Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Jodi L. McGill / The causative agent of tuberculosis (TB) in cattle is Mycobacterium bovis (M. bovis). γδ T cells are a unique subset of nonconventional T cells that play major roles in both the innate and adaptive arms of the immune system. Bovine γδ T cells have the capacity for multiple immune functions during infection with M. bovis. However, the alternative functions of γδ T cells as well as the responses of γδ T cells in vivo at the site of infection remain unclear. To identify novel functions for γδ T cells in response to M. bovis infections, RNA sequencing and transcriptomics analysis was completed on peripheral blood γδ T cells isolated from virulent M. bovis-infected cattle. Differentially expressed genes were confirmed with real-time PCR. In an attempt to model in vivo cell-to-cell interactions at the site of infection, γδ T cells were also isolated from naïve and M. bovis-infected calves and co-cultured with autologous, BCG-infected, monocyte-derived macrophages. γδ T cell chemokine and cytokine expression was analyzed via ELISA and real-time PCR. The characteristic lesions of bovine tuberculosis are well-organized pulmonary granulomas. To determine the relevance of the RNA-sequencing and in vitro co-culture results to in vivo infection, tissue samples from granulomatous lesions in the lungs and mediastinal lymph nodes of virulent M. bovis-infected cattle were collected 3 months after infection. mRNA transcripts for γδ T cells expression of-- IFN-γ, IL-17, IL-10, IL-22, and CCL2 were microscopically evaluated within the granulomas using an in situ hybridization system, RNAScope (Advanced Cell Diagnostics Inc.). Co-culture experiments and transcriptomics analysis revealed increased expression of chemokines and various cytokines by γδ T cells responding to M. bovis infection. The novel in situ hybridization assay revealed that cytokine expression by γδ T cells varied within the lesions, with significant levels of CCL2 and IFN-γ, and low expression of IL-10, IL-22, and IL-17 in situ at this time-point after infection. Co-culture experiments also revealed that γδ T cells from virulent M. bovis-infected cattle have the capacity to directly impact the viability of M. bovis in vitro. Our results suggest that γδ T cells accumulate within the granulomas, and influence host immunity to M. bovis by secretion of cytokines and chemokines, and direct cytotoxicity, in response to infected macrophages.

Le cytomégalovirus (CMV) est l’agent responsable de l’infection opportuniste la plus fréquemment rencontrée en transplantation d’organe. Chez les receveurs séronégatifs qui reçoivent un rein provenant d’un donneur séropositif, 50 % de ces patients peuvent développer une virémie, et 30 % une maladie. A court terme, malgré les traitements anti-viraux, elle est responsable d’une morbidité non négligeable. A long terme, le CMV est associé à une augmentation de la fréquence des sténoses artérielles, plus d’infections associées, plus de rejet aigu, plus de lésions de fibrose interstitielle et d’atrophie tubulaire, une moins bonne survie des greffons et des patients. La cohabitation et la coévolution du CMV avec l’homme depuis des milliers d’années ont aboutie à un état d’équilibre entre le virus et son hôte. Le virus s’est profondément adapté à son hôte afin d’échapper à la réponse immune. En réponse à cela, la réponse immunitaire anti-CMV occupe une part unique et majeure au sein de la réponse immune de l’hôte. Les lymphocytes T CD8+ spécifiques du CMV représentent par exemple 10.2% des lymphocytes T CD8+ mémoires. Avec l’âge, ils s’accumulent et peuvent représenter jusqu’à 30% du pool total de lymphocyte T CD8+. Le système immunitaire sous la contrainte du virus s’est donc refaçonné de façon à garder le contrôle du virus. Depuis 1999, un nouvel acteur de cette réponse immunitaire a été identifié : les lymphocytes T gamma delta Vdelta2-negative. Ces cellules sont impliquées habituellement dans la lutte contre les différents stress d’origine microbien et non microbien (tumeur). Elles interviennent plutôt localement (dans les épithéliums) par différents mécanismes et sont désormais considérées comme des effecteurs intermédiaires entre l’immunité innée et l’immunité adaptative. Leur expansion dans le sang est associée à la guérison de la maladie et à la résolution de l’infection à CMV. Elles ont par ailleurs in vitro une réactivité croisée contre des cellules infectées par le CMV et des cellules tumorales. Les lymphocytes T gamma delta Vdelta2-negative sont donc une représentation supplémentaire de l’énorme impact du CMV sur le système immunitaire de