Impact of Early-Life Exposures on Immune Maturation and Susceptibility to Disease.

Exiting from the largely sterile environment of the womb, the neonatal immune system is not fully mature, and thus neonatal immune cells must simultaneously mount responses against environmental stimuli while maturing. This dynamic process of immune maturation is driven by a variety of cell-intrinsic and extrinsic factors. Recent studies have focused on some of these factors and have shed light on the mechanisms by which they drive immune maturation. We review the interactions and consequences of immune maturation during the pre- and perinatal period. We discuss environmental signals in early life that are needed for healthy immune homeostasis, and highlight detrimental factors that can set an individual on a path towards disease. This early-life period of immune maturation could hold the key to strategies for setting individuals on trajectories towards health and reduced disease susceptibility.

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor ß, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RAR¿ and RARß), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL¿6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RAR¿ and RARß. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RAR¿ and RARß, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.

Influence of PTPN2 and pre-existing immunity on the generation of effector and memory CD8 T cells

Notre système immunitaire joue un rôle important pour la protection envers les maladies infectieuses. Au cours d'une réponse à une infection primaire, des cellules B et des cellules T spécifiques, dirigées contre le pathogène en question, sont générées et certaines d'entre elles deviennent des cellules dites mémoires. Leur fonction est de nous protéger contre une nouvelle infection avec le même pathogène, une infection secondaire. Dans certaines situations, comme c'est par exemple le cas avec la grippe, les pathogènes ne sont pas toujours complètement identiques et les cellules mémoires ne sont pas à même d'assurer leur rôle protecteur et d'empêcher une réinfection. Pourtant, on ne sait à l'heure actuelle que très peu comment une immunité acquise, mais non protectrice, influence le développement d'une réponse immunitaire ultérieure. Dans la première partie de cette thèse, nous avons étudié comment les cellules T mémoires cytotoxiques altèrent la réponse de cellules T cytotoxiques nouvellement induites. Au cours d'une réaction immunitaire dirigée contre une infection primaire, un vaste répertoire de lymphocytes T est créé, constitué de cellules T possédant divers degrés d'affinité pour le pathogène. Lors d'une infection secondaire, seules les cellules T ayant une forte affinité pour le pathogène participent à la réponse. Nous avons pu démontrer que ce phénomène de restriction du répertoire des cellules T est principalement causé par les cellules T mémoires qui sont à même de reconnaître un antigène pathogénique présent dans les deux infections. Dans un deuxième projet, nous avons étudié comment l'absence de PTPN2 influence la réponse des cellules T. Chez l'homme, une mutation dans le gène de PTPN2 est associée à des maladies auto-immunes et résulte en une activité réduite de cette phosphatase dans les lymphocytes T. Nous avons montré que la baisse d'activité de la phosphatase PTNP2 conduit à une meilleure expansion des cellules T ayant une qualité comparable à des cellules T auto-antigène spécifiques. De plus, nous avons observé que la survie de ces cellules T effectues ayant une phosphatase diminuée est nettement améliorée. Cela peut conduire à une réponse immunitaire plus efficace ou, éventuellement, à une pathologie auto-immune plus grave. En outre, nos résultats montrent qu'en manipulant l'activité de cette phosphatase, il est possible d'augmenter l'efficacité du transfert des cellules T dans un hôte receveur. Un tel transfert de cellules T est pratiqué chez des patients atteints de tumeurs. Nos travaux suggèrent que la manipulation de la phosphatase PTPN2 pourrait donc représenter une approche thérapeutique novatrice et prometteuse. -- Notre système immunitaire joue un rôle important pour la protection contre les maladies. Les cellules T CD8+ ont une importance primordiale pour le contrôle d'infections primaires causées par des virus ou bactéries, mais également contre certaines tumeurs. Par conséquent, mieux comprendre les exigences nécessaires à l'induction de bonnes réponses des cellules T CD8 pourrait nous permettre de construire des vaccins contre les pathogènes contre lesquels nous n'avons pour l'instant pas de vaccins mais aussi d'améliorer les réactions immunitaires dirigées anti-tumorales. Dans la première partie de cette thèse, nous avons étudié l'influence qu'une immunité préexistante a sur la réponse des cellules T CD8. Nous sommes souvent exposés à des pathogènes qui sont similaires mais pas identiques à ceux que nous avons rencontrés auparavant. De telles infections hétérologues ne sont pas l'objet de beaucoup d'études et certains exemples indiquent même qu'une immunité préexistante partielle peut mener à une aggravation de la maladie. Nous avons étudié le répertoire des lymphocytes T CD8 qui sont générés lors d'une rencontre avec un nouvel antigène, et ce en comparant infection primaire et secondaire. En utilisant le modèle expérimental d'infections à Listeria monocytogenes, nous avons pu montrer que lors d'une infection primaire, un répertoire diversifié comprenant des cellules T CD8 de forte et faible affinité est constitué. Au contraire, dans le cas d'une infection secondaire, le répertoire des cellules T est fortement limité et seulement les lymphocytes T de forte affinité sont impliqués dans la réponse immunitaire. Nous avons pu démontrer que ces Rangements sont provoqués par des cellules T CD8 mémoires capables de reconnaître un antigène présent dans les deux infections. Cette augmentation du seuil