La régulation de l’expression des gènes est critique pour l’adaptation des cellules à leur environnement et pour leur homéostasie. La transcription, qui représente une étape essentielle de l’expression des gènes, est contrôlée par plusieurs facteurs et cofacteurs. L’un de ces cofacteurs, TRRAP, correspond à la plus grosse sous-unité de deux complexes de remodelage de la chromatine, SAGA et TIP60. TRRAP interagit avec divers facteurs de transcription, tels que c-MYC et E2Fs et permet ainsi le recrutement de SAGA et TIP60 aux promoteurs des gènes. TRRAP est un membre d’une famille de kinases atypiques, les PIKKs. Des études antérieures ont défini la co-chaperonne TTT comme régulateur essentiel de la stabilité et l’activité des PIKKs. Contrairement aux autres PIKKs, TRRAP ne possède pas les résidus requis à son activité catalytique et représente donc la seule pseudo-kinase parmi les PIKKs. Bien que TTT interagit et stabilise TRRAP, son rôle sur l’activité de ce dernier reste inconnu. En utilisant un système de dégron inductible qui permet la dégradation rapide de protéines endogènes, nous avons démontré que TTT est requis pour l’assemblage de TRRAP dans ses complexes fonctionnels précédent son import nucléaire. De plus, à travers des analyses transcriptomiques, nous avons pu déterminer que TTT régule la transcription de plusieurs gènes TRRAP-dépendants dans des cellules de cancer colorectal. L’analyse du profile de fixation de TRRAP à l’échelle du génome grâce à la technique du CUT&RUN suivie d’un séquençage à haut débit (CUT&RUN-seq), a permis d’identifier les cibles directes de TRRAP, parmi lesquelles seule une fraction restreinte correspond à des cibles directes de MYC. Nous avons également découvert que TRRAP possède un rôle de répresseur direct sur la transcription d’une partie des gènes stimulés par l’interféron (ISGs) qui interviennent dans la réponse à l’interféron du système immunitaire innée. En outre, nos résultats suggèrent que TRRAP et sa co-chaperonne TTT participent à la tumorigenèse notamment en maintenant et régulant un programme transcriptionnel spécifique. / Gene expression regulation is critical for cells to adapt to external changes and maintain their homeostasis. Transcription is an essential step in gene expression and is controlled by numerous factors and cofactors. One such cofactor is TRRAP, the largest subunit of two distinct chromatin-modifying complexes, SAGA and TIP60. TRRAP interacts with a diverse range of transcription factors including c-MYC and E2Fs, and mediates the recruitment of SAGA and TIP60 to gene promoters. TRRAP is a member of the PIKK family of atypical kinases. Prior studies defined the TTT co-chaperone as an essential regulator of PIKK stability and activity. In contrast to its cognate kinases, TRRAP lacks catalytic residues and is the sole pseudokinase among PIKKs. Although TTT has been shown to stabilize and interact with TRRAP, the role of TTT on TRRAP function remains unknown. Using an inducible degron system that allows the rapid and acute depletion of endogenous proteins, we demonstrated that TTT is required to assemble TRRAP within its functional complexes prior its nuclear import. Additionally, through transcriptomic analyses we determined that TTT regulates a large number of TRRAP-dependent genes in colorectal cancer cells. Profiling of the genome-wide binding of TRRAP via CUT&RUN-seq identified the direct targets of TRRAP, of which only a small fraction overlaps with MYC targets. We also uncovered a direct inhibitory role of TRRAP on a subset of the interferon-stimulated genes, which mediate the interferon response in the innate immune system. Altogether, our data suggest that TRRAP and its chaperone TTT are involved in tumorigenesis through the maintenance of a specific transcriptional program.

