Ces dernières années ont été marquées par une progression importante dans la connaissance de la physiologie des cellules B in vivo et de leur différenciation en plasmocytes, grâce aux modèles murins et à l'imagerie intravitale. La transposition à l'Homme des connaissances acquises chez la souris soulève cependant des difficultés, telles que le manque d'outils pour visualiser les évènements qui se déroulent dans les organes lymphoïdes humains. Dans l'optique d'apporter une réponse à cette problématique, nous avons développé au sein du laboratoire un modèle in vitro en deux étapes, permettant la différenciation des lymphocytes B naïfs humains en plasmocytes. Ce modèle est particulièrement adapté pour étudier l'induction, au cours de la différenciation terminale B, des voies de signalisation, des facteurs de transcription et des grands processus cellulaires tels que la prolifération ou la mort programmée. A l'aide de ce modèle, nous avons mené deux études relatives à la différenciation B dans le centre germinatif: (i) La caractérisation sur le plan phénotypique et fonctionnel des populations cellulaires générées in vitro. Par une approche transcriptomique, nous avons comparé le profil d'expression et identifié les gènes et fonctions biologiques spécifiques à chacune de ces populations différenciées. Nous nous sommes concentrés sur l'étude de l'expression et de l'activité de l'enzyme AID, notamment au travers de la mesure des fréquences des hypermutations somatiques à chaque stade de la différenciation. (ii) L'étude des mécanismes cellulaires (cycle cellulaire, apoptose), génétiques et épigénétiques menant à la transition des cellules du centre germinatif vers le stade de plasmablastes. Cette étude a permis de caractériser au plus près une population cellulaire fondatrice à l'origine des cellules différenciées. En outre, nous avons mis en évidence l'importance de la méthylation de l'ADN, en particulier la 5-hydroxyméthylation, dans le contrôle de la différenciation terminale B. / Within the past few years there has been a significant increase in the knowledge of B cell physiology in vivo and their differentiation into plasma cells through the implementation of murine models and intravital imaging. However, the transposition of this knowledge from mouse to man raises difficulties such as the lack of tools available to visualize the ongoing events in human lymphoid organs. In order to overcome this issue, we have developed in our laboratory, a two-step in vitro model allowing naive B cell differentiation into plasma cells. This model is particularly suitable for studying the induction of signaling pathways, transcription factors and major cellular processes such as proliferation or programmed cell death. Using this model, we conducted two studies on B cell differentiation in the germinal center: (i) phenotypic and functional characterization of cell populations generated in vitro. This study involved a transcriptomic approach which identified and compared the expression profile of specific genes and their biological functions within each of these differentiated populations. Specifically, we focused on the expression and activity of the AID enzyme through the measurement of somatic hypermutation frequency at each stage of differentiation. (ii) The study of cellular (cell cycle, apoptosis), genetic and epigenetic mechanisms leading to the transition of the germinal center B cells into plasmablasts. This study allowed us to characterize a founder cell population of the in vitro generated plasmablasts. In addition, we have highlighted the importance of DNA methylation, in particular 5- hydroxymethylation in controlling terminal B cell differentiation.

Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the acute and persistent GC B cell reaction following respiratory IAV infection is lacking, as is the characterization of IAV-induced T follicular helper (TFH) cells that support GCs. Additionally, it remains unclear as to whether IAV-induced GC B cells are subject to control by regulatory T cells (Tregs). To address this, GC B cell and TFH cell responses were analyzed in mice following pulmonary challenge with IAV. Studies demonstrated that marked GC reactions were induced in lung-draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude, kinetics, and isotype switching patterns of the response was site-specific, and largely depended on the magnitude of IAV-induced TFH cell populations. TFH cell magnitude peaked prior to that of GC B cells in all tissues, and TFH cells purified from dLNs generated IL-21 and IFN-gamma upon activation, although CD4+CXCR5- T effector cells produced higher levels of all cytokines. IgA+ GC B cells were infrequent in most sites, but composed a significant subset of the switched GC population in NALT. Further, splenectomized mice withstood a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection. Additionally, GC B cell populations were analyzed at distal time points to assess the understudied, persistent GC B cell response after IAV infection. Our analysis demonstrated that persistent GC B cell populations in mouse lungs directly correlated with infectious dose, pathogenicity of the virus, as well as the presence of long-term CD4+ T cell help. Finally, experiments showed that Tregs contribute to the control of GCs induced in the spleen by IAV challenge. This was demonstrated by a marked increase in the number of total and switched GC B cell numbers when Tregs were either depleted or disrupted in vivo proximal to IAV exposure.

