Lymph nodes (LNs) are dynamic organs responsible for providing a supportive and centralized environment for the generation of immune response. Utilizing a highly organized network of non-hematopoietic stromal cells, the LN serves as the context in which the immune system collects and presents antigen, promotes innate and adaptive immune interaction, and generates protective cell-mediated and humoral immunity. In this way, proper organization and function of the LN environment is a critical component of effective immunity, and understanding its complexity has direct impact on the ability to generate and modulate primary immune response to specific antigens. To this end, the LN architecture, underlying stromal networks, and environmental and cellular responses to influenza vaccination were investigated. Using novel approaches to conduit imaging, details of the collagen network that comprises the LN scaffolding have been integrated into current understandings of LN architecture. The cellular compartment responsible for the maintenance of that scaffolding, fibroblastic reticular cells (FRCs), have been studied using an induced diptheria toxin receptor model. By specifically ablating the FRC population in mice, their role in the maintenance of T cell homeostasis has been confirmed in vivo. More surprisingly, a disruption of the FRC network resulted in a loss of B cell follicle structure within LNs, and a reduction in humoral immunity to influenza vaccination. These findings led to the identification of a new subset of FRCs which reside in B cell follicles, and serve as a critical source of the B cell survival factor BAFF. Turning towards the hematopoietic response to influenza vaccination, a highly unexpected lymph node resident dendritic cell (LNDC) response has been identified following vaccine antigen deposition within specialized sites in the LN medulla. Rapid migration of LNDCs into these sites optimizes exposure of the population to viral antigen, and de novo synthesis of a CXCL10 chemokine gradient by activated LNDCs ensures efficient antigen specific \(CD4^+\) T cell response, and protective humoral immunity - independent of migratory dendritic cell status. Altogether, these studies highlight a highly dynamic, responsive LN environment with direct influence on primary immune response - the understanding of which has broad implications in vaccine biology.

Dendritic cells (DCs) are regulators of the immune system and express a class of pattern recognition receptors known as C-type lectin receptors (CLRs) to recognize and respond to carbohydrates (glycans). Dendritic cells are hypothesized to be key mediators in the immune response to implanted materials and ligation of CLRs has been shown to have diverse effects on DC phenotype ranging from tolerogenic to pro-inflammatory. Thus, designing future biomaterials and combination products that harness the potential of CLR ligation on DCs has great promise. Additionally, many of the proteins which adsorb to biomaterials when implanted are glycosylated and thus understanding this interaction would provide further insight into the host response to currently implanted materials. However, DC responses to glycans presented from non-phagocytosable surfaces has not been well characterized and optimal factors for DC phenotype modulation by surface presented glycans are unknown. Additionally, studies relating DC response to glycan structures from soluble and phagocytosable displays to that of non-phagocytosable display have not been performed. This is of critical importance to the field because of the extremely limited supply of complex glycan structures that are able to be obtained. Because of this limited supply of glycans the trend in glycomics has been toward creation of glycan microarrays to assess initial candidates of interest for further study. However, the assumption that cell response to these glycoconjugate microarrays is equivalent to soluble or phagocytosable conjugates has not been validated. Therefore, the purpose of this study was to 1) determine the optimal molecular contextual variables of glycoconjugate presentation from a non-phagocytosable surface, namely, charge, density, and glycan structure for modulating DC phenotype; and 2) determine if modality of glycoconjugate presentation, i.e. soluble, phagocytosable, or non-phagocytosable will modulate DC phenotype differentially. To determine the effect of the molecular contextual variables primary human immature DCs (iDCs) were exposed to a range of adsorbed glycoconjugates in a 384 well plate and their subsequent phenotype assessed via a novel in house produced high throughput (HTP) assay. Bovine serum albumin (BSA) was modified to have a range of glycan densities and isoelectric points to determine which of these were optimal for DC phenotype modulation. Next, several poly-mannose structures were presented to DCs to determine if DC response was structure specific. Finally, contextual variables were modeled in a multivariate general linear model to determine underlying trends in DC behavior and optimal factors for glycan presentation from non-phagocytosable surfaces. To determine the effect of the modality of glycoconjugate display on DCs, optimized glycoconjugates from 1) were adsorbed to the wells of a 384 flat well plate, delivered at varying soluble concentrations, or adsorbed to phagocytosable 1 µm beads and subsequent DC phenotype assessed via the HTP assay. The cell response to the glycoconjugates was then validated to be CLR mediated and the DC response to glycan modality was modeled in another general linear model. Results from these studies show that highly cationized high density glycoconjugates presented from non-phagocytosable flat well display modulate DC phenotype toward a pro-inflammatory phenotype to the greatest extent. Additionally, significant impacts on DC phenotype in response to adsorbed conjugates can be seen when grouping glycan structure by terminal glycan motif. Finally, DC response to glycoconjugates were found to be CLR mediated and that each modality of glycan display is significantly different, in terms of DC phenotype, from the others. These results provide indications for the future design of glycan microarray systems, biomaterials and combination products. Furthermore, this work indicates that different mechanisms are involved in binding and processing of surface bound versus soluble glycoconjugates. With further study these differences could be harnessed for use in the next generation of biomaterials.

