Host immune responses must be tightly regulated by an intricate balance between positive and negative signals while fighting pathogens; persistent pathogens may usurp these regulatory mechanisms to dampen host immunity to facilitate survival in vivo. Here we report that Tim-3, a negative signalling molecule expressed on monocytes and T cells, is up-regulated on natural killer (NK) cells in individuals chronically infected with hepatitis C virus (HCV). Additionally, the transcription factor T-bet was also found to be up-regulated and associated with Tim-3 expression in NK cells during chronic HCV infection. MicroRNA-155 (miR-155), an miRNA that inhibits signalling proteins involved in immune responses, was down-regulated in NK cells by HCV infection. This Tim-3/T-bet over-expression and miR-155 inhibition were recapitulated in vitro by incubating primary NK cells or NK92 cell line with Huh-7 hepatocytes expressing HCV. Reconstitution of miR-155 in NK cells from HCV-infected patients led to a decrease in T-bet/Tim-3 expression and an increase in interferon-γ production. Blocking Tim-3 signalling also enhanced interferon-γ production in NK cells by improving signal transducer and activator of transcription-5 phosphorylation. These data indicate that HCV-induced, miR-155-regulated Tim-3 expression regulates NK cell function, suggesting a novel mechanism for balancing immune clearance and immune injury during chronic viral infection.

Immunotherapy has emerged as a current and future paradigm of cancer treatment, which utilizes the body’s immune system to eradicate cancer. Natural Killer (NK) cells as part of the innate immune system have immense potential in their anti-tumor cytotoxic activities and host cell surveillance properties. NK cells comprise approximately five to fifteen percent of peripheral blood lymphocytes and can be proliferated in vitro using recently developed methods with co-cultures with feeder cells (derived from engineered tumor cells) or plasma membrane (PM) particles, produced from the fore mentioned feeder cells, in combination with soluble cytokines. For efficient growth and maintenance of these NK cells, Interleukin-2 (IL-2) is utilized. IL-2 in solution, through receptor mediated signaling, stimulates proliferation of T-cells and NK cells. NK cells have lower responsiveness to IL-2 and consequently require a larger systemic dose to stimulate them as opposed to competing cell populations that have higher expression of receptors for IL-2, such as T-cells, which can have the effect of lower effective stimulation of NK cell growth. Such difference in the stimulatory capability of IL-2 toward NK cells and the short circulation lifetime of soluble IL-2 require higher dosages of soluble IL-2 for effective in vivo NK cell proliferation for therapeutic application against cancer, but is toxic. Therefore establishing another form of IL-2 delivery that improves its specific targeting to NK cells would be beneficial and may be crucial for novel therapeutic improvement. The Copik Laboratory has made an IL-2 fusion protein construct having a membrane anchor for expression of membrane-bound IL-2 on K562-41bbl-21 cells (K562-IL21). K562-IL21 cells are selectively recognized by NK cells and stimulate their proliferation and cytotoxicity. Hence, a K562-IL21 membrane–bound IL-2 form should be targeted to NK cells with IL-2 delivery. K562-IL21-2 cells were then used to prepare PM21-2 particles which have the potential to provide NK cell targeted, long-lived form of IL-2 for use as an injectable drug for in vivo adjuvant stimulation of NK cells. The presence of IL-2 on the in the PM21-2 particle product was verified by Western blot, and ELISA. Particle preparations from the modified K562 cells should possess characteristics that allow them to possibly replace soluble IL-2 and more specifically increase the numbers or anti-tumor activity of NK cell populations. The effect of PM21-2 particles was studied in in vitro culture based experiments, which tested the effectiveness the PM21-2 particles to induce selective NK cells expansion as compared to PM21 particles in the presence or absence of soluble IL-2.

