Currently, in most situations, the autologous immune system is unable to eradicate the residual leukemic burden persisting after chemo-radiotherapy, but a balance can be established between leukemic and immune cells leading to a clinical remission for several months or years. If this balance is broken, a clinical relapse can occur. The high incidence of relapses in human cancers demonstrates the frequent inefficacy of the immune system to control these residual cells. In this context, allogeneic hematopoietic stem cell transplantation (HSCT) has been proven to be the most effective way to reinforce the immune reaction against leukemia, graft-versus-leukemia (GVL) effect and, so, achieve a definitive eradication of the residual disease in a significant proportion of patients. Indeed, the whole concept of HSCT evolved from an organ transplant concept (to replace a defective ill organ with a new healthy one) to the concept of creating an extraordinary immunotherapeutic platform in which the donor immune system contributes to the eradication of the residual leukemic cells. Thus, the past and present issues remain those of finding the best immunomodulatory modalities to achieve a full engraftment, a powerful GVL effect and no or moderate graft-versus-host disease (GVHD). Different ways to reach this goal, such as post transplant cytokine modulation, specific or global cellular depletion of the graft and post transplant global or specific donor immune cell add-backs, are still extensively studied. Nevertheless, the persistent high relapse rate (RR) observed in leukemia patients after HSCT remains the most important cause of death before transplant-related toxicities. Moreover, since only about 40 to 70% (depending on the ethnic context) of patients with high-risk hematological malignancies, eligible for allogeneic HSCT, have a fully HLA-matched sibling or matched unrelated donor (MUD), a great deal of effort has been invested to make the use of an alternative haploidentical sibling donor feasible. The advantage of this procedure is the immediate availability of a donor for almost all patients. The aim of the work described in this thesis has been to implement a strategy to transplant a patient using a HLA haploidentical donor. The strategy is to try to improve DFS that could be applied both in the autologous or allogeneic context: first, by using nonspecific immune manipulation post transplant and then, by developing specific strategies directed against leukemia antigens. Particularly in the allogeneic situation, the aim was to increase the GVL effect without inducing or aggravating the deleterious GVHD. The first part of this thesis described our own clinical results, consisting of three consecutive phase I/II studies, in which we tried to determine the feasibility of giving prophylactic donor lymphocyte infusions (DLI) post transplant and the effect of replacing granulocyte colony-stimulating factor (G-CSF), typically used to speed up neutrophil recovery, with granulocyte macrophage colony-stimulating factor (GM-CSF), which is known for its immunomodulatory properties. The slow immune reconstitution in haploidentical transplant is chiefly responsible for the high incidence of early lethal viral and fungal infections, and most probably for early relapses; therefore, we sought to accelerate and strengthen the post transplant immune reconstitution without increasing the GVHD rate. Thus, we have studied the impact of post transplant growth factor administration and of unselected DLI in haploidentical transplant. We have also implemented, in our center, anti-cytomegalovirus (CMV) specific T cell generation and infusion to improve anti-CMV immune reconstitution. Since then, our results have been pooled in a multi-center analysis performed by the European Bone Marrow Transplantation group (EBMT) allowing us to compare our results with those of the entire group. We have also participated in the design of an ongoing study aimed at selectively depleting the graft from alloreactive T cells, and improving post transplant T cell add-backs. In our attempts to generate and expand ex vivo lymphocytes (directed against pathogens (CMV) and leukemia-associated antigens, Wilms' tumor gene 1 (WT1) and to use them in vivo, we found inconsistent results (in the case of WT1) using classical clinical grade dendritic cells (DC) generated and matured in bags, as was the case for the majority of the teams worldwide. This led us to question the full functionality of these DC and we undertook a thorough comparative analysis of DC generated and differentiated in bags and in plates (typical for most pre-clinical studies). This analysis showed us that one cannot transpose pre-clinical studies (using culture plates) directly to clinical protocols (generally using clinical grade culture bags) and that