Chronic HCV infection causes generalized CD8+T cell impairment, not limited to HCV-specific CD8+ T cells. Infiltrating monocyte-derived macrophages contribute to a micro- environment that could impact CD8+T cells trafficking through the liver. Macrophages can differentiate into pro-inflammatory (M1) and anti-inflammatory (M2a, M2b, and M2c) subsets. Whether macrophage subset generation in chronic HCV infection is altered and if that has a subsequent impact on CD8+T cell functions was not known. I have shown phenotypic alterations in both M1 and M2 macrophages in chronic HCV infection. In particular, M1 from advanced fibrosis patients show increased CD86 expression, reduced spontaneous TNF-α and increased spontaneous IL-10 production. In uninfected controls, co-culturing CD8+T cells with M1 macrophages significantly increased the percentage of CD107a+ and IFN-γ+ CD8+T cells in a contact-dependent manner. Similar autologous co-cultures between M1 and CD8+T cells from patients with chronic HCV infection showed that M1 significantly reduced the percentage of IFN-γ+ CD8+T cells, even though patients displayed elevated IFN-γ+CD8+ T cells at baseline prior to culture. Overall, I demonstrated the altered phenotype of macrophages generated from patients with chronic HCV infection. I also showed the ability of M1 macrophages to induce IFN-γ+CD8+T cells in normal donors and their opposite impact when the cells are derived from chronic HCV infected patients.

Le cancer du sein est le cancer féminin le plus fréquent et celui qui cause le plus de décès dans le monde. Il existe plusieurs types de cancer du sein qui diffèrent par l’expression des récepteurs aux œstrogènes, à la progestérone et du récepteur HER2. Le cancer du sein dit triple négatif (TNBC) n’exprime aucun de ces récepteurs. Le métabolisme des sphingolipides (SL) est fréquemment altéré dans les cancers du sein et semble participer à la progression tumorale. Tandis que les SL ont été décrits comme modulateurs des réponses immunitaires dans des modèles précliniques de cancer, il n’existe pas à ce jour d’étude chez l’Homme évaluant l’impact des altérations du métabolisme des SL sur le développement tumoral et la réponse immunitaire associée. Dans ce contexte, l’objectif de nos travaux est double. Le premier objectif est de caractériser une signature sphingolipidique dans des biopsies de tumeurs mammaires humaines de divers sous-types histologiques. Une analyse par spectrométrie de masse à haute résolution nous a permis d’identifier dans les TNBC des taux élevés de SL en -C24:0, qui pourraient constituer des biomarqueurs potentiels pour ce type de cancer. Le deuxième objectif est d’établir une signature sphingolipidique associée à la réponse immunitaire dans le cancer du sein. Nous avons analysé les lymphocytes infiltrant la tumeur (TIL) par cytométrie en flux et par immunohistochimie dans nos échantillons tumoraux. Ces analyses nous ont permis de montrer que, dans les TNBC, les taux de C16:0-céramide sont corrélés positivement avec la proportion de TIL CD8+ et négativement avec celle des Treg FoxP3+. Afin d’évaluer l’influence potentielle des SL sur la topologie d’infiltration des lymphocytes (intratumoral versus adjacent), nous avons aussi comparé les taux de SL dans les tumeurs ayant une forte ou une faible proportion de lymphocytes T CD8+ intratumoraux. Nous avons observé notamment des taux élevés de sphingosine-1-phosphate (S1P) dans les tumeurs faiblement infiltrées par les lymphocytes T CD8+ intratumoraux. Nos observations suggèrent que la production de C16:0-céramide soit en faveur d’un recrutement préférentiel des lymphocytes T CD8+ dans les TNBC, tandis que la S1P pourrait être un facteur d’immunoéchappement, impactant négativement sur la topologie des TIL CD8+ dans les tumeurs mammaires. L’ensemble de nos travaux indique que certains SL pourraient constituer des biomarqueurs originaux de TNBC. De plus, la reprogrammation de ce métabolisme pourrait augmenter (i) quantitativement et qualitativement l’infiltration intratumorale des lymphocytes, et (ii) potentiellement l’efficacité des immunothérapies dans le cancer du sein. / Breast cancer, the most common malignancy affecting women, is responsible for the majority of woman death by cancer worldwide. There are three different breast cancer types defined by the