Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques. In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level. To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs. The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. / <p>QC 20130927</p>

L’infection par le VIH-1 est caractérisée par une activation chronique du système immunitaire et par une réduction graduelle du nombre de lymphocytes TCD4+, qui contribuent à une détérioration lente du système immunitaire menant à la phase SIDA. Paradoxalement, ce sont majoritairement des lymphocytes T CD4+ non infectés qui sont détruits et la cause de ce phénomène reste encore inconnue. Certaines protéines virales, dont la protéine accessoire Vpr, sont soupçonnées de jouer un rôle dans ce processus. Synthétisée tardivement, Vpr est incorporée à l’intérieur des virions, en plus d’être relâchée sous forme soluble dans le milieu extracellulaire. La principale fonction biologique de Vpr est l’induction d’un arrêt de cycle en phase G2/M, via le recrutement du complexe d’ubiquitine E3 ligase CUL4A-DDB1VprBP et l’activation de la voie de dommage à l’ADN contrôlée par la kinase ATR. Une étude démontre que l’activation des voies de dommages à l’ADN conduit à l’expression de ligands du récepteur activateur NKG2D, exprimés par les cellules NK, déclenchant leurs fonctions cytolytiques. Chose intéressante, plusieurs études suggèrent que le VIH-1 régule positivement l’expression des ligands de NKG2D à la surface des lymphocytes T CD4+ infectés. Cependant, le facteur viral impliqué dans ce processus reste encore indéfini. Le but de cette thèse était d’évaluer le rôle de Vpr dans la modulation des fonctions cytolytiques des cellules NK et son implication potentielle dans la destruction des lymphocytes T CD4+. Nos travaux ont permis de démontrer que l’expression de Vpr, seule ou dans le contexte de l’infection, est suffisante afin d’augmenter spécifiquement l’expression du ligand de NKG2D, ULBP2, au niveau de lymphocytes T CD4+ primaires. Conséquemment, Vpr augmente ainsi la susceptibilité de ces cellules à une lyse par des cellules NK autologues. Nous démontrons que cette régulation positive d’ULBP2 repose sur la capacité de Vpr de recruter le complexe d’ubiquitine E3 ligase DDB1-CUL4AVprBP et l’activation de la voie de dommage à l’ADN ATR. Plus important encore, nous apportons des preuves que Vpr augmente également l’expression d’ULBP2 au niveau des cellules non infectées lors d’une infection de lymphocytes TCD4+ par le VIH-1. À cet effet, nous montrons que l’acheminement de Vpr au niveau de lymphocytes T CD4+ non infectés via des particules virales défectives est suffisant afin de réguler positivement ULBP2 et d’augmenter leur lyse par des cellules NK autologues. De plus, nous décrivons pour la première fois que Vpr, sous forme soluble, a la capacité d’induire des dommages à l’ADN et de réguler positivement ULBP2 suite à la transduction de différents types cellulaires, incluant des cellules T. Globalement, nos résultats démontrent que Vpr est un facteur viral clé impliqué dans la régulation positive des ligands de NKG2D induite par le VIH-1. Cette régulation positive d’ULBP2 pourrait alors contribuer à la destruction des lymphocytes T CD4+ infectés et non infectés via l’activation des fonctions cytolytiques des cellules NK. Une meilleure compréhension de la contribution de cette activité de Vpr dans la pathogenèse du VIH-1 a le potentiel de permettre le développement de nouvelles cibles ou stratégies thérapeutiques contre le VIH-1. / Chronic immune activation and gradual depletion of CD4+ T cells are hallmarks of HIV-1 infection, which are thought to contribute to the progressive deterioration of the host’s immune response that ultimately leads to AIDS. Paradoxically, the majority of CD4+ T cells that are destroyed are uninfected and causes for this bystander effect of infection on CD4+ T cells remains unclear. Some HIV-1 proteins, including the accessory protein Vpr, are suspected to play a role in this process. Vpr, expressed late during HIV-1 infection, is shown to be incorporated within the budding virions as well as secreted as soluble protein in the extracellular medium from the infected cells. The main biological function of Vpr is the induction of a G2/M cell-cycle arrest through the recruitment of the E3 ubiquitin ligase complex DDB1-CUL4AVprBP and activation of the ATR-mediated DNA damage pathway. One study showed that activation