No description available.

Numerous studies have demonstrated that tumour cells have the ability to alter immune function to create an immune suppressed environment. This allows tumour cells to escape immune surveillance and consequently the tumour can progress. Dendritic and T cells have critical roles in immune activation and tolerance and are thus major targets of tumour-mediated immune suppression. Understanding the mechanism(s) by which tumour cells modulate the immune system will facilitate the development of immune system-based therapies for cancer treatments. In this study we sought to determine the nature of, and cellular and molecular mechanisms underlying, changes in immune status during tumour progression using mouse models of prostate cancer. Detailed analysis of the immunological status in a mouse prostate dysplasia model (12T-7slow) revealed that immune suppression accompanied tumour progression. We found that T cells isolated from tumour-bearing hosts were hypo-responsive to antigen stimulation. Furthermore, we demonstrated that CD4+CD25+ regulatory T cells were responsible, at least in part, for this alteration. Anti-CD25 antibody treatment reduced, but did not prevent, tumour growth in either a transplanted prostate tumour model or a spontaneously developing prostate tumour model. In addition, an altered dendritic cell phenotype and an elevated frequency of CD4+CD25+ regulatory T cells were observed within the tumour mass. Similar alterations were observed in the prostate-specific Pten knockout mice which develop advanced prostate adenocarcinoma. Interestingly, evidence of immune activation, such as an increased frequency of activated T cells, was detected in the tumour microenvironment in both mouse prostate tumour models. To identify factors that may play critical roles in the altered immune cell phenotype observed in the tumour microenvironment, a global gene expression profiling analysis was carried out to evaluate the changes in immune-related gene expression patterns. This analysis provided additional evidence for the co-existence of immune suppression and immune activation. Moreover, subsequent analyses suggested that one differentially expressed transcript, interferon regulatory factor 7, and its target genes might be involved in modulating immune cells and/or tumour progression. Taken together, these studies have important implications for designing specific and effective anti-tumour immune therapy strategies that involve manipulation of tumour cells, dendritic cells and regulatory T cells.

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.

Advancements in the biomedical field are driven by the design of novel materials with controlled physical and bio-interactive properties. To develop such materials, researchers rely on the use of highly efficient reactions for the assembly of advanced polymeric scaffolds that meet the demands of a functional biomaterial. In this thesis two main strategies for such materials have been explored; these include the use of off-stoichiometric thiol-ene networks and dendritic polymer scaffolds. In the first case, the highly efficient UV-induced thiol-ene coupling (TEC) reaction was used to create crosslinked polymeric networks with a predetermined and tunable excess of thiol or ene functionality. These materials rely on the use of readily available commercial monomers. By adopting standard molding techniques and simple TEC surface modifications, patterned surfaces with tunable hydrophobicity could be obtained. Moreover, these materials are shown to have great potential for rapid prototyping of microfluidic devices. In the second case, dendritic polymer scaffolds were evaluated for their ability to increase surface interactions and produce functional 3D networks. More specifically, a self-assembled dendritic monolayer approach was explored for producing highly functional dendronized surfaces with specific interactions towards pathogenic E. coli bacteria. Furthermore, a library of heterofunctional dendritic scaffolds, with a controllable and exact number of dual-purpose azide and ene functional groups, has been synthesized. These scaffolds were explored for the production of cell interactive hydrogels and primers for bone adhesive implants. Dendritic hydrogels decorated with a selection of bio-relevant moieties and with Young’s moduli in the same range as several body tissues could be produced by facile UV-induced TEC crosslinking. These gels showed low cytotoxic response and relatively rapid rates of degradation when cultured with normal human dermal fibroblast cells. When used as primers for bone adhesive patches, heterofunctional dendrimers with high azide-group content led to a significant increase in the adhesion between a UV-cured hydrophobic matrix and the wet bone surface (compared to patches without primers). / <p>QC 20140116</p>

TLR3- and TLR4-mediated DC activation lead to distinct function of mature DC, in which MAPKs pathway is involved. JNK-associated leucine zipper protein (JLP) associates with JNK/p38 phosphorylation, however the role of JLP in DC is not well understood. We previously found either LPS or poly(I:C) up-regulated JLP expression in BMDC. Therefore we hypothesized JLP plays a differential role in TLR3- and TLR4-mediated BMDC maturation and functions. We demonstrated JLP facilitated LPS- and poly(I:C)-induced CD86 and CD40 up-regulation, LPS-induced CD80 up-regulation and poly(I:C)-induced MHC-II up-regulation. We found JLP down-regulated IL-12 production in LPS-stimulated BMDC, and up-regulated IL-6 production in poly(I:C)-stimulated BMDC. Our data also showed JLP negatively regulated MHC-II antigen presentation in LPS- and poly(I:C)-stimulated BMDC and JLP was involved in promoting LPS-activated BMDC survival, but not in poly(I:C)-activated BMDCs. Therefore our current data suggested a multi-functional role of JLP in the regulation of TLR3- and TLR4-mediated DC maturation.

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.