expression of oestrogen and progesterone and HER2 receptors. The so-called triple negative breast cancer (TNBC), doesn’t express any of these receptors. Sphingolipid (SL) metabolism is frequently altered in breast cancer, alterations are associated with tumour progression. Although SL can act as immune response modulators in preclinical cancer models, there are, to date, no study assessing the impact of SL metabolism alterations on tumor development and associated immune response in Humans. In this setting, our aim was double. Our first objective was to characterize the SL signature in human mammary tumour biopsies from different pathological subtypes. Using high-resolution mass spectrometry assay, we identified elevated levels of C24:0-SL in TNBC, which could be used as potential biomarkers of this cancer subtype. Our second objective was to identify a SL signature associated with immune responses in breast cancer tissues. Tumour-infiltrating lymphocytes (TIL) were analyzed by flow cytometry and immunohistochemistry. Those analyses showed that, in TNBC, levels of C16:0-ceramide are positively correlated with the proportion CD8+ TIL and negatively correlated with that of FoxP3+ Treg TIL. In order to evaluate the potential influence of SL on the topology of infiltrating lymphocytes (intratumoural versus adjacent), we compared the SL levels in tumors depicting high or low proportions of intratumoral CD8+ T cells. We observed higher levels of sphingosine-1-phosphate (S1P) in low infiltrated intratumoral CD8+ T cells. Our observations suggest that C16:0-ceramide production could favor CD8+ T cell recruitment in TNBC, whereas S1P could act as an immune escape factor, negatively impacting on CD8+ TIL topology in mammary tumours. Our whole work indicates that a set of specific SL could constitute original biomarkers in TNBC. Moreover, reprogramming SL metabolism could improve (i) the infiltration of tumours by lymphocytes, both in terms of quality and quantity, and (ii) possibly, the efficacy of immunotherapy in breast cancer.

Vaccines have become an important approach for the treatment of cancer. Cancer vaccines help the immune system to detect and eradicate tumor cells. Also, cancer vaccines are designed to stimulate an effective immune response that can create long-term immune memory to prevent tumor recurrence. This treatment approach involves the administration of a vaccine comprising or encoding an antigen and can often be combined with an adjuvant to further promote the immune response. The goal of this research was to study the effect of the polyanhydride composition of prophylactic cancer vaccine formulations on the tumor-specific immune response. To achieve this goal, three different amphiphilic polyanhydride copolymers were generated comprising different ratios of 1,6-bis-(p-carboxyphenoxy)-hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) or sebacic anhydride (SA) monomers. These copolymers were used to fabricate particles encapsulating a model antigen, ovalbumin (OVA), using a double emulsion solvent evaporation technique. The ability of the three different compositions of amphiphilic polyanhydride copolymers (50:50 CPTEG:CPH, 20:80 CPTEG:CPH, and 20:80 CPH:SA) encapsulating OVA to elicit immune responses was investigated. Further, the impact of soluble unmethylated oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODN), an immunologic adjuvant, on the immune response to the three formulations was also studied. The immune response to cancer vaccines was measured after treatment of C57BL/6J mice with two subcutaneous injections, seven days apart, of 50 μg OVA encapsulated in particles composed of different polyanhydride copolymers with or without 25 μg CpG ODN. In vivo studies showed that 20:80 CPTEG:CPH particles encapsulating OVA significantly stimulated the highest level of CD8+ T lymphocytes, generated the highest serum titers of OVA-specific IgG antibodies, and produced longer survival in comparison to formulations involving the other polyanhydride copolymers. The results also revealed that supplementing the vaccine formulations with CpG ODN did not enhance the immunogenicity of OVA. These results accentuate the crucial role of the copolymer composition of polyanhydrides in stimulating the immune response and improving cancer vaccine efficacy.

