Made available in DSpace on 2015-07-22T20:49:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-29 / O Lúpus Eritematoso Sistêmico (LES) é uma doença inflamatória crônica pertencente ao grupo das doenças reumáticas autoimunes sistêmicas, caracterizando- se por apresentar as mais variadas manifestações clínicas e laboratoriais. Seu mecanismo exato de etiopatogenia ainda permanece obscuro. Observações prévias avaliando o papel das células TREG CD4+ CD25+ nas doenças autoimunes, em que se tem detectado tanto alterações de freqüência como alterações funcionas e fenotípicas em modelos murinos e humanos, sugerem o papel significante dessa população celular na etiopatogenia da autoimunidade. No LES podemos observar a existência de uma complexa rede de interações que caracterizam a doença, em que muitos alvos para intervenção terapêutica podem ser considerados. Atualmente tem-se voltado bastante a atenção para o estudo das células TREG CD4+CD25+, a fim de que possam ser usadas como alvos potenciais para terapia imunomoduladora. Os dados sobre a freqüência e fenótipo das células TREG publicados ate o momento são controversos devido à heterogeneidade de marcadores fenotípicos e estratégias de análises utilizadas. Um alto nível de células efetoras ativadas contaminam as amostras de células selecionadas de acordo com as estratégias clássicas de identificação de células TREG no LES e este fenômeno é ainda mais acentuado quanto maior o grau de atividade da doença. Assim, o presente projeto pretendeu inicialmente validar uma estratégia de análise capaz de identificar e quantificar células TREG utilizando a combinação dos marcadores CD25 e CD127 associados à expressão de Foxp3 em pacientes com LES em atividade ou fora de atividade. Concluiu-se pelo painel CD4+CD25+/highCD127Æ/low como melhor marcador de células TREG em virtude de sua alta associação com Foxp3 tanto em sadios como em pacientes com LES. Num segundo momento avaliamos a freqüência de células TREG e células Tconvonde observamos níveis normais de células TREG e níveis elevados de células Tconv ativadas em pacientes com doença em atividade. Foi nosso objetivo, também, avaliar a expressão de marcadores fenotípicos importantes para biologia das células TREG. Foi avaliada a expressão dos marcadores: CTLA-4, GITR, PD-1, OX40, HLA-DR, CD95, CD45Ra, CD28, CD40L nas células CD4+CD25+/hiCD127Æ/low, em pacientes com LES em fase ativa e inativa. Avaliamos também a relação entre o balanço de células TREG versus células Tconv expressando estes marcadores mediante o calculo da razão de equilíbrio fenotípico TREG/Tconv. Em pacientes com doença ativa observamos níveis diminuídos de células TREG positivas para as moléculas CTLA-4 e CD28 e níveis elevados de células TREG CD40L+. Quando avaliada a razão TREG/Tconv observamos uma alteração no balanço TREG/Tconv positivas para GITR, HLA-DR, OX40, CD40L e CD45RO. Houve queda na razão TREG/Tconv para os marcadores GITR, HLADR, OX40 e CD45RO e ganho para o marcador CD40L em pacientes com LES quando comparado a controles sadios. Além da caracterização fenotípica ampla, o presente estudo tem um ponto original extra, que consiste na definição da população de células TREG a partir do fenótipo CD4+CD127lowCD25+, que tem se mostrado mais específico que o tradicional fenótipo CD4+CD25high altamente contaminado por células Teff. Estas informações, no futuro, poderiam levar a pistas importantes na busca de alternativas mais eficazes de imunoterapia, capazes de restabelecer os mecanismos normais de tolerância imunológica, evitando ou minimizando assim os danos causados pela resposta autoimune. / Systemic lupus erythematosus (SLE) is a chronic inflammatory disease that is part of the group of rheumatic autoimmune inflammatory diseases, being characterized by heterogeneous clinical and laboratory manifestations. The exact etiopathogenic mechanism underlying SLE still remains obscure. Previousr observations evaluating CD4+ CD25+ TREG cell function in auto-immune diseases detected alterations on frequency and on phenotypic and functional features in murine and human models that support the significant activity of this cell population on autoimmune pathophysiology. In SLE we can observe the existence of a complex interaction network that characterizes the disease, in which many targets for therapeutic intervention may be considered. The present study has focused on TREG cells, since they may represent putative targets for immunomodulatory therapy in this disease. Published data on frequency and phenotype of TREG cells is controversial due to heterogeneity of phenotypic markers and analytic strategies used. The present project aimed to validate an appropriate strategy to identify and quantify TREG in SLE. The CD4+CD25highCD127 low/- panel was validated as an appropriate strategy for identification of Foxp3+ TREG cells in healthy and in SLE patients. The frequency of TREG cells presented normal frequency in active and inactive