DC generated in bags are functionally deficient. We learned that, if we want to use a DC vaccine to improve the GVL effect in haploidentical transplant, we will have to be careful about the technique by which they are generated. To improve immunotherapeutic approaches, the understanding of the mechanisms underlying tumor tolerance and how to manipulate them is critical in the development of new effective immunotherapeutic clinical trials. This is why we currently focus on how to obtain effective in vivo anti-leukemia immune reactions using an ex-vivo manipulated product to trigger the immunotherapeutic response. More specifically, we are analyzing the impact of regulatory T cell (Tregs) depletion and function for an adequate anti-leukemic immune response. This pre-clinical work aims at improving the outcome of leukemia patients who have relapsed and been put back into second remission and at decreasing the RR after HSCT, especially in the field of haploidentical transplantation. In conclusion, haploidentical transplantation has become a valuable tool. The results are at least similar to those obtained using MUD when performed in the same group of patients. Specific immunomodulation post transplant can affect events such as GVHD and GVL, but clinically we are still at the level of nonspecific manipulations. It is our hope that ongoing pre-clinical work will enable us to perform specific anti-pathogen and anti-leukemia immune manipulation that will favorably influence the patient outcome. / Dans la majorité des situations, le système immunitaire autologue est incapable d’éradiquer les cellules leucémiques résiduelles qui échappent à la radiothérapie et à la chimiothérapie, cependant un équilibre peut s’établir entre les cellules leucémiques et immunitaires aboutissant à une rémission pouvant durer plusieurs mois ou années. Si cet équilibre se rompt, une rechute clinique peut se déclarer. Dans ce contexte, il est prouvé que la greffe allogénique de cellules souches hématopoïétiques est le moyen le plus efficace de renforcer les réactions immunitaires contre la leucémie par la réaction du greffon contre la leucémie et ainsi d’obtenir une éradication définitive de la maladie résiduelle chez un nombre significatif de patients. En effet, le concept global de l’allogreffe de cellules souches hématopoïétiques a évolué du concept de transplantation d’organe (remplacement d’un organe malade par un nouvel organe sain) vers celui de créer une extraordinaire plateforme d’immunothérapie à travers laquelle le système immunitaire du donneur contribue à l’éradication des cellules leucémiques persistantes. Donc, la problématique reste celle de trouver les meilleures modalités d’immunomodulation pour achever une prise du greffon, un effet anti-leucémique puissant du greffon, et l’absence ou un minimum d’effet du greffon contre l’hôte. Différentes stratégies existent pour atteindre cet objectif, comme l’utilisation de cytokines pour moduler la reconstitution immunitaire, des déplétions cellulaires globales ou spécifiques du greffon et l’infusion de cellules immunes «globales» ou spécifiques du donneur après greffe. Ces stratégies sont encore largement à l’étude. Néanmoins, la persistance d’un taux de rechute élevé observé chez les patients leucémiques, après allogreffe reste la cause principale de décès, avant celle liée à la toxicité de la greffe. De plus, étant donné que seulement environ 40 à 70% (dépendant de l’origine ethnique) des patients avec une hémopathie à haut risque, éligibles pour une greffe allogénique, ont un donneur familial ou non familial complètement HLA compatible, des efforts importants ont été développés pour rendre faisable l’utilisation de donneurs familiaux alternatifs, haploidentiques. L’avantage de cette approche est l’accès immédiat à un donneur pour quasiment tous les patients. Le but du travail décrit dans cette thèse a été l’implémentation d’une stratégie d’allogreffe utilisant un donneur haploidentique. Le travail vise également à développer de façon plus large des stratégies qui peuvent améliorer le taux de survie sans rechute, non seulement dans le contexte des greffes haploidentiques, mais également dans le cadre des greffes allogéniques en général, ainsi que dans les situations autologues : premièrement, par la manipulation immunitaire non spécifique après greffe et ensuite par le développement de stratégies spécifiques dirigées contre des antigènes leucémiques. En particulier dans la situation allogénique, le but a été d’augmenter l’effet du greffon contre la leucémie sans induire ou aggraver l’effet délétère du greffon contre l’hôte. La première partie de la thèse décrit les résultats cliniques de notre propre protocole de greffe haploidentique, qui a consisté en trois études consécutives de phase I/II. Dans ces études, nous avons voulu déterminer