Introduced species are the greatest threat to biodiversity after habitat loss. Understanding the processes that permit organisms to become successful invaders may provide opportunities to prevent or limit their dispersal and establishment and thereby alleviate some of their harmful effects. The goal of my dissertation research has been to investigate whether invasive species have distinctive interactions with parasites, and some of the mechanisms that may underlie that variation. I used one of the world's most successful vertebrate invaders as a case study: the house sparrow (Passer domesticus; Introduction). Previous research in the house sparrow suggested that loss of parasite diversity may contribute to invasion success. However, my work demonstrates that infection with common avian malaria parasites is primarily a function of environmental heterogeneity and is not a predictor of time since introduction for house sparrows that are currently expanding their range in Kenya (Chapter 1). Interestingly, in spite of a large proportion of the population being infected with avian malaria, a state that should reduce competitive ability of house sparrow populations, this species is still able to establish themselves among native competitors. Though there are a number of potential mechanisms that could explain this pattern, one of the most convincing explanations is that house sparrows, and perhaps other introduced species, have adaptive differences in immunity. As such, the findings of Chapter 1 inspired two studies in which my collaborators and I showed that house sparrows from two non-native populations seem capable of maintaining normal health, performance and behavior during immune challenge, a response often referred to as parasite tolerance. Specifically, in Chapter 2, we found that when Floridian house sparrows, established since ~1870, were challenged with synthetic pathogens that mimicked infection with a fungi, an RNA virus or Gram-negative bacteria, only individuals challenged by the synthetic bacteria showed measurable sickness behaviors and secretion of an inflammatory protein. In Chapter 3, we compared parasite tolerance in Kenyan house sparrows (introduced in ~2000) and a native congener, the grey-headed sparrow (P. griseus) to a common intestinal parasite of songbirds. We found that both species were tolerant in that they were able to maintain fat reserves, protein reserves and vertical flight ability during infection. However, house sparrows maintained burdens that were, on average, more than 10x those of grey-headed sparrows. Moreover, when examining nutrient allocation in the two species, house sparrows appeared to assimilate nutrients more efficiently than grey-headed sparrows and did not change how nutrients were allocated among immune and reproductive organs during experimental infection. Grey-headed sparrows, however, did shift nutrient allocation among immune and reproductive organs during experimental infection. Together, the larger nutrient pool and maintenance of nutrient allocation patterns in challenged house sparrows suggests that no physiological trade-offs occurred and that house sparrows experienced a lower cost of parasite exposure. In the fourth Chapter, I explored why house sparrows had such high coccidia burdens in comparison to their congeners. We suspected burden was a function of the frequency of exposure to coccidia. Consequently, we explored heterogeneity in foraging preferences and other behaviors in Floridian house sparrows and their role in coccidia burden. As expected, we found that house sparrows did not avoid contaminated food. In fact, they ate contaminated and uncontaminated foods indiscriminately. What was surprising was a lack of correlation between burden and consumption of contaminated foods and all of the behaviors we monitored (i.e., aggression, activity, feeding rates and defecation frequency). Overall, these data suggest that house sparrows do not benefit from typical parasite-avoidance behaviors. In sum, this dissertation research implies that house sparrows respond to parasite infection differently than many other known vertebrates, most likely in an effort to maximize efficient use of resources and, in so doing, augment competitive ability and invasion success.

Biomaterials are becoming the most commonly used therapeutic method for treatment of lost or damaged tissue in the body. Metallic materials are chosen for high strength orthopaedic and dental applications. Titanium (Ti) implants are highly successful in young, healthy patients with the ability to fully integrate to surrounding tissue. However the main population requiring these corrective treatments will not be healthy or young, therefore further research into material modifications have been started to improve outcomes in compromised patients. The body’s immune system will generate a response to any implanted material, and control the final outcome. Among the first and most influential, cells to interact with the implant will be macrophages. Throughout this study we have 1) established the ability of macrophages to recognize and differentially activate in response to material surface properties, 2) investigated the role of integrin binding in macrophage activation to material properties, and 3) confirmed the importance of macrophage activation in vivo following Ti implant placement. The generation of a hydrophilic implant surface promoted the greatest anti-inflammatory and pro-regenerative macrophage activation. Surface wettability will control protein adsorption which can activated different integrin binding on macrophages and may be responsible for changes in activation. When integrin β3 subunit binding was prevented hydrophilic surfaces no longer promoted an anti-inflammatory macrophage activation. Additionally, when macrophage levels were reduced using two separate ablation models, MaFIA mice and clodronate liposomes, hydrophilic surfaces no longer promoted anti-inflammatory T-cell populations and cytokine profiles. There were also fewer stem cells adhered to implant surfaces at 1, 3, and 7 days when macrophage populations were compromised.