l’hôte. Dans ce travail, nous avons pu étendre et approfondir leur rôle en transplantation d’organe. Nous avons tout d’abord décrit que les lymphocytes T gamma delta Vdelta2-negative avaient un phénotype et une cinétique d’expansion exactement superposable aux lymphocytes T CD8+ spécifiques du CMV in vivo. Nous avons ensuite observé que l’expansion des lymphocytes T gamma delta Vdelta2-negative induits pas l’infection à CMV s’associait à une survenue moindre de cancer à long terme chez les patients transplantés rénaux. Nous avons pu montrer que leur activation était sous la dépendance d’une interaction entre leur TCR et un ligand. Enfin, une autre voie d’activation dépendante du CD16, faisant intervenir les complexes immuns CMV-IgG anti-CMV a aussi été identifiée. Nos travaux depuis 10 ans ont donc démontré que les lymphocytes T gamma delta Vdelta2-negative occupaient une place majeure dans la réponse immune anti-CMV au même titre que les lymphocytes T CD8+. L’intégration de ces cellules à l’immunologie anti-CMV devrait permettre de mieux comprendre certains effets indirects induits par le virus, et pourrait être utile dans le suivi de la réponse immune anti-CMV en transplantation d’organe. L’identification de leur ligand pourrait permettre enfin de tester assez rapidement de nouveaux protocoles d’immunothérapie anti-virale ou anti-tumorale. / Cytomegalovirus (CMV) infection is the most frequent opportunistic infection encountered in solid-organ transplantation. Fifty percent of seronegative kidney transplant recipients (KTR) who receive a kidney from a seropositive donor may develop a CMV infection which causes a disease in 30% of cases. In the long term, CMV is associated with an increased incidence of arterial stenosis, more opportunistic infections, more acute rejection episodes, more interstitial fibrosis and tubular atrophy, and a poorer graft and patient survivals. For thousands years, the co-evolution between the CMV and the immune system allowed to a state of equilibrium between the virus and the host. The virus has deeply adapted to its host in order to escape the immune response. In response, the anti-CMV immune reaction takes up an important and unique place. For example, the CMV-specific CD8+ T cells represent an average 10.2% of the memory CD8+ T cell compartment in CMV-seropositive healthy individuals. With age, these cells accumulate and can represent around 30% of CD8+ T lymphocytes. Therefore under the long-lasting pressure of the virus, the immune system has redesigned in order to keep the control of the virus. Since 1999, a new actor was identified within the immune system: the Vdelta2-negative gamma delta T cells. These cells are involved against various microbial and non microbial stresses. They act locally in epithelia by different mechanisms and are now considered as intermediate effectors between innate and adaptive immunity. In vivo in KTR, their blood expansion is associated with the resolution of CMV infection. In vitro, they share a cross-reactivity against CMV-infected cells and tumor cells. Therefore, the Vdelta2-negative gamma delta T cells are new representatives of the huge impact of CMV on the host immune system. In this work, we were able to extend and get further insight into their role in organ transplantation. In vivo, we first described that Vdelta2-negative gamma delta T cells displayed a phenotype and an expansion kinetic similar to that of CMV-specific CD8+ T cells. Next, we observed that the CMV-induced Vdelta2-negative gamma delta T cells expansion was associated with a lower occurrence of cancer in long-term KTR. In vitro, experiments of transfer of gamma delta TCR allowed us to show that their activation against tumor ligands was TCR-dependent and that different tumor ligands could be recognized by each Vdelta2-negative gamma delta TCR studied. In addition, we observed that the recognition of CMV-infected cells was not only TCR-dependent, but under the dependence of a multi molecular complex involving co stimulatory signals. Finally, we also identified a new CD16-dependent pathway of activation in gamma-delta T cells, involving IgG-opsonised CMV. In summary, Vdelta2-negative gamma delta T cells take up a major place within the anti-CMV immune response in addition to CD8+ T lymphocytes. The integration of these cells to the anti-CMV immunology should provide a better understanding of some indirect effects of the virus and could be useful to monitor the immune response against CMV in solid-organ transplant recipients. Moreover, identification of their ligands could provide interesting tools for new protocols of anti-CMV and anti-tumor immunotherapy.

