Linfócitos T CD4+ (LTCD4) sofrem morte pelo reestímulo do TCR em um processo chamado de AICD (activation-induced cell death) no fim de uma resposta imune. Resultados prévios de nosso grupo de pesquisa mostraram que melatonina foi capaz de inibir o processo de AICD em LTCD4 em experimentos in vitro. Portanto, o objetivo deste trabalho é verificar se a melatonina é capaz de agir como estimulador, aumentando a resposta imune mediada pelos LTCD4 in vivo. Nós observamos que 3 e 9mg/Kg de melatonina aumentaram a resposta de DTH (Delayed-type Hypersensitivity), de forma diretamente proporcional à dose utilizada. O tratamento com melatonina estimulou um aumento da proliferação e do numero absoluto de LTCD4 específicos de antígeno. Em experimentos de diferenciação linfocitária in vitro, nós observamos que o tratamento com melatonina estimulou a produção de LTCD4 do perfil Th1 e Th2, no entanto inibiu a produção de linfócitos Th17. Em conclusão, nossos resultados sugerem um efeito estimulador da melatonina sobre a função de linfócitos T CD4+. / CD4+ T lymphocytes (LTCD4+) suffer cell death by a process known as activation-induced cell death (AICD) at the end of immune response. Previous results from our group showed that melatonin can inhibits AICD process in LTCD4 at in vitro experiments. Therefore, the aim of this work is verify if a melatonin can act as immune stimulator increasing a LTCD4-mediated immune response in vivo. We showed that 3 and 9 mg/Kg of exogenous melatonin added during immunization resulted in potentiation dose-dependent of Delayed-type hypersensitivity (DTH) response. The treatment with melatonin increased the absolute number LTCD4 antigen-specific, probably by increment of its proliferation. In experiment of T cell differentiation, we observed that the treatment with melatonin stimulated LTCD4 production of Th1 and Th2 profile however blocked the Th17 lymphocytes production. In conclusion, our results support the idea about a regulator role of melatonin on LTCD4 lymphocytes function for development of an immune response.

As infecções humanas com a filaria Mansonella ozzardi ocorrem em focos situados em regiões tropicais e subtropicais da América Central e do Sul e frequentemente coexistem com outras doenças endêmicas tropicais. Na Amazônia brasileira, as infecções são geralmente assintomáticas e a maior parte delas, consequentemente, deixam de ser diagnosticada. As filarioses crônicas, geralmente não tratadas, podem criar um ambiente imunorregulador, caracterizado pela expansão de linfócitos T produtores de IL-10, que mediam a supressão de respostas proliferativas de células T frente a antígenos específicos bem como a antígenos não-relacionados. Neste trabalho, utilizamos marcadores de ativação celular (CD69 e HLA-DR) e de atividade reguladora (CD39, CTLA-4, OX40, GITR, LAG3, PD-1, LAP-TGF-β e TNFRII) para caracterizar populações de células mononucleares de sangue periférico (PBMCs) em indivíduos infectados por M. ozzardi bem como em controles saudáveis de uma área endêmica deste parasito na Amazônia Brasileira. A análise de PBMCs, por citometria de fluxo multiparamétrica de 49 pacientes infectados por M. ozzardi, mostrou que pacientes e controles apresentam proporções similares de Treg clássicas circulantes, no entanto, indivíduos infectados apresentam um aumento da proporção de células CD4+ e células T reguladoras (Tregs) que expressam a molécula CD39. Células Treg CD39+ parecem definir uma população distinta entre as Treg, pois ao compararmos os marcadores de regulação e ativação entre Tregs CD39+ e CD39- encontramos proporções aumentas destes marcadores nas Treg CD39+. O bloqueio dessa molécula em condições de reestimulo celular aumenta a produção de citocinas inflamatórias e diminui a produção de IL-10 confirmando seu papel regulador. / Human infections with the filarial parasite Mansonella ozzardi are common in areas of tropical and subtropical Central and South America and often coexist with other endemic tropical diseases, such as malaria. In the Amazonian Basin of Brazil, infections are typically asymptomatic; most of them will remain undiagnosed. These chronic, untreated filarial infections are potentially associated with a regulatory immune environment, dominated by IL-10-producing T-cells, which mediate the suppression of T-cell proliferation in response to filarial and non-related antigens. Here, we