Thèses sur le sujet « Mucosal Associated Invariant T cell »

The C-type lectin CD161 is expressed by a large number of T lymphocytes, with approximately a quarter of both T cell receptor (TCR)αβ+ and TCRγδ+ T cells expressing this marker. Within CD8+ T cells, a large proportion of these are comprised of Mucosal Associated Invariant T (MAIT) cells, a novel innate-like lymphocyte subset characterised by expression of a semi-invariant TCR together with high levels of CD161 (CD161++). These cells display a phenotype reflective of type 17 CD4+ helper T cells (Th17), which are also hallmarked by CD161 expression. Both MAIT and Th17 cells arise from preprogrammed progenitors, identifiable within umbilical cord blood by expression of CD161. Thus, CD161 appears to identify cells of a pre-determined and distinct phenotype. Whether this reflects a common transcriptional programme, developmentally induced within these cells, and further whether this extends to other CD161 positive T cells, was examined here by mRNA microarray analysis. This analysis identified a shared transcriptional signature and common innate-like function of all CD161 expressing T lymphocytes, and independent of TCR expression or lineage. Furthermore, a population of CD8+ T lymphocytes expressing lower levels of CD161 which overlap phenotypically with CD161++CD8+ MAIT cells was identified by both mRNA microarray analysis and mass cytometry (CyTOF); the CD161+CD8+ T cell population. TCR repertoire analysis, flow cytometry and cell culture experiments were utilised to investigate the origin of this subset, and its phenotype and function in both health and disease investigated in depth. This revealed a pre-programmed, tissue-resident memory population with potent effector functions. Both CD161++ MAIT and CD161+CD8+ T cells expressed high levels of the drug efflux pump MDR1, previously described to confer drug resistance to certain malignant cells. The significance of expression of this pump was hence investigated to determine its potential affect on the success of a variety of clinical therapies.

Mucosal associated invariant T-cells (MAIT) are a phylogenetically conserved subset of alpha/beta T-cells with natural killer-like (NK) activity. MAIT are defined by the expression of an invariant T-cell receptor alpha (TCRα) chain; in mice and humans this chain uses the orthologous mVα19/hVα7.2-Jα33 genes respectively. Available evidence indicates that MAIT are restricted by MR1, a highly conserved MHC class I-related molecule, and that their development is dependent on B lymphocytes. They appear to constitute part of the innate immune response, but their precise functional role is poorly understood. This study aimed to characterise MAIT and MR1 in ruminants, and to further the knowledge and understanding of these unique cells. Using PCR primers based on partial database sequences, orthologous full-length TCRα chains were identified in circulating bovine and ovine T cells. The germline elements of the respective α chains were identified and their overall frequency of expression within the bovine TCRα repertoire determined. Experiments using the orthologous TCRα chain as a marker for MAIT cells to examine expression in bovine and ovine blood and various tissues showed that spleen and mesenteric lymph nodes contained the highest frequency of MAIT cells. Use of the same technique to study levels of this marker in cattle of different ages revealed very low numbers of MAIT cells in neonatal animals, followed by a marked increase in the first 3 weeks of life. Analyses of MAIT TCRα expression in different T cell subsets showed that, unlike mice and humans in which MAIT cells are predominantly within the CD4-/CD8- T-cell population, MAIT cells in bovine blood are predominantly CD8+. Full-length cDNAs were isolated for bovine and sheep MR1 and their sequences were found to display marked cross-species conservation. Using a specific PCR, MR1 was shown to be expressed in peripheral blood and by different lineages of Theileria-transformed cells. Alternatively-spliced transcripts of MR1 were detected in both cattle and sheep and several of these retained an intact open-reading frame. Constructs of bovine MR1 and an MR1/MHC chimera were prepared in a eukaryotic expression vector but these failed to give detectable cell surface expression following transfection into Cos-7, despite positive intracellular expression.