Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 219-242). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Introdução: a síndrome antifosfolípide (SAF) primária é uma vasculopatia autoimune mediada por autoanticorpos com trombose como sua principal manifestação clínica. A presença de anticorpos antifosfolípides (aPL), embora relevante para confirmar o diagnóstico, não parece ser suficiente para explicar completamente a fisiopatologia da doença e um segundo gatilho é usualmente necessário. Além das hipóteses de infecções virais e insulto inflamatório como possíveis desencadeantes, parece que os receptores toll like (TLR) e o Interferon (IFN) tipo I são possíveis protagonistas nesse processo, contribuindo para o início da trombose. Recentemente, dois pequenos estudos demonstraram que uma porcentagem relevante de pacientes com SAF primária tem uma regulação positiva de genes IFN em células mononucleares do sangue periférico (CMSP). Entretanto, 20% e 28% dos pacientes nessas duas coortes tiveram anticorpos anti-dsDNA positivos, um autoanticorpo altamente específico do lúpus eritematoso sistêmico (LES). Objetivo: avaliar se os pacientes com SAF bem caracterizados apresentam assinatura para interferon nas células mononucleares periféricas. Secundariamente foram avaliadas possíveis associações clínico laboratoriais com a assinatura de IFN. Métodos: foram selecionados 53 pacientes do sexo feminino com diagnóstico de SAF primária de acordo com os critérios de Sidney, com idade igual ou maior a 18 anos, selecionados no Ambulatório de SAF da Disciplina de Reumatologia do HCFMUSP, pareados por sexo e idade com 50 controles saudáveis. Um terceiro grupo com 29 paciente com antecedente de trombofilias não imunomediadas também foi incluido. Após a coleta de sangue as CMSPs foram purificadas por metodologia de Ficoll. A expressão gênica das CMSPs foi realizada através do TaqMan® RNA Assay em placas TLDA. Foram pesquisados 41 genes induzidos por IFN (GIIs). Uma análise de componente principal (ACP) foi realizada para determinar quais genes deveriam compor a assinatura de IFN. O teste de z-score foi utilizado para normalizar e calcular a assinatura de IFN para cada paciente. O cutoff da assinatura de IFN foi definido por uma curva ROC, e foi escolhido o ponto que maximizava a sensibilidade e especificidade. Características demográficas, clínicas e laboratoriais foram analisadas buscando por associações com a assinatura de IFN. Resultados: 11 genes estavam superexpressos nos pacientes com SAF em comparação aos controles. Após a análise de ACP foram escolhidos 6 genes que representavam mais de 95% do comportamento da amostra para compor a assinatura de IFN: DNAJA1, IFI27, IFI6, IFIT5, MX1 e TYK2. O cutoff encontrado pela curva ROC foi de 3,9 folds (AUC = 0,706, S = 0,49, E = 0,86, VPP = 0,79, VPN = 0,61). A assinatura de IFN estava presente em 49% dos pacientes com SAF primário vs. 14% dos controles saudáveis e 17% dos controles positivos (p < 0,001). Foi encontrada associação entre a assinatura de IFN e uma ocorrência mais precoce do primeiro evento clínico (p = 0,023), e com ocorrência de eventos obstétricos (em especial pré-eclâmpsia, p = 0,032). Não foi econtrada nenhuma associação entre a assinatura de IFN e número de eventos trombóticos, exames laboratoriais, comorbidades, antecedentes familiares de doenças autoimunes, e escores de risco de retrombose. De todos os tratamentos em uso a única associação encontrada foi entre uma menor assinatura de IFN e o uso de estatinas (p = 0,026). Conclusão: esse estudo indica que pacientes com SAF primária bem caracterizados apresentam uma assinatura de IFN tipo I, não observada em outras trombofilias não imunidade-mediadas ou em controles saudáveis. Também demonstrou-se que essa superexpressão de genes regulados por IFN tipo I está associada a um início mais precoce dos eventos e pré-eclâmpsia. Mais estudos são necessários para determinar se este subgrupo de pacientes se beneficiará de intervenções terapêuticas direcionadas à via de sinalização IFN tipo I / Introduction: primary antiphospholipid syndrome (PAPS) is an autoimmune vasculopathy mediated by autoantibodies with thrombosis as its main clinical manifestation. The presence of antiphospholipid antibodies, while relevant to confirm the diagnosis, does not seem to be sufficient to fully explain the pathophysiology and a second trigger is usually needed. Besides the hypotheses of viral infections and inflammatory insult as possible triggers, type I Interferon (IFN) has been pointed as a possible protagonist. Recently, two studies have demonstrated that a relevant percentage of PAPS patients have an up-regulation of IFN genes in peripheral blood mononuclear cells (PBMC). However, 20% and 28% of patients in these 2 cohorts, had antidsDNA positive antibodies, a highly specific Systemic Lupus Erythematosus (SLE) autoantibody. Objective: The aim of this study is to determine the prevalence of type I IFN signature in PBMC of patients with PAPS without specific SLE autoantibodies and search for it with clinical and laboratorial associations. Methods: 53 PAPS patients (according to Sydney´s criteria) were consecutively selected and age-matched with 50 healthy controls. A third group, with non-immune-mediated thrombophilia patients, was also included. The expression of 41 IFN induced genes was analysed using real time quantitative PCR (TaqMan Low Density Array). A principal component analysis (PCA) was used to determine which genes should compose the IFN signature and z-score was calculated. The IFN signature score cut-off was defined with a ROC curve, as the point that maximized both the specificity and sensitivity. Clinical and laboratorial features were analysed searching for associations with IFN signature. Results: 11 IFN genes were highly expressed in primary APS patients. After PCA, 6 genes remained in the IFN signature: DNAJA1, IFIT5, IFI27, MX1, IFI6, TYK2. The ROC cutoff was 3,9 folds (AUC = 0.706, S = 0.49, E = 0.86, VPP = 0.79, VPN = 0.61). The type I IFN signature was present in 49% of patients with primary APS compared to 14.0% of healthy controls and 17% of non-immune-mediated thrombophilia patients (p < 0.0001). The mean IFN score was significantly higher in PAPS patients (4.0 fold higher, p < 0.0001) than in controls. A higher IFN signature was associated with a younger age at the first APS event (p = 0.023) and with the presence of obstetric events, especially with preeclampsia (p = 0.032). There was no association between IFN signature and number of thrombotic events, laboratory exams, comorbidities, family history of autoimmune diseases, and thrombosis risk scores. Treatment with statins was associated with lower levels of IFN scores (p = 0.026). Conclusion: our result indicates that PAPS patients, without lupus specific antibodies, have an enhanced type I IFN gene signature, not observed in non-immune mediated thrombophilia. We also provide novel data demonstrating that this overexpression of type I IFN-regulated genes is associated with an earlier onset of APS events and preeclampsia. Further studies are necessary to determine if this subgroup of patients will benefit of interventions targeting the type I IFN signalling pathway

Les cellules dendritiques plasmacytoïdes (pDCs) sont caractérisées par leur capacité unique de sécrétion massive d’interféron de type I (IFN-I) suite à la stimulation des Tolllike récepteurs (TLR) 7 et 9. Un rôle fondamental des pDCs a été démontré dans le lupus érythémateux systémique via la production d’IFN-I. Les pDC expriment le récepteur de forte affinité aux immunoglobulines de type E (IgE), FcεRI, impliqué dans la régulation négative de la sécrétion d’IFN-I. L’objectif de notre étude est d’explorer, dans le contexte lupique, les effets du traitement par les IgE sur les fonctions des pDC, particulièrement sur la production d’IFN-I. In vitro, le traitement des pDC par des IgE monoclonales permet la surexpression du FcεRI à leur surface et diminue le taux de transcrits des TLR7/9 et de l’IRF7. De plus, les pDC traitées par des IgE diminuent leur production d’IFN-I et l’expression de marqueurs de maturation, induites par leur stimulation par des ligands des TLR7/9 et des complexes immuns lupiques. En outre, ces pDC pré-traitées par des IgE induisent la différenciation de LT4 naïfs allogéniques en LT4 produisant de l’IL-10. In vivo, les patients lupiques en phase quiescente de la maladie présentent des taux plus élevés d’IgE totales comparés aux patients en phase active (indépendamment d’allergies et d’infestations parasitaires). Chez les patientslupiques, le taux d’IgE totales est inversement corrélé au taux d’anti-ADN et à l’activité de la maladie (SLEDAI). L’ensemble de nos résultats suggère un rôle protecteur des IgE dans le lupus à travers la modulation de la réponse inflammatoire des pDC. / Plasmacytoid dendritic cells (pDCs) are characterized by their unique ability to produce large amounts of type I interferon (IFN-I) upon Toll-like receptors (TLR) 7 and 9 triggering. A fundamental role for pDCs has been shown in systemic lupus erythematosus (SLE) through IFN-I production. pDCs express the high affinity Fc receptor for immunoglobulin E (IgE), FcεRI, involved in the negative regulation of IFN-I secretion. The objective of our study is to investigate, in the context of SLE, the effects of IgE treatment on pDCs functions, especially on IFN-I production. In vitro, monoclonal IgE treatment of pDCs upregulate their surface expression of FcεRI and decrease transcripts levels of TLR7/9 and IRF7. IgE-treated pDCs decrease IFN-α secretion and downregulate maturation markers expression induced by TLR7/9 and immune complexes triggering. Moreover, the coculture of IgE pretreated pDCs with allogeneic naive LT4 promotes their differentiation into IL-10-secreting cells. In vivo, patients with quiescent SLE have higher IgE levels than patients with active disease (independently of allergy or parasitic infection). In SLE patients, IgE levels are inversely correlated to anti-DNA antibodies and disease activity (SLEDAI). All together, our data suggest a protective role for IgE in SLE through the modulation of the inflammatory response by pDC.

During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Efficient clonal expansion and differentiation of CD8 T cells are required to develop protective memory CD8 T cells. Antigen specific cells require 3 distinct signals for their activation: TCR engagement of peptide-MHC (signal 1), costimulation between B7 and CD28 (signal 2), and inflammatory cytokines including IL-12 or type 1 IFN (signal 3). CD8 T cells that encounter antigen and costimulation undergo programmed cell division, but these two signals alone are not sufficient for full effector cell differentiation and survival into memory. CD8 T cells need a third signal for efficient clonal expansion, differentiation into various effector populations, acquisition of cytolytic effector functions, and memory formation. The requirements for signal 3 cytokines in CD8 T cell activation have only been recently described; however, the timing of exposure to these signals has yet to be investigated. During the course of an immune response not all T cells will see antigen, costimulation, and inflammatory cytokines at the same time or in the same order. I sought to examine how the timing of signal 3 cytokines affected CD8 T cell activation. I questioned how the order of these signals effected CD8 T cell priming and subsequent activation, expansion and differentiation. In order to study the in vivo effects of out-of-sequence signaling on CD8 T cell activation, I utilized poly(I:C), a dsRNA analogue, which is known to induce a strong type 1 IFN response. Through the use of various congenic transgenic and polyclonal CD8 T cell populations, in conjunction with adoptive transfer models, specific T cells which had been exposed to poly(I:C) induced environments could be identified and tracked over time. I wanted to characterize how out-of-sequence signaling affected T cell activation immediately after cognate antigen stimulation (4-5hours), and after prolonged exposure to cognate antigen (days-weeks). Considering type 1 IFN can have both inhibitory and stimulatory effects on CD8 T cell proliferation, and when type 1 IFN provides signal 3 cytokine activity, it has positive effects on CD8 T cell expansion, I wanted to investigate the role of type 1 IFN as an out-of-sequence signal during CD8 T cell activation. We identified a transient defect in the phosphorylation of downstream STAT molecules after IFNβ signaling within poly(I:C) pretreated CD8 T cells. The inability of poly(I:C) pretreated CD8 T cells to respond to IFNβ signaling makes these cells behave in a manner more similar to T cells that only received 2 signals, rather than ones that received all 3 signals in the appropriate order. Consequently, poly(I:C) pretreated, or out-of-sequence, CD8 T cells were found to have defects in clonal expansion, effector differentiation and function as well as memory generation resulting in reduced efficacy of viral clearance. Out-of-sequence CD8 T cells showed suppression of CD8 T cell responses after prolonged exposure to cognate antigen, but naïve CD8 T cells pre-exposed to poly(I:C) exhibited immediate effector function within hours of cognate antigen stimulation, prior to cell division. Poly(I:C) pretreated naïve CD8 T cells acquired an early activated phenotype associated with alterations of transcription factors and surface markers. Changes in naïve CD8 T cell phenotype are thought to be mediated by poly(I:C)-induced upregulation of self-MHC and costimulatory molecules on APCs through direct type 1 IFN signaling. Inoculating with poly(I:C) enabled naive CD8 T cells to produce effector functions immediately upon stimulation with high density cognate antigen, reduced affinity altered peptide ligands (APLs), and in response to reduced concentrations of cognate antigen. Unlike conventional naïve CD8 T cells, poly(I:C) pretreated naïve CD8 T cells acquired the ability to specifically lyse target cells. These studies identified how the timing of activation signals can dramatically affect the acquisition of CD8 T cell effector function. This thesis describes how CD8 T cell exposure to activation signals in an unconventional order may result in altered response to antigen stimulation. Exposure of naïve CD8 T cells to type 1 IFN and costimulatory molecules in the presence of self-peptides enabled them to respond immediately upon antigen stimulation. Primed naïve CD8 T cells produced multiple cytokines in response to low-affinity, and low-density antigens, and gained ability to specifically lyse target cells. However, immediate effector function may come at the expense of clonal expansion and effector cell differentiation in response to prolonged antigen exposure as out-of-sequence CD8 T cells showed reduced proliferation, effector function and memory formation. The findings presented here may seem contradictory because out-of-sequence signaling can prime T cells to produce immediate effector functions and yet cause defects in T cell expansion and effector differentiation. However, these two models ascertained T cell function at different points after antigen exposure; one where functions were evaluated within hours after seeing cognate antigen, and the other showing T cell responses after days of antigen stimulation. Studies described in this thesis highlight the growing complexity of CD8 T cell activation. Not only do the presence or absence of signals 1-3 contribute to T cell activation, but the timing of these signals also proves to be of great importance. These studies may describe how both latecomer and third party antigen specific T cells behave when and if they encounter cognate antigen in the midst of an ongoing infection. Out-of-sequence exposure to IFN initially stimulates effector function but at the expense of efficient clonal expansion and subsequent memory formation. The immediate effector function that naïve T cells gain during out-of-sequence priming may explain how some individuals are more resistant to superinfections, whereas the impairment in proliferation describes a universal mechanism of virus-induced immune suppression, explaining how other individuals can be more susceptible to secondary infections. Ultimately, results identified here can be applied to developing better and more effective vaccines.

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.

As células tumorais prosperam como consequência da capacidade de resistir aos mecanismos de morte celular e de evasão da vigilância imunológica. Nós propomos que, em cânceres que possuem o supressor de tumor p53 selvagem, a remediação de ambas dessas defesas pode ser promovida pela transferência genica combinada de vetores adenovirais portadores dos transgenes de p19Arf (proteína supressora de tumor, parceira funcional de p53) e de interferon-beta (IFNbeta, citocina imunomoduladora). De fato, em resultados anteriores, notamos que a transdução combinada (p19Arf/IFNbeta), mas não os tratamentos individuais, em células de melanoma murino B16F10 resulta em aumento massivo de morte celular. Porém a capacidade destas células em processo de morte de desencadear imunidade antitumoral não foi analisada. Nesta tese e em estudos complementares, buscamos investigar os mecanismos moleculares de morte celular envolvidos na resposta imune estimulada por p19Arf/IFNbeta e explorar sua aplicação como imunoterapia do câncer. Inicialmente, em modelo de vacinação profilática, revelamos que o tratamento combinado em células B16F10 promove a expressão de IL-15, ULBP1, dos receptores de morte FAS/APO1 e KILLER/DR5, assim como uma resposta de células natural killer que rejeitam estas células tratadas quando inoculadas em camundongos imunocompetentes singênicos. Após desafio tumoral no flanco oposto, a progressão desses tumores foi fortemente reduzida devido ao engajamento de linfócitos T CD4+ e CD8+, que apresentaram produção aumentada das citocinas IFN-? e TNF-alfa e medeiam proteção antitumoral de longo prazo. Em seguida, explorando um contexto de imunização diferente, a transferência de gênica in situ foi realizada em carcinoma heterotópico de pulmão e exibiu proteção significativa contra um desafio tumoral secundário, apenas quando o tumor primário foi tratado com p19Arf/IFNbeta. Análise de transcriptoma destes tumores indicou uma assinatura quimiotáxica de neutrófilos e linfócitos T CD8+ através das quimiocinas CCL3, CXCL3 e da IL-1beta. Em apoio destas observações, análises mecanicistas in vitro revelaram que células tratadas com p19Arf/IFNbeta ativam programas apoptóticos de p53 e antivirais de IFNbeta, enquanto sucumbem a um processo de morte por necroptose que também libera moléculas de morte celular imunogênica (MCI), calreticulina, ATP e HMGB1. No entanto, procurando potencializar ainda mais o benefício terapêutico dos nossos vetores, exploramos sua associação com o quimioterápico imunogênico doxorrubicina (Dox), que também é indutor de MCI. E nesta associação, percebemos que a Dox aumenta não apenas os níveis de morte celular, mas também a imunogenicidade das células tratadas, proporcionando em um modelo de vacina terapêutica, um controle tumoral superior em camundongos que já portavam antes da vacinação tumores B16F10 ou MCA205. Além disso, a associação in situ destas terapias restaurou a eficácia de uma dose sub-terapêutica de Dox, que em contraste com sua dose terapêutica, não prejudica a função cardíaca. Finalmente, também exploramos a associação com o bloqueio dos pontos de controle imunológicos PD-1 ou CTLA-4, que no modelo de vacina terapêutica, sua associação induziu maior rejeição completa de tumores B16F10. Em conclusão, aqui apresentamos evidências sobre a capacidade da combinação p19Arf/IFNbeta de induzir morte celular e estimulação imunológica. E ressaltamos seu potencial como uma estratégia de imunoterapia do câncer / Cancer cells thrive as a consequence of resisting cell death mechanisms and escaping from immune surveillance. We propose that, in cancers that harbor the wild-type tumor suppressor p53, remediation of both of these defenses can be achieved by harnessing the adenoviral vector mediated gene transfer of p19Arf (tumor suppressor protein, p53 functional partner) together with interferon-beta (IFNbeta, immunomodulatory cytokine). Indeed, in our initial observations, it was noticed that combined-transduction (p19Arf/IFNbeta), but not the individual treatments, of B16F10 mouse melanoma cells results in massive cell death levels. Yet, the capability of these dying cells to unleash antitumor immunity was not investigated. Here in this thesis and in complementary studies, we sought to investigate the molecular mechanisms of cell death involved in the p19Arf/IFNbeta immune stimulation and explore its potential as a mediator of cancer immunotherapy. First, in a prophylactic B16F10 vaccine model, we revealed that the dual treatment led to the up-regulation of IL-15, ULBP1, FAS/APO1 and KILLER/DR5 death receptors, plus a natural killer cell response that completely rejects treated cells when inoculated in syngeneic immunocompetent mice. Whereas, upon a contralateral tumor challenge, progression was strongly reduced by engaging both CD4+ and CD8+ T cells, which displayed augmented production of IFN-? and TNF-alpha cytokines and provided long term antitumor protection. Next, exploring different immunization context, in situ gene transfer in a heterotopic lung carcinoma exhibited significant protection against a secondary tumor challenge only when the primary tumor was treated with p19Arf/IFNbeta. Transcriptome analysis of these treated tumors indicated a chemotaxic signature of neutrophils and CD8+ T cells with the involvement of CCL3, CXCL3 chemokines and IL-1beta. Moreover, in support of this evidence, mechanistic in vitro studies revealed that p19Arf/IFNbeta treated cells reactivate p53 apoptotic and IFNbeta antiviral programs, while succumbing to a necroptosis cell death processes that also releases immunogenic cell death (ICD) molecules, calreticulin, ATP and HMGB1. Yet, aiming to potentiate therapeutic benefit of our vectors, we explored their association with doxorubicin (Dox) immunogenic chemotherapy, which is also an inducer of ICD. And in this setting, this association with Dox enhances not only cell death levels but also immunogenicity of treated cells, providing superior tumor control in a therapeutic vaccine model, where mice were already bearing B16F10 tumors or MCA205 sarcomas before vaccination. Moreover, associated use of these therapies in situ rescued efficacy of a sub-therapeutic dose of Dox, which in contrast to its therapeutic dose, does not impair cardiac function. Finally, we also evaluated the association with PD-1 or CTLA-4 checkpoint blockade immunotherapy, which in the therapeutic vaccine model induced full tumor rejection in a greater number of mice. In sum, here we provide compelling evidence for the ability of the p19Arf/IFNbeta combined gene transfer to promote cell death and immunogenic stimuli and underscored its potential to be applied as a cancer immunotherapy strategy

As células tumorais prosperam como consequência da capacidade de resistir aos mecanismos de morte celular e de evasão da vigilância imunológica. Nós propomos que, em cânceres que possuem o supressor de tumor p53 selvagem, a remediação de ambas dessas defesas pode ser promovida pela transferência genica combinada de vetores adenovirais portadores dos transgenes de p19Arf (proteína supressora de tumor, parceira funcional de p53) e de interferon-beta (IFNbeta, citocina imunomoduladora). De fato, em resultados anteriores, notamos que a transdução combinada (p19Arf/IFNbeta), mas não os tratamentos individuais, em células de melanoma murino B16F10 resulta em aumento massivo de morte celular. Porém a capacidade destas células em processo de morte de desencadear imunidade antitumoral não foi analisada. Nesta tese e em estudos complementares, buscamos investigar os mecanismos moleculares de morte celular envolvidos na resposta imune estimulada por p19Arf/IFNbeta e explorar sua aplicação como imunoterapia do câncer. Inicialmente, em modelo de vacinação profilática, revelamos que o tratamento combinado em células B16F10 promove a expressão de IL-15, ULBP1, dos receptores de morte FAS/APO1 e KILLER/DR5, assim como uma resposta de células natural killer que rejeitam estas células tratadas quando inoculadas em camundongos imunocompetentes singênicos. Após desafio tumoral no flanco oposto, a progressão desses tumores foi fortemente reduzida devido ao engajamento de linfócitos T CD4+ e CD8+, que apresentaram produção aumentada das citocinas IFN-? e TNF-alfa e medeiam proteção antitumoral de longo prazo. Em seguida, explorando um contexto de imunização diferente, a transferência de gênica in situ foi realizada em carcinoma heterotópico de pulmão e exibiu proteção significativa contra um desafio tumoral secundário, apenas quando o tumor primário foi tratado com p19Arf/IFNbeta. Análise de transcriptoma destes tumores indicou uma assinatura quimiotáxica de neutrófilos e linfócitos T CD8+ através das quimiocinas CCL3, CXCL3 e da IL-1beta. Em apoio destas observações, análises mecanicistas in vitro revelaram que células tratadas com p19Arf/IFNbeta ativam programas apoptóticos de p53 e antivirais de IFNbeta, enquanto sucumbem a um processo de morte por necroptose que também libera moléculas de morte celular imunogênica (MCI), calreticulina, ATP e HMGB1. No entanto, procurando potencializar ainda mais o benefício terapêutico dos nossos vetores, exploramos sua associação com o quimioterápico imunogênico doxorrubicina (Dox), que também é indutor de MCI. E nesta associação, percebemos que a Dox aumenta não apenas os níveis de morte celular, mas também a imunogenicidade das células tratadas, proporcionando em um modelo de vacina terapêutica, um controle tumoral superior em camundongos que já portavam antes da vacinação tumores B16F10 ou MCA205. Além disso, a associação in situ destas terapias restaurou a eficácia de uma dose sub-terapêutica de Dox, que em contraste com sua dose terapêutica, não prejudica a função cardíaca. Finalmente, também exploramos a associação com o bloqueio dos pontos de controle imunológicos PD-1 ou CTLA-4, que no modelo de vacina terapêutica, sua associação induziu maior rejeição completa de tumores B16F10. Em conclusão, aqui apresentamos evidências sobre a capacidade da combinação p19Arf/IFNbeta de induzir morte celular e estimulação imunológica. E ressaltamos seu potencial como uma estratégia de imunoterapia do câncer / Cancer cells thrive as a consequence of resisting cell death mechanisms and escaping from immune surveillance. We propose that, in cancers that harbor the wild-type tumor suppressor p53, remediation of both of these defenses can be achieved by harnessing the adenoviral vector mediated gene transfer of p19Arf (tumor suppressor protein, p53 functional partner) together with interferon-beta (IFNbeta, immunomodulatory cytokine). Indeed, in our initial observations, it was noticed that combined-transduction (p19Arf/IFNbeta), but not the individual treatments, of B16F10 mouse melanoma cells results in massive cell death levels. Yet, the capability of these dying cells to unleash antitumor immunity was not investigated. Here in this thesis and in complementary studies, we sought to investigate the molecular mechanisms of cell death involved in the p19Arf/IFNbeta immune stimulation and explore its potential as a mediator of cancer immunotherapy. First, in a prophylactic B16F10 vaccine model, we revealed that the dual treatment led to the up-regulation of IL-15, ULBP1, FAS/APO1 and KILLER/DR5 death receptors, plus a natural killer cell response that completely rejects treated cells when inoculated in syngeneic immunocompetent mice. Whereas, upon a contralateral tumor challenge, progression was strongly reduced by engaging both CD4+ and CD8+ T cells, which displayed augmented production of IFN-? and TNF-alpha cytokines and provided long term antitumor protection. Next, exploring different immunization context, in situ gene transfer in a heterotopic lung carcinoma exhibited significant protection against a secondary tumor challenge only when the primary tumor was treated with p19Arf/IFNbeta. Transcriptome analysis of these treated tumors indicated a chemotaxic signature of neutrophils and CD8+ T cells with the involvement of CCL3, CXCL3 chemokines and IL-1beta. Moreover, in support of this evidence, mechanistic in vitro studies revealed that p19Arf/IFNbeta treated cells reactivate p53 apoptotic and IFNbeta antiviral programs, while succumbing to a necroptosis cell death processes that also releases immunogenic cell death (ICD) molecules, calreticulin, ATP and HMGB1. Yet, aiming to potentiate therapeutic benefit of our vectors, we explored their association with doxorubicin (Dox) immunogenic chemotherapy, which is also an inducer of ICD. And in this setting, this association with Dox enhances not only cell death levels but also immunogenicity of treated cells, providing superior tumor control in a therapeutic vaccine model, where mice were already bearing B16F10 tumors or MCA205 sarcomas before vaccination. Moreover, associated use of these therapies in situ rescued efficacy of a sub-therapeutic dose of Dox, which in contrast to its therapeutic dose, does not impair cardiac function. Finally, we also evaluated the association with PD-1 or CTLA-4 checkpoint blockade immunotherapy, which in the therapeutic vaccine model induced full tumor rejection in a greater number of mice. In sum, here we provide compelling evidence for the ability of the p19Arf/IFNbeta combined gene transfer to promote cell death and immunogenic stimuli and underscored its potential to be applied as a cancer immunotherapy strategy

The innate immune system provides the first line of defense against infection. Pathogens are detected though a variety of Pattern Recognition Receptors (PRRs), which activate downstream signaling cascades. Effector molecules such as cytokines and chemokines are released upon activation and aid in cell recruitment, control of pathogen replication, and coordination of the adaptive immune response. Nucleic acids that are released into the cytosol during viral and bacterial infection are recognized through a special class of PRRs, coined cytosolic nucleic acid sensors. Upon recognition, these receptors induce the production of type I interferons and other cytokines to aid in pathogen clearance. Although many cytosolic nucleic acid sensors have been discovered, it is unclear how they work in concert to mediate these responses. The Interferon Gamma Inducible protein (IFI)16 and its proposed mouse orthologue IFI204 are cytosolic DNA sensors that have been linked to the detection of cytosolic DNA during infection with Herpes Simplex Virus (HSV-1). IFI16 binds dsDNA that has been released into the cytosol during viral infection and engages the adaptor molecule Stimulator of Interferon Genes (STING) leading to TANK binding kinase-1 (TBK1) dependent phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of type I interferons and interferon stimulated genes. In addition to its role as a sensor, in chapter two of this thesis we describe a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in anti-viral immunity. In an effort to better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as other inducers of IFN such as cyclic-dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to cytosolic DNA ligands and DNA viruses. In contrast, expression of the NF-κB regulated cytokines such as IL-6 and IL-1β were unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of expression of IFN-α and IFN stimulated genes such as RIG-I in response to cyclic GMP-AMP (cGAMP), a second messenger produced in response to cGAS, as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in IFI16 knockdown cells suggesting that transcription of ISGs is dependent on IFI16. Since IFI16 knockdown compromised not only DNA virus driven pathways, we propose additional regulatory roles outside of DNA sensing. Collectively, these results indicate that IFI16 plays a role in the regulation of type I IFN gene transcription and production in response to both RNA and DNA viruses. The role of IFI16/IFI204 has been studied extensively in vitro, however the role of the receptors in vivo has yet to be determined. In chapter three of this thesis, we developed a mouse deficient in IFI204 to explore the role of IFI204 in in vivo immune responses to viruses. We investigated the ability of IFI204 deficient cells to induce type I interferons and other cytokines in response to a panel of DNA and RNA ligands in vitro. IFI204 deficient BMDMs displayed a partial defect in type I interferon induction in response to both DNA and RNA ligands and viruses as compared to WT mice. We also observed that this phenotype is time dependent, since there was no change in type I interferon induction after 12 hours post infection as compared to earlier time points. In contrast to these results, expression of the NF-κB regulated cytokines IL-6 and IL-1β were unaffected in IFI16 knockdown cells. These results suggest that IFI204 plays a partial role in the induction of type I interferons in response to both DNA and RNA ligands. Additionally, IFI204 may work in tandem with other receptors in a sequential manner to amplify the type I interferon response. We also studied the involvement of IFI204 in an in vivo model of HSV-1 infection. IFI204 knockout mice produce less brain and serum IFN-β, IL-6, and IL-1β 72 hours post intraperitoneal infection with HSV-1. Furthermore, IFI204 -/- mice are more susceptible to HSV-1 infection as compared to WT mice. These data indicate that IFI204 mediates the response to HSV-1 in vivo by inducing the production of cytokines that are necessary for the control of viral infection.

Dados recentes do nosso laboratório demonstram que somente a co-transdução, não a tradução individual, com vetores adenovirais portadores de Interferon-beta (IFN?) (citocina imuno modulatória) e p19Arf (parceira funcional da proteína supressora de tumor p53) resulta na morte celular massiva do melanoma murino B16F10. A capacidade desse tratamento combinado de induzir uma resposta imune antitumoral ainda não foi avaliada. Dessa maneira, o objetivo do presente trabalho foi investigar se células B16F0 tratadas por essa combinação são capazes de induzir uma resposta imune antitumoral em um modelo de vacinação profilático de melanoma. Para isso, essas células foram co-transduzidas com os vetores AdPGp19 e AdPGIFN? e 48 horas depois, inoculadas como agente vacinal no flanco esquerdo (sítio da vacina) de camundongos C57BL/6 imunocompetentes. Sete dias após a última vacinação, esses animais foram desafiados com células B16F10 naïve no flanco direito (sítio do desafio). A progressão tumoral do desafio foi significativamente reduzida, mesmo quando o desafio tumoral foi feito 73 dias após da vacinação. Porém, como os animais imunizados desenvolveram tumores no sítio da vacina, condições para o uso dessas células tratadas foram avaliadas, revelando que: o número de células e de aplicações usadas durante a vacinação tem influência no aparecimento desse tumores, e que apenas com o tratamento combinado os camundongos permanecem livres de tumor. A influência do sistema imune para este resultado foi revelada após protocolo de imunussupressão. Em seguida, o papel da p19Arf e do IFN? na proteção antitumoral da combinação foi estudado. In vitro, os efeitos antitumorais da combinação parecem ser mais influentes da reposição de p19Arf do que da expressão de IFN?, mas já in vivo, na presença do sistema imune, foram mais dependentes do IFN?. Com a combinação estes efeitos mostraram-se mais pronunciados, induzindo uma proteção antitumoral e maior sobrevida aos animais vacinados. Estes resultados indicam que a combinação p19Arf e IFN? pode ser aplicada como um agente imunoterápico e sugerem que a associação entre morte celular e imuno estimulação pode beneficiar o tratamento contra o câncer / Previously, we have shown in a mouse melanoma model of in situ gene therapy that co-transduction, but not individual application, with adenovirus vectors expressing the Interferon-beta (IFN?) (immune modulatory cytokine) and p19Arf (functional partner of the p53 tumor suppressor) transgenes results in massive cell death and reduced tumor progression. However, the capability of this combined treatment to stimulate an antitumor immune response has not been evaluated. Therefore, the aim of this work was to investigate, trough a prophylactic vaccine model, if B16F10 cells treated by the p19Arf and IFN? combination could induce such immune response. To do so, these cells were co-transduced by the AdPGp19 e AdPGIFN? adenoviral vectors and 48 hours after, inoculated as a vaccine agent in the left flank (vaccine site) of immune competent C57BL/6 mice. Seven days after the last vaccine, a tumor challenge was done with naïve B16F10 cells in the right flank (challenge site). Tumor progression was markedly reduced, even when challenge was done 73 days after the vaccination. However, since these animals developed tumors where the vaccine was applied, more appropriate conditions for the use of these treated cells were pursued, thus revealing that: the number of cells and inoculations can dictate tumor development, and also, that only with the combined treatment was tumor formation abolished. The influence of the immune system for this result was revelead by performing an immune supression protocol. Next, the roles of p19Arf and of IFN? were studied. In vitro, the antitumor effects were stronger upon the introduction of p19Arf than IFN?, but in vivo, in the presence of the immune system, the effects were more IFN? dependent. In fact, these effects were more pronouced with the combined treatment, inducing protection against tumor formation and progression and increasing survival in the vaccinated animals. Taken together, these results demonstrate the application of cells treated by the p19Arf e IFN? combination as an effective vaccine agent and also indicates that the association between cell death and immune stimulation may benefit the treatment of cancer

Le virus T-lymphotrope humain de type 1 (HTLV-1) est l’agent étiologique de deux principales pathologies : la leucémie/lymphome à cellules T de l’adulte (ATLL) et la paraparésie spastique tropicale/myélopathie associée à HTLV-1 (HAM/TSP). Ces deux maladies sont caractérisées par des phénotypes immunitaires opposés, puisque l’ATLL est associée à une immunosuppression et l’HAM/TSP à une réponse pro-inflammatoire. Les mécanismes qui déterminent l’évolution de l’infection chronique vers l’une ou l’autre de ces maladies sont peu connus. L’interféron de type 1 (IFN-I) a une fonction ambiguë dans l’organisme. Si cette cytokine contribue à la réponse immunitaire précoce, elle est également associée au développement de pathogenèses pour des infections virales persistantes. Les cellules dendritiques plasmacytoïdes (pDCs) ont la particularité de produire de grandes quantités d’IFN-I après la reconnaissance de cellules infectées par des virus. Nous avons montré que ceci était également vrai pour HTLV-1, puisque le contact entre une cellule infectée par HTLV-1 et la pDC est nécessaire à la production d’IFN-I. Cette production est induite par la particularité de HTLV-1 à s’accumuler en surface des cellules infectées, au sein d’une structure préalablement définie sous le terme de biofilm viral. La nature de la matrice extracellulaire dans laquelle est accumulée le virus régule la réponse IFN-I par les pDCs, la présence de l’antigène Galβ(1-3)GalNAc désialylé à la surface des cellules infectées contribuant à réduire cette réponse IFN-I. Nous avons également observé que des cellules infectées par le virus HTLV-2, virus phylogénétique proche de HTLV-1 mais peu pathogène, tendent à induire une plus faible production d’IFN-I, mais une meilleure maturation des pDCs. Nous avons enfin montré que la fréquence des pDCs dans le sang et leur capacité à répondre à un stimulus est similaire chez des patients HAM/TSP, des porteurs asymptomatiques et des individus sains. Ces résultats contrastent avec des études antérieures qui montrent une diminution de la fréquence des pDCs chez les patients ATLL et une diminution de leur activité chez les individus infectés. Le nombre et la fonction des pDCs pourraient ainsi contribuer à l’orientation de la pathogenèse vers l’ATLL ou l’HAM/TSP. / HTLV-1 (Human T-lymphotropic virus type 1) is the etiological agent of two main diseases: the adult T-cell leukemia/lymphoma (ATLL) and the HTLV-1 associated myelopathy/tropical spastic paraparesis, which are characterized by different immune phenotypes. While the ATLL is linked to an immunosuppressive state, the HAM/TSP is linked to a pro-inflammatory state in patients. The mechanisms contributing to the development of these two diseases in the HTLV-1 infected individuals are poorly understood. Type I interferon (IFN-I) has ambivalent functions in the organism. While this cytokine is an effector of early immune responses, several studies have reported a negative impact of this cytokine during chronic infections. The plasmacytoid dendritic cells (pDCs) are the main producers of IFN-I in vivo, and can produce high amounts of this cytokine after the recognition of virally infected cells. We have shown that pDCs are able to recognize HTLV-1-infected cells, thus leading to the production of IFN-I. pDCs’ triggering is mediated by the accumulated viral particles at the surface of the infected cells, within a carbohydrate-rich structure, previously described as the viral biofilm. The nature of the extracellular matrix itself seems to regulate IFN-I production by pDCs, since the exposition of an asialylated Galβ(1-3)GalNAc glycan at the surface of the HTLV-infected cells reduces the IFN-I production. We also observed that HTLV-2 (a close relative of HTLV-1)-infected cells, in contrast to HTLV-1-infected cells, tend to induce a lower production of IFN-I after being recognized by the pDCs but a greater maturation of the latter. Finally, we have shown that pDCs’ frequency in the blood and their ability to produce IFN-α after an ex vivo stimulation is equivalent in healthy donors, asymptomatic HTLV-1 carriers and HAM/TSP patients. This result contrasts with previous studies which demonstrated that blood circulating pDCs’ frequency is reduced in ATLL patients and that pDCs from HTLV-1 infected individuals have a reduced ability to produce IFN-α after stimulation. Thus, dysregulation of the frequency and functionality of pDCs could contribute to the development of one disease or the other.