Au cours du développement tumoral, le système immunitaire est constamment exposé aux antigènes tumoraux, mais le plus souvent dans un contexte non-inflammatoire qui favorise l'induction d'une tolérance envers ces antigènes. La tolérance peut être médiée par des mécanismes passifs (ignorance, anergie ou délétion des clones spécifiques de la tumeur) ou actifs, pour lesquels les lymphocytes T régulateurs (Tregs) jouent un rôle prépondérant. Les lymphocytes T (LT) CD4+ sont la source principale des Tregs mais présentent également des fonctions antitumorales directes et indirectes. Les connaissances actuelles sur le rôle des LT CD4+ au cours du développement tumoral proviennent en grande partie d'études de modèles murins de tumeurs transplantées. Cependant, l'inflammation initiée lors de l'inoculation de ces tumeurs due à une mort cellulaire importante favorise la présentation persistante par le MHC-II des antigènes tumoraux dans un environnement inflammatoire artificiel. Nous tentons ici de contourner ce problème en utilisant deux modèles murins différents: le premier est un modèle de tumeur transplantée dans lequel un néoantigène de classe II (DBY) est induit à distance du moment de l'inoculation de la tumeur. Le deuxième est un modèle d'adénocarcinome pulmonaire induit génétiquement exprimant l’epitope DBY, dans lequel la tumorigenèse est initiée par l'expression d’un oncogène associée à la délétion d’un gène suppresseur de tumeurs. La réponse antitumorale des LT CD4+ est suivie par le transfert de LT CD4+ spécifiques de DBY "Marilyn". Dans le modèle de tumeur transplantée, nous montrons que l'apparition d'un néoantigène dans une tumeur bien établie n'est pas ignorée par le système immunitaire. Bien au contraire, le néoantigène arrive au ganglion drainant la tumeur et induit une activation efficace des cellules Marilyn, qui prolifèrent, produisent de l'IFN-γ et recirculent jusqu'à la tumeur. En revanche, malgré une activation efficace des LT CD4+, les tumeurs ne sont pas rejetées. Dans le modèle génétiquement induit, nous montrons que des néoantigènes exprimés dès le début du développement tumoral, arrivent jusqu'au ganglion drainant la tumeur en quantité suffisante pour induire l'activation et la prolifération des LT CD4+, mais que cette activation est non-optimale et ne permet qu'une faible migration vers le site de la tumeur. En revanche, une partie des cellules Marilyn acquièrent l'expression de FOXP3 ainsi qu'une signature transcriptomique de Tregs et ce dès les stades précoces du développement tumoral, tandis que le reste des cellules Marilyn présentent un phénotype anergique (CD44hiCD73hiFR4hi). L'administration de CpG n'empêche pas la conversion en Treg des cellules Marilyn, malgré l'augmentation de la maturation des cellules dendritiques dans le poumon et le ganglion drainant la tumeur. La déplétion des Tregs de l'hôte en revanche inhibe cette conversion et favorise l'activation des cellules Marilyn en cellules effectrices compétentes, capables de migrer jusqu'au site tumoral. Enfin, les cellules Marilyn, lorsqu'elles sont activées hors du ganglion drainant la tumeur échappent à l'inhibition induite par la tumeur et deviennent des cellules effectrices compétentes. Ainsi, dans un modèle tumoral reproduisant le développement naturel progressif des tumeurs humaines, un état de tolérance est induit par la tumeur. Cette tolérance est dépendante des Tregs présents dans le ganglion drainant la tumeur, qui confèrent une tolérance aux LT CD4+ naïfs arrivant dans le ganglion. / During tumor development, the immune system is persistently exposed to tumor-associated antigens, frequently in a non-inflammatory context, favoring the establishment of tolerance. Passive (ignorance, anergy or deletion of tumor-specific T cells) or active mechanisms mediated by regulatory T cells (Tregs) may be involved in tolerance. CD4+ T cells are the main source of Tregs but they also display indirect and direct antitumor activity. So far, the contribution of CD4+ T cells during tumor development has been mainly addressed in murine transplanted tumor models. However, in these models the artificial inflammation associated with the presence of dying tumor cells at the time of tumor inoculation favors a long-lasting MHC-II-restricted tumor antigen presentation in an artificial inflammatory context. Here, we addressed this issue using two different models: a transplanted one in which the MHC-II neoantigen (DBY) is induced long after tumor implantation and a genetically engineered mouse (GEM) model of lung adenocarcinoma also expressing the DBY epitope, in which malignant transformation results from both the expression of an oncogene and the deletion of a tumor suppressor gene. Tumor-specific CD4+ T cell response was followed by transfer of naive DBY-specific Marilyn CD4+ T cells. In the transplanted tumor model, we found that the appearance of a neoantigen in established tumors was not ignored by the immune system. On the contrary, the neoantigen reached the tumor-draining lymph node (TdLN) and induced efficient priming of Marilyn cells that proliferated, produced IFN-γ, and recirculated to the tumor site. However, despite efficient induction of a tumor-specific CD4+ T cell response, tumors were not rejected. In the GEM model, we found that starting at the early tumor stages, neoantigens were expressed and reached the TdLN in sufficient amount to induce activation and proliferation of naive Marilyn T cells. However, this priming was suboptimal and resulted in a weak migration to the tumor site. Instead, some of the activated Marilyn cells acquired the expression of FOXP3 and a Treg gene signature while the remaining FOXP3- cells displayed a CD44hiCD73hiFR4hi anergic phenotype. CpG administration did not revert the Marilyn Treg conversion despite reinforcing dendritic cell maturation in the lung and the TdLN. Depletion of the host Treg compartment however, inhibited this conversion and favored Marilyn cell activation into full-blown effector cells able to migrate to the tumor site. Finally, Marilyn cells that were primed at distance of the TdLN, escaped tumor induced inhibition and became full effectors. Thus, in a tumor model reproducing the natural development of slowly growing human tumors, a tumor-associated dominant tolerance is established in the lymph node draining the tumor. This state of unresponsiveness is highly dependent on the presence of Treg cells in the TdLN, conferring tolerance to incoming tumor-specific naive CD4+ T cells.