Germinal center (GC) reactions are central features of T cell-driven B cell responses, and the site where antibody (Ab) producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity Abs. These processes require exquisite regulation not only to ensure the production of desired Abs, but to minimize unwanted autoreactive or low affinity Abs. To assess whether T regulatory cells (Treg) participate in the control of GC responses, immunized mice were treated with either an anti-glucocorticoid-induced TNFR-related protein (GITR) mAb or an anti-CD25 mAb to disrupt Treg activity. In both groups of treated mice, the GC B cell pool was significantly larger compared with control treated animals, with switched GC B cells composing an abnormally high proportion of the response. With these results indicating Tregs influence on GC dynamics, experiments were conducted to determine if Tregs were located in the GC, which subset of Treg was involved and by which mechanisms were their functions being effected. Within the spleens of immunized mice, CXCR5+ and CCR7- Tregs were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Studies demonstrated administration of either anti-TGF-β or anti-IL-10R blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Tregs is important in controlling the GC response. Blockade of two Treg methods of suppression, PD-1/PD-L1 pathway and CTLA-4, also resulted in disrupted GCs, indicating the possible use of them for suppression by Treg. Collectively, these findings indicate that Tregs contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.

Phosphoinositide 3-kinase is a family of lipid kinases that function by phosphorylating the D3 position of phosphoinositide (PI) lipids generating PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. These D3 phosphoinositides regulate various cellular processes through the recruitment of effector proteins containing lipid specific pleckstrin homology (PH) domains. PI phosphatases such as PTEN and SHIP function to restrain PI3K signaling by limiting the amount of D3 PI available for binding. Deletion of either PTEN or SHIP significantly alters B cell function and humoral immune responses. TAPP1 and TAPP2 are dual PH domain containing adaptors which selectively bind the phosphoinositide PI(3,4)P2 via their C-terminal PH domains. PI(3,4)P2 is a lipid messenger generated by PI3K and through the inositol phosphatase activity of SHIP. The function of PI(3,4)P2 remains incompletely understood. To identify the functional role of TAPP-PI(3,4)P2 interactions, we utilized a knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPPs. Our study assessed the effect of PI3K dependent KI mutation on B lymphocyte development, activation and antibody production. Flow cytometry analyses of lymphoid tissues found that TAPP KI mice develop relatively normal frequencies of mature B cell populations with the exception of peritoneal B1 cells, which are increased by approximately 50%. Strikingly, TAPP KI mice developed substantially elevated serum antibody levels. TAPP KI mice were able to generate high affinity antigen-binding antibodies upon immunization with NP-OVA in alum adjuvant; however, total immunoglobulin production was markedly increased under this immunization condition. We further assessed the germinal centre (GC) response, which are known to require PI3K signaling and a hallmark of T cell dependent (TD) antibody responses. TAPP KI mice generated larger germinal centers (GC) upon immunization, which was associated with increased GC B cell survival. We further assessed whether uncoupling of TAPPs from PI(3,4)P2 alters B cell signaling and functional responses in vitro. B cells purified from TAPP KI mice were found to have altered functional responses in vitro, with significantly increased survival and cell division following antigen receptor cross-linking. Consistent with increased cell survival, TAPP KI B cells show increased Akt phosphorylation on Ser473 and Thr308 after antigen receptor cross-linking. However, reconstitution of B cell deficient mice with either WT or TAPP KI B cells was found to generate similar GC responses, suggesting that activation of other cells may contribute to the enhanced in vivo responses. Consistently, when we examined the CD4+ T follicular helper cells, a subset providing critical cues to GC responses, we found increased expression of ICOS activation marker. Our results indicate the interactions of TAPP adapters with PI(3,4)P2 serve to restrain lymphocyte activation and limit antibody production, providing the first in vivo evidence that this interaction is important for immune function.