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like lymphocytes within the gut, liver and blood, expressing a semi-invariant T cell receptor (TCR) and high levels of the C-type lectin-like receptor, CD161. These cells recognise a metabolite of the microbial riboflavin synthesis pathway, presented by the highly conserved Major Histocompatibility Complex (MHC) class I-related protein, MR1, and are critical for the control of bacterial infections. The factors regulating the broad effector functions of MAIT cells have not been fully investigated. Utilising a novel flow cytometric killing assay, MAIT cells were shown here to require the induction of a cytotoxic phenotype through bacterial stimulation to efficiently kill target cells. Further in depth phenotypic analysis highlighted a distinct non-cytotoxic subset of CD4+ MAIT cells, with an altered cytokine-producing capacity, enriched within lymphoid tissues. Investigation into the potential role of these cells in psoriatic diseases revealed that MAIT cells within the synovial fluid of psoriatic arthritis patients are potently activated with increased IL-17 production, their frequency correlating with measures of clinical activity. MAIT cells also have an innate-like responsiveness to cytokines, a feature originally attributed to Natural Killer (NK) cells. Microarray analysis and mass cytometry experiments demonstrated that CD161 marks immature NK cells that have retained this ability to respond to innate cytokines during their differentiation, and is lost upon cytomegalovirus (CMV)-induced maturation in both healthy and human immunodeficiency virus (HIV)-infected patients. Thus, CD161 marks cells with innate-effector functions both in T cells and NK cells.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS), characterized by demyelination and axonal loss. It’s considered a multifactorial disease, whose etiology involves both environmental and genetic factors. The innate and the adaptive immune systems have long been considered the main players in CNS inflammation, plaque formation and, ultimately, neurodegeneration. In the last 10 to 15 years a lot of attention has been focused on a newly described subset of immune cells, called unconventional T cells, as possible modulators of the aberrant activation of the immune system. The family of unconventional T cells comprises mucosal associated invariant T (MAIT),  and NKT cells. MAIT cells are innate-like T cells, recognizing riboflavin metabolites presented by the evolutionary conserved antigen presenting molecule MR1. They are abundant at mucosal sites and their phenotype is deeply influenced by the microbiome. They can be activated either by direct TCR stimulation or by cytokines produced during inflammation. Once activated, they can produce both pro-inflammatory cytokines and cytotoxic molecule, like granzyme B. MAIT cells have been implicated in the pathogenesis of many autoimmune diseases, such as diabetes, inflammatory bowel diseases and rheumatoid arthritis. In this project we aim to elucidate the potential role of MAIT cells in the pathogenesis of MS, by using both the animal model of the disease, experimental autoimmune encephalomyelitis (EAE) and samples obtained from people with MS (pwMS).

Le diabète de type 1 (DT1) est une maladie auto-immune caractérisée par la destruction sélective des cellules β pancréatiques entraînant une hyperglycémie et nécessitant un traitement par insulinothérapie à vie. La physiopathologie du DT1 est complexe et fait intervenir les cellules immunitaires innées et adaptatives dans la pathogenèse et la régulation du DT1. Alors que le développement du diabète peut être associé à des facteurs génétiques, des facteurs environnementaux sont également impliqués dans le déclenchement de cette maladie. Des études récentes ont mis en évidence le rôle du microbiote intestinal dans le développement ou la protection du DT1. Des modifications du microbiote ont par ailleurs été observées chez les patients DT1 avant le déclenchement de la maladie. Plusieurs études ont également décrit des altérations de la muqueuse intestinale chez les souris NOD et chez les patients DT1. Les cellules MAIT sont des lymphocytes T de type inné reconnaissant la molécule de MR1 et exprimant un TCR Va semi-invariant (Vα7.2-Jα33 chez l'homme et Vα19-Jα33 chez la souris). Les cellules MAIT sont activées par des métabolites bactériens, dérivés de la synthèse de la riboflavine. Leur particularité est de produire rapidement diverses cytokines telles que le TNF-α, l’IFN-γ et l’IL-17 et le granzyme B. La localisation et la fonction des cellules MAIT suggèrent qu'elles pourraient jouer un rôle clé dans le maintien de l'intégrité intestinale et le développement des réponses auto-immunes dirigées contre les cellules β. Dans l’ensemble, nos résultats chez les patients DT1 et chez les souris NOD montrent une activation anormale des cellules MAIT chez les patients DT1. Ces anomalies peuvent être détectées avant le déclenchement de la maladie. L'analyse des tissus périphériques de souris NOD souligne le rôle des cellules MAIT dans deux tissus, le pancréas et la muqueuse intestinale. Dans le pancréas, la fréquence des cellules MAIT est augmentée. Dans ce tissu les cellules MAIT semblent participer à la destruction des cellules β. Contrairement au pancréas, les cellules MAIT situées dans la muqueuse intestinale semblent jouer un rôle protecteur grâce à leur production de cytokines IL-22 et IL-17. Nos données chez les souris NOD Mr1-/-, dépourvues de cellules MAIT, soulignent le rôle protecteur des cellules MAIT lors du développement du DT1 en participant au maintien de l'intégrité intestinale. En outre, la présence d'altérations intestinales à mesure que la maladie progresse chez les souris NOD souligne l'importance des cellules MAIT dans le maintien de l'homéostasie intestinale. De manière intéressante, les cellules MAIT pourraient représenter un nouveau biomarqueur de la maladie et permettre de développer des stratégies thérapeutiques innovantes basées sur l’activation locale des cellules MAIT