used markers of cell activation (CD69 and HLA-DR) and regulatory activity (CD39, CTLA-4, OX40, GITR, and TNFRII) to characterize peripheral-blood mononuclear cell (PBMCs) subpopulations in individuals infected with |M. ozzardi and in healthy controls living in an area of M. ozzardi endemicity in the Brazilian Amazon. Multiparameter flow cytometry analysis of PBMCs from 49 malaria patients showed that patients and controls have similar proportions of classic circulating Tregs, however, the proportion of CD4 + cells and Tregs expressing the CD39 (an ectonucleotidase that regulates the balance of immune responses through Phosphohydrolysis of ATP, an inflammatory molecule in adenosine, an anti-inflammatory molecule), is increased in infected patients. CD39+Treg cells seem to define a distinct population among Tregs, compare activation and regulatory markers between CD39+ and CD39- Tregs - we found increased proportions of these markers in the CD39+ Tregs. Blocking this molecule under cellular restimulation conditions increases production of inflammatory cytokines and decreases IL-10 production, improving its regulatory role.

A atopia caracteriza-se pela tendência de um indivíduo a produzir IgE em quantidade elevada, em resposta a um alérgeno específico, levando ao desenvolvimento de asma, rinite ou eczema. Todavia, a manifestação do fenótipo da alergia depende da interação de fatores genéticos e exposição a alérgenos ambientais. Desta forma, o alérgeno é processado e apresentado aos linfócitos T, que desenvolvem uma resposta imune Th2, exacerbada característica da atopia. A principal célula que está envolvida na comunicação entre a imunidade inata e adaptativa é a célula dendrítica (DC) cuja função é capturar, processar e apresentar o antígeno aos linfócitos. As DCs imaturas capturam o antígeno e migram do tecido para o órgão linfóide periférico, onde elas se diferenciam em DCs maduras e apresentam o antígeno aos linfócitos T naive. Assim, os linfócitos T naive podem se diferenciar em subtipos de linfócitos efetores como: linfócitos Th1 e Th2. Esses linfócitos auxiliam na produção de anticorpos pelos linfócitos B na resposta imune humoral contra patógenos específicos. O sistema imune humoral compreende cinco classes de imunoglobulinas: IgG, IgM, IgD, IgE e IgA e sua produção sofre influência da imunidade celular. Desta forma, alérgenos provenientes de ácaros como, Dermatophagoides pteronyssinus, podem levar à inflamação alérgica, alterando a produção de anticorpos. Tendo em vista evidências que demonstram a interação das DCs com anticorpos, propomos investigar sua influência na apresentação dos principais alérgenos da poeira domiciliar e sua modulação sobre a resposta imune em indivíduos alérgicos e não alérgicos. / Atopy is characterized by the trend of an individual to produce high amounts of IgE in response to a specific allergen, leading to the development of asthma, rhinitis or eczema. However, the manifestation of the allergy phenotype depends on the interaction of genetic factors and exposure to environmental allergens. In this way, the allergen is processed and presented to the T lymphocytes, which develop a Th2 immune response, exacerbated characteristic of atopy. The main cell that is involved in the communication between innate and adaptive immunity is the dendritic cell (DC) whose function is to capture, process and present the antigen to lymphocytes. Immature DCs capture the antigen and migrate from the tissue to the peripheral lymphoid organ, where they differentiate into mature DCs and present the antigen to naive T lymphocytes. Thus, naive T lymphocytes can differentiate into subtypes of effector lymphocytes such as Th1 and Th2 lymphocytes. These lymphocytes assist in the production of antibodies by B lymphocytes in the humoral immune response against specific pathogens. The humoral immune system comprises five classes of immunoglobulins: IgG, IgM, IgD, IgE and IgA and their production is influenced by cellular immunity. In this way, allergens from mites such as Dermatophagoides pteronyssinus, can lead to allergic inflammation, altering the production of antibodies. In view of evidence demonstrating the interaction of DCs with antibodies, we propose to investigate their influence on the presentation of the main house dust allergens and their modulation on the immune response in allergic and non-allergic individuals.

