No description available.

Dendritic cells (DCs) in different compartments can affect tolerance via distinct mechanisms. Thus, retinoid acid (RA) and integrins expressed by CD103+ dendritic cells in the gut play important roles in regional regulatory T cell induction and trafficking, while IL-10 expression by lung-associated tolerogenic dendritic cells is integral to tolerance in that compartment. Whether RA- and IL-10-differentiated DC (DCRA and DC10, respectively) can reciprocally induce tolerance in either compartment remains largely unexplored. We have shown that DC10 induce asthma tolerance in part by activating CD25+Foxp3+ Treg, but also by recruiting other cells (e.g., endogenous pulmonary DC) into an infectious tolerance pathway. Herein we began to assess whether DCRA can be equally tolerogenic, and whether they employ similar mechanisms, in an OVA/alum mouse model of asthma. On FACS analysis, we found that DCRA expressed significantly higher levels of CD40, CD86 and MHC II than DC10 (i.e., at levels equivalent to fully mature DC). DCRA secreted higher levels of TGF-β1 and IL-27 than DC10, but equivalent levels of IL-10. DCRA and DC10 suppressed in vitro Th2 response, but DCRA were more effective than DC10 at suppressing proliferation. Both DCRA and DC10 increased expression of Foxp3+ on effector T cells. DCRA promoted little expansion of Foxp3+ T cells. In contrast, DC10 promoted expansion of Foxp3+ T cells. Treatment of asthmatic mice with DC10 and DCRA reduced airway hyperresponsiveness and serum allergen-specific IgE and IgG1 levels. We previously showed that DC10-induced tolerance is critically dependent on their expression of IL-10. The results of this study showed that both DCRA and DC10 induce tolerance in asthmatic mice through different mechanisms.

Bovine herpesvirus-1 (BoHV-1) causes recurrent respiratory and genital infections in cattle; and predisposes them to lethal secondary bacterial infections. Vaccination is a primary strategy to prevent and reduce the severity of disease associated with BoHV-1, and to reduce virus transmission. While modified live (MLV) or killed (KV) BoHV-1 vaccines exist, these are expensive to produce, can cause disease (MLV) or may be ineffective (KV). Development of a DNA vaccine for BoHV-1 has the potential to address these shortcomings, but the very small amount of antigen expressed after DNA immunization presents a barrier to successful immunization of large animals. Engineering the vaccine to target this limited quantity of antigen to dendritic cells (DCs), the cells that prime immune responses, by attracting immature DCs (iDCs) to the vaccination site, is one way that DNA vaccine efficacy might be improved. Beta (β)-defensins are chemotactic peptides that, in studies with mice, improve induction of immune responses to DNA vaccines and this is due, at least in part, to their ability to attract iDCs to the site of vaccination. Accordingly, the objective of the studies described in this thesis was to determine whether using a bovine β-defensin in a DNA vaccine would enhance immune responses to the vaccine and subsequently protect cattle upon challenge with BoHV-1. First I characterized the bovine iDC and then used these cells to screen a panel of synthesized bovine β-defensins for chemotactic activity. The results showed that bovine neutrophil β-defensin (BNBD) 3, BNBD9 and enteric β-defensin (EBD) were equally the most chemotactic of the fourteen synthesized peptides for bovine iDCs. Because BNBD3 is the most abundant of the thirteen BNBDs and was able to attract CD1+ DCs when injected into the skin, I chose BNBD3 as the peptide I would use for the rest of the project. Next I constructed plasmids that expressed BNBD3; either alone or as a fusion construct with the BoHV-1 antigen truncated glycoprotein D (tgD), and then tested the effects of the plasmids as vaccines in both mice and cattle. In cattle, the addition of BNBD3 as a fusion strengthened the Th1 bias and increased cell-mediated immune responses to the DNA vaccine but not antibody response or protection from BoHV-1 infection. Given that inefficient humoral immune responses have been implicated in a lack of protection from BoHV-1 challenge, these results suggested that the successful BoHV-1 DNA vaccine would need to induce a much stronger humoral response. Lastly I assessed the ability of BNBD3 to improve humoral responses to pMASIA-tgD when complexed with the DNA vaccine and found that the vaccine complexed at a nanomolar peptide to DNA ratio of 125:1 increased humoral responses of mice. In vitro, treatment of mouse bone-marrow DCs with BNBD3 induced phenotypic and functional maturation/activation. This is an important aspect for vaccination in the skin, since after uptake, the DC must “mature” in order to traffic from the site of vaccination to the draining lymph node where induction of antigen-specific responses, by activated DCs, takes place. The findings in this thesis show that bovine β-defensins are chemotactic for bovine iDCs. I also show that using a bovine β-defensin as a fusion construct in a DNA vaccine enhances cell mediated but not humoral responses of cattle and yet this vaccine is protective against BoHV-1 challenge. I demonstrate that a bovine β-defensin, when used as a peptide to complex an antigen-encoding plasmid, can increase humoral responses. My work shows a multifunctional ability of bovine β-defensins to modulate and increase immune responses and suggests that bovine β-defensins likely have further untapped potential to enhance efficacy of DNA vaccines for large animals.

