Acute cardiovascular syndromes, including myocardial infarction or stroke, are the principal cause of death in the Western society. The main underlying pathology of cardiovascular diseases is... Show moreAcute cardiovascular syndromes, including myocardial infarction or stroke, are the principal cause of death in the Western society. The main underlying pathology of cardiovascular diseases is atherosclerosis, which is caused by the accumulation of lipids and inflammatory cells in the vessel wall, in so-called atherosclerotic plaques. Current therapies mainly target the disturbed lipid homeostasis, but recent clinical trials have shown a clear benefit in treating patients with anti-inflammatory drugs. However, more specific targeting is required to avoid unwanted side effects. In this thesis, we have generated a detailed atlas of all the cells present in human atherosclerotic plaques using a novel state-of-the-art technique called single-cell RNA sequencing. This data set can be applied as a powerful tool to select potential drug targets with a functional relevance for atherosclerosis. We showed that the majority of the immune cells in the human atherosclerotic plaque consisted of T cells. Subsequently, we identified a pro-inflammatory population of T cells that likely responds to a plaque-derived antigen, suggesting that atherosclerosis has an autoimmune-like component. Finally, we have applied our single-cell atlas to define and validate targets to intervene with the recruitment and activation of mast cells and other immune cells in atherosclerosis. Show less
A deeper understanding of the parameters driving response and resistance to immunotherapy is needed to improve the low response rates observed in breast cancer patients. Research into immunotherapy... Show moreA deeper understanding of the parameters driving response and resistance to immunotherapy is needed to improve the low response rates observed in breast cancer patients. Research into immunotherapy response has predominantly focused on T cells, however effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. By combining profiling of blood and tumors from metastatic breast cancer patients with mechanistic studies in mouse models, we uncovered the critical role of eosinophils in immunotherapy response, and we provide proof-of-principle for eosinophil engagement to enhance immunotherapy efficacy. Focusing on resistance mechanisms to immunotherapy, we demonstrate that neoadjuvant immunotherapy triggers persistent and systemic regulatory T cell activation which blunts therapeutic efficacy against metastatic spread of breast tumors. In addition, we demonstrate that neutrophils in the tumor microenvironment pose a barrier to immunotherapy response through T cell suppression. Lastly, we demonstrate that combining the immunomodulatory agent PD1-IL2v with cisplatin is a powerful approach to induce a broad activation of systemic and intratumoral adaptive and innate immunity, resulting in effective immunotherapy responses. Overall, this work identifies several key players and their interconnectivities in anti-tumor immunity and tumor-induced immunosuppression that may be therapeutically exploited to improve immunotherapy responses for breast cancer patients. Show less
A proper immune system is essential to fight off pathogens such as viruses, bacteria, and fungi. The immune system also plays a huge role in the protection against cancer, as it can eradicate tumor... Show moreA proper immune system is essential to fight off pathogens such as viruses, bacteria, and fungi. The immune system also plays a huge role in the protection against cancer, as it can eradicate tumor cells. All immune cells are derived from hematopoietic stem cells (HSC) that undergo differentiation in a highly regulated succession of developmental steps. Each of the cell types from the immune system perform a unique specialized role, and where most of these lineages develop in the bone marrow, the T cells that make part of our adaptive immunity, develop in the thymus within a specialized environment. To achieve this, the development of each of these cell types is regulated by a variety of transcription factors.In Chapter 2 of this thesis, we reviewed the complexity of one of the important signaling pathways of hematopoietic development, the Wnt pathway. While this serves as an introduction to the fundamental research we performed, it also shines light onto potential therapeutic targets within the Wnt pathway. For further study of the Wnt pathway, we generated a novel reporter mouse, which is described in Chapter 3 of this thesis. Here we developed a reporter mouse for the Axin2 gene with the fluorescent tag mTurquoise2 with CRISPR/Cas9 genome editing tools. Based on how the genetic engineering was done to create this reporter mouse, mice that are homozygous for this reporter knock-in are also a functional knockout for Axin2. For proper functional studies, the heterozygous mice should be used.The Axin2-mTurquoise2 mouse was used in Chapter 4 of this thesis to study Wnt involvement in hematopoiesis and T cell development. We observed an increase of canonical Wnt-signaling in thymocytes from mice that have a loss of Axin2 (Axin2-TQtg/tg mice). This confirms the Wnt dosage effect that was reported previously in literature. Conclusively, these results indicate that Axin2 is required to