Self-reactive CD4+ helper T cells (Th) are key causal agents in the pathogenesis of many autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a CD4+T cell model of the demyelinating autoimmune disease multiple sclerosis (MS). It has been shown that EAE is caused by CD4+ T-cells that produce pro-inflammatory cytokines IFN-γ (Th1) and IL-17 (Th17). As such, understanding how these Th cells are generated and controlled is essential. There is debate as to whether Th1 and Th17 cells act independently in EAE or if there is plasticity between these two subtypes, and whether the capacity to switch from Th1 to Th17 confers pathogenic capacity. T-bet was first described as the master transcription factor for Th1 cells, and is thought to have a critical role in EAE even though IFN-γ, the Th1 archetypal cytokine, has been shown to be redundant. More recent work has shown that T-bet is expressed in multiple immune cell types, and it remains unclear in what cells the expression of T-bet is required for EAE. Considerable efforts have been put into understanding the role of T-bet in EAE pathogenesis, with a view to modulate cells expressing T-bet for therapy. The hypothesis of this work was that T-bet has multifaceted roles in EAE, in initiating and directing an immune response in innate antigen presenting cells such as dendritic cells (DC) as well as programming pathogenic effector CD4+ T cell (Teff) response to antigen. T-bet-/- mice were studied using different models of EAE to dissect the role of T-bet in disease pathogenesis. Active immunisation of C57BL/6 mice with the immunodominant peptide from myelin oligodendrocyte glycoprotein (MOG35-55) showed that T-bet-/- mice developed EAE with an IL-17 dominated profile and critically, T-bet-/- mice were able to produce GM-CSF which has recently been described as a key cytokine for EAE. T-bet-/- cells were not able to transfer EAE in a model of passive transfer EAE, where CD4+ T cells were polarised towards a Th1 profile in vitro. Illustrating that T-bet is required in CD4+ T cells for Th1 mediated EAE. DC driven EAE showed that T-bet-/- DC were able to activate CD4+ T cells in vitro and cause EAE upon co-transfer into host mice with transgenic CD4+ T cells. Thus, it has been shown that T-bet is not required in EAE. This work represents a step further towards understanding the disease mechanisms involved in EAE and suggests T-bet is not an appropriate therapeutic target for the treatment of MS.

Linfócitos T CD4+ (LTCD4) sofrem morte pelo reestímulo do TCR em um processo chamado de AICD (activation-induced cell death) no fim de uma resposta imune. Resultados prévios de nosso grupo de pesquisa mostraram que melatonina foi capaz de inibir o processo de AICD em LTCD4 em experimentos in vitro. Portanto, o objetivo deste trabalho é verificar se a melatonina é capaz de agir como estimulador, aumentando a resposta imune mediada pelos LTCD4 in vivo. Nós observamos que 3 e 9mg/Kg de melatonina aumentaram a resposta de DTH (Delayed-type Hypersensitivity), de forma diretamente proporcional à dose utilizada. O tratamento com melatonina estimulou um aumento da proliferação e do numero absoluto de LTCD4 específicos de antígeno. Em experimentos de diferenciação linfocitária in vitro, nós observamos que o tratamento com melatonina estimulou a produção de LTCD4 do perfil Th1 e Th2, no entanto inibiu a produção de linfócitos Th17. Em conclusão, nossos resultados sugerem um efeito estimulador da melatonina sobre a função de linfócitos T CD4+. / CD4+ T lymphocytes (LTCD4+) suffer cell death by a process known as activation-induced cell death (AICD) at the end of immune response. Previous results from our group showed that melatonin can inhibits AICD process in LTCD4 at in vitro experiments. Therefore, the aim of this work is verify if a melatonin can act as immune stimulator increasing a LTCD4-mediated immune response in vivo. We showed that 3 and 9 mg/Kg of exogenous melatonin added during immunization resulted in potentiation dose-dependent of Delayed-type hypersensitivity (DTH) response. The treatment with melatonin increased the absolute number LTCD4 antigen-specific, probably by increment of its proliferation. In experiment of T cell differentiation, we observed that the treatment with melatonin stimulated LTCD4 production of Th1 and Th2 profile however blocked the Th17 lymphocytes production. In conclusion, our results support the idea about a regulator role of melatonin on LTCD4 lymphocytes function for development of an immune response.