la faisabilité de réaliser des infusions prophylactiques de lymphocytes du donneur après transplantation, et l’impact du remplacement du « granulocyte colony-stimulating factor » (G-CSF), largement utilisé pour permettre une récupération en polynucléaires neutrophiles plus rapide, par du « granulocyte-macrophage colony-stimulating factor » (GM-CSF), lequel est connu pour ses propriétés immunomodulatrices différentes. La reconstitution immunitaire très lente après greffe haploidentique est majoritairement responsable de l’incidence élevée de décès par infections virales et fungiques précoces, et très probablement des rechutes précoces. C’est pourquoi nous avons cherché à accélérer et à renforcer la reconstitution immunitaire post-greffe sans augmenter la fréquence de réaction du greffon contre l’hôte. Nous avons donc étudié l’impact de l’administration de facteurs de croissance et l’infusion de lymphocytes non sélectionnés du donneur en post greffe haploidentique. Nous avons également implémenté dans notre centre, la génération et l’infusion de lymphocytes T spécifiques anti-cytomégalovirus (CMV) afin d’améliorer la reconstitution immunitaire anti-CMV. D’autre part, nos résultats ont été regroupés dans une étude multicentrique menée par le groupe européen de transplantation de moelle osseuse (EBMT), ce qui nous a permis de comparer nos résultats avec ceux de l’entièreté du groupe. Nous avons parallèlement participé à la conception d’une étude actuellement en cours ayant pour but d’améliorer la reconstitution immunitaire après greffe par la déplétion sélective du greffon en lymphocytes T alloréactifs et par l’infusion après greffe de lymphocytes T du donneur également sélectivement déplétés en lymphocytes T alloréactifs. Afin d’optimaliser l’effet anti-leucémique du système immunitaire, nous avons débuté un protocole de vaccination par cellules dendritiques (DCs). Ces cellules dendritiques étaient chargées en lysat de blastes leucémiques dans le cas de patients présentant au diagnostic une leucémie aigue surexprimant l’oncogène 1 de la tumeur de Wilms (WT1). Néanmoins dans nos travaux de génération et d’expansion ex-vivo de lymphocytes T spécifiques de l’antigène WT1, utilisant les DCs de grade clinique, générées et maturées en poches, nous avons rencontré des résultats inconsistants, comme c’était le cas dans la majorité des protocoles cliniques internationaux de vaccination. Nous nous sommes alors posé la question de la fonctionnalité globale de ces cellules et nous avons entrepris une analyse comparative poussée des DCs générées et différenciées en poches ou en plaques. Les DCs générées en plaques sont celles utilisées dans la plupart des travaux précliniques. Cette analyse nous a montré que l’on ne pouvait pas directement transposer les résultats précliniques basés sur des DCs générées en plaques dans des protocoles cliniques basés sur des DCs générées en poches, car ces dernières présentent des déficits fonctionnels importants. Nous avons appris que si l’on voulait utiliser un vaccin à base de cellules dendritiques pour améliorer l’effet du greffon contre la leucémie dans les greffes allogéniques, nous devions être très attentifs quant au protocole utilisé pour la génération de ces vaccins cellulaires. Pour améliorer les approches immunothérapeutiques, la connaissance des mécanismes qui établissent la tolérance tumorale et des façons de manipuler ceux-ci, est critique dans le développement de nouveaux protocoles efficaces. C’est pourquoi nous nous concentrons actuellement sur les conditions nécessaires à l’obtention in vivo d’une réaction immune anti-leucémique efficace lors de l’utilisation d’un produit cellulaire manipulé ex vivo. Plus spécifiquement, nous analysons l’impact de la déplétion en lymphocytes T régulateurs (Tregs) sur la réponse anti-leucémique. Ce travail préclinique a pour but d’améliorer le devenir de patients leucémiques qui ont rechutés et ont été mis en seconde rémission, ainsi que de diminuer le taux de rechute après allogreffe, spécifiquement après greffe haploidentique. En conclusion, la transplantation haploidentique est actuellement un outil précieux pour de nombreux patients. Les résultats sont au minimum similaires à ceux qui sont obtenus par les greffes non-familiales HLA identiques lorsqu’elles sont pratiquées dans les mêmes groupes de patients. L’immunomodulation spécifique après greffe peut affecter des événements comme la réaction du greffon contre l’hôte et la réaction du greffon contre la leucémie, mais en pratique clinique nous en sommes encore au niveau de la manipulation aspécifique. Nous espérons que les travaux précliniques actuels vont nous permettre d’appliquer des stratégies spécifiques et d’obtenir une manipulation immune anti-leucémique qui aura une influence favorable significative sur le devenir des patients.

