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Orientador: Jose Andres Yunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-14T02:57:03Z (GMT). No. of bitstreams: 1 Ganazza_MonicaAparecida_M.pdf: 2462267 bytes, checksum: 471302ae0756c283d944a7545a0ff930 (MD5) Previous issue date: 2009 / Resumo: A leucemia linfóide aguda (LLA) é o câncer mais comum da infância. Os atuais protocolos de tratamento da LLA levam à cura em 70% dos casos e parte do sucesso se deve à aplicação de diferentes tratamentos para os pacientes estratificados em diferentes grupos de risco, segundo fatores prognóstico pré-tratamento (contagem leucocitária e idade ao diagnóstico). Contudo, pacientes considerados em remissão podem apresentar conteúdo substancial de células neoplásicas, chamada doença residual mínima (DRM), cuja proliferação está associada com a recaída da doença e que podem estar em níveis indetectáveis pelas técnicas convencionais de análise morfológica. Vários trabalhos têm mostrado que é possível prever a evolução clínica dos pacientes com base na DRM, porém, no protocolo do Grupo Brasileiro de Tratamento da Leucemia Infantil (GBTLI), a DRM não é utilizada como critério de realocação dos pacientes nos grupos de risco. Desta forma, o presente estudo objetivou (1) obter dados prospectivos de DRM com o uso de reação em cadeia da polimerase (PCR) para detecção de rearranjos de genes de imunoglobulina (Ig) e receptores de células T (TCR); (2) comparar resultados de DRM com fatores prognósticos ao diagnóstico e de resposta utilizados nos protocolos do GBTLI e (3) avaliar o valor preditivo da DRM em pacientes tratados pelo protocolo GBTLI-99. No total, foram estudadas 91 amostras de LLA pediátrica classificadas e tratadas pelo protocolo GBTLI-99. Duas metodologias foram empregadas, metodologia do grupo brasileiro (91 casos) e metodologia Biomed-1 (78 casos), as quais são baseadas em PCR com primers consenso para as regiões VDJ das imunoglobulinas e TCR seguido por análise de formação de homo/heteroduplex. Ambos métodos mostraram-se tecnicamente factíveis, reprodutíveis e capazes de detectar marcadores moleculares para a análise da DRM na maioria dos casos, a saber, 2 ou mais marcadores de DRM em 85,7 e 76,9% do total das amostras analisadas, respectivamente. As mesmas metodologias foram aplicadas para a análise da DRM nos dias 14 e 28 da terapia de indução e demonstraram concordância de 96% na detecção da DRM. Gênero masculino, idade <1 e _ 9 anos, imunofenótipo LLA-T, classificação em alto risco, ausência de CD10, presença de blastos no dia 14, presença de mais de 5% de blastos leucêmicos na medula óssea (MO) do D14 e resposta ao tratamento associaram-se com DRM positiva no D14. Já DRM positiva no D28 mostrou estar associada com imunofenótipo LLA-T, ausência de calla (CD10), presença de mais de 5% de blastos leucêmicos na MO no D28 e resposta ao tratamento. O valor preditivo da DRM, nas condições testadas, mostrou-se pouco útil para casos classificados como sendo de risco baixo pelo GBTLI/99. Para casos de alto risco, entretanto, a ausência de DRM no D14 caracteriza um grupo de pacientes com sobrevida similar a pacientes de risco básico. Novos protocolos para análise da DRM foram propostos baseados nas metodologias aplicadas no presente estudo visando otimizar o tempo e minimizar os custos / Abstract: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. The recent ALL treatment protocols can achieve the complete remission in 70% of cases and this success is due to different treatments for patients stratified into different risk groups , according to pre-treatment prognostic factors (white count cells and diagnosis age). Therefore, patients considered in remission may have substantial contents of neoplasic cells, the minimal residual disease (MRD). The proliferation of such neoplasic cells is associated with disease relapse and they can be undeteced by conventional methods. It has been demonstrated that the clinical evolution of patients based on MRD can be forecasted. Therefore, in the Brazilian Group for Childhood Leukemia Treatment (GBTLI), MRD is not used as a reallocation criterion of patients in risk groups. The present study aimed to (1) obtain MRD data through the use of polymerase chain reaction (PCR) for the detection of immunoglobulin (Ig) and T cell receptor genes rearrangements (TCR); (2) compare the results of MRD with prognostic factors at diagnosis and response factors used in the GBTLI protocols and (3) estimate the predictive value of MRD in patients treated by the GBTLI-99 protocol. In total, 91 samples of pediatric ALL classified and treated with the GBTLI-99 protocol were studied using two methodologies: Brazilian group methodology (91 cases) and