d'activation des cellules effectrices est majoritairement causée par les lymphocytes T CD8 mémoires non transférables. Ces observations indiquent que les vaccins visant à induire des cellules T anti-tumorales de faible affinité seraient inefficaces si le vaccin contient des épitopes contre lesquels il existe une mémoire immunologique. Les réponses immunitaires conduites par les cellules T contre les antigènes tumoraux dépendent des cellules T CD8 de faible réactivité contre les antigènes tumoraux puisque les cellules à forte réactivité sont éliminées par les mécanismes de tolérance. Nous basant sur l'existence dans la littérature de preuves indiquant que PTPN2 influence la réponse des cellules T de faible affinité, nous nous sommes intéressés à comprendre comment PTPN2 impacte les réponses des cellules T CD8 en général. Nous avons remarqué que des cellules T CD8 déficientes en PTPN2 exhibent une meilleure capacité à proliférer suite à une faible ou courte stimulation du récepteur des lymphocytes T. La phase effectrice est prolongée et la contraction retardée résultant ainsi à globalement plus de cellules effectrices. Ce phénomène est également accompagné d'une meilleure survie des cellules effectrices de différentiation terminale. Une fois transférées dans un nouvel hôte receveur, les cellules effectrices terminales KLRG1+CD127- déficientes en phosphatase PTPN2 peuvent survivre et se transformer en cellules mémoires CD127+ fonctionnelles. De façon inattendue, nous avons découvert que l'élimination de PTPN2 améliore l'efficacité du transfert et la formation des cellules mémoires ainsi que leur capacité protectrice. Manipuler l'activité de cette phosphatase apparaît donc comme une approche intéressante et prometteuse pour la thérapie cellulaire par transfert adoptif de lymphocytes T. Nos observations montrent que la manipulation d'un facteur intrinsèque, l'absence de PTPN2, peut, dans certaines circonstances, améliorer la réponse des cellules T. Une meilleure connaissance des mécanismes contrôlant la réponse des lymphocytes T CD8 pourrait donc permettre la manipulation de ces derniers et conduire à des réponses immunitaires plus vigoureuses. Si ces réponses sont déclenchées par l'utilisation de vaccins, il est nécessaire de considérer l'historique d'une exposition préalable à des agents pathogènes ou à des vaccins puisque celle-ci peut, comme nous l'avons démontré, influencer le répertoire des cellules T recrutées dans la réponse immunitaire et, par conséquent, modifier l'aptitude de notre système immunitaire à faire face à une infection. -- Our immune system plays an important role in the protection from disease. CD8 T cells are critical for the control of primary infections with most viruses and certain bacteria as well as against some tumors. Therefore, better knowledge of CD8 T cell responses might enable us to generate vaccines against pathogens for which currently no vaccines are available or to improve anti-tumor immune responses. In the first part of this thesis we addressed the issue how previously acquired immunity impacts on the response of CD8 T cells. We are often exposed to pathogens that are related but not identical to the previously encountered ones. Such heterologous infections are not well studied and there are some indications that partial pre-existing immunity may in some cases even lead to an enhancement of disease. We specifically studied the T cell repertoire of CD8 T cells that are responding to a newly encountered antigen in secondary compared to primary infections. Using the experimental model of Listeria monocytogenes infections, we showed that in primary infections a wide repertoire including high and low affinity CD8 T cells is recruited into the immune response. In contrast to this, in secondary infections, the T cell repertoire is severely restricted and only T cells of high affinity are responding. We were able to pinpoint this difference to the presence of memory CD8 T cells that recognize an antigen that is shared between the two subsequent infections. This increase in the activation threshold was most effectively mediated via non-transferable memory CD8 T cells. This would argue that vaccines targeting low affinity tumor-specific T cells would fail if the vaccine contains previously encountered CD8 T cell epitopes. T cell mediated immune responses to tumor antigen rely often on T cells which weakly react to tumor antigen as high affinity T cells are eliminated by tolerance mechanisms. Following indication in the literature that PTPN2 impacts on the response of such weakly antigen-reactive T cells, we investigated how PTPN2 impacts in general the response of CD8 T cells. We observed that CD8 T cells lacking PTPN2 show an enhanced expansion following weak or short-term T cell receptor stimulation. The effector phase is prolonged and contraction delayed thus resulting in overall more effector cells. This is accompanied by a better survival of terminal effector cells. When transferred into new recipients, KLRG1+CD127- terminal effector cells lacking PTPN2 can survive and convert into CD127+ functional memory cells. Surprisingly, we discovered that elimination of PTPN2 enhances the transfer efficacy and formation of memory cells as well as the protective capacity. Targeting PTPN2 might thus be a promising approach for adoptive T cell therapy. Our observations show how the manipulation of an intrinsic factor, the absence of PTPN2, can enhance T cell responses under certain circumstances. A better understanding of underlying mechanisms for the control of CDS T cell responses might enable the manipulation of these and allow for more powerful responses. If these responses are induced through vaccines it is imperative that the previous history of exposure to pathogens or vaccines is considered as it can, as we have shown in this thesis, influence the recruited T cell repertoire and thus possibly the ability to handle the infection.