acase@tulane.edu

acase@tulane.edu

Tim-1 (T cell immunoglobulin mucin domain 1) is a transmembrane protein expressed by many cell types, including activated T cells and B cells. Antibodies to Tim-1 have been shown to decrease severity of airway hyperreactivity and Th2 cytokine production in mice. Current literature suggests Tim-1 functions as a co-stimulatory molecule. We hypothesize that Tim-1 signals in lymphocytes, and that Tim-1 signaling modulates allergic airway disease. Chapter one provides a brief overview of current literature exploring identification of the Tim family of receptors, genetic associations between TIM-1 polymorphisms and human diseases, Tim-1 expression, Tim-1 ligands, studies of antibodies to Tim-1 in various mouse models of human disease, and signaling events downstream of Tim-1 engagement. Chapter two provides detailed experimental methodology. Chapter three details the characterization of Tim-1 deficient mice. Tim-1 deficient mice do not exhibit defects in lymphocyte or myeloid cell development, as determined by numbers of cells present in bone marrow, thymus, spleen, and lymph nodes. C57BL/6 Tim-1 deficient female mice appear to develop an increased number of lymph node cells and also develop anti-double stranded DNA antibodies. Chapter four explores the impact of Tim-1 deficiency in a murine allergic airway disease model, which demonstrated that Tim-1 deficient mice developed increased lung inflammation and increased antigen-specific Th2 cytokine production that was evident in mice backcrossed to both BALB/c and C57BL/6 backgrounds. These phenotypes were not evident using purified naïve CD4+ T cells polarized in vitro. As Tim-1 expression is not restricted to CD4+ T cells, adoptive transfer experiments were performed to determine whether the phenotype observed was due to the deficiency of Tim-1 on CD4+ T cells, non-CD4+ T cells, or Tim-1 deficiency on both CD4+ T cells and non-CD4+ T cells. Chapter five explores the impact of Tim-1 deficiency in a chronic Leishmania major intradermal infection model. Tim-1 deficient mice crossed to both BALB/c and C57BL/6 backrounds demonstrated similar parasite burden over the course of time, but in vitro restimulation of lymph node cells revealed a striking increase in cytokine production that extended to Th1, Th2, and Th17 lineages. Tim-1 signaling in murine B cell lines is explored in Chapter six. A Tim-1 monoclonal antibody conjugated to beads induces phosphorylation of Tim-1 and recruitment of the Src family kinase Fyn. This phosphorylation of Tim-1 is reduced in Fyn-deficient B cell lines. Chapter seven discusses the significance of these findings, relates current literature to these results, and provides some avenues for further exploration of Tim-1 function and signaling.