A endometriose (EDT) é caracterizada pela presença de tecido endometrial fora da cavidade uterina, e afeta mulheres em idade reprodutiva. Postulamos que alterações na frequência de células T reguladoras (Treg), natural killer (NK), supressoras mielóides (MDSC) e dendríticas (DC) no peritônio justificariam a redução da capacidade do sistema imune de reagir contra as células endometriais, permitindo sua implantação em locais ectópicos. Aqui, células Treg, NK, MDSC e DC foram quantificadas no fluido peritoneal (FP) e sangue de mulheres com EDT, a fim de associa-las ao desenvolvimento da doença; níveis de citocinas também foram avaliados. Na EDT, observou-se aumento na frequência de Treg, MDSC e DC no sangue e aparente redução destas no FP; ainda, a concentração de IL-12 foi menor no sangue comparadas ao grupo controle. Não foram observadas diferenças quanto às células NK e as outras citocinas analisadas. Os resultados indicam aumento da frequência de populações reguladoras em amostras de sangue de pacientes EDT, entretanto esses resultados não são refletidos no FP. / Endometriosis (EDT) is a gynecological disease characterized by the presence of endometrial cells out of the uterine cavity, which affects women in reproductive age. We postulated that alterations in the frequencies of regulatory T cells (Treg), natural killer cells (NK), myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) in the peritoneum could justify the reduced capacity of the immune system to react to these ectopic endometrial cells, allowing them to invade distant tissues. Here, Treg, NK, MDSC and DC were quantified in the peritoneal fluid (PF) and peripheral blood (PB) of women with EDT, in order to associate them with the development of EDT; cytokine levels were also assessed. In EDT, higher frequencies of Treg, MDSC and DC in the PB and apparent lower frequencies of these cells in the PF were observed; IL-12 concentration was smaller in PB of EDT compared to control. No differences between groups were observed for NK cells and the other cytokines evaluated. The results indicate higher frequencies of regulatory cells in PB samples of EDT patients, although these findings were not reflected in PF samples.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-02-16T19:08:17Z No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-22T17:20:04Z (GMT) No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Made available in DSpace on 2018-02-22T17:35:15Z (GMT). No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) Previous issue date: 2017-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is the most common etiologic agent in severe lower respiratory tract infections (LRTI) in children. RSV-associated LRTI is the main cause of bronchiolitis, pneumonia and exacerbation of asthma. This infection is responsible for the high rates of hospitalizations related to respiratory diseases worldwide, especially in children younger than 2 years. Currently, annual mortality rate due to RSV infections is worrying worldwide and is estimated at approximately two hundred thousand cases. The treatment strategies to RSV infections are limited. Ribavirin is an approved drug for use in RSV infections, but its use is limited due to adverse side-effects and risks posed to health professionals who handle it. Palivizumab is a monoclonal antibody which targets RSV F glycoprotein and its use is only indicated as a prophylactic measure. This treatment is already accepted in several countries for groups of high risk children (premature children, with chronic lung disease and with congenital heart disease). However, palivizumab has a high cost for public health and is not available in all countries. The development of an effective RSV vaccine to generate a long-lasting immunological memory response that prevents infection may be the best alternative because it will reduce high public health expenditures with antiviral drugs and monoclonal antibodies. The first attempt in the search for a vaccine against RSV was in the 1960s. This vaccine produced high levels of serum antibodies but could not protect against infection. Children who were vaccinated developed a more serious disease when later infected with the same virus. To date, there is no licensed vaccine for RSV, so the search for effective vaccines is an important focus of research. Natural RSV infections do not induce lasting protective memory, and multiple reinfections can occur lifetime. Nasal secretions from infected infants presented a small number of regulatory CD4 T cells (Treg) in peripheral blood, an increase in interleukin 4 (IL-4) production and T helper type 2 (Th2) response. Treg cells are important for controlling an exacerbated increase in immune responses. A reduction of the Tregs caused by the RSV infection generates an exacerbation of the pulmonary disease due to a Th2 