Type 1 diabetes (T1D) is an auto-immune disease characterized by the selective destruction of pancreatic islet β cells resulting in hyperglycemia and requiring a life-long insulin replacement therapy. The physiopathology of T1D is complex and still not entirely understood. Both innate and adaptive immune cells are involved in the pathogenesis and the regulation of T1D. While diabetes development can clearly be associated with genetic inheritance, environmental factors were also implicated in this autoimmune diseases. Recent studies have highlighted the role of the intestinal microbiota in the development or protection against T1D. Gut microbiota analyses in patients have shown differences before the onset of T1D. Moreover, several studies also described gut mucosa alterations in NOD mice and in T1D patients. MAIT (Mucosal Associated Invariant T) cells are innate-like T cells recognizing the MR1 molecule and expressing a semi-invariant receptor Vα chain (Vα7.2-Jα33 and Vα19-Jα33 in mice). MAIT cells are activated by bacterial metabolites, derived from the synthesis of riboflavin. Their particularity is to rapidly produce various cytokines such as TNF-α IFN-γ, IL-17 and granzyme B. The localization and the function of MAIT cells suggest that they could exert a key role in the maintenance of gut integrity, thereby controlling the development of autoimmune responses against pancreatic β cells. To summarize, our results in T1D patients and in NOD mice indicate an abnormal MAIT cell activation in this pathology, which occurs before disease onset. The analysis of peripheral tissues from NOD mice highlights the role of MAIT cells in two tissues, the pancreas and the gut mucosa. In the pancreas, MAIT cells frequency is elevated and they could participate to the β cells death. In contrast to the pancreas, in the gut mucosa MAIT cells could play a protective role through their cytokines production of IL-22 and IL-17. Our data in Mr1-/- NOD mice, lacking MAIT cells, reveal that these cells play a protective role against diabetes development and in the maintenance of gut mucosa integrity. Moreover, the presence of gut alteration as T1D progress in NOD mice underscores the importance of MAIT cells in maintaining gut mucosa homeostasis. Interestingly, MAIT cells could represent a new biomarker towards T1D progression and open new avenues for innovative therapeutic strategies based on their local triggering

Antimicrobial peptides (AMPs) are ubiquitous and display broad-spectrum antimicrobial activity that can control bacterial colonization of surfaces. Ceragenins are small-molecule mimics of AMPs and have several advantages over AMPs, including cost of manufacture and stability. A ceragenin, CSA-120, modified with an acrylamide group was directly incorporated into fluoropolymer coatings as a means of inhibiting bacterial biofilm formation. The ceragenin-containing coatings displayed improved performance. By conjugating a copper chelating group to the ceragenin, chelation of 64Cu by the conjugate was effective and provided a stable complex that allowed in vivo imaging. This conjugate may provide a means of identifying infection sites in patients presenting general signs of infection without localized symptoms. A combination nanoparticle comprised of a maghemite core for enhanced T2 MRI contrast diagnostics, a colloidal silver shell acting as an antimicrobial and therapeutic vehicle, and a ceragenin (CSA- 124) surfactant providing microbial adhesion was synthesized and characterized by multiple methods. Silver nanoparticles conjugated with ceragenin, CSA-124, as a potential Gram-positiveselective antimicrobial were synthesized and termed as CSA-SNPs. Herein, CSA-SNPs are characterized using multiple methods and the antimicrobial properties are determined through minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) and time-kill study. Prostanoids are a natural subclass of eicosanoids generated mainly from metabolic oxidation of arachidonic acid. Cyclopentenone prostaglandins (cyPGs) contain a highly reactive α,β-unsaturated carbonyl group in their cyclopentenone ring and possess three main potentially therapeutic properties: anti-inflammatory, antiproliferative and antiviral. We designed and synthesized EC and its derivatives in reducing secretion of pro-inflammatory cytokines IL-6 and IL-12. Mucosal-Associated Invariant T (MAIT) Cells are unique innate-like T cells and play a key role in host defense against bacterial and fungal infection as well as in human autoimmune diseases. The MAIT cells are activated through T-cell receptor αβ chain (TCR-αβ) binding with the MR1-ligand, which is vitamin B metabolites presented on MR1. Rribityllumazines, one of important MR1-ligand was synthesized in my study.

