In the recent years, it has been well established that primary respiratory viral infection-induced lung resident memory CD8 T cells (TRM) characterized by the expression of integrins CD49a and CD103, as well as the early-activation marker CD69, constitute the first line of defense against reinfection. On the other hand, viral vector-based respiratory mucosal (RM) vaccination, as well as parenteral vaccination followed by airway luminal manipulation induce lasting and protective lung T cell immunity towards pulmonary tuberculosis (TB). However, it remains poorly understood whether and how these TB vaccination strategies induce TRM in the lung. As such, within this thesis we will investigate generation of lung CD8 TRM upon different TB vaccination strategies and the underlying mechanisms regulating establishment of such cells. Here using distinct models of replication-deficient adenoviral vector-based TB vaccination, we find that RM vaccination leads to generation of lung CD8 TRM identified by the expression of CD69, CD103, and very late activation Ag 1 (VLA-1). These TRM-associated molecules are acquired by CD8 T cells in distinct tissues. In this regard, VLA-1 is acquired during T cell priming in draining mediastinal lymph nodes (dMLNs) and the others acquired after T cells entered the lung. Once in the lung, Ag-specific CD8 TRM continue to express VLA-1 at high levels through the effector/expansion, contraction, and memory phases of T cell responses. We also reveal that VLA-1 is not required for homing of these cells to the lung, but it negatively regulates them in the contraction phase. Furthermore, VLA-1 has a negligible role in the maintenance of such cells in the lung. Separately, we have observed that while parenteral intramuscular vaccination alone does not induce lung CD8 TRM, subsequent RM inoculation of an Ag-dependent, but not a non-specific inflammatory agonist induces lung CD8 TRM. Such generation of lung CD8 TRM needs CD4 T cell help. These findings not only fill the current knowledge gap, but also hold important implications in developing effective vaccination strategies towards mucosal intracellular infectious diseases such as acquired immunodeficiency syndrome (AIDS), TB and herpes virus infection. / Thesis / Doctor of Philosophy (PhD)

Résumé : L'activation des lymphocytes T CD8+ naïfs par un antigène nécessite deux signaux. Le premier signal est médié par le récepteur des cellules T (TCR) à la suite de son interaction avec le complexe majeur d'histocompatibilite (CMH) de classe I. Le deuxième signal est délivré par l'interaction des molécules de co-stimulation avec leur récepteur spécifique retrouvé sur les cellules présentatrices d'antigène professionnelles. Toutefois en réponse à la pression homéostatique, comme dans le cas de la lymphopénie, les lymphocytes T naïfs sont soumis à une prolifération sans stimulation antigénique par un processus appelé «prolifération induite par la lymphopénie (LIP)». LIP des lymphocytes T CD8+ naïfs nécessite IL-7, et un peptide du soi présenté pas le CMH de classe I. Des travaux récents de notre laboratoire ont montré que les cytokines homéostatiques IL-7 et IL-15 peuvent agir en synergie avec IL-21 et induire une prolifération antigène-indépendante des cellules CD8+ naives. Cette activation "sensibilise" les cellules à proliférer en réponse à une concentration sub-optimale de l'antigène spécifique au TCR. Également les cellules pré-stimulées avec des cytokines produisent davantage de cytokines effectrices telles que le TNFa et l'IFNy; et possèdent un potentiel plus élevé de cytotoxicity après stimulation avec un antigène spécifique. Dans mon projet de maitrise, j'ai étudié la possibilité que la pré-stimulation par les cytokines pouvait être un mécanisme important par lequel des cellules T CD8+ naïves qui sont potentiellement autoréactives soient stimulées pour provoquer une maladie auto-immune telle que le diabète de type 1. J'ai démontré cela en utilisant un modèle de souris transgéniques. Ces souris produisent des cellules T CD8+ qui expriment le TCR transgénique P14 (cellules PI4) , lesquelles reconnaissent un peptide antigénique dérivé de l'antigène glycoprotein (GP33) du virus de la chorioméningite lymphocytaire (LCMV). Nous avons montré qu'une pré-stimulation avec PIL-21 en présence de l'IL-7 ou l'IL-15, les cellules P14 acquièrent la capacité de répondre vigoureusement à des ligands peptidiques modifiés qui montrent une faible activité agoniste envers les cellules P14 naives. Les cellules P14 préstimulées qui sont re-stimulées avec des ligands peptidiques modifiés montrent des puissantes fonctions effectrices telles que la cytotoxicité et la production des cytokines effectrices TNFa et IFNy. Ces cellules induisent le diabète de type 1 lorsqu'elles ont été transférées dans des souris qui expriment l'antigène GP LCMV sous le contrôle du promoteur de l'insuline dans les îlots de Langerhans. Dans l'ensemble, nos résultats montrent que l'IL-15 et l'IL 21 produites durant une réponse inflammation chronique pourraient pré-stimuler des cellules T CD8+ potentiellement autoréactives, conduisant ainsi à des maladies auto-immunes. // Abstract : The activation of naive CD8[superscript +]T cells by an antigen requires two different signals. The first signal is mediated via the T cell receptor (TCR) following its interaction with the peptide presented on class-I major histocompability complex (MHC-I) molecules. The second signal is delivered via the co-stimulatory receptors upon recognition of their ligands on antigen presenting cells. However, in response to homeostatic pressure, as in lymphopenia, naive T cells undergo proliferation without antigenic stimulation through a process referred to as lymphopenia-induced proliferation (LIP). LIP of naive CD8[superscript +] T cells requires IL-7 and a self peptide presented by MHC-I, which implies that TCR signaling is needed for LIP of naive CD8[superscript +]T cells. Recent work from our laboratory has shown that with the homeostatic cytokines IL-7 and IL-15 synergize with IL-21 to induce antigen-independent proliferation of naive CD8[superscript +]T cells. Moreover, this cytokine-driven, antigen-independent proliferation "sensitizes" or "primes" naive CD8[superscript +] T cells to undergo robust proliferation in response to limiting concentrations of their cognate antigens. Cytokine-primed CD8[superscript +]T cells also abundantly produce effector cytokines, such as TNF? and IFN? and display a potent CTL activity following stimulation by antigen when pre-stimulated. In my project, I have investigated whether cytokine-induced priming could be an important mechanism by which potentially autoreactive naive CD8[superscript +]T cells are stimulated to cause autoimmune disease using a TCR transgenic mouse model of autoimmune type 1 diabetes (T1D). These mice harbor CD8[superscript +]T cells that express transgenic P14 TCR (P14 cells), which recognizes an antigenic peptide derived from the glycoprotein antigen (GP33) of lymphocytic choriomeningitis virus (LCMV). We show that, following priming with IL-21 in the presence of IL-7 or IL-15, P14 cells gain the ability to respond robustly to modified peptide ligands that possess weak agonistic activity towards unprimed P14 cells. Furthermore, cytokine-primed P14 cells stmulated with weak TCR ligands displayed potent effector functions such as cytotoxicity and production of effector cytokines, TNF? and IFN?. These cells also induced T1D when adoptively transferred to mice that expressed the LCMV GP antigen under the control of the insulin promoter in the islets. Collectively, our findings show that IL-15 and IL-21 produced during chronic inflammatory conditions could cause priming of potentially autoreactive CD8[superscript +]T cells, leading to autoimmune diseases. [symboles non conformes]

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Células dendríticas (DCs) são componentes centrais da imunidade celular, responsáveis pelo priming de linfócitos T naïve. A polarização de linfócitos T é restrita aos sinais fornecidos durante a apresentação do antígeno. Além desses sinais, a origem e natureza de DCs que induzem diferentes perfis de linfócitos T não é totalmente compreendida. Foi investigada a capacidade de células Natural Killer (NK) de modular estágios iniciais da diferenciação de monócitos em DCs e de impactar na sua função de primar e polarizar linfócitos T naïve. DCs derivadas de monócitos pré-co-cultivados com células NK favorecem o priming de linfócitos T CD8 do tipo Tc1/Tc17, com potente capacidade de produção de IFN-γ. Este fenômeno foi dependente de interações longas via NKp30 e da maquinaria citotóxica de células NK desencadeada nas etapas inicias da sua interação com monócitos. Esta interação pode ter implicações na compreensão da imunidade mediada por linfócitos T CD8 e pode ser explorada para imunoterapia em que a produção de IFN-γ por células T CD8 é necessária ou exacerbada. / Dendritic cells (DCs) are central components of cellular immunity, responsible for the priming of naïve T cells. The polarization of T cells is restricted to signals provided during antigen presentation. Besides such signals, the origin and nature of DCs that induce different T cell profiles is not fully understood. The ability of natural killer cells (NK) to modulate early stages of monocytes differentiation into DCs and to impact on DCs function to prime and polarize naïve T cells was investigated. DCs derived from monocytes co-cultured with NK cells support the priming of type Tc1/Tc17 CD8 T cells with potent IFN-γ production capacity. NK cell-mediated cytotoxicity triggered at early stages of NKp30-dependent long-lasting monocytes-NK-cells interactions, mediated the mechanism by which this phenomenon occurred. This interaction may have implications in the understanding of CD8 T cell-mediated immunity and can be exploited for immunotherapy in which IFN-γ production by CD8 T cells is required or exacerbated.

La ténascine-C (TNC), protéine de la matrice extracellulaire, favorise la progression tumorale et la métastase par des mécanismes pas totalement élucidés. J’ai utilisé un nouveau modèle de progression tumorale de la glande mammaire basé sur une approche de greffe de cellules tumorales orthotopiques syngéniques et j’ai ainsi identifié la TNC comme un régulateur important de la croissance tumorale. L’expression concomitante de la TNC par les cellules de l’hôte et les cellules tumorales induit une régression de la tumeur en induisant une signature de présentation d’antigène. Cette signature a été corrélée avec une meilleure survie des patientes atteintes de cancer du sein. D’autre part, la TNC exprimée par les cellules tumorales induit également l’expression de CXCL12 au sein de la tumeur, piégeant les lymphocytes CD8+ dans des travées de matrice enrichies avec le CXCL12 lié à la TNC. L’inhibition du récepteur de CXCL12, le CXCR4 provoque une régression tumorale qui s’accompagne d’un afflux important de lymphocytes T CD8+ et d’une augmentation de la mort cellulaire au sein du lit tumorale. La séquestration des lymphocytes T cytotoxiques par la TNC dans les travées de matrice peut avoir une implication importante dans le développement et l’utilisation des nouvelles immunothérapies ciblant l’activité des cellules effectrices du système immunitaire. / The extracellular matrix molecule tenascin-C (TNC) promotes tumor progression and metastasis by poorly understood mechanisms. I used a novel breast progression model based on a syngeneic orthotopic tumor cell grafting approach and identified TNC as an important regulator of tumor growth. I document that TNC promotes the battle between tumor regression and growth, where combined expression of tumor cell- and host-derived TNC induces tumor cell rejection. Tumor cell-derived TNC may elicit regression by induction of an antigen presenting signature (APS) expressed by the host, which correlates with better breast cancer patient survival. Tumor-cell derived TNC also triggers CXCL12 expression, thereby causing trapping of CD8+ T cells in the surrounding TNC matrix tracks. TNC binds CXCL12, and combined TNC/CXCL12 attracts and immobilizes CD8+ T cells. Inhibition of the CXCL12 receptor CXCR4 causes tumor regression that is accompanied by massive infiltration of CD8+ T cells and cell death inside the tumor cell nests. Altogether,TNC-triggered CXCL12 signaling may dampen CD8+ T cell function where physical trapping of CD8+ T cells in the TNC matrix may have implications for immune cell therapies. Our results and new tumor model, offer novel opportunities for preclinical cancer research and cancer patient therapy, by triggering the “good” and blocking the “bad” actions of TNC. In particular, overcoming the immune suppressive action of TNC, through inhibition of CXCR4, could be a useful approach.

Die Ätiologie und Pathogenese autoimmuner Lebererkrankungen sind nur unvollständig verstanden. Bei der primär sklerosierenden Cholangitis bzw. bei der autoimmunen Hepatitis sind Cholangiozyten der größeren Gallengänge und Hepatozyten die Zielzellen der Autoimmunreaktion in der Leber. Mausmodelle sind zur Analyse initialer pathophysiologischer Prozesse notwendig und tragen zum besseren Verständnis der immunologischen Vorgänge in der Leber bei. Mit Hilfe transgener Mauslinien, die das Modellantigen Ovalbumin gewebespezifisch in den Cholangiozyten (ASBT-OVA) oder in den Hepatozyten (TF-OVA) exprimieren, sowie adoptiven Transfers antigenspezifischer CD4+ und CD8+ T-Zellen wurden Untersuchungen zur Antigenpräsentation, T-Zell-Aktivierung und Toleranzinduktion in der Leber durchgeführt. Die Expression von Ovalbumin in Cholangiozyten resultierte in einer Aktivierung der CD8+ T-Zellen in der Leber und den Lymphknoten. Im Gegensatz dazu ignorierten naive CD4+ T-Zellen das Antigen und wurden nicht aktiviert. Die Expression von Ovalbumin in Hepatozyten resultierte in einer vollständigen Aktivierung der CD8+ T-Zellen zu Effektorzellen über Kreuzpräsentation durch professionelle antigenpräsentierende Zellen (APZ) in der Leber. Diese Aktivierung war transient und selbst-limitiert. Die Induktion von CD4+Foxp3+ regulatorischen T-Zellen trug entscheidend zur Limitierung der induzierten Autoimmunität und Kontrolle der Expansion von antigenspezifischen CD8+ T-Zellen bei. Naive CD4+ T-Zellen benötigten die Aktivierung durch APZ in einem anderen Organ, bevor sie in die Leber relokalisierten und wiesen keinen Effektorphänotyp auf. Beide Modelle repräsentieren nicht die chronische Eigenschaft humaner autoimmuner Lebererkrankungen, ermöglichen jedoch Untersuchungen zum besseren Verständnis der Rolle verschiedener T-Zell-Populationen in der Pathogenese autoimmuner Lebererkrankungen sowie der Antigenpräsentation und Aktivierung von T-Zellen durch hepatisches Antigen. / Aetiology and pathogenesis of autoimmune liver diseases are still incompletely understood. Cholangiocytes of the larger bile ducts and hepatocytes are the target structures of autoimmune reactions in the liver in primary sclerosing cholangitis and autoimmune hepatitis, respectively. Mouse models are necessary to analyse initial pathophysiological processes and contribute to a better understanding of immunological processes in the liver. With the help of transgenic mouse strains, in which the model antigen ovalbumin is expressed specifically in the cholangiocytes (ASBT-OVA) or in hepatocytes (TF-OVA), as well as the adoptive transfer of antigen specific CD4+ and CD8+ T cells, antigen presentation, T cell activation and tolerance induction in the liver, were analyzed. Expression of ovalbumin in cholangiocytes resulted in activation of CD8+ T cells in the liver and lymph nodes. In contrary, naïve antigen specific CD4+ T cells ignored the antigen expressed by cholangiocytes and were not activated. Expression of ovalbumin in hepatocytes resulted in complete activation of CD8+ T cells to become effector cells by crosspresentation depending on professional antigen presenting cells (APCs) in the liver. This activation was transient and self-limiting. Induction of CD4+Foxp3+ regulatory T cells played a crucial role in limiting autoimmunity and controlling the expansion of antigen specific CD8+ T cells in the liver. By contrast, naïve CD4+ T cells required activation by professional APCs in a different organ before relocating to the liver and did not display an effector phenotype. Both models do not represent the chronic characteristics of human autoimmune liver diseases, but help to gain a better understanding regarding the role of specific T cell populations in the pathogenesis of autoimmune liver diseases, as well as regarding antigen presentation and activation of T cells by hepatic antigen.

Konventionelle CD8 T-Zellen im intestinalen Epithel unterscheiden sich von T-Zellen gleicher Spezifität in anderen Organen. In dieser Arbeit sollten Mechanismen identifiziert werden, welche die Migration dieser T-Zellen in intestinale Gewebe bestimmen und/oder welche die funktionelle Adaption der T-Zellen an die mukosale Umgebung beeinflussen. Hierfür wurden Mäuse mit einem rekombinanten Listeria monocytogenes Stamm (LmOVA) infiziert, OVA-spezifische CD8 T-Zellen aus der Milz und dem intestinalem Epithel isoliert, und die mRNA-Expressionsprofile dieser Zellen mittels einer Mikroarray-Analyse verglichen. Eine Gruppe von NK-Rezeptoren zeigte auf OVA-spezifischen CD8 T-Zellen aus der intestinalen Mukosa eine geringere Expression. Dieser Unterschied war nach einer oralen Infektion wesentlich stärker ausgeprägt, als nach einer intravenösen Infektion. Die Präsenz der natürlichen Darmflora hatte nur einen sehr geringen Einfluss auf diese organspezifisch unterschiedliche NK-Rezeptor-Expression. Untersuchungen in transgenen CD4dnTGFbetaRII Mäusen wiesen auf TGFbeta als ein entscheidendes Element für die niedrigere NK-Rezeptor-Expression auf T-Zellen in der Darmmukosa hin. Die zweite Fragestellung war die, ob nach einer Lm-Infektion die primäre Aktivierung naiver CD8 T-Zellen und die Re-Aktivierung von Gedächtnis-T-Zellen auch außerhalb sekundärer lymphoider Organe stattfinden kann. Hierfür wurden Mäuse mit der immunmodulatorischen Substanz FTY720 behandelt und splenektomiert. Eine FTY720-Behandlung beeinträchtigte nach iv und nach ig Infektion weder die Ausbreitung der Lm, noch die Fähigkeit der Mäuse zu einer Elimination der Bakterien. Orale Infektionen sowie höher dosierte iv Infektionen führten auch in FTY720-behandelten und zusätzlich splenektomierten Mäusen zu einer T-Zell-Akkumulation und Proliferation in nicht-lymphoiden Organen. Nach einer niedrig dosierten iv Lm-Infektion wurden dagegen in FTY720-behandelten und splenektomierten Mäusen keine OVA-spezifischen T-Zell-Antworten ausgelöst. Insbesondere die Milz schien hier für die T-Zell Aktivierung und Proliferation erforderlich zu sein. Auch für eine Sekundärantwort nach einer iv Infektion mit LmOVA waren lymphoide Gewebe für die Ausbildung einer effektiven T-Zell-Antwort essentiell. / Conventional CD8 T cells in the intestinal epithelium differ from T cells with identical antigen specificity in other organs. One aim of this study was, to identify mechanisms, which determine gut-tropism of these T cells or which influence the functional adaptation of these cells to the mucosal environment. For this purpose, mice were infected with a recombinant Listeria monocytogenes strain (LmOVA). OVA-specific CD8 T cells were isolated from spleen and intestinal epithelium and the mRNA-expression profiles of these cells were compared in a microarray-analysis. One group of NK-receptors were substantially less expressed on Lm-specific CD8 T cells from the intestinal mucosa than on corresponding cells from the spleen. This difference was much more pronounced following oral infection than following iv infection. The presence of the natural gut-flora only slightly influenced the organ-specific NK-receptor expression in CD8 T cells. Experiments with transgenic CD4dnTGFbetaRII mice point to TGFbeta as a decisive factor for the low NK-receptor expression on T cells from the gut mucosa. In the second part of this study it was investigated, whether, following Lm infection, priming of naive CD8 T cells and re-activation of memory T cells could occur outside of secondary lymphoid organs. Mice were treated with the immunomodulatory drug FTY720 and splenectomized. Treatment with FTY720 did neither diminish bacterial dissemination into spleen, liver and MLN, nor impaired the ability of the mice to control the bacteria and to eradicate them from these organs. Oral infection and high dose iv infection led to T cell accumulation and proliferation in nonlymphoid organs of FTY720-treated and additionally splenectomized mice. Following low dose iv infection with LmOVA, no OVA-specific T cell responses were induced in FTY720-treated and additionally splenectomized mice. Especially the spleen seemed to be important for T cell activation and proliferation under these conditions. Also following a secondary iv infection with LmOVA, lymphoid tissues were essential for the generation of an effective T cell response.

L’infection par le VIH induit une activation immunitaire chronique qui est suspectée d’être un des moteurs de la pathogénèse du SIDA. L’identification des mécanismes de contrôle de cette activation immunitaire, ainsi qu’une meilleure compréhension du contrôle spontané de l’infection chez certains patients, sont des étapes essentielles vers la conception de thérapies innovantes. Nous avons utilisé le modèle d’infection du macaque cynomolgus (Macaca fascicularis) par le virus de l’immunodéficience simienne (SIV) pour étudier ces deux points fondamentaux de la physiopathologie de l’infection.Il est proposé que l’induction précoce de l’activation immunitaire chronique soit une conséquence de la surexpression des gènes induits par les interférons (ISG) en réponse aux interférons de type I (IFN-I). Ces IFN-I ( et ) sont notamment produits par les cellules dendritiques plasmacytoïdes (pDC) en réponse aux virus. Notre premier objectif a été d’étudier la dynamique de production des IFN-I et celle des pDC durant l’infection, en parallèle de celle de l’activation immunitaire chronique et de la virémie. Nos résultats montrent que les pDC sont activées dans les tissus et qu’elles sont responsables de la production d’IFN-I observée transitoirement au cours de la primo-infection. La dynamique des pDC (apoptose, activation, renouvellement) entraîne un épuisement de la capacité des pDC à produire de l’IFN-I, qui pourrait rendre compte à la fois de l’altération fonctionnelle des pDC et de l’arrêt de la production d’IFN-I. De manière surprenante, ce contrôle de la production d’IFN-I n’est pas suivi d’un contrôle de la surexpression des ISG, ce qui suggère l’existence d’autres mécanismes inducteurs des ISG et souligne l’origine multifactorielle de l’activation immunitaire chronique.Dans une seconde étude, nous avons analysé l’impact d’une déplétion in vivo des lymphocytes exprimant le CD8 chez des animaux contrôlant spontanément la réplication virale sur le long terme. Quatre des cinq animaux de l’étude n’expriment pas de complexe majeur d’histocompatibilité (CMH) précédemment associé au contrôle, et aucun d’entre eux ne présente de forte réponse LT CD8. La déplétion transitoire des cellules CD8 entraîne chez quatre des contrôleurs une augmentation transitoire de la virémie qui se stabilise ensuite à des valeurs similaires aux niveaux pré-déplétion lors du retour des cellules CD8+. Un de ces animaux contrôle sa virémie avant la restauration des LT CD8. Chez le cinquième animal, la déplétion des CD8 n’a pas été accompagnée d’une élévation de virémie. Globalement, le contrôle de la virémie après l’élévation transitoire n’a pas été accompagné d’une augmentation de leur fonction antivirale. En revanche, une expansion et une activation des LT CD4 consécutive à la déplétion des CD8 ont été remarquées et corrélées positivement avec la virémie plasmatique. Ces résultats suggèrent que les réponses LT CD8 ne sont pas les principales responsables du contrôle à long terme de la virémie chez ces animaux. Dans notre modèle, d’autres mécanismes, tels qu’un réservoir de virus limité ou un meilleur contrôle de l’activation immunitaire, semblent participer à ce phénotype de contrôle.En conclusion, ces résultats éclairent la contribution des pDC, des IFN-I et des LT CD8 dans la physiopathologie du VIH, et permettent de proposer un nouveau modèle d’étude des mécanismes immunologiques précoces mis en place chez les patients contrôleurs de la virémie à long terme. / HIV infection induces a chronic immune activation, which is suspected to be a driving force in the pathogenesis of AIDS. Identifying control mechanisms of this immune activation, and a better understanding of the spontaneous control observed in some patients, are essential steps towards the development of innovative therapies. We used the model of cynomolgus macaques (Macaca fascicularis) infected by the simian immunodeficiency virus (SIV) to study these two fundamental fields of HIV pathogenesis.It is proposed that early induction of chronic immune activation is a consequence of an interferon-induced genes (ISG) overexpression in response to type I interferons (IFN-I). These I IFN (α and β) are preferentially produced by plasmacytoid dendritic cells (pDC) in response to the virus. Our first objective was to study the dynamics of pDC and IFN-I production during infection, together with chronic immune activation and viremia analysis. Our results indicate that pDCs are activated in tissues and are responsible for the transient IFN-I production observed during primary infection. The dynamics of pDCs (apoptosis, activation, renewal) induces an impaired IFN-I production by pDC, which could account both the functional defect of pDCs and the arrest of IFN-I production. Surprisingly, control of IFN-I production is not followed by down-regulation of ISG, which suggests the existence of other mechanisms that induce ISG and emphasizes the multifactorial origin of chronic immune activation.In a second study, we analyzed the impact of a in vivo CD8 T cells depletion in animals which spontaneously control viral replication in the long term. Importantly, four of the five animals in the study do not express major histocompatibility complex (MHC) previously associated with control, and none of them display strong CD8 response. The transient depletion of CD8 cells results in four controllers in a transient increase in viremia, which then stabilizes at values similar to pre-depletion levels when CD8+ cells come back. One of these animals controls their viremia before the restoration of CD8. In the fifth animal, CD8 depletion was not followed by a rise in viremia. Overall, the control of viremia after the transient increase was not associated to an increase of the antiviral function of CD8 T cells. In contrast, CD4 T cells expansion and activation were noticed and positively correlated to plasma viremia. These results suggest that CD8 responses are not the main cause of long-term control of viremia in these animals. In our model, other mechanisms, such as smaller reservoir or better control of immune activation, seem to be involved in this controller phenotype.In conclusion, these results shed light on the contribution of pDCs, IFN-I and CD8 T cells in the pathogenesis of HIV, and allow us to propose a new model for studying early immunological mechanisms in HIV controllers.

Baixas doses de agentes desmetilantes do DNA (DNMTi) tem efeitos antitumorais por induzirem parada do ciclo celular e aumentarem a imunogenicidade se tornando mais visível para o sistema imune. Crescentes evidências sugerem que o tratamento com DNMTis podem aumentar a sinalização imune nas células tumorais por meio do mecanismo de mimetismo viral bem como aumento da expressão de antígenos associados ao tumor, destacando assim, o seu potencial clínico na combinação com outras terapias. Enquanto os efeitos dos DNMTis têm sido na maior parte investigados nas células tumorais, os efeitos sobre o sistema imune ainda permanecem desconhecido, especialmente sobre baixas doses. Assim sendo, nosso objetivo foi investigar os efeitos da administração de agentes desmetilantes do DNA sobre a função efetora dos linfócitos T CD8 e seu papel frente a resposta antitumoral. Para isso, injetamos células tumorais CT26 na concentração de 5x105 por animal no dia 0. Cada grupo de animais foi tratado ou não com 5-AZA-CdR (0,5 mg/kg). Os camundongos foram tratados diariamente intraperitonealmente do dia 5 ao dia 9. Como controle adicional, em um grupo de animais, foi utilizado anticorpo neutralizante para depletar células T CD8. O tratamento diminuiu o tamanho tumoral, aumentou o infiltrado de células T CD8 e aumentou a capacidade de produção de citocinas por essas células. Em adição, todos esses efeitos foram abolidos na ausência das células T CD8. Em nossos ensaios de estimulação ex vivo e de imunização com adenovírus, o tratamento gerou aumento da ativação celular, aumento da produção de citocinas, diminuição da proliferação e aumento de morte celular. Por fim em nossos ensaios de citotoxicidade, o tratamento aumentou a capacidade citotóxica dessas células. Nossos resultados mostram que o tratamento in vivo com DNMTis em tumores de camundongos levou ao aumento da infiltração de células T CD8 e que a diminuição do tamanho tumoral é dependente da ação das células T CD8. Uma vez que observamos a capacidade efetoras das células T CD8, o tratamento levou ao aumento da produção de citocinas e aumento da indução de morte celular de células alvo. Em resumo, nossos resultados demonstram que o tratamento com DNMTis aumenta a resposta antitumoral e aumentam o potencial citotóxico das células T CD8. / Low doses of the DNA methylation inhibitor (DNMTis) on cancer cells has durable antitumorigenic effects by inducing cell cycle arrest and immune-modulatory properties that increases their visibility by the immune system. Growing evidence suggest that DNMTis can upregulate immune signaling in cancer cells through viral mimicry and upregulation of tumour associated antigens, highlighting clinical potential to combine with immunotherapy. While the effects of DNMTis have been mostly studied in cancer cells, their effects on the immune system remains vastly unknown specially at clinically relevant low doses. Therefore, we aimed to investigate the effect of in vivo administration of DNA demethylating inhibitor on the effector function of CD8+ T cells and antitumor immune response. We injected 5x105 CT26 cells at day 0 in three group of mice (n=5 mice per group). Each group of mice were either mock treated or treated with Decitabine (5-AZA-CdR or DAC, at a dosage of 0,5 mg/kg). The mice were treated intraperitoneally daily from days 5 to 9. As a control, in one group of mice, it was used neutralizing antibody to deplete CD8+ T cells. The treatment significantly decreased tumor burden and in the absence of CD8+ T cells this effect was abolished. The treatment led to increase in CD8+ T cells infiltration into the tumor microenvironment and enhance secretion of effector cytokines. In addition, all these effects were abolished in the lack of CD8 T cells. Furthermore, using ex vivo and adenovirus immunization, the treatment led to enhance of cellular activation, decrease of proliferation and enhance of cell death. In addition, using cytotoxic assays, the treatment enhanced the killing capability of these cells. to characterize the CD8 T cells, we used ex vivo stimulation protocols. Remarkably, our results show that treatment in vivo with DNMTi in established murine tumors model led to increase CD8+ T cell infiltration and the decrease in tumor burden is dependent of CD8 T cells. Once the effector capacities of these CD8+ T cells were assessed, DNMTi treatment increased their effector cytokine production and increased killing of target cells. Altogether, our results show that treatment of CD8+ T cells enhance antitumor immune response and their cytolytic potential.

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