response. The M209-223 RSV peptide was identified to increase IFN-? production by peptide-specific CD4 T cells after challenge with the virus. The treatment with this peptide also induced an increase in pulmonary Treg frequency in infected mice. Recently, it has also been shown that Tregs aid in the development of a T CD8+ effector response, which is crucial for the control of RSV viral load. Our hypothesis is that the RSV M209-223 peptide impacts in the differentiation of CD4 T cells, increasing the population of specific Treg, reducing lung inflammation and modulating the anti-RSV immune response. This peptide in animal model induces the differentiation of specific Treg. Our findings suggest that vaccination with M209-223 peptide results in the differentiation of specific CD4 T cells into conventional effectors and Treg cells. Vaccination with this peptide decreased the expansion of a Th2 response in animals infected with RSV, protecting both the infection site and systemically. We believe that this approach could be an important component in vaccination strategies against this virus. / O v?rus sincicial respirat?rio (VSR) ? o agente etiol?gico mais comum nas infec??es graves do trato respirat?rio inferior (TRI) em crian?as. As infec??es do TRI associada com o VSR s?o a principal causa de bronquiolite, pneumonia e exacerba??o da asma. As TRI causadas pelo VSR s?o respons?veis pelas altas taxas das hospitaliza??es relacionadas ?s doen?as respirat?rias em todo o mundo, principalmente em crian?as menores de dois anos. Atualmente a taxa de mortalidade anual mundial devido ?s infec??es pelo VSR ? preocupante e ? estimada em aproximadamente duzentas mil crian?as. As estrat?gias de tratamento contra o VSR utilizadas s?o limitadas. A ribavirina ? um f?rmaco aprovado no uso para infec??es pelo VSR, por?m sua utiliza??o ? limitada devido aos efeitos secund?rios adversos e aos riscos que representam para os profissionais da sa?de que o manipulam. O palivizumabe ? um anticorpo monoclonal dirigido contra a glicoprote?na F do v?rus e sua utiliza??o ? apenas como medida profil?tica. Este tratamento j? ? aceito em v?rios pa?ses nos grupos de crian?as de alto risco (crian?as prematuras, com doen?a pulmonar cr?nica e com cardiopatia cong?nita). Entretanto o palivizumabe tem um alto custo para sa?de p?blica, n?o sendo disponibilizado em todos os pa?ses. O desenvolvimento de uma vacina eficaz contra o VSR pode ser a melhor alternativa, pois ao gerar resposta de mem?ria duradoura que previne a infec??o e reduz, desta forma, os altos gastos com a sa?de p?blica, com os f?rmacos antivirais e com os anticorpos monoclonais. A primeira tentativa na busca de uma vacina contra o VSR foi na d?cada de 60. A vacina produzida estimulou n?veis moderadamente elevados de anticorpos no soro, mas n?o conseguiu proteger contra ? infec??o. As crian?as que foram vacinadas desenvolveram uma doen?a mais grave quando mais tarde infectados com o v?rus. At? o presente momento n?o existe nenhuma vacina licenciada para o VSR. Desta forma, a busca de vacinas eficazes constitui um importante foco de pesquisa em todo mundo. As infec??es naturais pelo VSR n?o induzem mem?ria protetora duradoura, ocorrendo m?ltiplas reinfec??es ao longo da vida. Em crian?as infectadas, observou-se um n?mero reduzido de c?lulas T CD4+ regulat?rias (Treg) no sangue perif?rico, um aumento na produ??o de interleucina 4 (IL-4) e uma resposta T helper do tipo 2 (Th2) nas secre??es nasais. As c?lulas Treg s?o importantes para controlar um aumento exagerado da resposta imunol?gica. Por este fato acredita-se que quando h? uma redu??o das Tregs causada pela infec??o do VSR ocorre uma exacerba??o da doen?a pulmonar devido uma resposta Th2. Foi identificado que o pept?deo M209-223 do VSR aumenta a produ??o de IFN-? nas c?lulas T CD4+ ap?s o desafio com VSR. O tratamento com este mesmo pept?deo tamb?m apresentou um aumento na frequencia de c?lulas Treg ap?s infec??o prim?ria pelo VSR. Recentemente tamb?m foi demonstrado que as Tregs auxiliam no desenvolvimento de uma resposta efetora T CD8+, que ? crucial para o controle da carga viral do VSR. Nossa hip?tese ? que o pept?deo M209-223 do VSR influencia na diferencia??o das c?lulas T CD4+, aumentando a popula??o de c?lulas T efetoras e regulat?rias espec?ficas, reduzindo a inflama??o pulmonar e modulando a resposta imune. Os nossos resultados sugerem que a vacina??o com pept?deo M209-223 resulta na diferencia??o de c?lulas T CD4+ espec?ficas em efetoras convencionais, que produzem mais IFN-? e em c?lulas Treg. A vacina??o com este pept?deo diminuiu a expans?o de uma resposta Th2 nos animais infectados com o VSR, protegendo da inflama??o exacerbada tanto no local da infec??o como sistemicamente. Acreditamos que esta abordagem pode constituir um componente importante nas estrat?gias de vacina??o contra este v?rus.