This thesis by publication is a step forward in understanding the function of discrete immune cell populations in kidneys with chronic inflammation and fibrosis. We have successfully identified various human immune cells of the innate immune system as critical drivers of chronic kidney disease. The findings of this thesis sheds light on novel functions of innate immune cells and opens opportunities for the development of novel kidney therapies.

Le surpoids et l’obésité touchent plus de 1,9 milliard d’adultes à travers le monde et pourraient atteindre 3,3 milliards de personnes dans une dizaine d’année. L’obésité est associée à une inflammation tissulaire et systémique chronique de bas grade, qui contribuent à l’apparition de la résistance à l’insuline. Récemment, notre laboratoire a mis en évidence des anomalies d’une nouvelle population de lymphocytes T innés, les cellules MAIT (Mucosal Associated Invariant T) chez des patients obèses et/ou ayant un diabète de type 2 (T2D). Les cellules MAIT sont des lymphocytes T non conventionnels, qui expriment un récepteur des cellules T (TCR) avec une chaîne alpha invariante. Leur TCR reconnait la molécule d’histocompatibilité de classe 1 non classique MR1, présentant des métabolites dérivés de la voie de biosynthèse des vitamines B, notamment les vitamines B2 et B9. Dans cette étude, nous utilisons les modèles murins pour analyser le rôle des cellules MAIT dans le developpement du T2D. Au cours de l’obésité induite par un régime riche en graisse, les cellules MAIT du tissu adipeux viscéral (TA) et de l’iléon sont activées de façon précoce et anormale et produisent plus de cytokines pro-inflammatoires (i.e. IL-17, TNFa et l’IFN?). De plus, l’augmentation de la fréquence tissulaire des MAIT chez des souris Va19 transgéniques conduit à l’apparition de la résistance à l’insuline et à une intolérance au glucose, au cours de l’obésité. A l’inverse, les souris obèses déficientes en MAIT, MR1-/-, sont protégées contre ces anomalies métaboliques. Une fréquence élevée de MAIT est associée au changement de macrophages M2 (anti-inflammatoires) en M1 (inflammatoires) et à une infiltration des cellules NK et des LTaß-CD8 au niveau du TA. Par ailleurs, les MAIT contrôlent la fréquence des Treg, ILC2 et ILC3 dans l’iléon et des Treg, ILC2 et éosinophiles dans le tissu adipeux. La modification de la fréquence des ILC2 et ILC3 est associée à la production intestinale d’IL-33 et d’IL-25. De plus, nous montrons que le rôle délétère des MAIT dans le développement du T2D est associé à une dysbiose intestinale. Finalement, des expériences de transfert de flores intestinales montrent que cette dysbiose intestinale est en partie responsable des anomalies immunitaires et métaboliques
Obesity and type 2 diabetes are associated with low-grade chronic inflammation. Immune cells are recruited and activated in several tissues, including adipose tissue, thereby contributing to insulin-resistance and diabetes. Recent studies described gut microbiota dysbiosis as a consequence as well as a driver of obesity and type 2 diabetes. Mucosaassociated invariant T cells (MAIT) are innate-like T cells expressing a semi-invariant T-cell receptor restricted by the non-classical MHC class I molecule MR1 presenting bacterial ligands. In obese/T2D patients MAIT cells in blood and adipose tissue exhibit a pro-inflammatory profile. In the present study, we show that during high fat diet-induced obesity MAIT cells produce inflammatory cytokines in adipose tissue and the ileum and induce inflammation in these tissues by modifying other immune cell populations (i.e. macrophages, CD8 Taß cells, NK cells, LTreg, eosinophils and ILC2 in the adipose tissue and ILC2, ILC3 and LTreg in the ileum). These changes impair the function of both tissues leading to insulin resistance, glucose intolerance, impaired lipid metabolism and increased gut permeability. MAIT cells also impact gut microbiota dysbiosis during obesity and microbiota transfer experiments highlight a bidirectional crosstalk between MAIT cells and the gut microbiota leading to inflammation and gut leakage. Altogether these results reveal the major role of MAIT cells in promoting the development of type 2 diabetes during obesity

