Les cellules Natural Killer (NK), actrices majeures de la vigilance anti-tumorale, sont modulées par des facteurs nutritionnels et métaboliques. L'inhibition de leur activité favorise le développement tumoral. Un régime alimentaire hypercalorique induisant l'obésité est un facteur de risque de développer un cancer du sein. Au niveau du micro-environnement tumoral mammaire, la biodisponibilité en certaines molécules contrôle non seulement les cellules néoplasiques mais, également les cellules immunes infiltrées. Ainsi, la leptine, sécrétée à forte concentration par les adipocytes mammaires, pourrait favoriser la croissance tumorale et altérer les cellules NK. L'arginine fortement consommée par les cellules tumorales et les cellules suppresseurs dérivées des myéloïdes pourrait faire défaut aux cellules NK. L'objectif de cette thèse est de caractériser les activités fonctionnelles et métaboliques des cellules NK en situation de stress nutritionnel. Dans un premier temps, nous avons exploré, in vivo, l'impact d'un régime hypercalorique sur l'activité des cellules NK et sur le développement tumoral mammaire. Ensuite, nous avons cherché à identifier les potentielles altérations fonctionnelles des cellules NK en mimant, in vitro, les conditions retrouvées au niveau du micro-environnement tumoral telles que la présence de concentration élevée en leptine et la déplétion en arginine. Des souris Balb-c "nude" femelles ont été soumises à un régime hypercalorique (HC) versus une diète normo-calorique (NC) pendant 6 mois. Au bout de 5 mois, des cellules tumorales mammaires (MCF-7 ; groupes NCT et HCT) ou le véhicule (groupes NC et HC) ont été implantés au niveau de la quatrième paire de glandes mammaires. Sous régime HC, le développement tumoral s'accompagne d'une perte de masse grasse, de masse maigre et de poids corporel avec un volume et un poids de tumeur augmentés. Cette diète induit au niveau tumoral une sur-expression des ARNm d'enzymes impliquées dans la glycolyse et une sous-expression des acteurs du cycle de Krebs. Sous régime HC, l'expression de la caspase 3 clivée et des récepteurs des oestrogènes β et de la progestérone est réduite alors que celle du Ki67 est accrue. Les cellules NK des souris HC ont une cytotoxicité diminuée. Bien que la présence de tumeur stimule l'activité lytique des cellules NK, la cytotoxicité de ces cellules reste inférieure dans le groupe HCT comparativement à celle du groupe NCT. La leptine stimule, in vitro, de façon dose-dépendante l'activité métabolique des cellules NK. A fortes concentrations, elle active leur cytotoxicité vis-à-vis des cellules cibles MDA-MB-231. Cet effet passe par une stimulation de l'expression de TRAIL et de l'IFN-γ par les cellules NK. En revanche, vis-à-vis des cellules cibles MCF7, les cellules NK présentent une activité lytique réduite en présence de fortes concentrations de leptine, probablement en lien avec une réduction de l'expression de la perforine. En réponse à une déplétion en arginine dans le milieu de culture, la prolifération et la cytotoxicité des cellules NK sont abaissées. L'altération de la reconnaissance des cellules cibles par les récepteurs NKp46 et NKp30, la moindre transmission du signal activateur par la chaine ζ et la faible production d'IFN-γ peuvent expliquer l'inhibition de la cytotoxicité des cellules NK. Ainsi, un apport énergétique élevé favorise le développement tumoral mammaire notamment eninhibant la cytotoxicité des cellules NK. De plus, la leptine à fortes concentrations stimule ou réduit, in vitro, la cytotoxicité des cellules NK selon la nature des cellules cancéreuses mammaires cibles. Une déplétion en arginine, in vitro, quant à elle, inhibe la prolifération et la cytotoxicité des cellules NK. Ces travaux contribuent à mieux comprendre l'impact du micro-environnement sur la réponse antitumorale des cellules NK. / Natural killer (NK) cells are critical mediators of anti-tumor immunity. A high-calorie diet inducing obesity is associated with breast cancer development. NK cells are modulated by dietary and metabolic factors and a decrease in their lytic activity promotes mammary tumor development. In the breast microenvironment, high concentration of leptin can be secreted by mammary adipocytes and thereby could stimulate tumor growth and control immune cells. Arginine, strongly consumed by tumor and myeloid-derived suppressor cells, could be lacking to NK cells. The aim of this work is to characterize the functional and metabolic activities of NK cells in response to nutritional stress. Initially, we