Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Bacterial DNA is immunostimulatory, and the motifs responsible for this activity are unmethylated CpG dinucleotides. Following cellular uptake, CpG-containing oligodeoxynucleotides (CpG ODN) are trafficked to the endosome where they bind Toll-like receptor 9 (TLR9) to initiate a signaling cascade that culminates in the release of numerous pro-inflammatory cytokines. Because of their immunostimulatory properties, CpG ODN are being clinically evaluated as treatments and vaccine adjuvants for infectious diseases, cancer, and allergic disorders. α2-Macroglobulin (α2M) is a human plasma protein that binds and modulates the activity of a variety of cytokines, growth factors, enzymes, and antigens. Upon proteolytic activation, α2M is converted to its receptor recognized form, α2M*, and rapidly binds to and is internalized by immune competent cells expressing the α2M* endocytic receptor, LRP, and is then trafficked to the endosome. Based on these interactions, α2M seems to play an important role at sites of infection and inflammation by controlling the level of proteinase activity, modulating cytokine signals, and enhancing antigen processing for the adaptive immune response. Here, we report the first evidence that α2M* binds and forms stable complexes with nucleic acids. We have characterized the mechanisms and stoichiometry of this interaction, examined the pH and temperature stability of these complexes, and identified structural variables in the nucleic acids, namely length, base composition, and chemical modifications, that affect the nature of this interaction. We hypothesized that CpG ODN incorporation into α2M* may alter their immunostimulatory properties. Murine macrophages (MΦs) treated with α2M*-ODN complexes respond more rapidly and produce a greater cytokine response than those treated with free CpG ODN alone. Treating human PBMCs with α2M*-ODN complexes likewise demonstrated their enhanced ability to elicit immune responses. This was due to more rapid uptake and CpG ODN protection from degradation by extracellular nucleases. Co-incorporation of both protein ligands and CpG ODN into α2M* yields ternary complexes; these may permit the simultaneous delivery of both protein antigens and adjuvants to immune competent cells, potentially greatly enhancing the adaptive immune response and protective immunity. Based on the findings that incorporation into α2M* confers enhanced immunostimulatory activity of CpG ODN, this technology may be exploited to improve CpG ODN-based therapeutics by increasing efficacy, minimizing side effects, reducing dosing requirements, and reducing cost. / Dissertation

Corals are marine animals from the class Anthozoa and are widely distributed in tropical and subtropical seawaters. They are considered as an important source of lead compounds for drug discovery. For evaluating the medicinal activities of briarane-type diterpenoids (BrDs) from marine coral Briareum excavatum, the regulation of a group of briarane-type diterpenoids (BrDs) on dendritic cell (DC) function, TPA-induced dermatitis and type 1 diabetes was investigated. The results show that the BrD excavatolide K (BrD2) remarkably suppressed the activation of human DCs, especially the expression of IL-12 p40. This inhibitory effect was mediated apparently by interference with the rictor-mTOR/Akt-mediated signaling network, resulting in persistent-phase activation of NF-kB and Erk1/2 signalings. In addition, the 8,17-epoxide of BrDs was observed to play a crucial role in inhibition of IL-12 p40 expression. Replacement of the C-12 hydroxyl group with longer esters in BrDs gradually decreased this inhibitory activity in human DCs. BrD excavatolide B (BrD1) effectively suppressed the capacity of mouse bone marrow-derived DCs to induce an antigen-specific Th1, response via the inhibition of IL-12 expression. Moreover, excavatolide B prevented the onset of autoreactive T cell-mediated diabetes in NOD/SCID mice. Furthermore, excavatolide B remarkably suppressed TPA-induced vascular permeability and edema in test skin tissues. At the biochemical level, excavatolide B inhibited TPA-induced expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase-9, the key indicators of cutaneous inflammation. This inhibition is apparently mediated by interference with the Akt/NF-kB-mediated signaling network. Together, these studies demonstrate that BrDs from specific marine corals can effectively regulate defined molecular and cellular functions of dendritic cells, suppress TPA-induced dermatitis, and prevent type 1 diabetes in murine models suggesting that BrDs may warrant further investigation as natural immunomodulatory agents or therapeutics.

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

Dendritic Cells (DCs) are professional antigen presenting cells that play a crucial role in the induction of immune tolerance. Although DCs have been a potential target for immunotherapy, the amount of DCs in blood source is limited and ex vivo expansion has been inefficient. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide a great source in cell-based therapy because of their self-renewal ability and pluripotency. My project focuses on generating tolerogenic DCs (tDCs) from human pluripotent stem cells (i.e. hESCs and iPSCs) and their characterization. Specifically, hESCs and hiPSCs were first differentiated to hematopoietic progenitor cells (HPCs) using three different methods (i.e. bone-marrow stromal cell co-culture and two previously reported defined medium methods). The hESC/iPSC-differentiated hematopoietic progenitor cells (HPCs) were characterized by their surface phenotype using flow cytometry. Then the hESC/iPSC-differentiated immature DCs were further expanded and differentiated from the hESC/iPSCdifferentiated CD34+ HPCs with the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4). Tolerogenic properties were introduced by treating hESC-differentiated DCs with rapamycin. The treated DCs were characterized for their tolerogenicity by examining their expression of PDL1, PDL2, ICOS and CD40 etc., and their ability to promote regulatory T cells (Treg) differentiation. All these were compared with monocyte-derived tDCs. In summary, this study has examined the potential of using pluripotent stem cells-derived DCs as a cell source for immune tolerance induction therapy. / published_or_final_version / Anatomy / Master / Master of Philosophy

No description available.

Nucleotides, such as ATP and its derivatives, are released at high concentrations at sites of inflammation and modulate the immune response. When cultured in the presence of ADP or stable analogue ADP?S, DC surface expression of MHC-II and co-stimulatory molecules, CD40 and CD86 was unchanged. When DCs were pre-treated with ADP or ADP?S, there was no change in their ability to activate naïve CD4+ T cells. However, when CD4+ T cells were activated in the presence of ADP or ADP?S, activation and proliferation were significantly decreased. This correlated with a significant reduction in IL-2 secretion and CD25 surface expression, which may be due to decreased ERK and Akt phosphorylation. CD8+ T cell proliferation was unaffected by the addition of ADP or ADP?S, but secretion of IFN-? was significantly reduced. By demonstrating that ADP inhibits CD4+ T cell responses, we have identified a potential target of immune modulation by clinical intervention.