O tabagismo é o principal fator de risco para o desenvolvimento da doença pulmonar obstrutiva crônica (DPOC). A importância da imunidade adaptativa para este processo não está totalmente esclarecida, entretanto a inflamação persistente está associada à ocorrência de infecções e exacerbação da DPOC. O nosso objetivo foi desenvolver um modelo experimental de exacerbação da DPOC utilizando a exposição à fumaça de cigarro e instilação de lipopolissacarídeo (LPS), visando investigar os aspectos imunológicos. Camundongos machos (C57BL/6), foram divididos em 4 grupos. Grupo controle salina (CSAL) e grupo controle LPS (CLPS) que foram expostos ao ar filtrado durante 12 semanas e receberam duas instilações intratraqueais (50µl) de salina ou LPS (1mg/kg) com intervalo de 15 dias entre cada instilação e o grupo fumo salina (FSAL) e grupo fumo LPS (FLPS) que foram expostos à fumaça de cigarro e também receberam duas instilações intratraqueais de salina ou LPS com o mesmo intervalo de tempo entre as instilações e a mesma dosagem. Após 3 dias da última instilação os animais foram anestesiados, traqueostomizados e acoplados a um ventilador para pequenos animais para avaliação da mecânica respiratória. Em seguida, fizemos a coleta do lavado broncoalveolar (LBA) para avaliar o perfil inflamatório e posteriormente, os pulmões foram removidos e feitos cortes para análise do intercepto linear médio (Lm) subpleural e peribronquial, além da espessura epitelial. Também avaliamos a densidade de macrófagos, neutrófilos, células T CD4+, CD8+ e células T regulatória (Treg), células positivas para Stat3/5 e FosfoStat3/5 no parênquima pulmonar por imuno-histoquímica. Além disso, foram analisados por ELISA no homogenato pulmonar os fatores quimiotáticos (KC/CXCL1 e MIP-2/CXCL2) e interleucina (IL) (-17, -6, -10) e interferon gama (IFN-y). Observamos alteração da mecânica do sistema respiratório com aumento da resistência tecidual (Gtis) e elastância tecidual (Htis) nos animais expostos á fumaça de cigarro e desafiados com LPS. Nos grupos FSAL e FLPS observamos aumento de Lm (regiões subpleurais), e no grupo FLPS também observamos aumento de Lm nos espaços peribrônquicos e espessamento epitelial. A associação da fumaça de cigarro e o LPS, além de ter causado dano ao parênquima pulmonar mais difuso tanto em regiões subpleurais quanto em espaços peribrônquicos intensificou a resposta inflamatória com aumento de neutrófilos, macrófagos, células T CD4+, células positivas para Stat3, FosfoStat5 e CXCL2. A densidade das células Treg, os níveis de IL-17 e IL-6 aumentaram em ambos os grupos LPS, enquanto o nível de IL-10 aumentou apenas no grupo CLPS. O aumento das células positivas para Stat3 -5, FosfoStat3 -5, corrobora com valores mais elevados para as células IL-17 e Treg. Assim, nosso estudo demonstrou que embora a associação da fumaça de cigarro e o LPS tenha induzido a diferenciação das células Th17 e Treg, não observamos aumento da expressão de IL-10 e sim da expressão de IL-17 sugerindo que uma falha na produção de IL-10 desempenha um papel fundamental na exacerbação do processo inflamatório / The tobacco smoking is the main risk factor for the development of Chronic Obstructive Pulmonary Disease (COPD). The importance of adaptive immunity for this process is not completely clear, however there are studies attesting the association of infection with COPD exacerbation. We propose an experimental model of COPD exacerbation using a traditional method of cigarette smoke (CS) combining with lipopolysaccharide (LPS) instillations, focusing on the adaptive immunity response. C57BL/6 male mice were exposed to room air or to CS and after 3 months, they received two instillations of saline or LPS (Control/SAL; Control/LPS; CS/SAL and CS/LPS groups). Animals were anesthetized to perform the respiratory mechanics and inflammatory profile in broncho alveolar lavage (BAL) and lungs were removed to evaluate the mean linear intercept (Lm), the density of macrophages, neutrophils, CD4+ and CD8+ cells, regulatory T cells (Treg), signal transducer and activator of transcription (Stat) 3, 5 and phosphoStat 3, 5. The chemotactic factors (CXCL1 and CXCL2); interleukins (IL): -17, -6, -10 and INF-y were measured in lung using Enzyme Linked ImmunoSorbent Assay (ELISA). We observed a change in the mechanics of the respiratory system with increased tissue resistance (Gtis) and tissue elastance (Htis) in CS/LPS group. In addition, in CS/Sal and CS/LPS groups we observed increase of Lm (subpleural), and in CS/LPS group we observed the increase in Lm in peribronchial spaces. CS exposure and LPS challenge induced an increase in neutrophils, macrophages, CD4+ and CD8+ T cells. CS/LPS challenge association intensified this response and lung parenchyma damage. Treg density cells, IL-17 and IL-6 levels was increased in both LPS groups, while IL-10 level was increased only in Control/LPS group. The increase of Stat3, -5, PhosphoStat3, -5 positive cells corroborates with higher values for IL-17 and Treg cells. Although the CS/LPS challenge association induced both Th17 and Treg cells differentiation, there is no increase for IL-10 expression, suggesting that a failure in IL-10 production play a pivotal role in the inflammatory process exacerbation

