In the present study, we characterized the ability of a novel oncolytic rhabdovirus - Maraba MG1 to boost Natural Killer (NK) cell activity. In tandem, we addressed the ability of this enhanced NK cell functionality to reduce the incidence of post-cancer surgery micrometastases. Due to the potential safety barriers associated with the use of a live virus immediately prior to surgery in cancer patients, we generated a single cycle replication virus (MG1-Gless) and UV-inactivated MG1 to stimulate NK cell function and reduce post-operative metastases. Our in vivo data demonstrate that significant NK cell activation and a similar level of reduction in postoperative tumor metastases was achieved with live MG1, MG1-Gless and UV-inactivated MG1, concluding that viral replication is important, but not necessary for NK cell activation. Mechanistically, we observed that dendritic cells (DCs) are necessary intermediates for MG1-induced NK cell activation. Finally, we characterized and compared a panel of UV-inactivated MG1 (2mins to 2hrs) to better understand the requirements for NK cell activation. Our results suggest that intact viral particle and cellular recognition and association are essential for NK cell mediated anti-tumor responses. These findings provide the preclinical rationale to develop safe and viable virotherapy-based interventional protocols that might reduce the risk of metastatic disease after cancer surgery.

Suppression of anti-tumour immunity by the tumour microenvironment remains a major barrier to the development of broadly effective immunotherapies to treat solid tumours. Cytotoxic natural killer (NK) cells are vital to anti-cancer immunity and have shown clinical efficacy for treating hematologic malignancies. However, NK cell therapies have failed to be effective against solid tumours as cytotoxic NK cells become dysfunctional in the tumour microenvironment. While tumours hinder cytotoxic NK cells, they stimulate the tumour-promoting functions of regulatory NK cells. The mechanisms that dictate NK cell polarization and their fate in the tumour microenvironment remain poorly defined but harbour key therapeutic potential. Glucose-driven cellular metabolism has emerged as a central regulator of NK cell anti-tumour activity. Notably, tumour cells have deregulated metabolism, causing a metabolically hostile environment that is low in glucose and oxygen and high in metabolic waste. In the work presented, we demonstrate that NK cells expanded from cancer patients or healthy donors exert strong anti-tumour activity and dismantle the immunosuppressive tumour microenvironments of advanced ovarian and lung cancer. As a result, expanded NK cells were capable of sensitising initially non-responsive patient tumours to PD1 checkpoint-blockade therapy. Further, we uncover that the activity of cellular metabolic pathways plays a key role in NK cell functional fate in tumour microenvironment. We show that the tumour microenvironment induces paralysis of cytotoxic NK cell glucose metabolism to cause their dysfunction. However, reprogramming of NK cell metabolism through expansion arms expanded NK cells with enhanced metabolic flexibility which enabled their anti- tumour activity to be paradoxically strengthened by the tumour microenvironment. We further identify that regulatory NK cells have a distinct metabolic program compared to cytotoxic NK cells, including lower glucose-driven metabolism, that is amenable with the tumour microenvironment. Our work provides new mechanistic insight into how NK cell fate is regulated and how the pathological environment of a tumour capitalizes on this. This knowledge provides new therapeutic targets to intervene with the suppression of cytotoxic immunity in tumours. Further, this work identifies that expanded NK cells are a promising therapeutic candidate that exploit the metabolic hostility of the tumour microenvironment and synergize with other immunotherapies. / Thesis / Candidate in Philosophy / Harnessing the body’s natural immune defenses against cancer in the form of immunotherapy has emerged as a powerful treatment modality. Over the past decade, immune cell therapies have revolutionized the treatment of blood cancers like leukemia and lymphoma. Yet despite the potential, immune cell therapies have failed to be broadly effective against solid tumours because the anti-cancer activity of immune cells, such as Natural Killer (NK) cells, becomes severely impaired by the tumour environment. In this work, we identify that NK cells expanded from cancer patients and healthy donors overcome suppression by tumours and eliminate detectable tumour in pre-clinical models of advanced ovarian and lung cancer. These expanded NK cells also enhanced the functions of other immunotherapies. Further, we shed new light on how NK cells become dysfunctional in tumours. We uncover that NK cells undergo a metabolic energy crisis in tumours that causes their dysfunction, but that expanded NK cells have increased metabolic fitness which allows them to overcome this energy crisis and remain highly functional. Finally, we also characterize the metabolism of a subset of NK cells that are tumour-promoting and find that they harbour metabolic advantages to thrive in tumours. Overall, our work provides new insight as to how to overcome immunosuppression by tumours. This work identifies that expanded NK cells are a promising therapeutic candidate that exploit the hostility of tumours and synergize with other immunotherapies.