Les cellules MAIT (Mucosal-Associated Invariant T) constitue une nouvelle sous-population de LT phylogénétiquement conservée (homme, souris, vache), exprimant un répertoire du TCRα invariant, défini par l'utilisation de la chaîne canonique Vα7. 2/19-Jα33. Ces cellules sont localisées préférentiellement dans les ganglions mésentériques (MLN) et la lamina propria (LP) de l'intestin. Elles reconnaissent la molécule du CMH de classe Ib MR1 et l'expression de cette molécule par des LB est suffisante pour la sélection/expansion de ces cellules. L'étude de souris transgéniques iVα19-Jα33, a montré que le biais de répertoire Vß6/8, caractéristique de ces cellules, disparaît dans le thymus, les MLN et la LP en l'absence de la molécule MR1, et la sur-expression d'une chaîne du TCRß spécifique de ces cellules permet d'augmenter la sélection/expansion de ces cellules dans le thymus, les MLN et la LP. Enfin, leur sélection est dépendante de la présence de bactéries commensales gram+ ou Gram-
Mucosal-Associated Invariant T (MAIT) cells display unique T cell feature : they are phylogenetically conserved (human, mouse and cattle) and used an invariant TCRα chain, iVα7. 2/19-Jα33. These cells localized in the mesenteric lymph nodes (MLN) and the gut lamina propria (LP). They recognize the phylogenetically conserved class Ib MHC molecule MR1 and it expression on B cells is sufficient for selection/expansion of MAIT cells. The study of iVα19-Jα33 transgenic mice showed that the characteristic Vß6/8 biais of repertoire is loss in the thymus, the MLN and LP in the absence of MR1. Expression of a MAIT cells specific TCRß chain induces an increased selection/expansion of these cells in the thymus, MLN and LP. Finally, there selection/expansion is dependant upon the presence of commensal flora in the gut, as they are absent in germfree mice and both Gram+ and Gram- bacteria can induce MAIT selection/expansion

Les cellules MAIT (mucosal Associated Invariant T) sont des lymphocytes T dits innés qui expriment un répertoire de récepteurs à l’antigène restreint : une chaine α du TCR (iVα19-Jα33 chez la souris et iVα7. 2-Jα33 chez l’homme) associé à un nombre limité de chaine β (principalement Vβ6, Vβ8 chez la souris et Vβ2, Vβ13 chez l’homme). Les cellules MAIT sont sélectionnées par MR1 qui est une molécule du CMH de classe I non classique très conservée (90% d’homologie entre l’homme et la souris). De part leur développement spécifique, ces cellules ont des capacités particulières telles que leur localisation tissulaire, la taille clonale élevée et des capacités effectrices immédiates. Les cellules MAIT ont une réactivité antimicrobienne mais leurs fonctions restent à définir. Chez l’homme, les cellules MAIT sont des lymphocytes T CD161hiIL-18Rα+. Ces cellules présentent un phénotype mémoire effecteur (CD45RA-CD45RO+CD95hiCD62Llo) et l’expression de leurs récepteurs aux chimiokines (CCR9intCCR7-CCR5hiCXCR6hiCCR6hi) indique une localisation préférentielle dans les tissus et particulièrement dans l’intestin et le foie dans lequel les cellules MAIT représentent entre 20 et 45% des lymphocytes T. Les cellules MAIT expriment ABCB1 qui leur confère une résistance à la chimiothérapie et qui pourrait également leur permettre de résister aux xénobiotiques sécrétés par les bactéries intestinales. La plupart des cellules MAIT sont quiescentes et ne prolifèrent pas de façon extensive après stimulation. D’autre part, les cellules MAIT présentent des fonctions effectrices différentes selon le contexte de stimulation. Les cellules MAIT CD8-CD4- et CD8+ sont capables de sécréter de l’IFNγ, du TNFα après stimulation par des billes recouvertes d’anticorps anti-CD3 et anti-CD28 ou par des cellules présentatrices d’antigènes infectées par des bactéries. Cependant elles sécrètent également de l’IL-2 et de l’IL-17 après stimulation par la PMA-Ionomycine. Les cellules MAIT expriment également la granulysine et la perforine et dégranulent après stimulation par la PMA-Ionomycine. Par conséquent, les cellules MAIT présentent des capacités cytotoxiques, en induisant la lyse de cellules cibles infectées par des bactéries (monocytes, HELA-MR1 et HT10-80-MR1). Ainsi les cellules MAIT présentent différentes capacités fonctionnelles qui pourraient dépendre du type de cellules présentatrices d’antigène, de la nature et de l’intensité de la costimulation. Ces capacités pourraient leur permettre de jouer différents rôles dans l’immunité des muqueuses