Embora a fumaça do cigarro configure-se como o principal fator de risco para o desenvolvimento da DPOC, nem todos os fumantes desenvolvem a DPOC clinicamente significativa, sugerindo outros fatores intrínsecos ao indivíduo, como diferenças nas respostas imunológicas envolvidas na patogênese e progressão desta doença. Objetivos: Compreender melhor o papel da resposta imune adaptativa na progressão da DPOC. Métodos: Foram estudados amostras de tecidos pulmonares de 21 indivíduos não fumantes (grupo controle); 22 fumantes não obstrutivos (FNO) e 17 fumantes com DPOC. A densidade de células CD4+ e CD8+, células T regulatórias (Treg) FOXP3+, células B e células positivas para interleucinas IL-10 e IL-17, e citocinas como CCL19, BAFF e TGF-beta foram avaliadas em pequenas e grandes vias aéreas, e em tecidos linfoides associados ou não aos brônquios (BALT e iBALT, respectivamente). Resultados: Observamos um aumento das células T CD4+ e CD8+ em pequenas e grandes vias aéreas, BALT e iBALT em fumantes; no entanto, os valores mais elevados foram detectados em pequenas vias aéreas dos indíviduos DPOC. Além disso, observouse uma diminuição na expressão de TGF-? em pacientes com DPOC em comparação aos grupos de FNO e controle em pequenas e grandes vias aéreas ao passo que uma diminuição na densidade de Treg foi observado apenas em pequenas vias aéreas, com consequente diminuição da densidade de células positivas para IL-10 em pequenas e grandes vias aéreas. Em BALT observou-se uma resposta diferente, com um aumento na densidade de Treg no grupo DPOC, sem diferenças para as análises de IL-10. Houve um aumento da densidade de células positvas para IL-17 em pequenas e grandes vias aéreas e iBALT na DPOC. Conclusões: Observamos que a redução da atividade regulatória do processo inflamatório em pequenas vias aéreas e a progressão da obstrução em fumantes esteve associado à diminuição da densidade de células Treg e da expressão de IL-10 e aumento da expressão de IL-17. Além disso, verificou-se diferenças entre o perfil inflamatório nos compartimentos pulmonares estudados / Although cigarette smoke is configured as the primary risk factor for the development of COPD, not all smokers develop COPD clinically significant, suggesting that there are other factors intrinsic to the individual, such as differences in immune responses involved in the pathogenesis and progression of this disease. Objectives: To better understand the role of the adaptive immune response in the progression of COPD. Methods: Lung tissue samples from 21 nonsmokers were studied (control group); 22 non-obstructive smokers (NOS) and 17 COPD smokers. The density of CD4 + cells and CD8 + regulatory T cells (Treg) FOXP3 + cells, B cells and positive cells for interleukins IL-10 and IL-17, and cytokines as CCL19, BAFF and TGF-beta were evaluated in large and small airways, and lymphoid tissue associated with bronchi or not (BALT and iBALT, respectively). Results: We observed an increase in CD4 + T cells and CD8 + in small and large airways, BALT and iBALT in smokers; however, the highest amounts were detected in the small airways of COPD patients. Furthermore, there was a decrease in TGF-beta expression in COPD patients compared to FNO and control groups in large and small airways while a decrease in Treg density was observed only in small airway and consequent decrease in the density of cells positive for IL-10 in large and small airways. In BALT we observed a different response, with an increase in Treg density in COPD patients without differences in IL-10 analysis. There was an increase in cell density for IL-17 in large and small airways, and iBALT in COPD. Conclusions: We observed that reduction of inflammation regulatory activity in small airways obstruction and progression of smoking was associated with decreased Treg cell density and IL-10 expression and increased IL-17 expression. Furthermore, there are differences between the inflammatory profile in the lung compartments studied

Le respect de l’équilibre entre lymphocytes T effecteurs auto-réactifs et lymphocytes T régulateurs (LTreg) est primordial dans le maintien de la tolérance aux antigènes du soi. Les partenaires cellulaires et les mécanismes moléculaires impliqués dans la rupture de l’équilibre de cette balance ne sont pas ou peu connus dans les maladies auto-immunes. Ainsi, les travaux décrits dans cette thèse portent sur le dérèglement de la balance T effecteurs/ Treg dans deux modèles de maladies auto-immunes chez l’homme: le lupus érythémateux systémique et l’anémie hémolytique auto-immune (AHAI). Nous montrons une augmentation de l’expression de la molécule de costimulation OX40L (CD252, TNFSF4) à la surface des cellules présentatrices d’antigène circulantes et infiltrant les tissus chez les patients lupiques. Cette augmentation est corrélée à l’activité de la maladie chez l’adulte