Mature dendritic cells (DC) isolated from lymphoid tissues initiate antigen-specific T-dependent responses even though they are non-phagocytic and weakly pinocytic, whereas Langerhans cells (LC; immature DC) can process protein antigens but are poorly immunostimulatory. Thus antigens may be acquired by cells of this lineage at an immature stage but, to our knowledge, there have been no studies on the phagocytic capacity of these cells in vitro. Using a newly-developed flow cytometric assay to measure the association between fluorescent markers and LC in epidermal cell cultures, and light and electron microscopy, we have observed phagocytosis of a variety of particles by freshly-isolated LC. The cells readily phagocytosed zymosan, heat-killed S. cerevisiae, bacteria (S. aureus and C. parvum) and fluorescent latex beads, but were unable to take up IgG- or complement-coated sheep erythrocytes, as opposed to MØ. Similarly, many freshly-isolated splenic DC had some phagocytic activity. However, the capacity of both LC and splenic DC to phagocytose zymosan, bacteria and fluorescent latex beads was markedly decreased after maturation in culture, consistently with the fact that mature DC are poorly phagocytic. Zymosan binding and uptake were much greater in fresh LC from C57BL/6 compared to BALB/c mice, and the loss of phagocytic capacity for zymosan during maturation followed different kinetics in the two strains. Two receptors mediating uptake of zymosan in LC were identified based on the effect of different inhibitors. Both of these receptors, recognising mannose and β-glucan residues, appear to be differentially regulated in the two mouse strains and during culture of LC. Our findings support the notion that DC are capable of acquiring particulate antigens for presentation at an immature stage, through recognition units for carbohydrate determinants common to a variety of potentially pathogenic organisms.

No description available.

Dendritic cells (DC) are critical to an immune response by functioning as sensors of foreign antigen and presenting antigen to direct T cell responses. Under the influence of Flt3 ligand this heterogeneous group of cells originate from hematopoietic stem cells (HSCs) in the bone marrow and takes up residence in lymphoid organs, skin as well as mucosal surfaces where they are most likely to encounter pathogens. GM-CSF is another cytokine involved in DC development that is specifically involved in the maintenance of skin resident DC. Together Flt3L and GM-CSF as well as their respective receptors, Flt3 and GM-CSFR, are the most important factors identified to date for maintenance of DC homeostasis. The Src-like adaptor proteins, SLAP and SLAP-2, are negative regulators of antigen receptor signaling. SLAP and SLAP2 contain SH3 and SH2 protein interaction modules that facilitate interaction with proline-rich sequences and phosphotyrosine motifs, respectively. Through a unique C-terminal region, SLAP and SLAP2 also interact with the E3 ubiquitin ligase, c-Cbl. SLAP and SLAP2 enhance c-Cbl-mediated ubiquitylation and down-regulation of antigen receptors by binding both activated receptors and c-Cbl. SLAP and SLAP2 have also been shown to function as negative regulators of receptor tyrosine kinases (RTKs) and our group has shown that SLAP and SLAP2 bind to and inhibit the colony-stimulating factor-1 receptor (CSF-1R). In the work presented here, we identify a novel role for SLAP in regulation of the GM-CSF receptor of bone marrow (BM)-derived DC. We show that potentiated GM-CSF signaling, in the absence of SLAP and SLAP2, impairs BM-DC maturation such that these cells express minimal MHCII, secrete low amounts of IL-12 and are functionally impaired in their ability to stimulate T cell responses. SLAP and SLAP2 deficiency also has an effect on Flt3L-derived BM-DC development. For example, SLAP/SLAP2-/- BM-DC numbers are reduced in the presence of Flt3L as compared to wild-type BM-DC. To investigate the mechanism of reduced DC numbers, we examined splenic DC and found that DC numbers were similar in wild-type and SLAP/SLAP2-/- mice. In fact, SLAP/SLAP2-/- mice had proportionally more CD8α+ splenic DC in vivo than wild-type mice. Thus there may be cytokines affected by SLAP/SLAP2-deficiency in our cultures that are either dispensable or compensated for in vivo DC development. The work presented in this thesis has implications for the role of SLAP and SLAP2 in immune response to infections by the regulation of GM-CSFR and Flt3 in maintaining dendritic cell homeostasis.