Cytokines are small, secreted proteins that are important for cell signalling in theimmune system. Interferon gamma (IFN-γ) is one of the most potent cytokines thatnatural killer (NK) cells of the innate immune system secrete with both antiviral,antibacterial, and antitumoral activity. Analysis of NK cells, such as that of secretionof IFN-γ, is important for studying the immune response to cancer and for developingeffective immunotherapies. In this master thesis project, a method was developedfor determining the amount of IFN-γ secreted by NK cells when being confinedwith cancer cells in deep microwells. Antibody-coated microbeads was used tocapture secreted IFN-γ, which was fluorescently labeled and detected by imaging usingfluorescence microscopy. Microbead seeding into small microwells for single cellassays and into large microwells for embedding of beads into 3D tumor spheroidswas investigated. An analytical model based on experimental standard curves wasdeveloped for straightforward quantification of the amount of bound IFN-γ, with ademonstrated detection down to 2.10−18 moles per bead. The detection of IFN-γ wasevaluated for primary NK cells stimulated by PMA/ionomycin for different incubationtimes. The secretion rate of IFN-γ by IL-2 activated NK cells under PMA/ionomycinstimulation was estimated at 184 molecules per second. IFN-γ detection was alsoevaluated in cell cytotoxicity assays where NK cells were confined over time togetherwith cancer cells in microwells. Both assays showed a successful detection of IFN-γ secretion, demonstrating the potential of the developed method for immune cellanalysis. / Cytokiner är små proteiner som är viktiga för cellsignalering inom immunförsvaret.Interferon gamma (IFN-γ) är en av de mest potenta cytokinerna som naturligamördarceller (NK) i det medfödda immunsystemet utsöndrar med både antiviral,antibakteriell och antitumoral aktivitet. Analys av NK-celler, av till exempelutsöndring av IFN-γ, är viktigt för att studera immunsvaret vid cancer och för attutveckla effektiva immunterapier. I detta examensarbete har en metod utvecklatsför att bestämma mängden IFN-γ som utsöndras av NK-celler när de är tillsammansmed cancerceller i djupa mikrobrunnar. Antikroppsbelagda mikrokulor användesför att fånga utsöndrat IFN-γ, som sedan fluorescensinmärktes och detekteradesgenom fluorescensmikroskopi. Distributionen av dessa kulor studerades i småmikrobrunnar för encellsanalyser och i stora mikrobrunnar för inbäddning av kulornai 3D-tumörsfäroider. En analytisk modell baserad på experimentella standardkurvorutvecklades för enkel kvantifiering av mängden bunden IFN-γ, med en påvisaddetektion ner till 2.10−18 mol per kula. Detektionen av IFN-γ utvärderades för primäraNK-celler stimulerade med PMA/ionomycin för olika inkubationstider. Sekretionenav IFN-γ från IL-2-aktiverade NK-celler vid stimulering med PMA/ionomycinuppskattades till 184 molekyler per sekund. IFN-γ-detektion utvärderades ocksåför analyser av cell-cytotoxicitet där NK-celler var placerade tillsammans medcancerceller i mikrobrunnar över tid. Båda analyserna visade en framgångsrikdetektering av utsöndrad IFN-γ, vilket visar potentialen hos den utvecklade metodenför immuncellsanalys.

