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Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 induces B29-specific regulatory T cells, which suppressed experimental arthritis in mice by cross-recognition of their mammalian HSP70 homologs (1). The aim of this study is to characterize the B29 binding and specific CD4(+) T cell responses in the context of human MHC molecules (HLA). Binding of B29 peptide and its mammalian homologs to HLA molecules was examined with competitive binding assays. The effect of B29 immunization was assessed in proteoglycan-induced arthritis in HLA-DQ8 transgenic mice, followed by ex vivo restimulation with B29 to examine the T cell response. Human PBMC were used to investigate the presence of B29-specific T cells with immunoregulatory potential. We found a high to moderate binding affinity for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was functional, as B29 immunization resulted in suppression of arthritis and T cell responses in HLA-DQ8 transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4(+) T cells, which were cross-reactive with the mammalian homologs. With HLA-DR4(+) tetramers specific for B29 or mB29b we showed expansion of cross-reactive T cells, especially the human CD4(+) CD25(+) FoxP3(+) T cell population after in vitro stimulation with B29. These results demonstrated a conserved fine-specificity and functionality of the B29-induced regulatory T cell responses in the context of the human MHC. Based on these findings, a translational path of the B29 experimental findings into a clinical immunomodulatory therapeutic approach comes within reach. This article is protected by copyright. All rights reserved.

Major problems in the analysis of CD4+ effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+ effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+ T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+ T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis. © Society for Leukocyte Biology.

Summary This thesis can be roughly divided into two parts. In the first three chapters, we discussed and studied the role of Hsp70 in retinal pigment epithelial (RPE) cells, and the innate and adaptive immune response. We reviewed the relationship of T cell-mediated diseases and HSPs, and pointed out that the induction of Hsp70 may contribute to therapeutic tolerance (chapter 2). Then, we explored a new Hsp70 co-inducer, leucinostatin, and its role in canine RPE cells (chapter 3). Further, we studied the anti-inflammatory effects of Hsp70 in canine macrophages (chapter 4). In the last two chapters, we focus on various differently activated canine macrophage subsets originating from both primary monocytes and a monocyte-like cell line (030D cell) (chapter 5 and 6). We successfully polarized canine monocyte-derived macrophages (MDMs) into M1 and M2 cells and thoroughly characterized their features (chapter 5). Meanwhile, we demonstrated that 030D cells can be differentiated into M1 and M2 macrophages and that each subset shares the characteristics of the corresponding canine monocyte-derived macrophage subset.

More than a million animals are used annually in the European Union for regulatory batch testing of medicinal products, predominantly for potency assessment. With respect to inactivated poultry vaccines, most potency tests are still performed in vivo due to the lack of in vitro alternatives. It is important to move away from in vivo testing because of ethical concerns, variability, long duration and high costs of these tests, risks for personnel working with infected animals, and legal requirements to use in vitro methods instead of in vivo methods whenever possible. The primary aim of this thesis was to set up in vitro cell-based assays to assess the immunostimulatory capacity of inactivated poultry vaccines and to determine their applicability for routine potency testing. The studies presented in Chapters 2 and 3 of this thesis focus on the immunostimulatory properties of inactivated poultry vaccines by investigating their effect on chicken macrophages. The cell-based assays described involve the use of the macrophage-like cell line HD11. A phagocytosis assay with IgY-opsonized beads was used to investigate whether inactivated poultry vaccines against infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and egg-drop syndrome virus (EDSV) affect phagocytosis by macrophages. In addition, the use of nitric oxide production and cytokine gene expression are described to determine the immunostimulatory properties of an inactivated octavalent vaccine against IBV, NDV, EDSV and five strains of Avibacterium paragallinarum, the causative agents of infectious coryza characterized by an acute respiratory disease in chickens. The study presented in Chapter 4 describes a thorough morphological, phenotypical and functional characterization of chicken bone marrow-derived dendritic cell cultures. In Chapter 5, bone marrow-derived dendritic cells were exposed to an inactivated poultry vaccine against IBV and NDV to identify biomarkers of vaccine-induced immune responses by proteomic analysis. Furthermore, the mechanisms by which inactivated poultry vaccines activate dendritic cells were explored. Knowledge about the induction of T cell-mediated immunity by inactivated poultry vaccines is still limited. The study of Chapter 6 describes a T cell proliferation assay to evaluate cellular immunity in laying hens vaccinated with an inactivated vaccine against IBV and NDV and its potential as a quality test for this vaccine. In Chapter 7, a review describes the difficulties that are being encountered during the transition from in vivo to in vitro batch testing of vaccines for both human and veterinary use, as well as possibilities to overcome these difficulties. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic relevant aspects of in vivo vaccine-induced immune responses, the difficulties of adjuvanted vaccines and how to overcome these, the use of altered batches to validate in vitro test methods when non-compliant vaccine batches are limitedly available, and how cooperation between different stakeholders is key to move the transition forward. Finally, the findings of this thesis, as well as the next steps required for the transition in vitro potency testing of inactivated poultry vaccines, are discussed in Chapter 8.