The oxysterol, 25-hydroxycholesterol (25-HC), is important for sterol metabolism and emerging evidence suggests that 25-HC plays a more critical role in immunity and infection. However, the precise antiviral mechanism and the target of 25- HC remains unclear. Here efforts were made to investigate the link between viral infection and the triggering of the 25-HC associated interferon (IFN) response, and how this dynamically alters the endogenous level of 3-hydroxy- 3-methylglutaryl-CoA reductase (HMGCR), a key enzyme that catalyses the production of the precursor of cholesterol and oxysterols. In this thesis I have sought to specifically explore the temporal changes and role of HMGCR in DNA virus (cytomegalovirus) and RNA (Influenza) virus infections. I hypothesise that HMGCR is a target for 25-HC associated IFN-mediated host defence against viral infection. To characterise HMGCR and test this hypothesis, the following objectives were defined: (1). To establish an experimental system to quantitatively study the endogenous HMGCR protein level; (2). To investigate the mechanism of the down-regulation of HMGCR involved in the IFN-mediated innate immune response; (3). To study the behaviour of HMGCR in the influenza virus induced 25-HC associated IFN-mediated innate immune response; (4). To study the behaviour of HMGCR in the cytomegalovirus induced 25-HC associated IFN-mediated innate immune response. Chapter 3, describes establishing an experimental system for the quantification of endogenous HMGCR levels. Different protein detection methods, including a modified western blot protocol and immunostaining, were tested. The results of RNA interference of HMGCR demonstrate that under lipid-deficient condition with the supplementation of mevastatin (an HMGCR inhibitor) the modified western blot protocol specifically detects endogenous HMGCR. This chapter lays the foundational work for the temporal analysis and testing the role of HMGCR in infection. In Chapter 4, the mechanism of the degradation of HMGCR following 25-HC and IFN treatments, in wild-type and Ch25h−/− mouse bone marrow derived macrophages (BMDMs), was investigated. Similar to 25-HC, IFN-γ treatment results in the drop of both the transcript and protein abundance of HMGCR in wild-type BMDMs. Differential temporal analysis of RNA and protein alterations and the use of proteasome inhibitors reveals that both 25-HC and IFN-γ lead to a marked reduction of HMGCR protein via a proteasomal degradation mechanism within early times of treatments. Further, the immediate reduction of HMGCR levels induced by IFN-γ was completely abrogated in Ch25h−/− BMDMs. Hence, the reduction of HMGCR following IFN-γ treatment is due to the de novo synthesis in macrophages of 25-HC. However, the decrease of Hmgcr gene expression was observed in not only wild-type but also Ch25h−/− BMDMs, suggesting additional mechanisms for regulating Hmgcr RNA levels. These results demonstrate the mechanism of the down-regulation of HMGCR resulted from the induction of IFN response during viral infection, is only partially due the de novo synthesis of 25-HC. In chapter 5, influenza A virus was used to investigate the role of HMGCR in the IFN-mediated innate immune response. The inhibition of HMGCR by RNA interference inhibited viral growth, suggesting the requirement of HMGCR for optimal intracellular viral growth. Viral infection in wild-type murine BMDMs reduced the endogenous HMGCR levels. However, the reduction of HMGCR at early times was prevented in Ch25h−/− BMDMs. Intriguingly, the decrease of HMGCR at late time points was still observed in Ch25h−/− BMDMs. These results indicate that the down-regulation of HMGCR with influenza virus infection in BMDMs at early times is completely due to the de novo synthesis of 25-HC; whereas at late times alternative pathways or mechanisms exist. Additionally, human epithelial A549 cells and A549/PIV5-V cells that are deficient in STAT1 were used to study the role of IFN pathway in the down-regulation of HMGCR at late times during viral infection. Results from these studies show that at late times the reduction of HMGCR is due to IFN-independent mechanisms. Chapter 6, extends these investigations to the herpes virus murine cytomegalovirus and infection of BMDMs. HMGCR is known to be essential for cytomegaloviral infections and 25-HC, statin and RNAi inhibition of HMGCR restrict viral growth. 25-HC is shown to reduce HMGCR at immediate early times of infection. However, most notably, the down-regulation of HMGCR was also observed in Ch25h−/− BMDMs at late times with murine cytomegalovirus infected BMDMs. These results confirm that alternative pathways or mechanisms exist, playing roles in the crosstalk between cholesterol metabolism and innate immune response. Collectively, this study characterises the role of HMGCR in the 25-HC associated IFN-mediated host defence against viral infection. Results indicate that, in addition to the IFN-mediated host response, alternative pathways or other mechanisms also result in the down-regulation of HMGCR during viral infection. HMGCR is at the crossroad of different pathways or mechanisms, and is therefore not only targeted by 25-HC. Hence, further questions can be addressed from these results: (1). What are the alternative pathways or mechanisms for the down-regulation of HMGCR? (2). How do these pathways or mechanisms work in hosts’ immune system? Answering these questions can contribute to refining the pathway map of innate immunity and understanding the precise role of HMGCR, or even the sterol biosynthesis pathway, in hosts’ immune response against pathogens.