SLE. In contrast, the frequency of conventional non-regulatory T cells was increased in patients with active disease. We also evaluated the expression of important phenotypic markers for TREG cells biology, including CTLA-4, GITR, PD-1, OX40, HLA-DR, CD95, CD45RO, CD28 and CD40L in patients with active and inactive disease. In addition we evaluated the relationship between the balance of TREG cells versus conventional non-regulatory T cells expressing these markers by means of deriving the TREG/Tconv rate for each surface marker. In patients with active disease we observe reduced levels of TREG cells expressing CTLA-4 and CD28 molecules, and elevated levels of CD40L+ TREG cells. There was an imbalance in TREG/Tconv for GITR, HLA-DR, OX40, CD40L and CD45RO: samples from active SLE patients depicted a decreased TREG/Tconv ratio for GITR, HLA-DR, OX40 and CD45RO and an increased ratto for CD40L when compared with healthy controls. The knowledge on the role of TREG cells in SLE may bring important contribution in devising therapeutic alternatives for this disease. / TEDE / BV UNIFESP: Teses e dissertações

Apesar do sucesso do transplante na clínica, os importantes efeitos adversos dos imunossupressores, usados para prevenir e tratar a rejeição, apontam para a necessidade de novas terapias imunorreguladoras. A via oral tem sido efetiva na indução de imunorregulação, em diversos modelos experimentais, principalmente de doenças autoimunes. A Hsp60/65 é uma molécula com grande potencial imunoterapêutico, por sua capacidade de induzir respostas imunes pró-inflamatória e imunorreguladora. Testamos se a terapia oral com o probiótico Lactococcus lactis que expressa a Hsp65, combinada à administração de aloantígenos do doador (AloAg-doador), atua sinergicamente na indução de tolerância ao enxerto de pele semialogeneico murino, ou no aumento de sua sobrevida. Testamos diferentes combinações de terapia oral, assim como a influência da utilização de um anti-inflamatório, inibidor seletivo de COX-2 (celecoxibe). O transplante de pele foi realizado 10 dias após a última administração oral dos probióticos e aloantígenos do doador. Não observamos efeitos benéficos na sobrevida do enxerto no grupo de animais que receberam L.lactis que produz Hsp65, sozinho ou em combinação com AloAg-doador e/ou o anti-inflamatório. Em contraste, a terapia oral combinada com o probiótico L.lactis selvagem e AloAg-doador aumentou significativamente a sobrevida do enxerto (p=0,01), em comparação com o grupo não tratado. Nesse grupo que teve maior sobrevida do aloenxerto (L,lactis selvagem e AloAg-doador), também observamos maior quantidade de epitélio preservado (p=0,02) e maior expressão de TGF-beta (p=0,04), no enxerto, em comparação com o grupo sem tratamento. Não observamos diferenças significativas na expressão, in situ, de FOXP3 e IL-17, que foi baixa em todos os grupos experimentais. Concluímos que a Hsp65 não induziu efeito imunorregulador capaz de prolongar a sobrevida do enxerto. No entanto, a manipulação da microbiota com a terapia combinada com o L.lactis selvagem e a exposição a antígenos do doador, previamente, ao transplante, induz mecanismos imunorreguladores capazes de controlar, mesmo que parcialmente, as respostas inflamatórias dirigidas ao aloenxerto de pele, provavelmente, com a participação de TGF-beta / Despite the success of clinical transplantation, the significant side effects induced by immunosupressants used to prevent and treat rejection, indicate the need for novel immunoregulatory therapies. The oral route has been effective in inducing immunoregulation in several experimental models, mostly in pathological autoimmunity. Heat Shock protein 60/65 (Hsp) displays great immunotherapeutic potential due to its capacity to induce both pro-inflammatory and immunregulatory responses. We tested whether oral therapy with the probiotic Lactococcus lactis that expresses Hsp65, in combination with donor alloantigens (Donor-Allo-Ag), acted synergically, inducing immunotolerance or increasing graft survival, in a murine model of semiallogeneic skin transplantation. We tested different oral therapy combinations, as well as the association with a COX-2 selective nonsteroidal anti-inflammatory drug (celecoxib). Skin transplantation was performed 10 days after the last oral administration of probiotics and Donor-Allo-Ag. We observed no beneficial effect on graft survival in the group that received L.lactis that produce Hsp65, alone or in combination with Donor-Allo-Ag/and/or the anti-inflammatory drug. In contrast, combined oral therapy with wild type L.lactis and Donor-Allo-Ag significantly prolonged graft survival (p=0.01), in comparison to non-treated animals. In this prolonged-survival group (L.lactis and Donor-Allo-Ag), we also found higher extension of preserved epithelium (p=0.02) and higher expression of TGF-beta (p=0.04), within the graft, in comparison to non-treated animals. We found no significant differences in the intragraft expression of FOXP3 and IL-17, which was essentially absent or very low. We conclude that Hsp65 did not induce immunoregulatory effects capable of prolonging graft survival. However, the microbiota manipulation with the combined oral therapy with wild type L.lactis and Donor-Allo-Ag, prior to transplantation, induce immunoregulatory mechanisms capable of partially controlling the inflammatory responses to the graft, most likely involving the participation of TGF-beta