La thérapie génique peut être définie comme le transfert du gène thérapeutique dans le tissue d’intérêt à l’aide d’un vecteur. A ce jour, les vecteurs dérivés du virus adéno-associés (AAV) représentent les vecteurs de choix pour le transfert de gène in vivo. Cependant, les réponses immunitaires dirigées contre la capside AAV représentent l’obstacle majeur à l’efficacité du transfert de gène médié par le vecteur AAV. Ce travail de thèse a eu pour objectifs de prévenir et d’inhiber la réponse humorale dirigée contre le vecteur AAV. En administrant des nanoparticules contenant de la rapamycine (ou SVP[Rapa]) avec le vecteur AAV, nous avons démontré une inhibition spécifique des réponses humorale et cellulaire dirigées contre la capside AAV. De plus, cette stratégie nous a permis de re-administrer efficacement le vecteur AAV dans des modèles murins et chez le singe. Nos données ont aussi démontré l’élimination des anticorps préexistants dirigés contre le vecteur AAV en administrant SVP[Rapa] avec le bortezomib. Au cours de travail de thèse, nous avons aussi développée et testée l’efficacité des vecteurs AAV associés aux vésicules extracellulaires (vecteurs exo-AAV) à améliorer l’efficacité des vecteurs AAV. En utilisant les vecteurs exo-AAV, nous avons démontré une expression stable et durable du transgène. De plus, les vecteurs exo-AAV ont montré une résistance aux anticorps neutralisants préexistants. Ainsi, au cours de ce projet de thèse des stratégies efficaces ont été développées afin de prévenir et de contrôler les réponses immunitaires dirigés contre le vecteur AAV, permettant ainsi une re-administration efficace du vecteur AAV. / Gene therapy aims to achieve sustained expression of the therapeutic transgene by the introduction of vector cargo into target tissue. To date, viral vectors based on adeno-associated virus (AAV) represent the leading gene delivery tools in vivo. However, immune responses against AAV vector represent the biggest challenge for the widespread use of AAV-based products. In this PhD project, we aimed at the inhibition and prevention of humoral immune responses to AAV capsid. Using nanoparticles containing rapamycin (SVP[Rapa]) given at the time of vector administration, we demonstrated complete abrogation of anti-capsid humoral and cellular immune responses in antigen-specific manner. Using this strategy, we further demonstrated successful vector re-administration in murine models and in non-human primates. Our data also demonstrated elimination of pre-existing antibodies to AAV vector using a combination of SVP[Rapa] and bortezomib. In this PhD thesis project, we also developed and tested the ability of AAV vectors associated with extracellular vesicles (exo-AAV vectors) to enhance AAV vector potency. Using exo-AAV vectors, we demonstrated higher and sustained transgene expression at low vector doses. Exo-AAV vectors also exhibited resistance to neutralization by pre-existing anti-capsid neutralizing antibodies. Thus in this PhD project, powerful strategies have been developed to prevent and control immune responses against AAV vectors, enabling successful AAV vector re-administration.

No description available.