Viruses are obligate intracellular pathogens. Therefore, their successful replication, at every stage from attachment to assembly and egress, is dependent on host cell functions. The host cell in turn engages mechanisms to counteract virus replication. As a result, viruses have evolved mechanisms to evade these counteracting measures as well as ways to reshape the cellular environment into one that’s favourable for successful replication. Systematic studies offer a platform for unravelling virus-cell interactions and in particular can address three important aspects 1) increase our understanding of basic biology of the virus, 2) identify and characterise novel cellular functions 3) provide important leads for novel targets for antiviral therapy. In this study, I investigated two aspects of virus host interaction; the role of microRNAs (miRNAs) in virus infection and the role of interferon inducible genes in virus infection. Human cytomegalovirus (HCMV) is a β herpes virus that infects humans. HCMV maintains a persistent lifelong infection in the host involving a cycle of latency and reactivation. Infection of healthy individuals with HCMV results in relatively minor symptoms. In contrast, infection of individuals with a compromised immune system, as in the case of organ transplant recipients and AIDS patients, can cause significant morbidity and mortality. In common with other herpes viruses, HCMV expresses multiple small regulatory RNAs called miRNAs. HCMV encodes at least 14 miRNAs. Identifying the targets of these miRNAs will help us understand their functional importance during infection. Recently, a biochemical technique called Cross-Linking, Ligation and Sequencing of Hybrids (CLASH), was developed by Tollervey and colleagues, representing the most advanced systematic technique for the identification of miRNA targets. We adapted this approach to identify high confidence miRNA targets during HCMV infection. However, the protocol was sub-optimal and presented us with technical challenges. Although high quality data sets were not generated, the work was crucial for the establishment of the system which is now generating promising data. Virus-cell interactions can also be elucidated by probing for host factors that are important for virus replication. Type I interferon is a highly effective inhibitor of HCMV replication. Treatment of cells with interferon results in up regulation of multiple effectors known as interferon stimulated genes (ISGs). How these genes block HCMV replication is poorly understood. A library of more than 380 ISG expressing lentiviruses was screened to determine the effects of individual ISGs on HCMV replication. The screen was performed in primary human fibroblast cells and a glioblastoma cell line called U373s. Multiple inhibitory ISGs were identified including well characterised ISGs such as cGAS, STAT2, NOD2, DDX60 and HPSE as well as novel candidates TXNIP, ELF1, FAM46C, MT1H and CHMP5. Five ISGs were identified as HCMV replication enhancers including previously published ISGs BST2 and IFITM1 and novel enhancers ODC1, BCL3 and IL28RA. siRNA screens against top hits demonstrated that STAT2, CPT1A and cGAS are dominant inhibitory factors during HCMV infection and knockdown of these genes can partially rescue HCMV replication following interferon treatment. Finally, using a corresponding rhesus ISG library we show that rhesus SAMHD1 effectively inhibits HCMV replication while human SAMHD1 has no effect, suggesting that HCMV expresses a species-specific inhibitor of SAMHD1. This study defines interferon stimulated pathways important for HCMV replication and identifies multiple novel host factors that both restrict and enhance HCMV replication. These studies demonstrate the effectiveness of using systematic approaches for the identification of novel host virus interactions.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-10T11:11:21Z No. of bitstreams: 1 Edvaldo Cavalcanti de LIma Neto.pdf: 1484364 bytes, checksum: 176b6dea99569a96a24729551d8d6cab (MD5) / Made available in DSpace on 2017-02-10T11:11:21Z (GMT). No. of bitstreams: 1 Edvaldo Cavalcanti de LIma Neto.pdf: 1484364 bytes, checksum: 176b6dea99569a96a24729551d8d6cab (MD5) Previous issue date: 2006-02-20 / Viruses are among the most harmful pathogens found in aquaculture and are the most numerous and diverse of the pathogenic agents described to crustaceans. The knowledge about genes associated to immunity viral response, as well as the relationships between pathogen and host still very incomplete. Moreover, shrimp cell lines are not available, thus limiting primary systems of cellular culture to assist searches in genomic expression. The Expressed Sequence Tags (ESTs) analyses have become a common tool used to identify genes involved in some specific biological functions, particularly in organisms in which genomic data are not available. The Laboratory of Applied Genetics (LAGA) of the Universidade Federal Rural de Pernambuco (UFRPE) is one of the members of the Shrimp EST Genome Project (ShEST), assisting on sequencing and search for genes related to shrimp immune response against the viral infections. Libraries of different tissues (muscle, hepatopancreas, ocular peduncle)and different larval stages of Litopenaeus vannamei were sequenced using BigDye Terminator Sequencing Kit (Applied Biosystems) or ET-Dye Terminator (Amersham/GE Healthcare) and M13 universal primer (5'-TGTAAAACGACGGCCAGT-3'). The sequences were