Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers.<p> Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported.<p> To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection.<p> Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.<p> The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.

In addition to their activity against intracellular pathogens, it is now clear that CD8+ T-lymphocytes also mediate anti-tissue responses. In order to manipulate these responses in the setting of tumor immunity or autoimmunity, it is necessary that we understand the parameters that promote CD8+ activation. In the first section of this thesis, a transgenic mouse model was used to explore the effectiveness of peptide/adjuvant-based and dendritic cell (DC)-based vaccination techniques at eliciting CD8-mediated anti-pancreatic responses. It was found that, while peptide vaccines were unable to stimulate autoimmunity, the transfer of DCs promoted autoimmune diabetes in a manner that was dependent upon the toll-like receptor (TLR)-based maturation of the DCs. Furthermore, the diabetes induction was dependent upon the engagement of the immunodominant CD8+ population and a second T-cell specificity, indicating that polyclonal responses may be required for effective tissue destruction. In the second section of this thesis, I explored the requirements for CD28-signaling during the activation of naïve self-reactive CD8+ T-cells. The transfer of mature DCs was insufficient to promote diabetes in CD28-deficient animals, whereas infection with lymphocytic choriomeningitis virus could induce diabetes in the same animals. Anti-tissue responses were further explored in tumor-bearing mice following DC transfer and demonstrated that a critical determinant of the induction of anti-tissue immunity in the absence of CD28-derived costimulatory signals, was the persistence of antigen presentation. In the final section of this thesis, I explored the role of nuclear factor kappa B 1 (NF-κB1) in DC maturation using the DC transfer model described above. Surprisingly, NF-κB1-deficient DCs were capable of inducing diabetes without the need for external stimulation. Furthermore, the absence of NF-κB1 in unstimulated DCs was associated with dysregulated production of tumor necrosis factor alpha (TNF-α), and this cytokine was required for the proper upregulation of the cytotoxic effector molecule granzyme B in CD8+ T-cells that infiltrated the pancreatic islets. This work therefore presents a novel model of autoimmune tissue destruction, in which defined genes and pathways that contribute to DC-T-cell interactions can be explored in an in vivo non-TCR transgenic setting.

The self-assembly behavior of arborescent polystyrene-<em>graft</em>-poly(ethylene oxide) copolymers (PS-<em>g</em>-PEO) at the air-water interface and the solubilization/release properties of arborescent polystyrene-<em>graft</em>-poly(2-vinylpyridine) (PS-<em>g</em>-P2VP) copolymers were investigated. These amphiphilic dendritic molecules are covalently bonded unimolecular micelles incorporating a highly branched hydrophobic polystyrene core surrounded by a hydrophilic poly(ethylene oxide) or poly(2-vinylpyridine) shell. Molecules of PS-<em>g</em>-PEO copolymers spontaneously formed supramolecular assemblies at the air-water interface. The type of superstructures formed was found to depend upon copolymer composition, while the level of association was more directly related to the branching density of the polymers. At low surface pressures the PEO segments apparently remained adsorbed on the water subphase, but desorbed into water at very high surface pressures, in the condensed monolayer state. Controlled degradation of the PEO chains with UV light greatly enhanced molecular association, resulting in the formation of either large clusters or long ribbon-like superstructures. The PS-<em>g</em>-P2VP copolymers were found to efficiently solubilize and release hydrophobic small molecules in aqueous media. The partition coefficient and solubilization capacity of the copolymers for hydrophobic polyaromatic hydrocarbons increased with the polystyrene content of the copolymers, while the rate of solubilization decreased with increasing branching functionality of the copolymers. The release profiles for two model drugs displayed an initial burst in release followed by gradual approach to equilibrium. The diffusion coefficients of the drugs in the micelles increased with the branching functionality and the generation number of the micelles, presumably due to increased electrostatic repulsions of the protonated vinylpyridine units.

Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers.<p> Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported.<p> To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection.<p> Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.<p> The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.

abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Natural killer (NK) cells are innate lymphocytes with inherent ability to eliminate infected cells and produce several cytokines/chemokines. They express surface receptors to sense environment and interact with other immune cells including the Dendritic cells (DC). Reciprocally, DCs are also shown to activate NK-cells. NK/DC cross-talk is well-documented, yet the molecular interactions and the diverse NK-cell activities regulated by DC remain unclear. Several target proteins such as MHC-1, Qa-1 mediate NK-cell target recognition. One such antigen, Ocil/Clr-b functions as a cognate ligand of NKR-P1B/D, NK-inhibitory receptor. In first aim of my study, I documented that deficiency of Ocil/Clr-b expression not only augmented the sensitivity of DC towards NK-cell cytotoxicity but also regulated the development of mature NK-cells. Thus suggesting NKR-P1B/D:Ocil to be another receptor:ligand system, besides Ly49:MHC-1, that regulates NK-cell responsiveness. Src homology region 2-containing protein tyrosine phosphatase-1 (SHP-1) transmits inhibitory signals of the specific NK-inhibitory receptors, including NKRP-1B/D. SHP-1 silenced NK-cells showed unaffected target recognition towards prototypic target cells in this study. In addition, these cells also displayed an unexpected phenotype of self-killing in-vitro, thus implicated SHP-1 as an important regulator of some other unappreciated NK-cell functions. The data from my third study suggest that DCs are directly implicated in the induction of NK-cell migration. In summary, using a novel live-cell imaging microfluidic platform and conventional transwell migration assay this project established a clear molecular link between DC-derived soluble factors such as IP-10 and NK cell-chemokine receptor such as CXCR3. Previously, GM-CSF was shown as an inflammatory cytokine, involved in the development of DC as well as in mediating Th-1 immune responses. In this study I found that GM-CSF regulates NK-cell migration negatively. Lastly, the fourth aim of my thesis highlighted the critical role of immature-DC in the induction of maturation receptors (NK1.1 & Ly49) on differentiating NK-cells. I successfully established a multi-stage in-vitro NK-cell differentiation model and found that differentiating NK-cells required an active engagement with DCs, in addition to the soluble factors. I believe my PhD project findings would impact the existing knowledge to harness DC-based NK cell therapies in clinical settings. / October 2014

Recently, there has been considerable interest in developing specific cell-based immunotherapies using dendritic cells (DCs). Here the mechanisms underlying the tolerogenic properties of DCs in the suppression of experimental autoimmune uveoretinitis (EAU), the mouse model of human sight threatening autoimmune uveitis, were examined. Immature DCs have the ability to promote immune tolerance to self antigens and to prevent the development of autoimmune disorders including EAU. However there is a risk that immature DCs placed in the inflammatory environment would undergo maturation and instead of tolerance promote immunity. Therefore much effort has been directed to develop protocols that stabilize the tolerogenic DC properties which would ultimately ensure safety and effectiveness of DC-based vaccines. Previous work demonstrated that activation of DCs with lipopolysaccharide (LPS) increases the ability of these cells to prevent EAU. Here, it was demonstrated that LPS promotes the activation of both TRIF and MyD88 signalling pathways in DCs which after 24 h LPS treatment secreted a significant amount of IL-10, IFN-β, IL-1β, IL-6 and TNF-α while the level of secreted IL-12 is low. It was further shown that LPSinduced enhancement of the tolerogenic properties of DCs correlates with the state of endotoxin tolerance in the DCs, rendering them refractory to further stimulation. It was hypothesised that the LPS-induced enhancement of DC tolerogenicity is due to the reduced expression of the TLR4, which subsequently disables several signalling pathways and prevent DCs from initiating adaptive T cell immunity. In summary, the presented data provides valuable insights into the mechanisms involved in the suppression of autoimmune uveitis using a DC-based therapy.

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Estrogen receptor (ER) involvement through genomic mechanisms have been well established in different aspects of cognition and neuronal plasticity. However, much less is known about the rapid, non-genomic effects on these processes. G-protein coupled estrogen receptor (GPER) is a relatively recently discovered estrogen receptor that has previously been shown to affect learning and memory on a long term genomic timescale. This thesis investigated the rapid effects of the GPER selective agonist G-1 on four estrogen-sensitive learning paradigms: social recognition, object recognition, object placement and social transmission of food preferences (STFP) paradigm. The rapid effects of G-1 on neuronal plasticity were also examined through changes in hippocampal dendritic spine length and density. Results show that GPER activation may rapidly enhance social recognition, object recognition and object placement learning while may impair social learning in the STFP paradigm. G-1 treatment also resulted in increases of dendritic spine density in the stratum radiatum of the CA1 hippocampus. This suggests that GPER, along with the classical ERs, mediates the rapid effects of estrogen on learning and neuronal plasticity. Thus, GPER may be a viable target for enhancing cognitive function in estrogen related therapies.

No description available.