Tesi sul tema "Immunity"

L'immunité juridictionnelle des États a été pendant des siècles une question incontestée fondée sur le principe de l'égalité des Etats et sur leur indépendance absolue. Cette règle a été élaborée à une époque où tenter une action contre un État dans un pays étranger aurait été considéré comme une violation de sa souveraineté. Toutefois, les fonctions des Etats ont changé au cours des siècles. Désormais, les Etats s’engagent dans les activités commerciales comme une personne privée et jouent un rôle essentiel dans ce secteur.Alors, bien que le droit de l'immunité soit lié à l'octroi de l'immunité aux États pour leur permettre d'accomplir efficacement les fonctions publiques, le droit international moderne n'exige pas que les tribunaux d'un Etat s’abstiennent de connaître un litige simplement parce que l’État étranger n'a pas la volonté de défendre.Ce travail de recherche, est donc consacré à l’étude de l’immunité de juridiction et l’immunité d’exécution afin de montrer le droit international actuel sur la matière.Cette thèse vise à examiner une question spécifique qui a été mise en évidence au cours de ces dernières années. Comment et dans quelle mesure les États et ses démembrements devraient être soumis à des règles spécifiques de l'immunité d’Etat ?
The issue of jurisdictional immunity of states was for centuries an undisputed matter based on the principle of state equality and absolute independence of states. The rules were developed at a time when it was thought to be an infringement of a state's sovereignty to bring proceedings against it or its officials in a foreign country. However, the functions of states have changed over the centuries and nowadays states are involved in commercial activities as a private person and accordingly play an essential role in the commercial activities of the world. In fact, the issue of state immunities is an increasingly important and rapidly developing area of international law and practice. The state practice reflects the emerging global consensus that States and State enterprises can no longer claim absolute, unrestrained immunity from the proper jurisdiction of foreign courts, especially for their commercial activities. Therefore, although the law of state immunity is related to the grant of immunities to states to enable them to carry out their public functions effectively, modern international law does not require the courts of one state to refrain from deciding a case merely because a foreign state is an unwilling defendant. It is therefore important to know how a plea of state immunity may be made and to what type of dispute it applies

L’existence d’un partage des tâches a été démontrée au sein de nombreux systèmes biologiques et ce notamment en immunologie où il a été décrit dans le contexte de différentes sous-populations d’un même type cellulaire. Les cellules dendritiques plasmacytoïdes (pDC) jouent un rôle clé lors des infections virales. Les pDCs ont la capacité de sécréter de grandes quantités d’interférons de type I et de se différencier en cellules dendritiques matures capables d’activer une réponse immunitaire adaptative. Il a été proposé que ces fonctions innées et adaptatives soient séquentiellement induites après activation virale. Au cours de ma thèse, je me suis intéressée à ces deux fonctions principales des pDC et je suis arrivée à la description de différentes sous-populations de pDC activées : PD-L1+CD80- (P1), PD-L1+CD80+ (P2) and PD-L1-CD80+ (P3), démontrant qu’il existe un partage des tâches entre ces sous-types. P1 produit spécifiquement de l’IFN-α, indiquant une spécialisation en immunité innée, et promeut une réponse tolérogénique des cellules T CD4. Inversement, P3 induit une forte activation des cellules T CD4 naïves et une polarisation de type Th2, démontrant une spécialisation fonctionnelle dans l’immunité adaptative. P2 possède un profil fonctionnel intermédiaire. Plutôt qu’un lien séquentiel, nos résultats indiquent une exclusion réciproque des fonctions innées et adaptatives entre ces différents sous-types de pDC
Under microbial stimulation plasmacytoid pre-dendritic cells (pDC) secrete large amounts of type I interferon (IFN) and differentiate into mature dendritic cells capable of activating T cells. These innate and adaptive functions are thought to be induced sequentially in pDC through triggering of the IRF-7 and NFkB pathways, respectively. We found that viral activation of pDC induced their differentiation into three phenotypically distinct subsets: PD-L1+CD80- (P1), PD-L1+CD80+ (P2) and PD-L1-CD80+ (P3). P1 specifically produced IFN-α, indicating a specialization in innate immunity, while promoting weak activation and high IL-10 expression in CD4 T cells. Conversely, P3 showed increased expression of surface costimulatory molecules, improved migratory capacity, strong naïve CD4 T cell activation, and induction of Th2 differentiation. P2 had an intermediate functional profile. No conversion could be induced between subsets. We identified P1 in psoriatic skin, and blood from active lupus patients. Our results indicate reciprocal exclusion, rather than sequential link, of innate and adaptive pDC functions, with important implications in immune regulation and immunopathology

Il est connu depuis de nombreuses années que le métabolisme des cellules cancéreuses diffère de celui des cellules saines. La Restriction Calorique (RC) est connue pour prolonger la durée de vie et pour limiter l’oncogenèse. Ainsi, il a été montré que la RC et ses mimétiques comme le 2-deoxyglucose (2DG) augmentent l’efficacité de la chimiothérapie et peuvent aussi induire une immunité anti-tumorale. J’ai pu montrer qu’en régulant le métabolisme via la restriction calorique (ou des mimétiques) nous pouvions moduler l’expression de la protéine anti-apoptotique Mcl-1. Ainsi nous avons établi in vivo et in vitro que la RC restaure la sensibilité des cellules de lymphome à l’apoptose induite par un inhibiteur de Bcl-2/XL, l’ABT-737. Nous avons aussi établi que ces effets sont indépendants de la protéine p53 et/ou des « protéines BH3-only ». La deuxième partie de mon travail a été d’élucider les mécanismes moléculaires mis en place lors de la Chimiothérapie Hyperthermique Intra péritonéale (CHIP) pouvant expliquer les effets bénéfiques observés chez les patients atteints d’une carcinose péritonéale (CP). Une partie de ces bénéfices sont dus à la mise en place d’une immunité anti-tumorale. En utilisant des modèles in vivo et in vitro j’ai mis en évidence l’implication de la protéine du choc thermique 90 (Hsp90) dans l’effet observé. Ainsi, l’inhibition spécifique de la Hsp90 réverse les effets protecteurs de la CHIP, soulignant l’importance de cette protéine dans notre modèle d’immunité anti-tumorale
The link between cell metabolism and cancer at the cellular level has long been known. Caloric restriction (CR) is known to prolong lifespan and to protect from cancer incidence. The molecular mechanisms involved in these benefic effects have been evaluated and may offer new opportunities for therapeutic intervention. Moreover, CR and CR-mimetics such as 2-deoxyglucose (2DG) has been shown to enhance chemotherapy efficiency and to induce an anti-cancer immune response. During the period of my PhD I demonstrated how the modulation of metabolism through caloric restriction or through its mimetics could significantly reduce the expression of the anti-apoptotic protein Mcl-1 and sensitize lymphoma-bearing mice to apoptosis induced by a Bcl-2/XL inhibitor, ABT-737. We have demonstrated that CR can control Mcl-1 translation and sensitize cells to ABT-737-induced death regardless of the presence or absence of p53 and/or of the main “BH3-only proteins”. Then, I focused on deciphering the molecular mechanisms allowing the Hyper-thermic Intra-Peritoneal Chemotherapy (HIPEC) to be beneficial to patients suffering from peritoneal carcinomatosis. Part of the protective effect was mediated through the induction of an efficient anti-cancer immune response. Next, I showed the involvement of heat shock proteins 90 (Hsp90) in the observed effect. Indeed, when Hsp90 was blocked we lost the protection induced by the HIPEC-treated cells, therefore underling the role of Hsp90 in this HIPEC-dependent induction of anti-cancer immune response

Les anticorps bloquant les points de contrôle immunitaires modifient profondément la gestion des patients atteints de cancer. À la pointe de cette nouvelle classe d'agents anticancéreux, les anticorps anti-PD-1 / PD-L1 peuvent ainsi restaurer une réponse efficace des cellules T antitumorales. En conséquence, ces agents sont maintenant approuvés dans divers types de tumeurs, tels que le mélanome, le cancer bronchique non à petites cellules, le cancer du rein, les tumeurs ORL ou le cancer de la vessie. Ces nouvelles immunothérapies entraînent également de nouveaux profils de réponse tumorale tels que des réponses tumorales retardées ou des pseudoprogressions. Au fil de l’expérience acquise avec ces traitements, il a été observé chez certains patients un état de progression rapide de la maladie, ce qui pourrait suggérer que le blocage de points de contrôle immunitaire pourrait avoir un effet délétère en accélérant la maladie chez un sous-groupe de patients. Ce travail de thèse a permis de caractériser sur le plan clinique et biologique ce phénomène d’accélération de la croissance tumorale sous immunothérapie anti-checkpoint que nous avons définit “maladie hyperprogressive” (HPD). L’analyse transcriptomique d’échantillons tumoraux de ces patients a permis d’orienter vers un rôle spécifique de l’environnement myeloide
Immune checkpoint blocking antibodies are profoundly changing the management of patients with cancer. At the forefront of this novel anticancer agent class, anti-PD-1/PD-L1 antibodies can exhibit a significant activity by restoring an efficient antitumor T-cell response. As a result, these agents are now approved in various tumor types such as melanoma, squamous, and nonsquamous non–small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC) or bladder cancer. Interestingly, these new immunotherapies also result in novel tumor response patterns such as delayed tumor responses or pseudoprogressions. As experience grows with these therapeutics, anecdotal reports are relating rapid disease progressions, which could suggest that immune checkpoint blockade may have a deleterious effect by accelerating the disease in a subset of patients. This thesis work has made it possible to characterize clinically and biologically this phenomenon of accelerated tumor growth under anti-checkpoint immunotherapy, which we have defined as “hyperprogressive disease” (HPD). Transcriptomic analysis of tumour samples from these patients suggested a specific role for the myeloid environment

