In addition to leukocytes, secondary lymphoid organs are populated by non-hematopoietic stromal cells. This diverse group of cells supports lymphocyte migration and homing, facilitates antigen delivery, and promotes T cell survival. However, there is relatively little known about the specific molecules governing the roles that these cells play in regulating dendritic cell (DC) motility and lymph node architecture. Here, we examine the interaction between two molecules, CLEC-2 and podoplanin (PDPN), that are critical for DC migration and maintaining structural integrity of lymph nodes. Together, these studies identify novel functions of lymph node stromal cells and a unique function for PDPN in the immune system. In response detecting an potentially harmful antigen, DCs in peripheral tissues mature and travel to downstream lymph nodes by following chemokine gradients secreted by lymphatic endothelial cells (LECs) and fibroblastic reticular cells (FRCs) present in the lymph node paracortex. We discovered that, in addition to chemokines, DC migration requires CLEC-2 on DCs, as engagement of CLEC-2 with PDPN, which is expressed by LECs and FRCs, incites DC motility and is required for DC entry into the lymphatics, efficient arrival in the lymph node, and migration along the FRC network within the lymph node. Next, we examined the effect of this interaction with respect to the stromal cell. Through a combination approaches, we discovered that PDPN is a master regulator of contractility in FRCs. The fact that FRCs are contractile cells was previously reported, but our study is the first to identify a function for this contractility: upon blockade of PDPN-mediated contractility, lymph nodes became enlarged, the FRC network became more sparse, and there were increased numbers of lymphocytes in the lymph node. Importantly, during an immune response, these changes resulted in more proliferation of antigen-specific T cells and impaired contraction of the lymph node upon resolution of inflammation. Finally, we found that CLEC-2 binding PDPN recapitulated the effect of PDPN deletion. Thus, during an immune response, CLEC-2+ DCs would use PDPN to efficiently migrate to the lymph node and simultaneously cause FRCs to relax and prepare the lymph node for expansion.

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.

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.

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.

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.

Orientador: Silvana Regina Perez Orrico / Resumo: Diabetes mellitus (DM) é uma desordem metabólica importante caracterizada pela ausência de secreção e/ou ação deficiente da insulina, resultando em hiperglicemia crônica e persistente que é a principal característica do DM. A influência do diabetes sobre as doenças periodontais é amplamente documentada e reconhecida demonstrando que a desregulação da resposta imune associada ao diabetes afeta a interação microbiota-hospedeiro no periodonto, aumentando a severidade e extensão da destruição dos tecidos periodontais. Considerando o papel crucial das células dendríticas (DC) no desenvolvimento e regulação da resposta imune e da relativa escassez de informação sobre a influência específica de concentrações elevadas de glicose nas células imunes, investigou-se a hipótese de que a elevada glicose influencia a biologia de DCs. Especificamente, avaliamos a influência da alta de glicose sobre a proliferação e viabilidade, a apoptose, a maturação, a quimiotaxia, a migração para os linfonodos (homing), atividade fagocitária, expressão de mediadores inflamatórios e produção de espécies reativas de oxigénio (ROS). Foram utilizadas células dendríticas de linhagem murina (DC2.4), que foram expostas a concentração elevada (25 mM) ou normal (11,1 mM) de glicose, por períodos que variaram de 24 a 72 h. A exposição à alta glicose reduziu a proliferação mas não afetou a viabilidade, a sobrevivência ou a maturação de células DC2.4 induzida por LPS; no entanto a susceptibilidade à apoptose foi aume... (Resumo completo, clicar acesso eletrônico abaixo) / Diabetes mellitus (DM) is a major metabolic disorder characterized by the absence of secretion and/or impaired insulin action, resulting in chronic and persistent hyperglycemia which is the main characteristic of DM. The effect of diabetes on periodontal diseases is widely recognized and documented demonstrating that dysregulation of the immune response associated with diabetes affects microbial-host interaction in the periodontium, increasing the severity and extent of the destruction of the periodontal tissues. Considering the crucial role of dendritic cells (DCs) in the development and regulation of immune response and the relative scarcity of information on the specific influence of high glucose concentrations on immune cells, we investigated the hypothesis that high glucose influences the biology of DCs. Specifically, we assessed the influence of high glucose on proliferation and viability, apoptosis, maturation, chemotaxis, migration to the lymph nodes (homing), phagocytic activity, expression of inflammatory mediators and production of reactive oxygen species (ROS). We used a murine dendritic cell line (DC2.4), which was exposed to high (25 mM) or normal (11.1 mM) glucose concentrations for periods ranging from 24 to 72 h. Exposure to high glucose reduced proliferation but did not affect viability, survival or LPS-induced maturation of DC2.4 cells; however susceptibility to apoptosis was increased. High glucose also reduced phagocytic activity, chemotaxis and homing to lymph nodes in vivo. On the other hand, production of ROS and gene expression of inflammatory mediators were increased by high glucose. Collectively, the effects of high glucose on dendritic cell biology are consistent with a decrease on immune activating role of DCs and an exacerbation of local inflammation. / Doutor