Dendritic cells (DC) are critical to an immune response by functioning as sensors of foreign antigen and presenting antigen to direct T cell responses. Under the influence of Flt3 ligand this heterogeneous group of cells originate from hematopoietic stem cells (HSCs) in the bone marrow and takes up residence in lymphoid organs, skin as well as mucosal surfaces where they are most likely to encounter pathogens. GM-CSF is another cytokine involved in DC development that is specifically involved in the maintenance of skin resident DC. Together Flt3L and GM-CSF as well as their respective receptors, Flt3 and GM-CSFR, are the most important factors identified to date for maintenance of DC homeostasis. The Src-like adaptor proteins, SLAP and SLAP-2, are negative regulators of antigen receptor signaling. SLAP and SLAP2 contain SH3 and SH2 protein interaction modules that facilitate interaction with proline-rich sequences and phosphotyrosine motifs, respectively. Through a unique C-terminal region, SLAP and SLAP2 also interact with the E3 ubiquitin ligase, c-Cbl. SLAP and SLAP2 enhance c-Cbl-mediated ubiquitylation and down-regulation of antigen receptors by binding both activated receptors and c-Cbl. SLAP and SLAP2 have also been shown to function as negative regulators of receptor tyrosine kinases (RTKs) and our group has shown that SLAP and SLAP2 bind to and inhibit the colony-stimulating factor-1 receptor (CSF-1R). In the work presented here, we identify a novel role for SLAP in regulation of the GM-CSF receptor of bone marrow (BM)-derived DC. We show that potentiated GM-CSF signaling, in the absence of SLAP and SLAP2, impairs BM-DC maturation such that these cells express minimal MHCII, secrete low amounts of IL-12 and are functionally impaired in their ability to stimulate T cell responses. SLAP and SLAP2 deficiency also has an effect on Flt3L-derived BM-DC development. For example, SLAP/SLAP2-/- BM-DC numbers are reduced in the presence of Flt3L as compared to wild-type BM-DC. To investigate the mechanism of reduced DC numbers, we examined splenic DC and found that DC numbers were similar in wild-type and SLAP/SLAP2-/- mice. In fact, SLAP/SLAP2-/- mice had proportionally more CD8α+ splenic DC in vivo than wild-type mice. Thus there may be cytokines affected by SLAP/SLAP2-deficiency in our cultures that are either dispensable or compensated for in vivo DC development. The work presented in this thesis has implications for the role of SLAP and SLAP2 in immune response to infections by the regulation of GM-CSFR and Flt3 in maintaining dendritic cell homeostasis.

Early during an immune response, dendritic cells (DC) interact closely with CD4+ T cells, and cross-talk between these cells can come in the form of tumour necrosis factor (TNF) superfamily ligand-receptor interactions. These signals are critical for the maturation, function and survival of DC, and thereby dictate the capacity of DC to prime a robust T cell response. Among these cues, helper T cell-expressed CD40L interaction with DC-expressed CD40 is required to fully mature DC for cross-priming of help-dependent CD8+ T cell responses. The lymphotoxin-beta receptor (LTβR) is another TNF family receptor on DC, and it’s ligands LTα1β2 and LIGHT are expressed on activated T cells. Since abrogated LTβR signaling impairs T cell immunity, we have examined whether LTαβ represents another possible helper T cell-derived cue for full DC maturation. However the LT pathway controls lymphoid tissue organization and DC homeostasis, a second possible mechanism explaining the necessity of LTβR signaling for T cell immunity. Here we dissect the role of helper T cell-expressed LTβR ligands and DC-intrinsic LTβR signaling, independent of DC homeostasis or lymphoid organization, in DC function and T cell immunity. Absence of LTα1β2 and not LIGHT on helper T cells results in compromised T cell priming by DC ex vivo, and LTβ-/- CD4+ T cell responses are impaired in vivo. Ag-specific CD4+ T cell-expressed LTα1β2 and DC-intrinsic LTβR signaling are required for an optimal cytotoxic T lymphocyte (CTL) response in vivo. While CD40 induces IL-12 and is required for CTL function, DC-intrinsic LTβR signaling is necessary for CTL activation and expansion, early up-regulation of CD86 and IFNα/β production. Our results reveal non-redundant roles for distinct TNF family receptors in enabling DC to program different features in Ag-specific CD8+ T cells.

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.

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.