analyzed in automated sequencers MegaBace1000 (Amersham/GE Healthcare) and Applied Biosystems 377 (ABI377). These sequences were analyzed using the Basic Local Alignment Search Tool (BLAST). The results presented homology with interferon system proteins, like 2’,5’-Oligoadenylate binding protein, Toll like receptors, JAK/STAT and Interferon like protein. The 2’,5’-Oligoadenylate binding protein would be the first protein related to interferon system described in shrimps. A lot of sequences are still need to be investigated to complete the whole genome of Litopenaeus vannamei, nevertheless there are indications that this animal has a complex antiviral defense system, similar to interferon. / Os vírus estão entre os patógenos mais danosos encontrados na aqüicultura e são os mais numerosos e diversificados descritos para os crustáceos. O conhecimento sobre os genes associados à resposta imune viral, bem como a relação entre patógenos e hospedeiro ainda é escasso. Além disso, não há disponibilidade de linhas de célula para camarões, o que limita os sistemas primários de cultura celular, que por sua vez subsidiam pesquisas voltadas à expressão gênica. A análise de Seqüências Expressas Identificadas Expressed Sequence Tags (ESTs) tem se tornado uma prática comumente utilizada para identificar genes envolvidos em funções biológicas específicas, especialmente em organismos onde os dados genômicos ainda não estão disponíveis. O Laboratório de Genética Aplicada (LAGA) da Universidade Federal Rural de Pernambuco (UFRPE) é um dos participantes do projeto genoma EST do camarão Litopenaeus vannamei (ShEST) auxiliando no seqüenciamento e busca de genes de L. vannamei que atuam na resposta imune dos camarões contra as infecções virais. Foram seqüenciadas bibliotecas de diferentes tecidos (músculo, hepatopâncreas, pedúnculo ocular, etc) e diferentes fases larvais de Litopenaeus vannamei usando BigDye Terminator Sequencing Kit (Applied Biosystems) ou ET-Dye Terminator (Amersham/GE Healthcare) e o primer M13 universal (5'-TGTAAAACGACGGCCAGT-3'). As seqüências foram analisadas em seqüenciadorores automáticos MegaBace1000 (Amersham/GE Healthcare) e Applied Biosystems 377 (ABI377). Estas seqüências foram analisadas usando-se a Basic Local Alignment Search Tool (BLAST). Os resultados obtidos apresentaram homologia com proteínas do sistema interferon, como 2’,5’-Oligoadenylate binding protein, Toll like receptors (TLRs), JAK/STAT e interferon like protein (IntlP). A 2’,5’-Oligoadenylate binding protein pode ser a primeira proteína descrita para camarões que está relacionada ao sistema interferon. Muitas seqüências ainda precisam ser processadas para completar o genoma de Litopenaeus vannamei, contudo já há indícios de que este tenha um complexo sistema de defesa contra vírus, semelhante ao sistema interferon.

Listeria monocytogenes (LM) and Salmonella enterica serovar Typhimurium (ST) are intracellular bacterial pathogens that cause invasive disease in immune-altered individuals, including the immunocompromised and pregnant women. The mechanisms that modulate innate immunity to intracellular infection, particularly during pregnancy, are not well-understood. Functional selectin ligands play critical roles in leukocyte recruitment during inflammation. Increased control of LM infection in functional selectin ligand-deficient (FtDKO) mice is associated with increased levels of circulating innate immune cells, despite defective leukocyte migration compared to WT mice. Adoptive transfer of WT and FtDKO bone marrow (BM) cells to irradiated WT and FtDKO recipients demonstrates that BM reconstitution and the increased neutrophil phenotype of FtDKO mice is independent of functional selectin ligand expression within the host environment. Thus, functional selectin ligand deficiency enhances inherent innate immune resistance to intracellular infection. We then examined the impact of pregnancy-associated immunological changes on maternal susceptibility to intracellular infections. ST infection in pregnant mice results in profound systemic infection, increased fetal loss and enhanced serum and placental expression of pro-inflammatory cytokines. Pregnant mice showed decreased ratios of pro-inflammatory Th17 cells relative to anti-inflammatory regulatory T cells (Tregs) when compared to non-pregnant mice during infection. Functional inactivation of Tregs in vivo restored control of infection and normal Th17-to-Treg ratios, and reduced fetal loss. These indicate that modulation of Th17 and Treg responses impacts maternal and fetal protection from ST infection. Lastly, we examined the roles of type I interferons (IFNs) in modulating innate immunity to intracellular infections during pregnancy. Type I IFN receptor deficiency (IFNAR-/-) enhances immunity to LM and ST in the non-pregnant state by limiting pathogen-induced leukocyte death. We show that pregnant IFNAR-/- mice infected with LM retain increased protection from infection relative to WT controls. In contrast, protection conferred by IFNAR deficiency against ST infection in the non-pregnant state is abrogated during pregnancy. Distinctive maternal responses to LM and ST are associated with differential regulation of leukocyte distribution and cytokine expression in maternal systemic and/or placental compartments. Taken together, modulation of key mechanisms involved in leukocyte recruitment, immune-regulation and cytokine signaling impact host susceptibility to intracellular infections.