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Les cellules Natural Killer (NK), un sous-type de lymphocytes, sont cruciales dans l’immunité innée de par leur cytotoxicité directe envers les tumeurs, les cellules infectées et les cellules stressées. Elles contribuent également à l’orchestration de la réponse immunitaire adaptative de par leur capacité à produire des cytokines immunorégulatrices. Pour développer ces fonctions effectrices, les cellules NK requièrent l’intégration d’une multitude de signaux. De là, les cellules NK matures (mNK) sont considérées comme appartenant à quatre sous-types conséquents et concordants avec la polarisation vers la prolifération, la production de cytokines ou les propriétés cytotoxiques. Dans certaines maladies auto-immunes, tel le diabète de type 1 (T1D), un bloc dans leur maturation, et conséquemment des défauts en nombre et en fonctions, peut être observé. Ainsi, afin de découvrir la régulation génétique derrière la proportion et la maturation des cellules NK, des études de liaison génétique ont été produites sur des souris F2 (B10.BR x NOD.H2k) et F2.Rag (B6.Rag1-/- x NOD.Rag1-/-), où le fond génétique NOD est un modèle de T1D. Ces analyses ont révélé des locus potentiellement impliqués dans la régulation de la proportion, des nombres absolus ainsi que dans la maturation fonctionnelle des cellules mNK. Les chromosomes 8, 9 et 17 ont été liés à la proportion des cellules mNK tandis que les chromosomes 2, 4, 7, 10, 11 et 18 ont été liés aux sous-types fonctionnels de ces mêmes cellules. De là, nous avons validé l’influence du locus du chromosome 9 sur la proportion des cellules mNK en générant des souris sous-congéniques avec des insertions de segment génétique B10.BR dans des souris de fond génétique NOD. La proportion et le nombre absolu de cellules mNK ont ensuite été analysés par cytométrie en flux et comparés à ceux de souris contrôles. Pour la maturation fonctionnelle, nous avons retenus certains gènes candidats, et régulateurs associés, liés à un ou plusieurs sous-types de mNK, dont Tbx21, Zeb2, c-Myb, Trp53 et Pmaip1. Par association de voies de signalisation, nous sommes également allés vérifier certaines protéines associées, dont Bim et Eomesodermin. Nous avons alors validé leur implication par l’utilisation de modèles murins knock-out, qPCR, essais de prolifération et d’apoptose. Enfin, nos résultats supportent un rôle pour le locus du chromosome 9 dans la régulation de la proportion de cellules mNK ainsi qu’un rôle pour Trp53, Bim et Pmaip1 dans la maturation fonctionnelle de celles-ci. Nos études révèlent donc de nouveaux gènes candidats potentiels dans la régulation des cellules NK, dont les mécanismes pourront être approfondis dans la perspective de développement de thérapies cellulaires et génétiques dans le combat contre les cancers et les infections chroniques. / Natural Killer (NK) cells, a subset of lymphocytes, are crucial in innate immunity due to their direct cytotoxicity towards tumors, viral infected cells and stressed cells. NK cells also contribute to the orchestration of the adaptive response by their ability to produce immunoregulatory cytokines. To develop those effector functions, NK cells require the integration of multiple signals. As of now, mature NK cells (mNK) can be separated into a four-stage model of functional maturation that concords with a polarization either toward proliferation and cytokine production or cytotoxic functional properties. In autoimmune diseases, like type 1 diabetes (T1D), a block in their maturation, and consequently an impaired functionality and diminished numbers, can be observed. Thus, in order to uncover the genetic regulation behind the proportion and functional maturation of NK cells, a linkage analysis was performed by our lab on F2 (B10.BR x NOD.H2k) and F2.Rag (B6.Rag1-/- x NOD.Rag1-/- intercross) mice, where the NOD genetic background is a model of T1D. This analysis revealed loci that were potentially involved in the regulation of mNK cells proportion, absolute numbers or functional maturation. Loci on chromosomes 8, 9 and 17 were linked to the proportion of mNK cells while loci on chromosomes 2, 4, 7, 10, 11 and 18 were linked to different subsets of functional mNK cells. Hence, we validated the influence of the chromosome 9 locus on the proportion of mNK cells by generating congenic sub-strains of mice with insertion of B10.BR genetic segments and NOD genetic background. The proportion and absolute numbers of mNK cells were assessed by flow cytometry and compared to those of wild-type mice. Regarding the functional maturation of mNK cells, we considered potential candidate genes, and their upstream regulators, that were linked to one or more mNK subsets, namely Tbx21, Zeb2, Myb, Trp53 and Pmaip1. From those, we also looked further their associated pathway with proteins such as Bim and Eomesodermin. We proceeded to the in vivo validation of their implication by qPCR, proliferation and apoptosis assays and the use of knock-out mice. Indeed, our results support a role for a locus on chromosome 9 in the regulation of mNK cells proportion and for Trp53, Bim and Pmaip1 in NK cell functional maturation. As such, our study has revealed new candidate genes in NK cell regulation. Further explorations of the mechanisms by which those genes act could lead to the development of cellular and genetic therapies for cancers and chronic infections.