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. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Intestinal inflammatory disorders such as Crohn’s disease contribute significantly to human mortality and morbidity. Although the cells and molecules involved in suppression of intestinal inflammation have been extensively documented in mouse models, a full understanding of how these work together in the healthy and diseased gut remains elusive. It is known, however, that tight regulation over TH17 cells and regulatory T cells (Treg) is required to maintain immune homeostasis in the intestine. Activation of the cytokine transforming growth factor-β (TGFβ), which is secreted by immune cells as an inactive complex, plays a crucial role in the induction of both Treg and TH17 cells. Recent work has shown that the αvβ8 integrin is required for activation of TGFβ by murine dendritic cells (DC). Murine integrin αvβ8 is thus of fundamental importance for Treg and TH17 induction and, subsequently, for intestinal immune homeostasis. However, little is known about the signals controlling the expression of integrin αvβ8 on intestinal DC. Furthermore, whether this system is also important for regulation of the human system is entirely unknown. Here, expression of integrin αvβ8 is shown on human intestinal CD1c+CD103+SIRPα+ DC and CD1c+CD103-SIRPα+ DC, but not on CD141+CD103+SIRPα- DC. Expression of integrin αvβ8 is increased on DC from Crohn’s disease patients, and on DC from non-IBD donors treated with LPS. Both LPS and a number of probiotic bacteria were also able to induce integrin αvβ8 expression on human monocyte-derived DC (moDC), which suggested that the microbiota may play a role in the regulation of human integrin αvβ8. Importantly, we have also shown that TGFβ is activated by integrin αvβ8 on human moDC, and that integrin αvβ8 promotes expression of forkhead box P3 (FOXP3) in naïve human T cells in vitro. Together, these data suggest that integrin αvβ8-mediated activation of TGFβ by DC may play an important role in the regulation of human T cell responses in the human intestine, and that this pathway may be perturbed during intestinal inflammation.

As the principal antigen-presenting cells to T cells, dendritic cells (DCs) have a key role in the balance of immunity and autoimmunity. They are essential in two major, converse roles - eliciting T cell immune responses to pathogenic material, and maintaining peripheral tolerance to self-tissue by inhibiting self-reactive T cells. These functions involve the processing of pathogenic or self antigens and subsequent presentation of antigenic peptides on MHC to antigen-specific T cells. DC recognition of conserved pathogenic markers induces a mature phenotype that governs immunogenic presentation to T cells and, consequently, the adaptive immune response. In contrast, DC recognition of self tissue suppresses maturation, instead inducing a tolerogenic phenotype that induces self antigen-specific T cell to die, become anergised, or converted to T regulatory cells. Apoptotic cells are the major source of self-antigen for the maintenance of peripheral tolerance, and their defective clearance by DCs is implicated in autoimmunity. Apoptotic cells are thought to actively suppress maturation of DCs and inhibit the possible immune responses promoted by proinflammatory mediators released from necrotic cells. However, the immune function of apoptotic cells and their relative influence over necrotic cells are highly contested, partially due to the complex nature of immunogenicity arising from the sourcing and generation of apoptotic cells. In this investigation, various methods of inducing apoptosis and necrosis are evaluated. Definitive methods of inducing well-characterised cell death are then employed to compare the effects of apoptotic and necrotic cells on dendritic cells and in vitro and in vivo immune responses. Reported here are in vitro findings that support previous reports of the anti-inflammatory response of DCs to apoptotic cells, and the inflammatory response of DCs to necrotic cells. The previously-reported inhibitory effect of apoptotic cells on LPS-induced secretion of Th1 cytokines is supported here, but the inhibitory effect of apoptotic cells on LPS-induced upregulation of co-stimulatory molecules is contested. Novel findings describe the upregulation of DC expression of co-inhibitory molecules induced by both apoptotic cells and necrotic cells. Apoptotic cells, but not necrotic cells, had a suppressive effect on CpG-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokines. Apoptotic cells suppressed the capacity of untreated and CpG-treated, but not LPS-treated, DCs to elicit IFNγ production by T cells. Apoptotic cells, but not necrotic cells, induced regulatory T cells and partially restored their CpG-suppressed induction. Finally, apoptotic cell-modulation of DCs inhibited the induction of autoimmunity in a novel modification of an in vivo model of diabetes. Interestingly, novel evidence for the possibility of necrotic cell-induced tolerance by means of direct T cell killing is addressed.