Mucosal associated Invariant T (MAIT) cells are an innate like T lymphocyte population that express an antigen receptors with a very limited diversity. MAIT cells are selected on the strikingly conserved MHC-Ib molecule MR1. They follow a specific ontogenic pathway, which imparts them with selected tissue homing capacity, an elevated clonal size and immediate effector functions. MAIT cells have anti-microbial specificity but their functions remain unclear. MAIT cells are CD161hiIL-18Ra+ T-cells. We now show that they display an effector-memory phénotype and their chemokine receptor expression pattern indicates preferential homing to tissues and particularly to the intestine and the liver. MAIT cells can represent up to 45 % of the liver lymphocytes. MAIT cells express the multidrug resistance transporter (ABCB1), which confers resistance to chemotherapy. These features might also allow them to resist to xenobiotics secreted by gut bacteria. Most MAIT cells are non-cycling and do not extensively proliferate after stimulation. Nonetheless, MAIT cells display various effector functions depending on the context of this stimulation. MAIT cells secrete IFNg, TNFa and Granzyme B after stimulation with bacteria-infected antigenpresenting cells. However, IL-2 and IL-17 are additionally found after Ionomycine+PMA stimulation. MAIT cells display cytotoxic capacities, inducing the lysis of target cells infected by bacteria. Thus, MAIT cells display various functional capacities depending on the type of antigen-presenting cells and strength of co-stimulation. These features might enable them to react in different types of insults at mucosal surfaces

Tese de mestrado, Biologia Humana e Ambiente, Universidade de Lisboa, Faculdade de Ciências, 2019
A Organização Mundial de Saúde considera a doença da Hepatite B como a oitava causa de mortalidade a nível mundial e estima que aproximadamente 400 milhões de pessoas estarão infetadas nos dias de hoje. O vírus da hepatite B é caracterizado por ter um tamanho reduzido, possuir uma conformação genómica baseada em 4 “open reading frames” (ORFs), ser transmitido de forma singular e com um modo ação muito efetivo. Pacientes infetados com o vírus da hepatite B, dependendo da idade em que foram infetados, podem ser diferenciados em pacientes agudos ou devido ao historial de progressão da doença ser considerados pacientes crónicas, tendo um risco elevado de virem a contrair uma de duas patologias no futuro – cirrose hepática e cancro do fígado. Actualmente existem dois regimes terapêuticos utilizados contra a hepatite B crónica, sendo estes “Nucleos(t)ide analogues” (NUCs) e tratamentos a base de interferões. Contudo, apesar de promoverem uma cura funcional, estes tratamentos são considerados ineficazes a longo prazo contra a infeção, uma vez que não conseguem erradicar o ADN viral (cccADN) dos hepatócitos infetados e o seu consumo é imprescindível a partir do momento do seu diagnóstico. À semelhança de outras doenças virais crónicas, como HIV e Hepatite C também pacientes infectados com o virus da Hepatite B devido á exposição do mesmo por tempo indeterminado até ao final da vida, ficam sujeitos a uma continua disfunção a nivel celular. As células T, membros activos do nosso sistema imunitário, após combate contínuo perante a infeção, acabam por perder gradualmente a sua função contra o virus e acabam por entrar num estado de exaustão. Este processo está documentado em vários estudos científicos e sugere que não é necessariamente o grau e intensidade da infeção viral que aumenta, mas sim o próprio sistema imunitário que, por estar em constante esforço, acaba por ser ineficaz em erradicar o virus do corpo humano. No entanto, a comunidade científica está a direcionar esforços para a criação de novas medidas terapêuticas, que passam por fazer um targeting viral e utilizar mecanismos de imunoterapia que promovam a erradicação do vírus via a eliminação do ADN viral das células infetadas. Como exemplo, as células “mucosal-associated invariant T” (MAIT) são vistas como uma possível nova abordagem imunoterapêutica contra o vírus da hepatite B. As células MAIT constituem um subsistema das células T, sendo caracterizadas por serem inatas, não convencionais e reconhecerem metabolitos de riboflavina microbiana (Vitamina B2). Este reconhecimento acontece no seguimento da secreção de riboflavina microbiana por parte de diversas espécies bacterianas e fungos e são apresentadas as células MAIT por intermédio da molécula não-polimórfica do complexo de histocompatibilidade principal (“major histocompatibility complex”, MHC) classe I (MR1). Estas células estão ainda evolutivamente conservadas entre várias espécies e podem ser encontradas em larga escala nos tecidos da mucosa humana, sangue periférico e fígado, existindo uma correlação direta com a acumulação por parte destas células em locais de infeção e fornecimento de proteção contra infeções. Tendo por base a sua localização celular e a sua frequência no fígado, é sugerido que em ambientes de exaustão imunitária como é o caso da hepatite B, as células MAIT podem subsistir e estar funcionalmente ativas, imitando o papel imunitário das células T contra a infeção. Adicionalmente, as células MAIT dispõem de diversas funções effectoras quando estimuladas e, consequentemente, activas. A