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Recent developments in the expansion and manipulation of primary NK cells has allowed this source of effective anti-tumour cells to be exploited for cell-based cancer immunotherapies. While ex vivo expanded primary NK cells are highly effective in the treatment of haematological malignancies, their efficacy against the solid tumour has been limited due to the presence of immune-regulatory factors in the tumour microenvironment. These factors can abrogate NK cell function by down regulating the expression of NK activating receptors, thus preventing these highly cytotoxic effector cells from activating in response to tumour challenge. Our work explores whether the expression of a tumour specific chimeric antigen receptor (CAR) on ex vivo expanded primary NK cells would allow the lost activatory signalling to be recouped, and regain their efficacy against the solid tumour. Unfortunately, the use of primary NK cells as effectors in CAR based cell immunotherapies has been hampered by the technical limitations of producing large numbers of CAR positive primary NK cells. This has led many researchers to utilise the NK-92 cell line instead of primary cells. We demonstrate that ex vivo expanded primary CAR NK cells can be produced efficiently and demonstrate higher anti-tumour functionality than CAR NK-92. Finally, due to the intricacies of NK cell biology, they are able to effectively discriminate between healthy and malignant targets thus preventing their cytotoxic function from being directed towards the incorrect target. This could be a key advantage in the use of primary NK cells over T cells as effectors of CAR as the off-tumour/on-target adverse effects seen with CAR T cells has severely hampered this clinical strategy. We have shown that CAR T cells but not CAR NK cells are reactive towards phenotypically non-malignant, clinically relevant, healthy cells expressing the CAR target. / Thesis / Master of Science (MSc)

Objetivo: o objetivo deste estudo foi avaliar a prevalência de autoanticorpos e a dosagem da concentração de células Natural Killer (NK) no sangue periférico em pacientes com endometriose. Métodos: Entre dezembro de 2004 e dezembro de 2007 foram avaliadas 155 pacientes submetidas a videolaparoscopia, divididas em um grupo sem endometriose(n=55) e outro com endometriose (n=100). Foi coletada amostra de sangue periférico de todas as pacientes no momento da laparoscopia e nessa amostra foi realizada a quantificação do percentual de células NK em relação aos linfócitos periféricos (por citometria de fluxo), e a determinação dos seguintes auto-anticorpos: anticorpos antinucleares (ANA, por imunofluorescência indireta), anticorpos antitireoglobulina e antiperoxidase (anti-TG e anti-TPO, por eletroquimioluminescência), anticorpos anticardilipina e antifosfatidilserina (aCL e aPS IgG, IgM e IgA, todos por ensaio imunoenzimático). Além da presença de endometriose, essas pacientes também foram avaliadas quanto ao estadiamento, os locais de doença, relações com a fase do ciclo, e a classificação histológica dessa doença. Resultados: as pacientes com endometriose apresentaram percentual de células NK (média DP de 15,3 9,8%) superiores àquelas sem a doença (média DP de 10,6 5,8%), p<0,001. Quanto aos autoanticorpos, as portadoras de endometriose também apresentaram positividade para ANA mais frequentemente (33%) que as pacientes do grupo controle (12,7%), p=0,006. Quanto aos anti-TG, anti-TPO, anti-CL (IgG, IgM e IgA) e aPS ( IgG, IgM e IgA), não houve diferenças estatísticas quanto à sua positividade. As células NK também mostraram-se mais elevadas nas protadoras de endometriose em estádios avançados e naquelas com comprometimento de retossigmóide, grupo no qual encontramos o maior percentual de células NK com concentração média de 19,8 10,3%. Concentrações de células NK 12,5% podem ser usadas como marcadores de endometriose em retossigmóide, com sensibilidade de 73% e especificidade de 65%. Utilizando-se de um modelo estatístico de probabilidades, demonstramos que associação desse marcador (NK 12,5%) com a presença de sintomas como dor e/ou sangramento intestinal durante a menstruação nos possibilitou estimar uma probabilidade de comprometimento de retossigmóide de 60,4%. Conclusões: pacientes