Ebola virus (EBOV) is a member of the Filoviridae family of highly pathogenic viruses that cause severe hemorrhagic fever and is the causative agent of the 2014 West Africa outbreak. Currently, there are no approved filovirus vaccines or treatments to combat these sporadic and deadly epidemics. One target for EBOV antiviral therapy is to block viral entry into host cells. Recently, phosphatidylserine (PtdSer) receptors, primarily known for their involvement in the clearance of dying cells, were shown to mediate entry of enveloped viruses including filoviruses. The PtdSer receptors, T-cell immunoglobulin mucin domain-1 (TIM-1) and family member TIM-4, serve as filovirus receptors, significantly enhancing EBOV entry. TIM-dependent virus uptake occurs via apoptotic mimicry by binding to PtdSer on the surface of virions through a conserved PtdSer binding pocket within the amino terminal IgV domain. TIM-4 is expressed on antigen presenting cells (APCs), including macrophages and dendritic cells (DCs), which are critical in early EBOV infection. My studies are the first to define the molecular details of virion/TIM-4 interactions and establish the importance of TIM-4 for EBOV infection of murine resident peritoneal macrophages. In addition, previous work has utilized only in vitro models to establish the importance of the TIM proteins in EBOV entry. My studies are the first to demonstrate the importance of TIM-1 and TIM-4 for in vivo EBOV pathogenesis and to confirm them as relevant targets of future filovirus therapeutics. Macrophage phenotypes can vary greatly depending upon chemokine and cytokine signals from their microenvironment. Historically, macrophages have been classified into two major subgroups: classically activated macrophages (M1) and alternatively activated macrophages (M2). Macrophages are a critical early target of EBOV infection and my work primarily focused on interferon gamma-stimulated (M1) macrophages since this treatment profoundly inhibited EBOV infection of human and murine macrophages. Interferon gamma treatment blocked EBOV replication in macrophages, reducing viral RNA levels in a manner similar to that observed when cultures were treated with the protein synthesis inhibitor, cycloheximide. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited EBOV infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit infection of negative strand RNA viruses including EBOV. In addition and most exciting, using MA-EBOV, we found that murine interferon gamma, when administered either 24 hours before or after infection, protects lethally challenged mice and significantly reduces morbidity. Our findings suggest that interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option.

As a foodborne pathogen, Listeria monocytogenes (Lm) encounters many barriers to invasion and dissemination in the host that may change the nature of host response. Lm has been most commonly studied using intravenous (i.v.) inoculation, however, a method that delivers a bolus of bacteria directly to the bloodstream. Thus, little is known about what systemic and local mediators are triggered during the natural course of infection and how these may impact susceptibility. Our laboratory used foodborne transmission of Lm in mice to assess whether the method of transmission and the specific organ microenvironment could affect infection-induced secretion of type I interferon or prostaglandin E2. Type I interferon is a pro-inflammatory effector secreted in response to viruses that has been proposed to paradoxically down-regulate innate immunity to intracellular bacteria. In contrast to i.v. infection, type I interferon was not detrimental to the immune response when Lm were acquired orally. In fact, most of the anti-inflammatory effects of type I interferon in the spleen were attributable to i.v. but not foodborne infection. Importantly however, downregulation of the receptor for interferon gamma (IFNGR1), previously ascribed to the type I interferon response, was found to be a consequence of infection and unrelated to type I interferon. In the liver, robust recruitment and activation of neutrophils (PMN) is thought to be required for initiation of Lm immunity. Prostaglandin E2 (PGE2) is a lipid mediator most commonly associated with pain and fever that has also been demonstrated to have anti-inflammatory or tolerogenic effects. It is unknown, however, whether foodborne infection induces PGE2 in the liver and if PGE2 then down-regulates PMN activities. Recruitment of PMN to the liver following foodborne infection was robust in both susceptible and resistant animals. Bone marrow PMN from each killed Lm ex vivo with similar efficiency, thus suggesting that if PMN were dysfunctional during the course of natural infection, they were responding to cues in the microenvironment. Accordingly, significantly more PGE2 was made ex vivo by cells from the livers of susceptible animals than from resistant animals. When PGE2 was applied to naïve PMN prior to exposure to Lm, it consistently dampened the killing efficiency of these cells, suggesting that this lipid better known for its pro-inflammatory roles might have anti-inflammatory effects during Lm infection. Overall, these studies indicate that mediators produced as a result of infection may have very different roles dependent on route of inoculation, timing, and the specific organ examined.