comme chez l’enfant. Elle a pour conséquence l’induction de lymphocytes T effecteurs de type Tfh (T follicular helper) et le blocage des fonctions suppressives des Treg, deux acteurs majeurs dans la physiopathologie du lupus. Dans le second projet, nous montrons une augmentation de la proportion de T8reg circulants chez les patients affectés d’une AHAI à anticorps chauds en phase de rémission. Ces Treg expriment le CD25, le FoxP3 et exercent leur fonction suppressive par un mécanisme faisant intervenir l’IL10. De faibles doses d’IL-2 permettent l’expansion de cette population cellulaire in vitro. Ces résultats apportent de nouvelles connaissances dans la physiopathologie de ces deux maladies et offrent des perspectives thérapeutiques potentielles. / Respect of the balance between autoreactive T cells and regulatory T cells (LTreg) is important to maintain tolerance to self-antigens. Cellular partners and molecular mechanisms involved in the disruption of this balance are not or little known in autoimmune diseases.Thus, the work described in this thesis focuses on the disruption of the T effector/ Treg balance in two models of human autoimmune diseases: systemic lupus erythematosus and autoimmune hemolytic anemia (AIHA). We show an increased expression of the OX40L (CD252, TNFSF4) costimulatory molecule at the surface of both circulating and tissues-infiltrating antigen presenting cells in SLE patients. OX40L expression is correlated with disease activity in adults and in children and results in Tfh (follicular helper T) effector cells induction and Treg suppressive functions inhibition, two key mechanisms in the pathogenesis of lupus. In the second project, we show an increase of the circulating T8reg proportion in patients with a warm AIHA in a non-active state. These Treg express CD25, FoxP3 and exert their suppressive function by a mechanism involving IL-10. Low-dose IL-2 allows the expansion of this cell population in vitro. These results provide new insights into the pathophysiology of these diseases and offer potential therapeutic perspectives.

La réponse immune représente une réponse complexe à laquelle correspond une succession d’événements orchestrés finement. Parmi les mécanismes qui régulent la réponse immune, les lymphocytes T régulateurs (Tregs) assurent le maintien de la tolérance en périphérie et le contrôle des réponses immunes adaptatives. Ils représentent une population hétérogène et leurs mécanismes de suppression sont toujours l’objet d’intenses recherches. Suivant le contexte de suppression et leur nature, les lymphocytes Tregs réalisent une inhibition de l’activation des lymphocytes Th, soit directement, soit via la modulation de la fonction des cellules dendritiques (DC). Dans un modèle d’immunisation par des cellules dendritiques chargées de KLH, les lymphocytes Tregs naturels contrôlent sélectivement l’initiation des réponses de type Th1/CTL spécifiques de l’antigène. Le but de ce travail était de définir quels sont les acteurs potentiels du contrôle de cette réponse. A l’aide de l’anticorps PC61 dirigé contre le récepteur CD25 et éliminant les lymphocytes Tregs naturels, nous avons montré que le ligand de costimulation CD70 joue un rôle clé dans leur régulation de la réponse Th1/CTL (Article 1). Ainsi, dans des conditions normales, la cytokine IL-12 induit principalement l’initiation de la réponse Th1 in vivo, tandis qu’en l’absence de lymphocytes Tregs naturels, la voie CD70/CD27 est une voie alternative d’induction de l’IFN-γ. Cette voie d’activation pourrait être opérationnelle dans certains contextes infectieux lorsque les lymphocytes Tregs sont déstabilisés voire éliminés, par exemple lors d’infections par Toxoplasma gondii ou par les virus HTLV1, SIV ou HIV. Nous avons montré que les lymphocytes Tregs naturels diminuent l’expression du ligand CD70 sur les DC, de manière dépendante de son récepteur CD27. Ensuite, nous nous sommes intéressés à une deuxième population de lymphocytes T régulateurs, les lymphocytes Tregs ICOShigh induits in vivo par le traitement avec l’anticorps anti-CTLA-4. Dans le cadre d’une colite induite par l’agent alkylant TNBS et mettant en jeu une réponse Th1, cette population de lymphocytes Tregs amplifiée par le traitement à l’anticorps anti-CTLA-4 régule la réponse immune via la cytokine anti-inflammatoire IL-10 et l’enzyme immunosuppressive IDO (Article 2). Ainsi, les résultats obtenus nous ont permis de répondre à notre objectif et de définir certains mécanismes de suppression des lymphocytes Tregs naturels et des lymphocytes Tregs induits. Dans la dernière partie de ce travail, nous avons cherché à comparer les populations de lymphocytes Tregs naturels et ICOShigh présentes dans l’intestin d’une souris naïve. Une analyse transcriptomique a révélé que ces deux populations s’opposent sur les plans phénotypique et fonctionnel. Nous proposons un modèle dans lequel les deux populations de lymphocytes Tregs agiraient en synergie pour maintenir l’homéostasie intestinale. Les lymphocytes Tregs ICOShigh différenciés au niveau local et