Characterization of local and systemic immune-modulation in melanoma patients

Le mélanome cutané est un des cancers les plus agressifs et dont l'incidence augmente le plus en Suisse. Une fois métastatique, le pronostic de survie moyenne avec les thérapies actuelles est d'environ huit mois, avec moins de 5% de survie à cinq ans. Les récents progrès effectués dans la compréhension de la biologie de la cellule tumorale mais surtout dans l'importance du système immunitaire dans le contrôle de ce cancer ont permis le développement de nouveaux traitements novateurs et prometteurs. Ces thérapies, appelées immunothérapies, reposent sur la stimulation et l'augmentation de la réponse immunitaire à la tumeur. Alors que les derniers essais cliniques ont démontré l'efficacité de ces traitements chez les patients avec des stades avancés de la maladie, le contrôle de la maladie à long- terme est seulement atteint chez une minorité des patients. La suppression locale et systémique de la réponse immunitaire spécifique anti-tumorale apparaitrait comme une des raisons expliquant la persistance d'un mauvais pronostic clinique chez ces patients. Des études sur les souris ont montré que les vaisseaux lymphatiques joueraient un rôle primordial dans ce processus en induisant une tolérance immune, ce qui permettrait à la tumeur d'échapper au contrôle du système immunitaire et métastatiser plus facilement. Ces excitantes découvertes n'ont pas encore été établi et prouvé chez l'homme. Dans cette thèse, nous montrons pour la première fois que les vaisseaux lymphatiques sont directement impliqués dans la modulation de la réponse immunitaire au niveau local et systémique dans le mélanome chez l'homme. Ces récentes découvertes montrent le potentiel de combiner des thérapies visant le système lymphatique avec les immunothérapies actuellement utilisées afin d'améliorer le pronostic des patients atteint du mélanome. -- Cutaneous melanoma is one of the most invasive and metastatic human cancers and causes 75% of skin cancer mortality. Current therapies such as surgery and chemotherapy fail to control metastatic disease, and relapse occurs frequently due to microscopic residual lesions. It is, thus, essential to develop and optimize novel therapeutic strategies to improve curative responses in these patients. In recent decades, tumor immunologists have revealed the development of spontaneous adaptive immune responses in melanoma patients, leading to the accumulation of highly differentiated tumor-specific T cells at the tumor site. This remains one of the most powerful prognostic markers to date. Immunotherapies that augment the natural function of these tumor-specific T cells have since emerged as highly attractive therapeutic approaches to eliminate melanoma cells. While recent clinical trials have demonstrated great progress in the treatment of advanced stage melanoma, long-term disease control is still only achieved in a minority of patients. Local and systemic immune suppression by the tumor appears to be responsible, in part, for this poor clinical evolution. These facts underscore the need for a better analysis and characterization of immune- related pathways within the tumor microenvironment (TME), as well as at the systemic level. The overall goal of this thesis is, thus, to obtain greater insight into the complexity and heterogeneity of the TME in human melanoma, as well as to investigate immune modulation beyond the TME, which ultimately influences the immune system throughout the whole body. To achieve this, we established two main objectives: to precisely characterize local and systemic immune modulation (i) in untreated melanoma patients and (ii) in patients undergoing peptide vaccination or checkpoint blockade therapy with anti-cytotoxic T- lymphocyte-asisctaed protein-4 (CTLA-4) antibody. In the first and main part of this thesis, we analyzed lymphatic vessels in relation to anti-tumor