La toxine de pertussis (PTX) est une A-B protéine considérée comme l’un des principaux facteurs de virulence de Bordetella pertussis, l’agent bactérien responsable de la coqueluche. Aujourd’hui, cette maladie représente encore un réel danger pour les nouveaux-nés et les nourrissons non ou partiellement immunisés. Actuellement, la coqueluche provoque encore la mort d’environ 350.000 individus par an. La toxicité de la PTX est liée à l’activité enzymatique de sa sous-unité A capable d’inhiber les voies de signalisation associées aux protéines Gi. La partie B, quant à elle, permet l’entrée de cette sous-unité A dans le cytoplasme des cellules cibles en se liant spécifiquement à son ou ses récepteurs membranaires toujours inconnus de nos jours. Des études réalisées chez la souris et chez l’homme ont montré que les vaccins anticoquelucheux combinés à différents antigènes vaccinaux étaient capables de moduler leurs réponses humorales spécifiques. Par ailleurs, la PTX est couramment qualifiée d’agent immunostimulant. En effet, des modèles murins de vaccination permirent d’identifier des propriétés adjuvantes de la PTX coadministrée avec des antigènes non relevants. Le travail développé dans ce manuscrit étudie les effets de la PTX sur 2 types cellulaires primordiaux sollicités lors d’une vaccination : la cellule dendritique (DC) et le lymphocyte T CD4+ naïf. Les DC sont les seules cellules présentatrices d’antigènes aptes à initier une réponse immune primaire. Dans un premier temps, nous avons montré que la PTX était capable d’activer des DC générées in vitro à partir de monocytes. En effet, elles acquièrent un phénotype mature caractérisé par une augmentation de l’expression membranaire des molécules costimulatrices et du CMH de classe II, démontrant un effet direct et spécifique de la PTX sur les DC myéloïdes. Parallèlement, ces DC produisent du TNF-a, de l’IL-12p40 et de l’IL-12p70 et activent NF-kappaB, un facteur de transcription essentiel au processus de maturation. Nous avons obtenu des résultats similaires avec une toxine génétiquement modifiée qui est enzymatiquement inactive. A partir de sang total incubé avec la PTX, nous avons par ailleurs observé que les DC circulantes du nouveau-né étaient déficientes dans leur maturation et leur sécrétion d’IL-12p70 comparées aux DC de l’adulte. D’autre part, il a été décrit précédemment que la PTX exerçait des effets mitogènes sur les lymphocytes T humains et murins. Cependant, le rôle qu’elle joue sur la population des lymphocytes T CD4 naïfs reste peu connu. A l’issue de notre second travail, nous pouvons dès lors affirmer que la PTX est également capable d’activer des lymphocytes T CD4+CD45RA+ naïfs isolés à partir des cellules mononuclées du sang périphérique, et ce indépendamment de son activité enzymatique. En effet, ces lymphocytes T CD4+ naïfs stimulés par la PTX prolifèrent, synthétisent des quantités non négligeables d'ARN messagers codant pour l’IL-2 et le TNF-a, augmentent l’expression membranaire des molécules CD40L, CD69 et CD25 et expriment la protéine Foxp3. Cette activation s’accompagne de la translocation nucléaire de NF-kappaB et NFAT. Parallèlement à l’adulte, la PTX active les lymphocytes T CD4 néonataux. Néanmoins, ceux-ci prolifèrent moins bien et expriment plus faiblement le CD40L à leur surface. Enfin, la PTX induit la sécrétion de taux importants d’IFN-g par des T CD4+CD45RA+ naïfs adultes mis en présence de DC autologues. Nous terminerons en proposant l’hypothèse suivante : La PTX pourrait exercer ses propriétés adjuvantes par l’intermédiaire de différents mécanismes comprenant notamment la maturation des DC d’origine myéloïde et l’activation des lymphocytes T CD4+CD45RA+ naïfs. Ces 2 populations cellulaires sont en effet les principaux protagonistes impliqués dans la réponse immune primaire.