Phosphoinositide 3-kinase is a family of lipid kinases that function by phosphorylating the D3 position of phosphoinositide (PI) lipids generating PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. These D3 phosphoinositides regulate various cellular processes through the recruitment of effector proteins containing lipid specific pleckstrin homology (PH) domains. PI phosphatases such as PTEN and SHIP function to restrain PI3K signaling by limiting the amount of D3 PI available for binding. Deletion of either PTEN or SHIP significantly alters B cell function and humoral immune responses. TAPP1 and TAPP2 are dual PH domain containing adaptors which selectively bind the phosphoinositide PI(3,4)P2 via their C-terminal PH domains. PI(3,4)P2 is a lipid messenger generated by PI3K and through the inositol phosphatase activity of SHIP. The function of PI(3,4)P2 remains incompletely understood. To identify the functional role of TAPP-PI(3,4)P2 interactions, we utilized a knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPPs. Our study assessed the effect of PI3K dependent KI mutation on B lymphocyte development, activation and antibody production. Flow cytometry analyses of lymphoid tissues found that TAPP KI mice develop relatively normal frequencies of mature B cell populations with the exception of peritoneal B1 cells, which are increased by approximately 50%. Strikingly, TAPP KI mice developed substantially elevated serum antibody levels. TAPP KI mice were able to generate high affinity antigen-binding antibodies upon immunization with NP-OVA in alum adjuvant; however, total immunoglobulin production was markedly increased under this immunization condition. We further assessed the germinal centre (GC) response, which are known to require PI3K signaling and a hallmark of T cell dependent (TD) antibody responses. TAPP KI mice generated larger germinal centers (GC) upon immunization, which was associated with increased GC B cell survival. We further assessed whether uncoupling of TAPPs from PI(3,4)P2 alters B cell signaling and functional responses in vitro. B cells purified from TAPP KI mice were found to have altered functional responses in vitro, with significantly increased survival and cell division following antigen receptor cross-linking. Consistent with increased cell survival, TAPP KI B cells show increased Akt phosphorylation on Ser473 and Thr308 after antigen receptor cross-linking. However, reconstitution of B cell deficient mice with either WT or TAPP KI B cells was found to generate similar GC responses, suggesting that activation of other cells may contribute to the enhanced in vivo responses. Consistently, when we examined the CD4+ T follicular helper cells, a subset providing critical cues to GC responses, we found increased expression of ICOS activation marker. Our results indicate the interactions of TAPP adapters with PI(3,4)P2 serve to restrain lymphocyte activation and limit antibody production, providing the first in vivo evidence that this interaction is important for immune function.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 16, 2009). Includes bibliographical references.

Orientador: José Buratini Júnior / Resumo: Na produção in vitro (IVP), trabalha-se com uma população de oócitos heterogênea em relação ao estágio da maturação nuclear que estes oócitos se encontram, mais especificamente o estágio de vesícula germinativa (GV), uma vez que estes são obtidos de folículos em diferentes estágios de desenvolvimento. Visto que essa heterogeneidade impacta nos resultados da IVP, torna-se necessário que os processos de seleção de oócitos e de maturação in vitro sejam adequados e articulados, para que ocorra o desenvolvimento da competência oocitária para subsequente desenvolvimento. Neste estudo, objetivou-se avaliar a população de ovócitos obtida de folículos antrais grandes, testando a hipótese de que folículos dominantes saudáveis conteriam oócitos com grau intermediário de compactação da cromatina (oócitos em GV2). Em seguida, avaliou-se a população de oócitos obtida em dia aleatório do ciclo estral após OPU e testou-se o efeito de protocolo de sincronização combinando aspiração de folículos e tratamento com FSH para homogeneizar a população e controlar a qualidade dos oócitos. Os resultados sugerem que folículos dominantes saudáveis são predominantemente compostos por oócitos com níveis intermediários de compactação da cromatina e que protocolos de sincronização de