explored in vivo the impact of a high-calorie diet on NK cells activity and mammary tumor development. Then, we identified potential functional alterations in NK cells by mimicking the conditions found in the tumor microenvironment such as the presence of high leptin concentration and arginine depletion. Female Balb-c nude mice were fed a high-caloric diet (HC) versus a standard caloric diet (SC) for 6 months. After five months, mammary tumor cells (MCF-7, SCT, HCT) or MatrigelTM (SC, HC) were implanted into the fourth mammary fat pads. The tumor development in HC diet-fed mice was associated with a decrease in body weight, body fat and lean mass and an increase in volume and weight of tumors. This diet induced tumor over-expression, at the transcriptional level, of enzymes involved in glycolysis and a down-expression of citrate cycle actors. Protein tumor levels of cleaved caspase 3, estrogen β and progesterone receptors were reduced while Ki67 was increased in the HC diet-fed mice. NK cell cytotoxicity of HC diet-fed mice was reduced. Although the presence of tumor stimulated NK cell lytic activity, this later was lower in the HCT group compared to the one of SCT mice. In vitro, leptin stimulated, in dose-dependent manner, the metabolic activity of NK cells. High leptin concentrations enhanced NK cell cytotoxicity against the MDA-MB-231 target cells. This phenomenon involved the increase of expression of TRAIL and IFN-γ in NK cells. However, against the MCF-7 target cells, NK cell lytic activity was reduced in the presence of high concentrations of leptin, probably in link to the decreased perforin expression. NK cell proliferation and cytotoxicity were impaired in response to arginine depletion. This inhibition of NK cell cytotoxicity could be linked to a low target cells recognition by NKp46 and NKp30, a reduced activating signal transmission by ζ chain and a low production of IFN-γ. Thus, high energy intake promotes mammary tumor development in particular by inhibiting NK cell cytotoxicity. In vitro, high leptin concentrations stimulate or reduce NK cell cytotoxicity according to the breast cancer cell targets. Furthermore, arginine depletion inhibits NK cell proliferation and cytotoxicity in vitro. These findings provide insight into the microenvironment impacts on NK cell antitumor response in tumor development.

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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

Células dendríticas (DCs) são componentes centrais da imunidade celular, responsáveis pelo priming de linfócitos T naïve. A polarização de linfócitos T é restrita aos sinais fornecidos durante a apresentação do antígeno. Além desses sinais, a origem e natureza de DCs que induzem diferentes perfis de linfócitos T não é totalmente compreendida. Foi investigada a capacidade de células Natural Killer (NK) de modular estágios iniciais da diferenciação de monócitos em DCs e de impactar na sua função de primar e polarizar linfócitos T naïve. DCs derivadas de monócitos pré-co-cultivados com células NK favorecem o priming de linfócitos T CD8 do tipo Tc1/Tc17, com potente capacidade de produção de IFN-γ. Este fenômeno foi dependente de interações longas via NKp30 e da maquinaria citotóxica de células NK desencadeada nas etapas inicias da sua interação com monócitos. Esta interação pode ter implicações na compreensão da imunidade mediada por linfócitos T CD8 e pode ser explorada para imunoterapia em que a produção de IFN-γ por células T CD8 é necessária ou exacerbada. / Dendritic cells (DCs) are central components of cellular immunity, responsible for the priming of naïve T cells. The polarization of T cells is restricted to signals provided during antigen presentation. Besides such signals, the origin and nature of DCs that induce different T cell profiles is not fully understood. The ability of natural killer cells (NK) to modulate early stages of monocytes differentiation into DCs and to impact on DCs function to prime and polarize naïve T cells was investigated. DCs derived from monocytes co-cultured with NK cells support the priming of type Tc1/Tc17 CD8 T cells with potent IFN-γ production capacity. NK cell-mediated cytotoxicity triggered at early stages of NKp30-dependent long-lasting monocytes-NK-cells interactions, mediated the mechanism by which this phenomenon occurred. This interaction may have implications in the understanding of CD8 T cell-mediated immunity and can be exploited for immunotherapy in which IFN-γ production by CD8 T cells is required or exacerbated.