continuellement activés contrôleraient la réponse inflammatoire associée à la présence de la flore commensale. Les lymphocytes Tregs naturels, en quiescence dans les ganglions mésentériques, n’interviendraient qu’en cas d’infection par des pathogènes. L’étude des lymphocytes T régulateurs soulève un certain nombre de concepts clés de l’immunité : la spécificité des réponses, la distinction des microorganismes commensaux et pathogènes… Mieux connaître les lymphocytes Tregs dans un modèle murin permettra de mieux comprendre les réponses inflammatoires intestinales chroniques observées chez l'homme et d’envisager, à terme, de nouveaux traitements. / An immune response is complex and implies numerous sequential events. It is regulated by different mechanisms, among which regulatory T cells maintain peripheral tolerance and control adaptive immune responses. Regulatory T cells are very heterogeneous and suppress immune responses through different mechanisms, still under investigation. They can inhibit T cell activation directly or through the modulation of dendritic cell function, depending on their nature and the tissular context. In a dendritic cell-mediated immunization model, naturally occurring regulatory T cells selectively control the priming of antigen-specific Th1/CTL responses. Our goal was to define the potential actors of this control, targeted by natural regulatory T cells. Using the PC61 antibody which targets and depletes these cells, we showed that the costimulation ligand CD70 plays a key role in their control of Th1/CTL responses (first article). We showed that mainly IL-12 provokes Th1 development in normal conditions, wheras CD70 plays a major role in priming Th1 responses in the absence of natural Tregs. This pathway can be operational if regulatory T cells are destabilized or even depleted, for example during infection with Toxoplasma gondii or with HTLV1, SIV or HIV. We showed that natural Tregs downregulate CD70 expression on the surface of DCs. Next, we focused on another regulatory T cell population, induced in vivo by the anti-CTLA-4 mAb treatment. In a model of pro-Th1 colitis, induced by the alkylating agent TNBS, these ICOShigh regulatory T cells exert an IL-10 and IDO-dependant control over the immune response (second article). Thus, we succeeded in determining some control mechanisms of the immune response targeted by two populations of regulatory T cells. Finally, we compared two regulatory T cell populations: naturally occurring regulatory T cells and ICOShigh regulatory T cells from the intestines of naïve mice. A transcriptional analysis revealed two populations phenotypically and functionally distinct. We proposed a model in which these populations act synergistically and both maintain intestinal homeostasis. ICOShigh regulatory T cells might control commensal gut flora-specific inflammatory responses and quiescent natural regulatory T cells from mesenteric lymph nodes might control potential pathogen infections. As a conclusion, this study raises some immunological issues: specificity of immune responses, distinction between commensal and pathogenic microorganisms… A better knowledge of these regulatory populations will lead to a better understanding of human intestinal responses and in the medium term will lead to new therapeutic approaches and tools.

In einem komplexen tierexperimentellen Ansatz wurde das Potenzial der anti huCD4-Antikörper MAX16H5 und MAX12F6 zur Modulierung zellvermittelter Immun-reaktionen in vivo untersucht. Dafür kam ein mehrfach transgenes Mausmodell zur Anwendung, in dem das humane Zielmolekül und dessen physiologischer Ligand als Transgene exprimiert waren. Als T-Zell vermittelte Immunreaktion wurde eine Kon-taktreaktion (delayed type hypersensitivity, DTH) gegen DNFB etabliert und validiert. An der DTH wurde untersucht, ob und wie die verschiedenen Antikörper die Sen-sibilisierungs- und die Auslösungsphase beeinflussen. Die experimentellen Ergeb-nisse zeigen, dass die Antikörper epitop- und isotypabhängig die beiden Phasen der DTH unterschiedlich beeinflussen. Die Applikation der Antikörper während der Sensi-bilisierung führte zu einer unterschiedlich ausgeprägten Suppression der DTH. Dage-gen hatten sie gegensätzliche Effekte auf die Auslösung. Während nach MAX12F6-Behandlung eine stärkere und prolongierte DTH gemessen wurde, verlief die DTH-Reaktion nach MAX16H5-Applikation deutlich abgeschwächt. Mittels flowzytometri-scher Analysen konnte gezeigt werden, dass die Antikörper unterschiedliche Subpo-pulationen der T-Helferzellen depletieren. Darüber hinaus führte MAX16H5 offen-sichtlich zur Induktion regulatorischer T-Zellen. Die Daten erklären unterschiedliche Erfolge aus ersten klinischen Studien mit verschiedenen anti huCD4 Antikörpern. Auch eignet sich CD4 auch als diagnostisches Target zur in vivo Diagnostik T-Zell vermittelter Entzündungsreaktionen. Mit Antikörperfragmenten von MAX16H5 wurde ein immunszintigraphisches Verfahren entwickelt, das die spezifische Darstellung der mit der DTH einhergehenden Entzündungsreaktion ermöglicht.