of DNA damage pathways leads to the expression of specific ligands for the activating receptor NKG2D expressed on NK cells, thus triggering NK cell cytolytic function. Interestingly, several evidences suggest that HIV-1 upregulates expression of specific NKG2D ligands on infected CD4+ T cells. However, the viral factor involved in this process remains undefined. The aim of this thesis was to evaluate the role of Vpr in modulating NK cell cytolytic function and its potential involvement in CD4+ T cells depletion. Our work demonstrated that the expression of Vpr, alone or in the context of HIV-1 infection, is sufficient to specifically increase expression of the NKG2D ligand, ULBP2, on primary CD4+ T cells. Consequently, these CD4 T cells become more susceptible to autologuous NK cell-mediated lysis. Our studies have shown that this Vpr-mediated ULBP2 upregulation requires the recruitment of the E3 ubiquitin ligase complex DDB1-CUL4AVprBP and the activation of the ATR-mediated DNA damage pathway. More importantly, we provide evidence that Vpr augments ULBP2 expression on both infected and uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes. In that context, we show that delivery of Vpr into uninfected cells via defective viral particles is sufficient to upregulate ULBP2 and increase their susceptibility to autologuous NK cell-mediated killing. In addition, we describe for the first time that soluble Vpr has the ability to induce DNA damages and upregulate ULBP2 upon transducing target cells, including T cells. Overall, our results show that Vpr is a key HIV-1 factor involved in the upregulation of NKG2D ligands induced by HIV-1. This upregulation of UBP2 might contribute to depletion of infected and uninfected CD4 + T cells through activation of NK cell cytolytic functions. A better understanding of the contribution of this new activity of Vpr in HIV-1 pathogenesis has the potential to enable the development of new therapeutic targets or therapeutic strategies against HIV-1.

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Com o início da fortificação de farinhas com ácido fólico (AF) no Brasil, a partir 2004, a população passou a estar exposta de modo compulsório a maiores quantidades desta vitamina. Sabe-se que o AF na sua forma sintética pode não ser completamente convertido para formas metabolicamente ativas, levando ao aparecimento de uma fração não metabolizada no organismo. Este fato é mais preocupante nos indivíduos que além da fortificação, fazem uso terapêutico dessa vitamina, como em pacientes com anemias hemolíticas (esferocitose hereditária (EH), &#946;-talassemia heterozigótica (&#946;-TH), entre outras). O objetivo desse estudo foi avaliar se as concentrações séricas de AF não metabolizado (UMFA) afetam a metilação global do DNA; a expressão de RNAm de genes da DHFR, MTHFR, interferon-&#947;, TNF-&#945; e interleucina (IL)-8; e a citotoxicidade de células NK. Foram incluídos 27 pacientes com EH, 50 indivíduos com &#946;-talassemia heterozigótica (&#946;-TH) e 136 indivíduos saudáveis. Outros 30 indivíduos saudáveis foram incluídos num estudo de intervenção com 5mg/dia de AF durante 90 dias. Foram realizadas as análises de: ácido fólico sérico, eritrocitário e UMFA, vitamina B12, homocisteína total; expressões de RNAm dos genes de MTHFR, DHFR, IFN-&#947;, TNF-&#945; e IL-8; citotoxicidade das células NK; metilação global do DNA e citocinas séricas IL6, IL-8, IL10, IFN-&#947; e TNF-&#945;. Resultados: Os pacientes EH apresentaram maiores concentrações de AF (sérico, eritrocitário e UMFA) que seus controles, sendo que 55,5% (método microbiológico) e 74,1% (método LC-MS/MS) apresentaram concentrações suprafisiológicas da vitamina, e 74,1% apresentaram concentrações aumentadas de UMFA. No grupo &#946;-TH foi observado maiores concentrações de folato eritrocitário comparado ao controle e 22% dos indivíduos tinham concentrações suprafisiológicas de AF. No estudo de intervenção, após 45 e 90 dias de uso de AF as concentrações suprafisiológicas estavam presentes em 93,3% dos indivíduos e 100% deles apresentavam concentrações aumentadas de UMFA. Não foram observadas diferenças nas taxas de metilação global do DNA entre os grupos EH e &#946;-TH quando comparados aos seus controles e não foi verificada correlação entre metilação global e concentrações de UMFA. Tanto EH quanto &#946;-TH apresentaram maior expressão de RNAm de IL-8, quando comparados aos seus controles. No grupo de intervenção houve maior expressão de IL-8 após 45 dias de uso de AF quando comparado ao período pré-intervenção. Foi mostrada uma correlação inversa entre as concentrações de folato eritrocitário com o número de células NK no grupo EH e com a capacidade citotóxica das células NK no grupo total (EH + controle). No grupo intervenção foi observado menor número e menor capacidade citotóxica das células NK após 90 dias de uso de AF. Conclusões: O uso de AF 5mg/dia foi associado com aumento expressivo das concentrações de folato sérico, eritrocitário, UMFA, na expressão de RNAm de genes da citocina inflamatória IL-8 e redução do número e da citotoxicidade das células NK. Dessa forma, altas doses de AF podem resultar em alterações de componentes do sistema imune podendo prejudicar os mecanismos de vigilância celular das células NK. Os nossos achados sugerem que é importante o acompanhamento terapêutico dos pacientes que fazem uso de AF, especialmente aqueles indivíduos que fazem uso crônico desta vitamina por longo tempo, como os pacientes com anemias hemolíticas, mulheres que desejam engravidar e gestantes. / With the beginning of the fortification of flour with folic acid (FA) in Brazil, since 2004, the population has been exposed compulsorily to larger amounts of this vitamin. It is known that FA in its synthetic form can not be completely converted to metabolically active forms, leading to the appearance of an unmetabolized fraction in the body. This fact is more worrying in subjects beyond fortification, make therapeutic use of this vitamin, such as patients with hemolytic anemia (hereditary spherocytosis (HS), &#946;-thalassemia heterozygotic (&#946;-TH), among others). The aim of this study was to evaluate if serum concentrations of unmetabolized FA (UMFA) affect the global DNA methylation; mRNA expression of DHFR, MTHFR, interferon-&#947;, TNF-&#945; and interleukin (IL)-8 genes; and on cytotoxicity of NK cells. It was included 27 patients EH, 50 subjects &#946;-TH and 136 healthy subjects. Another 30 healthy subjects were included in an intervention study with 5 mg/FA daily during 90 days. Analyzes performed were: serum and erythrocyte folate, and UMFA, vitamin B12, tHcy; mRNA expression of MTHFR, DHFR, IFN-&#947;, TNF-&#945; and IL-8 genes; cytotoxicity of NK cells; global DNA methylation and serum cytokines IL6, IL-8, IL10, IFN-&#947; and TNF-&#945;. Results: EH patients presented higher FA concentrations (serum, erythrocytes and UMFA) than controls ones, and 55.5% (microbiologic method) and 74.1% (LC-MS/MS method) showed supraphysiologic concentrations of vitamin, and 74.1% presented increased concentrations of UMFA. In &#946;-TH group, it was observed higher erythrocyte folate concentrations compared with the control and 22% of subjects had supraphysiological concentrations of FA. In the intervention study, after 45 and 90 days of FA use, supraphysiological concentrations were present in 93.3% of subjects and 100% of them showed increased concentrations of UMFA. It was not observed differences in global methylation of DNA between EH and &#946;-TH groups when compared to their controls and it was not observed significant correlation between global DNA methylation and UMFA levels. Both EH and &#946;-TH showed higher mRNA expression of IL-8 gene, when compared to controls. In the intervention group, there was higher mRNA expression of IL-8 gene after 45 days of FA use when compared to pre-intervention period. It was demonstrated an inverse correlation between erythrocyte folate levels and the number of NK cells in EH group; and cytotoxic capacity of NK cells on total group (EH + control). In intervention group, it was observed fewer number and lower cytotoxic capacity of NK cells after 90 days of AF use. Conclusions: The use of AF 5mg daily was associated with a significant increase in serum and erythrocytes folate levels, accompanied by increase in UMFA levels, an increase in mRNA expression of IL-8 gene, and reduction of the number and the cytotoxicity capacity of NK cells. Thus, high doses of AF can result in modifications of the immune system components, which may damage the cell surveillance mechanisms of NK cells. Our findings suggest that is important the therapeutic follow up of patients that are using AF, especially those subjects with chronic use of this vitamin, such as patients with hemolytic anemia, women who wish to become pregnant and pregnant women.

Les cellules lymphoïdes innées (ILCs) sont des populations cellulaires d’identification récente, mais leur rôle in vivo chez l’homme reste mal connu. Dans une 1ère étude, nous avons pu montrer qu’un déficit sévère en NK au cours de déficits immunitaires communs variables est associé à un risque accru de manifestations non infectieuses et infectieuses bactériennes sévères, suggérant un rôle protecteur non redondant des cellules NK lorsque le système immunitaire adaptatif n’est pas fonctionnel. Dans une 2ème étude, nous avons montré que des patients atteints de déficits immunitaires combinés sévères ɣc et JAK3 déficients n’ont pas d’ILCs. Après allogreffe de moelle osseuse, le nombre d’ILCs circulantes reste indétectable, sans manifestation clinique notable associée. Ces résultats sont en faveur d’une redondance des fonctions des ILCs chez l’homme, lorsque les fonctions T et B sont conservées. Nous avons ensuite étudié les modifications phénotypiques et fonctionnelles des cellules NK au cours du purpura thrombopénique immunologique, et observé un défaut de production d’interféron-ɣ par les cellules NK circulantes et une augmentation de la cytotoxicité dépendante des anticorps des cellules NK spléniques. Une inhibition des fonctions des cellules NK par les immunoglobulines polyvalentes est également mise en évidence. Enfin, une étude des ILCs circulantes au cours de la maladie associée aux IgG4 ainsi qu’une revue de la littérature sur l’étude des ILCs au cours des pathologies inflammatoires sont rapportées. En conclusion, l’apparente redondance des ILCs chez l’homme ainsi que leur implication en pathologies inflammatoires en font de potentielles cibles thérapeutiques. / Innate lymphoid cells (ILCs) are recently identified components of the immune system, but their functions in vivo in humans are still elusive. In a first study, we show in patients with common variable immunodeficiency that non-infectious inflammatory complications and severe bacterial infections were more frequent in patients with severe NK cell lymphopenia, indicating potential non-redundant immune functions of NK cells when the adaptive immune response is not optimal. In a second study, we observe that in patients with ɣc and JAK3 severe combined immunodeficiencies, all ILC subsets are absent. After hematopoietic stem cell transplantation, ILCs remain indetectable with no susceptibility to disease, suggesting that ILCs might be redundant and dispensable in humans, if T and B cells functions are preserved. In the second part of this thesis, we study phenotypic and functional modifications of NK cell compartment in primary immune thrombocytopenia. Interferon gamma production by the peripheral blood NK cells of ITP patients is decreased. In contrast, splenic NK cells of ITP patients tend to be more efficient in antibody-dependent cell cytotoxicity. Intravenous polyvalent immunoglobulins lead to the inhibition of blood NK cell activation. Finally, we present the preliminary results of a study investigating the modifications of circulating ILCs in IgG4-related disease, and present an extensive litterature review concerning the role of ILCs in inflammatory diseases. In conclusion, the apparent redundancy of ILCs for protective immunity and their pathogenic role in inflammatory diseases make their targeting in humans for therapeutic purposes particularly promising.

Les cellules NK sont des lymphocytes ayant un rôle majeur dans les réponses immunes innées, particulièrement contre les cellules tumorales ou infectées par un agent pathogène. L'activation des cellules NK déclenche leur cytotoxicité cellulaire et/ou sécrétion de cytokines, telle que l'IFN-gamma. L'activité des cellules NK est régulée par une variété de facteurs intrinsèques et extrinsèques qui assurent leur tolérance et efficacité. L'analyse de la signalisation NK permettrait de démontrer leur potentiel clinique. Parmi les protéines impliquées dans la signalisation NK, les protéines adaptatrices ont été très peu analysées. Dans ce projet, je décris les adaptateurs Dok-1/Dok-2 (Downstream of kinase), lesquels sont fortement exprimés dans des cellules hématopoïétiques, notamment dans le lignage myéloïde et les cellules NK. La structure de Dok-1 et Dok-2 est pourvue de différents domaines d'interaction protéine-protéine leur permettant de recruter différentes molécules de signalisation intracellulaire. Au cours de mon travail de thèse, je démontre que Dok-1 et Dok-2 sont phosphorylées sur tyrosine suite à l'activation des NK. La surexpression de Dok-1/Dok-2 dans les cellules NK induit un frein à leur activation déclenchée par des récepteurs activateurs. En revanche, l'invalidation d'expression de Dok1 et Dok2 chez la souris entraine un défaut de développement/maturation NK, mais également une augmentation de leur sécrétion d'IFN-gamma induite par l'engagement de récepteurs activateurs. Ces résultats dévoilent ainsi que Dok-l et Dok-2 sont impliquées dans une boucle de rétrocontrôle négatif en aval des récepteurs activateurs des cellules NK chez l'homme et chez la souris. / NK cells are an important component of innate immunity by virtue of their ability to recognize microbe-infected, transformed (tumors) and allogenic cells, while sparing most autologous healthy cell. NK cell activation can elicit two different effector functions: cytotoxicity of target cell and/or secretion of a large array of cytokines and chemokines. NK cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Determining signaling events downstream of the NK receptors is in this line of establishing the clinical potential of NK cells. Among the proteins involved in NK signaling, the adaptor molecules are poorly understood. Here, I described two Downstream of tyrosine kinase (Dok) adaptors: Dok-1 and Dok-2; these two proteins are mainly expressed in hematopoietic cells, especially in myeloid lineage and NK cells. Dok-1 and Dok-2 adaptors contain a variety of protein-protein interaction motifs enabling them to recruit various intracellular signaling molecules. During my PhD work, I demonstrate that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK cell activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK cell developmental/maturation defect and leads to increased IFN-gamma production induced by activating receptors. Taken together, these results reveal that Dok-1 and Dok-2 adaptors are involved in an intrinsic negative feedback loop downstream of NK cell activating receptors in mouse and human.

L’immunothérapie basée dans l’utilisation des cellules NK pour le traitement de différents types de cancers humains est une stratégie très prometteuse. Les cellules dendritiques plasmacytoïdes (pDC) activées permettent de stimuler les cellules NK pour augmenter leurs propriétés anti-tumorales. Les cellules NK activées par les pDC sont capables de développer in vitro et in vivo une forte réponse cytotoxique contre différentes lignées de leucémie lymphoblastiques pre-B. En revanche, les faibles quantités de pDC obtenues à partir du sang périphérique limitent leur l’utilisation en clinique. L’expansion et la différenciation des pDC in vitro à partir des progéniteurs hématopoïétiques CD34, permet d’obtenir des pDC humaines en grande quantité. Récemment il a été démontré que l’utilisation des antagonistes du récepteur de l’aryl hydroxycarbone (AhR) augmente le nombre des pDC générées in vitro. Cependant, la capacité à activer les cellules NK des pDC différenciées in vitro en présence d’antagonistes de l’AhR n’a pas encore été étudiée. Dans cette étude, nous montrons que les pDC obtenues in vitro ont une expression de molécules d’activation et une sécrétion de IFN plus faibles que celles des pDC du sang périphérique, mais que leur capacité à stimuler des cellules NK est similaire. Ces résultats ouvrent donc la voie à l’utilisation des pDC générées in vitro comme agent immuno-therapeutique visant à stimuler les fonctions anti-tumorales des cellules NK. / NK cells immunotherapy is a promising treatment for different human cancers. An effective approach to stimulate NK cells has been the use of activated plasmacytoid dendritic cells (pDC). NK cell activated by pDC develops a strong cytotoxic response against pre-B acute lymphoblastic leukemia (ALL) cell lines in vitro and in vivo. However, the use of pDC in the clinic has limitations because of its low frequency. One suitable strategy is the differentiation of CD34+ progenitors using different cytokines and chemokines. Recently, it has been demonstrated that antagonists of aryl hydroxyl receptor (AhR) increase the number of pDC obtained after culture of CD34+ cells. Nevertheless, the ability of these in vitro differentiated pDC to induce NK cells activation has not been well documented. In this study, it was showed that activated in vitro differentiated pDC present different characteristics than adult pDC, like a lower expression of activation markers and IFNalpha secretion, but their capacity to stimulate NK cells was similar to that observed in adult pDC. In addition, NK cells activated by in vitro differentiated pDC showed a strong cytotoxicity against the pre-B ALL cell line REH suggesting its effectiveness to treat ALL patients.

Les cancers colorectaux et les cancers du sein sont deux des affections malignes les plus fréquentes dans les pays industrialisés. Lorsqu’elles sont diagnostiquées à un stade précoce, ces tumeurs sont traitées efficacement par la chirurgie associée ou non à la radiothérapie. Pour des tumeurs localisées ayant certains facteurs de pronostic péjoratifs, pour les tumeurs localement avancées, ou pour les tumeurs métastasiques, une chimiothérapie est instaurée. Cependant, la chimiothérapie à elle seule ne permet pas de guérir les patients aux stades avancés de ces 2 types de cancers. C’est pourquoi d’autres alternatives comme l’immunothérapie ou des traitement plus ciblés sont en cours d’étude ou viennent récemment d’être validés.Notre travail a porté sur deux modèles d’immunothérapie dans deux formes de cancers . 1/Dans un modèle expérimental chez le rat, un traitement par un analogue de lipide A permet de guérir tous les animaux porteurs de carcinomatose péritonéale macroscopique d’origine colique. 2/ Chez la femme porteuse de carcinome mammaire localement avancé et surexprimant l’oncoprotéine HER2, un traitement préopératoire comportant du trastuzumab permet d’obtenir la disparition complète des tumeurs et des métastases axillaires chez la moitié des patientes. A partir de prélèvements tissulaires issus de ces 2 modèles, nous avons pu montrer que des analyses histologiques et surtout immunohistochimiques pouvaient permettre d’appréhender les mécanismes de régression tumorale.Dans les régressions des carcinomatoses chez le rat, le rôle du monoxyde d’azote, de l’apoptose des cellules tumorales et de l’infiltration des tumeurs par les cellules dendritiques puis des macrophages a été proposé. Dans la régression des tumeurs du sein, le rôle des cellules NK, via un mécanisme d’ADCC a été suggéré. Ces travaux ont permis de montrer que, malgré ses limites, et en particuliers l’impossibilité de démonstration mécanistique, l’immunohistochimie peut permettre de proposer des hypothèses intéressantes, qui doivent être secondairement confirmées par des expérimentations complémentaires. / In the developed countries, colorectal and breast cancers are two of the most common malignancies. When the diagnosis is made at the beginning of the disease, surgery, associated or not with radiotherapy may cure the patients. For tumors that present some pejorative prognostic factors,for locally advanced tumors or for metastasic disease, chemotherapy has to be prescribed. However, in these 2 types of cancer, chemotherapy is not able to cure patients suffering from metastasic stage and alternative treatments as immunotherapy or targeted therapies are studied or are still validated.Our work was based on 2 different models of immunotherapy in 2 types of cancer. In an experimental model of carcinomatoses of colorectal cancer in rats, Lipid A injections are able to cure all the rats, even at a macroscopic stage and in half of the women affected by locally advance HER2-overexpressing breast cancer, trastuzumab based preoperative treatment are able to obtain the total disappearance of the tumor. When we look on tumor samples obtained in these two models, we can show that histology and even more immunohistochemistry are able to propose some mechanisms of regression of the tumors. In the rat model, we can show the role of nitric oxide, apotosis, dendritic cells and macrophages and in the breast cancer regression, we show the role of NK cells and ADCC. Theses works show that immunohistochemistry, even if insufficient, may propose some interesting hypotheses that have to be confirm by other experimentations.

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