Respiratory syncytial virus (RSV) is the primary viral pathogen responsible for lower respiratory tract disease in neonates and young children worldwide. By the age of two, virtually all children have been infected with RSV, and approximately 40% of them develop lower respiratory tract infections. In addition to acute morbidity, an association between RSV infection in early childhood and later development of recurrent wheezing and airway hyperresponsiveness (AHR) has been repeatedly demonstrated.<p>In this work we established a method for propagating pneumonia virus of mice (PVM) in a baby hamster kidney-21 (BHK-21) cell line. We also modified the standard plaque assay method and established a reliable and, most importantly, reproducible way to quantitate PVM. In our work we used PVM strain 15 to successfully establish an in vivo animal model for RSV disease in Balb/c and C57/Bl mice. Different susceptibility/resistance patterns to a pathogen exist for different mouse strains. In the case of Balb/c and C57/Bl mice, these patterns are well characterized for several pathogens including Leishmania major and adenovirus type 1. Our comparative study demonstrated clear differences in susceptibility to PVM strain 15 infection between Balb/c and C57/Bl mice; Balb/c mice being more susceptible.<p> In peripheral sites, dendritic cells (DCs) serve as sentinel cells that take up and process antigens. Numerous studies revealed that certain pathogens stimulate changes in DC phenotypic characteristics and thus contribute to functional alterations that lead to inappropriate T cell activation and disease augmentation. To examine effects of PVM on DCs, we infected bone marrow dendritic cells (BM-DCs) derived from both mouse strains with PVM, and evaluated their phenotypic and functional characteristics 24 hours post infection. Under these experimental conditions, PVM infected BM-DCs did not show a significant increase in the expression of costimulatory and major histocompatibility complex class II (MHC II) molecules compared to uninfected controls. Furthermore, there were no changes in the ability of PVM-infected DCs to take up soluble antigen. The production of IL-12p70, the pivotal cytokine in the development of a Th1-type response, by the PVM-infected BM-DCs was not significantly different from uninfected cells. In addition, there was no significant impact of PVM infection on the ability of DCs to induce naïve T cell proliferation.

Respiratory syncytial virus (RSV) is the primary viral pathogen responsible for lower respiratory tract disease in neonates and young children worldwide. By the age of two, virtually all children have been infected with RSV, and approximately 40% of them develop lower respiratory tract infections. In addition to acute morbidity, an association between RSV infection in early childhood and later development of recurrent wheezing and airway hyperresponsiveness (AHR) has been repeatedly demonstrated.<p>In this work we established a method for propagating pneumonia virus of mice (PVM) in a baby hamster kidney-21 (BHK-21) cell line. We also modified the standard plaque assay method and established a reliable and, most importantly, reproducible way to quantitate PVM. In our work we used PVM strain 15 to successfully establish an in vivo animal model for RSV disease in Balb/c and C57/Bl mice. Different susceptibility/resistance patterns to a pathogen exist for different mouse strains. In the case of Balb/c and C57/Bl mice, these patterns are well characterized for several pathogens including Leishmania major and adenovirus type 1. Our comparative study demonstrated clear differences in susceptibility to PVM strain 15 infection between Balb/c and C57/Bl mice; Balb/c mice being more susceptible.<p> In peripheral sites, dendritic cells (DCs) serve as sentinel cells that take up and process antigens. Numerous studies revealed that certain pathogens stimulate changes in DC phenotypic characteristics and thus contribute to functional alterations that lead to inappropriate T cell activation and disease augmentation. To examine effects of PVM on DCs, we infected bone marrow dendritic cells (BM-DCs) derived from both mouse strains with PVM, and evaluated their phenotypic and functional characteristics 24 hours post infection. Under these experimental conditions, PVM infected BM-DCs did not show a significant increase in the expression of costimulatory and major histocompatibility complex class II (MHC II) molecules compared to uninfected controls. Furthermore, there were no changes in the ability of PVM-infected DCs to take up soluble antigen. The production of IL-12p70, the pivotal cytokine in the development of a Th1-type response, by the PVM-infected BM-DCs was not significantly different from uninfected cells. In addition, there was no significant impact of PVM infection on the ability of DCs to induce naïve T cell proliferation.

Most currently defined hematopoietic progenitor pools are heterogeneous, contributing to uncertainty regarding the development of certain blood cells. The origins of plasmacytoid dendritic cells, for instance, have long been controversial and progenitors exclusively committed to this lineage have never been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-plasmacytoid dendritic cells were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-plasmacytoid dendritic cells. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the plasmacytoid dendritic cell lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

Pathogenic Gram-negative Yersinia species preferentially target and inactivate phagocytic cells of the innate immune defense by translocation of effector Yersinia outer proteins (Yops) into the cells via a type III secretion system. This indicates that inactivation and avoidance of the early innate immune response is an efficient way for Yersinia species to avoid elimination and to cause diseases ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to plague (Y. pestis). In this project, we aimed to study the interaction between enteropathogenic Y. pseudotuberculosis and phagocytic cells during early infection. In situ interaction studies on infected intestinal tissues showed that Y. pseudotuberculosis mainly interacts with dendritic cells (DCs) in lymphoid tissues of the intestine during initial infection. After massive recruitment of polymorphonuclear neutrophils (PMNs) to the infected tissues, wild-type (wt) bacteria also interacted with this phagocyte. In contrast to the wt, mutants lacking the anti-phagocytic effectors YopH and YopE are avirulent in mice and unable to spread systemically. Interestingly, our interaction assay showed that these mutants not only interacted with DCs, but also with PMNs during the initial stage of infection. Thus, indicating that Y. pseudotuberculosis can avoid interaction with PMNs during early infection and that this is Yop-dependent. In a phagocytosis assay Y. pseudotuberculosis was demonstrated to inhibit internalization by DCs in a YopE-dependent manner, while both YopH and YopE were shown to be involved in the blocking of phagocytosis by macrophages and PMNs. Thus, indicating that YopH has cell type-specific effects. To further investigate the role of DCs during initial stages of infection, a mouse DC depletion model (CD11c-DTRtg) was applied. However, the DTx-mediated depletion of DCs in CD11c-DTRtg mice induced neutrophilia and the model could not give a definite answer to whether DCs play an important role in either restricting or stimulating progression of Y. pseudotuberculosis infection. To investigate involvement of PMNs during early infection mice were injected with the depleting antibody α-Ly6G. In absence of PMNs wt, as well as yopH and yopE mutants became more virulent, which further supports the importance of these Yops for the ability of Y. pseudotuberculosis to disseminate from the initial infection sites in the intestine to cause systemic disease. In summary, our studies show that inhibiting internalization and maturation of DCs and avoiding phagocytosis by and interaction with macrophages and PMNs during the early stages of infection are important virulence strategies for Y. pseudotuberculosis to be able to colonize tissues, proliferate and disseminate systemically.

Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function. To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains. Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences. Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.

Prostate cancer (PC) is the most common visceral cancer amongst men in Australia and elsewhere in the world. The American Cancer Society estimated that in 2006, PC alone would account for about 33% of newly diagnosed cancer in men, and be responsible for 9% of cancer related deaths in men. In men with advanced, metastatic PC, hormone therapy is widely accepted as the treatment of choice and produces good initial responses in most patients. However, many patients will relapse and become resistant to further hormone manipulation. Currently used treatment modalities for these patients are intended to palliate symptoms and therefore improve quality of life; the long term survival benefit of currently used management strategies is marginal at best. The limited treatment modalities with survival benefit for patients with advanced PC results in the need for development of novel therapies. Enhancement of the natural anti-tumour defences of the immune system to recognise and destroy tumour cells appears as a favourable alternative to prior therapies. Use of autologous dendritic cells (DC) as stimulators of an anti-tumour response has shown promise in several phase I, II and III trials. The Mater Medical Research Institute has developed a novel system for the isolation of blood DC (BDC). This system utilizes an antibody selection system based on a human/mouse chimeric CMRF-56 (huCMRF-56) monoclonal antibody (mAb) which has been engineered from the prototype murine IgG1 CMRF-56 mAb. Pre-clinical studies have demonstrated that the huCMRF-56 mAb isolates a CMRF-56+ cell population comparable to that obtained with the murine IgG1 CMRF-56 mAb with a sufficient CMRF-56+ BDC purity and yield to warrant its use as a BDC based immunotherapy. The objective of this research project was to validate the use of the huCMRF-56 mAb isolated BDC preparations in PC immunotherapy. This was achieved by; i) determining the optimal concentration of