Viral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been... Show moreViral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8+ T-cells in the atherosclerotic lesion.\nThe presence, clonality, tissue enrichment, and phenotype of virus-associated CD8+ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-β sequencing and single-cell T-cell receptor (α and β) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8+ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined.\nVirus-associated CD8+ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P=0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8+ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8+ T-cells were phenotypically highly similar to other CD8+ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8+ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion.\nThis study suggests that virus-specific CD8+ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms. Show less
One of the main challenges in the development of an effective anti-cancer vaccine is the generation of an adequate and directed cellular immune response. Antigen presenting cells play an important... Show moreOne of the main challenges in the development of an effective anti-cancer vaccine is the generation of an adequate and directed cellular immune response. Antigen presenting cells play an important role in obtaining such immune responses as they express pathogen recognition receptors (PRRs), for example Toll-like receptors (TLRs) and Nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), and Fc receptors. This enables them to recognize pathogen associated molecular patterns (PAMPs). A promising strategy in immunotherapy is the use of PRR ligands or antibody-recruiting molecules (ARMs) that are covalently bound to an antigenic peptide. The research in this Thesis describes the design and synthesis of new carbohydrate ligands and conjugates for TLR4, NOD2 and mannose-6-phosphate receptor (MPR) in which these ligands are covalently bound to antigenic peptides. In the second part of this Thesis, Fc receptors are exploited as they bind to an immune complex, which is formed by binding of an antibody to an ARM. Therefore several C-glycosyl lysine building blocks are designed and synthesized of which C-rhamnose was conjugated several times to an antigenic peptide. Show less
The two branches of our highly advanced immune system work closely together to detect and eliminate pathological threats. The first line of defense is provided by the innate immune system via... Show moreThe two branches of our highly advanced immune system work closely together to detect and eliminate pathological threats. The first line of defense is provided by the innate immune system via detection of pathogenic or tumor cell fragments. Adaptive immune cells, on the other hand, recognize pathogens or malignant cells more specifically by scanning peptides, small protein fragments, presented by MHC molecules on other cells in our body. Specialized white blood cells (cytotoxic or killer T cells) can distinguish self- from non-self-peptides and directly eliminate cells that display signs of infection or mutation.The work described in this dissertation highlights how adaptive immunity can be used to our advantage, either from a therapeutic or diagnostic perspective. Immunotherapies that induce or promote anti-tumor or anti-viral responses have proven efficacious against infection and cancer. One strategy described is the development of chemically-modified epitopes as peptide vaccines, but small-molecule chemical drugs are also playing an increasing role in the field of cancer immunotherapy.In addition, monitoring of immune status and response to treatment, as well as mapping of epitopes, can aid diagnosis and design of treatment plans. The second part describes a novel method and application to visualize and monitor cytotoxic T cells. 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
Duchenne muscular dystrophy (DMD) is a severe progressive muscle wasting disorder. DMD is caused by reading frame disrupting mutations in the DMD gene resulting is an absence of the dystrophin... Show moreDuchenne muscular dystrophy (DMD) is a severe progressive muscle wasting disorder. DMD is caused by reading frame disrupting mutations in the DMD gene resulting is an absence of the dystrophin protein. Dystrophin is an important muscle protein as it provide stability upon muscle fiber contraction. Currently there is no therapy for the majority of the DMD patients. As part of the standard of care patient receive symptomatic treatment e.g. corticosteroids, respiratory and cardiac support. Various therapeutic approached are currently under development. Most advanced therapeutic approach is aimed to restore dystrophin production by using antisense oligonucleotides (AON): exon skipping. This thesis focusses on delivery of AON to skeletal and cardiac muscle for DMD. With the help of phage display technology combined with next generation sequencing analyses, muscle homing peptides have been identified. In this thesis is described how for the first time these homing peptides upon conjugation to a 2OMePS AON resulted in increased delivery and exon skipping in a mouse model for DMD. In Conclusion, muscle homing peptides have the potential to facilitate delivery of AONs and perhaps other compounds to skeletal and cardiac muscle. Show less