CD161 γδ T-cells have been implicated in the pathogenesis of Multiple Sclerosis however their role in health and chronic liver disease requires further exploration. In health, the majority of γδ T-cells expressed CD161 – a C-type lectin, and predominantly expressed the Vδ2 chain. The CD161+ γδ T-cells demonstrated a Th1-like pattern, expressing IFN-γ, TNF-α and Granzymes/Perforin when compared to the CD161- subset. The CD161+ γδ T-cells also expressed CCR6 and IL-18R thus also displaying a Th17-like pattern. These cells were also found in the lamina propria in the gut and rapidly expanded in the 1^st few weeks of life in the periphery. On gene array analysis, there were 409 genes expressed on the CD161+ γδ T-cells when compared to their CD161-ve counterparts including those coding for β2 receptors, CCL20, Acetycholinesterase, CCR1 and IL-18R. A potential clinical correlation to cardiac diseases was found when the upregulated genes were analysed. When the CD161+ γδ and CD161+ αβ T-cell populations were compared via gene-array, an association with a risk variant for coeliac disease was found. Thus in health, CD161+ γδ T-cells are not only a distinct subset of T-cells (confirmed by a FACS approach and gene array methods), but also the expression of CD161 may be linked to common genetic signals downstream in cell processes and disease pathogenesis, irrespective of T-cell subset population. In chronic liver disease there was a significant reduction in the periphery of CD161+ γδ T-cells in patients with chronic Hepatitis C (HCV) and an increase in patients with Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis when compared with healthy individuals. The CD161+ γδ T-cells appeared to be of a different phenotype in HCV infection. There was no overall significant localisation into of CD161+ γδ T-cells patients with chronic liver disease or specifically in HCV infection. There was however a CD161+ γδ T-cell enrichment in the liver in patients with Non-Alcoholic Fatty Liver disease. The CD161+ γδ T-cells were also found in Hepatocellular Carcinoma tissue. Overall it appears the CD161+ γδ T-cells are indeed a unique subset, playing a distinct role in health, as part of an early innate response, but also potentially involved in disease pathogenesis.

No description available.

Neuroblastoma is an aggressive solid tumor that develops from immature cells of the nervous system and is almost exclusively diagnosed in infants and young children. Over the past decade a multitude of immune based therapies have been explored as therapeutic candidates for patients with neuroblastoma. The anti-GD2 monoclonal antibody, 3F8, and more recently, natural kill (NK) cell based therapies have been accepted as hopeful therapeutic options for patients with Neuroblastoma. These options however have many drawbacks including dose limiting pain, the development of tolerance, reliance on MHC mismatch and possible reliance on the invariant NK (iNK) cells population. Gamma Delta T cells, a subpopulation of T cells composed of a T cell receptor (TCR) with a gamma and a delta chain instead of an alpha and a beta; chain, have been shown to recruit a more robust immune response then both 3F8 and NK cells through their activation of antigen presenting cells (APCs) and non-reliance on MHC mismatch. Gamma Delta T cells are also able to recruit NK cells as well as other cytotoxic lymphocytes. For these reasons, it is believed that Gamma Delta T cell based treatment alone or in combination with an anti-GD2 monoclonal antibody may have a greater efficacy than either NK cells or an anti-GD2 monoclonal antibody alone. The intent of this thesis is to explore and evaluate the current state of Gamma Delta T cell based immunotherapy against the backdrop of NK cell based immunotherapy for neuroblastoma.

Em 1993, a Organização Mundial da Saúde declarou a tuberculose (TB) como uma emergência global devido à sua relevância epidemiológica e a necessidade de seu controle. Atualmente, a TB ainda é considerada um problema de saúde pública e requer o desenvolvimento de vacinas e terapias que sejam mais eficazes na sua prevenção e tratamento. Nesse sentido, o Laboratório de Vacinas Gênicas da Faculdade de Medicina de Ribeirão Preto estuda há mais de dez anos a eficácia da vacina gênica DNAhsp65 na profilaxia e terapia da TB. Com o intuito de complementar o conhecimento existente sobre os mecanismos imunes desencadeados pela vacina DNAhsp65, assim como sua associação às drogas convencionais utilizadas no tratamento da TB, objetivou-se neste trabalho a caracterização de células natural killer (NK), T natural killer (NKT), e T ?? na imunoterapia da tuberculose experimental com a vacina DNAhsp65, no tratamento com as drogas rifampicina (RIF) e isoniazida (INH), e na associação DNAhsp65-drogas. Inicialmente, camundongos BALB/c foram infectados com Mycobacterium tuberculosis (Mtb) cepa H37Rv no dia 0 e nos dias 1, 7, 15, 30 e 70 após a infecção, foi promovida a eutanásia dos animais infectados ou não (controle) para análise das células T não convencionais no pulmão por citometria de fluxo. No dia 30 após a infecção, os animais infectados receberam os diferentes tratamentos: vacina DNAhsp65, vetor pVAX1, drogas RIF e INH, ou as drogas em associação à vacina. Dez dias após o fim dos tratamentos, foi promovida a eutanásia dos animais para análise das populações celulares no pulmão e linfonodo por citometria de fluxo, imunohistoquímica e PCR em tempo real. Os animais somente infectados com Mtb apresentaram aumento significativo no número das células NK (CD3-CD49b+), NKT (CD3+CD49b+) e T ?? (CD3+??