produção de citoquinas pro-inflamatórias, tais como IFN-ү e TNF-α, assim como degranulação, citotoxicidade, proliferação celular (marcador Ki-67) e respostas dependentes/independentes da presença do MR1, previamente referido, são algumas das funções effectoras relevantes levadas a cabo por estas células e que influenciam o seu desempenho junto do corpo humano. É importante mencionar que as células MAIT só recentemente comecaram a ser estudadas de uma forma mais abrangente estando associadas a diferentes patologias, como é o caso deste projecto, no qual nos focamos no papel destas células no contexto do virus da hepatite B. Contudo, na sua grande maioria, os marcos existentes referentes á função celular destas células remetem para infeções bacterianas, uma vez que estas células activamente reconhecem metabolitos produzidos por espécies bacterianas, nomeadamente no caso da Eschericia coli. O foco do nosso projecto passou pela caracterização de células MAIT, previamente isoladas de amostras de PBMCs de dadores saudáveis e doentes crónicos infetados com hepatite B, com o intuito de comparar o seu fenótipo, assim como a sua resposta funcional aquando estimulação com citoquinas recombinantes, neste caso as interleucinas IL-12 e IL-18. Além disso, estas células foram ainda avaliadas quanto à expressão de três painéis de anticorpos diferentes: 1) “Checkpoint Inhibitors” (PD-1, TIM3 e LG3), 2) “Activation Markers” (CD25, CD38, CD69 e HLA-DR) e 3) “Pro-Inflammatory Cytokines” (IFN-ү, TNF-α e IL-2), e analisadas através de citometria de fluxo. Ao mesmo tempo, o perfil expressivo de citoquinas e quimiocinas das células MAIT foi examinado na presença de uma linhagem celular humana (B lymphoblast - C1R) e aquando estimulação com um composto agonista do MR1 (Diclofenac). As células MAIT de 3 pacientes saudáveis e 3 pacientes crónicos infectados com o virus da hepatite B foram testadas. É sabido que os marcadores avaliados são expressos pelas células MAIT em pacientes saudáveis. Contudo, o intuito de avaliarmos os mesmos em pacientes infectados passou por perceber se, na presença de uma infeção crónica, estas células conseguiriam permanecer funcionais. Ainda assim, foi necessário perceber se o virus ou o tempo de infeção, que pode variar de paciente para paciente, promove uma alteração na frequência das células MAIT e na produção dos diversos marcadores testados. Embora os nossos dados preliminares sejam indicativos de que as células MAIT são ativamente funcionais em pacientes crónicos com hepatite B, comparativamente com os pacientes saudáveis testados, no futuro é necessária a recriação destas experiências num maior número amostral, uma vez que o nosso valor de N neste projeto é limitado. Não obstante, foi possível concluir com este projeto que células MAIT de pacientes infectado com HBV conseguem ser estimuladas e activadas pelas duas citoquinas recombinantes anteriormente referidas, IL-12 e IL-18, produzindo diversas citoquinas necessárias para que a sua actividade citotóxica seja equiparável as células MAIT de pacientes saudáveis. No entanto, foi verificado que a frequência dos marcadores de exaustão testados aparenta ser superior em pacientes crónicos. Contudo, seria importante testar células MAIT provenientes de amostras hepáticas de pacientes crónicos, com o objetivo de comparar com os resultados obtidos neste projecto, uma vez que o fígado é um órgão de alto risco em caso de infeção por hepatite B. Não descurando, os dados obtidos neste projeto mostram que as células MAIT poderão ter um papel imunoterapêutico promissor contra o vírus da hepatite B em combinação com terapias já existentes. O estudo cientifico realizado no âmbito de tese de mestrado demonstrou que as células MAIT não seguem o caminho clássico de exaustão celular e conseguem reter parcialmente a sua funcionalidade em ambientes desfavoráveis, como é o caso do fígado e sangue de pacientes crónicos com hepatite B. Por conseguinte, isto é indicativo de que as células MAIT são um alvo promissor quanto ao seu uso no âmbito da imunoterapia em infeções virais crónicas e no tratamento tumoral onde o local activo da infeção seja um ambiente resiliente. Para concluir, torna-se imeprativo mencionar que existem diversas questões para as quais não existem ainda respostas evidentes relativamente ao papel activo das células MAIT. Contudo, sabemos que estas células representam um facção substancial de células T especifícas circulatórias e teciduais no corpo humano. Deste modo, um maior número de estudos ciêntificos são fundamentais para compreender a contribuição das células MAIT para uma resposta imunitária contra o virus do HBV e outras infeções virais.

RESUMO: A imunoterapia alterou o tratamento padrão de múltiplos cancros. O tratamento atual para doentes com cancro pancreático, incluindo o adenocarcinoma ductal pancreático (PDAC), não melhorou substancialmente a sobrevivência global de pacientes. O PDAC é uma doença sistémica, das mais imunossupressoras e fatais, ampliando a necessidade de modalidades de tratamento mais eficazes. Foi explorado nesta tese um subconjunto de células imunitárias que estão a fazer a ponte entre as respostas imunitárias inatas e adaptativas. Estas células T invariantes associadas à mucosa (MAIT) são definidas pelo uso restrito do recetor da cadeia α Vα7.2 do recetor de células T (TCR) combinada com diferentes cadeias de TCR β. As MAIT produzem citoquinas pró-inflamatórias e foram encontradas em lesões primárias e metastáticas do cancro, no entanto a sua associação com a sobrevivência em doentes com PDAC ainda não foi definida. As células MAIT podem contribuir para o controlo de células malignas, quer através do reconhecimento direto de células tumorais restrito por MR1 e/ou pela produção de citoquinas anti-tumorais induzidas por espécies bacterianas presentes no microambiente tumoral. Um conjunto restrito de espécies bacterianas foram associadas a uma melhor sobrevivência em pacientes com PDAC. Diferentes protocolos de expansão utilizando combinações de citoquinas foram testados quanto à sua capacidade de expandir MAIT do tumor dos pacientes. A eficiência dos protocolos foi avaliada através da avaliação do número total de células e da frequência de MAIT. A capacidade funcional das células foi determinada pela produção de IFN-γ em resposta a células tumorais autólogas e/ou linhas celulares alogénicas de cancro. Foi demonstrado que células MAIT podem ser diretamente isoladas do tecido tumoral de pacientes com cancro e que são capazes de reconhecer células tumorais autólogas, produzindo IFN-γ. MAIT infiltrantes nos tumores detetam e reconhecem o tumor autólogo de forma restrita ao MR1 in vitro. Em resumo, estes dados fornecem a primeira evidência de que MAIT que infiltram o tumor podem ser expandidas a partir de lesões tumorais e as reconhecem, podendo ser utilizadas em imunoterapia para pacientes com cancro pancreático.
ABSTRACT: Immunotherapy has changed the standard of care for multiple cancers. The current treatment for patients with pancreatic cancer, including pancreatic ductal adenocarcinoma (PDAC), does not substantially improve overall survival. PDAC is a systemic disease, one of the most immunosuppressive and deadliest malignancies, hence the need for more effective treatment modalities. A subset of immune cells that are bridging innate and adaptive immune responses has been explored in this thesis. These mucosal-associated invariant T cells (MAIT) are defined by the restricted usage of the invariant Vα7.2 T cell receptor (TCR) α-chain combined with different TCR β chains. MAIT produce pro-inflammatory cytokines and have been found in primary and metastatic cancer lesions, however their association with survival in patients with PDAC has not yet been defined. MAIT cells may contribute to control malignant cells by either direct MR1 restricted recognition of cancer cells and/or by production of anti-cancer directed cytokines induced by bacterial species residing within the tumor. A restricted set of bacterial species has been associated with improved survival in patients with PDAC. Different expansion protocols using cytokines were tested for their capacity to preferentially expand MAIT from patients’ tumor. The protocols’ efficiency was assessed by evaluating the total cell number and MAIT’s frequency. Functional capacity was determined by IFN-γ production in response to autologous tumor cells and/or allogeneic cancer cell lines. It was demonstrated that MAIT can be directly isolated from patients’ tumor tissue and are able to recognize autologous tumor cells, producing IFN-γ. Tumor infiltrating MAIT revealed MR1 restriction when targeting autologous tumor cells in vitro. In summary, these data provide the first evidence that tumor infiltrating MAIT can be expanded from, and recognize, cancer lesions and may be used in cancer immunotherapy for patients with pancreatic cancer.