the huCMRF-56 mAb at which to perform isolations in order to obtain a CMRF-56+ cellular preparation with purity, yield and cellular composition, in terms of DC subsets, B cells, monocytes and contaminating cells, comparable to that obtained with the murine mAb, which has shown efficacy in in vitro studies; ii) Demonstrating that CMRF-56+ preparations obtained from PC patients using the huCMRF-56 BDC mAb are comparable to those from healthy donors (HD) and iii) demonstrating functional capacity of both freshly isolated and cryopreserved CMRF-56+ BDC isolated preparations to induce anti-tumour cytotoxic T lymphocyte (CTL) responses in both HD and PC patients. These studies utilised the appropriate immunostaining techniques and flow cytometry to determine the CMRF-56+ cell yield, CMRF-56+ BDC purity and viability of the preparation. To determine functional capacity CMRF-56+ preparations were isolated from HD and PC patients, and loaded with the prostate tumour associated antigens (TAA) and control antigen peptides: prostate specific antigen-3 (PSA-3), prostatic acid phosphatase- 5 (PAP-5), prostatic specific membrane antigen-2 (PSMA-2), flu matrix protein (FMP) and/or melanoma antigen recognised by T cells (MART). Co-culture experiments were set up with autologous peripheral blood mononuclear cells (PBMC) and induction of CTL responses were assessed using pentamer staining and ELISPOT assay. The working concentration of the huCMRF-56 mAb of 1.28mg/ml was determined to immunoselect a cellular preparation with maximal BDC purity, BDC yield and total viable cell number. Phenotyping studies of this preparation demonstrated it to be predominately comprised of antigen presenting cells (APC) and enriched for BDC with several BDC subsets immunoselected. CMRF-56+ preparations immunoselected from HD and PC donors were similar and comparable to previously described preparations immunoselected using the original murine CMRF-56 mAb. From antigen loaded CMRF-56+ preparations, specific major histocompatibility complex (MHC) class 1 restricted CD8 lymphocyte responses were generated against prostate TAAs in 2 of 5 HD and 1of 3 PC donors and against FMP in 1 of 3 PC and 5 of 5 HD. Cryopreserved antigen loaded CMRF-56+ BDC preparations from HD generated antigen specific FMP or MART-1 CTL responses in 3 of 4 HD, however anti-prostate TAA CTL responses were not observed. The humanised CMRF-56 mAb immunoselects a cellular preparation enriched in BDC and capable of effective uptake and presentation of antigen. The CMRF-56+ BDC enriched preparation, loaded with PC TAA is capable of inducing specific anti tumour responses in vitro. While further preclinical studies are required, the preparation, loaded with PC TAA shows promise as a potential immunotherapy for PC.

The intestinal barrier represents a complex environment, composed of different physical barriers and immune cells, which act to prevent the entrance of potentially harmful enteric pathogens and to maintain gut tolerance to food antigens and commensal bacteria. Thus, cross-talk between the different components of the intestinal barrier such as the mucus layer, dendritic cells (DC) and intestinal intraepithelial lymphocytes (IELs) may be important in maintenance of gut homeostasis. This thesis investigates how different components of the intestinal barrier regulate immune responses in the gut. Thus, expression of the transmembrane receptor integrin αvβ8 on DCs is shown to be required for the development of a specific IEL subset marked by expression of CD4 and CD8αα, suggesting that intestinal DC play important roles in regulating the IEL compartment. Moreover, considering that intestinal DCs are likely in close contact with intestinal mucus, it was hypothesized that interactions between DCs and mucins, the predominant proteins that form the mucus layer, may modulate DC function. To test this hypothesis, intestinal mucin was purified and used to treat human monocyte-derived DCs. It was found that that expression of the chemokine IL-8 and co-stimulatory DC markers CD86 and CD83 are significantly upregulated on human DCs in the presence of intestinal mucins. Additionally, IL-8 produced by mucin-treated DCs is able to recruit neutrophil-like cells in transmigration assays. These effects were not due to mucin sample contaminants such as LPS, DNA or contaminant proteins. Instead, mucin glycans seem to be important for the induction of these effects on moDCs. Thus, in contrast to recent published results, intestinal mucins appear capable of inducing important pro-inflammatory functions in DC. To investigate whether mucins modulated DCs found in the intestinal environment, intestinal mucins were used to treat murine intestinal DCs, and gene changes explored using microarray analysis. It was found that, amongst several genes modulated in intestinal DC, up-regulation of the mucosal cytokine IL-22 was induced by intestinal mucin. Therefore, interactions between different components of the intestinal barrier might be crucial for maintaining gut homeostasis. Understanding how different components of the intestinal barrier system work together to maintain homoeostasis may identify pathways that can be targeted to restore this balance in inflammatory disorders such as inflammatory bowel disease.

Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function. To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains. Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences. Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.

No description available.