fine-tune Wnt activity to the levels that are “just right” and cannot be maintained by Wnt activator Axin1 alone.Chapters 2, 3 and 4 focused on fundamental research on hematopoiesis and T cell development. Chapter 5 is more translational oriented and is an introductory review to thymic regenerative therapies. In Chapter 6 of this thesis, we describe the development of a combined cell and gene therapy effort to regenerate a functional thymus transplant from human Induced Pluripotent Stem Cells (iPSCs). We generated an iPSC-derived thymus by directed differentiation of human iPSCs towards thymic epithelial progenitor cells (TEPCs) using FOXN1, formation of 3-D structures from these cells which we named iPSC-derived TEPCs, or iTEPCs, and transplantation of these organoids into mice that lack a functional thymus. Functionality was demonstrated by reconstitution of functional T cells from iPSC-derived grafts, which was introduced by FOXN1 gene therapy (FOXN1 iTEPCs).Chapter 7 is the final translational research chapter of this thesis and investigated the use of iPSCs for the modeling of PIDs and the initial steps towards T cell regeneration in SCID patients. This chapter describes the iPSC generation, and its repair to use gene-corrected iPSCs from a RAG2 SCID patient to repair their disrupted immune system. The resulting iPSC model was used for disease modelling and provided novel insights into the T cell development in these RAG2-SCID patients, as we observed developmental blocks at every investigated stage of T cell development. The findings in this chapter also provide a proof-of-principle to treat a variety of SCID patients by utilizing ex vivo cell and gene therapy.Altogether, this thesis tackles two sides of the same coin: fundamentals of hematopoiesis and T cell development, and regenerative therapies for the immune system. The fundamental tools and findings in this thesis can lead to important insights to find new treatment options or improve existing therapies. Furthermore, we provide the basis for two potential therapies to treat patients with a variety of immune disorders, including DiGeorge Syndrome, SCID, age-related immune deficiencies and (post-transplant) leukemia patients that received ablative therapies. Show less
Immune checkpoint therapies that aim to (re)activate the immune response against cancer cells have shown promising results in a variety of tumor types. Yet, a large fraction of cancer patients does... Show moreImmune checkpoint therapies that aim to (re)activate the immune response against cancer cells have shown promising results in a variety of tumor types. Yet, a large fraction of cancer patients does not benefit from these therapies. While the presence of a substantial number of immune cells, and in particular T cells, in the tumor is generally related with a better clinical response to checkpoint therapies, the T cells in the tumor are diverse in their capacity to eliminate the tumor. In order to improve treatment outcome of cancer patients, we require a better understanding of the roles of different T cells in the response (and resistance) to immune checkpoint therapy. The development of single cell profiling technologies has provided us with a powerful tool to analyze the state and functionality of individual cells. In this thesis, I have used single cell profiling methods in combination with innovative experimental technologies to unravel the diversity of T cells in human tumors and define the changes in the profiles of T cells that occur in response to treatment with immune checkpoint therapy to dissect which T cells are important for therapy response. Show less
This thesis describes the research that was performed to unravel the function and development of human lymphoid tissue-resident (lt)NK cells in relation to the circulating CD56bright and CD56dim NK... Show moreThis thesis describes the research that was performed to unravel the function and development of human lymphoid tissue-resident (lt)NK cells in relation to the circulating CD56bright and CD56dim NK cells. Two methods, RNA sequencing and flow cytometry, both commonly used in the field of immunology, were employed at the bulk and single-cell level. We highlighted the complexity of single-cell analysis that could potentially lead to misinterpretation, but also the additional value over bulk approaches by uncovering the cellular heterogeneity and developmental trajectories. Finally, our workflow for single-cell analysis was applied on a patient more than 50 years after hematopoietic stem cell transplantation to perform combined analysis of the T cell phenotype and T cell receptor repertoire. Show less