Introdução: Os linfócitos T regulatórios CD4+CD25+Foxp3+ (Tregs) desempenham um papel fundamental na manutenção da tolerância aos antígenos próprios e no controle da magnitude da resposta imunológica. Alterações quantitativas ou funcionais foram descritas em diversos distúbios auto-imunes. O pênfigo foliáceo endêmico (PFE) é uma doença bolhosa cutânea de natureza auto-imune, que compartilha características clínicas e imunopatológicas com o pênfigo foliáceo clássico, mas apresenta achados epidemiológicos próprios. Auto-anticorpos circulantes e teciduais da classe IgG dirigidos contra caderinas desmossômicas (desmogleína 1), levam à perda de adesão entre os queratinócitos. Objetivo: O objetivo deste estudo foi avaliar se a perda de tolerância é associada com alterações quantitativas nos linfócitos Tregs CD4+CD25+Foxp3+ na pele de pacientes com PFE. Métodos: Amostras de pele de 22 pacientes e 10 controles saudáveis foram submetidos à análise imunoistoquímica com anti-CD4, anti-CD25 e anti-Foxp3. Fotomicrografias foram obtidas de campos consecutivos ao longo de toda epiderme e derme. A seguir, foi realizada quantificação dos linfócitos Foxp3+, CD4+, CD25+, CD4+Foxp3+ e CD25+Foxp3+ em cada compartimento, considerando-se a respectiva área de cada campo (m2). Valores significantemente estatísticos foram considerados como p<0,05. Resultados: Encontramos um infiltrado epidérmico aumentado de linfócitos imunomarcados CD25+(p=0,003), Foxp3+(p=0,04) e CD25+Foxp3+ (p=0,007), em comparação com os controles. O infiltrado dérmico exibiu uma maior expressão de linfócitos CD4+ (p<0,001) e CD25+ (p=0,008) em amostras de pele de pacientes com PFE, quando comparados aos controles. Conclusões: Nossos achados sugerem que a quebra de tolerância imunológica periférica nos pacientes com PFE não se correlaciona com uma diminuição dos linfócitos T reg CD4+CD25+Foxp3+ na pele afetada. Entretanto, uma maior expressão de linfócitos CD25+Foxp3+ no infiltrado epidérmico poderia representar outra população de linfócitos com atividade regulatória, a ser definida. / Background: CD4+CD25+Foxp3+ regulatory T cells (Tregs) play a crucial role in the maintenance of self tolerance and control of the magnitude of the immune response. Their quantitative or functional impairment has been reported in several autoimmune disorders. Endemic pemphigus foliaceus (EPF) is an autoimmune organ-specific blistering skin disorder that shares many clinical and immunopathological features with classic pemphigus foliaceus, but with unique epidemiological features. Circulating and tissue-bound IgG auto-antibodies react against desmosomal cadherins (desmoglein 1), causing loss of adhesion between keratinocytes. Aims: The purpose of this study was to evaluate whether the loss of tolerance is associated with impairment of CD4+CD25+Foxp3+ Tregs in the skin of EPF patients. Methods: Skin samples from 22 patients and 10 controls were submitted to immunohistochemistry with anti-CD4, CD25 and Foxp3. Photomicrographs were obtained from consecutive fields along epidermis and dermis; quantification of Foxp3+, CD4+, CD25+, CD4+Foxp3+ and CD25+Foxp3+ cells were performed in each compartment, taking into account the respective field area (m2). Significance was set at p<0.05. Results: We found an enhanced epidermal infiltrate of CD25+(p=0.003), Foxp3+(p=0.04) and CD25+Foxp3+(p=0.007) immunostained T cells in EPF patients, when compared to controls. Dermal infiltrate exhibited a higher expression of CD4+ (p<0.001) and CD25 p=0.008) T cells in EPF skin samples than in controls. Conclusions: Our findings suggest that the break of peripheral immunologic tolerance in EPF patients did not correlate with an impairment of CD4+CD25+Foxp3+ Treg cells present in the affected skin. However, higher expression of CD25+Foxp3+ cells in the epidermal infiltrate could be a counterpart of a diverse population of T cells, previously described as exerting a regulatory activity, yet to be defined