Manfouo Foutsop Germain (2010). Etude de lexpression des gènes cellulaires dérégulés dans des cellules T CD4+ exprimant Tat de HIV-1 : Focalisation sur la voie de signalisation TCR/CD3 (thèse de doctorat). Université de Liège-Gembloux Agro-Bio Tech, 157 pages, 11 tables, 29 figures. Résumé Nos travaux précédents ont suggéré que les gènes nef (negative factor) et tat (transactivateur de la transcription) du virus de limmunodéficience humaine de type 1 (HIV-1) pourraient induire la perte du récepteur des cellules T (TCR)/CD3 à la surface des lymphocytes T CD4+ infectés par le HIV-1, mais la contribution de tat dans cette perte navait pas été prouvée. Dans la première partie de ce travail nous avons utilisé un vecteur lentiviral exprimant les formes natives [1exon (72aa) ou 2exons (101aa)] ou des formes mutées du gène tat de HIV-1 pour transduire les cellules T CD4+. Ceci nous a permis de montrer que la production de la protéine Tat72aa ou Tat101aa entraîne une perte progressive du complexe TCR/CD3 en surface des cellules T CD4+. Nous avons constaté que cette perte de TCR/CD3 serait due à une diminution du taux des transcrits de CD3γ. Par ailleurs, nous avons montré que certains mutants du gène tat ont perdu leur activité transactivatrice et sont incapables dinduire la perte de TCR/CD3. Dans la deuxième partie des travaux, nous avons identifié par microarray, les gènes cellulaires dont les expressions sont communément altérées dans les cellules T CD4+ infectées par le HIV-1 ou transduites avec le gène tat-1exon ou tat-2exons. Plusieurs de ces gènes sont impliqués dans la voie de signalisation TCR/CD3. Nos travaux nous ont donc permis de montrer limplication du gène tat dans la dérégulation de la voie de signalisation TCR/CD3. Par conséquent, Tat pourrait induire une altération de la réponse immunitaire favorisant ainsi la progression de linfection par le HIV-1/SIDA. Manfouo Foutsop Germain (2010). A study of altered cellular gene expression in CD4+ T cells expressing HIV-1 Tat: Focus on the T cell receptor/CD3-directed pathway (PhD thesis). Université de Liège-Gembloux Agro-Bio Tech, 157 pages, 11 tables, 29 figures. Summary Our previous work has suggested that nef (negative factor) and tat (transactivator of transcription) are responsible for T cell receptor (TCR)/CD3 downmodulation on CD4+ T cells after infection with human immunodeficiency virus type 1 (HIV-1). However, the contribution of tat to this phenomenon has not been investigated. In the first part of this study we used lentiviral vector expressing the native tat gene [1exon (72aa) or two exon (101aa)] and various mutants to transduce CD4+ T cells. We found that Tat expression alone could provoke the progressive downmodulation of surface TCR/CD3 complexes due to the decrease of CD3γ transcripts. We also demonstrated that the majority of mutants lost their transactivation capability and their ability to downregulate the TCR/CD3 complex. In the second part of this work, we used gene expression arrays to identify cellular genes whose expression was altered in CD4+ T cells infected with HIV-1 or transduced with tat at different stages of receptor downregulation. We found progressive alteration of cellular gene expression in parallel with TCR/CD3 downregulation in the tat-transduced cells, which represented a subset of the changes detected in the HIV-1 infected cells, with significant number genes known to play a role in the TCR/CD3 signaling pathway. Our data demonstrate that tat deregulates the TCR/CD3 expression and signaling leading to altered immune function that likely favor HIV-1/AIDS disease progression. Copyright: Aux termes de la loi belge du 30 juin 1994, sur le droit dauteur et les droits voisins, seul lauteur a le droit de reproduire partiellement ou complètement cet ouvrage de quelque façon que ce soit ou dautoriser la reproduction partielle ou complète de quelque manière et sous quelque forme que ce soit. Toute photocopie ou reproduction sous autre forme est donc faite en violation de ladite loi et des modifications ultérieures.