Human natural killer (NK) cells are found in virtually all tissues where they act as a first line defense against malignant and virally infected cells. The development of NK cells from CD34+ hematopoietic progenitors is a complex process that involves navigating through different microenvironments and requires contact-dependent interactions with stromal cells. The molecular mediators of NK cell developmental subset trafficking, cell-cell interactions, and maturation have not been fully characterized. This thesis presents 3 studies that aim to uncover contact-dependent interactions that drive human NK cell development. Chapter 2 focuses on defining the adhesome profile of human NK cells from in vitro derived populations, tonsil, and peripheral blood. This study reveals that the tissue origin and developmental stage of NK cells influence the expression of adhesome-associated genes and proteins, as well as the content of cortical actin, which suggests a link between adhesome expression and actin regulation in NK cells. Chapter 3 presents the first comprehensive study on human NK cell development in pediatric tonsil using cyclic immunofluorescence microscopy and imaging mass cytometry. We reveal that NK cell subset localization and interactions are dependent on NK cell developmental stage and tissue residency. Chapter 4 demonstrates that neural cell adhesion molecule (NCAM) on stromal cells promotes maintenance of a mesenchymal-like state and subsequently the survival and proliferation of human NK cell precursors. Overall, this thesis provides new insights on previously unknown mediators of NK cell contact-dependent interactions and unveils the first road map of in situ NK cell development.

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Recent developments in the expansion and manipulation of primary NK cells has allowed this source of effective anti-tumour cells to be exploited for cell-based cancer immunotherapies. While ex vivo expanded primary NK cells are highly effective in the treatment of haematological malignancies, their efficacy against the solid tumour has been limited due to the presence of immune-regulatory factors in the tumour microenvironment. These factors can abrogate NK cell function by down regulating the expression of NK activating receptors, thus preventing these highly cytotoxic effector cells from activating in response to tumour challenge. Our work explores whether the expression of a tumour specific chimeric antigen receptor (CAR) on ex vivo expanded primary NK cells would allow the lost activatory signalling to be recouped, and regain their efficacy against the solid tumour. Unfortunately, the use of primary NK cells as effectors in CAR based cell immunotherapies has been hampered by the technical limitations of producing large numbers of CAR positive primary NK cells. This has led many researchers to utilise the NK-92 cell line instead of primary cells. We demonstrate that ex vivo expanded primary CAR NK cells can be produced efficiently and demonstrate higher anti-tumour functionality than CAR NK-92. Finally, due to the intricacies of NK cell biology, they are able to effectively discriminate between healthy and malignant targets thus preventing their cytotoxic function from being directed towards the incorrect target. This could be a key advantage in the use of primary NK cells over T cells as effectors of CAR as the off-tumour/on-target adverse effects seen with CAR T cells has severely hampered this clinical strategy. We have shown that CAR T cells but not CAR NK cells are reactive towards phenotypically non-malignant, clinically relevant, healthy cells expressing the CAR target. / Thesis / Master of Science (MSc)

Orientador: Erick da Cruz Castelli / Resumo: O gene KIR2DL4 codifica um importante receptor de células Natural Killer (NK). O único ligante de KIR2DL4 conhecido é a molécula HLA-G, expressa principalmente na placenta, modulando a ação das células NK durante a gestação. O gene KIR2DL4 parece ser bastante variável, quando considerado os bancos de dados que armazenam suas sequências conhecidas, porém não está claro o nível de diversidade deste gene em populações reais e heterogêneas. Polimorfismos presentes no gene KIR2DL4 poderiam influenciar a interação entre KIR2DL4 e HLA-G, modificando a ação das células NK. Neste estudo exploramos a variabilidade genética de KIR2DL4 em 157 indivíduos oriundos do Estado de São Paulo/Brasil. Devido à alta similaridade de sequências entre os genes KIR, erros de genotipagem são esperados quando se utiliza sequenciamento de segunda geração. Por este motivo, desenvolvemos uma abordagem para classificar cada leitura com base em sequências KIR conhecidas, endereçando-as ao gene mais provável. Também utilizamos o painel SNPforID 34-plex para avaliar a ancestralidade dessas amostras. Considerando o segmento completo desse gene, indo da região 5’URR até a 3’UTR, com aproximadamente 13kb, o gene KIR2DL4 se mostrou pouco polimórfico, com 152 pontos de variações identificados (MAF 1%). Esses pontos de variação estão organizados em 32 haplótipos estendidos que codificam 13 proteínas diferentes. Foram encontrados 11 haplótipos na região promotora, sendo que 8 possuem MAF maior que 1%. Na região codif... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: KIR2DL4 is the most unusual Killer Cell Immunoglobulin-Like receptor (KIR) family member in terms of structure, expression, and signaling properties. The only known KIR2DL4 ligand is HLA-G, and polymorphisms might disrupt this interaction. KIR2DL4 variability is not well explored in admixed populations. Here we explored KIR2DL4 exon variability in 157 individuals from the State of São Paulo/Brazil. Because of sequence similarity with other KIR genes, it is expected genotyping errors when using secondgeneration sequencing. We developed an approach to score each read based on known KIR sequences, addressing them to the most likely locus. We evaluated the SNPforID 34-plex panel to assess ancestry. The methodology was applied to survey the variability of a very admixed population, such as Brazilian, counting with 157 samples of São Paulo State. Considering a segment of about 13-kb, KIR2DL4 gene was conserved with few different and frequent sequences. Overall, 152 variable sites were detected, arranged in 32 haplotypes codifying 13 protein. We found 11 promoter haplotypes, 8 with a frequency greater than 1%. In the coding region we detected 70 haplotypes, four of which correspond to 50% of the coding sequences (KIR2DL4 * 0080204, * 008105, * 001, * 005). In the 3'UTR region, 14 haplotypes were identified with MAF greater than 1%. The KIR2DL4 coding region was the most variable segment. We observed that KIR2DL4 variability is strongly influenced by the sample ancestry background. K... (Complete abstract click electronic access below) / Mestre

Submitted by Filipe dos Santos (fsantos@pucsp.br) on 2017-09-13T13:44:48Z No. of bitstreams: 1 Thiara Ribeiro Santos.pdf: 5083039 bytes, checksum: a455035acf193849f87dc2fe74e782ca (MD5) / Made available in DSpace on 2017-09-13T13:44:48Z (GMT). No. of bitstreams: 1 Thiara Ribeiro Santos.pdf: 5083039 bytes, checksum: a455035acf193849f87dc2fe74e782ca (MD5) Previous issue date: 2017-08-28 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação São Paulo - FUNDASP / This research investigates the narratives of television series about serial killers, focusing most attention on the North American show Dexter, broadcast from 2006 to 2013, while at the same time comparing the context of the series to others of the same genre. In order to learn how the fascination of crime is narrativized in this type of program, our aim was to identify the similarities and differences, compare the narratives, and the main conflicts of each series, the protagonism of the serial killer, the relations between the subject-characters and complex issues related to actions and characteristics of each serial killer and the development of the narrative itself – all focused on the protagonist’s compulsion to kill, while taking into consideration their permanent invisibility and the social ties between them and the other characters; thus we look back in time to delve into the discursive processes of old movies with the same theme in order to understand how the world of series was established. We start with the assumption that a transformation has been made to the narrative structure and character of the serial killers in audiovisual productions over recent decades. Under this context, there is a need to understand how audiovisual languages are used within series, how they intensify normal versus abnormal opposition, how the viewers’ complicity is summoned, and how violence and perversion become elements of the social structure and eventually even agents of the actions. In our view, questioning what retains the tension in the narrative of the Serial Killers TV series is one way of assessing our processes of true meaning and civil morality. This theory is based on knowledge of the areas of Audiovisual, Semiotics, Psychoanalysis, Sociology and Philosophy. For the audiovisual area, we benefitted from input from Neale, Arlindo Machado and Sonia Rodrigues who have studied narrative strategies in dramatic series; the studies relating to cinema were enhanced by contributions from Shaviro and Naremore and the studies of series consolidated by Douglas L. Howard; investigations made about cinema and representation by Ismail Xavier also strengthened the analyses of this thesis. The analysis of the generative route toward the meaning of narratives was bolstered by the discursive-semiotic contributions from Eric Landowski and Ana Claudia de Oliveira, and the tensive-semiotics contributions from Claude Zilberberg. With regard to psychoanalysis, particularly to focus on the Lacanian concept of perversion and neurosis, we use texts by Nasio, Žižek, Bond, Philippe Julien and Antonio Quinet, which assist us in explaining the universe of our subject, the serial killer. For the sociological and ethical discussions about abnormality and perversity we adopt the thoughts of Dany-Robert Dufour and Simpson / A pesquisa investiga o tema das narrativas de séries televisivas sobre serial killers, centrando sua atenção na série norte-americana Dexter, produzida no período de 2006 a 2013, sem deixar de examiná-la no contexto de outras séries do mesmo gênero. Para saber como o fascínio do crime é narrativizado neste tipo de série buscamos identificar suas semelhanças e diferenças, comparando as narrativas, os conflitos principais, o protagonismo do serial killer, as relações entre os sujeitos-personagens e questões complexas ligadas às ações e características de cada serial killer e ao desenvolvimento da narrativa em si – todas centradas na compulsão de matar do protagonista, considerando sua permanência na invisibilidade e os laços sociais entre ele e os demais personagens; para isto buscamos recuperar historicamente o processo de construção discursiva de filmes antigos com a mesma temática para compreendermos como se estabeleceu o universo das séries. Partimos do pressuposto de que existe uma transformação da estrutura narrativa e dos personagens serial killers nos audiovisuais das últimas décadas. Há, nesse contexto, uma necessidade de entender como as séries se utilizam das linguagens do audiovisual, como tensionam a oposição normal versus anormal, como se dá a convocação da cumplicidade do espectador e como a violência e a perversão se tornam elementos de estruturação da organização social e eventualmente efetivadoras da sanção. A nosso ver, questionar sobre o que mantém a tensão da narrativa das séries é um modo de avaliarmos nossos processos de significação e moralidade civil. A fundamentação teórica se baseia nos saberes das áreas de Audiovisual, Semiótica, Psicanálise, Sociologia e Filosofia. Para o audiovisual, utilizamos Neale, Arlindo Machado e Sonia Rodrigues em seus estudos sobre as estratégias narrativas em séries dramáticas; os estudos sobre cinema contaram com os aportes de Shaviro e Naremore e os estudos de série com Douglas L. Howard; as investigações sobre cinema de Ismail Xavier também fortalecem as análises desta tese. Na análise do percurso gerativo de sentido das narrativas se fazem necessários os aportes da semiótica discursiva de Eric Landowski e Ana Cláudia de Oliveira, mas nos utilizamos também da semiótica tensiva de Claude Zilberberg. Quanto à psicanálise, para abordar o conceito lacaniano de perversão e neurose, nos utilizamos dos textos de Nasio, Žižek, Bond, Philippe Julien e Antonio Quinet, que nos apoiam na explanação do universo do sujeito serial killer. Para as discussões sociológicas e éticas sobre anormalidade e perversidade adotamos os pensamentos de Dany-Robert Dufour e Simpson

Objetivo: o objetivo deste estudo foi avaliar a prevalência de autoanticorpos e a dosagem da concentração de células Natural Killer (NK) no sangue periférico em pacientes com endometriose. Métodos: Entre dezembro de 2004 e dezembro de 2007 foram avaliadas 155 pacientes submetidas a videolaparoscopia, divididas em um grupo sem endometriose(n=55) e outro com endometriose (n=100). Foi coletada amostra de sangue periférico de todas as pacientes no momento da laparoscopia e nessa amostra foi realizada a quantificação do percentual de células NK em relação aos linfócitos periféricos (por citometria de fluxo), e a determinação dos seguintes auto-anticorpos: anticorpos antinucleares (ANA, por imunofluorescência indireta), anticorpos antitireoglobulina e antiperoxidase (anti-TG e anti-TPO, por eletroquimioluminescência), anticorpos anticardilipina e antifosfatidilserina (aCL e aPS IgG, IgM e IgA, todos por ensaio imunoenzimático). Além da presença de endometriose, essas pacientes também foram avaliadas quanto ao estadiamento, os locais de doença, relações com a fase do ciclo, e a classificação histológica dessa doença. Resultados: as pacientes com endometriose apresentaram percentual de células NK (média DP de 15,3 9,8%) superiores àquelas sem a doença (média DP de 10,6 5,8%), p<0,001. Quanto aos autoanticorpos, as portadoras de endometriose também apresentaram positividade para ANA mais frequentemente (33%) que as pacientes do grupo controle (12,7%), p=0,006. Quanto aos anti-TG, anti-TPO, anti-CL (IgG, IgM e IgA) e aPS ( IgG, IgM e IgA), não houve diferenças estatísticas quanto à sua positividade. As células NK também mostraram-se mais elevadas nas protadoras de endometriose em estádios avançados e naquelas com comprometimento de retossigmóide, grupo no qual encontramos o maior percentual de células NK com concentração média de 19,8 10,3%. Concentrações de células NK 12,5% podem ser usadas como marcadores de endometriose em retossigmóide, com sensibilidade de 73% e especificidade de 65%. Utilizando-se de um modelo estatístico de probabilidades, demonstramos que associação desse marcador (NK 12,5%) com a presença de sintomas como dor e/ou sangramento intestinal durante a menstruação nos possibilitou estimar uma probabilidade de comprometimento de retossigmóide de 60,4%. Conclusões: pacientes com endometriose apresentam maior concentração de células NK periféricas, além de maior prevalência de ANA positivo em relação àquelas sem endometriose. As células NK aumentam nas pacientes com endometriose predominantemente nos estádios avançados, com comprometimento de retossigmóide. Nesse sentido poderiam ser utilizadas como marcadores diagnósticos desse tipo de comprometimento da doença, principalmente se forem avaliadas em conjunto com os sintomas das pacientes / Objectives: The objective of this study was to evaluate the prevalence of autoantibodies and the percentage of natural killer (NK) cells in the peripheral blood of patients with endometriosis. Methods: Between December 2004 and December 2007, 155 patients submitted to videolaparoscopy were evaluated. Patients were divided into two groups: one group of women without endometriosis (n = 55) and another in which all the women had endometriosis (n = 100). Samples of peripheral blood were collected from all the patients at the time of laparoscopy and flow cytometry was used to determine the percentage of NK cells in relation to peripheral blood lymphocytes in these samples. In addition, the following autoantibodies were measured: antinuclear antibodies (ANA) by indirect immunofluorescence, anti-thyroglobulin and anti-thyroid peroxidase antibodies (anti-TG and anti-TPO) by electrochemiluminescence, and anticardiolipin and anti-phosphatidylserine antibodies (aCL and aPS IgG, IgM and IgA), all performed using immunoenzymatic assay. In addition to the presence of endometriosis, these patients were also evaluated with respect to staging, to the sites of the disease, any association with the phase of the menstrual cycle and the histological classification of the disease. Results: The patients with endometriosis had a higher percentage of NK cells (15.3 ± 9.8%; mean ± SD) compared to those without the disease (10.6 ± 5.8%; mean ± SD), (p<0.001). Evaluation of the autoantibodies showed that positivity for ANA was more common in the group of patients with endometriosis (33%) compared to the patients in the control group (12.7%), (p = 0.006). With respect to anti-TG, anti-TPO, aCL (IgG, IgM and IgA) and aPS (IgG, IgM and IgA), no statistically significant differences were found between the groups of patients with or without endometriosis. NK cell concentrations were also found to be higher in patients with advanced stages of endometriosis and in those in whom the rectosigmoid was affected by the disease, this being the group in which the highest percentage of NK cells was found, with mean concentrations of 19.8 ± 10.3%. NK cell concentrations 12.5% may be used as markers of endometriosis of the rectosigmoid, with sensitivity of 73% and specificity of 65%. Using a statistical model of probability, these findings showed that the association of this marker (NK 12.5%) with the presence of symptoms such as pain and/or intestinal bleeding during menstruation permitted an estimation to be made of a likelihood of 60.4% of rectosigmoid endometriosis. Conclusions: Patients with endometriosis have higher percentages of peripheral NK cells, as well as a greater prevalence of positive ANA compared to those without endometriosis. The concentration of peripheral NK cells increases in patients with endometriosis, predominantly in patients with advanced stages of the disease and those in whom the rectosigmoid is affected. Therefore, the concentration of NK cells in peripheral blood could be used as a diagnostic marker of this type of endometriosis, particularly when evaluated together with patients symptoms