Transplantation is the golden standard for the treatment of end-stage renal disease. During this process, the transplanted organ is often damaged. In this thesis, we investigated whether activation... Show moreTransplantation is the golden standard for the treatment of end-stage renal disease. During this process, the transplanted organ is often damaged. In this thesis, we investigated whether activation of the complement system, part of our innate immune system, plays a local role. We reviewed that properdin, the only known positive regulator of the complement system, was detected in serum, plasma and urine from patients with various complement-mediated renal diseases. In protocol biopsies obtained 10 days after transplantation, properdin was found deposited in addition to complement activation markers. Next, we showed that dendritic cells secrete properdin and a decrease in properdin levels during dendritic cell- T-cell interaction resulted in reduced T-cell proliferation and activation. We also showed that properdin is able to bind to surfaces of both viable and dead cells, contributing to complement activation. Macrophages can also produce properdin and negative regulators factor H and its splice variant FHL-1. Increasing knowledge on complement factor production by other cells than hepatocytes, including immune cells, hints towards a local role of the complement system in various processes. These findings contribute to a better understanding of the local role of the complement system and are important for the applications of (new) complement-inhibiting drugs. Show less
For many years, hematopoietic stem cell kinetics and/or cell signaling pathway activity has been studied through fluorescent in vitro or in vivo models. However, inaccurate measurement of the... Show moreFor many years, hematopoietic stem cell kinetics and/or cell signaling pathway activity has been studied through fluorescent in vitro or in vivo models. However, inaccurate measurement of the fluorescent proteins or lack of knowlegde about the genetic design of these models lead to incomplete conclusions. In the present thesis, in vivo fluorescent models are improved and new models are proposed together with analysis protocols to ensure precise measurement of fluorescent protein dynamics. Show less
The mechanisms involved in the autoimmune hypothesis of narcolepsy are investigated in this thesis. The role of HLA, auto- and cross-reactive T cells is explored and immune cell populations of... Show moreThe mechanisms involved in the autoimmune hypothesis of narcolepsy are investigated in this thesis. The role of HLA, auto- and cross-reactive T cells is explored and immune cell populations of interest are identified by a new technique, called mass cytometry. The second part of the thesis assesses unexplored clinical features of narcolepsy, such as weight gain and sleep state misperception. Show less
This thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after... Show moreThis thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after infection was assessed in population-based studies. The vaccine-induced changes in HPV-seroprevalence among the HPV unvaccinated Dutch population aged 0-89 years, where we compared the HPV-seroprevalence before the introduction of the HPV vaccine with data of approximately six years post-implementation of the national HPV vaccination program. Also, the HPV immune status of the Dutch Caribbean population just after introduction of HPV vaccination was determined. Moreover, the longitudinal relation between the hr-HPV antibody levels and the prevalence of HPV infections in three-dose vaccinated girls were studied. And more insight was gained into humoral and cellular immune responses after just a one-dose of the HPV vaccine. At last, the kinetics of innate and adaptive immune responses directly after vaccination different HPV vaccines were investigated. In the coming years some important changes are expected regarding HPV screening and vaccination. The effectiveness of the one-dose schedule will become clear as clinical trials end. In the Netherlands, a sex-neutral vaccination will be implemented soon. These changes will need to be monitored to provide scientific answers about the effectiveness and immunogenicity. Show less
In this thesis I have firstly applied gene transfer technologies to the redirection of T cell specificity, by trying to overcome limitations related to non-viral gene transfer systems. In the... Show moreIn this thesis I have firstly applied gene transfer technologies to the redirection of T cell specificity, by trying to overcome limitations related to non-viral gene transfer systems. In the second part of my PhD work, I focused on genetic screens, which, I applied to understanding molecular mechanisms of escape from T cell attack and to reveal mechanisms of PD-L1 regulation. The work presented in this thesis may on the one hand facilitate the clinical application of non-viral-based gene transfer systems in T cells. On the other hand, the more fundamental discoveries related to IFN-γ-mediated tumor cell killing and PD-L1 regulation may help to further understand resistance toward immunotherapies and how to overcome them. Show less
Atherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the... Show moreAtherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the vessel wall. Over time, this accumulation of lipids and immune cells induce morphological abnormalities in the vessel wall which cause the vessel lumen to narrow. This narrowing of the lumen (stenosis) causes ischemia in the downstream tissue. Prolonged ischemia causes myocardial ischemia and/or stroke. The research described in my thesis examines a well-recognized risk factor of atherosclerosis, being dyslipidemia, from an entirely new perspective. More specifically, it describes how dyslipidemia affects intrinsic metabolic processes in T cells, the conductors of the immune response characterizing atherosclerosis, and how this affects their function. My research has contributed to knowledge on the pathophysiology of atherosclerosis and might one day pave the way for the development of novel therapeutic approaches to treat cardiovascular disease. Show less
