Cytomegaloviruses (CMVs) are ubiquitous in nature, having evolved over many millenia with their hosts. While in healthy hosts most infections with CMV are asymptomatic, the virus can cause severe disease in immunocompromised hosts. Thus, the increase in organ transplantation and the HIV/AIDS pandemic have established human CMV (HCMV) as a clinically important pathogen. Indeed, HCMV infections are now the major cause of morbidity and mortality among immunocompromised patients, which has led to more research targeting CMV for effective anti-viral treatment. The discovery that cytomegaloviruses encode several genes which are involved in immune escape has prompted a new area of research, aimed at understanding immune escape mechanisms for exploitation as potential anti-viral therapeutics. By targeting the viral proteins directly, or their receptors in the host, it may be possible to treat CMV disease by agonistic/antagonistic therapy. The first part of this thesis describes the first demonstration of anti-NK1.1 staining in situ to identify NK cells using a modified in vivo perfusion/fixation method. Using this method, we have compared the acute NK1.1+ cellular response to wild-type MCMV infection in the visceral organs of genetically susceptible intra-NK complex recombinant BALB.B6-CT6 (Cmv1s, NK1.1+) mice with resistant C57B⁄J (Cmv1r, NK1.1+) and BALB.B6-Cmv1r mice (Cmv1r, NK1.1+). Expression of viral antigens and the consequences of infection on other cellular subsets, were also analyzed in this study. The data show that in susceptible mice (Cmv1s) MCMV infection is predominent in the marginal zone of splenic white pulp, resulting in local changes in various cellular constituents, including macrophages, NK cells and DC. In the liver, distinct foci of infection were comprised of large numbers of macrophages and NK1.1+ cells surrounding infected cytomegalic cells. In resistant mice (Cmv1r), 6 MCMV infection predominantly affected the red-pulp of the spleen and was associated with increased accumulation of NK1.1+ cells and macrophages at sites of viral infection

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

INTRODUÇÃO: A psoríase é doença inflamatória hiperproliferativa da pele, na qual mecanismos imunológicos são cruciais para o processo patogênico. O marcador CD57 denota inabilidade de replicação e imuno-senescência de células T CD8+, e sua expressão foi demonstrada em diversas condições inflamatórias. CD57 também pode ser expresso por células natural killer (NK), nas quais é considerado marcador de maturidade, por ser em geral adquirido pelas formas mais diferenciadas CD56+CD16+. A expressão de CD57 e outros receptores de células NK não foi amplamente investigada na psoríase. OBJETIVOS: Este estudo buscou examinar o fenótipo de células NK em biópsias de pele e células mononucleares do sangue periférico (CMSP) de pacientes com psoríase em relação a controles sadios. Este estudo investigou também o fenótipo e características funcionais de células T isoladas da pele lesional e não afetada de pacientes com psoríase. MÉTODOS: Foram isoladas células NK dos subtipos CD56+CD16- e CD56+CD16+ de pele lesional, não afetada e CMSP de pacientes com psoríase, comparadas com pele normal e CMSP de controles sadios. A expressão de CD57, NKG2A e NKG2C foi determinada nesses subtipos de células por citometria de fluxo. Células T CD4+ e CD8+ foram isoladas da pele lesional e não afetada de pacientes com psoríase, e a expressão de CD57 foi avaliada. Características funcionais de células T foram estudadas através da análise da secreção de diversas citocinas inflamatórias (IL-17A, IFN-\", IL-2, IL-33, TNF- #, IL-21, IL-22 and IL-27) produzidas por células T CD4+ e CD8+ isoladas por sorting celular, a partir de amostras de pele lesional e não afetada de pacientes com psoríase. RESULTADOS: Células NK isoladas das lesões de psoríase apresentaram um fenótipo particular, caracterizado por baixa expressão de CD57 e alta expressão de NKG2A na pele lesional e não afetada em relação aos controles. Em relação às células T, encontrouse frequência de células T CD4+CD57+ e CD8+CD57+ significativamente maior na pele não afetada em relação à pele lesional de pacientes com psoríase. Células T CD4+ isoladas por sorting celular a partir de amostras de pele lesional produziram níveis maiores de IL-17A, IL-22 e IFN-\" em relação às amostras de pele não afetada. Células T CD8+ isoladas da pele lesional secretaram maiores níveis de IL-17A, IFN-\", TNF-# e IL- 2 em relação à pele não afetada. CONCLUSÕES: Esses dados sugerem que células NK presentes nas lesões de psoríase apresentam fenótipo imaturo, que foi previamente associado a maiores capacidades funcionais, e poderiam ser implicadas na patogênese da psoríase. Em relação às células T, as características fenotípicas sugerem menor sobrevivência de células com baixa capacidade replicativa na pele lesional, pelo ambiente inflamatório local ou pelo alto turnover celular da psoríase / INTRODUCTION: Psoriasis is a hyper-proliferative inflammatory disease of the skin in which immunological mechanisms play a direct role in disease pathogenesis. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and its expression is increased in a number of inflammatory conditions. CD57 is also expressed by NK cells and is considered a marker of NK cell maturity, being acquired by more differentiated CD56+CD16+ NK cells. The expression of CD57 and other NK cell markers in psoriasis has not been thoroughly investigated. OBJECTIVES: This study sought to examine the phenotype of NK cells in skin biopsies and peripheral blood mononuclear cells (PBMC) from patients with psoriasis and healthy controls. We also investigated the phenotype and functional characteristics of T cells from psoriasis patients, comparing lesional and unaffected skin. METHODS: CD56+CD16- and CD56+CD16+ NK cells were isolated from lesional skin, unaffected skin and PBMC of psoriasis patients, and normal skin and PBMC from healthy controls. The expression of CD57, NKG2A, and NKG2C was assessed by flow cytometry. CD57 expression was also determined on T cells from lesional and unaffected skin by flow cytometry. We assessed functional characteristics of T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-\", IL- 2, IL-33, TNF-#, IL-21, IL-22 and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin of psoriasis patients, by multiplex assays. RESULTS: NK cells in psoriasis skin lesions exhibited a distinct phenotype, with CD57 expression significantly reduced and NKG2A expression increased on NK cells in lesional and unaffected skin compared to controls. In relation to T cells, we observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly increased in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriasis lesional skin produced higher levels of IL-17A, IL-22 and IFN-\" compared to unaffected skin. CD8+ T cells isolated from lesional skin produced higher levels of IL- 17A, IFN-\", TNF-# and IL-2 compared to unaffected skin. CONCLUSIONS: These data suggest that NK cells in psoriasis lesions exhibit an immature phenotype, that has been previously associated with higher functional abilities, and could implicate NK cells in psoriasis pathogenesis. For T cells, the findings of this study suggest lower survival of cells with low replicative ability in lesional skin, due to the local inflammatory environment or to the high cellular turnover in psoriasis

Orientador: Aureo Tatsumi Yamada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T22:06:48Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_M.pdf: 2003445 bytes, checksum: 597310400704b8df5b1e07544bd6caca (MD5) Previous issue date: 2008 / Resumo: Durante a gestação em animais que possuem placentação hemocorial, a hipóxia no primeiro terço da prenhez é um dos fatores cruciais para indução da angiogênese e o adequado desenvolvimento da placenta. Contudo, esta hipóxia se contrapõe à intensa atividade das células que requerem elevado metabolismo, gerando um estresse fisiológico para estas células presentes na interface materno-fetal. Presume-se que estas células necessitem de mecanismos apropriados de citoproteção para sua sobrevida enquanto comprometidos ativamente no suporte funcional do útero gestante. Neste sentido, o presente trabalho teve como objetivo investigar a expressão e a distribuição da proteína de choque térmico 70 (HSP70) na interface materno-fetal durante a gestação normal em camundongos e a sua possível variação em condição de estresse adicional induzido experimentalmente através da lesão embrionária. Sítios de desenvolvimento embrionário/fetal de camundongos prenhes do dia de gestação (dg) 6 ao 17 e, após 30 minutos, 1, 6 e 12 h dos animais submetidos à lesão cirúrgida do embrião (LCE) no dg 9 foram coletados para: - processamento histotécnico convencional de embebição em parafina destinados às análises citoquímicas (lectina DBA e reação de TUNEL) e imunocitoquímicas (anti-HSP72/73, anti-PCNA); - embebição em resina Lowilcryl- K4M para imunocitoquímica ultraestrutural (anti-HSP72/73); - obtenção de homogenados teciduais destinados à SDS-PAGE das frações protéicas e Westernblot (anti-HSP72/73) e, - extração de RNA de homogenados teciduais e de células uNK isoladas para análise de transcritos (HSP72 e 73) com amplificação pelo RTPCR. As análises imunocitoquímicas demonstraram que as células uNK eram as únicas células que expressavam de forma constante as isoformas HSP72/73 ao longo da gestação, sendo confirmada a expressão dos transcritos gênicos das isoformas HSP72/73 nas células uNK isoladas pelo RT-PCR. A imunomicroscopia eletrônica detectou marcação conspícua nas mitocôndrias das células uNK. A análise quantitativa demonstrou que a lesão do embrião reduz o número de células uNK positivas para HSP72/73 e, o SDS/PAGE/Western-blotting identificou as isoformas HSP72 e 73 presente nos homogenados teciduais do útero com uma perceptível redução na intensidade da banda correspondente ao HSP73 nas amostras de pós-lesão, sem afetar significativamente a isoforma HSP72. As análises realizadas com a dupla marcação de TUNEL e PCNA demonstrarm redução de células uNK PCNA positivas no útero submetido a lesão embrionária e aumento de núcleos marcadas positivamente pelo TUNEL. Estes resultados demonstram de forma inédita a expressão de HSP72/73 nas células uNK, sendo inédita também