Les cellules dendritiques (DC) tiennent une place centrale dans l'initiation des réponses immunitaires et dans le contrôle du développement des tumeurs. La sous-population cDC1 suscite aujourd'hui en grand intérêt de par ses fonctions d'activation de réponses cytotoxiques par présentation croisée d'Ag aux lymphocytes T (LT) CD8+ ainsi que son implication dans l'immunité antitumorale et la réponse aux immunothérapies chez la souris. Le rôle des cDC1 chez l'Homme est cependant peu décrit. Les cDC1 murines et humaines sont aussi connues pour produire de larges quantités d'interféron (IFN) de type III (IFN-III), aussi appelés IFN-λ. Tout comme les IFN-I avec lesquels ils partagent la même voie de signalisation, les IFN-III ont un rôle antiviral bien décrit. Des modèles murins ont également suggéré un rôle antitumoral, mais ces IFN n'ont jamais été étudiés dans un contexte de cancer chez l'Homme. Il est donc crucial de comprendre les mécanismes expliquant l'impact pronostique positif des cDC1 ainsi que le rôle des IFN-III dans l'immunité antitumorale, en particulier pour le développement de nouvelles approches thérapeutiques. Nous avons démontré pour la première fois l'infiltration des tumeurs humaines de sein et d'ovaire par diverses sous-populations de DC. Les cDC1 sont particulièrement enrichies par rapport au sang des patientes et forment de nombreuses interactions avec les LT CD8+ dans les tumeurs. Une approche de bio-informatique a permis de révéler que les cDC1 représentent l'unique population de DC associée à une meilleure survie des patients dans la majorité des cancers du TCGA. De façon intéressante, la signature de réponses aux IFN-I et III est enrichie dans les tumeurs de sein fortement infiltrées par les cDC1 mais pas par les autres sous-populations. L'expression des gènes codant pour l'IFN-λ1 ou le récepteur aux IFN-III est également associée à une meilleure survie sans rechute dans le cancer du sein. De plus, nous avons démontré la capacité des cDC1 à produire de l'IFN-III sans aucune réactivation ex vivo. Ce résultat indique clairement que dans un contexte de réponse immunitaire antitumorale chez l'Homme, la synthèse d'IFN-III est une spécificité des cDC1 comparées aux autres sous-populations. La présence d'IFN-III dans les surnageants tumoraux a été confirmée au niveau protéique et démontrée comme étant fortement corrélée avec l'IL-12p40, les CXCR3-L, le CX3CL1 et le TNF-α. Ces données soulèvent alors l'hypothèse de l'association entre l'IFN-III, produit dans le microenvironnement tumoral par les cDC1, et la présence de cytokines et chimiokines impliquées dans le recrutement et l'activation de lymphocytes cytotoxiques tels que les LT CD8+ ou cellules NK. Notre étude apporte des informations détaillées quant à la nature des différentes sous-populations de DC infiltrant les tumeurs humaines de sein et d'ovaire et démontrent pour la première fois la production d'IFN-III par les cDC1. L'association de ces cellules et des IFN qu'elles produisent avec une meilleure survie des patientes confirme l'intérêt de développer de nouvelles immunothérapies ciblant les cDC1, en particulier dans le cancer du sein / Dendritic cells (DCs) represent a promising target for the development of new immunotherapies because of their central role in the initiation and the control of immunity. The rare cDC1 population is under considerable scrutiny because their murine counterparts called CD8α+ DCs are essential for cross-presentation to CD8+ T cells, antitumor immunity and response to immunotherapies. In contrast, the role of human cDC1 in cancer has not been investigated as extensively as in mice. They were identified in several tumors and