[Truncated abstract] HIV infection compromises the ability of the host to mount effective immune responses. In untreated HIV disease, immune activation drives high rates of cell turnover and apoptosis, ultimately leading to abnormal and dysregulated cellular function. Immune activation may also induce the expansion of CD4+ regulatory T (Treg) cell populations capable of suppressing anti-HIV responses. Treatment with antiretroviral therapy (ART) allows the recovery of CD4+ T cell numbers in most patients. Persistent deficiencies in the number and function of CD4+ T cells seen in a proportion of individuals may reflect elevated numbers of Treg cells or an imbalanced regulatory-to-effector cytokine milieu. Furthermore, some patients develop paradoxical illnesses associated with the recovery of cellular function, known as immune restoration disease (IRD). The first part of this thesis addresses the role of CD4+ Treg cells in untreated and treated HIV disease. The second part addresses the phenotype of immune cells that express IL-10 and its receptor in untreated and treated patients, and the role of IL-10 in mycobacterial IRD. Firstly, several cell surface markers were evaluated to find a flow cytometry assay that could be used routinely to identify CD4+ Treg cells in HIV-infected patients. I tested CD25, GITR, CTLA-4, NRP-1 and LAG-3, but their expression did not mirror the expression of FoxP3, an intracellular transcription factor specific to CD4+ Treg cells (Chapter 2). Two published studies then described the use of CD127 to identify CD4+FoxP3+ Treg cells in humans. Using CD127, I determined the proportions and numbers of CD4+ Treg cells in untreated HIV-infected patients and in patients in their first year of ART. Proportions of CD4+ Treg cells correlated with the proportions of activated (HLA-DRHI) CD4+ T cells and with plasma HIV RNA levels in untreated patients, but showed an inverse correlation with CD4+ T cell count. In both untreated and treated patients, the proportions and numbers of FoxP3+ cells that expressed CD8 were significantly higher than in uninfected donors. This was clearest in patients with CD4+ T cell counts below 300/'L (Chapter 3). This body of work suggests that the frequencies of CD4+ Treg cells are directly related to the level of HIV-associated immune activation. Phenotyping of FoxP3+CD4+ Treg cells in untreated and treated patients and in uninfected donors revealed that co-expression of CD45RO, CD28, CTLA-4 and markers of activation were similar in all HIV-infected patients and controls. ii FoxP3+CD8+ T cells exhibit lower levels of CD45RO, CD28 and CTLA-4, but higher expression of PD-1 and CD57 (Chapter 4). This suggests that FoxP3+CD8+ T cells may have a reduced functional capacity. It is unclear whether they have regulatory activity by virtue of FoxP3 expression. ... Both patients produced higher levels of IFN? compared with IL-10 in response to mycobacterial antigens. In contrast, patients who experienced uneventful immune reconstitution produced higher levels of IL-10 (Chapter 6). Part 1 of this thesis highlights the importance of using specific cellular markers to identify CD4+ Treg cells, and confirms CD127 as a valuable marker for routine monitoring of blood Treg cells. Part 2 of this thesis demonstrates the important regulatory role of IL-10 in patients receiving ART.

[Truncated abstract] The design and development of effective anti-viral immunotherapies requires a comprehensive understanding of the cellular and molecular processes that are involved in the generation and regulation of immune responses. The fundamental objective of the immune system is to successfully complete the task of eliminating/controlling the invading pathogen without causing overt pathology. Cytomegaloviruses (CMVs) are large DNA viruses that are able to evade immune attack and persist lifelong within the host. In a healthy host, CMV causes an asymptomatic infection, but in instances of decreased immune functions, such as in newborns, acquired immunodeficiency syndrome (AIDS) patients and transplant recipients, the infection can result in serious morbidity and mortality. Thus, human CMV (HCMV) is a clinically important pathogen and an understanding of the pathogenesis, mechanisms of immune subversion and, importantly the cascade of immune events that ensue following infection is highly relevant. The studies presented in this thesis have provided useful insight into various aspects of viral immunity and it is hoped that they will assist in the design of more effective therapies against viruses of clinical importance. Genetic variability in humans can greatly influence anti-viral immune responses and the outcome of viral infection. ... Furthermore, these studies provide novel evidence that NK cells are also crucial for the control of virus in some organs of susceptible mice during early acute infection. The data reveals that both NK cells and CD8+ T cells utilise perforin- and IFN-? dependent control of MCMV. Furthermore, these studies provide novel evidence that protection mediated by Ly49H+ NK cells in resistant mice is dependent on perforin. Chapter 3 focuses on the biological relevance of Grz during MCMV infection. These studies found that GrzA and GrzB are essential components of the machinery involved in limiting MCMV during acute infection. These analyses also provide the first evidence suggesting that GrzM plays a role, albeit minor, in controlling MCMV replication. Furthermore, the current studies suggest that Grz can mediate direct antiviral activities independent of the induction of cell death in conjunction with perforin. Interestingly, in the absence of both GrzA and GrzB (GrzAB), mice were as susceptible to MCMV infection as perforin-deficient mice. However, unlike perforin-deficient mice, GrzAB-deficient mice controlled and survived the infection. In Chapter 4 the roles of perforin, GrzA and GrzB in anti-viral immunity and immunopathology during MCMV infection were examined. These studies show that NK cell-derived perforin is required to eliminate infected targets as well as activated effector cells, suggesting that NK cells are crucial not only in defensive immunity but also in limiting the immune activation that follows MCMV infection. In summary, the studies presented in this thesis define the significant role played by specific effector molecules in limiting MCMV replication during different stages of this viral infection. Furthermore, these studies provide novel evidence that perforin, GrzA and GrzB play distinct roles in defensive immunity and limiting immunopathology during MCMV infection.

Deficiencies in immune responses can lead to increases in the rate of infections and chronic diseases, such as cancer. Critical to the adaptive immune response is the activation of the T helper (Th)/CD4+ cell, the subsequent production of interleukin 2 (IL-2), expression of IL-2 and transferrin receptors (IL-2R, TfR) and transcription of genes resulting in DNA synthesis and T cell clonal expansion. The CD4+ lymphocyte response is impaired with ageing. Recent evidence suggests that moderate, regular aerobic training may increase the responsiveness of CD4+ lymphocytes to antigenic and mitogenic challenge, and thereby improve immune function in the older individual. Large volumes of chronic endurance training, and also high intensity training, may adversely affect the immune response, leading to immunosuppression and increased risk of infections. Impaired immune function and increased rates of URTI are found in athletes who undergo large volumes of training, often at high intensity. Purpose: To investigate if long-term aerobic training improved the immune response in men and women aged 65 to 75 years and, and to investigate if long-term endurance training depressed the immune response in male athletes aged 23 to 36 years. Methods:T helper lymphocyte proliferation was assessed monthly, by inducing the expression of CD25 (IL-2R ) and CD71 (transferrin) receptors with phytohemagglutinin (PHA). Percentage of CD4+ cells positive for the receptors, and the receptor density, were measured using two colour flow cytometry. Concentrations of intracellular calcium (Ca2+) and iron (Fe3+) were also measured monthly to determine the effect of endurance training on intracellular Ca2+ ([Ca2+]i) and Fe3+ ([Fe3+]i) within the CD4+ lymphocyte signal transduction pathway. Results: After twelve months of moderate aerobic training the percentage of CD4+ lymphocytes positive for CD25 increased in males aged 65 to 75 years, but not in females. There was no training effect on the density of CD25 in either gender, nor was there a training-induced increase in [Ca2+]i, total intracellular [Ca2+] from endoplasmic reticulum stores ([Ca2+]t) or [Fe3+] in this age group. Significant month to month variations in leucocyte, erythrocyte and haemoglobin concentration, mean corpuscular haemoglobin concentration, haematocrit, platelets, CD25 expression, CD71 expression, [Ca2+] and [Fe3+] were documented for both trained and untrained male and female groups. Aerobic capacity increased significantly with training for both men and women, with increases in peak, peak power and peak ventilation (p less than 0.05). Twelve months of chronic endurance training produced significantly lower haemoglobin, mean corpuscular haemoglobin and platelet concentration for six ([Hb]) and nine months ([MCHC], platelets) of the year in Ironman-distance triathletes, compared to sedentary males aged 23 to 36 years. There was no evidence of immunosuppression in the trained group, with no significant differences between groups in the percentage of CD4+ cells positive for CD25. The trained group showed a significantly higher density of CD25 receptors in October, January and June, suggesting a better immune response during these months. Endurance training did not effect [Ca2+] or [Fe3+]. The trained group did not show a reduced leucocyte concentration, and reported significantly fewer cases of URTI in twelve months than their sedentary counterparts. The 23 to 36 years age group showed seasonal changes in haematological and immunological indices similar to older individuals, indicating that autumn, late winter and late spring are periods of reduced immuno-competency. Conclusion: Twelve months of moderate intensity training significantly increased functional capacity in older men and women, and the percentage of CD4+ lymphocytes expressing CD25 in older men, thereby improving the lymphoid immune response. Twelve months of endurance training significantly increased CD25 density in CD4+ lymphocytes in Ironman triathletes compared to sedentary young males. The monthly changes in immune variables in young and older subjects suggested that autumn, late winter and late spring might be periods where individuals were more at risk of succumbing to infections due to decreased lymphocyte responsiveness. Summer months appeared to be a period of increased lymphocyte responsiveness and proliferation.