For decades a large amount of research has dealt with membrane interactions of peptides and proteins as well as peptide-peptide interactions to understand the mechanisms of essential biological... Show moreFor decades a large amount of research has dealt with membrane interactions of peptides and proteins as well as peptide-peptide interactions to understand the mechanisms of essential biological processes such as protein-driven vesicle budding and fission, cell penetration and lysis by peptides, and of course protein-driven membrane fusion. The advance of these fields, in combination with recent progress in cell biology, has inspired chemists to mimic these biological processes with simple model systems. However, it becomes apparent that these model systems are more complex than initially thought and the lessons that were learned from natural systems can also be applied here. The work reported in this thesis applied and extended classical methods for the study of peptide-peptide and peptide-membrane interactions to study the properties of the fusogenic coiled-coil forming lipopeptides in different membrane model systems or in solution. Hypotheses were constructed and tested based on the current biochemical and biophysical models of natural membrane fusion. Taken together, the results reported here led to a new perspective on lipopeptide mediated vesicle fusion. Show less
Fusion of lipid bilayers in cells facilitates the active transport of chemicals. Non-viral membrane fusion is regulated by a cascade of proteins as the process is highly regulated both in space and... Show moreFusion of lipid bilayers in cells facilitates the active transport of chemicals. Non-viral membrane fusion is regulated by a cascade of proteins as the process is highly regulated both in space and time. In eukaryotic cells, the so-called SNARE protein complex is at the heart of fusion. However, little is known about the actual mechanism at the molecular level. Inspired by the SNARE protein complex, our group previously developed a model system composed of a pair of lipidated complementary coiled coil peptides enabling targeted liposome-liposome fusion. This model system possesses all the key characteristics of membrane fusion similar to SNARE mediated fusion. The tetrameric coiled-coil of SNAREs is mimicked by a complementary pair of coiled coil forming peptides composed of three heptad repeat units (denoted __coil-E__ and __coil-K__). A flexible poly(ethylene glycol) spacer is conjugated to the N-terminus ensuring rotational freedom of the peptides. Lipidation warrants the anchoring of the peptides in the membrane by means of a phospholipid anchor (DOPE), mimicking the transmembrane domain of SNAREs. In order to develop future applications of this model system, the mechanism of membrane fusion needs to be studied in more detail and this has been the goal of this thesis. Show less
Protein-protein interactions are essential for various biological processes including cell metabolism, muscle contraction, and signal transduction. The dissertation describes a study of the... Show moreProtein-protein interactions are essential for various biological processes including cell metabolism, muscle contraction, and signal transduction. The dissertation describes a study of the interaction between the proteins cytochrome c and cytochrome c peroxidase by electron paramagnetic resonance spectroscopy (EPR). A spin label containing an unpaired electron was placed at the surface of one of the proteins. The combination of spin labelling and EPR provided novel information on the structure and dynamics of the proteins. Show less