immune responses in tissues from vaccinated patients using a combination of immunohistochemistry (IHC) techniques, whole slide scanning/analysis, and an automatic quantification system. Strikingly, we found that increased lymphatic vessel density was associated with high expression of immune suppressive molecules, low functionality of tumor-infiltrating CD8+ T cells and decreased cytokine production by tumor-antigen specific CD8+ T cells in the blood. These data revealed a previously unappreciated local and systemic role of lymphangiogenesis in modulating T cell responses in human cancer and support the use of therapies that target lymphatic vessels combined with existing and future T cell based therapies. In the second objective, we describe a metastatic melanoma patient who developed pulmonary sarcoid-like granulomatosis following repetitive vaccination with peptides and CpG. We demonstrated that the onset of this pulmonary autoimmune adverse event was related to the development of a strong and long-lasting tumor-specific CD8+ T cell response. This constitutes the first demonstration that a new generation tumor vaccine can induce the development of autoimmune adverse events. In the third objective, we assessed the use of Fourier Transform Infrared (FTIR) imaging to identify melanoma cells and lymphocyte subpopulations in lymph node (LN) metastasis tissues, thanks to a fruitful collaboration with researchers in Brussels. We demonstrated that the different cell types in metastatic LNs have different infrared spectral features allowing automated identification of these cells. This technic is therefore capable of distinguishing known and novel biological features in human tissues and has, therefore, significant potential as a tool for histopathological diagnosis and biomarker assessment. Finally, in the fourth objective, we investigated the role of colony- stimulating factor-1 (CSF-1) in modulating the anti-tumor response in ipilimumab-treated patients using IHC and in vitro co-cultures, revealing that melanoma cells produce CSF-1 via CTL-derived cytokines when attacked by cytotoxic T lymphocytes (CTLs), resulting in the recruitment of immunosuppressive monocytes. These findings support the combined use of CSF-1R blockade with T cell based immunotherapy for melanoma patients. Taken together, our results reveal the existence of novel mechanisms of immune modulation and thus promote the optimization of combination immunotherapies against melanoma. -- Le mélanome cutané est un des cancers humains les plus invasifs et métastatiques et est responsable de 75% de la mortalité liée aux cancers de la peau. Les thérapies comme la chirurgie et la chimiothérapie ont échoué à contrôler le mélanome métastatique, par ailleurs les rechutes sous ces traitements ont été montrées fréquentes. Il est donc essentiel de développer et d'optimiser de nouvelles stratégies thérapeutiques pour améliorer les réponses thérapeutiques de ces patients. Durant les dernières décennies, les immunologistes spécialisés dans les tumeurs ont démontré qu'un patient atteint du mélanome pouvait développer spontanément une réponse immune adaptative à sa tumeur et que l'accumulation de cellules T spécifiques tumorales au sein même de la tumeur était un des plus puissants facteurs pronostiques. Les immunothérapies qui ont pour but d'augmenter les fonctions naturelles de ces cellules T spécifiques tumorales ont donc émergé comme des approches thérapeutiques très attractives pour éliminer les cellules du mélanome. Alors que les derniers essais cliniques ont démontré un progrès important dans le traitement des formes avancées du mélanome, le contrôle de la maladie à long-terme est seulement atteint chez une minorité des patients. La suppression immune locale et systémique apparaitrait comme une des raisons expliquant la persistance d'un mauvais pronostic clinique chez ces patients. Ces considérations soulignent la nécessité