Biomed-1 methodology (78 cases), both using the consensus PCR primers for the immunoglobulin and TCR VDJ regions followed by homo/heteroduplex formation. These methods were feasible, reproducible and able to detect molecular markers for MRD analysis in most cases, since they provided the detection of two or more markers in 85,7% and 76,9% of the samples analyzed, respectively. The same methodologies were applied in the MRD analysis on days 14 and 28 of induction therapy and the results are in agreement in 96% of the MRD detection. Male, age (<1 e _ 9 years), immunophenotyping (T-ALL), high risk classification, calla (CD10) absence, presence of blasts in peripheral blood on day 14, presence of more than 5% of leukemic blasts in the bone marrow (BM) on day 14 of treatment response were associated with positive MRD on D14. Already, positive MRD on day 28 of treatment were associated with immunophenotyping (T-ALL), CD10 absence, presence of more than 5% of leukemic blasts in the BM on D28 and treatment response. MRD predictive value on the conditions tested proved to be useful for the patients classified by GBTLI/99 as basic risk. However, for high risk cases, the absence of MRD on D14 defines a new group of patients with similar survival to basic risk patients. New protocols to the MRD analysis were proposed based on methodologies applied in this study to optimize the time and minimize the costs / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Introdução: A micose fungóide foliculotrópica (MFF) é subtipo de linfoma cutâneo de células T que atinge especialmente o folículo piloso e parece ter prognóstico mais reservado. Informações clínicas sobre a população acometida por linfomas cutâneos no Brasil são escassas. O fenômeno de imunoprivilégio (IP) diz respeito à habilidade de alguns órgãos em permanecer protegidos contra reações inflamatórias. Tem sido sugerido que o folículo piloso normal represente um local de IP. Nesse estudo aventou-se a possibilidade de haver uma quebra no equilíbrio desse fenômeno na MFF, com alteração na expressão de moléculas do complexo maior de histocompatibilidade (MHC) e na expressão de MHC não-clássicos (HLA-G), com algum papel no mecanismo do foliculotropismo. Os objetivos foram: descrever o perfil clínico-epidemiológico de paciente com MFF, descrever a histologia e imunofenótipo dos casos de MFF e investigar os mecanismos envolvidos na predileção dos linfócitos atípicos pelo folículo piloso. Metodologia: Os prontuários de pacientes com diagnóstico de MFF provenientes do ambulatório de Linfomas Cutâneos da Faculdade de Medicina da Universidade de São Paulo (FMUSP) foram revisados (n=33). O material histológico de biópsias de pele dos pacientes com MFF provenientes dos ambulatórios de Linfomas Cutâneos da FMUSP e da Northwestern University foi analisado por meio de escala semi-quantitativa (n=43). Na coloração de hematoxilina-eosina foram avaliados os seguintes parâmetros: infiltrado neoplásico epidérmico, infiltrado neoplásico dérmico, presença de acantose/espongiose, de mucinose folicular, de fibroplasia do tecido conjuntivo, de eosinófilos, de plasmócitos, o tamanho celular e o grau de dano folicular. Analisou-se a positividade do infiltrado neoplásico para os seguintes marcadores celulares: CD1a, CD56, TIA-1 e CD117. As expressões do complexo de histocompatibilidade HLA-G e do MHCII no infiltrado celular e no epitélio folicular foram investigadas no grupo de pacientes com MFF e comparadas com o grupo de pacientes com micose fungóide clássica (MFC) e pele normal. A expressão do complexo de histocompatibilidade MHCII também foi investigada na epiderme. Resultados: A mediana das idades ao diagnóstico foi de 46 anos com 61% dos pacientes classificados como portadores de estágio avançado. A proporção entre homens e mulheres foi de 1,54 e a mediana de duração de doença antes do diagnóstico foi de três anos. Ao final de três anos de acompanhamento, 67% dos casos estavam vivos com a doença. O prurido foi relatado em 82% dos casos. Histologicamente, encontrou-se associação entre a presença de eosinófilos e de plasmócitos com fibroplasia do tecido conjuntivo. Observou-se diminuição da expressão do HLA-G no epitélio folicular nos grupos MFF e MFC em relação à pele normal. Observou-se aumento da expressão do MHCII no epitélio folicular na MFF em comparação à pele normal e na epiderme na MFC quando comparada à MFF. Conclusões: Dados clínicos da população estudada assemelharam-se aos dados da literatura como estágio avançado ao diagnóstico e prognóstico reservado. Cerca de metade dos casos de MFF foi positiva para o marcador citotóxico TIA-1. Demonstrou-se haver um provável colapso do imunoprivilégio folicular nos linfomas cutâneos com expressão diminuída de moléculas HLA-G em comparação à pele normal. O aumento da expressão do MHCII poderia relaciona-se com o foliculotropismo na MFF e com o epidermotropismo na MFC / Introduction: Folliculotropic mycosis fungoides (FMF) is a subtype of cutaneous T cells lymphoma affecting mainly the hair follicle and seems to have a less favorable prognosis. Clinical information on the population affected by cutaneous lymphomas in Brazil is scarce. The immune privilege (IP) phenomenon involves the ability of some body sites remaining protected from inflammatory reactions. It has been suggested that normal hair follicle represents an IP location. We hypothesized that a collapse of this phenomenon would occur in FMF, with changes in the expression of classical major histocompatibility molecules (MHC) and in the expression of nonclassical MHC molecules (HLA-G) with a role in folliculotropism mechanism. The objectives of this study were to describe the clinical and epidemiological profile of patients with MFF, describe the histology and immunophenotype of cases of MFF and investigate the expression of MHC molecules. Methods: The medical records of patients from the outpatient Cutaneous Lymphoma Clinic of the University of Sao Paulo Medical School (FMUSP) diagnosed with MFF were reviewed (n = 33). The histological material from skin biopsies of patients with MFF from the Cutaneous Lymphomas Clinic of FMUSP and Northwestern University was stained and evaluated by semi-quantitative scale. In hematoxylin-eosin staining the following parameters were evaluated: epidermal neoplastic infiltrate, dermal neoplastic infiltrate, acanthosis/spongiosis, follicular mucinosis, connective tissue fibroplasia, presence of eosinophils and plasma cells, cell size and degree of follicular damage. We analyzed the positivity of the neoplastic infiltrate for the following cellular markers: CD1a, CD56, TIA-1, and CD117. Finally, the expression of histocompatibility complex HLA-G and MHC II in the neoplastic infiltrate and the follicular epithelium was investigated in MFF group and compared to patients with classical mycosis fungoides (CMF) and to normal skin. MHCII expression in the epidermis was also investigated. Results: The median age at diagnosis was 46 years, with 61% classified as advanced stage disease. The ratio between men and women was 1.54, the median disease duration before diagnosis was three years. After a median time of follow-up of three years, 67% of the cases were alive with disease. Pruritus was reported in 82% of the cases. Histologically, an association between the presence of eosinophils and plasma cells with fibroplasia of collagen was found. There was a decrease of HLA-G expression in the follicular epithelium in MFF and CMF groups compared to normal skin. There was an increase of MHCII expression in the follicular epithelium in FMF group compared to normal skin. There was an increased MHCII expression in the epidermis in CMF compared to FMF. Conclusions: Clinical data from the studied population were similar to the previous literature in relation to advanced stage at diagnosis and prognosis. There was a relationship between the presence of eosinophils and plasma cells in neoplastic infiltrate and the connective tissue fibrosis. Near half of the cases of FMF was positive for the cytotoxic marker TIA-1. A possible hair follicle immune privilege collapse was suggested by a decreased expression of HLA-G molecules in FMF and CMF compared to normal skin. Increased MHCII expression appears to be involved in the folliculotropism of FMF and epidermotropism of CMF

Tick-borne encephalitis virus is the cause of much morbidity and sometimes a fatal infection. A vaccine based on formaldehyde inactivated virus is currently the only available way of preventing disease. This vaccine gives a high rate of seroconversion but there are reports of vaccination breakthrough, even in people who have demonstrated a neutralizing antibody response. The T cell response to inactivated TBE vaccine is largely unknown, but could be of importance for the effect of the vaccine. This study characterizes aspects of the T cell response by investigating the expression of two transcription factors, T-bet and GATA-3 with RT-PCR. T-bet is expressed in CD4+ T cells of the Th1 type, while GATA-3 is expressed in CD4+ T cells of the Th2 type. Our data show that vaccination with inactivated TBE vaccine leads to increase in expression of the T-bet gene when cells of vaccinated subjects are cultured with TBE virus. In contrast, the expression of GATA-3 remains unaffected by vaccination. Thus, this study suggests that the inactivated TBE vaccine leads to a Th1-like immune response in humans.

Le rôle des lymphocytes T CD4+ dans la progression des tumeurs et dans l'immunité anti-tumorale est de plus en plus reconnu chez l'homme et chez la souris. Les mécanismes effecteurs de l’immunité T CD4+ contre le cancer ont été étudiés principalement dans des systèmes de tumeurs transplantées. Dans ces modèles, de nombreuses cellules tumorales meurent au moment de l'implantation, ce qui conduit à la libération de l'antigène tumoral (Ag) dans un contexte inflammatoire. Ceci contraste avec la croissance lente et non-destructrice des tumeurs humaines aux stades précoces. Nous avons montré que la présentation de l’Ag retreint par le CMH-II DBY, libéré par des cellules tumorales mortes injectées en sous-cutané, peut persister durant plusieurs semaines dans le ganglion drainant. L’activation précoce d’une réponse immune dirigée contre l’Ag lors de l’injection de lignées tumorales peut gêner l’étude des relations entre les tumeurs et le système immunitaire. Par conséquent, nous avons généré une lignée tumorale dans laquelle l’expression de DBY peut être induite in vivo à distance de l’implantation. Nous avons également utilisé un modèle de tumeurs pulmonaires endogènes se développant dans des souris transgéniques KrasG12D, p53flox et exprimant DBY de manière spécifique. Notre objectif était d'étudier dans ces deux modèles l'histoire naturelle de la réponse des lymphocytes T CD4+ spécifiques de la tumeur. Dans le système de tumeur transplantée «Ag inductible », nous avons montré que DBY est présenté de manière efficace à des cellules naïves T CD4+ spécifiques dans les ganglions drainant la tumeur. Les réponses prolifératives et effectrices sont similaires dans les systèmes où DBY est exprimé de façon inductible in vivo ou constitutive. Le récepteur de co-stimulation ICOS, ainsi que les récepteurs co-inhibiteurs PD-1 et BTLA sont régulés positivement sur les lymphocytes T CD4+ spécifiques en réponse à l’Ag. La production de cytokines en réponse à une restimulation in vitro révèle un profil effecteur mixte TH1 /TH17. Notamment, un petit pourcentage de lymphocytes T co-expriment les marqueurs de cytotoxicité LAMP-1 et granzyme B. Ainsi, lorsqu’un Ag apparaît à distance de l'implantation de la tumeur, il n’est pas ignoré et n’induit pas de tolérance immune. D'autres mécanismes doivent être considérés pour expliquer l'absence de rejet de tumeur efficace malgré l’activation et la migration des cellules effectrices T CD4+ dans les tumeurs. Les travaux réalisés sur le modèle de tumeurs pulmonaires endogènes sont en cours. A ce stade, nous avons observé que, comme dans les tumeurs transplantées, l’Ag est présenté aux LT CD4+ naïfs qui prolifèrent dans le ganglion drainant et ne se différencient pas en cellules T régulatrices, même aux stades très avancés de la maladie. La capacité des tumeurs à induire une réponse T effectrice semble liée à leur stade de développement. Durant la phase tumorale précoce, les LT spécifiques de DBY produisent de l’IFN-γ et du granzyme B. En revanche, des LT spécifiques produisant de l'IL-17 sont retrouvés dans les poumons de souris ayant des tumeurs invasives de stade terminal. Bien que l'IL-17 puisse favoriser la progression des tumeurs, y compris dans les modèles induits par l’oncogène KrasG12D, de nouvelles données suggèrent que les cellules effectrices TH17 possèdent un haut degré de plasticité et peuvent présenter une activité anti-tumorale. Notre modèle pourrait être utile pour tester de nouvelles stratégies de ciblage de l’IL-17 dans l'immunothérapie du cancer. / The role of CD4+ T cells in both tumor progression and immunity is being increasingly acknowledged in humans and mice. CD4+ T cell immunity against cancer has been mostly studied using murine transplanted tumor systems. In these models, many tumor cells die at the time of surgical implantation, leading to the release of tumor antigen (Ag) in an inflammatory context. This contrasts with the slow and non-destructive growth of early stage human tumors. Here, we show that the presentation of a MHC class II-restricted model (male, DBY) Ag released by dying fibroblastic tumor cells may last more than 3 weeks in the tumor draining lymph node (dLN). This artificial, early and long lasting priming precludes the study of the interactions between the immune system and tumors at the steady state. We therefore generated a cell line that could be induced to efficiently express DBY as a neoAg after implantation. We also took advantage of a previously described mouse model of genetically engineered, KrasG12D p53flox lung adenocarcinoma to generate a “spontaneous” tumor model expressing DBY. Our aim was to study in these two models the natural history of the tumor-specific CD4+ T cell response. In the transplanted tumor system, we show that the Ag reaches the dLNs and is efficiently presented to naïve specific CD4+ T cells. The proliferative and effector responses were similar in the inducible and constitutively expressed Ag tumor systems. The ICOS co-stimulatory receptor, and the PD-1 and BTLA co-inhibitory receptors were upregulated on the Ag specific CD4+ T cells in the dLN. We did not observe de novo induction of tumor-specific regulatory T cells. Finally, the pattern of secreted lymphokines in the dLN, spleen and tumor after in vitro Ag restimulation was similar, with a mixed TH1/TH17 response. Notably, a small percentage of DBY-specific effector T cells also displayed a cytolytic phenotype marked by the co-expression of granzyme B and LAMP-1. Thus, when the neo-Ag appears at distance of tumor implantation, the tumor was not ignored and did not induce tolerance of naïve CD4+ T cells. Other mechanisms have to be thought to explain the absence of tumor rejection despite efficient priming and migration of effector CD4+ T cells into tumors. Similarly to the strong proliferative response mentioned above, the DBY tumor Ag was efficiently presented in LNs draining “spontaneous” lung tumors, and induced activation and proliferation of adoptively transferred naive T cells. After priming they did not convert into Tregs, even in end-stage disease. This work is still ongoing, but preliminary results show that activated DBY-specific T cells from the dLN and the lungs produced IFN-γ and granzyme B during early stages of the disease. In contrast, IL-17 secreting cells were found exclusively in the lungs from mice with late-stages invasive tumors. Although IL-17 may enhance tumor progression, including in models driven by the Kras oncogen, emerging data strongly suggest that TH17 effector cells demonstrate a high grade of plasticity and can display anti tumor activity. Little is known about the antigen specificity of IL-17 production in lung cancer patients, and our model could be useful to test new strategies targeting either positively or negatively tumor Ag-specific TH17 cells in cancer immunotherapy.

Le cancer du sein représente un problème de santé publique. Les efforts déployés pour définir de nouvelles stratégies thérapeutiques ont mis en lumière les mécanismes développés par la tumeur pour inhiber l'instauration d'une réponse immune adaptative efficace. La tumeur favorise la mise en place d'une immunosubversion, caractériisée par la fort infiltration d'une sous-population de lymphocytes T CD4 régulateurs (Treg). Les Treg jouent un rôle central dans les mécanismes de contrôle de réactions pro-inflammatoires et protègent contre le développement de pathologies auto-immunes. Nous avons observé que le Treg sont recrutés spécéfiquement dans l'environnement tumoral via l'axe de chimiotactisme CCR4/CCL22 suite à la détetion des cellules transformées par les NK et les macrophages. D'autre part, plusieurs publications récentes démontrent un rôle des cellules dendritiques plasmacytoïdes (pDC) dans l'indcution de Treg chez l'homme et la souris. Nos observations montrent que des facteurs solubles sécrétés dans l'environnement tumoral inhibent la fonction clef des pDC, ce qui favorise fortement leur capacité à induire l'expansion de Treg et de T CD4 prodcuteur d'IL-10. Ces cellules T immunosuppressives expriment fortement le récepteur de co-simulation ICOS, prolifèrent in situ, exercent un important pouvoir supresseur sur les autres popuylations T et leur présence est associées à un impact péjoratif sur la survie des patientes. Par l'utilisation de l'Ac bloquant anti-COS 314.8 nous avons démontré le rôle essentiel d'ICOS dans la prolifération des Treg et l'induction de T CD4 sécréteurs d'IL-10 par les pDC dasn les tumeurs, offrant la perspective d'une nouvelle immunothérapie visant à éradqiuer les Treg intra-tumoraux. / Tumor immunosbversion favors disease progression and is mediated by increased IL-10 secretion, reduced type-I IFN production and regulatory T cell (Treg) accumulation among CD4+ T cell in breast tumor. We showed that the presence of high number of both Treg and/or plasmacytoid DC (pDC) a subpopulation of antigen presenting celles correlates with poor prognosis in breast carcinoma. We previoously demonstrated that CCR4+ Treg are recruited from the periphery trough CCL22 production by breast tumor cells. Tumor-asssociated Treg (Ta-Treg) are highly activated (GITRhighHDLA-DRhighCD39high), show a selective expression of high levels of ICOS and proliferate in situ (Ki-67+). Tumor associated (Ta-) pDC express a partially activated phenotype but their type-1 interferon (IFN) production is strongly impaired in human tumors. pDc secretion of type-I IFN is linked to their capacity to induce anti-viral and anti-tumor immunity in mice models. We shown that 1) Ta-Treg and Ta-pDC colocalize in breast tumor section and 2) TapDC favor Ta-Treg proliferation and IL-10 secretion by CD4+ T cells in absence of type-I IFN. Ogf importance, targeting ICOS with a neutralizing antibody suppresses Ta-Treg proliferation as well as IL-10 secretion inpDC/CD4+ T cell co-culture, demonstrationg a riole of ICOS-ICOS-L interaction in Ta-Treg proliferation mediated by Ta-pDC. At the end, we report that high ICOS expression in breast tumor sections is associated with reduced patient's overall and disease free survival. Altogether these observations suggest that ICOS in breat cancer may represent a therapeutic target to restore anti-tumor immunity

Le syndrome de Sézary (SS) est un variant leucémique et érythrodermique de lymphomes T cutanés. Il est caractérisé par la présence d’une population clonale de LT CD4+, présentant un noyau cérébriforme atypique, dans la peau, les ganglions lymphatiques et le sang périphérique. Après un bilan clinique, le diagnostic de cette pathologie est confirmé par l’analyse immunohistochimique d'une biopsie cutanée. Néanmoins, la cytomorphologie des cellules de Sézary circulantes n’est pas uniquement associée au SS. Notre laboratoire a identifié CD158k comme marqueur membranaire spécifique des cellules de Sézary. Ce récepteur offre un intérêt dans le diagnostic du SS et dans le suivi de l’évolution de la pathologie. Ainsi, nos résultats montrent qu’un immuno-marquage CD3+ CD158k+, analysé en cytométrie en flux, est une technique spécifique et sensible de détection de la cellule de Sézary par rapport à la cytomorphologie. Alors que dans plus de 30% des cas le SS passe inaperçu durant l’examen cytomorphologique, une analyse en cytométrie en flux permet la mise en évidence de cellules tumorales résiduelles. La présence systématique de CD158k à la surface des cellules de Sézary nous a conduit à rechercher l’expression d’autres KIRs. Sur les lymphocytes tumoraux circulants d’un patient ainsi que sur la lignée cellulaire correspondante, l’expression des formes activatrices et inhibitrices des récepteurs CD158a/h et CD158b/j est détectée. A la différence des lymphocytes NK et T CD8+, le récepteur présentant une fonction inhibitrice (KIR-L) ne l’emporte pas sur celui ayant une fonction activatrice (KIR-S) dans la cellule de Sézary. En fait, les KIR-L, à l’exception de CD158k, sont trouvés non fonctionnels dans la cellule tumorale. Ainsi, l’engagement des formes activatrices CD158h ou CD158j permet une régulation positive de la voie de signalisation CD3-dépendante de JNK et de la prolifération tumorale. Une étude fonctionnelle de la population T CD4+ KIR+, équivalent normal de la cellule de Sézary, a aussi été réalisée. Nous avons mis en évidence une expression préférentielle de la forme activatrice ou inhibitrice des récepteurs KIR homologues, selon le donneur. D’autre part, les KIRs activateurs ou inhibiteurs, exprimés à la surface des LT CD4+, jouent un rôle de co-récepteur vis-à-vis du TCR. Ainsi, une régulation positive ou négative de la prolifération et de la voie de signalisation CD3-dépendante de ERK est observée en fonction du type de récepteur co-engagé. Il est bien établi que les KIR-S s’associent à la molécule adaptatrice KARAP/DAP12 pour la transduction d’un signal d’activation. Dans les cellules T CD4+ saines et tumorales, la protéine recrutée par ces récepteurs est encore non identifiée. Notre étude sur la population T CD4+ CD158j+ de sujets sains montre l’implication de la protéine HS1 dans la signalisation mise en place par le récepteur KIR activateur. La réalisation de ce travail a permis de mieux comprendre les mécanismes mis en place à partir des KIRs dans les cellules T CD4+. Ce travail ouvre de nouvelles perspectives concernant le rôle de ces récepteurs dans les mécanismes permettant l'expansion tumorale des cellules de Sézary / Sézary syndrome (SS) is a leukemic and erythrodermic variant of cutaneous T-cell lymphomas. It is characterized by the presence of a clonal CD4+ T lymphocyte population in the skin, lymphnodes and peripheral blood. After clinical assessment, diagnosis of this disease is confirmed by immunohistochemistry analysis of a skin biopsy. However, the cytomorphology of circulating Sézary cells is not just associated to SS. Our laboratory has identified CD158k as a phenotypic marker for Sézary cells. This receptor can be used in the diagnosis of the SS and in monitoring the evolution of the disease. Our results show that the CD3/CD158k immunostaining, analysed by flow cytométrie, is more specific and sensitive than cytomorphology to detect atypical circulating cells. While more than 30% of the SS is misdiagnosed by the cytomorphologic identification, flow cytometry analysis allows the detection of residual tumor cells. Given the systemic expression of CD158k on Sézary cells, we next investigated the expression of additional KIRs. On circulating malignant lymphocytes from one patient and the corresponding cell line, the expression of inhibitory and activating forms of CD158a/h and CD158b/j receptors was detected. In contrast to NK cells and CD8+ T lymphocytes, the inhibitory receptor signaling (KIR-L) does not outweigh the activating receptor signaling (KIR-S) in the Sézary cell. In fact, KIR-L, except CD158k, are found not functional in the tumor cell. Thus, CD158h or CD158j engagement results in an enhanced CD3-induced cell proliferation and JNK activation. A functional study of CD4+ KIR+ T lymphocyte population, the normal equivalent of Sézary cells, was then performed. We observed an exclusive expression of the activating or the inhibitory form of KIR receptors, depending on the donor. Activating or inhibitory KIRs, expressed on the CD4+ T cell surface, act as coreceptors. Thus, a positive or negative regulation of the CD3-induced cell proliferation and ERK activation is observed by triggering the KIR-S or -L respectively. It is well known that stimulatory KIR initiates intracellular signals through their association with the adaptor protein KARAP/DAP12. However, in normal and malignant CD4+ T cells the protein recruited by these receptors is still not identified. Our study on CD4+ CD158j+ T lymphocyte population from healthy individuals showed the involvement of HS1 protein as a potential adaptor molecule in the activating KIR signaling pathway. This work has provided insight into the mechanisms of KIRs signaling in CD4+ T cells and opens new perspectives on the role of these receptors in proliferation of Sézary cells

La régulation transcriptionelle fine assurée par les Eléments Cis Régulateurs (ECR, eg. promoteurs et «enhancers») et les facteurs protéiques associés, est à la base de la mise en place et le maintien de l'identité tissulaire. Les modifications de la chromatine corrèlent avec l’activité d’ECRs et constituent l’épigénome de la cellule. Au cours de ma thèse, je me suis intéressée aux transitions des modifications des histones (H3K4me1/me2/me3, H3K36me3, H3K27me3 and H3K9me2) accompagnant le développement précoce de la cellule T. Pour cela, j’ai utilisé un modèle murin reproduisant une étape cruciale de la thymopoïèse - la sélection β - et la technique d’Immunoprecipitation de la chromatine couplée à des puces à ADN (ChIP-chip). Au sein des enhancer connus, nos analyses ont mis en évidence une nouvelle signature épigénétique liée à leur activité. De plus, nous montrons que l'étendue d'enrichissement d’H3K4me2 au sein des régions géniques des gènes exprimés, constitue une signature épigénétique des gènes tissus spécifiques. Tout ceci a permis de mieux comprendre le rôle de l’épigénétique dans l'établissement et le maintien de l'identité cellulaire.Le traitement anti-cancer moderne est basé sur les analyses de différents marqueurs d'agressivité (MA) et par la suite, de l’établissement de la thérapie personnalisée. Durant la dernière partie de ma thèse, j’ai participé à un projet collaboratif avec le laboratoire de Thérapie Cellulaire de l’Institut Paoli Calmettes à Marseille, qui visait l’isolation des MA des Leucémies Aiguës Myéloïdes à caryotype normal (LMAcn) grâce aux études de profilage épigénétique (H3K27me3) des blastes des patients atteints de LMAcn. / Precise transcriptional regulation underlies the establishment and maintenance of cell type specific identity and is governed by dedicated DNA sequences (i.e., cis regulatory elements (CREs): eg.: promoters, enhancers) and transcription factors. Chromatin modifications (eg.: histone modifications, DNA methylation) impinge on CREs activity and constitute the epigenome of the cell.During my PhD, I was interested in the transitions of a set of histone modifications (H3K4me1/me2/me3, H3K36me3, H3K27me3 and H3K9me2), during one of the major checkpoints of thymopoiesis - the β-selection. I used a dedicated mouse model and Chromatin Immunoprecipitation coupled with microarrays (ChIP-chip) technique. Our data evidenced a previously unappreciated epigenetic signature linked to enhancer activity. In parallel, computational analyses of the patterns of gene body enrichment of H3K4me2 highlighted an epigenetic signature linked to the regulation of the tissue specific gene expression. Altogether, this enabled to deepen the relationship between chromatin states and regulation of cell type specific identity.Modern anticancer treatment is based on the analyses of a number of cancer aggressiveness markers (CAM) and results in a highly personalized therapy. Epigenetic profiling can constitute a powerful tool for CAM’s isolation. In the second part of the presented work, I participate in a collaborative project (with Cellular Therapy Centre at the Paoli Calmettes Institut, Marseille) aiming to isolate new CAM for Acute Myeloid Leukemia with normal karyotype (AMLnc) patients. For this purpose I performed epigenetic (H3K27me3) profiling of blasts of AMLnc.

La nature des cellules dendritiques (DC) engagées lors d'une vaccination conditionne la qualité de la réponse immunitaire adaptative. L'immunisation par la peau est particulièrement efficace car elle cible de nombreuses sous-populations de DC cutanées telles que les cellules de Langerhans (LC). Cependant, les relations entre ces DC et les cellules effectrices associées à la réponse humorale ne sont pas connues. L'objectif de ma thèse est d'identifier les mécanismes cellulaires précoces impliqués dans l'initiation de la réponse humorale, dans un contexte de vaccination intradermique (i.d.) avec un antigène particulaire. En étudiant la distribution spatiale et temporelle des particules synthétiques de PLA adsorbées par la protéine p24 du VIH, nous avons observé leur prise en charge par les DC cutanées mais également par les DC résidentes des ganglions drainant de la peau. Cependant, l'étude de la réponse immunitaire a démontré que seules les cellules cutanées, et en particulier les LC, induisent la polarisation des lymphocytes T CD4+ folliculaires (TFH) et le développement des lymphocytes B sécrétant des IgA. L'immunisation i.d. a également généré l'infiltration de cellules inflammatoires au niveau du site d'injection et du ganglion. En utilisant un modèle murin Ccr2-/-, nous avons démontré que les cellules dépendantes de CCR2+ interfèrent avec la formation des TFH. Enfin, l'étude du micro-environnement ganglionnaire suggère que TNF est favorable à la polarisation des TFH. En conclusion, ces résultats soulignent l'importance de cibler les DC cutanées lors de la vaccination afin de proposer de nouvelles stratégies vaccinales. / The quality of the adaptive immune response to a vaccine is driven by the nature of dendritic cells (DCs) engaged during vaccination. Skin immunization is particularly efficient as it targets the numerous cutaneous DCs, including Langerhans cells (LCs). However, the relationship between DCs and effector cells associated with humoral immunity has not been elucidated. The main objective of my thesis was to identify cellular mechanisms implicated in the initialization of the humoral immune response, in the context of intradermal (i.d.) vaccination with particle-based antigens. In examining the spatial and temporal distribution of synthetic PLA particles adsorbed with the HIV-p24 protein, we observed their uptake by both cutaneous DCs and also skin-draining lymph node (dLNs) resident DCs. However, our immune response study highlighted that only skin cells, and in particular LCs, were able to stimulate polarization of follicular helper T cells (TFH) and the development of IgA-secreting B lymphocytes. I.d. vaccination also induced an inflammatory cell infiltration at both the injection site and in dLNs. Using a Ccr2-/- mouse model, we have shown the CCR2+ dependant cells can interfere in TFH polarization. Finally, the study of the dLN micro-environment suggested TNF can promote TFH formation. In conclusion, these findings highlight the importance of targeting skin DC in vaccination to propose new vaccine strategies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22341号 / 医博第4582号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 羽賀 博典, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM