic cells (DC) are increasingly used in vitro to activate cytotoxic T lymphocyte (CTL) immune responses. Because T cell activation protocols consist of multiple restimulation cycles of peripheral blood lymphocytes with antigen-loaded mature DC, continuous generation of DC is needed throughout the experiment. Therefore, cryopreservation of DC loaded with antigen is a valuable alternative for weekly generation and modification of DC. Recently, we described an antigen loading method for DC based on electroporation of defined tumor antigen mRNA. In this study, we demonstrate that mRNA-electroporated DC can efficiently be prepared for cryopreservation. Using an optimized maturation and freezing protocol after mRNA electroporation, we obtained high transgene-expressing viable mature DC. In addition, we showed that these modified cryopreserved DC retain stimulatory capacity in an influenza model system. Therefore, cryopreservation of mature mRNA-electroporated DC is a useful method for continuous availability of antigen-loaded DC throughout T cell activation experiments.