Il est connu depuis de nombreuses années que le métabolisme des cellules cancéreuses diffère de celui des cellules saines. La Restriction Calorique (RC) est connue pour prolonger la durée de vie et pour limiter l’oncogenèse. Ainsi, il a été montré que la RC et ses mimétiques comme le 2-deoxyglucose (2DG) augmentent l’efficacité de la chimiothérapie et peuvent aussi induire une immunité anti-tumorale. J’ai pu montrer qu’en régulant le métabolisme via la restriction calorique (ou des mimétiques) nous pouvions moduler l’expression de la protéine anti-apoptotique Mcl-1. Ainsi nous avons établi in vivo et in vitro que la RC restaure la sensibilité des cellules de lymphome à l’apoptose induite par un inhibiteur de Bcl-2/XL, l’ABT-737. Nous avons aussi établi que ces effets sont indépendants de la protéine p53 et/ou des « protéines BH3-only ». La deuxième partie de mon travail a été d’élucider les mécanismes moléculaires mis en place lors de la Chimiothérapie Hyperthermique Intra péritonéale (CHIP) pouvant expliquer les effets bénéfiques observés chez les patients atteints d’une carcinose péritonéale (CP). Une partie de ces bénéfices sont dus à la mise en place d’une immunité anti-tumorale. En utilisant des modèles in vivo et in vitro j’ai mis en évidence l’implication de la protéine du choc thermique 90 (Hsp90) dans l’effet observé. Ainsi, l’inhibition spécifique de la Hsp90 réverse les effets protecteurs de la CHIP, soulignant l’importance de cette protéine dans notre modèle d’immunité anti-tumorale
The link between cell metabolism and cancer at the cellular level has long been known. Caloric restriction (CR) is known to prolong lifespan and to protect from cancer incidence. The molecular mechanisms involved in these benefic effects have been evaluated and may offer new opportunities for therapeutic intervention. Moreover, CR and CR-mimetics such as 2-deoxyglucose (2DG) has been shown to enhance chemotherapy efficiency and to induce an anti-cancer immune response. During the period of my PhD I demonstrated how the modulation of metabolism through caloric restriction or through its mimetics could significantly reduce the expression of the anti-apoptotic protein Mcl-1 and sensitize lymphoma-bearing mice to apoptosis induced by a Bcl-2/XL inhibitor, ABT-737. We have demonstrated that CR can control Mcl-1 translation and sensitize cells to ABT-737-induced death regardless of the presence or absence of p53 and/or of the main “BH3-only proteins”. Then, I focused on deciphering the molecular mechanisms allowing the Hyper-thermic Intra-Peritoneal Chemotherapy (HIPEC) to be beneficial to patients suffering from peritoneal carcinomatosis. Part of the protective effect was mediated through the induction of an efficient anti-cancer immune response. Next, I showed the involvement of heat shock proteins 90 (Hsp90) in the observed effect. Indeed, when Hsp90 was blocked we lost the protection induced by the HIPEC-treated cells, therefore underling the role of Hsp90 in this HIPEC-dependent induction of anti-cancer immune response

This thesis concerns the attempt to establish human rights exceptions to foreign state immunity. The problem has multiple facets. Firstly, suits against foreign governments should be distinguished from suits against foreign officials. Further, in the latter context there is a distinction both between criminal and civil cases and between cases against individuals with immunity ratione personae and those with immunity ratione materiae. Individuals suffering extraterritorial jus cogens violations have been increasingly seeking justice against foreign governments and officials. Restrictive immunity largely displaced absolute immunity in the Western and developing world during the latter half of the Twentieth Century. This restrictive immunity only retained immunity for acta jure imperii. Many common-law nations entered into treaties and enacted foreign state immunity legislation purportedly embodying the restrictive doctrine, but these treaties and statutes actually accord a complete immunity to foreign states, subject only to specific, enumerated exceptions. Drafted mostly from the 1960s to the 1980s, they are to some extent from a bygone era. The chief issue of the time was whether state-owned trading entities should be immune from suit. The rights of private traders were upheld with the recognition of inter alia the commercial activity exception. In modern times, the human rights or jus cogens exception is now an important battleground. Research into attempts to establish such an exception to immunity was split into: (1) the origin and history of foreign state immunity and foreign official immunity; (2) the human rights dimension to foreign state immunity; and (3) the human rights dimension to foreign official immunity. In each part, representative cases were selected to best draw out the developmental contours. To aid holistic understanding of these cases, the litigation is followed from first instance to the exhaustion of appeals. The main findings of the thesis were, in regard to: (1) it is arguable whether sufficient uniformity in practice established absolute immunity as a binding norm and, even if it did, this could only have been during 1920-1976; (2) foreign state immunity statutes were mostly drafted before human rights cases against foreign states became an issue, as such they are not designed to cope and a jus cogens amendment may be necessary; (3) the High Court of Australia has the chance to break with older UK precedent, favoured by Canada, by paying closer regard to the discussion of the US Supreme Court on the matter of whether the definition of foreign state should include a foreign official.

Le monoxyde d'azote (NO) est une molécule produite par l'ensemble des cellules des voies aériennes. Sa synthèse à partir de la L-arginine fait appel à un groupe d'enzymes : les NO synthases (NOS). Il existe trois isoformes de NOS exprimées par des cellules ayant des fonctions diverses conférant ainsi au NO produit une spécificité d'action dépendante de la cellule et de la « situation » ayant entraîné sa synthèse. Les travaux effectués dans le cadre de cette thèse explorent des rôles différents (bronchoréactivité, immunitaires) du NO produit par les voies aériennes ainsi que la mesure du NO dans le contexte de l'anesthésie – réanimation. L'objectif du premier travail était de « revisiter » la fonction régulatrice du tonus bronchique dans le cadre d'une pathologie respiratoire, l'objectif du second travail était méthodologique (étude des sources anatomiques du NO expiré chez le patient sous ventilation mécanique), et l'objectif du troisième travail était d'utiliser la mesure validée dans le second travail pour évaluer les fonctions immunes du NO en réanimation.Premier travail. La compétition entre les NO synthases et les arginases pour leur substrat commun, la L-arginine, pourrait être impliquée dans régulation de la réactivité et du remodelage bronchique chez le patient atteint de BPCO. Le but du premier travail était d'évaluer la relation entre expression de cette balance enzymatique, et les effets pharmacologiques de l'inhibition des NOS et des arginases sur la réactivité bronchique ex vivo à l'acétylcholine de patients sans et avec une BPCO peu sévère. Pour cela, nous avons étudié les bronches de 22 patients. Des études immunohistochimiques nous ont permis de mettre en évidence une expression bronchique NOS-2 plus importante chez les patients BPCO comparée aux patients non BPCO. De plus, les études pharmacologiques réalisées en cuve à organe ont permis de mettre en évidence une tension bronchique de base plus importante chez les BPCO, corrélée à l'expression de la NOS-2 et au degré d'obstruction bronchique (VEMS). L'utilisation d'inhibiteur des NOS diminuait cette tension de base. Nous avons démontré ainsi qu'une augmentation de l'expression NOS-2 chez le BPCO était impliquée dans l'augmentation du tonus bronchique de base et dans l'obstruction bronchique.Second travail. Les variations de NO expiré après chirurgie cardiaque avec circulation extracorporelle (CEC) demeurent controversées. Le but de ce deuxième travail était de déterminer quelle source de NO expiré (bronchique ou alvéolaire) était modifiée après CEC, et d'étudier les effets de la ventilation mécanique pendant la CEC sur ces variations. Nous avons étudié ainsi 32 patients ventilés ou non durant une chirurgie cardiaque avec CEC. Nous avons observé une diminution significative du NO expiré d'origine bronchique après la CEC. Cette diminution n'était pas observée lors du maintien durant la CEC d'une ventilation avec pression expiratoire positive (PEEP). Ce travaille permettait de conclure que la diminution du NO bronchique après la CEC pouvait être liée à une occlusion des petites voies aériennes. Cette atteinte de petites voies aériennes était prévenue par la PEEP.Troisième travail. Enfin, dans ce troisième travail, nous avons émis l'hypothèse que la mesure du NO produit par les voies aériennes (NO expiré et nasal) pouvait constituer un marqueur prédictif de survenue d'infection nosocomiale (fonctions immunitaires du NO). Dans une étude observationnelle chez 45 patients de réanimation ventilés (15 patients ont développé une infection nosocomiale), le NO nasal était le seul marqueur significativement plus bas chez les patients développant une infection nosocomiale (le NO expiré, les dosages d'IL6 et d'IL10 ainsi que le score SOFA n'étaient pas différents entre les deux groupes). Un NO nasal inférieur à 148 ppb 72 heures après l'admission du patient, permettait de prédire la survenue d'une infection nosocomiale avec une sensibilité et une spécificité de 80% et de 70% respectivement et un odds ratio de 2.7. Le développement de ce bio marqueur simple à mesurer permettrait de mettre en place des stratégies préventives (immunonutrition avec de la L-arginine)
In the respiratory tract, NO is produced by a wide variety of cell types and is generated via oxidation of l-arginine that is catalyzed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (NOS-1), inducible NOS (NOS-2), and endothelial NOS (NOS-3). NO derived from the constitutive isoforms of NOS (NOS-1 and NOS-3) and other NO-adduct molecules (nitrosothiols) have been shown to be modulators of bronchomotor tone. On the other hand, NO derived from NOS-2 seems to be a proinflammatory mediator with immunomodulatory effects. This thesis explores the physiological and pathophysiological role of endogenous nitric oxide in the airways, and the clinical aspects of monitoring nitric oxide in exhaled air of patients with respiratory disease.First Study: competition between nitric oxide synthases (NOSs) and arginases for their common substrate l-arginine could be involved in the regulation of cholinergic airway reactivity and subsequent airway remodeling. The aims of this study were to evaluate the relationships between the expression of this enzymatic balance and the effects of NOS and arginase inhibition on bronchoconstrictive response to acetylcholine of patients without and with early chronic obstructive pulmonary disease (COPD). Twenty-two human bronchi were investigated for immunohistochemistry and modulation of acetylcholine-induced airway constriction. Significantly increased expression of NOS2 in immunoblots of bronchial tissue and staining in smooth muscle cells was evidenced in patients with COPD compared with control subjects. Forced expiratory volume in 1 s (FEV1) and NOS2 expression were negatively correlated. Pharmacological experiments demonstrated that resting tension was elevated in COPD compared with control subjects and was positively correlated with the expression of NOS2. The sole effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine was to decrease sensitivity in COPD patients, whereas NG-nitro-L-arginine methyl ester unexpectedly decreased resting tension because of a non-cGMP-dependent effect. In conclusion, an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity.Second Study: the change in exhaled NO after cardio-pulmonary bypass remains controversial. The aims were to determine whether exhaled NO sources (alveolar or bronchial) are modified after bypass, and whether mechanical ventilation (MV) settings during bypass modify exhaled NO changes. Thirty-two patients were divided into three groups: without MV during bypass and positive end-expiratory pressure (PEEP) (n=12), dead space MV without PEEP (n=10) and dead space MV with PEEP (n=10). Alveolar NO concentration and bronchial NO flux were calculated before and 1h after surgery using a two-compartment model of NO exchange developed in spontaneous breathing patients. Whereas a significant decrease in bronchial NO was found after bypass in the two groups without PEEP during bypass, this decrease was not observed in patients with dead space ventilation with PEEP. Alveolar NO was not significantly modified whatever the ventilation settings. In conclusion, the impairment of bronchial NO seemed related to airway closure since dead space mechanical ventilation with PEEP prevented its decrease.Third Study: the development of biomarkers able to predict the occurrence of nosocomial infection could help manage preventive strategies, especially in medical patients whose degree of acquired immunosuppression may be variable. We hypothesized that the NO fraction present in the airways (upper and lower) of critically ill patients under mechanical ventilation could constitute such a biomarker. We conducted an observational study in a medical intensive care unit. Forty-five patients (26 men; 72 [25th-75th percentiles] years [56-82]; Simplified Acute Physiology Score II, 63 [50-81], 14 infected) under mechanical ventilation (>3 days) underwent on day 1 and day 3 of their stay: nasal and exhaled (partitioned in bronchial and alveolar sources) bedside NO measurements, determination of urine NO end products and plasma cytokine (IL-6, IL-10) concentrations, and Sequential Organ Failure Assessment score calculation. Nosocomial infection incidence was recorded during the 15 subsequent days. Fifteen patients (33%) acquired a nosocomial infection. Nasal NO was the only marker significantly different between patients with and without subsequent infection (day 1, 52 ppb [20-142] vs. 134 [84-203], P = 0.038; day 3, 98 ppb [22-140] vs. 225 [89-288], P = 0.006, respectively). Nasal NO fraction 148 ppb or less at day 3 had an 80% sensitivity, a 70% specificity, and an odds ratio of 2.7 (95% confidence interval, 1.9-3.8) to predict acquisition of nosocomial infection. Nasal NO seems to be a relatively sensitive and specific biomarker of subsequent nosocomial infection acquisition