transcriptomic analyses revealed their association with a favorable patient outcome. They also represent a major source of type III interferon (IFN-III), also called IFN-λ, playing a crucial role in viral infections, similarly to IFN-I that share the same signaling pathway. Its antitumor activity was also reported in mouse models, therefore raising questions regarding the use of IFN-IIl in clinical oncology. We believe that understanding the underlying mechanisms of cDC1 favorable prognostic impact and the role of IFN-III in antitumor immunity will be central to design new therapeutic approaches. Here, we demonstrated the infiltration of human primary breast and ovarian tumors by several DC populations and the enrichment of cDC1 compared with patient blood. We also showed for the first time close contacts between cDC1 and T cells in breast tumors. An in silico approach using MCPcouter on the TCGA data sets revealed that cDC1 represented the only DC subset associated with a prolonged overall survival in the majority of solid tumors. Interestingly, type I/III signature was strongly enriched in tumors highly infiltrated only with cDC1. Furthermore, we observed by feature intracytoplasmic flow cytometry analysis a spontaneous production of IFN-λ1 that is restricted to cDC1 in the absence of any ex vivo stimulation in one third of tumors. This result clearly indicates that IFN-λ1 production is a distinct of cDC1 compared with other DC subsets, even in a human tumor context. Notably, a high expression level of genes coding for IFN-III or its receptor was correlated with an increased relapse-free survival in breast cancer. We confirmed the presence of the IFN-λ1 protein in more than 50% of tumors and observed its abundancy compared with other IFN subtypes. IFN-λ1 was strongly correlated with IL-12p40, CXCL9, CXCL10, CXCL11, CX3CL1 and TNF-α. These results raised the hypothesis that IFN-λ1, produced by cDC1 in the TME, could be associated with the production of cytokines and chemokines involved in the recruitment and activation of cytotoxic lymphocytes (NK cells and CD8+ T cells). Our study provides detailed information about the DC compartment infiltrating human breast and ovarian tumors, revealing their potential implication in the antitumor immunity. By focusing on the pathways associated with each DC subset, our findings shed new light on the link between DC population called cDC1 and IFN-I/III signature in tumors. Our clear demonstration of IFN-III production by cDC1 and of its positive impact on the prognosis of cancer patients provides valuable evidences to support the development of new therapeutic strategies targeting cDC1 to amplify the response to immunotherapies, especially in breast cancer

Die Todesrezeptoren der TNF-Familie sind neben der Vermittlung von Apoptosesignalen auch in der Lage, nicht-apoptotische intrazelluläre Signalwege zu beeinflussen. Der Caspase-8-Inhibitor cFLIPlong inhibiert dosisabhängig die Prozessierung der Initiator-Caspase-8 am TRAIL-DISC (death inducing signalling complex) und hemmt die Aktivierung des NF-kappa-B-Signalweges über die Modulation der Rekrutierung und Spaltung des für die NF-kappa-B-Aktivierung notwendigen RIP (receptor interactin protein)am DISC. / TNF-derived death-receptors are not only involved in transducing apoptosis-signalling but also modulate non-apoptotic pathways. Cellular FLICE-inhibitory protein cFLIPlong is able to block processing of initiator-caspases in the TRAIL-DISC dependent on the FLIP/Casp-8- level. It also interacts with NF-kappa-B-signalling pathways by modulating the recruitment and processing of receptor-interacting-protein RIP in the TRAIL-DISC-complex.