aspiração do folículo combinadas ao tratamento com FSH podem ser úteis para controlar a qualidade do oócito para OPU / IVP. / Abstract: In vitro production (IVP), a heterogeneous oocyte population is employed in relation to the stage of nuclear maturation that these oocytes are found, more specifically the germinal vesicle (GV) stage, once they are obtained from follicles in different stages of development. Since this heterogeneity impacts the results of IVP, it is necessary that the processes of oocyte selection and in vitro maturation are adequate and articulated, so that occurs development of oocyte competence for subsequent development. The objective of this study was to evaluate the oocyte population obtained from large antral follicles, testing the hypothesis that healthy dominant follicles would contain oocytes with an intermediate degree of chromatin compaction (GV2 oocytes). Then we evaluated the population of oocytes obtained at random day of the estrous cycle after OPU and tested the synchronization protocol combining follicle aspiration and FSH treatment to homogenize the population and control the quality of oocytes. The results suggest that healthy dominant follicles are predominantly composed of oocytes with intermediate levels of chromatin compaction and that follicle aspiration synchronization protocols combined with FSH treatment may be useful to control oocyte quality for OPU / IVP. / Mestre

INTRODUÇÃO: Os linfomas B atualmente podem ser agrupados de acordo semelhanças moleculares e imunoistoquímicas com o linfócito do centro germinativo (CG) ou linfócito ativado (LA/pós CG), sendo este de pior prognóstico. O objetivo deste trabalho foi analisar a expressão de CD10, BCL-6 e MUM1 em pacientes portadores de LBPM e correlacionar com prognóstico. MÉTODOS: análise retrospectiva das variáveis clínicas e de tratamento de 44 pacientes portadores de LBPM. Estudo imunoistoquímico de CD10, BCL-6 e MUM1 em 29 pacientes com material disponível. RESULTADOS: idade mediana foi de 28 anos e 70% eram do sexo feminino. A positividade para CD10, BCL-6 e MUM1 foi de: 24%, 65% e 58%. De acordo com o modelo de Hans, 38% foi classificado como CG e 62% como pós CG. A sobrevida global em 5 anos e sobrevida livre de doença foi de 47% e 81%, respectivamente. Resposta Completa após quimioterapia de primeira linha (p=0,0001), radioterapia de mediastino (p=0,004) e IPI (0,039) tiveram associação com a sobrevida. A positividade para MUM1 esteve associado a pior sobrevida global (p=0,014). Aplicando o modelo de Hans não foi observada nenhuma associação com sobrevida. Na análise multivariada apenas Resposta (RR 4,28 (IC 95% 1,3-13,6) e MUM1 (RR 3,54 (1,1-11,5) correlacionaram com a sobrevida. CONCLUSÃO: Para este grupo de pacientes com características clínicas homogêneas, resposta completa e expressão de MUM1 estiveram associados à sobrevida. A classificação deste linfoma em CG e pós-CG utilizando CD10, BCL-6 e MUM1 não se correlacionou com evolução. Estudos futuros com casuística maior são necessários para melhor definir os fatores prognósticos do LBPM / INTRODUCTION: Primary Mediastinal Large B Cell Lymphoma (PMLBCL) is a distinct clinico-pathologic entity that differs from other Diffuse Large B Cell Lymphomas (DLBCL). Classification of DLBCL in GC and post-GC according can identify two subgroups of lymphomas with distinct prognosis. The aim of this study is to analyze the expression of CD10, BCL-6 and MUM1 in PMLBCL and correlate with prognosis. METHODS: retrospective analysis of clinical variables of 44 patients with PMLBCL and expression of CD10, BCL- 6 and MUM1 in 29 patients with available tissue. RESULTS: median age was 28 years and 70% of the patients were female. CD10, BCL-6 and MUM1 was positive in 24%, 65% and 58%, respectively. According to Hans classification, 38% were classified as GC and 62% as post-GC. Five year OS and DFS was 47% and 81%, respectively. In univariate analysis Complete Response (p=0.0001), Radiation therapy (p=0.004), IPI (0.039), and MUM1 expression (0.014) correlated with OS. No correlation was seen with Hans classification and survival. CONCLUSION: for this group of patients with homogeneous clinical features, response to therapy and MUM1 expression were associated with prognosis. The Hans algorithm proposed for aggressive lymphomas was not a predictive tool for survival in PMLBCL. Further studies are necessary to validate our finding and identify better prognostic variable for PMLBCL