Tesis sobre el tema "T-cell subpopulations"

Leukocytes express a family of high molecular weight glycoproteins called leukocyte common antigens (CD45) which have tyrosine phosphatase activity and are involved in phosphotyrosine signal transduction. Antibodies to different CD45 isoforms distinguish functionally different CD4+ T cell subsets in humans, rats, and mice. Selected protein isoforms are expressed through a process of exon splicing which is cell-type and differentiation-state specific. Splicing of the three variable exons, A, B, and C, which encode amino acids located near the extracellular amino terminus of the protein, potentially results in generation of eight different mRNA transcripts. The purpose of this study was to determine the relative levels of all eight different CD45 transcripts present in a panel of murine CD4+ T cell lines and normal murine and human CD4+ T cell subsets separated with antibodies to CD45 variable exons. I show, as expected, that the broad features of CD45 surface isoform expression in these cells can be accounted for by the relative amounts of the eight differentially spliced transcripts. Unexpectedly, all the differences in CD45 isoform expression among the CD4+ T cell subpopulations that I measured could be accounted for by differences in the overall level of variable exon expression. I did not see differences among T cell populations in the relative expression of particular variable exons. Exon B was always found in greater abundance than exons C or A. Of the dual exon species, only AB and BC were found in CD4+ T cells. The AC species was undetectable. Human CD4+ T cells, especially those in the naive subset, express higher levels of CD45 variable exons than murine CD4+ T cells. In unrelated studies, I have generated a rat-mouse hybridoma which secretes a rat IgG antibody reactive with mouse CD45. I show that the monoclonal antibody, 25D10, defines a novel epitope consistent with a post-translational modification of CD45, similar but distinct from the epitope recognized by monoclonal antibody RA3.6B2 (anti-B220). This conclusion is based on evidence that it precipitates similar molecular weight bands from cells as does a framework monoclonal antibody to CD45, yet has a distinct cell surface expression as determined by flow cytometric analysis. It stains activated Th cell lines at a higher intensity than resting Th cells, stains 60-70% of splenocytes, and 25-30% of lymph node cells. It stains all class II positive cells but not freshly isolated CD4+, CD8+ T cells or CD45 transfected fibroblasts.

We have examined CD8+ sub-populations to determine whether these subsets are critical to the production of CD8+ T cell nonlytic factors. The production of the beta-chemokines MIP-1alpha, MIP-1beta and RANTES, and the chemoattractant cytokine IL-16 were measured in cells derived from 24 HIV-infected and 25 uninfected subjects. Asymptomatic HIV+ subjects (CD4 > 200/ul) produced significantly higher levels of MIP-1alpha and MIP-1beta from CD8+ T cells and some sub-phenotypes. Higher RANTES levels were produced by CD28-, CD38- and HLA-DR+/- sub-phenotypes. However, IL-16 was only modulated in the CD38+ subset in comparison to total CD8+ T cells. Infection of CD8+ T cells and sub-populations resulted in generally increased levels of chemokine and IL-16 production, which dissipated over a 15 day time course. Moreover, CD8+ antiviral factor (CAF) activity, another major component of CD8+ T cell nonlytic suppression factors, was not associated with chemokine production. However, significantly higher levels of CAF were produced by CD38+ and HLA-DR+ sub-populations. In addition, we also showed that in CD8+ T cell populations, the production of MIP-1alpha, MIP-1beta and IL-16 was inversely correlated with virus copy number. These findings shed light on the noncytotoxic responses of CD8+ T cells in controlling the natural course of HIV infection.

Les monocytes sont des leucocytes circulants dont la caractérisation est longtemps restée difficile. La dissection de l’ensemble de ces cellules en sous-populations fonctionnelles chez l’homme reste à ce jour insuffisante. Les monocytes sont cependant des précurseurs circulants de plusieurs populations de cellules dendritiques et de macrophages tissulaires, et occupent donc à ce titre une place prépondérante dans la mise en place des réponses immunitaires normales et pathologiques.À l'heure actuelle, trois sous-populations sont décrites chez l’homme : les monocytes classiques CD14+CD16neg, les non-classiques CD14dimCD16+ et les intermédiaires CD14+CD16+. Fonctionnellement, ces sous-populations sont diverses et hétérogènes et dotées de propriétés pro- et anti-inflammatoires apparemment redondantes. En pathologie, une augmentation du ratio entre CD16+ et CD16neg monocytes a été décrite en situation inflammatoire, suggérant un rôle des premières dans le développement et/ou l’amplification de l’inflammation. Parmi les monocytes non-classiques, des cellules capables de détecter des altérations de l’endothélium et ayant donc des propriétés spécifiques de surveillance du lit vasculaire ont été identifiées et caractérisées. Dans le but d’obtenir une meilleure définition des populations monocytaires et de les subdiviser en sous-populations où l’identification des fonctions de ces cellules serait plus accessible, je me suis attachée, dans ce travail de thèse, à analyser les différentes populations de monocytes humains circulants de manière aussi exhaustive que possible et avec les outils d’analyse biologiques et informatiques actuels. Les résultats, obtenus par l’analyse en cytométrie de flux de PBMC de 28 donneurs sains après marquage des cellules par vingt anticorps dirigés contre les molécules de surface, ont révélé l’existence d’une population de monocytes de plus grande taille. Ces « large » monocytes se subdivisent également en populations CD16neg et CD16+ (monocytes la14+16neg et la14+16+). Les monocytes restant ou « small » se composent de sm14+16neg largement majoritaires, de sm14+16+, et de sm14dim16+ auxquels se rajoutent des monocytes sm14lo16neg dont nous confirmons l’existence. L’expression des divers marqueurs sélectionnés a été faite par des méthodes d’analyse classiques, manuelles, ainsi que par l’utilisation d’algorithmes d’analyse non-supervisée. Les résultats ont montré les particularités d’expression propres à chaque population mais ont aussi indiqué que l’hétérogénéité phénotypique à l’intérieur de ces six populations de monocytes reste importante. Cependant, des profils d’expression qui sont partagés par plusieurs donneurs sains ont été identifiés. L’expression des molécules d’adhésion telles que CD49d, CD62L, CD162, ainsi que CD43 a été particulièrement utile pour cette identification. Quatre groupes phénotypiques majeurs ont ainsi été définis chez les 28 donneurs sains analysés
Monocytes are circulating leukocytes which characterization has long been difficult. Dissection of these cells into functional subpopulations in humans is still insufficient. Monocytes are however circulating precursors of several populations of dendritic cells and tissue macrophages, and play a prominent role in the development of immune response in steady state and pathology. At present, three monocyte subpopulations are described in humans: classical CD14+CD16neg, non-classical CD14dimCD16+ and intermediates CD14++CD16. Functionally, these subpopulations are diverse and heterogeneous and with apparently redundant pro - and anti-inflammatory properties. In pathology, an increase in the ratio of CD16 + to CD16neg monocytes has been described in inflammatory situation, suggesting a role of the former in the development and amplification of inflammation. Among the non-classical monocytes, cells that can detect changes in the endothelium and having then specific properties of vascular bed monitoring have been identified and characterized. In order to get a better definition of monocyte populations and break them down into subpopulations in which the identification of the cell functions would be more accessible, I endeavoured in this thesis work to analyse different populations of circulating human monocytes as comprehensively as possible and with state of the art analytical and computer tools. The results of flow cytometry analysis of PBMC from 28 healthy donors after cell staining with twenty antibodies directed against surface molecules revealed the existence of a population of monocytes of larger size

Les monocytes sont des leucocytes circulants, précurseurs de cellules dendritiques et de macrophages, dont le phénotype est hétérogène. L’association entre les sous-populations de monocytes humains décrites dans la littérature et les fonctions dans la réponse immunitaire reste difficile. Les différentes populations de monocytes humains sont classées en fonction de l’expression de molécules de surface CD14 et CD16. Trois populations ont ainsi été identifiées : les monocytes classiques CD14+ CD16neg, les monocytes non-classiques CD14dim CD16+ et les monocytes intermédiaires CD14+ CD16+. Les fonctions attribuées à ces populations sont diverses et enchevêtrées. En particulier, les propriétés pro- et anti-inflammatoires associées à ces populations de cellules sont redondantes et conflictuelles suivant les auteurs. En situation inflammatoire, l’augmentation de la fraction de monocytes CD16+ suggère leur implication dans le développement et/ou dans l’amplification de l’inflammation. L’objectif de cette thèse porte sur une meilleure définition des populations monocytaires afin de pouvoir les subdiviser en sous-populations dont les fonctions seraient mieux définies. Ce travail s’est inscrit dans un projet à long terme du laboratoire visant à l’analyse phénotypique aussi exhaustive que possible des monocytes, utilisant les outils d’analyses biologiques et informatiques actuels. Cela a permis de mettre en évidence l’existence d’une population de monocytes de plus grande taille. Ces « large » monocytes se subdivisent en populations CD16neg et CD16+ (respectivement nommées la14+16neg et la14+16+). Les monocytes communément analysés, de taille plus petite ou « small », se subdivisent comme attendu en trois sous-populations identifiées ici comme monocytes sm14+16neg largement majoritaire, sm14dim16+ et sm14+16+....Enfin, l’analyse des phénotypes de monocytes circulants en situation pathologique a été effectuée chez des grands brûlés. Ces patients ont une susceptibilité accrue à l’infection due, entre autres, à des réponses immunitaires innées déficientes. Les résultats obtenus chez 18 patients prélevés à l’admission et à 7 et 28 jours plus tard permettent d’identifier différents profils de modifications phénotypiques des monocytes et leur évolution en fonction de l’état clinique. Cette étude a également permis de mettre en évidence l’existence d’une population de cellules à forte granulosité, amplifiée de façon importante chez les patients et dont les fonctions sont en cours d’analyse
Monocytes are circulating leucocytes, precursors of dendritic cells and macrophages, whose phenotype is heterogeneous. The association between human monocyte subsets described in the literature and the functions in the immune response remains difficult.The different populations of human monocytes are classified according to the expression of surface markers CD14 and CD16. Three populations have thus been identified: the classical monocytes CD14+ CD16neg, the non-classical monocytes CD14dim CD16+ and the intermediate monocytes CD14+ CD16+. The functions assigned to these populations are diverse and entangled. In particular, the pro- and anti-inflammatory properties associated with these populations are redundant and conflicting depending on the authors. In an inflammatory situation, the increase of the CD16+ monocyte fraction suggests their involvement in the development and/or the amplification of inflammation.The aim of this thesis is to improve the definition of monocytes populations so that they can be subdivided into subpopulations whose functions are better defined. This work was part of a long term laboratory project which has the goal to the most comprehensive phenotypic analysis of monocytes, using current biological and computer analysis tools. This highlighted to demonstrate the existence of a larger monocyte population. These "large" monocytes are subdivided into CD16neg and CD16+ populations (respectively named la14+16neg and la14+16+). Monocytes commonly analyzed, of smaller size or "small", are subdivided as expected in three subpopulations identified here as monocytes sm14+16neg largely in the majority, sm14dim16+ and sm14+16+.Finally, the analysis of the phenotypes of circulating monocytes in pathological situation was conducted in burn victims. These patients have an increased susceptibility to infection due, among other things, to deficient innate immune responses. These results obtained in 18 patients taken at admission and at 7 and 28 days later permitted to identify different phenotypic modification profiles of monocytes and their evolution depending on the clinical state. This study has also highlighted the existence of a population of cells with high granulosity, greatly amplified in patients and whose functions are being analyzed

De grands bouleversements sont apparus au début du 21ème siècle dans la compréhension de la physiopathologie du cancer avec l'énoncé de la théorie de l'immunoédition complétant le concept de l'immunosurveillance. La communauté scientifique s'accorde désormais sur le fait que le système immunitaire et particulièrement les lymphocytes T (LT) CD8+ tiennent une place essentielle dans le contrôle de la croissance tumorale. Toutefois, par pression de sélection, la cellule tumorale développe des mécanismes de résistance aux attaques du système immunitaire, neutralisant ainsi l'effet cytotoxique des LT. Restaurer leurs fonctions antitumorales est une stratégie thérapeutique qui a fait ses preuves avec l'immunothérapie. Cependant, ces traitements ne sont pas toujours efficaces et peuvent être optimisés par une meilleure compréhension de l'immunité antitumorale. Dans ce but, nous nous sommes intéressés au déroulement de la réponse antitumorale des LT CD8+ en nous attardant sur les LT résidents mémoires (Trm) particulièrement efficaces contre la tumeur et au fort impact pronostic, qui pourrait être une cible thérapeutique pertinente. L'induction d'une réponse antitumorale efficace requière une présentation antigénique optimale conduisant à l'activation du LT CD8+ et à sa migration dans la tumeur via le réseau chimiokine/récepteur. Dans le premier travail, le récepteur de chimiokine CXCR6 a été identifié comme molécule de homing fortement exprimée par les LT CD8+ Trm du poumon. Sa chimiokine CXCL16 peut être sécrétée par les cellules présentatrices d'antigènes, les cellules épithéliales et tumorales mais le rôle de l'axe CXCR6/CXCL16 dans l'immunosurveillance des cancers n'est pas connu à ce jour. Pour en comprendre les mécanismes, des expériences de vaccinations antitumorales par voie intranasale (i.n.) réalisées dans des modèles de souris déficientes en CXCR6 ont permis de mettre en évidence l'impact de CXCR6 dans l'établissement d'une infiltration optimale en LT CD8+ spécifiques et Trm dans le lavage broncho-alvéolaire et au sein des tumeurs des voies aérodigestives supérieures. L'axe CXCR6/CXCL16 pourrait représenter un outil thérapeutique intéressant pour les vaccins anticancéreux ou pour les thérapies de transferts adoptifs de LT modifiés dont l'infiltration intra-tumorale est limitée. Les Trm ont la particularité d'exprimer des intégrines (CD103, CD49a) impliquées dans leur interaction avec le microenvironnement tumoral. Ils présentent un phénotype original microenvironnement-dépendant qui leur confère des avantages en termes d'activités cytotoxiques dans les tumeurs et expliquant leur impact pronostic favorable. Une meilleure connaissance de leur phénotype et de leurs mécanismes d'induction permettrait d'optimiser la réponse antitumorale. Le travail 2 s'est concentré sur l'étude de deux intégrines principales CD103 et CD49a dans les cancers pulmonaires par des techniques multiparamétriques d'immunofluorescence in situ et de cytométrie en flux. Les résultats montrent que leur expression expliquait l'infiltration des LT CD8+ et leur contact avec la cellule tumorale, en lien avec leur forte implication dans la survie des malades. Nos données suggérèrent également la possibilité d'un priming local pulmonaire nécessaire à l'induction du phénotype Trm par des modèles de vaccinations i.n. et d'un lien entre les structures lymphoïdes tertiaires et les Trm. Ces travaux ont montré l'importance de l'analyse de l'immunité locale avec les LT CD8+ Trm pour la compréhension de cette réponse antitumorale. Etudier le phénotype Trm a permis de mettre en lumière leur rôle crucial et leur potentiel comme cible thérapeutique. Une meilleure connaissance des mécanismes sous-jacents à l'induction des Trm permettra à terme de les cibler pharmacologiquement pour optimiser les thérapies et donc la survie des malades
With the immunoediting theory, new concept in the cancer physiopathology has appeared in the beginning of the 21st century. It is now established that the immune system and CD8+ T cells play a crucial role in tumor growth control. However, by selective pressure, the tumor cell develops mechanisms to avoid immune destruction and to inhibit T cells cytotoxicity. Reinvigorating antitumor functions is a well-proven therapeutic strategy with immunotherapy. Nevertheless, patients do not always respond to these treatments which could be optimized. In this context, we had studied antitumor response induction by focusing on CD8+ T cells and especially on resident memory T cells (Trm), new cytotoxic cells correlated with a good prognosis and which could be a relevant therapeutic target. A potent antitumor response requires an optimal antigenic presentation to prime CD8+ T cells and favor their migration into the tumor through chemokine network. In a first study, we identified a chemokine receptor CXCR6, highly expressed by lung CD8+ Trm. Its chemokine CXCL16 is produced by antigen presenting cells, epithelial and tumor cells, but the role of the CXCR6/CXCL16 axis in cancer immunosurveillance is not known yet. To understand its mechanisms, antitumor vaccinations strategies by intranasal (i.n.) route had been set up in CXCR6-deficient mice and had shown the role of CXCR6 in promoting the infiltration of specific CD8+ T cells and Trm in lung tissue and head and neck tumors. The CXCR6/CXCL16 axis could represent an interesting therapeutic tool for antitumor vaccines or adoptive cell transfer in which tumor infiltration is a challenge. Trm have the particularity to express integrins (CD103, CD49a) involved in the interaction with the tumor microenvironment. They exhibit an original and an heterogenous phenotype, microenvironment-dependent. Their phenotype is involved in their cytotoxic activities, highlighting their high prognostic impact and their potential to be a suitable therapeutic target. Better understanding Trm phenotype complexity and their induction mechanisms are crucial to further optimize antitumor response. The second work of this thesis focused on the expression of two main integrins CD103 and CD49a in lung cancer by an in situ multiparametric immunofluorescence technique and by flow cytometry. The results showed that their expression determine their contact with the tumor cells and their involvement in patient survival. Our data obtained by i.n. vaccination models and by tertiary lymphoid structures analysis suggest the possibility of a priming in the lung to induce the Trm phenotype. Our work shows the necessity of analyzing local immunity and CD8+ Trm T cells for a better understanding of antitumor response. Studying Trm phenotype has highlighted their crucial role and their potential to be a relevant therapeutic target. Identifying and targeting their mechanisms of induction might optimize therapies and patient's survival

L’allogreffe de cellules souches hématopoïétiques (CSH) est un des seuls traitements curatifs des hémopathies bénignes ou malignes et des déficits immunitaires primitifs. Cependant, les infections notamment virales ainsi que la réaction du greffon contre l’hôte comptent parmi les complications les plus fréquentes des allogreffes associées à une morbidité et une mortalité élevées. Les infections virales surviennent souvent en l’absence de reconstitution immunitaire spécifique dans un contexte d’immunosuppression liée à la GVHD elle-même ou à la prophylaxie ou au traitement de la GVHD. Les traitements médicamenteux anti-viraux préconisés présentent une efficacité inconstante dans ce contexte d’immunodéficience et ne sont pas dénués de toxicité. L’alternative thérapeutique prometteuse est l’immunothérapie adoptive cellulaire notamment celle qui consiste en l’injection de lymphocytes T spécifiques anti-viraux isolés par technique immunomagnétique (VSTs). Cependant, ces lymphocytes T peuvent être la cible des traitements immunosuppresseurs administrés pour la GVHD mais également par eux-mêmes être potentiellement la cause de la survenue ou de la réactivation d’une GVHD. Nous avons montré dans ce travail que l’efficacité des VSTs, qui repose sur leur expansion in vivo lors de la rencontre avec le virus circulant, est principalement permise par les sous-populations lymphocytaires les plus immatures, même si elles ne sont présentes qu’en faible proportion. Nous défendons dans ce travail le fait que l’efficacité des VST ainsi que leur persistance repose prioritairement sur la présence des sous-populations lymphocytaires T les plus immatures et ce quel que soit le degré de compatibilité HLA entre les VSTs et le receveur. De plus, leur sensibilité modérée aux corticoïdes, que nous avons étudiée in vitro, ne justifie pas la modulation de l’immunosuppression lors de l’injection des ADV-VSTs, comme observé in vivo dans le protocole clinique multicentrique de phase I/II que nous avons mené entre 2012 et 2015. En effet, ce protocole clinique ne rapporte aucune GVHD de novo après injection d’ADV-VSTs ; en revanche, la modulation de l’immunosuppression peut potentiellement être incriminée dans la réactivation de GVHD dans les semaines suivant l’injection des ADV-VSTs. La réalisation d’un essai comparatif de phase II permettra de prouver très clairement le rôle des VSTs dans la réactivation de GVHD
Hematopoietic stem cell transplantation (HSCT) is one of the only curative treatments for benign or malignant hematological diseases and primary immune deficiencies. However, viral infections and graft-versus-host disease (GVHD) are among the most frequent complications after HSCT associated with high morbidity and mortality. Viral infections often occur in the absence of specific immune reconstitution in the context of immunosuppression related to GVHD itself or to the prophylaxis or treatment of GVHD. The recommended anti-viral drug treatments have an inconsistent efficacy in this context of immunodeficiency and are not devoid of toxicity. The promising therapeutic alternative is adoptive immunotherapy, in particular the infusion of specific anti-viral T lymphocytes isolated by immunomagnetic technique (VSTs). However, these T lymphocytes may be targeted by immunosuppressive treatments administered for GVHD, but also may be the cause of the onset or reactivation of GVHD. We have shown in this work that the efficacy of VSTs, which is based on their in vivo expansion when they encounter the circulating virus, is mainly allowed by the most immature lymphocyte subpopulations, even in a small proportion. We argue in this work that the efficacy of VSTs and their persistence is mainly based on the presence of the most immature T lymphocyte subpopulations and this regardless of the degree of HLA compatibility between the VSTs and the recipient. Moreover, their moderate sensitivity to corticosteroids, which we have studied in vitro, does not justify the modulation of immunosuppression at the time of infusion of ADV-VSTs, as observed in vivo in the multicenter phase I / II clinical trial we conducted between 2012 and 2015. Indeed, this clinical trial does not report any de novo GVHD after ADV-VSTs infusion. On the other hand, modulation of immunosuppression may potentially be incriminated in the reactivation of GVHD within weeks of ADV-VST infusion. A Phase II comparative trial will bring the evidence of efficacy and will clearly determine the role of VSTs in the reactivation of GVHD

Investigation of the T-cell receptor (TCR) repertoire in patients with Relapsing-Remitting Multiple Sclerosis (RRMS) before and after two effective immunomodulatory treatments: Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT). The TCR repertoire was investigated on peripheral T-cell subpopulations (naive and memory) by TCRbeta sequencing and, therefore, with high-dimensional bioinformatic analysis.

Thesis summary The process of B cell lymphogenesis in swine remains uncertain. Some reports indicate that pigs belong to a group of animal that use ileal Peyers's patches (IPP) for the generation of B cells while others point to the possibility that the bone marrow is functional throughout life. The functional subpopulations of B cells in swine are also unknown. Together with other ruminants, and also birds, γδ T cells in swine may account for >70% of all T cells which is in apparent contrast with humans and mice. The purpose of this thesis was to address these discrepancies and unresolved issues. The results disprove the existing paradigm that the IPP is primary lymphoid tissue and that B cells develop in IPP in an antigen-independent manner. On the other hand, it shows that bone marrow is fully capable of B cell lymphogenesis and remains active at least for the same period of time as it had been speculated for the IPP. This thesis also identified functionally different subsets of porcine peripheral B cells, and shows that CD21 molecules can be expressed in differential forms. Finally, this thesis identifies two lineages of γδ T cells that differ in many functional and phenotype features. This finding may explain why γδ T cells constitute of minority of lymphocytes in circulation of humans and mice.

A unique class of T lymphocytes has been identified in the spleen of normal mice. This splenic T cell population, which expresses the αβ TCR associated with the CD3 complex yet lacks the CD4 and CD8 coreceptor molecules, constitutes approximately 0.1-0.3% of total nucleated cells and 17- 22% of CD4[sup]- CD8[sup]- HSA[sup]- Mac-1[sup]- cells in the normal murine spleen. Phenotypic analysis of splenic TCR αβ[sup]+ CD4[sup]- CD8[sup]- HSA[sup]- Mac-1[sup]- (DNαβT) cells demonstrated the expression of Lyt-1, Pgp-1, ICAM-1 and the transferrin receptor whereas minimal to no B220 or YE1/19 expression was observed. Amongst splenic DNαβT cells there is a higher frequency of V[sub]β8 usage in comparison with mature SP T cells. Similar to TCR αβ[sup]+ DN thymocytes, splenic DNαβT cells proliferate in the presence of IL-7 and this response is enhanced by the addition of IL-1. CD4[sup]+ and CD8[sup]+ SP T cells, in contrast do not respond to IL-7 alone or in combination with IL-1. Splenic DNαβT cells are considered to be mature based on the expression of the heat stable antigen. Furthermore, this splenic T cell subpopulation expresses a functional T cell receptor as suggested by their responsiveness to crosslinking of the TCR associated CD3 complex which is comparable to that of mature SP T cells. In contrast, splenic TCR γδ[sup]+ DN T cells were nonresponsive to anti-CD3 crosslinking despite the expression of the TCR/CD3 complex. The addition of IL-1, however, appeared to restore the responsiveness of TCR γδ[sup]+ DN towards the activation of the TCR/CD3 complex. The functional maturity of the splenic DNαβT cell population was confirmed by their ability to express cytokine specific mRNA. DNαβT cells constitutively express IFN[sub]γ message whereas IL-4 mRNA was induced by crosslinking of the TCR associated CD3 complex. SP T cells, in contrast, express messages for IFN[sub]γ and IL-4 as well as IL-2 only in response to anti-CD3 crosslinking. The above results support the hypothesis that splenic DNαβT cells represent a unique class of mature T cells which are related to the phenotypically similar cells found in the thymus yet are distinct from the classical type of mature T cell which expresses the SP phenotype.

RESUMO: As subpopulações linfocitárias, e em particular as células T reguladoras (Tregs), têm sido alvo de intensa investigação nos últimos anos. As Tregs são células reconhecidas pelo seu papel imunorregulador e responsáveis pela expressão de citocinas anti-inflamatórias como, por exemplo, a IL-10 e o TGF-β. A redução de níveis circulantes de Tregs tem sido associada a várias doenças auto imunes, particularmente em casos de doença activa. No caso da uveíte não-infecciosa (NIU), os resultados têm sido contraditórios, sendo necessários estudos adicionais para que se possa entender o papel definitivo da frequência e função das subpopulações de células Tregs na sua patogénese. O presente estudo tem como objectivo analisar as subpopulações linfocitárias, em especial a subpopulação Treg, bem como os perfis de citocinas inflamatórias e anti-inflamatórias circulantes de doentes com NIU activa, comparando-os com os de controlos normais. Há ainda a realçar a análise adicional de um subgrupo de doentes com NIU após tratamento e resolução da uveíte. Em doentes com uveíte infecciosa (IU) foram ainda analisadas amostras de humor aquoso (HAq), tendo em vista a caracterização de um perfil de citocinas. Em doentes com NIU activa não foram encontradas diferenças significativas nos níveis periféricos de Tregs (incluindo naïve e de memória) relativamente aos controlos. Em doentes avaliados ao longo do tempo, os nossos resultados mostraram um aumento das percentagens de Tregs totais e de memória, numa primeira avaliação, mas novamente sem diferença relativamente aos controlos após o tratamento. Ainda assim é interessante observar que este aumento inicial nas percentagens de Tregs totais e de memória não foi acompanhado de um aumento significativo na expressão de CD39, o que pode significar a perda da normal função supressora destas células. Relativamente aos níveis séricos de citocinas em doentes com NIU e controlos, os nossos resultados mostraram uma tendência para elevação da IL-17 no grupo com uveíte, bem como correlações positivas entre o ratio de citocinas inflamatórias (TNF-α + IFN-ɣ + IL-17) /anti-inflamatórias (IL-10 + TGF-β) e o ratio IL-17/IL-10 com as contagens absolutas de Tregs de memória. Para além disso, níveis séricos mais elevados de IL-17 mostraram uma associação com concentrações mais elevadas de Tregs naïve e de memória e ainda com níveis séricos mais elevados de TNF-α e de IFN-ɣ.Após tratamento, houve uma diminuição significativa nos níveis circulantes de IL-17 e de TNF-α no grupo de doentes com NIU em remissão, bem como uma redução significativa, entre avaliações, no ratio de citocinas inflamatórias/anti-inflamatórias. Por fim, no grupo de doentes com IU, os nossos resultados mostraram um aumento das concentrações de IL-10, TNF-α e IFN-ɣ no humor aquoso de doentes com infecção intraocular comprovada por vírus varicella zoster (VVZ). Já no sangue periférico, não se encontraram diferenças significativas entre perfis de citocinas de doentes e controlos saudáveis. Os nossos resultados não apoiam a utilização isolada da frequência periférica total de Tregs como um biomarcador de uveíte activa. Pensamos que este estudo é pioneiro na avaliação de subpopulações Treg naïve e de memória, e sua respectiva expressão CD39, no sangue periférico de doentes com NIU. No entanto, são necessários mais estudos para que o papel individual de cada uma destas subpopulações Treg na patogénese e eventual tratamento da NIU possa ser esclarecido. Relativamente aos perfis de citocinas em doentes com uveíte de causa infecciosa e não-infecciosa, este trabalho de investigação permite sublinhar a importância da citocina IL-17 na NIU activa e uma eventual associação entre níveis intraoculares elevados de IL-10 e a uveíte associada a infecção por VVZ. Estes resultados podem vir a contribuir para a identificação de biomarcadores de inflamação intraocular activa e na investigação de novos alvos terapêuticos.
ABSTRACT: Lymphocyte subpopulations, particularly regulatory T-cells (Tregs), have been extensively studied in the last few years. Tregs are T-cells with an immunosuppressive role, responsible for the expression of regulatory cytokines like IL-10 and TGF-β. A reduction of circulating Treg levels has been associated with various auto-immune diseases, especially in active disease. As for non-infectious uveitis (NIU), results have been conflicting, and further studies are needed to better understand the role of the frequency and function of total Treg and subsets in NIU. The present thesis aimed to analyze the lymphocyte subpopulations, especially Tregs, as well as inflammatory and anti-inflammatory cytokines in patients with active NIU and compare them with normal controls. One subgroup of NIU patients was further evaluated after treatment and uveitis resolution. Later on, in infectious uveitis (IU) patients, aqueous humour (AqH) samples were also analyzed for cytokine characterization. In active NIU, we found no significant differences in Treg levels (including naïve and memory subsets) between patients and controls. In NIU patients evaluated over time, our results showed an increased percentage of both total and memory Tregs in patients with active inflammation, without significant difference from controls after treatment. Nevertheless, it is interesting that this initial increase in total and memory Treg percentages was not associated with an increased CD39 expression, which may lead us to speculate whether these cells maintained their normal suppressive function. When comparing serum cytokine levels in NIU patients and controls, our results showed a tendency for IL-17A elevation in the NIU group and positive correlations between the inflammatory (TNF-α + IFN-ɣ + IL-17A) /anti-inflammatory (IL-10 + TGF-β) cytokine ratio and the IL-17/IL-10 ratio with the absolute counts of memory Tregs. We also found that higher IL-17A levels were associated with higher serum concentrations of memory and naïve Tregs as well as higher TNF-α and IFN-ɣ levels. After treatment, lower levels of IL-17A and TNF-α were present in patients in uveitis remission. Furthermore, the inflammatory/anti-inflammatory ratio also showed a significant reduction between evaluations. As for cytokine levels in IU patients, our results showed that while there were no significant differences between patients and controls regarding serum cytokine profiles, increased concentrations of IL-10, TNF-α, and IFN-ɣ in the AqH samples were found in patients diagnosed with varicella-zoster virus (VZV)- associated uveitis. Our results do not support the role of total Treg frequency alone as a uveitis biomarker, and since, to our knowledge, these are the first studies addressing the role of Treg naïve and memory subsets and their respective CD39 expression in the peripheral blood of NIU patients, further studies should be addressed to elucidate the possible role of each subset in NIU pathogenesis and treatment. As for cytokine profiles in infectious and non-infectious uveitis, our results highlight the importance of IL-17 in active NIU and the possible association between elevated intraocular IL-10 levels and VZV associated infection. We hope that this work may contribute to finding new biomarkers for active intraocular inflammation and new therapeutic targets for future research.