During aging, the immune system undergoes changes that bring the tightly regulated immune system out of balance. The age-related changes to the immune system have collectively been termed ‘immunosenescence’. As a consequence of immunosenescence, the risk of acquiring infectious diseases and the course of disease is prolonged and more severe at old age. T cells are imperative for protection against pathogens and various molecules on different subsets of T cells are involved in maintaining a balanced immune system. T cells express molecules that initiate cellular activation and proliferation, but there are also molecules that act as a breaking mechanism on these responses to prevent over-activation. During aging, this system becomes dysregulated. To explain reduced response to vaccination, susceptibility to infectious disease, and increased disease severity observed at older age, it is crucial to identify the underlying age-related changes of T cells. In this thesis, we set out to identify novel age-related changes of the T-cell population in mice and humans that may contribute to understanding the impact of aging on T cells. We investigate T-cell phenotype, responses, and proliferation in both mice and humans under healthy conditions and during respiratory viral infection in humans. In mice and humans we found that the activation and proliferation of several T-cell subsets slow down at older age. These findings may serve as a new hallmark of T-cell aging and contribute to understanding the reduced response of T cells after vaccination or infection. Additionally, we report an upsurge of regulatory T-cell frequencies in older wildtype mice. Using a mouse model for the accumulation of DNA damage (Ercc1-deficient mice), we found that the accumulation of regulatory T cells with age may in part be explained by the accumulation of DNA damage with age. Furthermore, we show that T cells with regulatory functions also accumulate in humans. We discovered a novel CD8+ T-cell subset with immunosuppressive capacities, which we named KIR+RA+ T cells based on their expression of KIR and CD45RA. We found KIR+RA+ T cells to be highly activated in (older) individuals that are suffering from respiratory viral infection, including Influenza A virus and the novel SARS-CoV-2. Importantly, activation of KIR+RA+ T cells was associated with prolonged presence of symptoms in older adults infected with Influenza A virus, indicating that these regulatory KIR+RA+ T cells may aggravate respiratory disease following viral infection, possibly by suppressing anti-viral CD8+ T cells. Our findings highlight the importance of studying the altered response kinetics of T cells at older age and the rise of regulatory-cell features in the T-cell population, which both may contribute to impaired protective immunity against infectious diseases in older individuals. Ultimately, these fundamental findings may provide new starting points for other fundamental, translational, and clinical studies that focus on vaccination of older adults, infectious disease resistance, and healthy aging.

This thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and atherosclerosis. For each of these treatment strategies, the liposomal formulation was tailored to obtain the desired therapeutic effect. Chapter 2 reviews some of the most important physicochemical properties (size, shape, and rigidity) that determine the immunological effects of liposomes and other nanoparticles. In chapter 3 we present a detailed study on the effect of the rigidity of anionic liposomes, as measured by atomic force microscopy, on antigen-specific regulatory T-cell (Treg) responses. In chapter 4, we show that our optimized anionic liposomes can induce potent antigen-specific Treg responses, and can be used to delay atherosclerosis progression in a mouse model. Chapter 5 also focuses on liposomal treatment of atherosclerosis, but here targeted liposomes were prepared to successfully deliver a small molecule to foam cells in atherosclerotic plaques. In Chapter 6, we used cationic liposomes in combination with an adjuvant for cancer immunotherapy in mice. Finally, we summarize the overall findings in chapter 7 and discuss perspectives of using liposomes for vaccination and targeted drug delivery.

Antibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render tuberculosis (TB) untreatable. Efforts to develop novel antibiotics have so far been unsuccessful, calling for additional approaches for treatment of bacterial infections. Intracellular pathogens like Mtb and Salmonella can survive in the host by manipulating host cell signaling. This provides opportunities for novel therapeutic strategies by targeting the host, rather than the bacterium (host-directed therapy). In this thesis we report the development and application of novel (in vitro and in vivo) methods for identifying host genes and proteins involved in host control of intracellular bacteria, as well as chemical compounds that target host molecules as a basis for drug development for host-directed therapies. As a result, we report the identification of RTK inhibitors, the novel kinase inhibitor 97i, the human kinase family PCTAIRE and the host protein DRAM1 as promising leads for further drug development for host-directed therapeutic strategies for intracellular bacterial infections.