Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

The Fas pathway is critical for the maintenance of normal T cell homeostasis. Humans and mice with defects in this pathway exhibit the accumulation of large numbers of peripheral lymphocytes and lupus-like autoimmunity. A major feature of these organisms is the accumulation of non-NK TCRαβ+CD4-CD8- “double negative” (DN) T cells. While regulatory T cells (Tregs) with the DN phenotype have been extensively characterized in Fas-sufficient mice and humans, limited data exist on the role of DN T cells as Tregs in Fas-deficient animals. In fact, most of the literature suggests that the DN T cells accumulating in Fas-deficiency states are pathogenic, contributing to secondary lymph node enlargement and autoimmune disease. In this body of work, data are presented that illustrate that Fas-deficient lymphoproliferative (LPR) DN T cells can act as Tregs in an interferon γ (IFNγ)- and Fas ligand (FasL)-dependent fashion toward Fas-sufficient T cells. LPR DN T cells needed to be able to secrete and respond to IFNγ in order to upregulate surface FasL, in order to ameliorate GVHD mediated by CD4+ T cells in vivo and to suppress the proliferation of and kill activated CD4+ T cells in vitro. FcRγ, a key molecule involved in innate immune responses, can substitute for CD3ζ in the T cell receptor (TCR) of mouse and human T cells in certain circumstances; in doing so, it is essential for the regulatory function of TCR transgenic DN Tregs. FcRγ-deficient LPR mice were found to have exacerbated T cell accumulation and early mortality. We show that while FcRγ expression was required for LPR DN T cells to regulate CD4+ and CD8+ T cells responding to alloantigens in vitro and in vivo, it does not control autologous lymphoproliferation in LPR mice by supporting the function of a regulatory cell, nor does it affect the rate of proliferation of LPR T cells in vivo. Instead, FcRγ-expressing LPR CD4+, CD8+ and DN T cells were found to be undergoing apoptosis at a high rate in vivo, and in contrast to their FcRγ-deficient counterparts, FcRγ+ LPR DN T cells were capable of undergoing TCR restimulation-induced cell death (RICD). The data presented in this thesis therefore show that LPR DN T cells can exhibit IFNγ-, FasL- and FcRγ-dependent regulatory function, and also illustrate a previously unknown function for FcRγ in controlling the expansion of Fas-deficient T cells. The implications of these data for autoimmune lymphoproliferative syndromes, and normal T cell homeostasis, are discussed.

The Fas pathway is critical for the maintenance of normal T cell homeostasis. Humans and mice with defects in this pathway exhibit the accumulation of large numbers of peripheral lymphocytes and lupus-like autoimmunity. A major feature of these organisms is the accumulation of non-NK TCRαβ+CD4-CD8- “double negative” (DN) T cells. While regulatory T cells (Tregs) with the DN phenotype have been extensively characterized in Fas-sufficient mice and humans, limited data exist on the role of DN T cells as Tregs in Fas-deficient animals. In fact, most of the literature suggests that the DN T cells accumulating in Fas-deficiency states are pathogenic, contributing to secondary lymph node enlargement and autoimmune disease. In this body of work, data are presented that illustrate that Fas-deficient lymphoproliferative (LPR) DN T cells can act as Tregs in an interferon γ (IFNγ)- and Fas ligand (FasL)-dependent fashion toward Fas-sufficient T cells. LPR DN T cells needed to be able to secrete and respond to IFNγ in order to upregulate surface FasL, in order to ameliorate GVHD mediated by CD4+ T cells in vivo and to suppress the proliferation of and kill activated CD4+ T cells in vitro. FcRγ, a key molecule involved in innate immune responses, can substitute for CD3ζ in the T cell receptor (TCR) of mouse and human T cells in certain circumstances; in doing so, it is essential for the regulatory function of TCR transgenic DN Tregs. FcRγ-deficient LPR mice were found to have exacerbated T cell accumulation and early mortality. We show that while FcRγ expression was required for LPR DN T cells to regulate CD4+ and CD8+ T cells responding to alloantigens in vitro and in vivo, it does not control autologous lymphoproliferation in LPR mice by supporting the function of a regulatory cell, nor does it affect the rate of proliferation of LPR T cells in vivo. Instead, FcRγ-expressing LPR CD4+, CD8+ and DN T cells were found to be undergoing apoptosis at a high rate in vivo, and in contrast to their FcRγ-deficient counterparts, FcRγ+ LPR DN T cells were capable of undergoing TCR restimulation-induced cell death (RICD). The data presented in this thesis therefore show that LPR DN T cells can exhibit IFNγ-, FasL- and FcRγ-dependent regulatory function, and also illustrate a previously unknown function for FcRγ in controlling the expansion of Fas-deficient T cells. The implications of these data for autoimmune lymphoproliferative syndromes, and normal T cell homeostasis, are discussed.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 181-228.

Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

Nas últimas décadas, verificou-se diferenças nas classificações da World Health Organization (WHO) de 2001 e da European Organization for Research and Treatment of Cancer (EORTC) de 1997 para os linfomas cutâneos primários. Em 2005, representantes dessas classificações se reuniram e em consenso estabeleceram a classificação WHO-EORTC que foi adotada pela última classificação da WHO de 2008. O presente estudo visa a avaliar a aplicabilidade dessa nova classificação em casuística retrospectiva de um único centro de referência no diagnóstico e tratamento de linfomas cutâneos. Assim, todos os casos de linfoma cutâneo de células T, excluindo-se micose fungóide (MF) e síndrome de Sézary (SS), no período de 1986 a 2009, foram analisados em relação aos aspectos clínicos, histopatológicos e imunofenotípicos, incluindo-se a realização de novas reações imunoistoquímicas. Os casos foram, então, classificados de acordo com critérios estabelecidos na classificação WHO de 2008. Houve, assim, 33 casos de linfomas cutâneos de células T não-MF e não-SS, sendo 08 (24,2%) de linfoma cutâneo de grandes células anaplásicas, 05 (15,2%) de papulose linfomatóide, 06 (18,1%) de linfoma extranodal de células NK/T tipo nasal, 05 (15,2%) de neoplasia de células dendríticas plasmocitóides blásticas, 05 (15,2%) de linfoma/leucemia de células T do adulto e 04 (12,1%) de linfoma de células T periféricas, sem outra especificação. Portanto, a classificação WHO de 2008 é aplicável à maioria dos casos de linfoma cutâneo de células T não-MF e não-SS. Entretanto, permanecem casos não classificáveis, alguns dos quais com curso clínico agressivo / Recent years have witnessed differences between the World Health Organization (WHO) 2001 and the European Organization for Research and Treatment of Cancer (EORTC) 1997 classification systems of primary cutaneous lymphomas (PCLs). In 2005, a joint WHO-EORTC classification system for PCLs has been reached and was adopted by last WHO 2008 classification. This study was performed to assess the applicability of this new classification to a single referral center. All cutaneous T-cell lymphoma (CTCL) cases, excluding mycosis fungoides (MF) and Sezary syndrome (SS), who were referred from 1986 to 2009 were included. The clinical features, histological and immunohistochemical stainings were reviewed, and additional stains were performed as needed. The cases were then reclassified according to the WHO 2008 classification. There were 33 cases of non-MF and non-SS CTCL, included 08 (24.2%) CD30+ anaplastic large-cell lymphomas, 05 (15.2%) cases of lymphomatoid papulosis, 06 (18.1%) extranodal NK/T-cell lymphoma nasal type, 05 (15.2%) blastic plasmacytoid dendritic cell neoplasm, 05 (15.2%) adult T-cell lymphoma/leukemia and 04 (12.1%) peripheral T-cell lymphomas, unspecified. The new WHO 2008 classification is applicable to most nonMF and non-SS CTCL cases. However, there is still a substantial subset of T-cell PCLs which cannot be classified beyond the unspecified peripheral T-cell category, some of which may have an aggressive course

Le virus Epstein-Barr (VEB) est fortement associé au développement de syndromes lymphoprolifératifs (SLP) en greffe pédiatrique. Ce virus a la capacité d’immortaliser les lymphocytes B et de provoquer leur prolifération incontrôlée chez l’hôte immunodéprimé. Plusieurs études démontrent que le cycle lytique du virus jouerait un rôle primordial dans la genèse des SLP en produisant des particules virales pouvant infecter les cellules B adjacentes. Chez un individu immunodéprimé, ces cellules B nouvellement infectées peuvent donner naissance à une expansion lymphocytaire. Le projet présenté dans ce mémoire fait partie d’un programme de recherche visant à élucider le rôle de l’infection productive par le VEB dans le développement des SLP. L’objectif précis de ce projet est de développer un anticorps monoclonal chimère contre la glycoprotéine gp350 du VEB dans le but de neutraliser le virus et d’ainsi prévenir son entrée dans les cellules B. Notre laboratoire a construit une version chimère de l’anticorps monoclonal murin 72A1, lequel se lie à la gp350 et bloque l’infection. Les premiers essais ont révélé la présence de chaînes non fonctionnelles (aberrantes) dans l’hybridome produisant l’anticorps 72A1. La construction de la chaîne légère authentique est maintenant complète alors que celle de la chaîne lourde est toujours en cours. Le processus de caractérisation de l’anticorps chimère inclura des essais de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC). Dans cette optique, une lignée cellulaire exprimant de façon stable la gp350 a été établie. Notre anticorps chimère anti-gp350 pourrait éventuellement être utilisé comme thérapie préventive chez les greffés présentant un risque élevé de SLP en empêchant l’infection des cellules B adjacentes. / The Epstein-Barr virus (EBV) is associated with B-cell post-transplant lymphoproliferative disease (PTLD). EBV has the unique property of immortalizing B lymphocytes, thereby causing their uncontrolled proliferation in an immunocompromised host. Certain evidence suggests that EBV productive infection may play a primary role in the genesis of PTLD by generating virus particles which can infect bystander B cells. In an immunocompromised individual, these infected B cells may then give rise to expanding B-cell clones. The project presented in this thesis is part of a research program seeking to elucidate the role of EBV productive infection in the genesis of PTLD. The specific aim of this work was to design a chimeric monoclonal antibody against the EBV envelope glycoprotein gp350 in order to neutralize the virus, thereby preventing entry into B cells. Our laboratory constructed a chimeric version of the murine monoclonal antibody, 72A1, which binds to gp350 and blocks infection. The initial cloning attempts revealed the presence of nonfunctional (aberrant) transcripts in the hybridoma line producing the 72A1 antibody. The chimeric version of the authentic light chain is now completed while the chimeric heavy chain construction is ongoing. As part of the characterisation process for the chimeric antibody, a cell line stably expressing surface gp350 was generated. This gp350-expressing cell line will be used for antibody dependent cellular cytotoxicity assays (ADCC). This anti-gp350 chimeric antibody could be useful as a preventive therapy in transplant patients at high risk for PTLD by blocking the infection of bystander B cells.

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP. / Epstein-Barr virus (EBV) is an opportunistic pathogen in immunocompromised transplant patients. In these patients EBV infection can lead to malignant B-cell lymphoproliferation, called post-transplant lymphoproliferative disease (PTLD). This thesis project aimed to investigate the role of lytic EBV infection in the genesis of PTLD. The first experimental objective was to provide in vitro proof that EBV could induce productive replication upon acute in vitro infection of B cells. Data obtained through study of viral DNA replication and transcription during the first 96 hours post-infection indicate that lytic infection does occur. These results provided justification for proceeding to the second experimental objective which involved the characterization of an anti-gp350 human-mouse chimeric antibody for its capacity to recognize and neutralize EBV. Results showed that this antibody did possess neutralization activity. Further study of this anti-gp350 chimeric antibody in SCID mice is necessary in order to evaluate its in vivo efficacy against PTLD.

Résumé en français : Notre équipe étudie les mécanismes de l’apoptose induite par la nutline-3, une molécule capable de se fixer sur MDM2 et d’activer la p53, dans différents types de lymphomes associés au virus d’Epstein-Barr (EBV) comme le lymphome de Burkitt (LB) ou syndromes lymphoprolifératifs post-transplantation (PTLD). Nos résultats montrent que la nutline-3 induit l’apoptose des cellules de LB EBV (-) alors que les cellules EBV (+) en latence de type III sont résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans ce phénomène de résistance afin de mettre en place des stratégies pour les contourner. Une première étude initiée par les résultats d’une analyse transcriptomique, effectuée après traitement avec la nutline-3 de deux lignées qui ne diffèrent que par leur statut EBV, nous a permis de montrer que : 1) l’autophagie est induite en réponse au traitement dans les cellules EBV (+) en latence de type III ; 2) ces cellules expriment fortement Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie contribue à la résistance de ces cellules à l’apoptose. Par ailleurs, nos résultats indiquent que la protéine anti-apoptotique Bcl-2 est également impliquée dans la résistance de ces cellules et que l’utilisation d’ABT-737, un inhibiteur de Bcl-2, restaure leur sensibilité à la nutline-3. L’efficacité de ce composé a donc été évaluée in vivo, seul ou en combinaison avec des traitements conventionnels (Cyclophosphamide pour le LB et Rituximab pour les PTLD). Les résultats obtenus lors de ces études pré-cliniques montrent que : 1) ABT-737 réduit considérablement la croissance tumorale et augmente la survie de souris xénogreffées avec des cellules d’une lignée lymphoblastoïde (LCL, utilisées comme modèle pour les PTLD) alors qu’il n’a pas d’effets chez les souris xénogreffées avec une lignée de LB ; 2) la combinaison BT-737/Cyclophosphamide permet de limiter la croissance tumorale durant le traitement mais n’améliore pas la survie des souris xénogreffées avec une lignée de LB ; 3) l’association ABT-737/Rituximab est très efficace et induit une rémission complète chez 70% des souris xénogreffées avec la lignée de LCL / - Résumé en anglais : Our team is working on the mechanisms of apoptosis induced by nutlin-3, a small molecule which binds to MDM2 and activates p53, in different lymphomas associated with Epstein-Barr virus such as Burkitt lymphoma (BL) or Post-transplant lymphoproliferative disorder (PTLD). Our results show that nutlin-3 strongly induce apoptosis in EBV (-) cells whereas EBV (+) latency III cells are much more resistant. The aim of my PhD project was to study the mechanisms involved in the resistance of EBV (+) latency III cells to apoptosis and to develop new therapeutic strategies to bypass these mechanisms. A transcriptomic analysis was realized after treatment with nutlin-3 of two cell lines which only differs by their EBV status. Based on the results obtained, a study was performed which allow us to show that: 1) autophagy is induced after nutlin-3 treatment in EBV (+) latency III cells; 2) these cells strongly expressed beclin-1 and present a constitutively high level of autophagy; 3) autophagy is involved in the resistance of apoptosis observed in these cells. Furthermore, our results demonstrate that Bcl-2 also contributes to the resistance of EBV (+) latency III cells and that treatment with ABT-737, a Bcl-2 inhibitor, restores their susceptibility to nutlin-3 treatment. We thus assessed the efficiency of this compound in vivo, in monotherapy or associated with conventional treatments (Cyclophosphamide for BL and Rituximab for PTLD). Results obtained during these pre-clinical studies show that: 1) ABT-737 reduces tumor growth and increase the overall survival of mice xenografted with a lymphoblastoïd cell line (LCL, used as a model for PTLD studies) but has no effects on mice xenografed with BL cell lines; 2) the association ABT-737/Cyclophosphamide reduces tumor growth during treatment but doesn’t improve the overall survival of mice xenografed with BL cell lines; 3) the association ABT-737/Rituximab is very efficient and induces 70% of complete remission in mice xenografted with LCL.