de mieux analyser et caractériser les voies immunitaires non seulement au niveau local dans le microenvironement tumoral mais aussi au niveau systémique dans le sang des patients. Le but de cette thèse est d'obtenir une plus grande connaissance de la complexité et de l'hétérogénéité du microenvironement tumoral dans les mélanomes mais aussi d'investiguer la modulation immunitaire au delà du microenvironement tumoral au niveau systémique. Afin d'atteindre ce but, nous avons établi deux objectifs principaux : caractériser précisément la modulation locale et systémique du système immunitaire (i) chez les patients atteints du mélanome qui n'ont pas reçu de traitement et (ii) chez les patients qui ont été traités soit par des vaccins soit par des thérapies qui bloquent les points de contrôles. Dans la première et majeure partie de cette thèse, nous avons analysé les vaisseaux lymphatiques en relation avec la réponse immunitaire anti-tumorale dans les tissus des patients vaccinés grâce à des techniques d'immunohistochimie et de quantification informatisé et automatique des marquages. Nous avons trouvé qu'une densité élevée de vaisseaux lymphatiques dans la tumeur était associée à une plus grande expression de molécules immunosuppressives ainsi qu'à une diminution de la fonctionnalité des cellules T spécifiques tumoral dans la tumeur et dans le sang des patients. Ces résultats révèlent un rôle jusqu'à là inconnu des vaisseaux lymphatiques dans la modulation directe du système immunitaire au niveau local et systémique dans les cancers de l'homme. Cette recherche apporte finalement des preuves du potentiel de combiner des thérapies visant le système lymphatique avec des autres immunothérapies déjà utilisées en clinique. Dans le second objectif, nous rapportons le cas d'un patient atteint d'un mélanome avec de multiples métastases qui a développé à la suite de plusieurs vaccinations répétées et consécutives avec des peptides et du CpG, un évènement indésirable sous la forme d'une granulomatose pulmonaire sarcoid-like. Nous avons démontré que l'apparition de cet évènement était intimement liée au développement d'une réponse immunitaire durable et spécifique contre les antigènes de la tumeur. Par là- même, nous prouvons pour la première fois que la nouvelle génération de vaccins est aussi capable d'induire des effets indésirables auto-immuns. Pour le troisième objectif, nous avons voulu savoir si l'utilisation de la spectroscopie infrarouge à transformée de Fourier (IRTF) était capable d'identifier les cellules du mélanome ainsi que les différents sous-types cellulaires dans les ganglions métastatiques. Grâce à nos collaborateurs de Bruxelles, nous avons pu établir que les diverses composantes cellulaires des ganglions atteints par des métastases du mélanome présentaient des spectres infrarouges différents et qu'elles pouvaient être identifiées d'une façon automatique. Cette nouvelle technique permettrait donc de distinguer des caractéristiques biologiques connues ou nouvelles dans les tissus humains qui auraient des retombées pratiques importantes dans le diagnostic histopathologique et dans l'évaluation des biomarqueurs. Finalement dans le dernier objectif, nous avons investigué le rôle du facteur de stimulation des colonies (CSF-1) dans la modulation de la réponse immunitaire anti-tumorale chez les patients qui ont été traités par l'Ipilimumab. Nos expériences in vivo au niveau des tissus tumoraux et nos co-cultures in vitro nous ont permis de démontrer que les cytokines secrétées par les cellules T spécifiques anti-tumorales induisaient la sécrétion de CSF-1 dans les cellules du mélanome ce qui résultait en un recrutement de monocytes immunosuppresseurs. Dans son ensemble, cette thèse révèle donc l'existence de nouveaux mécanismes de modulation de la réponse immunitaire anti-tumorale et propose de nouvelles optimisations de combinaison d'immunothérapies contre le mélanome.