com endometriose apresentam maior concentração de células NK periféricas, além de maior prevalência de ANA positivo em relação àquelas sem endometriose. As células NK aumentam nas pacientes com endometriose predominantemente nos estádios avançados, com comprometimento de retossigmóide. Nesse sentido poderiam ser utilizadas como marcadores diagnósticos desse tipo de comprometimento da doença, principalmente se forem avaliadas em conjunto com os sintomas das pacientes / Objectives: The objective of this study was to evaluate the prevalence of autoantibodies and the percentage of natural killer (NK) cells in the peripheral blood of patients with endometriosis. Methods: Between December 2004 and December 2007, 155 patients submitted to videolaparoscopy were evaluated. Patients were divided into two groups: one group of women without endometriosis (n = 55) and another in which all the women had endometriosis (n = 100). Samples of peripheral blood were collected from all the patients at the time of laparoscopy and flow cytometry was used to determine the percentage of NK cells in relation to peripheral blood lymphocytes in these samples. In addition, the following autoantibodies were measured: antinuclear antibodies (ANA) by indirect immunofluorescence, anti-thyroglobulin and anti-thyroid peroxidase antibodies (anti-TG and anti-TPO) by electrochemiluminescence, and anticardiolipin and anti-phosphatidylserine antibodies (aCL and aPS IgG, IgM and IgA), all performed using immunoenzymatic assay. In addition to the presence of endometriosis, these patients were also evaluated with respect to staging, to the sites of the disease, any association with the phase of the menstrual cycle and the histological classification of the disease. Results: The patients with endometriosis had a higher percentage of NK cells (15.3 ± 9.8%; mean ± SD) compared to those without the disease (10.6 ± 5.8%; mean ± SD), (p<0.001). Evaluation of the autoantibodies showed that positivity for ANA was more common in the group of patients with endometriosis (33%) compared to the patients in the control group (12.7%), (p = 0.006). With respect to anti-TG, anti-TPO, aCL (IgG, IgM and IgA) and aPS (IgG, IgM and IgA), no statistically significant differences were found between the groups of patients with or without endometriosis. NK cell concentrations were also found to be higher in patients with advanced stages of endometriosis and in those in whom the rectosigmoid was affected by the disease, this being the group in which the highest percentage of NK cells was found, with mean concentrations of 19.8 ± 10.3%. NK cell concentrations 12.5% may be used as markers of endometriosis of the rectosigmoid, with sensitivity of 73% and specificity of 65%. Using a statistical model of probability, these findings showed that the association of this marker (NK 12.5%) with the presence of symptoms such as pain and/or intestinal bleeding during menstruation permitted an estimation to be made of a likelihood of 60.4% of rectosigmoid endometriosis. Conclusions: Patients with endometriosis have higher percentages of peripheral NK cells, as well as a greater prevalence of positive ANA compared to those without endometriosis. The concentration of peripheral NK cells increases in patients with endometriosis, predominantly in patients with advanced stages of the disease and those in whom the rectosigmoid is affected. Therefore, the concentration of NK cells in peripheral blood could be used as a diagnostic marker of this type of endometriosis, particularly when evaluated together with patients symptoms

Depuis la formulation de la théorie de l’immuno-surveillance en 1957 par Burnet et Thomas, le monde scientifique s’est efforcé d’identifier les cellules immunitaires impliquées dans ce processus. Les lymphocytes Natural Killer (NK) constituent une composant majeure de l’immuno-surveillance innée dans plusieurs cancers hématologiques et solides. L’activité des lymphocytes NK passe principalement par une grande variété de récepteurs avec un rôle activateur ou inhibiteur. Parmi les récepteurs activateurs présents à la surface des lymphocytes NK, le récepteur NCR3/NKp30 a un rôle majeur dans la toxicité directe contre la cellule cible et dans l’activation des cellules dendritiques.Les tumeurs stromales