L’indoléamine-2,3-dioxygénase (IDO) est une enzyme du catabolisme du tryptophane suspectée de jouer un double rôle lors des infections en contribuant aux défenses innées de l’hôte et en régulant la réponse immunitaire. IDO est exprimée au cours de l’infection par le virus de l’hépatite C (VHC). Cependant, les mécanismes moléculaires conduisant à l’expression de IDO lors de l’hépatite C et l’impact de IDO sur la réplication virale et sur la réponse immunitaire ne sont pas connus. Dans ce travail de thèse, nous avons montré que le VHC stimule l’expression de IDO dans les hépatocytes.L’expression de IDO était transitoire et coïncidait avec l’expression des interférons (IFNs) de types I et III et avec la transcription de gènes stimulés par les IFNs. L’expression de IDO était également augmentée dans les hépatocytes exposés à la présence de lymphocytes T CD4+ activés et producteurs d’IFN-γ. L’expression hépatique de IDO diminuait la réplication virale, ce qui suggère que IDO limite la diffusion du VHC dans le foie au cours de l’hépatite C. Grâce à des expériences de silencing, nous avons montré que IDO contribue à l’effet antiviral de l’IFN-α sur le VHC. L’expression de IDO était régulée par l’activation des facteurs de transcription IRF-1 et STAT-1 dans les hépatocytes infectés par le VHC. En plus de son effet antiviral sur le VHC, l’expression hépatique de IDO inhibait significativement la prolifération des lymphocytes T CD4+ activés, suggérant un rôle immunorégulateur de IDO au cours de l’hépatite C. Nos données suggèrent donc que IDO joue un double jeu lors de l’hépatite C, en limitant la réplication virale et en régulant la réponse immunitaire adaptative de l’hôte. Notre travail ouvre la voie à des expérimentations in vivo et à des études cliniques visant à préciser la place des inhibiteurs pharmacologiques de IDO dans l’arsenal thérapeutique contre le VHC. / Indoleamine-2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that plays a dual role during infectious diseases by contributing to the innate defenses against pathogens and by regulating the immune response. IDO is expressed in patients with hepatitis C virus (HCV) infection. However, the molecular mechanism of IDO induction in HCV infection and its role in the antiviral immune response remain unknown. Using primary human hepatocytes, we have shown that HCV infection stimulates IDO expression. IDO gene induction was transient and coincided with the expression of type I and type III interferons (IFNs) and IFN-stimulated genes (ISGs) in HCV-infected hepatocytes. IDO expression was also stimulated when the hepatocytes were incubated with IFN-γ-secreting CD4+ T cells. Expression of IDO prior to HCV infection significantly impaired HCV replication in hepatocytes, suggesting that IDO limits the spread of HCV in the liver. By using siRNA-mediated IDO knockdown experiments, we have shown that IDO contributes to the IFN-α-antiviral effect on HCV replication. IDO expression was regulated by IRF-1 and STAT-1 in HCV-infected hepatocytes. Hepatic IDO expression also had a significant inhibitory effect on CD4+ T cell proliferation, suggesting an immunoregulatory role of IDO during HCV infection. Our data suggest that hepatic IDO plays a dual role during HCV infection by retarding viral replication and also regulating host immune responses. This work paves the way for in vivo experiments and clinical studies aiming to determine the relevance of pharmacological inhibition of IDO during HCV infection.

[EN]It is well illustrated that the generation of Tregs is the main mechanism responsible for maintaining immune tolerance during developmental exposure to non-inherited maternal antigen (NIMA). Moreover, the presence of NIMA-specific Tregs in the uterus of pregnant mice promote reproductive fitness by enforcing maternal tolerance to overlapping paternal antigens expressed by the fetus during next-generation pregnancies. However, the reason why perinatal T cell lineage is biased towards immune tolerance is poorly understood. Due to the fact that terminal deoxynucleotidyl transferase (TdT) is not expressed in neonatal T cells in the mouse, neonatal T cells have a less diverse TCR repertoire. This is known to limit their specificity and to increase their affinity for MHC/peptide complexes. At the start of the present work, we postulated that expressing high affinity TCR might be the reason that forces the development of antigen-specific Tregs in neonates. We undertook our study with the aim to investigate the mechanisms underlying mouse NIMA-specific Treg development in the perinatal period. Using 2W1S-OVA+ heterozygous mouse model in which 2W1S antigen was transformed into surrogate NIMA for half of the offspring, we observed an increased frequency of 2W1S-specific Tregs in NIMA-2W1S-exposed animals. Moreover, we also observed that periphery-derived NIMA-2W1S Tregs had a less diverse TCR repertoire and were phenotypically distinct from thymus-derived SELF-2W1S-specific Tregs. In order to investigate whether the lack of diversity was responsible for the development of neonatal NIMA-specific Tregs, we generated transgenic mice where TdT expression was enforced in T cells before birth. We found that transgenic TdT added clonal TCR diversity but did not prevent the development of T cell clones with neonatal type TCR repertoire and did not modify the frequency of neonatal NIMA-specific Tregs. On the contrary, TdT expression increased significantly generation