The breakthrough of immunotherapy for cancer has introduced promising new options, but nonetheless only a minority of cancer patients show significant clinical benefit. This situation has inspired... Show moreThe breakthrough of immunotherapy for cancer has introduced promising new options, but nonetheless only a minority of cancer patients show significant clinical benefit. This situation has inspired two avenues of research to find solutions to this problem: mechanistic studies to decipher the working mechanisms of immunotherapies and to investigate why many patients do not respond, and studies developing combination treatments to achieve clinical benefit in situations where immunotherapy alone is not sufficient. This thesis explores both these avenues by investigating applications of visible light in immunotherapy of cancer in pre-clinical models. We developed optical imaging platforms for visualization of immune cells and immunotherapies, which can shed light on the immunological events after administration of immunotherapy. In addition, we investigated novel therapies based on the combination of tumor ablation by Photodynamic Therapy and different types of immunotherapy. Our findings may prove useful in understanding success and failure of immunotherapy, and provide new combination treatment options when the efficacy of monotherapy is insufficient. Show less
Immunotherapy of cancer has established itself in recent years as a promising novel approach to treat cancer patients. One of the experimental approaches is based on therapeutic vaccination. We... Show moreImmunotherapy of cancer has established itself in recent years as a promising novel approach to treat cancer patients. One of the experimental approaches is based on therapeutic vaccination. We have previously developed vaccines consisting of synthetic long peptides (SLP) which successfully eradicated premalignant lesions in 50% of patients. To further improve these vaccines, a Toll-like receptor ligand (TLR-L) was conjugated to SLP which enables targeting of the SLP to relevant antigen-presenting cells while concomitantly activating these cells. In fact, the research described in this thesis shows that TLR-L SLP conjugates induce enhanced antitumor immunity. Furthermore, optimization of the TLR-L led to even furher improved antitumor responses in mice. Using human cancer patient-derived lymph node cells, we show that lymph node-derived T cells are favorably activated by the TLR-L SLP conjugates. Finally, we combine multiple innate immune stimulatory agonists (TLR2-L and NOD2-L) in one molecule to establish synergistic immune activation. Overall, the research described in this thesis demonstrates the potency of TLR-L SLP conjugates as cancer vaccines, which could strongly contribute to the treatment of cancer patients. Show less
Parasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these... Show moreParasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these infections cause significant morbidity in a large proportion worldwide. However, helminth infections, by means of their ability to modify host immune responses can also provide protection against inflammatory diseases (inflammatory bowel disease, diabetes, and asthma). It is important to better understand the underlying mechanisms of these Yin (positive) and Yang (negative) consequences of helminth infections. The general objective of this thesis is to track helminths at different levels. On the one hand to improve the detection of helminth infections, essential for the studying helminths and the interaction with their human host. Moreover, a more sensitive diagnostics is instrumental for monitoring the distribution of helminth infections and to evaluate the helminth infections elimination program. On the other hand, to understand the mechanistic insights of the interplay between helminths and the host immune system results in priming of Th2 and regulatory T cell responses. This could contribute to the identification of targeted pathways to manipulate immune responses, as part of developing therapeutics to treat inflammatory disorders characterized by deregulated Th2 and/or Treg responses. Show less
The objective of this PhD thesis is to understand mast cell (and basophil) functions and their role in autoimmune disease by focusing on three main aims: 1. To characterize the interaction between... Show moreThe objective of this PhD thesis is to understand mast cell (and basophil) functions and their role in autoimmune disease by focusing on three main aims: 1. To characterize the interaction between innate and Fc receptor triggers on mast cell and basophil function 2. To analyze the interaction between mast cells and CD4+ T cells 3. To understand the function of mast cells in chronic inflammation In this thesis I showed that mast cells can significantly contribute to chronic inflammation through their activation by Fc receptors and TLRs, as well as their interaction with CD4+ T cells, thereby increasing our understanding of their role in allergy and autoimmunity and providing several therapeutic targets to prevent mast cell-mediated immune responses. Show less
Seasonal influenza epidemics lead to severe flu in 3 to 5 million individuals and emerging pandemic influenza strains pose an even greater threat to society. We describe a clinical trial in which... Show moreSeasonal influenza epidemics lead to severe flu in 3 to 5 million individuals and emerging pandemic influenza strains pose an even greater threat to society. We describe a clinical trial in which vaccine-specific responses were measured during two consecutive influenza seasons, including the season in which the 2009 pandemic virus emerged, and showed that these vaccines induced both humoral and cellular responses. However, these responses are unlikely to be protective against newly emerging strains, due to the variable nature of influenza virus. Therefore, the other chapters in this thesis describe concepts within peptide-based vaccination strategies, which is one method to induce responses to highly conserved sequences of influenza virus. We evaluated a concept based on long peptides directed to highly conserved B and T cell epitopes and showed that this vaccine was capable of inducing both humoral and cellular immune responses. Furthermore, the vaccine provided partial protection against infection in an animal model. We also described another successful strategy to enhance the immunogenicity of minimal peptides by modification of these peptides and proceeded with formulations to improve delivery of these minimal peptides. Altogether, the findings in this thesis may contribute to the development of the next generation influenza vaccines. Show less
Current seasonal influenza vaccines rely on the induction of antibodies to neutralize the virus. However, influenza viruses frequently undergo genetic mutations due to antigenic drift and shift,... Show moreCurrent seasonal influenza vaccines rely on the induction of antibodies to neutralize the virus. However, influenza viruses frequently undergo genetic mutations due to antigenic drift and shift, altering the surface proteins hemagglutinin and neuraminidase to which antibodies usually bind. This could render vaccine-induced antibody responses ineffective, resulting in an ineffective influenza vaccine. Influenza vaccines based on the induction of T cell responses might be cross-reactive, since they target conserved influenza epitopes that do not tend to mutate. However, the peptide antigens that are able to induce such T cell responses are often poorly immunogenic. In this thesis, several formulation strategies are described that could improve the immunogenicity of influenza T cell peptide antigens. Using combinations of delivery systems and immunostimulators, the peptide antigens were able to induce influenza-specific T cell responses in mice. Furthermore, a model was developed that could predict the in vitro adjuvanticity of liposomes according to the liposomal lipid composition. In addition, the recent advances in influenza vaccine development are discussed. Finally, an alternative delivery system, the Bioneedle, was evaluated for the delivery of several influenza vaccines. Show less
The main underlying mechanism resulting in cardiovascular complications is the formation of atherosclerotic lesions in arteries. The progression of the lesion is hallmarked by a chronic... Show moreThe main underlying mechanism resulting in cardiovascular complications is the formation of atherosclerotic lesions in arteries. The progression of the lesion is hallmarked by a chronic inflammatory immune response in the arterial wall. Activated T helper cells differentiate into subsets, each producing a specific set of cytokines. T helper cell subsets contribute to the formation of the lesions, with a pro-atherogenic role for T helper 1 and a protective role for regulatory T cells. Cytokines of the IL-12 family play a crucial role in de differentiation of T helper cells. In this thesis I studied the role of IL-12 family members IL-27, IL-30 and IL-35 in atherosclerotic lesion formation. Furthermore the contribution of IL-37, part of the IL-1 family, was studied. In addition to modifications in lesion size and composition, I focus on the effects of the cytokines on T cell activation and differentiation. Show less
Uveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of... Show moreUveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of infiltrating macrophages and T cells is associated with a poor prognosis rather than a good one. The clear link between inflammation and this malignancy provides a paradigm for macrophage plasticity and function. Macrophages in uveal melanoma have an M2-like phenotype and are associated with the loss of one specific chromosome - monosomy 3. The central players involved in this process and discussed include macrophages, T lymphocytes, chemokines and cytokines, including the macrophage-attraction molecules. When a tumor acquires the ability to release significant amounts of macrophage-attraction molecules it causes the expansion of a population of myeloid immature cells that may not only help the tumor to suppress immune reactions but also aid in the construction of new blood vessels for tumor growth. A better understanding of the molecular basis of a local myelomonocytic cell population will bring a better understanding of the immunopathology of this disease and will lead to therapeutic interventions in uveal melanoma. This thesis focuses on the roles of the local inflammatory microenvironment in the development and progression of uveal melanoma. Show less