La tesi té com a objectiu investigar la resposta immune dels búfals d’aigua (Bubalus bubalis) durant malalties infeccioses (per exemple, mastitis, brucel·losi i tuberculosi) i malalties de produccions, com malalties metabòliques relacionades amb el peripart o l’estrès. Tenint en compte la relació entre l’entorn microbià i el sistema immunitari, també s’ha identificat el contingut de la microbiota de llet. El sistema immunitari del búfalo d’aigua ha estat poc caracteritzat fins ara, per no parlar de la microbiota, que era desconeguda. Els búfals d’aigua són sensibles a les mateixes malalties que els remugants, com per exemple mastitis, tuberculosi, brucel·losi, però no es coneix l’impacte d’aquestes malalties sobre el sistema immune dels búfals. El búfal d’aigua presenta diferències anatòmiques (per exemple a la glàndula mamària i a nivell de la pell) i fisiològiques (malalties infeccioses i periparts) en comparació amb els remugants. Per tant, és evident que tant el sistema immune com la microbiota podrien presentar diverses diferències. A la meva tesi doctoral vaig tractar d’abordar algunes d’aquestes qüestions. En primer lloc, es va investigar la microbiota de llet dels búfals d’aigua en relació amb la mastitis. En segon lloc, l’avaluació de la resposta immune, en termes d’expressió gènica i miRNAs, es va tractar en animals afectats per brucel·losi i tuberculosi. Finalment, es va realitzar la caracterització del període de transició mesurant proteïnes de fase aguda per avaluar l’estat de la inflamació durant el període de transició.
El objetivo de la tesis es investigar la respuesta inmunitaria de los búfalos de agua (Bubalus bubalis) durante las enfermedades infecciosas (por ejemplo, mastitis, brucelosis y tuberculosis) y las enfermedades de producción, como las enfermedades metabólicas relacionadas con el periparto o el estrés. Debido a que el sistema inmunitario está relacionado con el entorno microbiano, también se ha caracterizado la composición de la microbiota de la leche. Hasta el momento, el sistema inmunitario del búfalo de agua no estaba bien caracterizado y la microbiota de la leche era desconocida. Aunque los búfalos sean sensibles a las mismas enfermedades que los rumiantes (como por ejemplo mastitis, tuberculosis, brucelosis), se desconoce el impacto de estas enfermedades en el sistema inmunitario de los búfalos. En comparación con los otros rumiantes, el búfalo de agua presenta diferencias anatómicas (por ejemplo, en la glándula mamaria y en la piel) y fisiológicas (enfermedades infecciosas y peripartos). Por lo tanto, es evidente que tanto el sistema inmune como la microbiota podrían presentar varias diferencias. En mi tesis doctoral, he abordado algunos de estos problemas. En primer lugar, se investigó la microbiota de leche de los búfalos de agua durante la enfermedad de la mastitis. En segundo lugar, se evaluó la respuesta inmunitaria en animales afectados por brucelosis y tuberculosis, mediante técnicas de expresión génica y miRNAs. Finalmente, se caracterizó el período de transición midiendo las proteínas de fase aguda para evaluar el estado de inflamación.
The thesis aims to investigate the immune response of water buffaloes (Bubalus bubalis) during infectious diseases (e.g. mastitis, brucellosis and tuberculosis) and productions diseases, such as peripartum related metabolic diseases or stress. Given the relationship between the microbial environment and the immune system, the microbiota content of milk has been identified as well. The immune system of water buffalo has been poorly addressed so far, not to mention the microbiota, which was unknown. Dairy water buffaloes are sensitive to the same diseases as dairy ruminants, such as for example mastitis, tuberculosis, brucellosis, but the impact of these diseases on water buffaloes’ immune system are unknown. Water buffalo presents anatomical (e.g. at mammary gland and skin level) and physiological (peripartum and infectious diseases) differences as compared to cow and other dairy ruminants. Therefore, it is evident that both the immune system and microbiota could present several differences. In my PhD thesis, I tried to address some of these issues. Firstly, the milk microbiota of water buffaloes was investigated in relation to mastitis disease. Secondly, the evaluation of the immune response, in terms of gene expression ad miRNAs, was carried out in animals affected by brucellosis and tuberculosis. Finally, the characterization of the transition period was performed measuring acute phase proteins to assess the inflammation status during the transition period.

Ants and other social Hymenoptera (social bees and wasps) have a remarkable mating strategy. Social Hymenoptera live in societies where reproduction is monopolized by a fertile caste consisting of males and queens. On the other hand, the logistical tasks of the colony are carried out by a sterile female caste known as workers. Reproductive individuals mate during a single bout early in their life and will never engage in additional reproductive events later on. Males die soon after mating while queens store millions of sperm cells in a specialized organ, the spermatheca. Queens will use this sperm stock to fertilize eggs during the rest of their life that can last up to several decades. With a record of 28.5 years in the black garden ant, ant queens have the longest lifespan recorded to date among the social Hymenoptera. In my thesis, I addressed three aspects of ant queen reproduction. First, I tested the effect of mating on the expression of several genes involved namely in fecundity, longevity and immunity. I found that mating induces an up-regulation of the yolk precursor vitellogenin and of the antimicrobial peptide defensin. Second, I measured the intensity of different immune responses in male and queen genital organs in order to determine which immune pathways are activated to protect sperm. Antimicrobial peptide genes are expressed in the genital tract of both sexes and the queen spermatheca is capable of strongly inhibiting bacterial growth. The immune melanization response is, however, overall inactive in the organs tested probably because its unspecific mode of action and cytotoxic by-products are likely to damage sperm cells. Immunity thus seems to be closely regulated in organs that are in contact with sperm. Third, I determined if activation of the queen immune system had an impact on the survival of sperm stored in the spermatheca. There is no detectable effect in young newly mated queens whereas, in one year old queens, immune activation induces a significant reduction in sperm viability. Life stage thus seems to influence queen ability to preserve sperm viability in the event of an immune challenge. In addition, one year old queens have higher sperm viability than newly mated queens suggesting queens are able to displace dead sperm cells from their spermatheca. Finally, I relied on the well-established sequence of behaviors inherent to the early life of ant queens to try to uncover the largely unknown roles of inotocin, the insect ortholog of the vertebrate hormones oxytocin and vasopressin, in regulating insect behavior. I measured gene expression of the inotocin receptor and found that it is highly expressed during social and reproductive behaviors, which is consistent with previous results in vertebrates. Inotocin might thus also be involved in modulating these behaviors in insects, but further studies are needed to be able to fully understand this complex signaling system. Overall, I show that reproduction and immunity are closely linked in ant queens and that the latter provide promising models for investigating the roles of hormones in insects.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

A cross-linking model for the activation of the A cell or immune accessory cell as a function of certain extracellular conditions is developed to determine the valency of the specific factor receptor on the A cell surface. It is found that such a determination can be made based on the FWHM of cross-linking curves which differ by a full order of magnitude between the bivalent receptor case and the monovalent receptor case. This determination can be made provided one can obtain accurate values for the equilibrium constants which characterize the system and provided that activation and IL-1 secretion is a linear function of cross-linking. It is also found that a determination of valence can be made if the equilibrium constants are such that substantial one receptor bridge formation takes place (one antibody molecule bound on both ends by the same receptor). This one-receptor bridge formation only takes place if the receptor is bivalent, and it presents itself in the cross-linking curve in a very distinctive manner. A second network model described as an ecological competition model of steady state lymphocyte populations is presented. This model, known as the symmetrical network theory is analysed numerically by integration of the differential equations and shown to provide a reasonable qualitative picture of the immune system's stable steady states, and offer a glimpse of state switching.
Science, Faculty of
Physics and Astronomy, Department of
Graduate

This thesis covers three main areas of research. The first is a longitudinal sero-epidemiological study of a dairy herd which suffered an abortion storm linked to infection with N. caninum during August/September 1995. The main aims were to study the long term antibody response in cattle which have suffered N. caninum associated abortion, and to assess the rates of congenital infection, abortion and repeat abortion on the farm during the subsequent 3 year period. The second area of study investigated Neospora antigens recognised by Neospora antibody positive sera using western blot. Diagnosis of infection with N. caninum depends on detection of anti-N.caninum antibody in serum, but animals which have previously aborted due to neosporosis can become sero-negative by Neospora IFAT and ELISA several months post-abortion. The third area investigates cell mediated immune responses to N. caninum and the antigens involved in induction of T cell responses. N. caninum can induce repeat abortion in some individuals unlike the closely related coccidian parasite Toxoplasma gondii which induces life long protective immunity after primary infection. Cellular immune responses are important in the development of immunity to T.gondii and therefore are likely to be important in preventing repeat abortions in 95% of the cattle which abort due to neosporosis. This study showed that experimental infection of calves with N. caninum NC1 tachyzoites stimulated a cell mediated response detectable in peripheral blood using a simple proliferation assay. This response was characterised by the production of the T cell cytokine IFNγ which is produced by CD4+, CD8+ and natural killer cells and is known to be important for protection against other intracellular parasites.

Identifying immune biomarkers in healthy humans that indicate an increased susceptibility to upper respiratory tract illness (URTI) is necessary to develop improved diagnostic and treatment strategies. URTI is associated with substantial socio-economic and personal cost. Small to moderate reductions in the severity and duration of illness could lead to substantial reductions in these costs. This thesis investigated the relationship between the immune system and URTI in healthy individuals utilising exercise as a model of stress. Chapter 2 (Section 2.2) reviews the effects of exercise on the immune system and URTI, with a particular focus on the way in which exercise can be used to better understand the role of the salivary antimicrobial proteins (AMPs) lactoferrin and lysozyme in host defence. Determining mucosal immune status, that is the condition of the immune system at body surfaces interfacing with the external environment, is necessary to understand the role of the immune system in host defence. Exercise-related disturbances in the immune system may increase susceptibility to URTI, particularly when prolonged intense exercise is undertaken frequently. Th e link between exercise-induced disturbances in immunity and URTI risk suggests that exercise may be a useful model by which to study the relationship between immunity and illness in healthy individuals.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Physiotherapy and Exercise Science
Griffith Health
Full Text

IFITM1, 2 et 3 sont des protéines transmembranaires qui sont régulées à la hausse après stimulation interféron. Ces protéines sont capables d’inhiber un large spectre de virus. Le mécanisme d’action admis indique que la présence des IFITMs dans la membrane lipidique des cellules cibles diminue l’entrée des virus en bloquant la fusion de la membrane virale avec la membrane cellulaire.J’ai pris part en début de thèse à un travail qui a permis à notre équipe de mettre en évidence une deuxième configuration antivirale des protéines IFITMs contre le VIH-1 (Virus de l’Immunodéficience Humaine). En effet la présence des IFITMs dans les cellules productrices de virus et non seulement dans les cellules cibles permet deux choses: l’incorporation des IFITMs dans les particules virales et la baisse d’infectivité des virus produits. Suite à cette première étude, nous nous sommes posés deux problématiques: 1) comprendre le mécanisme d’inhibition du VIH-1 par les IFITMs et 2) déterminer le niveau de conservation de cette nouvelle configuration. Mon travail de thèse s’est concentré sur la première et l’utilisation d’un panel de mutants d’IFITM3 a permis: de dissocier l’activité anti VIH-1 et l’incorporation virale et d’identifier des domaines protéiques régulant l’habilité d’IFITM3 à interférer avec la production de particules virales infectieuses. J’ai également participé à travail collaboratif mis en place par notre équipe qui nous a permis de montrer que le mécanisme d’inhibition que nous avons mis en évidence pour le VIH-1 était un mécanisme conservé qui permettait de réduire l’infectivité de nombreux autres virus
IFITM1, -2 and -3 are transmembrane proteins, upregulated after type I interferon response and have been shown to inhibit a broad spectrum of viruses. The commonly admitted restriction in the field denotes that the presence of IFITM proteins in the lipidic membranes of target cells decreases viral entry by impeding the viral to cell membrane fusion, essential for the liberation of the core viral into the cytoplasm.I took part at the beginning of my thesis to a teamwork that allowed us to discover a new antiviral mechanisms for these proteins, at least for HIV-1. According to this mechanism, the presence of IFITMs in virus producing cells results in the production of viral particles that incorporate IFITMs and display decreased infectivity.Since then, my PhD work has consisted in: 1) understanding the molecular mechanism by which IFITMs inhibit HIV virion particles and 2) determine the conservation of this novel mechanism of inhibition against other viruses.First, I focused on IFITM3 and tested a large panel of mutants to identify the protein domain(s) required for either incorporation into virions and/or for the antiviral activity. This work allowed me to identify unknown domains in IFITM3 important for the antiviral effect of IFITM3 in virus-producing cells. Second, I have participated to a large collaboration initiated by our team to analyze the antiviral effects that IFITMs exerted on several viruses. Our results indicate that the novel mechanism of inhibition by IFITMs that we have described for HIV is conserved among different classes of viruses

La vaccination est l'un des plus grands progrès réalisés en santé publique. Toutefois, malgré de nombreuses connaissances sur le système immunitaire, de nombreux pans d’ombre empêchent la conception de vaccins contre des pathogènes complexes. Pour pallier ce problème, une meilleure compréhension des modes d'action des vaccins est requise. En particulier, la plupart des vaccins nécessitent plusieurs immunisations pour induire une mémoire immunitaire adaptative au long terme, mais l'impact du délai entre primo-vaccination, induisant une mémoire primaire, et rappel(s) la restimulant pour générer une mémoire secondaire, est peu défini. De plus, la réponse immunitaire innée, induite à chaque immunisation et façonnant l'immunité adaptative, reste peu caractérisée dans ce contexte vaccinal. En vaccinant des macaques cynomolgus avec le virus de la vaccine modifiée Ankara, selon un schéma de primo-vaccination suivie d’un rappel homologue à deux mois, et en utilisant la cytométrie de masse couplée à des analyses bio-informatiques, nous avons caractérisé la réponse innée induite par chaque immunisation. Les réponses innées diffèrent entre primo-vaccination et rappel, avec induction par la primo-vaccination d’une modification phénotypique tardive des cellules innées, suggérant une meilleure capacité à répondre au rappel. De surcroît, la réduction à deux semaines du délai entre primo-vaccination et rappel abroge la mobilisation de ces cellules innées phénotypiquement modifiées et altère la qualité de la réponse humorale. En définitive, en plus de la réponse innée précoce, ce projet a mis en évidence l'induction par la primo-vaccination d'un vraisemblable entraînement inné tardif, un concept émergent traduisant la capacité de mémorisation des cellules innées via des modifications épigénétiques. Ce vraisemblable entraînement, non seulement des monocytes et cellules tueuses naturelles, mais aussi des cellules dendritiques et surprenamment des neutrophiles, est corrélé à la qualité de la mémoire immunitaire adaptative, de manière hautement dépendante du délai entre primo-vaccination et rappel. Ces résultats contribuent à ouvrir la voie vers l’optimisation rationnelle des futurs vaccins, via l'optimisation des calendriers vaccinaux et la valorisation de l'entraînement inné
Vaccination is one of the best achievements made in public health. However, designing vaccines against complex pathogens is currently challenging. The immune system is indeed uncompletely characterized, despite large amount of accumulated knowledges. A better understanding of vaccine-induced immunity is then required to optimize vaccine design. In particular, while most vaccines require several immunizations to induce a long-lasting adaptive immune memory, little is known on the impact of the delay beween the prime inducing a primary memory and the boost restimulating it to induce a secondary memory. Also, the innate immunity induced by each immunization and shaping the adaptative immunity is poorly characterized in this vaccine context.We studied the innate immune responses in cynomolgus macaques immunized with the modified vaccinia virus Ankara, following an homologous prime-boost vaccination at two months apart. We applied mass cytometry and bioinformatic analyses to characterize the innate response induced by each immunization. We showed that prime and boost vaccination triggered distinct innate responses. Actually, prime induced late phenotypic modifications of innate cells. These phenotypic changes suggest a stronger ability to react to the boost. Moreover, reducing the delay between prime and boost to two weeks impeded the mobilization of these phenotypically modified innate cells, and qualitatively altered humoral response.In conclusion, beyond the early innate responses, these results highlight the late induction by the prime of "likely trained" innate cells. This emerging concept corresponds to the ability of innate cells to display memory features based on epigenetic modifications. This "likely training" occured not only on monocytes and NK cells, but also on dendritic cells and strikingly on neutrophils. It was deeply connected with adaptive immune memory establishment, in a prime-boost delay dependant fashion. These findings contribute to pave the way towards to the rationale design of future vaccines, via vaccine schedule optimization and harnessment of innate training

Immunitat Olímpica és el nom donat al mecanisme de protecció dels drets dels esportistes que proposa aquesta tesi doctoral, sustentat jurídicament tant pels Principis Fonamentals de l'Olimpisme, especialment els supòsits de “no discriminació” recollits en la Carta Olímpica i en els estatuts de les federacions internacionals, com pels instruments del Dret Internacional que conformen la Carta Internacional dels Drets Humans. El treball demostra mitjançant l’exposició de casos com aquests drets han estat i són vulnerats, amb respostes desiguals per part de les organitzacions esportives i disparitat jurídica entre la justícia ordinària i l'esportiva. La solució precisa d'una major harmonització i interactuació del Dret Privat i el Dret Públic, i de la voluntat de l'esport i la política.
Inmunidad olímpica es el nombre dado al mecanismo de protección de los derechos de los deportistas que propone esta tesis doctoral, sustentado jurídicamente tanto por los Principios Fundamentales del Olimpismo, especialmente los supuestos de "no discriminación" recogidos en la Carta Olímpica y en los estatutos de las federaciones internacionales, como por los instrumentos del Derecho Internacional que conforman la Carta Internacional de los Derechos Humanos. El trabajo demuestra mediante la exposición de casos cómo esos derechos han sido y son vulnerados, con respuestas desiguales por parte de las organizaciones deportivas y disparidad jurídica entre la justicia ordinaria y la deportiva. La solución precisa de una mayor armonización e interactuación del Derecho Privado y el Derecho Público, y de la voluntad del deporte y la política.
Olympic Immunity is the name given to the mechanism of data protection for sports performers set down in this doctoral thesis. It is judicially based both on the basic Olympic Ideals (on the assumption of non-discrimination stipulated in the Olympic Charter and in the international sports federations statute law), as well as part of the International Human Rights that make up the International Declaration. This paper shows, by means of case studies, how these rights have been breached in the past and how they are still being breached today. It aims to show the inequality present in sporting organizations while examining the judicial differences between ordinary justice and sporting justice. The required solution being to establish communicative links between Private Law and State Law, as well as a will to change in the sporting world and in Politics.
Immunité Olympique est le nom que cette thèse doctorale propose pour désigner le mécanisme de protection des droits des sportifs. Il est appuyé juridiquement autant par les Principes Fondamentaux de l’Olympisme, notamment celui de la “non-discrimination” de la Charte Olympique et des statuts des fédérations internationales, que par les instruments du Droit International recueillis dans la Charte Internationale des Droits de l’Homme. Ce travail prouve, à travers des études de cas, comment ces droits ont été et sont encore bafoués, à cause de réponses inégales de la part des organisations sportives et de disparités entre la justice ordinaire et la justice sportive. La solution requiert une plus ample harmonisation et une interaction plus étroite entre Droit Privé et Droit Public, ainsi qu’une volonté sportive et politique.

The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection.

The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection.

More than a century after the identification of the tubercle bacillus and the first attempts at vaccination, tuberculosis (TB) still remains one of the world’s most serious infectious diseases. TB, caused by the bacterium Mycobacterium tuberculosis, is typically a disease of the lung, which serves both as port of entry and as the major site of disease manifestation. The currently used vaccine, BCG, is administered parenterally and induces a systemic immune response. However, it fails to protect against pulmonary TB, thereby raising the question whether vaccination targeting the mucosal immunity in the lungs could be favourable.

The respiratory mucosal surfaces represent the first line of defence against a multitude of pathogens. Secretory IgA, in mucosal secretions has an important function by blocking entrance of pathogenic organisms and preventing infections. Additionally, a role for IgA in modulation of immune responses is currently being revealed. In this work, we investigated the relevance of mucosal IgA in protection against mycobacterial infections using mice deficient for IgA and the polymeric Ig receptor, the receptor responsible for mucosal secretions of IgA. Gene-targeted mice were more susceptible to mycobacterial infections in the respiratory tract and displayed reduced production of proinflammatory, and protective, factors such as IFN-γ and TNF-α in the lungs. The mechanisms explaining the defective proinflammatory responses in the lungs of deficient mice might involve impaired signalling through Fcα receptors, or homologous receptors, which could lead to inadequate activation of pulmonary macrophages. This could subsequently result in suboptimal induction and production of cytokines and chemokines important for attraction and migration of immune cells to the site of infection.

Induction of optimal adaptive immune responses to combat mycobacterial infections requires prompt innate immune activation. Toll-like receptors (TLRs) are vital components of the innate branch of the immune system, ensuring early recognition of invading pathogens. Using TLR-deficient mice we demonstrated an important role for TLR2, and partly TLR4, in protection against mycobacterial infection in the respiratory tract. TLR2-deficient mice failed to induce proper proinflammatory responses at the site of infection, and macrophages derived from the knockout mice displayed impaired anti-mycobacterial activity.

Experimental evidence has concluded that the immune response upon an infection can influence the outcome of succeeding infections with other pathogens. Concurrent infections might additionally interfere with responses to vaccinations and have deleterious effects. We developed an in vitro model to study the effect of a malaria infection on a successive M. tuberculosis infection. Our results demonstrate that a malaria blood-stage infection enhances the innate immune response to a subsequent M. tuberculosis infection with a Th1 prone profile. Reduced infectivity of malaria-exposed dendritic cells implies that a malaria infection could impose relative resistance to ensuing M. tuberculosis infection. However, a prolonged Th1 response may interfere with malaria parasite control.

The outcome of this work emphasizes the importance of generating effective immune responses in the local mucosal environment upon respiratory mycobacterial infections. It furthermore puts new light on the immunological interaction between parasites and mycobacteria, which could have implications for future vaccine research.

This thesis aimed to the identification of immune biomarkers of mycobacterial infection for better diagnosis of tuberculosis (TB) and also focused on new vaccination strategies with a particular emphasis on the immune responses in the respiratory tract using murine models. Since the lung is the natural habitat for the M. tuberculosis, we reasoned that immune responses detected locally in the lungs would be good correlates of infection (Paper I). Likewise, immune responses induced in the respiratory tract following immunization would be more effective against mycobacterial infection. We showed that cytokines (IL-12, TNF, and IFN-γ) and cytokine receptors (sTNFR1 and sTNFR2) together with specific antibodies in the respiratory tract correlated better with the bacterial burden in the organs. In Paper II, we investigated the role of the BCG vaccination as a priming vaccine in a heterologous prime-boost immunization protocol. The results showed that the neonatal BCG vaccination primed the immune system for a relevant antigen and showed a generalized adjuvant effect. Using this immunization protocol, protective immune responses in the lungs were generated independently of the route used for the booster immunization. In Paper III, We showed that exposure to mycobacterial antigens during the gestational period led to antigen transportation from the mother to the fetus and this resulted in an early priming of the fetal immune system. Immunization with the same antigen during the postnatal life increased antigen-specific recall IFN-γ responses and protection against infection. We examined the role of innate immunity for the induction of acquired immune responses upon immunization with mycobacterial antigens using TLR2 deficient mice (Paper IV). Our data indicated that suboptimal innate immune responses in the TLR2-/- mice might compromise the induction of acquired immune responses. Overall, the current findings suggested that a better understanding of the mucosal immunity would be useful for the improvement of diagnostic procedures and the development of efficient vaccines against TB.
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript

Network decontamination is a well known mobile agent problem with many applications. We assume that all nodes of a network are contaminated (e.g., by a virus) and a set of agents is deployed to decontaminate them. An agent passing by a node decontaminates it, however a decontaminated node can be recontaminated if any of its neighbours is contaminated. In the vast literature a variety of models are considered and different assumptions are made on the power of the agents. In this thesis we study variation of the decontamination problem in mesh and tori topologies, under the assumption that when a node is decontaminated, it is immune to recontamination for a predefined amount of time t (called immunity time). After the immunity time is elapsed, recontamination can occur. We focus on three different models: mobile agents (MA), cellular automata (CA), and mobile cellular automata (MCA). The first two models are commonly studied and employed in several other contexts, the third model is introduced in this thesis for the first time. In each model we study the temporal decontamination problem (adapted to the particular setting) under a variety of assumptions on the capabilities of the decontaminating elements (agents for MA and MCA, decontaminating cells for CA). Some of the parameters we consider in this study are: visibility of the active elements, their ability to make copies of themselves, their ability to communicate, and the possibility to remember their past actions (memory). We describe several solutions in the various scenarios and we analyze their complexity. Efficiency is evaluated slightly differently in each model, but essentially the effort is in the minimization of the number of simultaneous decontaminating elements active in the system while performing the decontamination with a given immunity time.

State participation in the arbitration of transnational commercial disputes is steadily increasing. It is estimated that arbitration of state contract disputes presently constitute one quarter of the disputes submitted to ICC arbitration. Where a state party is involved in an arbitration, the sovereign immunity doctrine - which in some cases exempts foreign states from the jurisdiction of municipal courts - may have adverse effect on the arbitration process. The thesis explores the impact of the immunity doctrine on the arbitration of state contract disputes. State practice in selected jurisdictions is used to illustrate the methods adopted in an effort to mitigate the impact of the immunity doctrine on commercial arbitration. In this respect, focus is placed on both jurisdictional immunity and immunity from execution. The thesis concludes that the private party may avoid unnecessary litigation by requiring the state party to expressly waive its immunity both during the recognition and enforcement stages. The waiver should be included in the arbitration agreement.
Law, Peter A. Allard School of
Graduate

Humoral immune function was studied in surgical patients. The antibody response to vaccination with a protein antigen, tetanus toxoid (TT), was reduced among all patients, especially those with reduced delayed type hypersensitivity (DTH) and increased degree of physiological derangement. The antibody response to a polysaccharide antigen, pneumococcal polysaccharide (PPS), was normal. In trauma patients, the antibody response to TT was normal. The in vitro production of specific and total immunoglobulin (Ig) by blood mononuclear cells was studied. Patients that failed to produce a serum antibody response to TT also failed to produce anti-TT in vitro. Anti-PPS production was normal. More total Ig was produced by patients, especially those with reduced DTH responses. Some patients showed a reduction, rather than the normal increase, in Ig synthesis with mitogen stimulation. These data show evidence of humoral immune deficiency to protein antigens, and in vivo activation of the B cell system.

MCLK1 (a.k.a. COQ7) is an evolutionarily conserved mitochondrial hydroxylase necessary ubiquinone biosynthesis. Mclk1+/- mice display a 50% reduction in this protein, as well as an array of phenotypes including increased longevity, decreased ubiquinone in the inner mitochondrial membrane, decreased mitochondrial respiration, and increased mitochondrial oxidative stress. We report here by various measures that Mclk1+/- mutants also exhibit an enhanced immune response in vivo. That is, Mclk1+/- mice mount a more extreme inflammatory response to bacterial lipopolysaccharide stimulation and bacterial infection, evidenced by increased measures of plasma cytokine levels. These mice also demonstrate resilience to tumour development, evidenced by a prolonged, dose-dependent delay in tumour onset following tumour cell xenograft. Furthermore, we report that Mclk1+/- mice react differently than wild-type mice following rapamycin injection. That is, circulating cytokine levels are attenuated in mutants but increased in wild-type mice following rapamycin administration. Despite their more extreme immune response, we demonstrate that Mclk1+/- mutants sustain less tissue damage as a result of infection or old age. Finally, using mouse models of high mitochondrial or cytoplasmic oxidative stress, we report that the Mclk1+/- phenotype is not simply due to increased reactive oxygen species in the mitochondria. These findings suggest characteristics that may contribute to the increased lifespan of these mice, though the causes of these characteristics require further investigation.
MCLK1 (COQ7) est une enzyme hydroxylase conservée au cours de l'évolution et nécessaire pour la biosynthèse de l'ubiquinone. Les souris Mclk1+/- présentent une réduction de 50% du niveau de cette protéine, ainsi qu'une gamme de phénotypes, tels qu'un accroissement de la longévité, une réduction de la quantité d'ubiquinone dans la membrane interne mitochondriale, une réduction de la respiration mitochondriale, et une augmentation du stress oxydatif mitochondrial. Différentes mesures ont démontrées que les souris Mclk1+/- arborent également une meilleure réponse immunitaire suite à la stimulation par des lipopolysaccharides bactériens (LPS) ainsi que par l'infection bactérienne, comme en témoigne une augmentation du niveau de plusieurs cytokines plasmatiques en réponse à ces stimulations. Les mutants Mclk1+/- sont aussi plus résistants au développement de tumeurs, comme en témoigne le délai dans l'apparition de tumeurs après une xénogreffe de cellules tumorales. En outre, nous avons découvert que les souris Mclk1+/- réagissent différemment par rapport aux souris de type sauvage à un traitement avec la rapamycine. Nous avons observé que suite à l'administration prolongée de rapamycine suivi par une injection de LPS, le niveau de cytokines circulantes diminue chez les souris mutantes alors que chez les souris de type sauvage ce niveau augmente. Malgré leur réponse immunitaire plus intense, nous avons démontré que les souris Mclk1+/- subissent moins de dommages tissulaires à la suite d'une infection ou du processus de vieillissement. Enfin, en utilisant des modèles murins de stress oxydatif mitochondrial ou cytoplasmique augmenté, nous avons aussi établi que le phénotype Mclk1+/- ne résulte pas simplement de l'augmentation des radicaux libres dans les mitochondries.

Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

Preterm birth (PTB) is a major problem in the UK and worldwide, leading to high mortality and significant long-term morbidity. A complex interaction between ascending genital tract infection and the maternal immune system is the likely underlying pathogenesis. We hypothesized that impaired cervical immunity leads to ascending infection and PTB i.e. women at high risk of PTB have a distinct cervical innate immune phenotype, which reflects genotypic variance. The systemic and mucosal expression and function of human beta defensins (HBDs), antimicrobial peptides with a range of immunomodulatory properties were investigated. The study included: (1) a comprehensive antenatal survey of lower genital tract (LGT) infections and dysbiosis in women at increased risk of PTB; (2) a case control study, investigating the relationship between HBD genotype and PTB; (3) an observational study investigating the relationship between cervical innate immune phenotype and (a) the risk of PTB and (b) HBD genotype; and (4) an in vitro investigation to examine endocervical HBD expression in response to progesterone. The prevalence of sexually transmitted LGT infections was low in this cohort of women at increased risk of PTB. Bacteria associated with vaginal dysbiosis and chorioamnionitis were more prevalent, and antenatal Group B streptococcus or Peptostreptococcus micros carriage were associated with both altered antimicrobial immunity and PTB. HBD expression varied systemically and in the cervix according to genotype, and the cervicovaginal antimicrobial killing activity correlated with both HBD protein levels and genotype in women who delivered at term. The relationship between HBD genotype and protein expression was altered in women who delivered preterm; increased cervicovaginal HBD1 levels were associated with PTB. These findings suggest that women who deliver preterm have altered cervicovaginal antimicrobial immunity, which may contribute to the pathogenesis of infection related PTB.

The studies submitted herein have contributed to our understanding of ruminant immunology, host-parasite interactions during ruminant infection with nematode parasites, and potential vaccine strategies to combat parasitic gastroenteritis (PGE). PGE of sheep and cattle, caused by T. circumcincta and O. ostertagia respectively, is a major problem for the global farming industry both in terms of productivity and animal welfare. To date control of these parasites has relied on the use of anthelmintic drugs however the emergence of widespread anthelmintic resistance is driving the search for alternative methods of control. As ruminants do acquire immunity in the field, vaccination is one such alternative under investigation. The first three papers contributing to this thesis used modern immunological tools alongside a locally developed surgical technique to revisit a model of nematode infection in sheep, investigating the composition and kinetics of the ovine local immune response to infection with Teladorsagia circumcincta via cannulation of the efferent gastric lymph duct. A protective local secondary immune response was observed in sheep which had previously experienced infection with T. circumcincta, but was absent from naive sheep. This immune response consisted initially of a rise in TE and BE cell activity peaking at 3 and 5 days post challenge respectively, followed by a secondary parasiteEspecific IgA response from 5 days post challenge which correlated with stunting of parasite growth. Significant parasite loss occurred by 2 days post challenge, prior to detection of the secondary immune response, suggesting critical early events in the host-parasite interaction and the potential importance of larval antigens in these interactions. No difference was observed in either the manifestations of immunity, or the magnitude and quality of the immune response, between adult sheep and lambs. The fourth and fifth papers describe vaccine trials carried out in bovine and ovine hosts using detergent soluble proteins derived from 4th larval stage Ostertagia ostertagi and Teladorsagia circumcincta respectively as antigens. Substantial reduction in total faecal egg output of up to 85% was observed in the calf trials, but not in the sheep trials which attained a maximum reduction of 29% in total faecal egg output. The sixth paper is a transcriptomic study carried out using the Roche 454 sequencing platform to investigate the immediate responses of Teladorsagia circumcincta upon encountering ovine host tissue of either immune or naive status. Following larval exsheathing and 4 hours of exposure to either immune or naive abomasal environments the transcript level of several genes was observed to differ. Genes which were most upregulated in response to encountering the immune environment included a peptidyl-glycine alpha-amidating mono-oxygenase homologue and a small heat shock protein. The studies described herein represent a body of work carried out using up-to-date tools and technologies. The first three papers confirmed the existence of critical early events in the host-parasite interaction, pointing to the potential use of larval antigens as vaccine candidates described in the trials in papers 4 and 5, and leading to the in-depth transcriptomic analysis described in paper 6. Papers 4 and 5 demonstrated that while Teladorsagia circumcincta and Ostertagia ostertagi have similar life cycles and host-site predilection, and both the ovine and bovine host can develop immunity to incoming parasitic larvae in the field, important differences may exist in either the proteome of the fourth stage larvae and/or the nature of the host response. Paper 6 revealed that changes in T. circumcincta transcript levels in response to ovine-host immune status can be detected early in the host-parasite interaction.

The innate immune system is capable of responding to tissue injury by detecting the abnormal exposure of intracellular, often ubiquitously expressed, molecules referred to as damage-associated molecular patterns (DAMPs). DAMPs are normally sequestered inside healthy cells but become exposed to the extracellular environment upon loss of membrane integrity during cell death. Exposed DAMPs are then recognised by receptors of the innate immune system. One such DAMP receptor is DNGR-1 (CLEC9A), which is expressed on CD8+ DCs, a rare but specialised subset of DCs involved in regulating T cell responses. Loss of DNGR-1 on CD8+ DCs impairs cross-presentation of dead-cell associated antigens to CD8+ T-cells indicating that DNGR-1 couples DAMP recognition to the generation of cytotoxic T cell immune responses. Prior to the work presented in this thesis, the DAMP ligand for DNGR-1 had not been identified. Using a variety of experimental approaches, I demonstrated that this ligand corresponds to filamentous actin (F-actin), a component of the cytoskeleton of all cells. Given its extreme evolutionary conservation, abundance and ubiquitous expression, as well as its association with tissue damage in a range of inflammatory conditions, actin possesses ideal DAMP characteristics. Thus, I further hypothesised that actin may engage receptors other than DNGR-1 and act as a universal and evolutionarily ancient sign of cell damage that is more generally detected by metazoans as a means of inducing sterile inflammation and/or tissue repair. In order to test this hypothesis, I made use of the Drosophila melanogaster model system. I found that actin injection into flies stimulates strong activation of the stress-induced JAK/STAT pathway without triggering immune defence pathways. Given the conservation of innate defence mechanisms in invertebrates and vertebrates, it is tempting to speculate that understanding the recognition of actin in Drosophila melanogaster will provide useful insights into the induction of inflammation in mammals.

Background: Cutaneous Leishmaniasis caused by the Leishmania mexicana complex is associated with unpleasant or disfiguring lesions, for which there only limited treatment options. The life cycle of L. mexicana consists of two stages which involve different immune evasion mechanisms: promastigote and amastigote. Understanding parasitic interactions with host cells and developing a protective vaccine could improve the management and treatment of the disease. Aims: • To construct and compare the immunogenicity of 3 Leishmania genes in 3 different plasmids. • Study the effect of long term in vitro culture on the virulency of L. mexicana and its interaction with host cells. • To analyse the influence of Leishmania infection on MHC class I expression by susceptible human cell lines (U937 macrophages, U937 and MonoMac-6 monocytes), and Toll-like receptor, cytokines and chemokines gene expression profiles. Methodology: Three Leishmania genes (L. mexicana GP63, L. donovani centrin1 and L. donovani centrin3) were cloned into three plasmids (pcRT7/CT-TOPO; VR1012; and pcDNA3.1/Hygro(-)). The immunogenicity of the prepared DNA constructs and their empty counterparts was assessed in Balb/c mice using gene gun immunisation method. An in vivo model of attenuated L. mexicana was produced by growing the parasite in vitro for up to passage 20, and testing the infectivity of these parasites in vivo and in vitro. The influence of infecting target cells with virulent and avirulent L. mexicana at different growth stages on MHC class I expression was determined by flow cytometry. Gene expression profiles were determined by qPCR analysis of extracted mRNA. Results: All tested Leishmania DNA constructs were highly immunogenic compared to the controls, as assessed using ELISPOT and cell proliferation assays. A novel survival assay developed in this study illustrated that macrophages derived from immunised mice were resistant to Leishmania infection. The parasite at passage 1 was highly infectious (virulent), but this progressively decreased to be completely avirulent at passage 20. This was associated with a significant down regulation of virulence-associated genes (GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3) at passage 20, and was also accompanied by morphological changes. The avirulent parasite was unable to transform to the pathogenic amastigote stage in infected target cells. The gene expression profile of toll-like receptors (TLR-1, TLR-2, TLR-4, and TLR-9), cytokines (IL-1, IL-6, IL-10, IL-12β, TNF-α, and TGF-β), and chemokines (CCL-1, CCL-2, CCL-3, CCL-4, CCL-5, and CCL-22) in target cells was were induced and inhibited according to the virulence status of the parasite. Similar and significant down regulation of MHC class 1 was induced by infection of target cells for 24 hours with both virulent and avirulent L. mexicana parasite, however after 48 hours of infection only cells infected with avirulent but not virulent parasite have significantly restored their MHC class I expression. Conclusions: Since no differences in the immunogenicity of the three plasmids encoding the same Leishmania gene were observed, immunogenicity is not dependent on the plasmid type. The failure of the avirulent L. mexicana parasite to infect Balb/c mice, and its inability to produce the pathogenic amastigote stage in vitro suggests that it might have potential as a vaccine candidate. This was also supported by the up regulation of Th2 mediators following the infection with virulent compared avirulent parasites. The level of MHC class I down regulation was dependent on parasite growth stage, virulency and infection dose.

Plant pathogens constitute a major threat to global food security. The use of naturally resistant crop varieties can limit crop losses, however new races of pathogen can arise that are able to overcome these defences. Plant breeding for race-specific resistance typically relies on disease-resistance genes, which generally encode proteins with nucleotide-binding and leucine-rich repeat domains (NB-LRRs). NB-LRRs are a large of proteins found in both plants and animals, with plant NB-LRRs further classified by the presence of N-terminal coiled-coil or toll-interleukin receptor domains. Although qualitative models exist to describe R-protein regulation and activation, these are predominantly based on genetic and molecular studies. Biochemical investigations into R-protein function have been hampered by difficulties obtaining sufficient yields of material. When suitable material has been identified, biochemical studies have been used to complement well-established in planta assays to validate numerous hypotheses. This work describes the screening processes undertaken to obtain R-protein domains suitable for downstream experiments. Using E. coli for high-throughput screening of constructs from a single R-protein, traditional construct design to investigate multiple R-protein domains and expanding our expression hosts to eukaryotic systems we successfully purified four coiled-coil domains and a single NBARC domain for use in downstream experiments. Characterisation of this NBARC domain by circular dichroism and small-angle X-ray scattering indicates that the protein is well-folded and stable in solution, allowing in vitro investigations. In testing models for R-protein regulation we were able to confirm previous findings, such as low levels of ATPase activity, however we were unable to find evidence for a commonly cited method of signal repression. A preliminary crystal structure of the NBARC domain shows significant similarity to Apaf-1, and highlights the importance of conserved motifs in NBARC architecture. The tools presented here should prove a valuable resource to complement existing models to better understand the structure, biochemistry, and ultimately regulation of plant R-proteins.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2009.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Härtill 4 uppsatser.

Many types of immune cells rely on secretory lysosomes to execute their function. These specialised organelles perform the catabolic function of conventional lysosomes and possess the added capacity to undergo regulated exocytosis. In particular, the cytotoxic function of natural killer (NK) cells depends on the rapid, polarised secretion of secretory lysosomes, known as lytic granules, for the targeted destruction of malignant and virally infected cells. Dysregulation of the biogenesis and exocytosis of secretory lysosomes is the cause of several severe immunological diseases such as Chédiak-Higashi syndrome (CHS). In CHS, mutations in the LYST gene result in enlarged secretory lysosomes that cannot undergo exocytosis. However, the mechanisms underlying LYST function remain elusive. Here, characterisation of the giant lysosome phenotype of beige cells reveals altered phosphoinositide metabolism and increased autophagy, indicating a potential role for LYST in regulating these pathways. Furthermore, RNA interference of LYST recapitulated the giant lysosome phenotype, suggesting a strategy for analysis of the pathways in which LYST functions. Pharmacological inhibition of the phosphoinositide kinase PIKfyve induces a giant vesicle phenotype resembling that in beige cells. However, on further analysis PIKfyve inhibition was shown to induce giant endosomes as opposed to the giant lysosomes found in beige cells. Despite these differences, PIKfyve inhibition, like LYST mutations, reduced NK cell granule exocytosis at a late stage in the pathway. Thus, LYST and PIKfyve act upon different intracellular compartments but inhibit the formation and exocytosis of NK cell granules. These results extend our knowledge of the exocytosis of secretory lysosomes within the immune system and suggest future strategies to define the pathways by which lysosomal biogenesis and exocytosis is regulated.

The involvement of human papilloma virus (HPV) in the aetiology and progression of cervical intraepithelial neoplasia (CIN) is still unresolved. This study was designed to assess the immunological responses to HPV types in patients presenting with varying degrees of CIN and in control groups, using in vitro measures of cellular and humoral responses. Lymphocyte proliferation assays (LPA) were performed using peripheral blood mononuclear cells (PBM) and various papillomavirus (PV) antigens. Twenty -five per cent (23/92) of patients with CIN responded to antigens derived from purified BPV, HPV -1 or HPV -2 with or without detergent disruption. The responses correlated with a past history of skin warts rather than cervical abnormalities, and the percentage of responders was similar to that in laboratory personnel (30 %) and lower than that in a group with recalcitrant common warts (50 %). Antigens specific to HPV -16 and HPV -18, in the form of bacterially expressed fusion proteins derived by the transcription and translation of the E6 and E4 open reading frames (ORF), occasionally produced specific positive responses, provided contaminating E.coli B galactosidase sequences had been removed during purification. Responses were low and suggested that the numbers of memory T cells specific to PV antigens were low and at the lower limit of detection of LPA. An indirect ELISA was developed to detect circulating IgG to PV antigens in colposcopy patients. Fifty per cent of patients had antibodies to disrupted HPV -1, HPV -2 or both, suggesting that a predominantly type- specific response was being detected. No correlation of immune responses with a degree of dysplasia or the presence of koilocytes in cervical biopsies was noted, but a high incidence of forgotten or inapparent past infection with cutaneous HPV types was found. In situ hybridisation (ISH) methods using non -radioactively labelled, cloned probes and synthetic oligonucleotide probes were developed for use on paraffin sections. Synthetic probes allowed a quicker, less destructive hybridisation protocol, with the sensitivity of detection being (y) improved by an anti -biotin -immunogold conjugated immunoglobulins -silver enhancement (IGSS) detection system. Double staining of PV antigen and nucleic acid on the same section was achieved. Synthetic oligonucleotides offer an exciting new tool for diagnostic virology, worthy of exploitation in many systems. Implantation of human foreskin infected with HPV -11 was shown to provide an animal model, albeit technically difficult, in which HPV could be produced, but a more practical technique of productive HPV infection in vitro is still required if the biology and pathogenesis of HPV infections is to be clarified further.

Intestinal helminths are highly prevalent worldwide, infecting approximately a third of the world’s population, causing significant host morbidity. With no current vaccines, a limited number of effective chemotherapeutic drugs available and the emergence of drug-resistant helminths, it is essential to further our understanding of the mechanisms of antihelminth immunity. Our current understanding of antihelminth immunity places the type 2 immune response at the forefront of protection, with type 2 cytokines orchestrating and activating a plethora of immune and non-immune cells to mediate parasite expulsion. The naturally occurring intestinal helminth Heligmosomoides polygyrus establishes a chronic infection in many inbred naïve mice, with resistance to a challenge infection established following drug-cure. This experimental model allows us to identify novel mechanisms of drug-induced resistance, relative to susceptibility. In this thesis, we utilised next generation sequencing technology to identify two novel mechanisms of antihelminth immunity. Firstly, we determined that the enzyme phospholipase A2 group 1B (PLA2g1B) is an endogenous anthelmintic, upregulated in intestinal epithelial cells of resistant mice. We demonstrated that PLA2g1B was essential for resistance to H. polygyrus and that PLA2g1B directly cleaves phospholipids off infective H. polygyrus larvae. Secondly, we identified that the microRNAs miR-99a-5p, miR-148a-3p and miR-155-5p were upregulated in mice resistant to H. polygyrus during infection and were also essential for functional immunity. In summary, we have identified and characterised two novel mechanisms of antihelminth immunity and propose a model of tissue memory, essential for acquired resistance to H. polygyrus.

Hard corals underpin the existence and biodiversity of tropical reefs, however they are declining globally at an alarming rate, largely because of increases in disease prevalence and thermal bleaching events. Despite extensive documentation of coral reef demise, no substantive investigations into the coral host's biological mechanisms for disease resistance, bleaching mitigation or wound healing have been conducted. Coral immunology may therefore provide important insights into understanding the capacity of corals to resist global coral declines. The principal objectives of this thesis were therefore, firstly, to investigate the presence of well-characterised invertebrate innate immunity effector responses, including the melanin-synthesis pathway, the coagulation pathway, immune cell activation and antioxidants, within a number of species from the anthozoan orders Scleractinia, Alcyonacea and Zoantharia. The second objective was to establish the extent to which coral immunity effector responses are activated following physical injury and infection. Finally, the potential influence of warmer seawater temperatures on coral immunity levels and activation were investigated. These objectives were addressed using anthozoans from the Great Barrier Reef Australia, the Caribbean and Hawai'i, using both colourimetric enzyme assays and histological techniques. Principal findings include establishing the presence of each of the four invertebrate immunity effector responses investigated within corals. A coagulation enzyme, three types of enzymes involved in melanin synthesis and several immune cells were identified for the first time, indicating that anthozoan immune systems are as complex as phylogenetically higher invertebrates. Furthermore, an antioxidant property of coral fluorescent proteins (FP) was established and, in combination with the presence of FPs in coral tissues with increased immune activity, these findings suggest a further potential function of these colourful proteins. Comparative baseline levels of a suite of coral immunity components were found to explain among-family variation in both coral bleaching and disease susceptibility, suggesting immunity as a physiological link between, and mitigator of, these two threats. Furthermore, the ability of hard corals to activate an immune response was demonstrated by a discernable increase in all measured effector responses, both enzymatic and cellular, with both injury and infection. Similarly, the cells and phases of wound healing were characterised within a hard coral for the first time, demonstrating conservation of mechanisms from the phylogenetically basal corals to humans. Additionally, within one coral species, elevated seawater temperature increased baseline levels of immunity but suppressed responses to injury, explaining links between warming sea surface temperatures and increasing disease prevalence that have been found for several coral diseases, but also suggesting the potential for corals to acclimatise to ocean warming. Overall, this thesis presents novel findings in a number of areas of coral innate immunity, thereby laying the foundations for this new field of research for scleractinian corals. Coral immunology is an emerging field that is highly relevant to understanding the capacity of corals and coral reefs to persist in a warming world. Preliminary tools developed here will significantly advance the capacity to quantify coral health and, therefore, to better predict the future state of the world’s coral reefs.

Nutrition plays an important role in modulating livestock species immunity. Therefore, this thesis aimed at evaluating the effects of different dietary molecules used in animal nutrition on mammalian and avian immunity. Both, in vitro functional analyses and OMIC technologies (proteomics and miRNAomics) were implemented herein for an integral characterization of the molecules’ impact on the animals’ immune response. Specifically, in this thesis the in vitro impact of the n-6 conjugated linoleic acid (CLA), citrus pectin (CP), and porcine milk exosomes and n-3 polyunsaturated fatty acids (PUFA) on bovine, chicken and porcine mononuclear cells immune response was evaluated, respectively. In the first study, the in vitro activity of CLA on bovine monocytes apoptosis and immune activities, including chemotaxis, phagocytosis, killing capability, and extracellular reactive oxygen species (ROS) production was assessed. Anti-apoptotic effects and an increase in extracellular ROS production during experimental pro-inflammatory conditions were observed, only when using the mixture of the two main isomers of CLA in equal proportions (50:50). The present results demonstrated for the first time that CLA does have immunomodulatory effects on some functions of bovine monocytes in vitro and that the CLA (50:50) mixture is more effective than the CLA isomers individually. The proteomics analysis performed on bovine peripheral blood mononuclear cells (PBMC) revealed that CLA (50:50) mixture does modulate bovine PBMC proteome, supporting the antiapoptotic and immunomodulatory effects observed in the previous in vitro study on bovine monocytes, and propose a potential cytoprotective role of CLA (50:50) mixture against oxidative stress. In the second study, the in vitro activity of CP on chicken monocytes viability, apoptosis, chemotaxis and phagocytosis was assessed. The study demonstrated for the first time that CP inhibits monocytes’ chemotaxis and phagocytosis in vitro, suggesting a potential anti-inflammatory activity. The proteomics analysis carried out on chicken PBMC provided a proteomics background to the anti-inflammatory activity of CP, demonstrating that the in vitro reduction of phagocytosis and chemotaxis is associated with changes in proteins related to the actin cytoskeleton. In the third study, the in vitro activity of porcine milk exosomes on porcine monocytes viability, apoptosis, chemotaxis, phagocytosis, killing capability and extracellular ROS production was assessed. Milk exosomes were successfully purified from sows’ milk and characterized using their size, concentration, morphology, and exosome protein markers. This study reported for the first time that porcine milk exosomes can be internalized by porcine monocytes in vitro and that they can modulate the cell's immune response, by decreasing their chemotaxis and phagocytosis; and increasing their ROS production under resting and pro-inflammatory conditions. The proteomics analysis performed on porcine PBMC demonstrated for the first time that porcine milk exosomes can modulate porcine PBMC proteome in vitro. Moreover, the gene ontology (GO) functional analyses revealed that porcine milk exosomes enrich biological processes related to innate immune-related processes and exosome uptake processes, supporting the immunomodulatory effects and the exosome internalization observed in the previous in vitro study. In the last study, the in vitro activity of the n-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on porcine monocytes viability, apoptosis, chemotaxis, phagocytosis, and intracellular, extracellular and total ROS production was assessed. The results of the study showed that DHA and EPA at the highest concentration (200 µM) decreased porcine monocytes' viability. In addition, it was reported for the first time that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes' chemotaxis and potentiating their intracellular oxidative burst, respectively. The proteomics and miRNAomics analyses were not performed for this study. Instead, a first glance on the results from the bioinformatic analyses of the miRNAomics data of all the rest of the studies is presented herein. In conclusion, this thesis provides both, a phenotypical and molecular characterization of the in vitro impact of these dietary molecules on bovine, porcine and chicken immune responses.

Le mélanome est la forme la plus agressive des cancers de la peau. Si sa prise en charge à des stades précoces est de bon pronostic, l’espérance de vie des patients chute dramatiquement pour les stades métastatiques. Malgré les avancées thérapeutiques spectaculaires récentes, le problème majeur réside dans la résistance aux traitements et la récidive et le défi principal est désormais de tendre vers un contrôle efficace et durable. Les anticorps monoclonaux (AcM) ont la capacité de cibler et éliminer spécifiquement la cellule tumorale tout en recrutant des cellules du système immunitaire, permettant de développer et/ou renforcer l’immunité de l’hôte avec le développement d’une réponse immune anti-tumorale de type vaccinale. Dans un modèle de tumeur solide de mélanome murin après greffe sous-cutanée des cellules B16F10, nous avons étudié le potentiel immunomodulateur de l’AcM TA99 qui cible un antigène de surface TYRP-1 surexprimé dans les mélanocytes tumoraux. Nos résultats montrent qu’environ 30% des souris sont protégées sur le long-terme et présentent une réponse immunitaire humorale et cellulaire mémoire. Par ailleurs, l’analyse de l’infiltrat immunitaire chez les souris qui échappent au traitement par l’AcM TA99 et qui développent une tumeur à plus ou moins long terme, montre une surexpression de PD-1 et Tim3 associée à une perte de fonctionnalité des cellules effectrices au sein de la tumeur. Ce même phénotype a été observé sur des biopsies de patients atteints de mélanome métastatique. Nous montrons aussi dans le cadre de ce travail que, le blocage de l’axe PD1/PD-L1 par inoculation d’un AcM anti-PD1 au moment de l’échappement, potentialise la réponse immunitaire anti-tumorale et entraîne une augmentation de la survie. Cependant, l’absence de régression complète suggère la mise en place d’autres voies immunosuppressives. En effet nous avons observé une surexpression des ectonucleotidases CD39 et CD73 dans le micro-environnement tumoral suggérant l’implication de l’adénosine dans la résistance au traitement de l’AcM TA99 plus l’α-PD-1. Ce résultat ouvre des perspectives intéressantes pour le blocage concomitant la voie de l’adénosine et de l’axe PD1/PD-L1. Une autre stratégie a consisté à améliorer les effets immunomodulateurs précoces de l’AcM TA99 en le combinant avec l’oxaliplatine, chimiothérapie favorisant la mort immunogénique. Bien que les combinaisons thérapeutiques testées dans cette étude montrent des effets in vivo encourageants avec un délai significatif dans la survie globale, aucune augmentation significative de la réponse anti-tumorale sur le long terme n’a pu être observée, suggérant la mise en place d’autres voies immunosuppressives non redondantes ou des stratégies de combinaisons non adaptées. Une analyse dynamique approfondie, tant phénotypique que fonctionnelle, des différents acteurs cellulaires du micro-environnement tumoral sera une étape clé dans la mise en place de combinaisons pertinentes en association avec l’AcM TA99. Ce travail prend d’autant plus d’intérêt qu’un essai clinique de phase I (IMC-20D7S) utilisant le flanvotumab (équivalent humain de l’AcM TA99) réalisé chez 27 patients atteints de mélanome métastatique, montre des effets cliniques intéressants sans effets secondaires sévères, ouvrant la voie au développement de combinaisons thérapeutiques associées à cet AcM
Melanoma is the most aggressive form of skin cancer. Although early management is of good prognosis, the survival of patients decrease dramatically for metastatic stages. Despite the recent spectacular therapeutic advances, the major problem lies in resistance to treatment and relapse and the main challenge now is to develop an effective and sustainable control. Monoclonal antibodies (mAbs) have the ability to specifically target and eliminate tumor cells while recruiting cells from the immune system, to develop and / or enhance the immunity of the host with the development of a vaccinal immune response. In a solid tumor model of murine melanoma after subcutaneous transplantation of B16F10 cells, we investigated the immunomodulatory effect of TA99 mAb targeting a TYRP-1 surface antigen overexpressed in tumor melanocytes. Our results showed that about 30% of mice are protected in the long term and have an antitumoral humoral and cellular immune response. Moreover, the analysis of the immune infiltrate in mice that escape to the treatment with TA99 mAb and develop a tumor, shows an overexpression of PD-1 and Tim3 associated with a loss of effector cell functions within the tumor. This same phenotype has been observed in biopsies of patients with metastatic melanoma. Thus, blocking the PD-1 / PDL-1 axis by inoculation of an anti-PD1 mAb at the time of tumor escape potentiates the anti-tumor immune response and results in increased survival. However, the absence of complete regression suggests the establishment of other immunosuppressive pathways. Indeed we have observed an overexpression of CD39 and CD73 ectonucleotidases in the tumor microenvironment suggesting the involvement of adenosine in the resistance mechanisms observed and opening interesting perspectives for the concomitant blocking of this pathway and the PD1 / PDL-1 axis. Another strategy has been to improve the early immunomodulatory effects of TA99 mAb by combining it with oxaliplatin, a chemotherapy that promotes immunogenic death. Although the therapeutic combinations tested in this study showed encouraging in vivo effects with a significant delay in overall survival, no significant increase in the long-term anti-tumor response was observed, suggesting the establishment of other non-redundant immunosuppressive mechanisms or unsuitable combinations strategies. Both phenotypic and functional analysis of the different cellular actors of the tumor microenvironment will be a key step in the implementation of relevant combinations in association with the TA99 mAb. This work is highlighted by a phase I clinical trial (IMC-20D7S) using flanvotumab (human equivalent of mAb TA99) in 27 patients with metastatic melanoma that shows interesting clinical outcome without severe side effects, opening the way for the development of therapeutic combinations associated with this mAb

Les syndromes neurologiques paranéoplasiques (SNP) sont des maladies neurologiques rares, associés à une réponse immunitaire efficace contre un cancer sous-jacent exprimant un antigène également exprimé par des cellules du système nerveux central (SNC). Le cancer déclenche alors une réponse auto-immune secondaire qui provoque la destruction des cellules du SNC. Certains travaux récents suggèrent que l'immunité à médiation cellulaire associée à des auto-anticorps reconnaissant des antigènes intracellulaires pourrait jouer un rôle majeur, bien qu'encore mal compris, dans la physiopathologie des SNP. Les exemples de SNP les plus représentatifs sont le syndrome Hu, qui conduit à la perte de diverses populations de neurones du SNC et l'ataxie cérébelleuse subaiguë (PCD en anglais, pour Paraneoplastic Cerebellar Degeneration), caractérisée par la perte sélective des cellules de Purkinje du cervelet. Alors que le syndrome Hu se développe en général chez des patients présentant des tumeurs du poumon à petites cellules qui expriment l'antigène HuD spécifique des neurones, la majorité des patients souffrant de PCD présente un cancer gynécologique qui exprime la protéine CDR2, également exprimée dans les cellules de Purkinje. Afin de mieux cerner la physiopathologie des SNP et de tester l'implication de l'immunité cellulaire, notamment des lymphocytes T, nous avons durant ma thèse développé et analysé deux modèles murins, l'un pour le syndrome Hu et l'autre pour la PCD. Ces modèles reposent sur l'utilisation de souches de souris génétiquement modifiées : la souris CamK-HA, qui exprime l'hémagglutinine (HA) du virus de la grippe dans la plupart de ses neurones du SNC et la souris L7-HA dans laquelle la protéine HA est exprimée exclusivement par les cellules de Purkinje du cervelet. Dans ces souris, une réponse anti-tumorale est provoquée par l'injection de cellules tumorales 4T1 exprimant HA (4T1-HA). Afin le faciliter le suivi des réponses cellulaires contre l'antigène HA, nous avons injecté des lymphocutes T CD4+ et/ou T CD8+ naïfs isolées à partir de souris transgéniques pour des récepteurs de lymphocytes T spécifiques de HA. Nos résultats montrent que seul le transfert in vivo des cellules tumorales 4T1-HA, et non celui des cellules 4T1 témoins, peut conduire à l'activation, la prolifération et la différentiation des deux types de lymphocytes T spécifiques pour l'antigène HA. De plus, nous avons observé que les populations de lymphocytes T CD4+ et CD8+ sont toutes deux requises, non seulement pour une réponse anti-tumorale efficace, mais aussi pour le déclenchement d'une réaction auto-immune collatérale chez la souris CamK-HA. Enfin, nous avons montré qu'il était nécessaire d'injecter en parallèle des anticorps contre le récepteur inhibiteur CTLA-4 chez la souris L7-HA, afin de permettre la migration des lymphocytes T spécifiques de HA dans le cervelet. Chez ces souris L7-HA, nous avons en outre démontré que les lymphocytes T CD8+ cytotoxiques sont les effecteurs principaux de la maladie. Ces nouveaux modèles murins représentent donc des outils précieux pour une meilleure compréhension des mécanismes moléculaires responsables du développement des SNP. De plus, ils pourraient permettre de tester et de valider de nouvelles approches thérapeutiques visant à bloquer la pénétration dans le SNC d'effecteurs immunitaires potentiellement pathogènes, tout en préservant l'efficacité de la réponse anti-tumorale en périphérie
Paraneoplastic neurological disorders (PNDs) are rare human autoimmune diseases that mostly affect the central nervous system (CNS). They are triggered by an efficient immune response against a neural self-antigen that is ectopically expressed in neoplastic tumor cells and naturally expressed in CNS cells. Due to this shared antigenic expression, the immune system reacts not only to tumor cells but also to neural cells resulting in neurological damage. Growing data point to a major role of cell-mediated immunity in PNDs associated to autoantibodies against intracellular proteins. However, its precise contribution in the pathogenesis remains unclear. Two illustrative examples of possibly cell-mediated PNDs are the Hu-syndrome, characterized by inflammation and widespread los of neurons, and paraneoplastic cerebellar degeneration (PCD), characterized by the selective loss of Purkinje cells. PCD develops mostly in patients with gynecologic carcinomas that express the Purkinje neuron-specific CDR2 protein whereas most patients with the Hu-syndrome harbor small cell lung cancer expressing the neuron-specific protein HuD. In this context, our study aimed to investigate the impact of anti-tumor cellular immune responses in the development of these PNDs. To this end, we developed two animal models mimicking the Hu-syndrome and PCD. We used a tumor cell line expressing the hemagglutinin (HA) of influenza virus to induce an anti-tumor response in CamK-HA mice, which express HA in CNS neurons and L7-HA mice, which express HA only in cerebellar Purkinje neurons. To promote and track the T cell response against the HA antigen, naïve HA-specific CD8+ and/or CD4+ T cells, originating from TCR-transgenic animals, were transferred into these mice. We demonstrate that HA-expressing tumors, but not control tumors, induce in vivo activation, proliferation and differentiation of naïve HA-specific CD4+ and CD8+ T cells into effector cells. Moreover, the collaboration between these two T cell subsets was needed to control tumor growth and induce CNS inflammation in CamK-HA mice. In L7-HA mice the additional injection of the antibody against the inhibitory receptor CTLA-4 was necessary to allow T cells to enter the cerebellum to cause inflammation and the subsequent destruction of Purkinje neurons. Furthermore, in L7-HA mice we demonstrate that cytotoxic CD8+ T cells are the main effectors driving the disease. Thus, these two new mouse models provide further insights into the cellular mechanisms of PND whereby a potent anti-tumor immunity triggers a cancer-associated autoimmune disease, and may therefore help to develop new therapeutic strategies against PND