From T cell “exhaustion” to anti-cancer immunity

The immune system has the potential to protect from malignant diseases for extended periods of time. Unfortunately, spontaneous immune responses are often inefficient. Significant effort is required to develop reliable, broadly applicable immunotherapies for cancer patients. A major innovation was transplantation with hematopoietic stem cells from genetically distinct donors for patients with hematologic malignancies. In this setting, donor T cells induce long-term remission by keeping cancer cells in check through powerful allogeneic graft-versus-leukemia effects. More recently, a long awaited breakthrough for patients with solid tissue cancers was achieved, by means of therapeutic blockade of T cell inhibitory receptors. In untreated cancer patients, T cells are dysfunctional and remain in a state of T cell "exhaustion". Nonetheless, they often retain a high potential for successful defense against cancer, indicating that many T cells are not entirely and irreversibly exhausted but can be mobilized to become highly functional. Novel antibody therapies that block inhibitory receptors can lead to strong activation of anti-tumor T cells, mediating clinically significant anti-cancer immunity for many years. Here we review these new treatments and the current knowledge on tumor antigen-specific T cells.

Analysis of a Plant Complex Resistance Gene Locus Underlying Immune-Related Hybrid Incompatibility and Its Occurrence in Nature

Mechanisms underlying speciation in plants include detrimental (incompatible) genetic interactions between parental alleles that incur a fitness cost in hybrids. We reported on recessive hybrid incompatibility between an Arabidopsis thaliana strain from Poland, Landsberg erecta (Ler), and many Central Asian A. thaliana strains. The incompatible interaction is determined by a polymorphic cluster of Toll/interleukin-1 receptor-nucleotide binding-leucine rich repeat (TNL) RPP1 (Recognition of Peronospora parasitica1)-like genes in Ler and alleles of the receptor-like kinase Strubbelig Receptor Family 3 (SRF3) in Central Asian strains Kas-2 or Kond, causing temperature-dependent autoimmunity and loss of growth and reproductive fitness. Here, we genetically dissected the RPP1-like Ler locus to determine contributions of individual RPP1-like Ler (R1R8) genes to the incompatibility. In a neutral background, expression of most RPP1-like Ler genes, except R3, has no effect on growth or pathogen resistance. Incompatibility involves increased R3 expression and engineered R3 overexpression in a neutral background induces dwarfism and sterility. However, no individual RPP1-like Ler gene is sufficient for incompatibility between Ler and Kas-2 or Kond, suggesting that co-action of at least two RPP1-like members underlies this epistatic interaction. We find that the RPP1-like Ler haplotype is frequent and occurs with other Ler RPP1-like alleles in a local population in Gorzów Wielkopolski (Poland). Only Gorzów individuals carrying the RPP1-like Ler haplotype are incompatible with Kas-2 and Kond, whereas other RPP1-like alleles in the population are compatible. Therefore, the RPP1-like Ler haplotype has been maintained in genetically different individuals at a single site, allowing exploration of forces shaping the evolution of RPP1-like genes at local and regional population scales.

