Portal cd4+ and cd8+ t lymphocyte correlate to intensity of interface hepatitis in chronic hepatitis C

BACKGROUND: The pathogenesis of chronic hepatitis C is still a matter of debate. CD4+ and CD8+ T lymphocytes (TL) are typically observed within the portal and periportal spaces of affected livers, but their functional role in hepatitis C progression has not been fully elucidated. METHODS: CD4+ and CD8+ TL were quantified by immunohistochemistry in portal and periportal spaces of 39 liver biopsies from patients with chronic hepatitis C. They were associated to demographic data, histological parameters, laboratory findings of patients and hepatitis C genotypes. RESULTS: There was high numbers of CD4+ and CD8+ TL from which the density of CD4+ T was higher than CD8+ TL in portal and periportal spaces. CD4+ and CD8+ TL were directly correlated to intensity of interface hepatitis. CD8+ TL correlated to serum enzyme levels. CONCLUSION: The high numbers of CD4+ and CD8+ TL in portal and periportal spaces and their correlation to interface hepatitis suggest that hepatitis C evolution depends on the action of intrahepatic T lymphocytes, lending support to the notion of an immune-mediated mechanism in the pathogenesis of chronic hepatitis C.

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

Distinct mechanisms control human naive and antigen-experienced CD8+ T lymphocyte proliferation.

Human Ag-specific CD8(+) T lymphocytes are heterogeneous and include functionally distinct populations. In this study, we report that at least two distinct mechanisms control the expansion of circulating naive, memory, and effector CD8(+) T lymphocytes when exposed to mitogen or Ag stimulation. The first one leads to apoptosis and occurs shortly after in vitro stimulation. Susceptibility to cell death is prominent among primed T cell subsets, and it is inversely correlated with the size of the ex vivo Bcl-2(high) population within these subsets. Importantly, the Bcl-2(high) phenotype is associated to the proportion of responsive CD8(+) T cells, independently of their differentiation stage. The second one depends on the expression of newly synthesized cyclin-dependent kinase inhibitor p16(INK4a) that occurs in a significant fraction of T cells that had been actively cycling, leading to their cell cycle arrest upon stimulation. Strikingly, accumulation of p16(INK4a) protein preferentially occurs in naive as opposed to primed derived T lymphocytes and is not related to apoptosis. Significant levels of p16 are readily detectable in a small number of ex vivo CD8(+) T cells. Our observations reveal that activation-induced p16 expression represents an alternative process to apoptosis, limiting the proliferation potential of activated naive derived T lymphocytes.

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival.

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.

Increased rates of CD4+ and CD8+ T lymphocyte turnover in simian immunodeficiency virus-infected macaques

Defining the rate at which T cells turn over has important implications for our understanding of T lymphocyte homeostasis and AIDS pathogenesis, yet little information on T cell turnover is available. We used the nucleoside analogue bromodeoxyuridine (BrdUrd) in combination with five-color flow cytometric analysis to evaluate T lymphocyte turnover rates in normal and simian immunodeficiency virus (SIV)-infected rhesus macaques. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labeling and elimination rates of both CD4+ and CD8+ BrdUrd-labeled cells were increased by 2- to 3-fold as compared with normal controls. In normal and SIV-infected animals, the rates of CD4+ T cell BrdUrd-labeling and decay were closely correlated with those of CD8+ T cells. The elimination rate of BrdUrd-labeled cells was accelerated in both naive and memory T lymphocytes in SIV-infected animals. Our results provide direct evidence for increased rates of both CD4+ and CD8+ T cell turnover in AIDS virus infection and have important implications for our understanding of T cell homeostasis and the mechanisms responsible for CD4+ T cell depletion in AIDS.

Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.

Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions

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Early Effects on T lymphocyte Response to Iron Deficiency in Mice. Short Communication

Iron deficiency is a common nutritional disorder, affecting about 30% of the world population. Deficits in iron functional compartments have suppressive effects on the immune system. Environmental problems, age, and other nutrient deficiencies are some of the situations which make human studies difficult and warrant the use of animal models. This study aimed to investigate alterations in the immune system by inducing iron deficiency and promoting recuperation in a mouse model. Hemoglobin concentration, hematocrit, liver iron store, and flow cytometry analyses of cell-surface transferrin receptor (CD71) on peripheral blood and spleen CD4+ and CD8+ T lymphocyte were performed in the control (C) and the iron-deficient (ID) groups of animals at the beginning and end of the experiment. Hematological indices of C and ID mice were not different but the iron stores of ID mice were significantly reduced. Although T cell subsets were not altered, the percentage of T cells expressing CD71 was significantly increased by ID. The results suggest that iron deficiency induced by our experimental model would mimic the early events in the onset of anemia, where thymus atrophy is not enough to influence subset composition of T cells, which can still respond to iron deficiency by upregulating the expression of transferrin receptor.

High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors.

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.

Mechanisms regulating the proliferative potential of human CD8+ T lymphocytes overexpressing telomerase.

In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division, eventually leading to a state of cellular senescence. Ectopic expression of telomerase results in the extension of their replicative life spans without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Using CD8+ T lymphocyte clones overexpressing telomerase, we investigated the molecular mechanisms that regulate T cell proliferation. In this study, we show that early passage T cell clones transduced or not with human telomerase reverse transcriptase displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in human telomerase reverse transcriptase-transduced cells with extended life spans. These cells, despite maintaining high expression levels of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. Strikingly, late passage T cells overexpressing telomerase accumulated the cyclin-dependent inhibitors p16Ink4a and p21Cip1 that have largely been associated with in vitro growth arrest. We conclude that alternative growth arrest mechanisms such as those mediated by p16Ink4a and p21Cip1 still remained intact and regulated the growth potential of cells independently of their telomere status.

Study of the mechanisms regulating the proliferative potential of human CD8+T lymphocytes over-expressing telomerase reverse transcriptase (hTERT)

R��sum�� La plupart des cellules issues du sang ont une dur��e de vie limit��e. Dans les cellules somatiques humaines, y incluant les lymphocytes T, la taille des t��lom��res diminue progressivement �� chaque division cellulaire, pouvant aboutir �� des instabilit��s chromosomiques. L'expression ectopique du g��ne de la transcriptase r��verse de la t��lom��rase (hTERT) dans les cellules restaure l'activit�� de la t��lom��rase, et permet un rallongement de leur vie r��plicative. Malgr�� l'absence de signes caract��ristiques de transformation, nous ne savons pas encore si les cellules somatiques qui surexpriment hTERT sont physiologiquement indiscernables des cellules normales. Certaines ��tudes r��centes proposent que la t��lom��rase joue plusieurs r��les additionnels dans d'autres ph��nom��nes biologiques tels que la r��paration de l'ADN, la survie et la croissance des cellules. Dans notre ��tude, nous avons utilis�� des clones issus de lymphocytes T cytotoxiques surexprimant la t��lom��rase afin d'��tudier les m��canismes mol��culaires qui r��glent leur prolif��ration et leur s��nescence. Nous avons montr�� que les ��jeunes �� cellules T exprimant ou non hTERT r��v��lent des taux de croissance identiques suite �� des r��ponses de stimulation induites par des mitog��nes. De plus, aucun changement global dans leur expression des g��nes n'a pu ��tre mis en ��vidence. Curieusement, nous avons observ�� des r��ponses r��duites dans la prolif��ration des cellules transduites avec la t��lom��rase qui pr��sentaient une ��longation des t��lom��res et une dur��e de vie prolong��e. Ces cellules, malgr�� le maintien d'un niveau ��lev�� de l'expression de g��nes impliqu��s dans la progression du cycle cellulaire, ont ��galement montr�� une expression accrue de plusieurs g��nes trouv��s en commun avec nos lymphocytes T vieillissants n'exprimant pas de t��lom��rase. En particulier, les cellules ayant une dur��e de vie prolong��e gr��ce �� l'expression de la t��lom��rase accumulaient ��galement certains inhibiteurs du cycle cellulaire tels que p16Ink4a et p21Cip1, associ��s �� l'arr��t de la croissance cellulaire. En r��sum��, nos r��sultats indiquent la pr��sence fonctionnelle de m��canismes alternatifs pouvant contr��ler la croissance r��plicative de ces cellules; ils sont donc encourageants dans l'optique d'une utilisation �� moindre risque de lymphocytes T ��immortalis��s �� �� des fins th��rapeutiques pour traiter les tumeurs malignes ou les infections. Summary Most mature blood cells have a finite life span. In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division eventually leading to chromosomal instability. Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene in cells restores telomerase activity and results in the extension of their replicative life span. Despite lack of transformation characteristics, it is yet unknown whether somatic cells that over-express telomerase are biologically and physiologically indistinguishable from normal cells. Recent data suggest that telomerase might mediate additional functions in DNA repair, cell survival and cell growth. Using CD8+ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T cell proliferation and senescence. Here we show that early-passage T cell clones transduced or not with hTERT displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in hTERT-transduced cells with elongated telomeres and extended life span. These cells, despite maintaining high expression level of genes involved in cell cycle division and progression, also showed increased expression of several genes associated with normal aging T lymphocytes. In particular, late passage T cells over-expressing telomerase accumulated the cyclin-dependent inhibitors p16INK4a and p21CIP1 that have largely been associated with in vitro growth arrest. Whether tumor-reactive CD8+ T cells that ectopically express telomerase could now be used for adoptive transfer therapy in cancer patients remains unclear at this point. Nevertheless, our results regarding the safe and effective use of hTERT-transduced lymphocytes are encouraging, since they indicate that alternative growth arrest mechanisms such as p 16 and p21 are still functional in these cells and regulate to some extend their growth potential.

Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions.

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8+ T-lymphocyte subsets, RA+CCR7-27+28+ and RA+CCR7-27+28-, in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon gamma, and tumor necrosis factor alpha. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage.

Caract��risation de DKK1 comme antig��ne tumoral et manipulation des lymphocytes T CD8 : utilisation de la voie de Wnt en immunoth��rapie du cancer

L���immunoth��rapie tumorale �� m��diation cellulaire est un traitement qui utilise le syst��me immunitaire des patients afin d���induire une r��ponse des lymphocytes T CD8+ (T CD8+) contre la tumeur. Cette r��ponse est produite suite �� la reconnaissance des antig��nes par les T CD8+. Ces cibles sont appel��es antig��nes tumoraux (TAA) et d��finies comme des prot��ines exprim��es par les cellules canc��reuses mais absentes des tissus normaux. Par une approche bio-informatique, notre laboratoire a identifi�� Dickkopf-1 (DKK1), une prot��ine inhibitrice de la voie de Wnt, comme un TAA potentiel. Une immunoth��rapie �� m��diation cellulaire efficace requiert l���identification de TAA candidats pertinents. Le traitement de patients par immunoth��rapie pourrait ��galement ��tre am��lior��es par l���augmentation de la puissance d���action anti-tumorale ainsi que la persistante des T CD8+ sp��cifiques aux TAA. Ce projet de doctorat se divise en deux parties : 1- La caract��risation de l���expression de DKK1 dans les cancers communs et la d��termination de son immunog��nicit�� afin de valider sa candidature comme TAA. 2- La reprogrammation des T CD8+, de patients atteints d���un cancer commun, vers un ph��notype moins diff��renti�� afin d���augmenter leur potentiel anti-tumoral et leur persistance. Dans le premier objectif, nous avons caract��ris�� l���expression de DKK1 dans le cancer du sein et dans d���autres cancers communs. Le profil d���expression de DKK1 a ��t�� ��tudi�� par RT-PCR et par ELISA dans plusieurs lign��es cellulaires de cancer et dans les tissus normaux. L���expression de DKK1 a aussi ��t�� ��tudi��e dans des ��chantillons cliniques provenant de cancers du sein, du poumon et du rein. Trente pourcents (30%) des tumeurs provenant d���un cancer du sein exprimaient DKK1. La moiti�� des tumeurs DKK1(+) ��tait triple n��gative, donc pas de r��cepteurs d�����strog��ne et de progest��rone et ��tait Her-2/neu(-) (ces patientes ont des possibilit��s de traitements tr��s restreintes). De plus, 50% des ��chantillons cliniques de tumeurs du poumon et 30% des tumeurs de rein exprimaient DKK1. Les observations effectu��es dans le cancer du poumon ont ��t��, par la suite, corrobor��es par d'autres groupes qui ont montr�� une corr��lation entre l'expression de DKK1 et un mauvais pronostic. Apr��s avoir confirm��e l���expression de DKK1 dans les cancers communs, justifiant ainsi sa candidature comme TAA, nous avons ��valu�� l���immunog��nicit�� de DKK1. Pour ce faire, nous avons effectu�� des stimulations in vitro de cellules mononucl����es du sang p��riph��rique (PBMC) de patient(e)s atteint(e)s d���un cancer du sein ou du poumon avec des peptides d��riv��s de DKK1 pouvant ��tre pr��sent��s par les complexes majeurs d���histocompatibilit�� (CMH) HLA-A*0201. Des clones de T CD8+ reconnaissant un peptide de DKK1 ont ��t�� identifi��s et isol��s. Par essai multiplex et cytom��trie de flux intracellulaire, la polyfonctionnalit�� d���un ces clones T CD8+ sp��cifiques �� DKK1 a ��t�� ��tudi��e et a r��v��l��e un profil effecteur, renfor��ant ainsi la candidature de DKK1 comme TAA. Dans l���ensemble, les r��sultats obtenus dans cette premi��re partie de th��se sugg��rent une possible utilisation de DKK1 en immunoth��rapie contre les cancers communs, attribuable �� son expression dans ces cancers et la possibilit�� de faire prolif��rer des T CD8+ effecteurs sp��cifiques �� DKK1 �� partir de sang de patients. Dans la seconde partie de cette th��se, je d��crirai la manipulation in vitro des T CD8+ de patients atteints d���un cancer commun, afin d���augmenter la force et la dur��e de leurs fonctions anti-tumorales. Il a ��t�� d��montr�� que des lymphocytes moins diff��renti��s sont capables d���une r��ponse immunologique plus efficace et durable. Nous avons bas�� ce projet sur l���utilisation d���un inhibiteur pharmacologique de la GSK-3���, pour activer de la voie de Wnt chez les T CD8+ et ainsi leur conf��rer un ph��notype moins diff��renti��, partageant des caract��ristiques de la cellule na��ve et de la cellule m��moire. Des cultures de T CD8+, sp��cifiques �� des antig��nes viraux, en pr��sence de l���inhibiteur ont permis d���augmenter la s��cr��tion d���interf��ron (IFN)-��� et leur activit�� cytotoxique. Ces r��sultats indiquent un effet de l���activation de la voie de Wnt sur la fonction des T CD8+. Ces observations sont rapport��es pour la premi��re fois chez les T CD8+ humains et sugg��rent une nouvelle strat��gie, applicables �� l���immunoth��rapie du cancer, afin de prolonger la persistance des cellules ainsi que leur activit�� anti-tumorale. En conclusion, ces travaux de recherche ont men�� �� la r��alisation d���une ��tape tr��s importante dans la validation de la candidature de DKK1 comme TAA pour les cancers communs, soit la d��monstration de son expression dans ces cancers et son absence dans les tissus normaux d��riv��s d���organes importants. Ces travaux ont ��galement men�� �� la d��monstration de l���immunog��nicit�� de DKK1, par l���identification d���un peptide de DKK1 reconnu par les T CD8+. De plus, l�����tude de la polyfonctionnalit�� des T CD8+ sp��cifiques �� DKK1 a r��v��l��e un profil effecteur favorable pour l���obtention d���une r��ponse anti-tumorale efficace. Ces d��couvertes pourraient servir �� l�����laboration d���une strat��gie d���immunoth��rapie �� m��diation cellulaire pour les cancers communs. Pour sa part, l�����tude ph��notypique et fonctionnelle de la modulation de la voie de Wnt dans les T CD8+ a donn�� lieu �� l���observation d���un ph��notype encore jamais rapport�� chez l���humain, conf��rant aux T CD8+ un aspect moins diff��renti�� avec des caract��ristiques propre �� un ph��notype m��moire. Ces r��sultats sont pertinents dans l���am��lioration de l���immunoth��rapie du cancer, passant par l���augmentation de la persistance des lymphocytes. En r��sum��, les r��sultats pr��sent��s dans cette th��se de doctorat fournissent des ��vidences ind��niables quant �� la validation de DKK1 comme TAA pour une immunoth��rapie �� m��diation cellulaire des cancers communs. Ces r��sultats fournissent ��galement des preuves quant �� la pertinence de la reprogrammation des T CD8+ par l���activation de la voie de la voie de Wnt, afin de g��n��rer des lymphocytes m��diateurs plus efficaces pour ce type de th��rapie.

��tude de la diff��renciation des lymphocytes T CD8+ effecteurs et m��moires : r��le de la cellule pr��sentatrice d���antig��ne et de la voie de signalisation Notch

Lors d���une infection par un pathog��ne, des lymphocytes T CD8+ na��fs (LTn) sp��cifiques de l���antig��ne sont activ��s, prolif��rent et se diff��rencient en LT effecteurs (LTe). Les LTe produisent diff��rentes cytokines et acqui��rent une activit�� cytotoxique menant �� l�����limination du pathog��ne. Seulement 5 �� 10 % des LTe survivront et se diff��rencieront en LT m��moires (LTm), qui sont capables de r��pondre plus rapidement lors d���une seconde infection par le m��me pathog��ne, contribuant au succ��s de la vaccination. Toutefois, la compr��hension de l���ensemble des m��canismes r��gulant le d��veloppement des LTe et des LTm demeure incompl��te. Afin de mieux comprendre les signaux requis pour la diff��renciation des LT CD8+ lors de la r��ponse immune, nous avons pos�� deux hypoth��ses. Nous avons d���abord propos�� que diff��rentes cellules pr��sentatrices d���antig��ne (CPA) fournissent diff��rents signaux au moment de la reconnaissance antig��nique influen��ant ainsi le devenir des LT CD8+. Vu leur potentiel d���utilisation en immunoth��rapie, nous avons compar�� la capacit�� d���activation des LT CD8+ par les lymphocytes B activ��s via le CD40 (CD40-B) et les cellules dendritiques (CD). Nous avons montr�� que l���immunisation avec des CD40-B induit une r��ponse effectrice mais, contrairement �� l���immunisation avec des CD, pratiquement aucun LTm n���est g��n��r��. Les LTe g��n��r��s sont fonctionnels puisqu���ils s��cr��tent des cytokines, ont une activit�� cytotoxique et contr��lent une infection avec Listeria monocytogenes (Lm). Nous proposons qu���une s��cr��tion 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 d��faut de diff��renciation des LTm observ�� lors de la vaccination avec les CD40-B. Ensuite, nous pos�� l���hypoth��se que, parmi les signaux fournis par les CPA au moment de la reconnaissance antig��nique, la voie de signalisation Notch influence le d��veloppement des LTe, mais aussi des LTm CD8+ en instaurant un programme g��n��tique particulier. D���abord, gr��ce �� un syst��me in vitro, le r��le de la signalisation Notch dans les moments pr��coces suivant l���activation du LT CD8+ a ��t�� ��tudi��. Ce syst��me nous a permis de d��montrer que la voie de signalisation Notch r��gule directement l���expression de la mol��cule PD-1. Ensuite, gr��ce �� des souris o�� il y a d��l��tion des r��cepteurs Notch1 et Notch2 seulement chez les LT CD8+ matures, un r��le de la voie de signalisation Notch dans la r��ponse immune des LT CD8+ a ��t�� d��montr��. Nos r��sultats d��montrent que suite �� une infection avec Lm ou �� une immunisation avec des CD, la signalisation Notch favorise le d��veloppement de LTe, exprimant fortement KLRG1 et faiblement CD127, destin��s �� mourir par apoptose. Toutefois, la signalisation Notch n���a pas influenc�� la g��n��ration de LTm. De fa��on tr��s int��ressante, l���expression des r��cepteurs Notch influence la production d���IFN-��� en fonction du contexte d���activation. En effet, suite �� une infection avec Lm, l���absence des r��cepteurs Notch n���affecte pas la production d���IFN-��� par les LTe, alors qu���elle est diminu��e suite �� une immunisation avec des CD sugg��rant un r��le d��pendant du contexte pour la voie de signalisation Notch. Nos r��sultats permettent une meilleure compr��hension des signaux fournis par les diff��rentes CPA et de la voie de signalisation Notch, donc des m��canismes mol��culaires r��gulant la diff��renciation des LT CD8+ lors de la r��ponse immunitaire, ce qui pourrait ultimement permettre d���am��liorer les strat��gies de vaccination.