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

Das Neuroblastom (NB) ist ein solider, extrakranieller Tumor neuroektodermalen Ursprungs, der sich im Kleinkindalter manifestiert. Ein etabliertes Zielantigen für die passive Immuntherapie beim NB ist das Disialogangliosid GD2. Aufgrund der geringen oder fehlenden Expression von MHC Klasse I Molekülen sowie der Tatsache, dass die Lyse von NB-Zellen durch verschiedene Mechanismen der natürlichen Zytotoxizität von NK-Zellen vermittelt werden kann, stellt eine auf NK-Zellen basierende Therapie einen vielversprechenden Ansatz zur Behandlung dieser Erkrankung dar. Auf dieser Grundlage wurde eine NK-Zelllinie generiert, die einen GD2-spezifischen chimären Antigenrezeptor (CAR) exprimiert (NK-92-scFv(ch14.18)-zeta). Die Hauptbestandteile dieses CARs sind ein Einzelkettenantikörper, welcher die variablen Regionen des GD2-spezifischen Antikörpers ch14.18 enthält, und die CD3ζ-Kette als signaltransduzierende Komponente. Im Rahmen dieser Arbeit konnte gezeigt werden, dass NK-92-scFv(ch14.18)-zeta in der Lage sind, auch Chemotherapie-resistente GD2-positive NB-Zelllinien effektiv abzutöten und dass dabei die Interaktion des CARs mit GD2 den Hauptmechanismus darstellt. Die anti-tumorale Wirkung von NK-92-scFv(ch14.18)-zeta in vivo wurde in einem Chemotherapie-resistenten GD2-positiven Xenograft-Mausmodell gezeigt. Die wiederholte Applikation von NK-92-scFv(ch14.18)-zeta in Kombination mit IL-2 resultierte in einem signifikant verlangsamten Tumorwachstum und einem verbesserten Überleben. Die Ergebnisse dieser Arbeit belegen, dass GD2-spezifische NK-92 das Potential für eine zukünftige klinische Anwendung besitzen. Demnach stellt der Einsatz einer solchen GD2-spezifischen NK-Zelllinie, die unter GMP-Bedingungen expandiert werden kann und zu jeder Zeit in einer standardisierten Qualität verfügbar wäre, eine vielversprechende Alternative zur Behandlung von Hochrisikopatienten dar, deren Erkrankung nicht mehr auf die Standardtherapie anspricht. / Neuroblastoma (NB) is a solid extracranial childhood malignancy of neuroectodermal origin. The Disialoganglioside GD2 is an established antigen for passive immunotherapy of NB. Cellular therapy of NB with natural killer (NK) cells is especially appealing because MHC class I expression is absent or low in most NB, rendering this tumor sensitive to NK cell recognition. Additionally, natural cytotoxicity of NK cells, mediated by interaction of activating NK cell receptors and their respective ligands on tumor cells, has been shown to play a role in lysis of NB cells. It is therefore tempting to assume that a combination of passive immunotherapy with GD2-specific antibodies and adoptive transfer of NK effector cells would result in an improved NB therapy. To achieve this goal an NK cell line expressing a GD2-specific chimeric antigen receptor (CAR) was engineered: NK-92-scFv(ch14.18)-zeta. This CAR consists of a GD2-specific scFv-fragment, which was generated from ch14.18, and the CD3ζ-chain as intracellular signal-transducing domain. Within this thesis, GD2-specificity of NK-92-scFv(ch14.18)-zeta as well as efficacy towards GD2-expressing NB cell lines, including relapse cell lines that exhibit partial or multidrug resistance were demonstrated. Blocking the interaction between the CAR and GD2 resulted in almost complete abrogation of NK-92-scFv(ch14.18)-zeta-mediated lysis of GD2-positive NB cell lines in vitro, indicating that this interaction is the main mechanism of activation of NK-92-scFv(ch14.18)-zeta. Importantly, repeated application of NK-92-scFv(ch14.18)-zeta in combination with IL-2 significantly decreased tumor growth and prolonged survival of mice in an aggressively growing drug-resistant xenograft NB mouse model. These findings suggest that GD2-specific NK-92 has potential for a future clinical application as NB-specific effector cells that would be ready on demand in a standardized quality.

