The research described in this thesis focused on the use of bioorthogonal antigens to investigate immunological processes in antigen presenting cells. Bioorthogonal antigens are antigenic proteins... Show moreThe research described in this thesis focused on the use of bioorthogonal antigens to investigate immunological processes in antigen presenting cells. Bioorthogonal antigens are antigenic proteins produced through recombinant expression in a methionine auxotrophic E. coli strain. This allows for the replacement of methionine residues with the bioorthogonal non-canonical amino acid, azidohomoalanine (Aha), that resembles methionine. Aha contains an azide group that enables the selective and rapid visualization or enrichment of the antigen after a biological experiment using alkyne-modified fluorophores or alkyne-containing resins, respectively, via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The research involved studying the effects of post-translational modifications (PTMs), antigen complexation and glycosylation of antibodies in immune complexes on the uptake, proteolysis, and T cell activation by dendritic cells (DCs) of Aha-containing antigens. Additionally, a new method was developed to enrich low abundant bioorthogonal antigenic fragments from complex mixtures. This method can be used in future studies to identify processed Aha-containing fragments from immune cells that are preserved for T cell presentation. Show less
Throughout this thesis, I have endeavored to apply an engineer’s mindset in my pursuit to better understand the marvelously convoluted immune system. In doing so, my colleagues and I have generated... Show moreThroughout this thesis, I have endeavored to apply an engineer’s mindset in my pursuit to better understand the marvelously convoluted immune system. In doing so, my colleagues and I have generated a number of new ‘hardware’(i.e., genetically engineered) tools and ‘software’ modules (i.e., custom analyses and models) that enable the investigation of several otherwise difficult-to- study concepts. Although we have used these modules here to study immune responses, I hope they may be utilized as tools and approaches to crack outstanding questions in other fields of research. 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
Bovenkamp, F.S. van de; Dijkstra, D.J.; Kooten, C. van; Gelderman, K.A.; Trouw, L.A. 2021
C1q is the recognition molecule of the classical pathway of the complement system. By binding to its targets, such as antigen-bound immunoglobulins or C-reactive protein, C1q contributes to the... Show moreC1q is the recognition molecule of the classical pathway of the complement system. By binding to its targets, such as antigen-bound immunoglobulins or C-reactive protein, C1q contributes to the innate defense against infections. However, C1q also plays several other roles beyond its traditional role in complement activation. Circulating levels of C1q are determined in routine diagnostics as biomarker in several diseases. Decreased C1q levels are present in several autoimmune conditions. The decreased levels reflect the consumption of C1q by complement activation and serves as a biomarker for disease activity. In contrast, increased C1q levels are present in infectious and inflammatory diseases and may serve as a diagnostic biomarker. The increased levels of C1q are still incompletely understood but are suggested to modulate the adaptive immune response as C1q is known to impact on the maturation status of antigen-presenting cells and C1q impacts directly on T cells leading to decreased T-cell activity in high C1q conditions. In this review, we provide a comprehensive overview of the current literature on circulating levels of C1q in health and disease, and discuss how C1q can both protect against infections as well as maintain tolerance by regulating adaptive immunity. Show less
Persistent infections with high-risk type human papillomaviruses (hrHPVs) can progress to cancer. HrHPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years... Show morePersistent infections with high-risk type human papillomaviruses (hrHPVs) can progress to cancer. HrHPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years despite the fact that KCs are well equipped to detect and initiate immune responses to invading pathogens. HrHPV interferes with the innate immune response by affecting several signaling pathways that otherwise would prompt anti-viral mechanisms in the host cell. Furthermore, hrHPV interferes with the production of cytokines that are involved in the attraction of immune cells to the infected epithelium. In addition, hrHPV hides itself from the immune system by suppressing the antigen presentation machinery and employs means to hamper the response of KC__s to signals from adaptive immune cells. In this thesis we show that hrHPV attenuates innate immune signaling (Chapter 2) and CD40-mediated (Chapter 3) and IFN_ and/or TNF_-induced (Chapter 4) adaptive immune signaling. HrHPV exploits the cellular proteins UCHL1 (Chapter 2) and IFRD1 (Chapter 4) that act on multiple points in the IRF and NF_B signaling pathways. Moreover, hrHPV downregulates cellular IFITM1 to resist the growth inhibitory effects of IFN_ and/or TNF_ (Chapter 5). Our data provide important new insights on how hrHPV can persist in the face of host immunity. Show less
HPVs need to avoid immune responses of the host in order to establish persistent infection. HPVs achieve this by dampening innate immunity of keratinocytes, the major cell type targeted by HPV. As... Show moreHPVs need to avoid immune responses of the host in order to establish persistent infection. HPVs achieve this by dampening innate immunity of keratinocytes, the major cell type targeted by HPV. As there is reduced production of danger signals including antimicrobial molecules, proinflammatory cytokines and chemokines by keratinocytes, HPV infection remains undetected by the immune system. However, our further data showed that PRR signaling is not completely blocked by hrHPV. Thus, the activation of innate and adaptive immunity at the site of HPV infection is slowed down but not prevented. In order for cancers to grow out they need to suppress the local effector cells. We focused on the role of the PD-1 receptor and its ligands PD-L1 and PD-L2, our data showed that the majority (81%) of the tumors from cervical cancer patients do not express PD-L1. Furthermore, PD-L1 expression was not associated with patient survival. Finally, we presented evidence that the chemokine receptor CXCR7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients. Show less