The mammalian immune system protects, amongst others, against invading pathogens and consists of an innate and adaptive component. The innate system is the first line of defense in which pattern... Show moreThe mammalian immune system protects, amongst others, against invading pathogens and consists of an innate and adaptive component. The innate system is the first line of defense in which pattern recognition receptors, like TLR2, NOD1 and NOD2 receptors, detect pathogen associated molecular patterns (PAMPS) that are specific for pathogens. PAMPs exhibit a broad structural variety and the exact molecular structures of ligands that bind to the corresponding PRRs are mostly unknown. The research presented in this Thesis is directed to the design, synthesis and immunological evaluation of new NOD1, NOD2 and TLR2 ligands as well as conjugates in which these ligands are covalently bound to an antigenic OVA-derived peptide. The designed NLR and TLR ligand-antigen conjugates contribute to the insight of the processes of the mammalian immune system at molecular level. Further elucidation of pattern recognition receptor acti vation and antigen presentation with these conjugates may eventually result in synthetic vaccine modalities. Show less
Dit proefschrift beschrijft de ontwikkeling van nieuwe methoden voor de synthese van hybride biomoleculen die samengesteld zijn uit een peptide- en een nucle_nezuurfragment. Zulke hybride moleculen... Show moreDit proefschrift beschrijft de ontwikkeling van nieuwe methoden voor de synthese van hybride biomoleculen die samengesteld zijn uit een peptide- en een nucle_nezuurfragment. Zulke hybride moleculen komen in de natuur voor en hebben belangrijke functies. In dit proefschrift wordt aandacht besteed aan twee klassen van hybride biopolymeren; de klasse van nucleoproteinen, die een belangrijke rol speelt in virale replicatie processen en de klasse van ADP-geribosyleerde eiwitten, die het resultaat zijn van een post-translationeel modificatie proces. Show less
This Thesis aims at the development of novel subunit selective inhibitors of the proteasome. -Three vinyl sulfone analogues of three epoxyketone containing inhibitors described in literature are... Show moreThis Thesis aims at the development of novel subunit selective inhibitors of the proteasome. -Three vinyl sulfone analogues of three epoxyketone containing inhibitors described in literature are synthesised and characterised. The nature of the electrophile determines subunit selectivity of the inhibitor. -Ten Michael acceptors are coupled to three peptoid tails yielding thirty peptide-like compounds. None of the peptoid Michael acceptors are efficient proteasome inhibitors. Using the Staudinger-Bertozzi ligation followed by affinity purification, tryptic digestion of the isolated proteins and LC/MS/MS identification, the targets of two compounds in HEK293T and RAW264.7 cells are determined. -Syringolins are potent proteasome inhibitors found in nature. A syringolin structural motif was built in peptoid epoxyketones and vinyl sulfones yielding a 16-membered library of proteasome inhibitors. The distance between the urea and electrophile dictates subunit selectivity. -Three cyclooctynes are compared to the Staudinger-Bertozzi two-step labelling strategy. Cyclooctynes quantitatively convert azide labelled proteasomes at a lower concentration than the Staudinger-Bertozzi phosphane but cyclooctyne two-step labelling is associated with tremendous background labelling. -A library of peptoid vinyl sulfones with basic amino acid side chains is synthesised. A compound with two benzylamine residues is a potent and _2 selective proteasome inhibitor. Attachment of a BODIPY fluorophore yielded a _2 selective probe. Show less
The minor histocompatibility (mH) antigen HA-1 is expressed by cells of hematopoietic origin only. The sequences of the immunogenic CTL epitope VLHDDLLEA (HA-1H) and the VLRDDLLEA (HA-1R)... Show moreThe minor histocompatibility (mH) antigen HA-1 is expressed by cells of hematopoietic origin only. The sequences of the immunogenic CTL epitope VLHDDLLEA (HA-1H) and the VLRDDLLEA (HA-1R) counterpart differ by one aminoacid. Selectively infusing HA-1H specific donor cytotoxic T cells may mediate a strong GvL effect with a low risk for GvHD. However, this HA-1H specific immunotherapy is currently feasible only for HLA-A2 HA-1HH or HA-1HR patients who relapsed following an SCT from an HLA-A2 HA-1RR donor. In addition, the therapy is not guaranteed for all of these patients. For instance, the anonymous umbilical cord blood (UCB) donors cannot be traced again for use of DLI or adoptive immunotherapy following transplantation. Moreover, the success rate of HA-1H specific HLA2 restricted CTL induction is donor dependent. This thesis describes the following important features, which may lead to extension of the patient population that may benefit from HA-1 specific immunotherapy: - The instable natural presentation of HA-1R in HLA-A2. - A novel HLA-B60 restricted HA-1H epitope. - Hematopoietic-specific CTLs from UCB directed against HA-1H in the context of HLA-A2. - HA-1 specific TCR transfer directing non- HA-1 TCR expressing adult and UCB CD8+ T cells to hematopoietic-specific cytolytic activity. Show less