System-wide approaches to uncover Th2 cell lineage commitment

T helper (Th) cells are vital regulators of the adaptive immune system. When activated by presentation of cognate antigen, Th cells demonstrate capacity to differentiate into functionally distinct effector cell subsets. The Th2 subset is required for protection against extracellular parasites, such as helminths, but is also closely linked to pathogenesis of asthma and allergies. The intracellular molecular signal transduction pathways regulating T helper cell subset differentiation are still incompletely known. Moreover, great majority of studies regarding Th2 differentiation have been conducted with mice models, while studies with human cells have been fewer in comparison. The goal of this thesis was to characterize molecular mechanisms promoting the development of Th2 phenotype, focusing specifically on human umbilical cord blood T cells as an experimental model. These primary cells, activated and differentiated to Th2 cells in vitro, were investigated by complementary system-wide approaches, targeting levels of mRNA, proteins, and lipid molecules. Specifically, the results indicated IL4-regulated recruitment of nuclear protein, and described novel components of the Th2-promoting STAT6 enhanceosome complex. Furthermore, the development of the activated effector cell phenotype was found to correlate with remodeling of the cellular lipidome. These findings will hopefully advance the understanding of human Th2 cell lineage commitment and development of Th2-associated disease states.

Reactive oxygen species in inflammation

Chronic inflammation is the underlying cause of many common disabling conditions such as rheumatoid arthritis (RA), multiple sclerosis, coeliac disease, type I diabetes and coronary artery disease. NOX2 complex derived reactive oxygen species (ROS) are known to regulate joint inflammation in rats and mice, and additionally recent genetic evidence associates phagocyte ROS and the development RA in humans. Ncf1mutated mice have lost the functionality of their NOX2 complex and thus have no phagocyte ROS production. These mice suffer from exacerbated arthritis. The immune suppressive effect of the NOX2 complex derived ROS is mediated by monocytes/macrophages that downregulate the activation of autoreactive T cells. The aim of this thesis was to study how ROS modulate immune responses in different arthritis models and in tumor development. Additionally, genome wide gene expression profiling was carried out to assess the global effects of NOX2 complex derived ROS. Firstly, these results confirmed the potent anti-inflammatory nature of phagocyte ROS in arthritis models that were driven by the adaptive immune system. Secondly, arthritis models with predominantly innate immunity induced pathophysiology were moderately enhanced by phagocyte, more specifically, neutrophil derived ROS. Thirdly, the ROS induced immune suppression mediated by the adaptive immune system allowed development of bigger implanted tumors, while phagocyte ROS production did not affect the development of spontaneously growing tumors. Lastly, genome wide gene expression analysis revealed that both humans and mice with abrogated phagocyte NOX2 complex ROS production had an enhanced type I interferon signature in blood, reflecting their hyperinflammatory immune status.

Frequency of B cells in normal mice which recognize self proteins

The mechanism whereby the immune system avoids self-aggression is one of the central issues of Immunology. The discovery of natural autoantibodies, mainly of IgM isotype, and of idiotypic interactions between antibodies indicates that elements of the immune system interact with self constituents and with themselves. Results of studies with soluble antibodies have indicated that the pool of circulating IgM represents the end result of a highly selective process of B cells activation and differentiation by self proteins resulting in the formation of a network. The objective of the present work was to determine the frequency of self-reacting B cells in normal mice. We were able to detect B cells that recognize self proteins present in extracts of different organs in normal adult, 2-3-month old, BALB/c and C57BL/6 mice with an ELISA spot assay. About 1% of total IgM-secreting cells among small, LPS-stimulated spleen cells reacted with organ extracts, whereas among large spleen cells the frequency was 5- to 10-fold lower. Immunization induced an increase in the frequency of IgM-secreting cells. The present results provide cellular evidence for the results of studies done at the serological level. The physiological role of these self-recognizing cells, as well as their participation in autoimmune processes, remain to be established

Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-g. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

Cytokines in innate and acquired immunity to Trypanosoma cruzi infection

Resistance to Trypanosoma cruzi infections is critically dependent on cytokine-mediated activation of cell-mediated immune effector mechanisms. This review focuses on the role of IL-10, TNF-a, IFN-g and IL-12 in controlling T. cruzi replication by the innate and specific immune systems of the vertebrate host. A study performed on mice with disrupted recombinase-activating genes (RAG/KO), which lack T and B lymphocytes, revealed the importance of IL-12, IFN-g and TNF-a in the resistance against T. cruzi mediated by the innate immune system. In addition, data from experiments using IL-10 KO, RAG/KO and double RAG/IL-10 KO mice indicating an in vivo regulatory role of IL-10 in innate and T. cruzi-specific immunity are discussed

Monounsaturated fats and immune function

Animal studies suggest that olive oil is capable of modulating functions of cells of the immune system in a manner similar to, albeit weaker than, fish oils. There is some evidence that the effects of olive oil on immune function in animal studies are due to oleic acid rather than to trace elements or antioxidants. Importantly, several studies have demonstrated effects of oleic acid-containing diets on in vivo immune responses. In contrast, consumption of a monounsaturated fatty acid (MUFA)-rich diet by humans does not appear to bring about a general suppression of immune cell functions. The effects of this diet in humans are limited to decreasing aspects of adhesion of peripheral blood mononuclear cells, although there are trends towards decreases in natural killer cell activity and proliferation. The lack of a clear effect of MUFA in humans may be attributable to the higher level of monounsaturated fat used in the animal studies, although it is ultimately of importance to examine the effects of intakes which are in no way extreme. The effects of MUFA on adhesion molecules are potentially important, since these molecules appear to have a role in the pathology of a number of diseases involving the immune system. This area clearly deserves further exploration

Gap junctions in the cardiovascular and immune systems

Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

The neuroimmune-endocrine axis: pathophysiological implications for the central nervous system cytokines and hypothalamus-pituitary-adrenal hormone dynamics

Cytokines are molecules that were initially discovered in the immune system as mediators of communication between various types of immune cells. However, it soon became evident that cytokines exert profound effects on key functions of the central nervous system, such as food intake, fever, neuroendocrine regulation, long-term potentiation, and behavior. In the 80's and 90's our group and others discovered that the genes encoding various cytokines and their receptors are expressed in vascular, glial, and neuronal structures of the adult brain. Most cytokines act through cell surface receptors that have one transmembrane domain and which transduce a signal through the JAK/STAT pathway. Of particular physiological and pathophysiological relevance is the fact that cytokines are potent regulators of hypothalamic neuropeptidergic systems that maintain neuroendocrine homeostasis and which regulate the body's response to stress. The mechanisms by which cytokine signaling affects the function of stress-related neuroendocrine systems are reviewed in this article.

Immunological effects of donor lymphocyte infusion in patients with chronic myelogenous leukemia relapsing after bone marrow transplantation

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.