INTRODUÇÃO: O fato do vírus da Hepatite C (HCV) estabelecer uma infecção crônica persistente, na maioria dos casos, mesmo sendo reconhecido e alvejado pelos sistemas imune inato e adaptativo sugere que o mesmo tenha desenvolvido estratégias eficazes para driblar a ação desses sistemas. O HCV interfere na fase inicial de ativação da resposta imune adaptativa alterando a função das células dendríticas (DCs), o que provavelmente leva a uma ativação deficiente das células natural killer (NKs) e de linfócitos T. Portanto, a realização de estudos sobre DCs e NKs na infecção pelo HCV se torna de fundamental importância para a compreensão da patogênese e persistência desta infecção. MÉTODOS: Foram selecionados indivíduos com resolução espontânea da infecção pelo HCV, indivíduos com infecção crônica e indivíduos saudáveis. A técnica de citometria de fluxo foi utilizada para a determinação da frequência e do fenótipo de células dendríticas e NKs nesses indivíduos. Além disso, foi avaliada a atividade citotóxica das células NKs sob estímulo de IL-12 e IL-18, e também da linhagem K-562. RESULTADOS: A frequência de DC mielóides (mDC) expressando CD86, nos indivíduos crônicos, foi elevada e uma correlação positiva com a carga viral foi observada. Na análise do ensaio funcional foi observado que as populações de células NKs CD7+ CD57+ apresentaram maior expressão da molécula CD107a e baixa produção de IFNy nos indivíduos com infecção crônica. A constante exposição das células imunes ao IFN-alfa, induzido durante a infecção pelo HCV, resulta na polarização do fenótipo citotóxico, caracterizado por células NK ativadas com elevado poder de degranulação, mas com deficiente produção de IFN-y. CONCLUSÕES: As frequências das células DCs e NKs eram semelhantes em todos os indivíduos. A expressão da molécula CD86 na superfície das mDCs pode ter sido induzida pela presença do HCV, uma vez que foi observada correlação positiva com a carga viral. Células NK citotóxicas, altamente diferenciadas e incapazes de produzir IFN-y foram as mais frequentes na infecção crônica pelo HCV. A baixa produção de IFN-y por parte dessas células é um dos fatores envolvidos na deficiente ativação de uma resposta imune adaptativa capaz de controlar a infecção pelo HCV / INTRODUCTION: Hepatitis C virus (HCV) develops a chronic persistent infection in most of the cases, even being recognized and targeted by the innate and adaptive immune systems, suggests that the virus have developed effective strategies to circumvent the action of these systems. HCV interferes in the initial activation of the adaptive immune response by altering the function of dendritic cells (DCs), which probably leads to a deficient activation of natural killer cells (NK) and T lymphocytes. Therefore, studies of DCs and NK in HCV infection are very important for understanding the pathogenesis and the persistence of this infection. METHODS: We selected subjects with spontaneous resolution of HCV infection, with chronic infection and healthy subjects. Flow Cytometry was used to determine the frequency and phenotype of dendritic cells and NK cells of these individuals. In addition, we evaluated the NK cell cytotoxic activity in response to stimulation of IL-12 and IL-18 and in co-cultivation with the cell line K-562. RESULTS: In individuals with chronic infection, the frequency of myeloid (m) DC cells expressing CD86 was elevated and a positive correlation between these cells and viral load was observed. It was observed in chronic infected individuals that NK cells co-expressing CD7 and CD57 showed higher expression of CD107a and low production of IFN gamma. The constant exposure of immune cells to IFN-alfa induced during HCV infection results in the polarization of cytotoxic phenotype characterized by activated NK cells with high power degranulation, but with impaired production of IFN-y. CONCLUSIONS: The frequency of DCs and NK cells were similar in all individuals. The expression of CD86 molecule on the surface of mDCs may have been induced by the presence of HCV, since a positive correlation was observed with viral load. Cytotoxic NK cells, highly differentiated and unable to produce IFN-y, were the most frequent in chronic HCV infection. The low production of IFN-y by these cells is one of the factors involved in the poor activation of an adaptive immune response able to control HCV infection

A endometriose (EDT) é caracterizada pela presença de tecido endometrial fora da cavidade uterina, e afeta mulheres em idade reprodutiva. Postulamos que alterações na frequência de células T reguladoras (Treg), natural killer (NK), supressoras mielóides (MDSC) e dendríticas (DC) no peritônio justificariam a redução da capacidade do sistema imune de reagir contra as células endometriais, permitindo sua implantação em locais ectópicos. Aqui, células Treg, NK, MDSC e DC foram quantificadas no fluido peritoneal (FP) e sangue de mulheres com EDT, a fim de associa-las ao desenvolvimento da doença; níveis de citocinas também foram avaliados. Na EDT, observou-se aumento na frequência de Treg, MDSC e DC no sangue e aparente redução destas no FP; ainda, a concentração de IL-12 foi menor no sangue comparadas ao grupo controle. Não foram observadas diferenças quanto às células NK e as outras citocinas analisadas. Os resultados indicam aumento da frequência de populações reguladoras em amostras de sangue de pacientes EDT, entretanto esses resultados não são refletidos no FP. / Endometriosis (EDT) is a gynecological disease characterized by the presence of endometrial cells out of the uterine cavity, which affects women in reproductive age. We postulated that alterations in the frequencies of regulatory T cells (Treg), natural killer cells (NK), myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) in the peritoneum could justify the reduced capacity of the immune system to react to these ectopic endometrial cells, allowing them to invade distant tissues. Here, Treg, NK, MDSC and DC were quantified in the peritoneal fluid (PF) and peripheral blood (PB) of women with EDT, in order to associate them with the development of EDT; cytokine levels were also assessed. In EDT, higher frequencies of Treg, MDSC and DC in the PB and apparent lower frequencies of these cells in the PF were observed; IL-12 concentration was smaller in PB of EDT compared to control. No differences between groups were observed for NK cells and the other cytokines evaluated. The results indicate higher frequencies of regulatory cells in PB samples of EDT patients, although these findings were not reflected in PF samples.

Uterine NK (uNK) cells are a distinct subset of NK cells in the decidua of humans and rodents during pregnancy, which are essential for remodelling of the spiral arteries supplying the feto-placental unit. Similarly to peripheral NK cells, uNK cells express Natural Killer receptors (NKRs) that engage MHC class I molecules. Evidence from human genetic association studies suggests that, in the presence of allogeneic cognate paternal MHC class I ligands, inhibitory uterine NKRs are associated with disorders of pregnancy arising from impaired decidual vascular remodelling. Conversely, enhancement of human uNK cell activity through activating NKRs is associated with high birth weight. Evidence from mouse models corroborates that uNK cell activity is modulated by interactions between NKRs and MHC class I, but has largely focussed on the effect of paternal MHC. In this study, the contribution of maternal immunogenetic regulation of NK cell function to reproductive outcome was assessed independently of parental MHC disparity in mice. To evaluate the role of NKR genes in isolation, I used congenic B6.BALB-TC1 (TC1) mice that differ from C57BL/6 (B6) mice only within the region of chromosome six encoding NKRs that recognise MHC class I. Absence of a major inhibitory NKR for self-MHC, Ly49I, in TC1 mice causes a compensatory shift in the NKR repertoire expressed and preserves a majority subpopulation of educated NK cells. B6 and TC1 splenic and uterine NK cells are similarly functionally reactive and mature, and no significant differences could be detected in spiral arterial remodelling or fetal growth between these strains in MHC-syngeneic matings. This supports data from human immunogenetic studies showing that maternal uterine NKRs are not associated with differences in pregnancy outcome in the absence of novel paternal MHC class I ligands, and highlights the importance of maternal and paternal co-regulation of uNK cell activity during pregnancy. No mouse models of uNK cell activation are currently available with which to corroborate human immunogenetic associations between activating uterine NKRs and high birth weight. Male m157-transgenic (m157-Tg) mice, which ubiquitously express viral m157 glycoprotein ligands for the activating NKR Ly49H, were mated with B6 females. Exclusive expression of m157 glycoprotein by trophoblast improved placental efficiency, but did not enhance fetal growth. Some fertility clinics surmise that uNK cell activation initiates the pathogenesis of spontaneous abortion. It has been suggested that this may occur due to reduced expression by human uNK cells of miR-483-3p, which stimulates endogenous insulin-like growth factor (IGF)-1 production and uNK cell cytotoxicity in vitro. It is demonstrated here that neither miR-483-3p nor IGF-1 regulate murine NK cell development, maturation or function. No discernible reproductive phenotype is evident in miR-483 deficient females. It can be inferred that post-transcriptional control by miR-483 is not biologically relevant to murine NK cell function. Although m157-Tg mice may provide an interesting model to further study uNK cell-mediated placental adaptations, it remains important to identify a murine model of enhanced uNK cell function to corroborate human immunogenetic associations with high birth weight and to challenge the supposition that uNK cell activation is harmful to pregnancy.