a constatação em leucócitos, sugerindo um papel citoprotetor para estas células importantes na manutenção da gestação. A redução de células uNK HSP72/73 positivas no útero gestante desencadeada pela lesão embrionária, consubstancia a hipótese da atuação da HSP 72/73 como chaperona citoprotetora nas células uNK sendo crítica a atuação da isoforma HSP73 presente na mitocôndria através da regulação negativa das vias de morte celular por apoptose nas células uNK / Resumo: Durante a gestação em animais que possuem placentação hemocorial, a hipóxia no primeiro terço da prenhez é um dos fatores cruciais para indução da angiogênese e o adequado desenvolvimento da placenta. Contudo, esta hipóxia se contrapõe à intensa atividade das células que requerem elevado metabolismo, gerando um estresse fisiológico para estas células presentes na interface materno-fetal. Presume-se que estas células necessitem de mecanismos apropriados de citoproteção para sua sobrevida enquanto comprometidos ativamente no suporte funcional do útero gestante. Neste sentido, o presente trabalho teve como objetivo investigar a expressão e a distribuição da proteína de choque térmico 70 (HSP70) na interface materno-fetal durante a gestação normal em camundongos e a sua possível variação em condição de estresse adicional induzido experimentalmente através da lesão embrionária. Sítios de desenvolvimento embrionário/fetal de camundongos prenhes do dia de gestação (dg) 6 ao 17 e, após 30 minutos, 1, 6 e 12 h dos animais submetidos à lesão cirúrgida do embrião (LCE) no dg 9 foram coletados para: - processamento histotécnico convencional de embebição em parafina destinados às análises citoquímicas (lectina DBA e reação de TUNEL) e imunocitoquímicas (anti-HSP72/73, anti-PCNA); - embebição em resina Lowilcryl- K4M para imunocitoquímica ultraestrutural (anti-HSP72/73); - obtenção de homogenados teciduais destinados à SDS-PAGE das frações protéicas e Westernblot (anti-HSP72/73) e, - extração de RNA de homogenados teciduais e de células uNK isoladas para análise de transcritos (HSP72 e 73) com amplificação pelo RTPCR. As análises imunocitoquímicas demonstraram que as células uNK eram as únicas células que expressavam de forma constante as isoformas HSP72/73 ao longo da gestação, sendo confirmada a expressão dos transcritos gênicos das isoformas HSP72/73 nas células uNK isoladas pelo RT-PCR. A imunomicroscopia eletrônica detectou marcação conspícua nas mitocôndrias das células uNK. A análise quantitativa demonstrou que a lesão do embrião reduz o número de células uNK positivas para HSP72/73 e, o SDS/PAGE/Western-blotting identificou as isoformas HSP72 e 73 presente nos homogenados teciduais do útero com uma perceptível redução na intensidade da banda correspondente ao HSP73 nas amostras de pós-lesão, sem afetar significativamente a isoforma HSP72. As análises realizadas com a dupla marcação de TUNEL e PCNA demonstrarm redução de células uNK PCNA positivas no útero submetido a lesão embrionária e aumento de núcleos marcadas positivamente pelo TUNEL. Estes resultados demonstram de forma inédita a expressão de HSP72/73 nas células uNK, sendo inédita também a constatação em leucócitos, sugerindo um papel citoprotetor para estas células importantes na manutenção da gestação. A redução de células uNK HSP72/73 positivas no útero gestante desencadeada pela lesão embrionária, consubstancia a hipótese da atuação da HSP 72/73 como chaperona citoprotetora nas células uNK sendo crítica a atuação da isoforma HSP73 presente na mitocôndria através da regulação negativa das vias de morte celular por apoptose nas células uNK / Abstract: During the pregnancy of animals developing hemochorial placenta, the hypoxia in the first third of pregnancy is one of the crucial factor for induction of angiogenesis and adequate placental development. However, this hypoxia is contradictory to the great dynamism and metabolism of cells required in the pregnant uterus, conditioning a physiological stress for the cells present at the maternal-fetal interface. It is presumed these cells demand appropriate cytoprotective mechanism for their survival while are committed to actively support the pregnancy. In this way, the present work aimed to investigate the expression and distribution of the chapelone isoforms heat shock protein 72 and 73 (HSP72/73) at the maternal fetal-interface through the pregnancy in mice and its possible variations under additional stressing condition induced experimentally by embryo lesion. Embryo/fetus developing sites of pregnant mice from gestational days (gd) 6 to 17 and, after 30min, 1h and 6h of surgical embryo lesion (SEL) on gd 9 mice, were collected for: - conventional paraffin embedding for cytochemical (DBA lectin and TUNEL reaction) and immunocytochemical (anti-HSP72/73, anti-PCNA) analysis; - LR-white resin embedding for ultrastructural immunocytochemistry (anti- HSP72/73); - uterine tissue homogenates for SDS-PAGE of proteins fractions and Western-blot (anti-HSP7273) and; - RNA extratction form uterine tissue homogenates and isolated uNK cells for transcripts (HSP72 and 73) amplification by RT-PCR. The immunocytcchemical analysis showed the uNK cells as the only cell expressing constantly the HSP72/73 isoforms throughout the gestation, being confirmed the expression of both gene isoforms by RT-PCR in uNK cells. The immunoelectron microscopy detected conspicuous labeling in the mitochondria of uNK cells. The quantitative analysis demonstrated that embryo-lesion reduced the number of HSP72/73 positive uNK cells in the uterus and, SDS/PAGE and Westernblot identified the HSP72 and 73 isoforms present in the tissue homogenates with low reactive intensity of the band corresponding to HSP73 in the after-lesion samples, without affecting significantly the HSP72 isoform. The analysis of TUNEL and PCNA double labelling showed decreasing of PCNA positive-uNK cells in the uterus after embryo-lesion and increasing of TUNEL positive nuclei. These results confirms the expression of HSP72 and HSP73 isoforms in the uNK cells through the gestation and to date, this is also the first report showing HSP70 in leukocytes, suggesting a cytoptotective function to this cell while working actively as important cells supporting the pregnancy. The decreasing of HSP72/73 positive uNK cells in the pregnant uterus triggered by embryo lesion consubstantiate the hypothesis of HSP72/73 working as cytoprotective chaperone in the uNK cells, and the HSP73 isoform in the mitochondria seems to be critical on down-regulation of apoptotic cell depth pathway / Abstract: During the pregnancy of animals developing hemochorial placenta, the hypoxia in the first third of pregnancy is one of the crucial factor for induction of angiogenesis and adequate placental development. However, this hypoxia is contradictory to the great dynamism and metabolism of cells required in the pregnant uterus, conditioning a physiological stress for the cells present at the maternal-fetal interface. It is presumed these cells demand appropriate cytoprotective mechanism for their survival while are committed to actively support the pregnancy. In this way, the present work aimed to investigate the expression and distribution of the chapelone isoforms heat shock protein 72 and 73 (HSP72/73) at the maternal fetal-interface through the pregnancy in mice and its possible variations under additional stressing condition induced experimentally by embryo lesion. Embryo/fetus developing sites of pregnant mice from gestational days (gd) 6 to 17 and, after 30min, 1h and 6h of surgical embryo lesion (SEL) on gd 9 mice, were collected for: - conventional paraffin embedding for cytochemical (DBA lectin and TUNEL reaction) and immunocytochemical (anti-HSP72/73, anti-PCNA) analysis; - LR-white resin embedding for ultrastructural immunocytochemistry (anti- HSP72/73); - uterine tissue homogenates for SDS-PAGE of proteins fractions and Western-blot (anti-HSP7273) and; - RNA extratction form uterine tissue homogenates and isolated uNK cells for transcripts (HSP72 and 73) amplification by RT-PCR. The immunocytcchemical analysis showed the uNK cells as the only cell expressing constantly the HSP72/73 isoforms throughout the gestation, being confirmed the expression of both gene isoforms by RT-PCR in uNK cells. The immunoelectron microscopy detected conspicuous labeling in the mitochondria of uNK cells. The quantitative analysis demonstrated that embryo-lesion reduced the number of HSP72/73 positive uNK cells in the uterus and, SDS/PAGE and Westernblot identified the HSP72 and 73 isoforms present in the tissue homogenates with low reactive intensity of the band corresponding to HSP73 in the after-lesion samples, without affecting significantly the HSP72 isoform. The analysis of TUNEL and PCNA double labelling showed decreasing of PCNA positive-uNK cells in the uterus after embryo-lesion and increasing of TUNEL positive nuclei. These results confirms the expression of HSP72 and HSP73 isoforms in the uNK cells through the gestation and to date, this is also the first report showing HSP70 in leukocytes, suggesting a cytoptotective function to this cell while working actively as important cells supporting the pregnancy. The decreasing of HSP72/73 positive uNK cells in the pregnant uterus triggered by embryo lesion consubstantiate the hypothesis of HSP72/73 working as cytoprotective chaperone in the uNK cells, and the HSP73 isoform in the mitochondria seems to be critical on down-regulation of apoptotic cell depth pathway / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Introdução: A resistência a infecção pelo HIV-1 depende de fatores virais, genéticos e imunológicos do hospedeiro, incluindo os componentes da resposta imune inata e adaptativa. As células Natural Killer (NK) e as células T CD8+ são as principais células efetoras que medeiam atividade citotóxica contra células transformadas ou infectadas, que exercem importante papel protetor nos indivíduos expostos e não infectados por HIV-1 (ENI). Objetivo: Avaliar a expressão de receptores de ativação e inibição/exaustão nas células NK e T CD8+, e a capacidade das células NK em secretar citocinas e componentes citotóxicos após estimulação via receptores Toll-like (TLRs), e a resposta de células T CD8+ a peptídeos da Gag do HIV-1 em indivíduos ENI e seus parceiros infectados por HIV-1. Resultados: No grupo ENI foi observado aumento da frequência de células NK CD56bright que expressam moléculas de ativação NKG2D e CD95 na população CD56dim, enquanto no grupo HIV-1 foi mais prevalente a expressão de MIC A/B em ambas populações de células NK, com redução da expressão de NKG2D na população CD56dim. Além disto, foi observado expansão da população de células NK CD56dim que expressam CD94, NKG2C e principalmente de CD57 foi mais prevalente nos indivíduos ENI, com correlação positiva com títulos de anticorpos IgG anti-citomegalovírus humano. Nos indivíduos ENI foi observado que a ativação via TLR-3, TLR-7 ou TLR-7/8 foi capaz de potencializar a expressão de marcadores de desgranulação e de citotoxicidade, CD107a e granzima B, principalmente na população CD56dim, e de IFN-y e TNF nas populações CD56bright e CD56dim. Além disto, somente o grupo ENI, foi detectado aumento da freqüência de células NK secretoras de CD107a, granzima B, IFN-y e TNF, após estimulação com acetato de miristato de forbol e ionomicina. A frequência de expressão de alelos de KIR (killer cell immunoglobulin-like receptors) foi similar entre os grupos analisados. Elevada frequência de células T CD8+ CD38+ e CD8+PD-1+ (programmed cell death protein 1) foi detectado nos grupos ENI e HIV-1, cuja alteração foi observada em todas as fases de maturação celular. Os indivíduos ENI mostraram presença de resposta antígeno-específica de células T CD8+ secretoras de CD107a, granzima B, IFN-y e TNF, semelhante ao grupo HIV-1. Conclusão: Os resultados mostraram que no grupo ENI, as células NK expressam um perfil de ativação, com potente resposta aos estímulos de resposta inata e células NK com perfil de memória. Presença de células TCD8+ antígeno-específica foi evidenciada no grupo ENI, com perfil semelhante, mas de menor magnitude ao detectado no grupo infectado por HIV. Em conjunto, os achados mostraram que no grupo ENI a resposta inata está potencialmente ativa, e que em associação a resposta T CD8+ antígeno-específica podem contribuir para a resistência a infecção pelo HIV-1 / Introduction: Resistance to human immunodeficency virus 1 (HIV-1) is dependent on viral, genetic and immunological host factors, including components of innate and adaptive immune response. Natural Killers cells (NK) and CD8+ T cells are main effectors cells mediating cytotoxic role against transformed or infected cells, playing a crucial role in HIV-1 exposed uninfected individuals (EU). Aim: To evaluate the expression of activation and inhibitory/exhaustion receptors on NK cells and CD8+ T-cells, and to determine the NK cells ability to cytokines and cytotoxic molecules secretion upon Toll-like receptors (TLRs) pathway activation as well as CD8+ T-cells response to HIV Gag peptides in EU individuals and HIV-1 infected partner. Results: Increased frequency of NK CD56bright cells expressing NKG2D and CD95 on CD56dim cells have been observed in EU group, while HIV-1 group was more prevalent MIC A/B expression in both NK cells subsets, with reduced expression of NKG2D in CD56dim cells. Moreover, expansion of NK CD56dim cells expressing CD94, NKG2C, and CD57 was prevalent on ENI group, which positive correlation with anti-human cytomegalovirus IgG serum titers. EU individuals showed that TLR-3, TLR-7 or TLR-7/8 pathway activation was able to enhance CD107a and granzyme B expression in CD56dim cells, and IFN-y and TNF expressions levels in both CD56bright and CD56dim NK cells. Moreover, only in EU group, high frequency of NK cells expressing CD107a, granzyme B, IFN-y and TNF were detected upon phorbol myristate acetate and ionomicyn stimulation. Frequency of KIR alleles (killer cell immunoglobulin-like receptors) was similar between groups. High frequency of CD8+CD38+ and CD8+PD-1+ (programmed cell death protein 1) T-cells were observed in EU and HIV-1 groups, in all stages of cellular differentiation. EU subjects showed presence of antigen-specific response by CD8+ T-cells secreting CD107a, granzyme B, IFN-y and TNF similar to HIV-1 group. Conclusion: The results showed that NK cells in EU subjects express activating profile, with potent ability to innate immune stimuli, as well as NK cells with memory profile. Presence of antigen-specific CD8+ T-cells was detected in EU group, with similar profile, but in less magnitude than HIV-1 group. Taken together, the findings showed an enhanced innate immune response in EU subjects, in association with antigen-specific CD8+ T-cell response can contribute to resistance to HIV-1 infection

Orientadores: Carmino Antonio de Souza, Jeane Eliete Laguila Visentainer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T20:55:46Z (GMT). No. of bitstreams: 1 Marangon_AmandaVansan_D.pdf: 2274304 bytes, checksum: 433df5157fab3588b51689f24c4b86cf (MD5) Previous issue date: 2014 / Resumo: O Linfoma difuso de grandes células B (LDGCB) representa o subtipo mais prevalente de linfoma maligno não-Hodgkin, sendo responsável por 30-40% de todos os casos de LNH. O LDGCB não tem etiologia e patogênese bem definidas, porém o seu desenvolvimento parece estar relacionado a respostas imunes ineficazes, devido a frequente associação desse linfoma com estados de imunossupressão. Os fatores genéticos envolvidos no desenvolvimento e evolução da doença não são bem entendidos. Nesse contexto, o objetivo desse trabalho foi avaliar a influência dos genes KIR, dos ligantes HLA e do polimorfismo em genes de citocinas na susceptibilidade ou resistência ao desenvolvimento de LDGCB, bem como na evolução clínica e resposta ao tratamento. Para tanto, foram selecionados 112 pacientes com diagnóstico de LDGCB e 292 doadores de sangue e medula óssea como grupo controle. As tipificações dos genes KIR e dos ligantes HLA foram realizadas com a técnica de PCR-SSOP e a tipificação de citocinas foi realizada com a técnica PCR-SSP. As análises estatísticas foram realizadas pelo pacote estatístico "R" versão 3.0.2 para o programa Windows e os valores de P<0,05 foram considerados significativos. A distribuição dos genes KIR nos grupos estudados mostrou uma menor frequência do gene KIR2DL2 nos pacientes quando comparados aos controles (45,5% vs 58,1%; P=0,036), essa associação mostrou-se significativa também na combinação de KIR2DL2 com C1 (33,0%vs 45,9%; P=0,026) sugerindo um papel de proteção desse gene ao desenvolvimento de LDGCB. Em relação à evolução clínica da doença, os ligantes HLA-Bw4 e HLA-Bw4 80I foram mais frequentes nos pacientes com estádios mais avançados da doença (64,7% vs 40,9%; P=0,020 e 44,1% vs 25,0%; P=0,046, respectivamente) sugerindo que a presença desses ligantes pode ser fator de prognóstico ruim ao LDGCB. Em relação à resposta terapêutica, o gene KIR2DL3 foi associado positivamente ao tratamento do LDGCB, pois esse gene foi mais frequente nos indivíduos com resposta completa que nos indivíduos não respondedores (88,3% vs 71,0%; P=0,044). A respeito dos genes reguladores de citocinas, o genótipo IFN-gama-874/A:A foi associado positivamente ao LDGCB, sendo encontrado mais frequente nos pacientes que nos controles (50,9% vs 27,9%; P=0,001). Contrariamente os genótipos: IFN-gama-874/T:A, IL10-819/C:C e IL10-592/C:C foram menos frequentes nos pacientes que nos controles (P=0,001; P=0,025; P=0,025). Ademais, o genótipo IL10-1082/G:G foi relacionado a maior sobrevida livre de progressão. Os resultados encontrados sugerem que os genes KIR, os ligantes HLA e os genes de citocinas parecem ter envolvimento na proteção, susceptibilidade, evolução clínica e resposta ao tratamento do LDGCB / Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of malignant non-Hodgkin lymphoma and affects approximately 30-40 % of all cases. The DLBCL has no clearly defined etiology and pathogenesis, but its development seems to be related to ineffective immune responses due to frequent association of lymphoma with immunosuppression. Genetic factors involved in the development and progression of the disease are not well understood. The aim of this study was to evaluate the influence of KIR genes, HLA ligands and cytokine polymorphisms in the susceptibility or resistance to the development of DLBCL, as well as influence in the clinical course and response to treatment. To this end, we selected 112 patients with DLBCL and 292 bone marrow donors as control group. The typing of KIR genes and HLA ligands were performed by PCR-SSOP and typing of cytokine genes was performed by PCR-SSP technique. Statistical analyzes were performed by the statistical package " R " version 3.0.2 for Windows program. P values < 0.05 were considered significant. The distribution of KIR genes in both groups showed a lower frequency of the KIR2DL2 gene in patients compared to controls (45.5% vs 58.1% P=0.036), this association was significant also in combination KIR2DL2 with C1 (33.0% vs 45.9%, P=0.026) suggesting a protective role of this gene to the development of DLBCL. Regarding the clinical course of the disease, HLA-Bw4 and HLA-Bw4 80I ligands were more frequent in patients with more advanced stages of the disease (64.7% vs 40.9%, P=0.020 and 44.1% vs 25 0%, P=0.046, respectively) suggesting that the presence of these ligands may be poor prognostic factor to DLBCL. In regard to treatment response, the KIR2DL3 gene was positively associated with the treatment of DLBCL, because this gene was more frequent in individuals with complete response than in nonresponders individuals (88.3% vs 71.0%, P=0.044 ). Regarding the cytokine genes , IFN -gamma-874/A genotype:A/A was positively associated with DLBCL, it was more frequently in patients than in controls (50.9% vs 27.9%, P=0.001). On other hand genotypes: IFNG -874 /T:A, IL10-819/C:C and IL10 -592 /C:C were less frequent in patients than in controls (P=0.001, P=0.025, P=0.025 respectively). Moreover, the genotype IL-1082/G:G was related to increased progression-free survival. The results suggest that the KIR genes, HLA ligands and cytokine genes seem to be involved in the protection, susceptibility, clinical course and response to treatment of DLBCL / Doutorado / Clinica Medica / Doutora em Clínica Médica

La formation et le développement d'une tumeur sont provoqués par une série de défauts qui se produisent à l'intérieur de la cellule cancéreuse et dans son microenvironnement. Ces anomalies permettent à la cellule de développer ses propres stratégies de croissance, de prolifération, de différenciation et de métabolisme. Toutes ces adaptations, ainsi que la création d'un micro-environnement unique favorisent la croissance de la tumeur et inhibent la réponse immunitaire anti-tumorale. Le métabolisme des cellules cancéreuses et l'évasion immunitaire sont des points très sensibles dans le développement des cancers et peuvent être utilisés en clinique. Les études récentes suggèrent que ces deux phénomènes sont liés, et que le métabolisme des cellules cancéreuses peut amener à l'échappement immunitaire par la tumeur. Le métabolisme des cellules tumorales a tendance à éviter l'activité mitochondriale et la phosphorylation oxydative, et est principalement basée sur la glycolyse pour la production d'énergie (effet Warburg). Mon travail de thèse est divisé en deux parties. Dans la première partie nous avons proposé un concept thérapeutique novateur avec une nouvelle thérapie combinatoire pour le traitement de cancers hématologiques. Cette thérapie est basée sur l'induction de changements métaboliques par le dichloroacétate (DCA), et elle est associée avec la chimiothérapie conventionnelle (doxorubicine, vincristine) pour réactiver les fonctions de p53. Les tumeurs avec p53 mutantes sont résistantes à cette combinaison. Dans ce cas, nous avons constaté que le DCA peut coopère avec 17-AAG (l'inhibiteur de Hsp90) pour éliminer spécifiquement les cellules cancéreuses. En conséquence, une meilleure compréhension des signaux et des mécanismes par lesquels le DCA sensibilise les cellules tumorales à la chimiothérapie est nécessaire pour en comprendre le mode d'action. En outre, l'identification de ce mécanisme permettra d'élucider les voies métaboliques impliquées dans la survie des cellules cancéreuses. La deuxième partie de ma thèse se concentre sur la biologie des cellules NK. Les cellules NK sont des lymphocytes du système immunitaire inné et possèdent une cytotoxicité naturelle contre les cibles, c'est à dire les cellules tumorales. L'utilisation optimale des cellules NK en clinique nécessite leur expansion et leur activation in vitro. Les cellules NK s'activent en présence de cytokines ou par le contact avec les cellules cibles. L'activation des cellules NK induit la prolifération, mais celle-ci dépend aussi de la présence d'autres cellules immunitaires. L'activation, par les cytokines et par les cellules cibles, induit un différent ARNm/microARN profil d'expression. L'analyse détaillée des isoformes de la protéine tyrosine phosphatase CD45 a permis de caractériser de nouvelles populations de cellules NK anti-tumorales humaines. L'identification de différentes populations de cellules NK est très importante pour la compréhension de leur physiologie et pour l'amélioration de leur utilisation en immunothérapie clinique. Cela peut également donner des informations précieuses sur l'état physiologique de l'hôte. En effet, l'augmentation des cellules CD45RAdim et CD45RO + dans le compartiment des cellules NK matures identifie clairement les patients avec des hémopathies malignes. Nous pensons que leur détection peut être utilisée comme un outil de diagnostic et également pour évaluer l'efficacité des traitements anti-tumoraux, car ces populations de cellules NK spécifiques devraient diminuer lors de l'élimination de cellules tumorales cibles. Dans l'avenir, nous voulons combiner le traitement du métabolisme de la tumeur avec la thérapie anti-tumorale basée sur les cellules NK. Sur la base de nos données préliminaires, nous pouvons proposer le traitement des cellules cancéreuses par des médicaments métaboliques pour augmenter la sensibilité et la reconnaissance par les cellules NK activées. / Tumor formation and development are caused by a range of defects that occur inside the cancer cell and in the external cellular microenvironment. These abnormalities allow developing tumors to establish their own strategies of growth, proliferation, differentiation and metabolism. All these adaptations, as well as the creation of a unique microenvironment, promote tumor growth and suppress the anti-cancer immune response. Tumor cell metabolism and immune evasion are sensitive points of cancer development that can be targeted in clinic. Recent studies suggest that these two phenomena are related and that cancer cell metabolism may propel tumor immune escape. Tumor cell metabolism tends to avoid mitochondrial activity and oxidative phosphorylation (OXPHOS), and largely relies on glycolysis to produce energy (Warburg effect). My thesis work is divided into two parts. The first one proposes an innovative therapeutic strategy, which is the use of different combinatorial therapy depending on the p53 status for the treatment of hematological cancers. This is based on the induction of metabolic changes by dichloroacetate (DCA), combined with conventional chemotherapy (doxorubicin, vincristine) to reactivate wild type p53 functions. Mutant p53 tumors are resistant to this combination approach. However, we found that DCA synergized with the Hsp90 inhibitor 17-AAG to specifically eliminate these cells. Therefore, a clearer understanding of the signals and mechanisms by which DCA sensitize cancer cells to chemotherapy was needed to understand its mode of action. We uncovered it in our work. In addition, identification of this mechanism will help to elucidate metabolic pathways involved in cancer cell survival.The second part of my thesis is focused on the study of NK cell biology. NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity against targets, i.e. tumor cells. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and the encounter with target cells activate NK cells, induce their proliferation, and cause clearly different mRNA/miRNA expression profile. Detailed analysis of the leucocyte-specific phosphatase CD45 isoforms allowed us to characterize new human anti-tumor NK cell populations. The identification of the different NK cell populations is important for understanding their physiology and for improving their therapeutic use in the clinic. It can also give valuable information about the host physiological status. Indeed, the increase of CD45RAdim and CD45RO+ cells in the mature NK cell compartment clearly identifies patients with hematological malignancies. We thus hypothesize that their detection could be used as a diagnostic tool, and also to assess the efficacy of antitumor treatments, because these specific NK cell populations should decrease upon removal of the targeted tumor cells. Our future goal is to use a novel combinatorial therapy in hematological cancers that will combine metabolic drugs and NK cell-based therapy. Based on our preliminary data, we propose that the treatment of cancer cells with metabolic drugs could increase their sensitivity and recognition by activated NK cells.

Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l’enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues.L’objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d’incubation, elles sont fortement tuées après 25h.Cette cytotoxicité est contact-dépendante mais ni la voie de l’exocytose des granules cytotoxiques ni celle des récepteurs de mort n’y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l’amplifie partiellement. Interférer avec les dérivés de l’oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l’X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l’expression par les NK des composants clefs d’une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l’existence d’une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires. / Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and anti-tumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood.In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h.Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with X-linked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway.

Natural killer (NK) cells are an intensively studied part of immune system, possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. Recent research in their C-type lectin-like receptors repertoire has shown that ligands of some of these previously orphan receptors lie within their own family, describing a lectin-lectin interaction. This is the case of human inhibitory receptor NKRP1 (gene KLRB1) and its ligand LLT1 (gene CLEC2D). Previous studies have shown that overproduction of LLT1 in cancer cells or lower production of NKRP1 in NK cells is connected to cancerous manifestations. This master's thesis shows a successful production of the extracellular part of LLT1 utilizing a mammalian expression system based on transient transfection of modified human embryonic kidney (HEK) cell lines. It was found that the five cystein residues contained within the lectin domain of LLT1 tend to cause misfolding and formation of aggregates. Stabilization of the domain was achieved by restoration of the sixth cystein residue at the evolutionary conserved...

Dendritic cells (DCs) are professional antigen-presenting cells, which display an extraordinary capacity to induce T-cell responses. Recent findings revealed that DCs also play a crucial role in the activation of natural killer (NK) cells representing important effectors in the innate immune defense against viruses and tumors. Here, we summarize various studies investigating the bidirectional crosstalk between human DCs and NK cells. In this context, it has been reported that DCs efficiently enhance CD69 expression, proliferation, interferon (IFN)-γ secretion and cytotoxic activity of NK cells. Cell membrane-associated molecules as well as soluble factors such as interleukin-12, tumor necrosis factor-α and type I IFNs contributed to DC-mediated NK cell activation. Reciprocally, the ability of human NK cells to enhance the immunostimulatory capacity of DCs was shown. Thus, NK cells promoted the maturation of DCs and markedly augmented their capacity to produce proinflammatory cytokines and to stimulate T-cell responses. The NK cell-mediated effects on DCs were dependent on cell membrane-associated molecules such as NKp30 and soluble factors such as tumor necrosis factor-α and IFN-γ. In conclusion, the reciprocal activating interaction between human DCs and NK cells may play a pivotal role in the immune defense against viruses and tumors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.