of SELF-specific Tregs to levels similar to that of NIMA-specific Tregs. Taken together, our data indicate that the developmental pathways of NIMA- and SELF-specific Treg repertoire are different in terms of inducing and maintaining neonatal tolerance. / [FR]Il est bien illustré que la génération périnatal de Treg est le principal mécanisme responsable du maintien de la tolérance immunitaire fœtale qui se développe suite à l'exposition aux antigènes maternels non-hérités (NIMA). De plus, la présence de Tregs spécifiques des NIMA dans l'utérus des femmes enceintes favorise la capacité de reproduction en renforçant la tolérance maternelle aux mêmes antigènes paternels exprimés par le fœtus pendant les grossesses de prochaine génération. Cependant, la raison pour laquelle la lignée des cellules T fœtales est biaisée en faveur de la tolérance immunitaire est mal comprise. Chez la souris, en raison du manque d'expression de la désoxynucléotidyl transférase terminale (TdT), les cellules T néonatales ont un répertoire de TCR moins diversifié. Ceci est connu pour limiter leur spécificité et augmenter leur affinité pour les complexes CMH / peptide. Au début du présent travail, nous avons émis l'hypothèse que l'expression de TCRs de haute affinité pourrait être la raison qui force le développement de Treg spécifiques chez les nouveau-nés. Nous avons plus particulièrement entrepris notre étude dans le but d'étudier les mécanismes sous-jacents au développement de Tregs spécifiques des NIMA chez la souris pendant la période périnatale. En utilisant le modèle de souris hétérozygotes pour 2W1S-OVA+ dans lequel l'antigène 2W1S a été transformé en NIMA pour la moitié de la progéniture, nous avons observé une fréquence accrue de Tregs spécifiques de 2W1S chez les animaux exposés au NIMA. De plus, nous avons également observé que les Treg NIMA-2W1S dérivés de la périphérie avaient un répertoire de TCRs moins diversifié et étaient phénotypiquement distincts des Tregs spécifiques de SELF-2W1S dérivés du thymus. Afin de déterminer si le manque de diversité était responsable du développement de Tregs néonataux spécifiques de NIMA, nous avons généré des souris transgéniques où l'expression de TdT était appliquée dans les cellules T avant la naissance. Nous avons constaté que le TdT transgénique ajoutait une diversité de TCR clonale, mais n'empêchait pas le développement de clones de cellules T avec un répertoire TCR de type néonatal et ne modifiait pas la fréquence des Treg néonataux spécifiques du NIMA. Au contraire, l'expression de TdT a augmenté de manière significative la génération de Tregs spécifiques de SELF-2W1S à des niveaux similaires à ceux des Treg spécifiques de NIMA-2W1S. Prises ensembles, nos données indiquent que les voies de développement du répertoire des Tregs néonataux spécifiques de NIMA et SELF sont différentes en termes d'induction et de maintien de la tolérance néonatale. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Background: Extracorporeal photopheresis (ECP) induces immunological changes that lead to a reduced risk of transplant rejection. The aim of the present study was to determine optimum conditions for ECP treatment by analyzing a variety of toleranceinducing immune cells to optimize the treatment. Methods: Ten ECP treatments were applied to each of 17 heart-transplant patients from month 3 to month 9 post-HTx. Blood samples were taken at baseline, three times during treatment, and four months after the last ECP treatment. The abundance of subsets of tolerance-inducing regulatory T cells (Tregs) and dendritic cells (DCs) in the samples was determined by flow cytometry. A multivariate statistical model describing the immunological status of rejection-free heart transplanted patients was used to visualize the patient-specific immunological improvement induced by ECP. Results: All BDCA+ DC subsets (BDCA1+ DCs: p < 0.01, BDCA2+ DCs: p < 0.01, BDCA3+ DCs: p < 0.01, BDCA4+ DCs: p < 0.01) as well as total Tregs (p < 0.01) and CD39+ Tregs (p < 0.01) increased during ECP treatment, while CD62L+ Tregs decreased (p < 0.01). The cell surface expression level of BDCA1 (p < 0.01) and BDCA4 (p < 0.01) on DCs as well as of CD120b (p < 0.01) on Tregs increased during the study period, while CD62L expression on Tregs decreased significantly (p = 0.04). The cell surface expression level of BDCA2 (p = 0.47) and BDCA3 (p = 0.22) on DCs as well as of CD39 (p = 0.14) and CD147 (p = 0.08) on Tregs remained constant during the study period. A cluster analysis showed that ECP treatment led to a sustained immunological improvement. Conclusions: We developed an immune monitoring assay for ECP treatment after heart transplantation by analyzing changes in tolerance-inducing immune cells. This assay allowed differentiation of patients who did and did not show immunological improvement. Based on these results, we propose classification criteria that may allow optimization of the duration of ECP treatment.

L’injection de cellules immunologiquement compétentes à un hôte histo-incompatible amène une réaction qui peut se traduire par la maladie du greffon-contre-l’hôte (GVHD). La GVHD demeure une barrière importante à une utilisation plus répandue de la greffe allogénique de cellules hématopoïétiques (AHCT), pourtant un traitement efficace pour traiter de nombreuses maladies. Une meilleure compréhension des mécanismes qui sous-tendent cette pathologie pourrait en faciliter le traitement et la prévention. L’Interféron-gamma (IFN-γ) et le Transforming Growth Factor-béta (TGF-β) sont deux cytokines maîtresses de l’immunité impliquées dans la fonction et l’homéostasie des cellules greffées. Nous démontrons chez la souris que l’IFN-γ limite la reconstitution lympho-hématopoïétique de façon dose-dépendante en mobilisant des mécanismes d’apoptose et en inhibant la prolifération cellulaire. Le TGF-β est quant à lui généralement connu comme un immunosuppresseur qui contrôle l’immunité en utilisant plusieurs voies de signalisation. Le rôle relatif de ces voies en AHCT est inconnu. Nous avons étudié une de ces voies en greffant des cellules provenant de donneurs déficients pour le gène SMAD3 (SMAD3-KO), un médiateur central de la voie canonique du TGF-β, à des souris histo-incompatibles. Bien que l’absence de SMAD3 ne cause aucune maladie chez nos souris donneuses, l’injection de cellules SMAD3-KO amène une GVHD du colon sévère chez le receveur. Cette atteinte est caractérisée par une différenciation Th1 et une infiltration massive de granulocytes témoignant d’un rôle central de SMAD3 dans la physiologie des lymphocytes T CD4 et des cellules myéloïdes. Nous avons focalisé ensuite nos efforts sur le rôle de SMAD3 chez les lymphocytes T CD4 en sachant que SMAD3 était actif chez les lymphocytes T CD4 tolérants. Nous avons découvert que SMAD3 était rapidement inactivé après une activation des cellules T, suggérant que l’inactivation de SMAD3 était fonctionnellement importante pour briser l’état de tolérance. Des études de micro-puces d’ADNc nous ont montré que SMAD3 contrôlait en effet l’expression de nombreux transcrits de gènes connus comme étant reliés à la tolérance et/ou à des processus biologiques dont les rôles dans le maintien de la tolérance sont plausibles. / The injection of immuno-competent cells into a histo-incompatible host can result in the development of Graft-versus-Host disease (GVHD). GVHD is the most significant barrier to a more widespread use of allogeneic hematopoietic cell transplantation (AHCT), a potent treatment for several diseases. A better understanding of the pathophysiological underpinnings of GVHD would facilitate the design of rational approaches to treat and prevent this complication of AHCT. Gamma-interferon (IFN-γ) and Transforming Growth Factor-beta (TGF-β) are master cytokines of immunity and have a role in the function and homeostasis of transplanted cells. Using a murine model, we show that IFN-γ curtails lympho-hamatopoitic reconstitution in a dose-dependent fashion by increasing apoptosis and by limiting donor cell proliferation. TGF-β is an immunosuppressive cytokine that controls immune cells through multiple signaling pathways. The relative contribution of these pathways in AHCT is unknown. We specifically studied the role of one of these pathways by transplanting SMAD3 deficient cells (SMAD3-KO) in histo-incompatible hosts. SMAD3 is a key mediator of the so-called canonical TGF-β signaling pathway. Although SMAD3-KO donor mice are healthy, the injection of SMAD3-KO cells leads to severe GVHD in the hosts, characterized by intestinal involvement associated with Th1 skewing and massive granulocyte infiltration. These findings hint at a crucial role for SMAD3 in CD4 T-cell and myeloid cell biology. We then focalized on the role of SMAD3 in CD4 T cells knowing that SMAD3 is active in tolerant, resting CD4 T cells. We found that SMAD3 was rapidly inactivated upon T cell activation, suggesting that SMAD3 inactivation was functionally important to break the state of tolerance. Our cDNA microarray experiments show that indeed, SMAD3 regulates the transcript levels of multiple genes known to be involved in T cell tolerance and in biological processes plausibly related to immune tolerance.

Le facteur VIII (FVIII) joue un rôle essentiel dans la coagulation sanguine. Lorsque le FVIII fait génétiquement défaut, une pathologie hémorragique grave survient: l'hémophilie A (HA) congénitale. La complication majeure de la prise en charge de ces patients est l'apparition d'allo-anticorps (alloAcs) dirigés contre le FVIII thérapeutique administré. Dès lors, la seule thérapeutique efficace est l'induction de tolérance immune (ITI) qui vise à les éradiquer. Cependant, ce traitement échoue dans 30% des cas, sans qu'aucun facteur ne permette actuellement de prédire l'échec de ce traitement contraignant et coûteux. des facteurs immunologiques prédictifs de l'efficacité de l'ITI ont été recherchés chez 25 patients par analyse du répertoire épitopique et isotypique des Acs anti-FVIII à l'aide de la technologie x-MAP. Des biomarqueurs individuels (Acs anti-A2 et -A1 du FVIII), et des combinaisons originales ont été identifiés (0,841 < AUC < 0,946). Des manifestations hémorragiques peuvent apparaitre chez des patients non hémophiles, dues à des autoAcs anti-FVIII (HA acquise). Dans certains cas, les autoAcs se développent au moment du postpartum. peu de données sont disponibles sur cette réponse immune. Dans une seconde étude portant sur 73 cas, nous avons découvert un profil immunologique (autoAcs anti-A1) différenciant les HA du postpartum et les autres HA acquises. Les profils d'IgG anti-FVIII que nous avons établis s'avèrent prometteurs pour prédire l'efficacité de l'ITI et engendrer une cartographie précise de la réponse autoimmune chez les patients atteints d'HA acquise. / Factor VIII (FVIII) plays a critical role in blood coagulation. When FVIII s genetically defective, a serious hemorrhagic disease occurs: congenital hemophilia A (HA). The main complication of the management of these patients is the appearance of alloantibodies (alloAbs) directed against administred therapeutic FVIII. therefore, the only effective treatment is the immune tolerance induction (ITI), which aims to eradicate these alloAbs. However, this treatment fails in up to 30% of cases, without any factor currently able to predict the failure of this constraining and expensive treatment. Immunological factors predictive to the efficacy of ITI were investigated in 25 patients by analysis of epitopic and isotypic IgG profile of anti-FVIII Abs using x-MAP technology. Individual biomarkers (anti-FVIII A1 and -A2 Abs), and original combinations were identified (0,841 < AUC < 0,946). Hemorrhagic manifestations can occur in non-hemophiliac patients, due to anti-FVIII autoAbs (acquired HA). In some patients, the autoAbs appear in postpartum period but few data are available on the immune response due to the rarity of the disease. In a second study of 73 cases, we found a different immunological profile between patients with postpartum HA and the other acuired HA patients. IgG profiles of anti-FVIII we have established are promising for predicting the effectiveness of ITI and generate an accurate mapping of autoimmune response in patients with acquired HA.

Le système immunitaire protège un organisme du développement de pathogènes et participe activement au maintien de la tolérance immunitaire. Les cellules dendritiques (DC) sont des cellules spécialisées dans l’équilibre pro et anti-inflammatoire de la réponse immunitaire. Les DC jouent un rôle important dans de nombreux contextes pathologiques notamment la transplantation d’organes, en oncologie et dans les pathologies inflammatoires. Elles sont modulables grâce à divers facteurs, intrinsèques et extrinsèques. Parce qu’elles sont capables d’induire une réponse tolérogène, ces cellules représentent des cibles intéressantes pour moduler la réponse immunitaire dans le contexte de la transplantation d’organes et des pathologies inflammatoires. Certains agents pathogènes utilisent des mécanismes d’échappement au système immunitaire en favorisant l’induction d’une tolérance immunitaire. Cette modulation est réalisée par le ciblage des récepteurs de reconnaissance des pathogènes (PRR) sur la présence des DC, induisant la synthèse d’une cytokine antiinflammatoire IL-10, un des inducteurs de la tolérance immunitaire. Notre stratégie a été de construire un anticorps bispécifique ciblant deux PRR différents à partir d’une banque d’anticorps anti-PRR. Notre travail montre que cet anticorps bispécifique est capable d’orienter les DC vers d’un profil tolérogène. Cet anticorps bispécifique induit un phénotype de DC semi-mature avec un profil de sécrétion pro-tolérogène avec de l’IL-10 et peu de cytokines inflammatoires. Le profil de tolérance immunitaire induite par ces cellules reste à explorer. Nos travaux ouvrent de perspectives intéressantes sur l’association des PRR en vue d’obtenir la modulation des cellules de l’immunité. / The immune system protects an organism from the development of pathogens and actively participates in maintaining immune tolerance. Dendritic cells (DC) are specialized cells in the balance and anti-inflammatory immune response. DC play an important role in many pathological contexts, including organ transplantation, oncology and inflammatory diseases. Various factors, both intrinsic and extrinsic, can modulate. Because they are capable to inducing a tolerogenic response, these cells represent interesting targets for the immune response in the context of organ transplantation and in inflammatory pathologies. Some pathogens use mechanisms of escape to the immune system by promoting the induction of immune tolerance. This modulation is achieved by targeting the pathogen recognition receptors (PRRs) present on the surface of DC, inducing the synthesis of an anti-inflammatory cytokine IL-10, one of the main inducers of immune tolerance. Our strategy was to construct a bispecific antibody targeting two different PPRs from an anti-PRR antibody library. Our work shows that this bispecific antibody is able to direct the DC to a pro-tolerogenic profile. This bispecific antibody induces a semi-mature DC phenotype with a secretion profile of pro-tolerogenic cytokines such as IL-10 and few inflammatory cytokines. The immune tolerance profile of these DC remains to be explored. Our work opens interesting perspectives on the association of PRRs in order to obtain the modulation of the cells of the immunity.

O timo é um orgão linfóide primário, no qual ocorre a indução da tolerância imunológica central aos antígenos do próprio que são expressos pelos tecidos periféricos (PTAs). A medula tímica é formada por células tímicas medulares epiteliais (mTECs) que expressam centenas desses PTAs que representam virtualmente todos os órgãos e tecidos do corpo. Esse fenômeno foi denominado de expressão gênica promíscua (PGE) a qual é parcialmente regulada pelo modulador da transcrição Autoimmune regulator (Aire). Os precursores de células T oriundos da medula óssea migram para o timo (agora são denominados de timócitos) e na medula desse órgão, passam pela seleção negativa mediada pelas mTECs. As células sobreviventes evoluem para células T maduras e funcionais que migram para a periferia com capacidade de reconhecimento das moléculas de MHC e tolerantes aos PTAs. Além de controlar a transcrição de genes PTAs, Aire também controla a expressão de microRNAs (miRNAs), relacionados com a integridade e funcionalidade do microambiente tímico. A seleção negativa no timo é um processo essencial para a manutenção da autotolerância imunológica e o desbalanço desse processo está associado com o desenvolvimento de doenças autoimunes como, por exemplo, o diabetes mellitus do tipo 1 (DM1).Tendo em vista essas premissas, nosso trabalho se fundamentou em duas hipóteses: 1) Variações na expressão do gene Aire podem perturbar a expressão de genes PTAs e miRNAs no timo, causando alterações na PGE, 2) A expressão balanceada de genes como Aire e/ou PTAs nas mTECs, é fundamental para a integridade da tolerância central. O desbalanço na expressão desses genes, está associado com a emergência do diabetes mellitus tipo 1 no camundongo. Para testar nossa primeira hipótese efetuamos o silenciamento de Aire (Aire knockdown) por meio de eletrotransfeção de RNA interferente (siRNA) anti-Aire in vivo no timo de camundongos BALB/c. Análises do transcriptoma (mRNAs) e miRNoma (miRNAs) das mTECs, revelaram que silenciamento parcial e transitório de Aire foi suficiente para afetar a expressão de PTAs Aire dependentes bem como a de miRNAs. Redes de interação miRNA-mRNA, revelaram que o controle pós-transcricional da PGE também é afetado pelo silenciamento de Aire. Os resultados encontrados revelam que Aire e miRNAs podem formar uma via essencial durante a indução da tolerância central. Para testar nossa segunda hipótese comparamos o transcriptoma de mTECs de camundongos BALB/c (linhagem não-autoimune) com mTECs de camundongos non-obese diabetic NOD (modelo animal utilizado nos estudos de DM1 autoimune). Nossos resultados revelaram que a expressão transcricional de autoantígenos relacionados ao DM1 está desbalanceada em camundongos NOD já numa fase precoce, quando esses animais ainda não apresentavam a doença clínica (fase pré-diabética). Inesperadamente, os níveis transcrionais de Aire apresentaram-se equivalentes no timo dessas duas linhagens, porém os níveis da proteína AIRE estavam reduzidos no timo da linhagem NOD. Esses resultados sugerem a participação de algum mecanismo de atenuação póstrascricional de Aire nessa linhagem provavelmente envolvendo atuação de miRNAs. Isso poderia explicar o desbalanço de PTAs Aire-dependentes e a repressão autoantígenos relacionados ao DM1. Concluímos que nossos resultados, além de abrir novas perspectivas para pesquisas nesta área, contribuem com melhor compreensão dos mecanismos moleculares desencadeados por Aire e por miRNAs no controle da expressão de autoantígenos no timo o que é importante para a tolerância imunológica central. / The thymus is a primary lymphoid organ, in which occurs in the induction of central immune tolerance to self peripheral tissue antigens (PTAs). The thymic medulla is formed by medullary thymic epithelial cells (mTECs) expressing hundreds of such PTAs representing virtually all organs and tissues of the body. This phenomenon has been termed promiscuous gene expression (PGE), which is partially regulated by the Autoimmune regulator (Aire) gene. The T cell precursors derived from the bone marrow migrate to the thymus (now termed thymocytes). A part of these thymocytes are eliminated by negative selection mediated mTEC cells. The surviving cells to evolve and functional mature T cells that migrate to the periphery and are capable of recognizing MHC molecules and are tolerant to PTAs. In addition to controlling the transcription of PTA genes, Aire also controls the expression of microRNAs (miRNAs). The negative selection in the thymus is a process essential to the maintenance of immunologic self-tolerance and imbalance of this process is associated with the development of autoimmune diseases such as type 1 diabetes mellitus (DM1) . Given these assumptions, our work was based on two hypothesis: 1) Changes in the expression of the Aire gene can disrupt the expression of PTA genes and miRNAs in the thymus, causing changes in PGE, 2) The balanced expression of Aire / or PTA genes in mTECs is fundamental for central tolerance. The imbalance in the expression of these genes is associated with the emergence of type 1 diabetes in mice. To test our first hypothesis we made Aire silencing (Aire knockdown) through electrotransfection of anti - Aire interfering RNA (siRNA) in vivo in the thymus of BALB/c mice. Analysis of the transcriptome (mRNAs) and miRNome (miRNAs) of mTECs revealed that partial and transient silencing of Aire was enough to affect the expression of Aire - dependent PTAs as well as miRNAs. miRNA -mRNA interaction networks revealed that the posttranscriptional control of PGE is also affected by the silencing of Aire. The results show that Aire and can form an miRNA pathway essential for the induction of central tolerance. To test our second hypothesis we compared the transcriptome of mTECs of BALB/c mice (non-autoimmune strain) with mTECs from non - obese diabetic NOD (animal model used in studies of autoimmune DM1) . Our results indicate that the transcriptional expression of DM1-related autoantigens are unbalanced in NOD mice in an very early stage, when these animals have not had clinical disease (pre-diabetic period). Unexpectedly, the transcriptional levels of Aire in the thymus was equivalent in these two strains, but the AIRE protein levels were reduced in thymus of NOD strain. These results suggest that some mechanism of post-transcriptional attenuation of Aire is acting in this lineage probably involving action of miRNAs . This could explain the imbalance of Aire - dependent PTAs and repression autoantigens related to DM1. Our results open perspectives for research in this area, contributing to better understanding the molecular mechanisms triggered by Aire and miRNAs in control of the expression of autoantigens in the thymus, which is important for the central immune tolerance.