Les cellules Natural Killer (NK) sont capables de lyser les cellules tumorales sans la nécessité de reconnaitre un antigène tumoral spécifique. Cette propriété leur confère un avantage par rapport aux lymphocytes T et les rend intéressantes à utiliser en tant que cellules effectrices pour l’immunothérapie adoptive. A ce jour, le potentiel thérapeutique des cellules NK n’a pas été complétement exploré notamment dans le contexte du traitement de la rechute post-allogreffe de cellules souches hématopoïétiques. Actuellement, les patients en rechute post-greffe sont traités avec des injections de lymphocytes du donneur (DLI) parfois issues de petites fractions du greffon de cellules souches hématopoïétiques congelées. Les cellules souches périphériques étant fréquemment utilisées comme source de cellules souches et parfois utilisées comme DLI, nous avons souhaité évaluer l’impact du G-CSF sur le phénotype et les fonctions des cellules NK présentes dans ces fractions. Dans cet objectif, nous avons comparé différentes sources de cellules NK isolées à partir de sang de donneurs sains, de sang mobilisé de donneurs sains ou de patients et observé l’évolution des différentes sous-populations de cellules NK issues de ces prélèvements au décours d’une expansion en présence d’IL-15. Nos résultats ont montré que l’administration de G-CSF diminuait la proportion de cellules NK CD56brightCD16+ au profit d’une population CD16-, diminuait la prolifération des cellules NK lors de l’expansion en culture, et modifiait les propriétés fonctionnelles des cellules NK. / The ability of natural killer (NK) cells to kill tumor cells without the need to recognize a tumor-specific antigen provides advantages over T cells and makes them appealing for a use as effectors for adoptive immunotherapy. However, the full therapeutic potential of NK cell-based immunotherapy has not been fully investigated in the context of leukemic relapse after hematopoietic stem cell transplantation. Today, patients relapsing after hematopoietic stem cell transplantation are often treated with donor lymphocyte infusion (DLI) based on small cell fractions frozen at the time of the stem cell transplantation. Since peripheral blood stem cells are increasingly used as stem cell source and as source of cells for DLI, we aimed to evaluate the impact of G-SCF mobilization on NK cell phenotype and functions. Therefore, we compared the expansion capacity, the phenotype and the function of NK cells from blood for healthy donors, from allogeneic HSCT healthy donors or from autologous HSCT from patients. We also determine the impact of G-CSF on NK cell subset repartition before and after expansion in presence of IL-15. Our results showed that G-CSF administration to patients decreases CD56brightCD16+ NK cell population, proliferation and function. Overcoming this impairment in lymphoid capacity may be important to facilitate post-transplant immunotherapy.

INTRODUÇÃO: O objetivo do estudo foi avaliar o perfil de marcadores imuno-histoquímicos relacionados às células NK em biópsias de aloenxertos renais com diagnóstico anatomopatológico de rejeição aguda (mediadas por células T ou anticorpos) e estabelecer relações desses marcadores com parâmetros morfológicos de lesão à microcirculação e sobrevida do enxerto. MÉTODOS: Estudo retrospectivo histórico que revisou 74 biópsias realizadas entre janeiro de 2009 e dezembro de 2012, de pacientes com rejeição aguda mediada por células T (n=36), rejeição aguda mediada por anticorpos com expressão positiva (n=19) ou negativa (n=19) para o marcador C4d, juntamente com levantamento de dados clínicos e laboratoriais pertinentes ao estudo. Foram realizadas reações imuno-histoquímicas, para os marcadores CD56, CD57, CD16, CD68, CD3, CD8 e CD4 com ênfase para os marcadores CD56 e CD16. Foi feita análise das células positivas em toda a cortical da biópsia nos compartimento intersticial, glomerular e vascular. Testes estatísticos foram aplicados conforme os pressupostos definidos no objetivo da pesquisa. RESULTADOS: No compartimento intersticial, células CD56+ (P = 0,004) e CD57+ (P < 0,001) foram expressas em maior quantidade em biópsias negativas para dosagem sérica de anticorpos específicos anti-doador (DSA) com diagnóstico de rejeição aguda mediada por células T. CD56 intersticial foi associado estatisticamente com presença de glomerulite (g >= 1) (P = 0,02) e ausência / leve capilarite peritubular (ptc <= 1) (P = 0,003). Células intersticiais positivas para o marcador CD56 com média superior a 0,56 céls/mm2 tiveram uma pior sobrevida do enxerto renal (P = 0,028). Biópsias com contagem inferior ou igual a 0,56 cél/mm2 tiveram associação estatisticamente significante para ausência ou leve capilarite peritubular (P = 0,012) e com contagem superior a 0,56 céls/mm2, foram associadas à presença de glomerulite (P = 0,002). Foi observado maior número de células positivas para o marcador CD16 no compartimento glomerular em biópsias positivas para dosagem sérica de DSA com diagnóstico de rejeição aguda mediada por anticorpos (P = 0,03) e em biópsias com presença de glomerulite (P = 0,009). Presença de maior número de células CD16+ no compartimento intersticial associou-se com capilarite peritubular (P = 0,0001). CONCLUSÕES: Maior expressão de células CD56 positivas no compartimento intersticial das biópsias foi significantemente associada com escores relacionados à lesão na microcirculação, especialmente glomerulite, com rejeição aguda mediada por células T e pior sobrevida do enxerto renal. Células CD16 positivas, no compartimento glomerular foram associadas com rejeição aguda mediada por anticorpos e glomerulite. As variações na expressão dos marcadores de células NK nos diferentes compartimentos da biópsia renal podem sugerir presença de envolvimento das células NK em diferentes vias do sistema imune nas rejeições agudas de aloenxertos renais / INTRODUCTION: The aim of this study was to investigate the immunohistochemical profile of markers related to NK cells from renal allograft biopsies with morphological diagnosis of acute rejection (T-cells or antibodies mediated rejection) and to study associations of those markers with types of rejection, microcirculation injury morphological parameters and graft survival. METHODOLOGY: Historical retrospective study that reviewed 74 biopsies performed between January 2009 and December 2012 in patients with acute T-cell-mediated rejection (n=36) and acute antibody-mediated rejection with (n=19) or without evident C4d deposition (n=19). The study was performed with relevant clinical and laboratory data. Immunohistochemical reactions were performed for CD56, CD57, CD16, CD68, CD3, CD8 and CD4 markers with highlights for CD56 and CD16. Counting of positive cells throughout cortical biopsy was performed in glomerular, interstitial and vascular compartments. Statistical tests were applied according to assumptions set out the goal of the study.RESULTS: DSA-negative biopsies-from patients with acute T-cell mediated rejection (aTCMR) had an increased expression of CD56+ and CD57+ cells (P = 0.004 and P < 0.001) in the interstitial compartment in comparison with donor-specific antibodies ( DSA)-positive biopsies from patients acute antibody-mediated rejection with and without C4d deposition. Interstitial CD56+ cells had an increased expression for presence of glomerulitis (g >= 1) (P = 0.02) and peritubular capillaritis (ptc >= 2) (P = 0.003). Interstitial CD56 + cells with mean superior to 0.56cells/mm2 had worse allograft survival (P = 0.028). CD56+ cells in the interstitial compartment with mean inferior or equal to 0.56cells/mm2 associated with absence or mild peritubular capillaritis (P = 0.012) and mean superior to 0.56cells/mm2 was associated with presence of glomerulitis (P = 0.002). CD16+ cells was increased in the glomerular compartment in DSA-positive biopsies (P = 0.03) and in the presence of glomerulitis (P = 0.009). Interstitial CD16+ cells associated with peritubular capillaritis (P = 0.0001).CONCLUSION: CD56+ cell infiltrates in the interstitial compartment were significantly associated with microcirculation injury scores, especially glomerulitis, acute T-cell mediated rejection and clinical outcomes. CD16+ cell infiltrates in glomerular compartment was associated with acute antibody-mediated rejection and glomerulitis. Our findings showed variations in expression of NK cells markers in renal biopsy different compartments which might suggest the involvement of NK cells in different immune system pathways in acute renal allograft rejection

OBJETIVOS: Comparar a expressão placentária de células natural killer deciduais (dNK), e a expressão sérica e placentária de suas citocinas reguladoras em gestações gemelares com pré-eclâmpsia (grupo pré-eclâmpsia, GPE) e sem comorbidades (grupo-controle, GC). MÉTODOS: Estudo transversal do tipo caso-controle, desenvolvido na Clínica Obstétrica do HC-FMUSP no período de julho de 2015 a junho de 2017. Foram obtidas amostras das regiões deciduais placentárias, para avaliação, por meio de técnica de imuno-histoquímica, da expressão de células dNK e suas interleucinas (IL) 10, 12 e 15, em pacientes que contemplaram os critérios de inclusão e concordaram em participar do estudo. Além disso, estas pacientes tiveram amostra sérica colhida no terceiro trimestre para dosagem de IL-10, IL-12 e IL-15 - por meio de kit comercial Milliplex®, que utiliza a tecnologia Luminex® xMAP®, da EMDMillipore (Merck Millipore Co., Alemanha) - e de fatores relacionados à angiogênese, como soluble fms-like tyrosine kinase-1 (sFlt-1) e placental growth factor (PlGF) - por meio de ensaio com imunoanalisador COBAS e411 (Roche Diagnostics, Alemanha). Os valores obtidos para as análises placentárias e séricas foram comparados entre o GPE e o GC, e a significância estatística estabelecida foi p < 0,05. RESULTADOS: Foram selecionadas 30 pacientes, sendo 20 no GC e 10 no GPE. Não se observaram diferenças significativas com relação às características maternas, gestacionais e de desfechos perinatais entre os dois grupos, exceto pela idade gestacional de início do pré-natal, menor no GPE (12,5 vs. 20,0 semanas, p = 0,015). Quanto à avaliação placentária, houve maior expressão de IL-15 no GPE (p = 0,001), e não houve diferença entre os grupos quanto à expressão placentária local de células dNK (p = 0,999), IL-10 (p = 0,063) e IL-12 (p = 0,135). Com relação às interleucinas séricas maternas, demonstrou-se aumento significativo nos níveis de IL-10 (22,7 vs. 11,9 pg/mL, p = 0,024) e IL-15 (15,9 vs. 7,4 pg/mL, p = 0,024) no GPE com relação ao GC, sem diferença entre os grupos para IL-12 (102,5 vs. 61,5 pg/mL, p = 0,373). A dosagem dos fatores relacionados à angiogênese demonstrou maiores níveis séricos maternos de sFlt-1 (15920 vs. 7978 pg/mL, p = 0,009) e da razão sFlt-1/PlGF (88,71 vs. 24,63, p = 0,002), e menores valores de PlGF (193,0 vs. 340,6 pg/mL, p = 0,036) no GPE. CONCLUSÃO: Observou-se maior concentração sérica materna tanto de fatores pró quanto anti-inflamatórios no GPE, quando comparado ao GC. Entretanto, não foram observadas diferenças entre os grupos quanto à expressão placentária de IL-10, importante fator anti-inflamatório. Estes achados podem sugerir que a tentativa sérica materna de equilibrar estas interleucinas não alcançou resposta localmente na placenta, contribuindo para o desenvolvimento da doença no GPE / OBJECTIVES: To compare the placental expression of decidual natural killer cells (dNK) and serum and placental expression of their regulatory cytokines in twin pregnancies with preeclampsia (preeclampsia group, PEG) and uncomplicated twin pregnancies (control group, CG). METHODS: This was a case-control study, developed in a tertiary referral center, from July 2015 to June 2017. Samples of the placental decidual region were obtained and analyzed by immunohistochemistry technique for the expression of dNK cells and interleukins (IL) 10, 12 and 15, in patients who met the inclusion criteria. In addition, maternal serum sample was collected in the third trimester for the dosage of IL-10, IL-12 and IL-15 - by means of a commercial Milliplex® kit using Luminex® xMAP® technology from EMDMillipore (Merck Millipore Co., Germany) - and angiogenesis factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) - by COBAS e411 immunoassay (Roche Diagnostics, Germany). The values obtained for the placental analyzes and maternal circulating factors were compared between PEG and CG and the statistical significance was set at p < 0.05. RESULTS: Thirty patients were selected, 20 in CG and 10 in PEG. There were no significant differences in maternal, gestational and perinatal outcomes between the two groups, except for the gestational age at the onset of prenatal care, which was lower in PEG (12.5 vs. 20.0 weeks, p = 0.015). PEG showed strong immunostaining for IL-15 (p = 0.001) when compared to CG, with no difference between the groups concerning the placental expression of dNK (p = 0.999), IL-10 (p = 0.063), and IL -12 (p = 0.135). Relating to maternal circulating interleukins, a significant increase in IL-10 (22.7 vs. 11.9 pg/mL, p = 0.024) and IL-15 (15.9 vs. 7.4 pg/mL, p = 0.024) was observed in PEG, with no difference between the groups for IL-12 (102.5 vs 61.5 pg/mL, p = 0.373). We also demonstrated higher maternal levels of sFlt-1 (15920 vs. 7978 pg/mL, p = 0.009) and sFlt-1/PlGF ratio (88.71 vs. 24.63, p = 0.002) and lower levels of PlGF (193 vs. 340.6 pg/mL, p = 0.036) in PEG. CONCLUSION: A higher maternal serum concentration of both pro- and anti-inflammatory factors was observed in the PEG. However, no difference was found between the groups regarding the placental expression of IL-10, an important anti-inflammatory factor. These findings may suggest that the maternal serum attempt to balance these interleukins did not reach local placental response, which contribute to the development of the disease in the PEG

OBJETIVOS: Comparar a expressão placentária de células natural killer deciduais (dNK), e a expressão sérica e placentária de suas citocinas reguladoras em gestações gemelares com pré-eclâmpsia (grupo pré-eclâmpsia, GPE) e sem comorbidades (grupo-controle, GC). MÉTODOS: Estudo transversal do tipo caso-controle, desenvolvido na Clínica Obstétrica do HC-FMUSP no período de julho de 2015 a junho de 2017. Foram obtidas amostras das regiões deciduais placentárias, para avaliação, por meio de técnica de imuno-histoquímica, da expressão de células dNK e suas interleucinas (IL) 10, 12 e 15, em pacientes que contemplaram os critérios de inclusão e concordaram em participar do estudo. Além disso, estas pacientes tiveram amostra sérica colhida no terceiro trimestre para dosagem de IL-10, IL-12 e IL-15 - por meio de kit comercial Milliplex®, que utiliza a tecnologia Luminex® xMAP®, da EMDMillipore (Merck Millipore Co., Alemanha) - e de fatores relacionados à angiogênese, como soluble fms-like tyrosine kinase-1 (sFlt-1) e placental growth factor (PlGF) - por meio de ensaio com imunoanalisador COBAS e411 (Roche Diagnostics, Alemanha). Os valores obtidos para as análises placentárias e séricas foram comparados entre o GPE e o GC, e a significância estatística estabelecida foi p < 0,05. RESULTADOS: Foram selecionadas 30 pacientes, sendo 20 no GC e 10 no GPE. Não se observaram diferenças significativas com relação às características maternas, gestacionais e de desfechos perinatais entre os dois grupos, exceto pela idade gestacional de início do pré-natal, menor no GPE (12,5 vs. 20,0 semanas, p = 0,015). Quanto à avaliação placentária, houve maior expressão de IL-15 no GPE (p = 0,001), e não houve diferença entre os grupos quanto à expressão placentária local de células dNK (p = 0,999), IL-10 (p = 0,063) e IL-12 (p = 0,135). Com relação às interleucinas séricas maternas, demonstrou-se aumento significativo nos níveis de IL-10 (22,7 vs. 11,9 pg/mL, p = 0,024) e IL-15 (15,9 vs. 7,4 pg/mL, p = 0,024) no GPE com relação ao GC, sem diferença entre os grupos para IL-12 (102,5 vs. 61,5 pg/mL, p = 0,373). A dosagem dos fatores relacionados à angiogênese demonstrou maiores níveis séricos maternos de sFlt-1 (15920 vs. 7978 pg/mL, p = 0,009) e da razão sFlt-1/PlGF (88,71 vs. 24,63, p = 0,002), e menores valores de PlGF (193,0 vs. 340,6 pg/mL, p = 0,036) no GPE. CONCLUSÃO: Observou-se maior concentração sérica materna tanto de fatores pró quanto anti-inflamatórios no GPE, quando comparado ao GC. Entretanto, não foram observadas diferenças entre os grupos quanto à expressão placentária de IL-10, importante fator anti-inflamatório. Estes achados podem sugerir que a tentativa sérica materna de equilibrar estas interleucinas não alcançou resposta localmente na placenta, contribuindo para o desenvolvimento da doença no GPE / OBJECTIVES: To compare the placental expression of decidual natural killer cells (dNK) and serum and placental expression of their regulatory cytokines in twin pregnancies with preeclampsia (preeclampsia group, PEG) and uncomplicated twin pregnancies (control group, CG). METHODS: This was a case-control study, developed in a tertiary referral center, from July 2015 to June 2017. Samples of the placental decidual region were obtained and analyzed by immunohistochemistry technique for the expression of dNK cells and interleukins (IL) 10, 12 and 15, in patients who met the inclusion criteria. In addition, maternal serum sample was collected in the third trimester for the dosage of IL-10, IL-12 and IL-15 - by means of a commercial Milliplex® kit using Luminex® xMAP® technology from EMDMillipore (Merck Millipore Co., Germany) - and angiogenesis factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) - by COBAS e411 immunoassay (Roche Diagnostics, Germany). The values obtained for the placental analyzes and maternal circulating factors were compared between PEG and CG and the statistical significance was set at p < 0.05. RESULTS: Thirty patients were selected, 20 in CG and 10 in PEG. There were no significant differences in maternal, gestational and perinatal outcomes between the two groups, except for the gestational age at the onset of prenatal care, which was lower in PEG (12.5 vs. 20.0 weeks, p = 0.015). PEG showed strong immunostaining for IL-15 (p = 0.001) when compared to CG, with no difference between the groups concerning the placental expression of dNK (p = 0.999), IL-10 (p = 0.063), and IL -12 (p = 0.135). Relating to maternal circulating interleukins, a significant increase in IL-10 (22.7 vs. 11.9 pg/mL, p = 0.024) and IL-15 (15.9 vs. 7.4 pg/mL, p = 0.024) was observed in PEG, with no difference between the groups for IL-12 (102.5 vs 61.5 pg/mL, p = 0.373). We also demonstrated higher maternal levels of sFlt-1 (15920 vs. 7978 pg/mL, p = 0.009) and sFlt-1/PlGF ratio (88.71 vs. 24.63, p = 0.002) and lower levels of PlGF (193 vs. 340.6 pg/mL, p = 0.036) in PEG. CONCLUSION: A higher maternal serum concentration of both pro- and anti-inflammatory factors was observed in the PEG. However, no difference was found between the groups regarding the placental expression of IL-10, an important anti-inflammatory factor. These findings may suggest that the maternal serum attempt to balance these interleukins did not reach local placental response, which contribute to the development of the disease in the PEG