L’importance respective des lymphocytes T régulateurs naturels générés dans le thymus ou induits en périphérie dans la régulation immunitaire et la résolution de l’inflammation est désormais bien établie. Nous avons contribué à mettre en évidence une nouvelle voie d’induction de lymphocytes T régulateurs périphériques à partir de cellules T humaines CD4+CD25- naïves et mémoires. Nous avons montré que l’engagement de la molécule ubiquitaire transmembranaire CD47 sur la cellule T par un anticorps monoclonal ou par le peptide 4N1K (peptide dérivé du domaine carboxy-terminal de la thrombospondine-1 et spécifique du site de liaison à CD47) induisait des lymphocytes T CD4+ régulateurs exerçant une fonction suppressive sur les lymphocytes T effecteurs. Les propriétés suppressives induites par la thrombospondine-1 confortent les fonctions anti-inflammatoires de cette protéine de la matrice extracellulaire. L’inhibition exercée par les lymphocytes T régulateurs induits dépend du contact intercellulaire entre les cellules T régulatrices et leurs cibles, et est indépendante du TGF-. Nos résultats démontrent également le rôle de CD47 sur le lymphocyte T CD4+ dans la réponse immunitaire spécifique de l’antigène in vivo. En effet, les souris BALB/c déficientes pour CD47 présentent un biais de la sécrétion d’anticorps et de cytokines de type Th1, alors que les souris BALB/c sont décrites comme exprimant un profil de production de cytokines de type Th2. Nos travaux mettent en évidence le rôle de CD47 dans l’inhibition du développement d’une réponse cellulaire et humorale de type Th1 in vivo, confirmant de précédentes études in vitro réalisées avec des cellules T CD4+ humaines. Nous présentons également le rôle inhibiteur de l’engagement de CD28 in vitro sur la différenciation en cellules Th17 des lymphocytes T CD4+ naïfs isolés de souris BALB/c. Le mécanisme proposé est dépendant de la production de l’IL-2 et de l’IFN- et indépendant de la présence de lymphocytes T régulateurs. Notre étude du rôle de deux molécules transmembranaires CD47 et CD28 exprimées sur la cellule T CD4+, contribue à une meilleure connaissance des mécanismes impliqués dans la tolérance immunologique, la résolution de l’inflammation et la différenciation des cellules T "helper" CD4+. / Nowadays, the importance of natural regulatory T cells and adaptive regulatory T lymphocytes in immune regulation and resolution of inflammation are well established. We report a previously unknown pathway to generate adaptive regulatory T cells in the periphery from naive and memory human CD4+CD25- T cells. We show that the stimulation of the broadly expressed transmembrane proteins CD47 on T cells by a monoclonal antibody or by the 4NK1 peptide (carboxy-terminal peptide of thrombospondin-1 (TSP) specific of the binding site of CD47) induced regulatory T cells that exerted an inhibitory function on effector T cells. Our study on the suppressive proprieties of the TSP corroborates with reported anti-inflammatory activities of this extracellular matrix protein. The suppressive function of TSP induced regulatory T cells was contact-dependent and TGF--independent. Our data further demonstrate the role of CD47 expression on T cells in the antigenic-specific immune response in vivo. We report that the CD47-deficient BALB/c mice displayed a Th1-biased antibody and cytokine responses, instead of the Th2 cytokine profile observed in unmanipulated BALB/c mice. Our study outlines the role of CD47 as a self-control mechanism to negatively regulate type 1 cellular and humoral immune responses and most importantly confirm in vivo previous in vitro studies with human CD4+ T cells. We also report that soluble anti-CD28 monoclonal antibody suppressed in vitro differentiation of naïve CD4+ T cells isolated from BALB/c mice into IL-17-producing cells by mechanism that are IL-2 and IFN-γ-dependent but independent of the presence of regulatory T cells. Our studies highlight the suppressive function of two transmembrane molecules CD47 and CD28 expressed by CD4+ T cells in vitro and in vivo in human and mice. They thus may contribute to a better understanding of the mechanisms involved in the induction of immune tolerance, the resolution of inflammation and the differentiation of the T helper cells.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal