Interest in cellular immunotherapy has increased with the recognition of the pivotal role that dendritic cells (DC) play in the adaptive immune system. The preparation of DC to present tumour antigens and subsequent induction of tumour specific T cells have been widely documented. This thesis studied the ability of cord blood (CB) stem cells to differentiate into functional CD34+DC, followed by the optimisation of electroporation of RNA into these cells. Total RNA derived from a leukaemic cell line and a primary human leukaemic sample was electroporated into CD34+DC DC and we were able to generate anti-leukaemic cytotoxic T lymphocytes (CTL). The CTL specifically targeted leukaemia but not normal cells. While the in vitro data showed promising results of the CTL specificity, a NOD-SCID model of human ALL was established to allow the CTL to be tested in vivo. We established a reproducible model of human ALL in NOD-SCID mouse using four primary human ALL samples. The adoptively transferred anti-leukaemic CTL into the ALL bearing NOD-SCID mice showed that ALL engraftment was significantly delayed. However, the addition of total RNA loaded CD34+DC DC did not enhance the in vivo CTL effect. Lastly, by dissecting the CTL response, we found that the polyclonal CTL were targeting survivin, HM1.24 and CT-7 antigens. The CTL clones generated from these polyclonal CTL showed high specificity for leukaemia but not normal cells. In conclusion, these preliminary data support the use of total RNA electroporated CD34+DC as a means of inducing anti-leukaemic CTL, and have demonstrated the efficacy of the CTL in a NOD-SCID model of ALL. This study has also provided insight into the polyclonal CTL response and future studies will likely continue along this path.

O sistema nervoso simpático (SNS) inerva a maioria dos órgãos linfoides e durante situações de estresse, por meio da liberação da noradrenalina de seus ramos eferentes, emitem sinais capazes de modular as repostas imunes. Nossa hipótese foi de que esta via poderia alterar a função de células dendríticas (DCs) derivadas de monócitos humanos, uma vez que receptores adrenérgicos já foram demonstrados em DCs murinas e pelo fato de que as estas células são chave na iniciação de respostas imunes adaptativas, bem como indutoras de tolerância. Desta forma, DCs diferenciadas a partir de monócitos sanguíneos provenientes de doadores saudáveis tratadas com ligantes adrenérgicos foram analisadas quanto a expressão de marcadores de membrana, atividade fagocítica, apresentação antigênica em ensaio de reação mista de linfócitos, expressão gênica de marcadores de diferenciação e ativação, bem como produção de citocinas. Os resultados revelaram que as DCs apresentam transcritos apenas para o adrenoceptor β2, e esta expressão é similar à de macrófagos, mas inferior a de linfócitos. A análise dos marcadores fenotípicos de membrana, atividade fagocítica, apresentação antigênica e produção de citocinas não mostraram alterações nas células tratadas com agonistas adrenérgicos. No entanto, o tratamento com ligantes adrenérgicos foi capaz de alterar a expressão dos genes CD40, CD80, CD83, CXCL1, TGFB1, FCGR3A, CCR7 e CCL5 em DCs e macrófagos estimulados com LPS ou TNF-α. Embora os efeitos dos ligantes adrenérgicos não tenham sido fortemente evidenciados nos testes realizados, os resultados sugerem que pequenas alterações podem ser provocadas pela ligação das catecolaminas em DCs, sugerindo que estas possam ser moduladas pelo SNS, modificando as respostas imunes / The sympathetic nervous system (SNS) innervates most of the lymphoid organs and during stress situations, by releasing norepinephrine from its efferent branches, emit signals capable of modulating immune responses. Our hypothesis was that this pathway could alter the function of human monocyte-derived dendritic cells (DCs), since adrenergic receptors have already been demonstrated in murine DCs, and by the fact that these cells are key in initiating adaptive immune responses as well as tolerance inducers. Thus, differentiated DCs from blood monocytes from healthy donors treated with adrenergic ligands were analyzed for expression of membrane markers, phagocytic activity, antigenic presentation in a mixed lymphocyte reaction assay, gene expression of differentiation and activation markers and cytokine production. The results revealed that DCs present transcripts only for the β2 adrenoceptor, and this expression is similar to that observed in macrophages, but lower than what it is found in lymphocytes. The analysis of phenotypic membrane markers, phagocytic activity, antigenic presentation and cytokine production did not revealed any changes in the cells treated with adrenergic agonists. However, treatment with the adrenergic ligands was able to alter the expression of CD40, CD80, CD83, CXCL1, TGFB1, FCGR3A, CCR7 and CCL5 genes in DCs and macrophages stimulated with LPS or TNF-α. Although the effects of adrenergic ligands have not been strongly demonstrated in the tests performed, the results suggest that small changes can be caused by the binding of catecholamines in DCs, suggesting that they can be modulated by the SNS, modifying immune responses.

O sistema nervoso simpático (SNS) inerva a maioria dos órgãos linfoides e durante situações de estresse, por meio da liberação da noradrenalina de seus ramos eferentes, emitem sinais capazes de modular as repostas imunes. Nossa hipótese foi de que esta via poderia alterar a função de células dendríticas (DCs) derivadas de monócitos humanos, uma vez que receptores adrenérgicos já foram demonstrados em DCs murinas e pelo fato de que as estas células são chave na iniciação de respostas imunes adaptativas, bem como indutoras de tolerância. Desta forma, DCs diferenciadas a partir de monócitos sanguíneos provenientes de doadores saudáveis tratadas com ligantes adrenérgicos foram analisadas quanto a expressão de marcadores de membrana, atividade fagocítica, apresentação antigênica em ensaio de reação mista de linfócitos, expressão gênica de marcadores de diferenciação e ativação, bem como produção de citocinas. Os resultados revelaram que as DCs apresentam transcritos apenas para o adrenoceptor β2, e esta expressão é similar à de macrófagos, mas inferior a de linfócitos. A análise dos marcadores fenotípicos de membrana, atividade fagocítica, apresentação antigênica e produção de citocinas não mostraram alterações nas células tratadas com agonistas adrenérgicos. No entanto, o tratamento com ligantes adrenérgicos foi capaz de alterar a expressão dos genes CD40, CD80, CD83, CXCL1, TGFB1, FCGR3A, CCR7 e CCL5 em DCs e macrófagos estimulados com LPS ou TNF-α. Embora os efeitos dos ligantes adrenérgicos não tenham sido fortemente evidenciados nos testes realizados, os resultados sugerem que pequenas alterações podem ser provocadas pela ligação das catecolaminas em DCs, sugerindo que estas possam ser moduladas pelo SNS, modificando as respostas imunes / The sympathetic nervous system (SNS) innervates most of the lymphoid organs and during stress situations, by releasing norepinephrine from its efferent branches, emit signals capable of modulating immune responses. Our hypothesis was that this pathway could alter the function of human monocyte-derived dendritic cells (DCs), since adrenergic receptors have already been demonstrated in murine DCs, and by the fact that these cells are key in initiating adaptive immune responses as well as tolerance inducers. Thus, differentiated DCs from blood monocytes from healthy donors treated with adrenergic ligands were analyzed for expression of membrane markers, phagocytic activity, antigenic presentation in a mixed lymphocyte reaction assay, gene expression of differentiation and activation markers and cytokine production. The results revealed that DCs present transcripts only for the β2 adrenoceptor, and this expression is similar to that observed in macrophages, but lower than what it is found in lymphocytes. The analysis of phenotypic membrane markers, phagocytic activity, antigenic presentation and cytokine production did not revealed any changes in the cells treated with adrenergic agonists. However, treatment with the adrenergic ligands was able to alter the expression of CD40, CD80, CD83, CXCL1, TGFB1, FCGR3A, CCR7 and CCL5 genes in DCs and macrophages stimulated with LPS or TNF-α. Although the effects of adrenergic ligands have not been strongly demonstrated in the tests performed, the results suggest that small changes can be caused by the binding of catecholamines in DCs, suggesting that they can be modulated by the SNS, modifying immune responses.

Το Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ) αποτελεί νόσημα με διαταραχή σε επίπεδο αρχέγονου αιμοποιητικού κυττάρου (stem cell) που χαρακτηρίζεται από μη αποδοτική αιμοποίηση και κυτταροπενίες του περιφερικού αίματος που περιλαμβάνουν μία ή περισσότερες αιμοποιητικές σειρές. Διάφορες ανοσολογικές διαταραχές των ασθενών με ΜΔΣ, όπως, αυξημένη ευαισθησία σε βακτηριακές λοιμώξεις, αυτοάνοσα φαινόμενα και υψηλή συχνότητα κακοηθειών του λεμφικού ιστού, υποδεικνύουν αδυναμία των ασθενών με ΜΔΣ για ανοσολογική απάντηση, οι αιτίες των οποίων παραμένουν άγνωστες μέχρι σήμερα. Τα Δενδριτικά Κύτταρα (ΔΚ) είναι κύτταρα του ανοσολογικού μηχανισμού που προέρχονται από το μυελό των οστών. Ως αντιγονοπαρουσιαστικά κύτταρα (APC), είναι εξειδικευμένα για τη πρόσληψη, επεξεργασία, μεταφορά και παρουσίαση του αντιγόνου στα Τ λεμφοκύτταρα. Στη παρούσα μελέτη πραγματοποιήθηκε ανάλυση διαφορετικών ποσοτικών και λειτουργικών παραμέτρων των ΔΚ από ασθενείς με Μυελοδυσπλαστικό Σύνδρομο, in vivo ή in vitro. Αρχικά διερευνήθηκε ο αριθμός, ο φαινότυπος, η ικανότητα ενδοκύττωσης και η αλλογενής διεγερτική δυνατότητα των ΔΚ, προερχόμενων από μονοκύτταρα του περιφερικού αίματος (ΜοΔΚ) ασθενών με ΜΔΣ και υγιών μαρτύρων, σε διαφορετικά στάδια διαφοροποίησης. Τα μονοκύτταρα των ασθενών με ΜΔΣ χαρακτηρίστηκαν από μειωμένη ικανότητα διαφοροποίησης σε ΔΚ, λόγω του μειωμένου αριθμού των διαφοροποιημένων κυττάρων και τη χαμηλή έκφραση του CD1a αντιγόνου επιφανείας. Τα ΜοΔΚ των ΜΔΣ ασθενών παρουσίασαν χαμηλή έκφραση του υποδοχέα της μανόζης και μειωμένη ικανότητα ενδοκύττωσης. ΜοΔΚ των ΜΔΣ ασθενών επέδειξαν μειωμένη απάντηση ύστερα από διέγερση με TNF-α, καθώς η έκφραση των CD83, CD80 και CD54 αντιγόνων και η αλλοδιεγερτική ικανότητα ήταν μειωμένη, ενώ η επίδραση με LPS είχε ως αποτέλεσμα να εμφανίσουν φαινοτυπικά χαρακτηριστικά και ικανότητα διέγερσης των Τ-κυττάρων, όμοια με τα ΜοΔΚ των φυσιολογικών μαρτύρων. Σε δύο από τους ασθενείς με σύνδρομο 5q-, σχεδόν όλα τα μονοκύτταρα και τα ΜοΔΚ περιείχαν τη χρωμοσωμική διαταραχή, υποδηλώνοντας την προέλευσή τους από τον παθολογικό κλώνο. Στη συνέχεια διερευνήθηκε το δυναμικό πολλαπλασιασμού και διαφοροποίησης των CD34+ προγονικών κυττάρων του μυελού ασθενών με ΜΔΣ σε δενδριτικά κύτταρα (CD34-ΔΚ) σε υγρή καλλιέργεια παρουσία κυτοκινών. Παράλληλα, έγινε ανάλυση των κυκλοφορούντων ΔΚ περιφερικού αίματος στους ίδιους ασθενείς. Τα CD34+ προγονικά κύτταρα παρουσίασαν χαμηλή δυνατότητα ανάπτυξης ΔΚ in vitro, καθώς ο αριθμός των παραγόμενων ΔΚ ανά CD34+ κύτταρο ήταν χαμηλότερος συγκριτικά με τα δείγματα των υγιών μαρτύρων. Παρά την αυξημένη απόπτωση των προγονικών κυττάρων του μυελού των ΜΔΣ ασθενών, η επιβίωση και ο πολλαπλασιασμός των CD34+ κυττάρων στην καλλιέργεια, δεν συσχετίστηκε με την απόπτωση και αποτελεί αξιοσημείωτη παρατήρηση. Φαινοτυπικά, τα CD34-ΔΚ των ΜΔΣ ασθενών δεν διέφεραν από τα ΔΚ που παρήχθησαν από τα CD34+ κύτταρα του μυελού των φυσιολογικών μαρτύρων καθώς επέδειξαν όμοια έκφραση των CD83, CD80, CD40, HLA-DR και CD54 αντιγόνων. Κυτταροεπιλεγμένα CD1a+ κύτταρα ασθενών είχαν όμοια διεγερτική ικανότητα αλλογενών Τ κυττάρων με τα CD34-ΔΚ των φυσιολογικών ατόμων. Το ποσοστό των κυκλοφορούντων μυελοειδών- και πλασματοκυτταροειδών- ΔΚ στους ασθενείς με ΜΔΣ ήταν σημαντικά μειωμένο συγκριτικά με τους υγιείς μάρτυρες. Στους ασθενείς με 5q έλλειψη, τόσο τα CD34-ΔΚ, όσο και τα ΔΚ του αίματος, είχαν τη χρωμοσωμική ανωμαλία. Τα παραπάνω αποτελέσματα υποδηλώνουν ότι η διαδικασία παραγωγής δενδριτικών κυττάρων από το μυελό (‘δενδριτοποίηση’) των ασθενών με ΜΔΣ, είναι μέρος της κλωνικής διαταραχής με αποτέλεσμα την μη αποδοτική παραγωγή ΔΚ από τα προγονικά κύτταρα του μυελού και το χαμηλό ποσοστό των κυκλοφορούντων πρόδρομων ΔΚ. Όλες οι ΔΚ υποομάδες προέρχονται από τον παθολογικό κλώνο και χαρακτηρίζονται από ποσοτικές και ποιοτικές ανωμαλίες. Το σύνολο αυτών των διαταραχών που παρατηρήθηκαν στα ΔΚ πολύ πιθανόν να συμβάλει στη διαταραγμένη ανοσολογική απάντηση έναντι παθογόνων οργανισμών, στην επιβίωση και στην επικράτηση του παθολογικού κλώνου, όπως επίσης και στην εμφάνιση αυτοάνοσων φαινομένων, που παρατηρούνται στους ασθενείς με ΜΔΣ. / Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis and blood cytopenias involving one or several myeloid lineages. Various immune disturbances in MDS such as increased susceptibility to bacterial infections, autoimmune phenomena and high incidence of lymphoid malignancies reveal an underlying defect of the immune response in MDS patients, the reasons for which still remain unclear. Dendritic cells (DCs) are bone marrow derived cells. As the most potent antigen presenting cells (APC), they are specialized for the uptake, processing, transport and presentation of Ag to T cells. In the present study different quantitative and functional parameters of DCs in patients with MDS were analyzed either in vivo or in vitro. The number, phenotype, endocytic ability, and allostimulatory capacity of DCs derived from peripheral blood monocytes (MoDCs) were investigated in patients with MDS and healthy controls at different stages of differentiation using the maturation stimuli-TNF-á and LPS. Monocytes in MDS showed low potential to differentiate into DCs, as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of Mannose receptor and reduced endocytic capacity. When stimulated with TNF-á, MoDCs obtained from MDS patients showed a diminished response with low CD83, CD80 and CD54 expression and allostimulatory capacity, whereas in the presence of LPS MDS-MoDCs acquired phenotypic characteristics and ability to stimulate T-cells similar to MoDCs derived from controls. In two patients with 5q- syndrome the vast majority of both monocytes and MoDCs were positive for the 5q deletion, suggesting that they originate from the malignant clone. Second, we investigated the potential of bone marrow CD34+ progenitors in patients with MDS to proliferate and differentiate into DCs in a liquid cytokine supplemented culture system and also analyzed the status of blood DC subsets in those patients. CD34+ progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34+ cell was significantly lower compared to controls. Interestingly, although the increased apoptotic level of bone marrow progenitors in MDS, the survival and proliferation of CD34+ cells in culture was not correlated to the degree of apoptosis. Phenotypically the MDS CD34-DCs did not differ from DCs obtained from normal BM CD34+ cells, exhibiting similar expression of CD83, CD80, CD40, HLA-DR, and CD54. FACsorted CD1a+ cells from MDS patients were as efficient stimulators of allogeneic T cells as normal CD34-DCs. The percentage of both circulating DC subsets, MDCs and PDCs in MDS patients was extremely diminished compared to controls. In cases with the 5q deletion both CD34-DCs and blood DCs harbor the cytogenetic abnormality. The results indicate that “dendritopoiesis” in MDS is affected by the transformation process resulting in ineffective production of DCs from bone marrow progenitors with low circulating blood precursors. All DC subsets were derived from the malignant clone and exhibited quantitative and qualitative abnormalities. This constellation of DCs defects probably contribute to the defective immune response against pathogens, escape and expansion of the malignant clone, as well as autoimmune phenomena, observed in MDS patients.

L'eczéma allergique de contact (EAC) est une pathologie inflammatoire cutanée de plus en plus fréquente. Il s’agit d’une sensibilisation vis-à-vis de substances chimiques, appelées haptènes, qui sont en contact répété avec la peau. A l’heure actuelle, le pouvoir sensibilisant d’une molécule est évalué principalement grâce à des modèles animaux. Cependant, dans un contexte européen exigeant le développement de méthodes alternatives, la compréhension et la reproduction in vitro des mécanismes de l’EAC permettent le développement de nouvelles stratégies. Le but de ce travail, réalisé sur des cellules dendritiques (DCs) et la lignée humaine U937, est de mettre en évidence les événements précoces de la signalisation intracellulaire à l’origine de l’activation des DCs induite par des allergènes, et notamment par le composé mercurique thimerosal. Les résultats obtenus démontrent que l’induction d’un stress oxydant par les allergènes est un mécanisme induit précocement. L’utilisation d’antioxydants montre que ce mécanisme participe de manière directe à l’initiation du processus d’activation des DCs (expression du CD86 et sécrétion d’IL-8) et de l’apoptose. Le stress oxydant, caractérisé par une production d’ERO associée à une chute du potentiel membranaire mitochondrial et une déplétion en glutathion intracellulaire, est un acteur majeur de la signalisation induite par les allergènes et notamment dans la réponse induite par le thimerosal et le DNCB. Plus particulièrement, ce travail démontre le rôle des groupements thiols dans l’initiation de la transduction du signal aboutissant à l’activation des DCs. Par ailleurs, une signalisation calcique, dépendante du stress oxydant, a également été mise en évidence en réponse aux allergènes. Ces résultats mettent en valeur l’importance de l’étude de la réactivité des allergènes vis-à-vis des groupements thiols et de la nécessité de tenir compte du potentiel oxydant (rédox) des haptènes ainsi que du métabolisme cellulaire dans la mise en place de modèles prédictifs in vitro. / Allergic Contact Dermatitis (ACD) resulting from skin sensitization is a frequent inflammatory skin disease linked to the use of chemicals, called haptens. At this time, the sensitizing potential of a new chemical is evaluated on animal models. However, new European legislation requires alternative methods for skin sensitization. In this context, a better knowledge of ACD and the capacity to reproduce in vitro its mechanisms lead to the development of new alternative methods. The aim of this study performed with human dendritic cells (DCs) and the human cell line U937 was to determine the early events involved in dendritic cell activation induced by contact sensitizers and especially by the mercury compound thimerosal. Data show that oxidative stress induced by sensitizers is an early signaling event leading to DC activation (expression of CD86 and IL-8 release) and to apoptosis. Using antioxidants, our data show that oxidative stress, characterized by ROS production in correlation with the depletion of the mitochondrial membrane potential and of intracellular glutathione, is a key player in signal transduction induced by sensitizers, especially in the response of DCs towards thimerosal and DNCB. More specifically, these studies demonstrate that thiol groups play a direct role in the initiating events leading to DC activation. In addition, calcium influx was detected in DCs exposed to sensitizers, in correlation with oxidative stress. These data highlight the great interest in the development of a hapten-protein binding assay based on thiol groups and the necessity to better understand the redox status of chemicals as well as cell metabolism for predicting skin sensitization.

Lors d’une infection par un pathogène, des lymphocytes T CD8+ naïfs (LTn) spécifiques de l’antigène sont activés, prolifèrent et se différencient en LT effecteurs (LTe). Les LTe produisent différentes cytokines et acquièrent une activité cytotoxique menant à l’élimination du pathogène. Seulement 5 à 10 % des LTe survivront et se différencieront en LT mémoires (LTm), qui sont capables de répondre plus rapidement lors d’une seconde infection par le même pathogène, contribuant au succès de la vaccination. Toutefois, la compréhension de l’ensemble des mécanismes régulant le développement des LTe et des LTm demeure incomplète. Afin de mieux comprendre les signaux requis pour la différenciation des LT CD8+ lors de la réponse immune, nous avons posé deux hypothèses. Nous avons d’abord proposé que différentes cellules présentatrices d’antigène (CPA) fournissent différents signaux au moment de la reconnaissance antigénique influençant ainsi le devenir des LT CD8+. Vu leur potentiel d’utilisation en immunothérapie, nous avons comparé la capacité d’activation des LT CD8+ par les lymphocytes B activés via le CD40 (CD40-B) et les cellules dendritiques (CD). Nous avons montré que l’immunisation avec des CD40-B induit une réponse effectrice mais, contrairement à l’immunisation avec des CD, pratiquement aucun LTm n’est généré. Les LTe générés sont fonctionnels puisqu’ils sécrètent des cytokines, ont une activité cytotoxique et contrôlent une infection avec Listeria monocytogenes (Lm). Nous proposons qu’une sécrétion plus faible de cytokines par les CD40 B ainsi qu’une interaction plus courte et moins intime avec les LT CD8+ comparativement aux CD contribuent au défaut de différenciation des LTm observé lors de la vaccination avec les CD40-B. Ensuite, nous posé l’hypothèse que, parmi les signaux fournis par les CPA au moment de la reconnaissance antigénique, la voie de signalisation Notch influence le développement des LTe, mais aussi des LTm CD8+ en instaurant un programme génétique particulier. D’abord, grâce à un système in vitro, le rôle de la signalisation Notch dans les moments précoces suivant l’activation du LT CD8+ a été étudié. Ce système nous a permis de démontrer que la voie de signalisation Notch régule directement l’expression de la molécule PD-1. Ensuite, grâce à des souris où il y a délétion des récepteurs Notch1 et Notch2 seulement chez les LT CD8+ matures, un rôle de la voie de signalisation Notch dans la réponse immune des LT CD8+ a été démontré. Nos résultats démontrent que suite à une infection avec Lm ou à une immunisation avec des CD, la signalisation Notch favorise le développement de LTe, exprimant fortement KLRG1 et faiblement CD127, destinés à mourir par apoptose. Toutefois, la signalisation Notch n’a pas influencé la génération de LTm. De façon très intéressante, l’expression des récepteurs Notch influence la production d’IFN- en fonction du contexte d’activation. En effet, suite à une infection avec Lm, l’absence des récepteurs Notch n’affecte pas la production d’IFN- par les LTe, alors qu’elle est diminuée suite à une immunisation avec des CD suggérant un rôle dépendant du contexte pour la voie de signalisation Notch. Nos résultats permettent une meilleure compréhension des signaux fournis par les différentes CPA et de la voie de signalisation Notch, donc des mécanismes moléculaires régulant la différenciation des LT CD8+ lors de la réponse immunitaire, ce qui pourrait ultimement permettre d’améliorer les stratégies de vaccination. / Following an infection with a pathogen, antigen-specific naive CD8+ T lymphocytes (Tn) will proliferate and differentiate into effector (Te) cells. Those Te cells will produce different cytokines and acquire a cytotoxic activity, leading to pathogen clearance. Only 5 to 10 % of Te cells will survive and differentiate into memory CD8+ T lymphocytes (Tm) able to respond rapidly following a second encounter with the same pathogen, contributing to the success of vaccination. However, the mechanisms regulating Te and Tm cells development remain incompletely understood. To better understand the signals required for CD8+ T lymphocytes during an immune response, we proposed two hypotheses. First, we propose that different antigen presenting cells (APCs) can deliver different signals to CD8+ T lymphocytes at the time of priming leading to different outcome. Given their potential for use in immunotherapy, we compared the ability of CD40 activated B lymphocytes (CD40-B) and dendritic cells (DCs) to activate CD8+ T lymphocytes. We have shown that CD40-B cell immunisation leads to an effector response but very few Tm cells are generated compared to DC immunisation. The Te cells generated following CD40-B cell immunisation are functional because they secrete cytokine, are cytotoxic and control a Listeria monocytogenes (Lm) infection. We propose that CD40-B cells secrete less cytokines and interact during shorter period of time with the CD8+ T lymphocytes, without engulfment, contributing to the decreased Tm generation observed following immunisation with CD40-B cells. Second, among the signals provided by APC at the time of CD8+ T lymphocyte priming, we have hypothesised that the Notch signalling pathway influences Te and Tm cell differentiation by inducing a particular genetic program. Using an in vitro system, we first studied the role of the Notch signalling pathway in the hours following CD8+ T lymphocyte priming. We demonstrated that Notch signalling directly regulates PD-1 expression. Then, studying mice where Notch1 and Notch2 receptor genes are deleted only in mature CD8+ T lymphocytes, we characterised the role of the Notch signalling pathway on Te and Tm differentiation during an immune response. Our results show that following Lm infection or a DC immunisation, the Notch signalling pathway promotes the differentiation of short lived effector cells Te cells (KLRG1highCD127low) meant to die by apoptosis. However, the Notch signalling pathway did not influence the generation of CD8+ Tm cells. Most interestingly, IFN- regulation by the Notch signalling pathway depends on the activation context. Indeed, following Lm infection, lack of Notch receptors does not impact IFN- secretion by Te cells while it is significantly decreased following a DC immunisation suggesting a context dependant role for the Notch signalling pathway. Our findings provide a better understanding of the key signals provided by APC as well as the Notch signalling pathway, and thus the molecular mechanisms leading to CD8+ lymphocyte effector and memory generation which is crucial as this knowledge may ultimately lead to improved vaccination.