Thesis (Ph. D.)--Cornell University, August, 2006. / Vita. Includes bibliographical references (leaves 116-143).

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Pharmaceutical Sciences. Title from pdf file main screen (viewed on February 17, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 91-108).

Thesis (Ph. D.)--University of Nevada, Reno, 2000. / Includes bibliographical references. Online version available on the World Wide Web.

Dendritic cells (DCs) are a migratory group of bone-marrow-derived leukocytes specialized for uptake, transport, processing and presentation of antigens to T cells. Exposure of DCs to bacterial pathogens can induce DC maturation characterized by cytokine production, up-regulation of co-stimulatory molecules and an increased ability to activate T cells. DCs have the ability to restrict growth of L. pneumophila (Lp), an intracellular Gram-negative bacillus that causes a severe form of pneumonia known as Legionnaires' disease, in murine ER-derived organelles (121) but replicate in human DCs (145). Even in human cells, however, lysis of the DCs does not occur for at least 24 hours which may allow DCs time to participate in the transition from innate to adaptive immunity (145). The primary polyphenol in green tea extract is the catechin (-)-epigallocatechin-3-gallate (EGCG) which accounts for most of the numerous reported biological effects of green tea catechins, including anti-bacterial, anti-tumor, and neuroprotective effects. Primary murine bone marrow derived DCs from BALB/c mice were treated in vitro with Lp, or stimulated for comparison with Escherichia coli lipopolysaccharide (LPS). CD11c, considered an important marker of mouse DCs, and surface expression of co-stimulatory molecules CD40, CD80, CD86, as well as class I/ II MHC molecules was determined by flow cytometry. Treatment of the cells with EGCG inhibited the microbial antigen induced up-regulation of CD11c, CD40, CD80, CD86 and MHC I/ II molecules. EGCG also inhibited, in a dose dependent manner, induced production of the Th1 helper cell activating cytokine, IL-12, and the chemokines RANTES, MIP1a, and MCP-1. However, EGCG upregulated TNFa production. In addition, EGCG inhibited both Lp and LPS induced expression of both TLR2 and TLR4 as well as LPS-induced NF-kB activation; all of which are important mediators of DC maturation. The modulation of phenotype and function of DCs by EGCG has implications for host interaction with microbial pathogens like Lp, which involve TLR interaction.

Allergic asthma is a chronic inflammatory airway disease that is dominated by Th2 immune responses, with accumulation of eosinophils, IgE and IgG1 production, and airway hyperresponsiveness. We reported previously that treatment of OVA-asthmatic mice with allergen-presenting IL-10-differentiated dendritic cells (DC) (DC10) leads to progressive and long-lasting full-spectrum asthma tolerance. However, little has been done in investigating a role for antigen-specific B cells in DC10-induced tolerance. In this study, we characterized the surface markers of DC10 and found that these cells expressed lower levels of CD40, CD80, MHC II, PD-L1 and PD-L2 relative to immunostimulatory LPS-differentiated DCs (DCLPS). Co-culturing DC10 or DC10-induced regulatory T cells (iTreg) with CD4+ Th2 effector T cells from asthmatic mice led to a marked suppression of DCLPS-induced T effector cell proliferation. Moreover, DC10 treatment of asthma phenotype mice down-regulated airway eosinophilic inflammation as determined 48 h after a recall allergen challenge, and reduced pulmonary parenchymal tissue OVA-specific IgG1-secreting (OVA-IgG1) plasma cell numbers. The number of lung OVA-specific IgG1 plasma cells decreased by 46.7% over a 2 week period in the absence of repeated allergen challenge, while the numbers of bone marrow OVA-specific IgG1 plasma cells stayed relatively stable over a 6 week period, as determined 48 h after a single allergen challenge of asthmatic mice. DC10 treatment had a significant impact on the serum of IgG1/IgE response. To address the question of how DC10 influence OVA-IgG1 plasma cells responses, we co-cultured enzymatically-dispersed lung total cells from asthmatic mice with or without DC10, and found that the DC10 significantly suppressed OVA-IgG1 plasma cell antibody production. To determine whether DC10 required input from T cells to accomplish this, we co-cultured CD4 T cell-depleted, B cell-enriched populations from the lungs of asthmatic mice with or without DC10, and found that DC10 strongly (65.4+/-3.5%) suppressed OVA-IgG1 plasma cells in CD4 T cell-depleted lung cell cultures. To assess whether DC10-induced Treg also suppress IgG1-secretion, we co-cultured lung CD4+ T cells from untreated or DC10-tolerized asthmatic mice with total lung cells from asthmatic donors, and found that the DC10-induced Tregs effectively (52.2+/-8.7%) suppressed OVA-IgG1 plasma cell responses. In summary, DC10 treatment strongly down-regulate OVA-specific IgG1 plasma cell responses of asthmatic mice, both in vivo and in vitro by at least two mechanisms: directly via DC10 as well as indirectly through DC10-induced Tregs.

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

Cells detect and respond to environmental changes using intracellular networks, and defects in the wiring of these networks contribute to diseases. For example, Toll-like receptors (TLRs) sense microbial molecules and trigger pathways critical for host defense. Genetic defects in components of the TLR and other pathogen-sensing pathways have been linked to human diseases. Hence, rational targeting of these pathways should help to manipulate immune responses associated with infections, autoimmunity, or vaccines. A fundamental challenge is to dissect the intracellular networks mobilized by pathogen-sensing pathways. Here we present approaches to dissect the TLR network of innate immune dendritic cells (DCs), focusing on two regulatory layers: signaling and transcription. First, we present a strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We apply this approach to derive regulatory networks that control the transcriptional response to TLR engagement by microbial molecules. Our approach revealed the regulatory functions of 125 transcription factors (TFs), chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 “fine-tuners” that help explain how TLR pathways achieve specificity. Second, we report the systematic discovery of signaling components in TLR responses. By combining transcriptional profiling, genetic and small molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known members of the TLR signaling pathways. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Lastly, we expand these approaches to integrate functional and physical interactions linking the ‘signaling-to-transcription’ TLR network. By combining our perturbation-based approach with measurements of physical interactions, including phosphorylation, protein complexes, and TF binding to DNA, we uncover 30 signaling regulators mechanistically linked to 19 downstream TFs. The integration of these datasets into a model reveals the organization of the TLR response. Overall, these studies illustrate the power of combining systematic measurements and perturbations to elucidate complex intracellular circuits and discover potential therapeutic targets.

Topical antigen (Ag) application mimics natural Ag exposure across the skin. Soluble Ag introduced through this route requires cross-presentation by dendritic cells (DCs) to generate CD8 T cell responses, including skin-homing T cells. DCs process Ag for display to other immune cells, and stimulate T cells to release cytokines or directly lyse infected cells. Some T cells are further stimulated to express homing markers allowing them to enter non-lymphoid tissue such as the skin or the gut.

The field of immunology has advanced tremendously over the last 40 years, with seminal findings that have guided the development of powerful new therapies. However, the ability to induce safe and long-lasting antigen-specific tolerance has remained elusive. A therapy that could prevent the immune system from aberrantly destroying self-tissues, without impairing its capacity to eliminate dangerous pathogens, would be transformative for the treatment of autoimmune diseases. In addition, such a therapy could also greatly advance the field of organ transplantation by inducing antigen-specific tolerance to prevent graft rejection. / Engineering and Applied Sciences