gastrointestinales (GIST) et le Neuroblastome (NB) sont deux tumeurs sensibles à l’immuno-surveillance par les lymphocytes NK. Dans une étude récente notre équipe a démontré que l’épissage alternatif du gène NCR3/NKp30 peut être déterminant dans la fonction NK et dans la survie des patients atteints de GIST.Afin de caractériser les lymphocytes infiltrant le GIST, nous avons effectué une recherche visant à analyser l’infiltrat des lymphocytes CD3+, des lymphocytes T régulateurs (Treg) et des lymphocytes NK dans des tumeurs GIST localisés, et corréler ces résultats à la survie des patients. Nous avons mis en évidence que, avant traitement, les lymphocytes NK sont surtout localisés au niveau des fibres trabéculaires qui entourent la tumeur, alors que les lymphocytes T sont localisé à l’intérieur de la tumeur en contact avec les cellules tumorales qui expriment HLA-I.Nous avons aussi observé que les cellules NK ont un phénotype plutôt CD56bright et migrent à l’intérieur de la tumeur après traitement par Imatinib. L’analyse de survie a mis en évidence que les lymphocytes NK et T peuvent prédire la survie sans progression (PFS). Ces résultats mettent en évidence l’importance de l’infiltrat immunitaire dans la prédiction du risque de rechute dans le GIST et surlignent l’importance de viser une réponse immunitaire dans les protocoles thérapeutiques.Nous avons ensuite déterminé la proportion de lymphocytes NK dans le sang périphérique et dans la moelle dans une cohorte de Neuroblastome (NB) localisé et métastatique : une infiltration plus important par les NK CD56bright a été observé chez les patients présentant une maladie métastatique et chez les patients avec une réponse mineure au traitement d’induction. De plus, les NK présents dans les échantillons de moelle osseuse infiltrés par les neuroblastes, présentaient une expression plus basse du récepteur NKp30. L’expression du ligand de NKp30, B7-H6, a été mise en évidence sur les neuroblastes infiltrant la moelle osseuse, et sa forme soluble, sB7-H6, a été retrouvée être positivement corrélée à l’extension de maladie et inversement à la réponse au traitement d’induction. L’analyse de l’épissage alternatif du gène NCR3/NKp30 a permis de mettre en évidence l’impact des isoformes NKp30 sur la survie sans progression chez les patients atteints de NB de haut risque en maladie minimale résiduelle après chimiothérapie d’induction. En particulier, les patients présentant un taux élevé de l’isoforme pro-inflammatoire (NKp30b) par rapport à l’isoforme immunosuppressive (NKp30c), présentent une meilleure survie sans évènement. Nous avons aussi démontré le rôle des monocytes dans l’amplification de la réponse NKp30 dépendant. Les résultats de notre recherche dans le GIST et dans le NB, deux maladies différentes mais toutes les deux sensibles aux lymphocytes NK, surlignent l’importance d’intégrer de nouvelles options thérapeutiques aptes à cibler le système immunitaire. / Since Burnet and Thomas formulated in 1957 the cancer immunosurveillance theory, the scientific world has made tremendous progress to identify the immune cells involved in this process. Natural Killer (NK) cells have emerged as a major component of the innate immunosurveillance of several hematological and solid malignancies. The activity of NK-cells is mainly mediated through their wide variety of receptors with activating and inhibitory functions. Among the versatile receptors present on NK cells, the activating receptor NCR3/NKp30 is a major receptor involved in both direct killing of target cells and mutual NK and dendritic cell activation.Gastrointestinal stromal tumors (GIST) and Neuroblastoma (NB) are known to be tumors sensitive to NK immunosurveillance. In a recent study we showed that alternative splicing of NCR3/NKp30 gene can affect NK cell function and GIST patient’s outcome.In order to better characterize the GIST tumor-infiltrating lymphocytes, we analyzed the CD3+, T regulatory (Treg) and NK lymphocytes infiltration within primary localized GIST tumors and we determined their prognostic value. We described that, before treatment, NK cells are mainly localized in fibrous trabeculae while T lymphocytes are in the tumor nests in HLA-I positive tumor cells contact. Moreover infiltrating NK cells displayed a secreting CD56bright phenotype, and accumulate in tumor nests after Imatinib (IM) treatment. Importantly CD3+ and NK lymphocytes independently predicted progression free survival (PFS). These results highlight the importance of the immune infiltrate in re-define the GIST risk stratification and allow enhancing the immune response in the therapeutic decisions.We next investigated the proportions of NK cells in blood and bone marrow (BM) in a cohort of localized and metastatic NB; a high proportion of CD56bright NK cells was associated with metastatic NB and with poor response to induction treatment within the metastatic NB. Moreover, infiltrated BM presented NKp30 down regulation. The expression of the NKp30 ligand, B7-H6, was found on BM neuroblasts, while the soluble protein, sB7-H6 correlated with resistance to treatment. Furthermore the transcriptional status of NKp30/NCR3 dictated the event-free survival rates of HR-NBs with minimal residual disease post-induction chemotherapy: in particular patients presenting a high proportion of the immunosuppressive isoform (NKp30c) compared to the pro-inflammatory isoform (NKp30b), presented a worse outcome. We further demonstrated the significant role of monocytes to amplify the NKp30 activation response.These researches in GIST and NB, two different but at the meantime NK-sensitive diseases support the effort to define new immunological therapeutic approaches and to determine their optimal use.

Le microenvironnement tumoral, et notamment le stress hypoxique, joue un rôle immunosuppressif permettant l’échappement des cellules tumorales à la surveillance du système immunitaire. Des études récentes ont montré que l’échange de microvésicules (MVs) entre les cellules tumorales et les cellules du système immunitaire peut être responsable de l’établissement d’un microenvironnement immunosuppressif. Dans ce contexte, nous avons étudié l’effet des MVs issues des cellules tumorales hypoxiques sur la cytotoxicité des cellules «Natural Killer» (NKs). Nos résultats démontrent clairement que les cellules NKs sont capables d’internaliser les MVs issues des cellules tumorales normoxiques et hypoxiques. Cependant, seules les MVs hypoxiques sont capables de diminuer significativement la cytotoxicité des cellules NKs. Ainsi, nous avons déterminé que les MVs dérivées des cellules tumorales hypoxiques séquestrent deux immunomodulateurs, le TGF- et le miR-23a. Nous avons montré que le transfert de TGF- et miR-23a aux cellules NKs était responsable de la diminution respective de l’expression du récepteur activateur NKG2D à leur surface et de la protéine membranaire associée aux lysosomes (LAMP-1/CD107a) impliquée dans la dégranulation des granules cytotoxiques. Dans la deuxième partie de cette étude nous avons montré que les cellules tumorales soumises à un stress hypoxique étaient capables de déjouer un système immunitaire fonctionnel et d’échapper ainsi à la surveillance immunitaire des cellules NKs. En effet, nos résultats ont clairement démontré que la résistance des cellules tumorales hypoxiques à la lyse par les cellules NKs n’était pas liée à un défaut de reconnaissance, mais plutôt à l’activation d’un mécanisme de résistance intrinsèque dans les cellules tumorales. Ce mécanisme de résistance implique l’activation de l’autophagie qui opère dans les cellules tumorales pour dégrader le granzyme B, une protéase à sérine secrétée par les cellules NKs dont l’internalisation par les cellules tumorales cibles est nécessaire pour induire leur mort. Les expériences d’imagerie cellulaire combinées à des approches biochimiques ont confirmé que le niveau de granzyme B dans les cellules tumorales hypoxiques était significativement mois élevé par rapport à celui des cellules tumorales normoxiques. Ces résultats suggèrent fortement que le granzyme B est destiné à être dégradé par autophagie dans les cellules tumorales hypoxiques. En effet, l’inhibition génétique et pharmacologique de l’autophagie dans les cellules tumorales hypoxiques était suffisante pour contrecarrer la dégradation de granzyme B et ainsi restaurer la sensibilité des cellules tumorales hypoxiques à la lyse par les cellules NKs. Nos résultats ont clairement établi que l’inhibition de l’autophagie pouvait améliorer la réponse immunitaire antitumorale dépendante des cellules NK. Nous avons validé ce concept in vivo chez la souris en utilisant deux modèles syngéniques de cancer du sein et de mélanome. L’ensemble de nos travaux indiquent clairement que le stress hypoxique, qui est une caractéristique majeure du microenvironnement tumoral, peut favoriser l’établissement d’un microenvironnement immunosuppressif par plusieurs mécanismes qui ne s’excluent pas mutuellement. En effet, le stress hypoxique modifie les caractéristiques des cellules tumorales et active des mécanismes de résistance à la surveillance immunitaire. De plus, les cellules tumorales modifiées peuvent éduquer et exporter leur phénotype hypoxique aux cellules immunitaires présentes dans le microenvironnement afin d’affaiblir leur pouvoir cytotoxique. Nos résultats ouvrent ainsi la voie à la mise en place de nouvelles applications cliniques en immunothérapie anticancéreuse basées sur la réactivation des lymphocytes cytotoxiques et l’inhibition simultanée de l’autophagie. / The tumor microenvironment, including hypoxic stress plays an immunosuppressive role in tumor cell escape from immune surveillance. Recent studies have shown that the exchange of microvesicles (MVs) between tumor cells and cells of the immune system could be responsible for the establishment of an immunosuppressive microenvironment. In this context, we investigated the effect of MVs derived from hypoxic tumor cells on the cytotoxicity of Natural Killer (NK) cells. Our results clearly demonstrated that NK cells are able to internalize MVs derived from both normoxic and hypoxic tumor cells. However, only hypoxic MVs are able to significantly reduce the cytotoxicity of NK cells. Thus, we revealed that MVs derived from hypoxic tumor cells sequester two immunomodulators, TGF- and miR-23a. We have shown that the transfer of TGF- and miR-23a to NK cells was responsible for the respective reduction of the expression of NKG2D activating receptor on their surface and lysosomal-associated membrane protein (LAMP-1 / CD107a) involved in degranulation of cytotoxic granules.In the second part of this thesis we have shown that tumor cells subjected to hypoxic stress were able to outmaneuver a functional immune system and thus escape NK-mediated immune surveillance. Indeed, our results clearly demonstrated that the resistance of hypoxic tumor cells to NK-mediated lysis was not related to the impairment of recognition by NK cells, but rather to the activation of an intrinsic resistance mechanism in tumor cells. We showed that the resistance mechanism involves the activation of the autophagy which operates in the tumor cells to degrade the granzyme B, a serine protease secreted by NK cells and internalized by target tumor cells to induce cell death. Cell imaging experiments combined to biochemical approaches have confirmed that the level of granzyme B in hypoxic tumor cells was significantly higher compared to normoxic tumor cells. The analysis of the subcellular distribution of granzyme B reveals that it is predominantly present in the endosomes and autophagosomes of hypoxic tumor cells. These results strongly suggest that granzyme B is subjected to be degraded by autophagy in hypoxic tumor cells. Genetic and pharmacological inhibition of autophagy in hypoxic tumor cells was sufficient to block the degradation of granzyme B and thus restore the sensitivity of hypoxic tumor cells to NK-mediated lysis. Our results clearly demonstrated that inhibition of autophagy could improve NK-mediated antitumor immune response. We validated this concept in vivo using two syngeneic mice model of breast cancer and melanoma.Taken together, our work clearly shows that hypoxic stress, which is a major feature of the tumor microenvironment, can promote the establishment of an immunosuppressive microenvironment by several mechanisms which are not mutually exclusive. Thus, hypoxic stress changes the characteristics of tumor cells and activates the mechanisms of resistance to immune surveillance. In addition, tumor cells can educate and export their hypoxic phenotype to the immune cells in the microenvironment in order to impair their cytotoxicity. Our findings pave the way for the development of new clinical applications in cancer immunotherapy based on the reactivation of cytotoxic lymphocytes and simultaneous inhibition of autophagy.

Objetivo: o objetivo deste estudo foi avaliar a prevalência de autoanticorpos e a dosagem da concentração de células Natural Killer (NK) no sangue periférico em pacientes com endometriose. Métodos: Entre dezembro de 2004 e dezembro de 2007 foram avaliadas 155 pacientes submetidas a videolaparoscopia, divididas em um grupo sem endometriose(n=55) e outro com endometriose (n=100). Foi coletada amostra de sangue periférico de todas as pacientes no momento da laparoscopia e nessa amostra foi realizada a quantificação do percentual de células NK em relação aos linfócitos periféricos (por citometria de fluxo), e a determinação dos seguintes auto-anticorpos: anticorpos antinucleares (ANA, por imunofluorescência indireta), anticorpos antitireoglobulina e antiperoxidase (anti-TG e anti-TPO, por eletroquimioluminescência), anticorpos anticardilipina e antifosfatidilserina (aCL e aPS IgG, IgM e IgA, todos por ensaio imunoenzimático). Além da presença de endometriose, essas pacientes também foram avaliadas quanto ao estadiamento, os locais de doença, relações com a fase do ciclo, e a classificação histológica dessa doença. Resultados: as pacientes com endometriose apresentaram percentual de células NK (média DP de 15,3 9,8%) superiores àquelas sem a doença (média DP de 10,6 5,8%), p<0,001. Quanto aos autoanticorpos, as portadoras de endometriose também apresentaram positividade para ANA mais frequentemente (33%) que as pacientes do grupo controle (12,7%), p=0,006. Quanto aos anti-TG, anti-TPO, anti-CL (IgG, IgM e IgA) e aPS ( IgG, IgM e IgA), não houve diferenças estatísticas quanto à sua positividade. As células NK também mostraram-se mais elevadas nas protadoras de endometriose em estádios avançados e naquelas com comprometimento de retossigmóide, grupo no qual encontramos o maior percentual de células NK com concentração média de 19,8 10,3%. Concentrações de células NK 12,5% podem ser usadas como marcadores de endometriose em retossigmóide, com sensibilidade de 73% e especificidade de 65%. Utilizando-se de um modelo estatístico de probabilidades, demonstramos que associação desse marcador (NK 12,5%) com a presença de sintomas como dor e/ou sangramento intestinal durante a menstruação nos possibilitou estimar uma probabilidade de comprometimento de retossigmóide de 60,4%. Conclusões: pacientes com endometriose apresentam maior concentração de células NK periféricas, além de maior prevalência de ANA positivo em relação àquelas sem endometriose. As células NK aumentam nas pacientes com endometriose predominantemente nos estádios avançados, com comprometimento de retossigmóide. Nesse sentido poderiam ser utilizadas como marcadores diagnósticos desse tipo de comprometimento da doença, principalmente se forem avaliadas em conjunto com os sintomas das pacientes / Objectives: The objective of this study was to evaluate the prevalence of autoantibodies and the percentage of natural killer (NK) cells in the peripheral blood of patients with endometriosis. Methods: Between December 2004 and December 2007, 155 patients submitted to videolaparoscopy were evaluated. Patients were divided into two groups: one group of women without endometriosis (n = 55) and another in which all the women had endometriosis (n = 100). Samples of peripheral blood were collected from all the patients at the time of laparoscopy and flow cytometry was used to determine the percentage of NK cells in relation to peripheral blood lymphocytes in these samples. In addition, the following autoantibodies were measured: antinuclear antibodies (ANA) by indirect immunofluorescence, anti-thyroglobulin and anti-thyroid peroxidase antibodies (anti-TG and anti-TPO) by electrochemiluminescence, and anticardiolipin and anti-phosphatidylserine antibodies (aCL and aPS IgG, IgM and IgA), all performed using immunoenzymatic assay. In addition to the presence of endometriosis, these patients were also evaluated with respect to staging, to the sites of the disease, any association with the phase of the menstrual cycle and the histological classification of the disease. Results: The patients with endometriosis had a higher percentage of NK cells (15.3 ± 9.8%; mean ± SD) compared to those without the disease (10.6 ± 5.8%; mean ± SD), (p<0.001). Evaluation of the autoantibodies showed that positivity for ANA was more common in the group of patients with endometriosis (33%) compared to the patients in the control group (12.7%), (p = 0.006). With respect to anti-TG, anti-TPO, aCL (IgG, IgM and IgA) and aPS (IgG, IgM and IgA), no statistically significant differences were found between the groups of patients with or without endometriosis. NK cell concentrations were also found to be higher in patients with advanced stages of endometriosis and in those in whom the rectosigmoid was affected by the disease, this being the group in which the highest percentage of NK cells was found, with mean concentrations of 19.8 ± 10.3%. NK cell concentrations 12.5% may be used as markers of endometriosis of the rectosigmoid, with sensitivity of 73% and specificity of 65%. Using a statistical model of probability, these findings showed that the association of this marker (NK 12.5%) with the presence of symptoms such as pain and/or intestinal bleeding during menstruation permitted an estimation to be made of a likelihood of 60.4% of rectosigmoid endometriosis. Conclusions: Patients with endometriosis have higher percentages of peripheral NK cells, as well as a greater prevalence of positive ANA compared to those without endometriosis. The concentration of peripheral NK cells increases in patients with endometriosis, predominantly in patients with advanced stages of the disease and those in whom the rectosigmoid is affected. Therefore, the concentration of NK cells in peripheral blood could be used as a diagnostic marker of this type of endometriosis, particularly when evaluated together with patients symptoms

Les patients admis en réanimation semblent pouvoir présenter des infections sévères à CMV, en dehors de tout traitement immunosuppresseur, mais l’incidence exacte de ces infections est difficile à évaluer. Nous avons réalisé un dépistage systématique du CMV chez 242 patients consécutifs, considérés non immunodéprimés avant leur admission en réanimation. Nous avons ainsi identifié que 16% des patients développaient au cours de leur séjour en réanimation une infection à CMV. La mortalité des patients ayant présenté une infection à CMV était supérieure à ceux n’ayant pas présenté l’infection. La physiopathologie des réactivations à CMV chez ces patients est incomprise. Nous avons sélectionné les patients de réanimation non-immunodéprimés et séropositif pour le CMV à l’admission, et nous avons comparé le statut des NK circulants de 15 patients ayant présenté une réactivation CMV dépistée par antigénémie circulante (les cas) à celui de 15 patients contrôles appariés aux premiers pour l’âge, le sexe, et la gravité à l’admission. Dans la période précédant la réactivation CMV, alors que les capacités de cytotoxicité sont comparables, nous mettons en évidence une déficience NK en termes de capacité de production d’interféron gamma chez les cas en comparaison des contrôles, et également de témoins sains. Le sepsis, cad le tableau clinique résultant de la réponse à toute infection, est dans sa forme la plus sévère, l’une des principales causes d’admission en service de réanimation. Nous avons réalisé un « immunomonitoring » complet des NK circulantes à la phase initiale d’états pro-inflammatoires chez 42 patients admis en réanimation pour des motifs infectieux (sepsis sévère ou choc septique) ou des tableaux de SIRS non infectieux. Les patients septiques présentaient une capacité réduite de dégranulation en comparaison des patients non-septique (SIRS). Ils présentaient également une réduction de la sécrétion d’interféron-&#61543;, toujours en comparaison des patients SIRS. / Patients admitted to the intensive care unit (ICU) seem to have severe infections with CMV, without any immunosuppressive therapy, but the exact incidence of these infections is difficult to assess. We conducted a systematic screening of CMV in 242 consecutive patients non-immunosuppressed before ICU admission. We identified that 16% of patients developed CMV infection during their stay in ICU. The mortality of patients with CMV infection was higher than those without such infection. The pathophysiology of CMV reactivation occurring in critically ill patients with no previous immunosuppression is misunderstood. We selected non-immunocompromised ICU patients, CMV seropositive at admission, and we compared the status of circulating NK of 15 patients with CMV reactivation detected by antigenemia (cases) to 15 controls matched for age, gender, and severity on admission. In the period preceding the CMV reactivation, whereas capabilities cytotoxicity (degranulation) are comparable, we show impaired NK in terms of production of gamma interferon in cases compared to controls, and also healthy. Levels of IL-10 were significantly higher in cases with a strong correlation between the levels of this cytokine and severity of CMV reactivation as measured by the number of cells positive antigenemia. Sepsis, the clinical picture resulting from the response to any infection, in its severest form, is one of the main causes of admission in ICU. We conducted a complete immunomonitoring of circulating NK in the initial phase of pro-inflammatory state in 42 patients admitted to intensive care for reasons of infection (severe sepsis or septic shock) or of non-infectious SIRS (systemic inflammatory response syndrome). Septic patients showed a reduced ability of degranulation compared with non-septic patients (SIRS). They also showed a reduction in the secretion of gamma interferon, compared with patients with SIRS.

Les cellules Natural Killer (NK) sont des lymphocytes du système immunitaire inné capables de tuer des cellules cibles et de produire des cytokines telles que l'interféron-&#947;. Au cours de mon travail de thèse, j'ai utilisé des approches de génétique directe et inverse dans le but d'étudier les mécanismes impliqués dans la régulation des capacités effectrices des cellules NK. La tolérance des cellules NK au soi est en partie assurée par les récepteurs inhibiteurs de surface qui sont spécifiques des molécules du complexe majeur d'histocompatibilité de classe I (CMH-I) exprimées par les cellules du soi. Cependant, des cellules NK qui ne sont pas capables de détecter l'expression du CMH-I ne sont pas autoréactives. Dans la première partie de ce travail de thèse, nous avons cherché à déterminer, chez la souris, les mécanismes de la tolérance NK, indépendante de la reconnaissance du CMH-I, qui est associée à une hyporeactivité des cellules NK. En utilisant des techniques de spectrométrie de fluorescence par corrélation à spot variable (svFCS), nous avons montré que dans les cellules NK hyporéactives les récepteurs activateurs et inhibiteurs sont confinés à la membrane plasmique par des réseaux structurés d'actine. A l'inverse, la reconnaissance par les cellules NK du CMH-I, qui « éduque » les cellules NK pour qu'elles acquièrent leurs capacités effectrices maximales, est associée une relocalisation des récepteurs activateurs au sein de nanodomaines. Ces résultats suggèrent que ce serait le confinement particulier des récepteurs activateurs à la membrane des cellules NK qui assure la tolérance au soi. / Natural killer (NK) cells are bone marrow-derived innate immune lymphocytes able to kill cellular targets and secrete cytokines such as interferon-&#947;. During my PhD work, I used reverse and forward genetic approaches to dissect the mechanisms involved in the regulation of NK cell effector capabilities at steady state. NK cell tolerance to self is partly ensured by major histocompatibility complex class I (MHC- I)-specific inhibitory receptors on NK cells, which detect MHC-I expression on self-cells and prevent NK cell activation. However, NK cells that do not detect self MHC-I are not autoreactive. In the first part of this PhD work, we sought to determine the mechanism at the basis of this MHC-I independent NK cell tolerance. Using spot variation fluorescence correlation spectroscopy (svFCS), we showed that MHC-I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC-I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We thus propose that the confinement of activating receptors at the plasma membrane is essential to ensuring self-tolerance of NK cells.