+) logo na primeira semana após a infecção, e esta diferença em relação aos animais controle permaneceu em até 70 dias após a infecção. Entre as células NK presentes no pulmão, observou-se predominância da subpopulação CD11bhighCD27low em todos os animais. Nos animais infectados, verificou-se aumento significativo das subpopulações de NK: CD11bhighCD27high e CD11blowCD27high, nos dias 7 e 15 e somente no dia 15 após a infecção, respectivamente. Entre a população de células T ?? presentes no pulmão, houve predomínio do fenótipo CD27- em animais controles e infectados nos diferentes tempos experimentais. Quanto aos animais infectados com Mtb e tratados com DNAhsp65, verificou-se aumento significativo de células T ?? produtoras de IFN-? e IL-17 no pulmão, e apesar de não ter sido observada diferença na freqüência de células NK e NKT neste grupo, as células NK apresentavam maior expressão da molécula FasL relacionada à morte celular induzida por apoptose. Nos grupos drogas e DNAhsp65-drogas observou-se aumento da freqüência de células T ?? no pulmão, assim como aumento de células NK produtoras de IL-10 e que expressavam o marcador de ativação CD69. Os resultados deste trabalho mostram mais uma vez a eficácia da vacina DNAhsp65 e da associação DNAhsp65- drogas no tratamento de animais infectados com Mtb e sugerem que células T não convencionais como as células NK, NKT e T ?? podem participar na modulação da resposta immune na TB. Estes achados devem ser levados em consideração no desenho de novas estratégias terapêuticas e também profiláticas para a TB. / In 1993, the World Health Organization declared tuberculosis (TB) as a global emergence due to its epidemical relevance and the need to improve its control. Nowadays, TB still remains a public health problem and requires the development of more effective vaccines and therapies. In this sense, the Laboratory of Genetic Vaccines from the School of Medicine of Ribeirão Preto studies, for more than ten years, the efficiency of the genetic vaccine DNAhsp65 in TB prophylaxis and therapy. In order to complement the knowledge about the immune mechanisms triggered by DNAhsp65 vaccine and by its association with conventional drugs used in TB, our aim in this work was to characterize natural killer (NK), natural killer T (NKT) and gamma-delta (??) T cells in the immunotherapy of experimental tuberculosis with the DNAhsp65 vaccine, in the treatment with rifampicin and isoniazid drugs and in the association DNAhsp65-drugs. Initially, BALB/c mice were infected with Mtb strain H37Rv on day 0, and on days 1, 7, 15, 30 and 70 after infection, infected animals or not (control) were euthanized for lung cell analysis by flow cytometry. On day 30 after infection, infected animals received the following treatment: DNAhsp65 vaccine, pVAX1 vector, rifampicin and isoniazid drugs, or drugs in association with DNAhsp65. Ten days after the end of treatment, animals were euthanized for lung and lymph node cell analysis by flow cytometry, immunohistochemistry and real time PCR. Infected animals showed a significant increase of NK (CD3-CD49b+), NKT (CD3+CD49b+) and ?? (CD3+??+) T cells in the first week of infection and this difference compared to control animals remained until 70 days after infection. Within the lung NK cell population, we observed a predominance of CD11bhighCD27low phenotype in all animals. In infected animals, we verified a significant increase of the following NK cell subpopulations: CD11bhighCD27high and CD11blowCD27high on days 7 and 15, and only on day 15 after infection, respectively. Within the lung ?? T cell population, there was a predominance of CD27- ?? T cell in control and infected animals in the different experimental times. In infected animals and subsequently vaccinated with DNAhsp65, we verified a significant increase in ?? T cells producing IFN-? and IL-17 in the lungs. Although we have not seen any differences in NK and NKT cells in this group, NK cells showed higher expression of FasL molecule related to induced cell death by apoptosis. In DNAhsp65-drugs and drugs groups, we observed an increase in lung ?? T cells frequency, as well as increase in NK cells producing IL-10 and expressing CD69, an activation marker. Our results confirm the effectiveness of DNAhsp65 vaccine and its association with drugs in Mtb infected animals and suggest a modulation in the immune response through unconventional T cells such as NK, NKT and ?? T cells. These findings should be taken into consideration in the design of new therapeutic and prophylactic strategies for TB.

Em 1993, a Organização Mundial da Saúde declarou a tuberculose (TB) como uma emergência global devido à sua relevância epidemiológica e a necessidade de seu controle. Atualmente, a TB ainda é considerada um problema de saúde pública e requer o desenvolvimento de vacinas e terapias que sejam mais eficazes na sua prevenção e tratamento. Nesse sentido, o Laboratório de Vacinas Gênicas da Faculdade de Medicina de Ribeirão Preto estuda há mais de dez anos a eficácia da vacina gênica DNAhsp65 na profilaxia e terapia da TB. Com o intuito de complementar o conhecimento existente sobre os mecanismos imunes desencadeados pela vacina DNAhsp65, assim como sua associação às drogas convencionais utilizadas no tratamento da TB, objetivou-se neste trabalho a caracterização de células natural killer (NK), T natural killer (NKT), e T ?? na imunoterapia da tuberculose experimental com a vacina DNAhsp65, no tratamento com as drogas rifampicina (RIF) e isoniazida (INH), e na associação DNAhsp65-drogas. Inicialmente, camundongos BALB/c foram infectados com Mycobacterium tuberculosis (Mtb) cepa H37Rv no dia 0 e nos dias 1, 7, 15, 30 e 70 após a infecção, foi promovida a eutanásia dos animais infectados ou não (controle) para análise das células T não convencionais no pulmão por citometria de fluxo. No dia 30 após a infecção, os animais infectados receberam os diferentes tratamentos: vacina DNAhsp65, vetor pVAX1, drogas RIF e INH, ou as drogas em associação à vacina. Dez dias após o fim dos tratamentos, foi promovida a eutanásia dos animais para análise das populações celulares no pulmão e linfonodo por citometria de fluxo, imunohistoquímica e PCR em tempo real. Os animais somente infectados com Mtb apresentaram aumento significativo no número das células NK (CD3-CD49b+), NKT (CD3+CD49b+) e T ?? (CD3+??+) logo na primeira semana após a infecção, e esta diferença em relação aos animais controle permaneceu em até 70 dias após a infecção. Entre as células NK presentes no pulmão, observou-se predominância da subpopulação CD11bhighCD27low em todos os animais. Nos animais infectados, verificou-se aumento significativo das subpopulações de NK: CD11bhighCD27high e CD11blowCD27high, nos dias 7 e 15 e somente no dia 15 após a infecção, respectivamente. Entre a população de células T ?? presentes no pulmão, houve predomínio do fenótipo CD27- em animais controles e infectados nos diferentes tempos experimentais. Quanto aos animais infectados com Mtb e tratados com DNAhsp65, verificou-se aumento significativo de células T ?? produtoras de IFN-? e IL-17 no pulmão, e apesar de não ter sido observada diferença na freqüência de células NK e NKT neste grupo, as células NK apresentavam maior expressão da molécula FasL relacionada à morte celular induzida por apoptose. Nos grupos drogas e DNAhsp65-drogas observou-se aumento da freqüência de células T ?? no pulmão, assim como aumento de células NK produtoras de IL-10 e que expressavam o marcador de ativação CD69. Os resultados deste trabalho mostram mais uma vez a eficácia da vacina DNAhsp65 e da associação DNAhsp65- drogas no tratamento de animais infectados com Mtb e sugerem que células T não convencionais como as células NK, NKT e T ?? podem participar na modulação da resposta immune na TB. Estes achados devem ser levados em consideração no desenho de novas estratégias terapêuticas e também profiláticas para a TB. / In 1993, the World Health Organization declared tuberculosis (TB) as a global emergence due to its epidemical relevance and the need to improve its control. Nowadays, TB still remains a public health problem and requires the development of more effective vaccines and therapies. In this sense, the Laboratory of Genetic Vaccines from the School of Medicine of Ribeirão Preto studies, for more than ten years, the efficiency of the genetic vaccine DNAhsp65 in TB prophylaxis and therapy. In order to complement the knowledge about the immune mechanisms triggered by DNAhsp65 vaccine and by its association with conventional drugs used in TB, our aim in this work was to characterize natural killer (NK), natural killer T (NKT) and gamma-delta (??) T cells in the immunotherapy of experimental tuberculosis with the DNAhsp65 vaccine, in the treatment with rifampicin and isoniazid drugs and in the association DNAhsp65-drugs. Initially, BALB/c mice were infected with Mtb strain H37Rv on day 0, and on days 1, 7, 15, 30 and 70 after infection, infected animals or not (control) were euthanized for lung cell analysis by flow cytometry. On day 30 after infection, infected animals received the following treatment: DNAhsp65 vaccine, pVAX1 vector, rifampicin and isoniazid drugs, or drugs in association with DNAhsp65. Ten days after the end of treatment, animals were euthanized for lung and lymph node cell analysis by flow cytometry, immunohistochemistry and real time PCR. Infected animals showed a significant increase of NK (CD3-CD49b+), NKT (CD3+CD49b+) and ?? (CD3+??+) T cells in the first week of infection and this difference compared to control animals remained until 70 days after infection. Within the lung NK cell population, we observed a predominance of CD11bhighCD27low phenotype in all animals. In infected animals, we verified a significant increase of the following NK cell subpopulations: CD11bhighCD27high and CD11blowCD27high on days 7 and 15, and only on day 15 after infection, respectively. Within the lung ?? T cell population, there was a predominance of CD27- ?? T cell in control and infected animals in the different experimental times. In infected animals and subsequently vaccinated with DNAhsp65, we verified a significant increase in ?? T cells producing IFN-? and IL-17 in the lungs. Although we have not seen any differences in NK and NKT cells in this group, NK cells showed higher expression of FasL molecule related to induced cell death by apoptosis. In DNAhsp65-drugs and drugs groups, we observed an increase in lung ?? T cells frequency, as well as increase in NK cells producing IL-10 and expressing CD69, an activation marker. Our results confirm the effectiveness of DNAhsp65 vaccine and its association with drugs in Mtb infected animals and suggest a modulation in the immune response through unconventional T cells such as NK, NKT and ?? T cells. These findings should be taken into consideration in the design of new therapeutic and prophylactic strategies for TB.

Le cytomégalovirus (CMV), un β-herpes virus, est considéré comme un modèle d'immuno-évasion virale. Il s'agit d'un agent pathogène opportuniste fréquent chez les patients immunodéprimés et une cause majeure de malformations congénitales lors de l'acquisition in utero. Le CMV code pour des protéines (i) qui empêchent la présentation de l'antigène aux lymphocytes T αβ notamment par l'inhibition de l'expression des molécules HLA-I et (ii) qui suppriment les fonctions des cellules NK en imitant ou en régulant à la baisse les ligands des récepteurs NK (NKR). Ces mécanismes d'évasion ne devraient pas affecter les lymphocytes T γδ dont la reconnaissance antigénique est indépendante du HLA-I, et d’ailleurs leur réponse au CMV a été largement rapportée dans de nombreux contextes physiopathologiques. Notre objectif était de comprendre comment les mécanismes d’immuno-évasion du CMV affectent la réponse des lymphocytes T γδ. Nous avons utilisé des adénovirus recombinants exprimant chacun des quatre gènes du CMV impliqués dans l’inhibition de l’expression du HLA-I, et un mutant du HCMV déficient pour ces 4 gènes (CMV-∆US). Nous avons observé une induction de l'expression de HLA-I par l'adénovirus control, et une inhibition par US2, US3 et US11. Lors de l'utilisation de CMV-∆US, les cellules infectées exprimaient beaucoup plus de HLA-I que les cellules infectées par CMV-WT. De façon intéressante et à l’opposé des lymphocytes T αβ, les lymphocytes T γδ produisent plus d'IFNy en présence de fibroblastes infectés par le CMV-WT, qu’avec des fibroblastes infectés par CMV-∆US. Ces résultats indiquent que les molécules HLA-I régulent les lymphocytes T γδ grâce à des mécanismes qui sont en cours d'investigation dans notre équipe. Les processus d'échappement immunitaire développés par le CMV pourraient ainsi favoriser la réponse des lymphocytes T γδ par rapport à celle des lymphocytes T αβ et expliquer le rôle important des cellules T γδ dans le contrôle du virus chez les individus immunodéprimés. D'autre part, les acides nucléiques et les protéines du HCMV ont été trouvés dans les tissus tumoraux, mais la relation précise entre le HCMV et le cancer reste un sujet de débat. La plupart du temps, HCMV est décrit comme un virus oncomodulateur avec un rôle pro-tumoral. Notre objectif était d'utiliser le modèle de la souris pour tester in vivo l'impact de CMV de souris (MCMV) sur la croissance des cellules tumorales. Nous avons observé que MCMV pourrait inhiber la croissance de tumeurs sous-cutanées de côlon MC38 chez les souris immunodéficientes. Encore plus surprenant lorsque l'on considère la spécificité d’espèce des CMV, l'infection par le MCMV inhibe de la même façon la croissance des cellules cancéreuses du côlon humain HT29, qui n’est pas affectée par le HCMV. In vitro, les protéines MCMV précoces (IE-1) sont détectées dans des cellules cancéreuses humaines et murines après l'infection. Cependant, peu de cellules cancéreuses sont retrouvées positives pour le MCMV dans les tumeurs HT29 prélevées sur des souris infectées par le MCMV. De manière surprenante, le MCMV inhibe la prolifération des cellules cancéreuses de côlon humain contrairement au HCMV. De plus, la transcription de l'interféron β humain est induite après une infection par le MCMV. Cette induction n'a pas été observée après l'infection par le HCMV. En conclusion, nos données suggèrent un potentiel effet anti-tumoral de MCMV sur les cellules cancéreuses du côlon humain (HT29), qui pourrait être au moins partiellement médiée par l'interféron β. Ces résultats ouvrent la voie à l'utilisation potentielle du MCMV en tant que traitement du cancer du côlon humain. / Cytomegalovirus (CMV), a Beta Herpes virus, is considered as a paradigm for viral evasion.It is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. CMV encodes molecules to prevent antigen presentation to αβ T cells through inhibition of MHC Class I expression and to suppress NK cell functions by mimicking or down-regulating ligands of NK receptors (NKR). These evasion mechanisms are not expected to affect γδ T cells and, as a matter of fact, their response to CMV has been widely reported in many different physiopathological contexts as well as in CMV-seropositive healthy donors (Dechanet et al, 1999)( Scheper, 2013). Our aim was to understand how CMV induce γδ T cell response. We used recombinant adenoviruses expressing each of the four US genes, and a mutant HCMV deleted for these 4 genes (CMV-DUS). We observed an induction of HLA-I expression by the control adenovirus, and an inhibition by US2, US3 and US11. When using CMV-DUS, infected cells expressed much more native HLA-I than CMV-WT infected cells. Interestingly and in sharp contrast to αβ T cells, γδ T cell were activated to produce IFNg when cultured with fibroblasts infected with CMV-WT, but not when fibroblasts were infected with CMV-DUS. These results indicate that HLA-I molecules regulate γδ T cells through mechanisms that are under investigation in our team. The immune escape processes developed by CMV could thus promote γδ over αβ T cell response and explain the important response of γδ T cells to the virus in immunosuppressed individuals.

Chez les vertébrés gnathostomes, le système immunitaire repose en grande partie sur les lymphocytes T qui se développent dans un organe conservé évolutivement : le thymus. Chez les mammifères, cet organe constitue une cible privilégiée pour les œstrogènes. La question soulevée ici est donc de savoir si c’est également le cas chez les poissons téléostéens. Dans ce but, la distribution des différents sous-types de récepteurs aux œstrogènes a d’abord été étudiée dans le contexte d’une description de l’anatomie fonctionnelle du microenvironnement thymique. Par la suite, l’expression de gènes relatifs à la fonction thymique et aux différents sous-types de lymphocyte T a été analysée dans le thymus, le rein-antérieur et la rate de bars exposés au 17ß-œstradiol. De plus, la capacité de flambée oxydative a été évaluée sur des leucocytes du rein-antérieur et de rate à la suite d’expositions in vivo et in vitro. Finalement, la variation du nombre de thymocytes a été examinée sur des bars capturés durant trois ans. La thèse fournit de nouvelles connaissances concernant l’évolution des fonctions immunomodulatrices des œstrogènes sur la différenciation des cellules T. En effet, en plus d’une organisation morpho-fonctionnelle fortement conservée, la distribution des sous-types de récepteurs aux œstrogènes ainsi que les effets œstrogéniques apparaissent conservés au cours de l’évolution. Nos résultats suggèrent que, chez le bar comme chez les mammifères, les œstrogènes (1) stimulent une voie alternative de maturation des lymphocytes T ayant des propriétés similaires aux cellules immunitaires innées, (2) augmentent la tolérance immunitaire et (3) régulent la plasticité du thymus. / Jawed vertebrates have developed an efficient adaptive immune system partly based on T lymphocytes. They develop in an evolutionarily conserved organ, the thymus. In mammals, endogenous oestrogens are well known to regulate thymus function and plasticity. The question is, therefore, whether this is also the case in lower vertebrates, such as teleosts. To achieve these aims, firstly the distribution of oestrogen receptor subtypes was investigated on the background of a detailed description of the functional anatomy of the thymic microenvironment. Secondly, thymic function- and T cell-related gene expression was analysed in the thymus, the head-kidney and the spleen of sea bass exposed to 17ß-oestradiol. Moreover, the oxidative burst capacity in the two latter organs was evaluated in vivo and in vitro in leucocytes of the head-kidney and spleen following exposure to oestrogen. Eventually, age- and size-dependent variations in thymocyte number were examined in sea bass caught at various time points over three years. The thesis provides new insights into the evolution of the immunomodulatory function of oestrogen with respect to the thymic and peripheral T cell differentiation in vertebrates. As a matter of fact, in addition to a highly conserved morpho-functional organisation, the distribution of oestrogen receptor subtypes as well as the oestrogenic effects appear to be evolutionarily conserved. Our results suggest that in sea bass, similar to mammals, oestrogen (1) stimulates a thymic alternative pathway of T cell maturation with innate-like properties, (2) enhances immune tolerance by promoting Treg differentiation, and (3) actively regulate thymic plasticity.