We performed a retrospective study in 185 patients with myelogenous leukemias who had received hematopoietic cells from unrelated donors. The aim of this study was to answer the question wether the benefit of KIR ligand incompatibility seen in haploidentical tranplantations can also be seen using unrelated donors. We could not detect a significant difference in survival between patients with a KIR ligand incompatibility and those with either fully matched or partially mismatched unrelated donors in this patient cohort. / In der Therapie von Leukämien ist die Knochenmark- bzw. Stammzelltransplantation eine tragende Säule. Für den Transplantationserfolg ist eine Übereinstimmung der Haupthistokompatibilitätsantige (HLA-Antigene der Klassen I und II) zwischen Spender und Empfänger von zentraler Bedeutung. Diese Notwendigkeit ergibt sich aus der sogenannten MHC-Restriktion in der T-Zellrezeptorerkennung. Ob auch NK-Zellrezeptoren und deren Liganden in der Spenderauswahl berücksichtigt werden sollten, ist bisher unzureichend untersucht. Insbesondere trifft das für die KIR-Rezeptoren zu, die wie die T-Zellrezeptoren ebenfalls HLA-Antigene als Liganden besitzen. Velardi et al. haben 2002 erstmalig gezeigt, daß in der Therapie myeloischer Leukämien die Transplantation von Blutstammzellen verwandter Spender mit KIR-Liganden-Inkompatibilität von klinischem Vorteil ist. Ob KIR-Liganden-Inkompatibilität auch bei Knochenmark-/ Stammzelltransplantationen Unverwandter Bedeutung erlangen könnte, war zu Studienbeginn offen und blieb auch infolge diskrepanter Untersuchungsergebnisse von verschiedenen Arbeitsgruppen im Verlauf der Studie widersprüchlich. Im Rahmen dieser Arbeit wurde diese Fragestellung, die auch Teil einer internationalen Studie war, an 185 Spender-Empfänger-Paaren retrospektiv untersucht. Dabei wurde bei den Paaren einerseits die KIR-Liganden-Kompatibilität auf der Grundlage der HLA-C-Supertypen erschlossen (nach Velardi et al.). Andererseits konnte sie im internationalen Studienprogramm direkt aus dem KIR-Genotyp des Spenders und dem HLA-C-Supertyp des Empfängers ermittelt werden. Die Untersuchungen ergaben folgende Resultate: bei Vorliegen von KIR-Liganden-Inkompatibilität hat die Verwendung von ATG als Bestandteil der GvHD-Prophylaxe keinen Einfluß auf das klinische Ergebnis. Die Vermutungen von Giebel et al. wurden damit nicht gestützt. Die Bestimmung des KIR-Liganden-Status mit Hilfe der Rückschlußmethode allein aus dem HLA-Typ ist unzuverlässig. Für eine exakte Differenzierung ist die gleichzeitige KIR-Genotypisierung erforderlich. KIR-Liganden-Inkompatibilität ist bei unverwandten Knochenmark-/ Stammzelltransplantationen nicht von klinischem Vorteil. Auch ein gezieltes Aussuchen HLA-C-inkompatibler Spender auf der Grundlage einer KIR-Genotypisierung stellt derzeit keine therapeutische Option dar.