Le développement d'un vaccin anti-paludique est limité par notre connaissance incomplète des effecteurs agissant contre P.falciparum. Nous avons mis en évidence que les cellules T Vγ9Vδ2 sont activées par la forme intra-érythrocytaire (schizonte) et par les phosphoantigènes de P.falciparum, et peuvent inhiber la croissance du parasite in vitro par un mécanisme dépendant de la granulysine ciblant la forme invasive du parasite (mérozoïte). Ces résultats suggérent que les lymphocytes T Vγ9Vδ2 jouent un rôle dans le contrôle précoce de la charge parasitaire. Cependant, le mécanisme médiant l’interaction entre les schizontes, les mérozoïtes les cellules T Vγ9Vδ2 et reste élusif. L'objectif de cette thèse est d’étudier les interactions entre les stades sanguins de P. falciparum et les cellules T Vγ9Vδ2, afin de mieux comprendre leurs activités anti-parasitaires, dans le but à long terme d’une utilisation clinique. Dans ce travail, nous étudions l'importance du contact direct avec les parasites dans l’activation et les activités anti-parasitaires des cellules T Vγ9Vδ2, par des approches de microscopie confocale et de cytométrie en flux. Nous suggérons que les cellules T Vγ9Vδ2 forment peu ou pas de contacts avec les mérozoïtes, et très peu de contacts avec le schizonte. De plus, nous montrons que, contrairement à une lignée cellulaire tumorale cible (Daudi), le contact avec les schizontes n'affecte pas l'activation des cellules T Vγ9Vδ2, suggérant que les phosphoantigenes du parasite sont libérés dans le milieu. Nous démontrons que les phosphoantigènes produits par la voie DOXP sont probablement libérés par un processus actif, dépendant des new permeation pathways (NPP). Ensembles, ces résultats suggèrent que l'activation et l'activité antiparasitaire des cellules T Vγ9Vδ2 n’est pas dépendante du contact, mais est médié par des facteurs solubles. / The limited knowledge of immune effector against Plasmodium falciparum precludes the development of a malaria efficient vaccine. We have recently evidenced that Vγ9Vδ2 T cells act as a new immune effector early in malaria infection. These cells are activated by the mature intraerythrocytic form (schizont) and by parasite-derived antigens (phosphoantigens). After activation, they inhibit in vitro parasite growth by targeting the extraerythrocytic invasive form (merozoite), by a granulysin-dependent mechanism. However, the mechanism by which Vγ9Vδ2 T cells are activated by schizonts and target merozoites remains elusive. The aim of this PhD project is to describe the interactions between P.falciparum blood stages and γδ T cells, in order to better understand their anti-parasitic activities and in the long term goal to manipulate these cells to prevent malaria. In the work, we investigate the importance of cell to parasite contact in Vγ9Vδ2 T cell activation and anti-parasitic activity by time-lapse and fixed confocal imaging, and cytometry. We suggest that Vγ9Vδ2 T cells form little or no contacts with merozoites, and very few contacts with the mature intraerythrocytic (schizont) form of the parasite. Moreover, we show that cytotoxic activities are elicited by schizonts, but that contrary to a known tumor cell line (Daudi cells), contact has no effect on the level of activation, suggesting that parasite-derived phosphoantigens are secreted in the microenvironment. We pursue the characterization of the parasite-derived phosphoantigens and demonstrate that they are produced by the DOXP pathway. Lastly, we show that phosphoantigens are most likely released by an active process, dependent on the new permeation pathways. Altogether, these results shed light on an unconventional mode of activation by P.falciparum blood stages and antiparasitic activity of Vγ9Vδ2 T cells, which is not contact-dependent, but rather is mediated by soluble factors.