Infektionen durch C. albicans auf den Schleimhäuten sind eine häufige Erkrankung bei Patienten mit einer Schwächung der T-Zellimmunität. Blutstrominfektionen mit der Hefe C. albicans (Candidämie) stellen, vor allem bei Patienten auf Intensivstationen, eine nach wie vor bedrohliche Komplikation mit hoher Letalität dar. Das pH-regulierte Antigen 1 (Pra1) ist ein Protein, das von C. albicans produziert wird, auf der Oberfläche des Pilzes gebunden vorkommt und auch vom Pilz in den Überstand sezerniert wird. Im humanen System bindet das Protein an T-Zellen an das Oberflächenprotein CD46. Es ist des Weiteren bekannt, dass das Pra1 an bestimmte Immunzellen der Maus (Monozyten und Phagozyten) binden kann. Eine Bindung an T-Zellen der Maus ist bisher nicht beschrieben. Eine genaue Charakterisierung der Interaktion von Pra1 mit Immunzellen der Maus ist interessant, da die Maus als biologischer Modellorganismus zur Erforschung der Infektion mit C. albicans dient. In dieser Arbeit konnte gezeigt werden, dass rekombinantes Pra1 (rPra1) auch an Maus-CD4+ T-Zellen binden kann. Es wurden Einflussfaktoren auf die gefundene Bindung von Pra1 an CD4+ T- Zellen gesucht. Als ein Einflussfaktor wurde Zink identifiziert. Pra1 kann an freies Zink binden und durch Zugabe von ZnCl2 während der Inkubation von Pra1 mit T-Zellen kann das Signal von gebundenem Pra1 an CD4+ T-Zellen erhöht werden. Aspf2, ein Protein aus Aspergillus fumigatus mit großer Homologie zu Pra1, kann nicht an diese Zellen binden. Im in-vivo-Experiment mit Tieren, die mit C. albicans infiziert wurden, konnte kein wildtypisches sezerniertes Pra1 gebunden an T-Zellen nachgewiesen werden. Zellkulturüberstände von C. albicans zeigten nach Inkubation in vitro mit T-Zellen ein Signal für gebundenes Pra1 an CD4+ T-Zellen. Die Bindungskinetik von Pra1 an T-Zellen zeigte eine über die Zeit der Inkubation konstante Zunahme des Signals von zellgebundenem rPra1 an CD4+ T-Zellen. In der off-Kinetik fand sich eine Abnahme des Signals über die Zeit bis an die Grenze der Nachweisbarkeit. Der Bindungspartner von Pra1 auf T-Zellen konnte nicht identifiziert werden. Die strukturell und funktionell verwandten Oberflächenproteine Crry, CD59a und CD55 wurden auf Bindungsfähigkeit an T-Zellen in entsprechenden Knockout- Mäusen getestet, konnten jedoch als Rezeptor für Pra1 ausgeschlossen werden. Durch die Bindung von sezerniertem Pra1 an neutrophile Granulozyten wird die Fähigkeit dieser Zellen zur Phagozytose eingeschränkt. Die Bindung von Pra1 an CD4+ T-Zellen führt zur Kostimulation der T-Zellen, also zur verstärkten Zellaktivierung und Proliferation. Durch die Zugabe von 10 μM Zinkchlorid wird die kostimulatorische Aktivität von Pra1 verstärkt. Während der Zellaktivierung von Effektor-Memory-CD4+ T-Zellen reduziert rPra1 die Sekretion von IFN-γ. Diese Reduktion von IFN-γ-produzierenden Zellen entsteht nicht durch einen Einfluss von Pra1 während der Zellaktivierung von naiven CD4+ T-Zellen zu Th1-Zellen und auch nicht durch die Auslösung von Apoptose in IFN-γ-produzierenden Th1-Zellen. Die Bindung von Pra1 an CD4+- T-Zellen, die über den T-Zell-Rezeptor aktiviert werden, reduziert in vitro die Sekretion des Zytokins. Zusätzlich werden weitere Zytokine in ihrer sezernierten Menge reduziert wie IL-2 und TNF-α. / Infections caused by C. albicans on mucosal surfaces are a common disease in patients suffering from suppression of the T cell immune defense. Blood stream infections by the yeast C. albicans (candedemia) represent still a severe complication in patients in intensive care units with high rates of lethality. The pH-regulated antigen 1 (Pra1) is a protein produced by C. albicans which is present on the surface of the fungi and is secreted into the supernatant of fungal cultures as well. Pra1 can bind to human T cells via the surface protein CD46. It is known, that this protein can also bind to certain immune cells of mice (monocytes and phagocytes). Binding to T cells of mice is not yet known. A characterization of the interaction of Pra1 with immune cells of mice would be valuable, because mice act as a biological model system for the investigation of infections with C. albicans. In this paper, it could be shown that recombinant Pra1 (rPra1) can bind to mouse CD4+ T cells as well. After the finding that rPra1 can bind to CD4+ T cells, different parameters determining this binding have been studied. Zinc was found to be one influencing factor on the binding. Pra1 can bind free zinc ions and by the addition of ZnCl2 while incubating T cells with Pra1 the signal of bound Pra1 to CD4+ T cells could be increased. Aspf2, a protein from Aspergillus fumigatus with high homology to Pra1, was not able to bind to these cells. In in-vivo-experiments with animals infected with C. albicans, no wild-typic secreted Pra1 was found bound to T cells. Supernatant from C. albicans cultures produced, after incubation in vitro, a signal for cell-bound Pra1 on CD4+ T cells. Kinetics of the binding of rPra1 to T cells showed a constant increase of signal over the time of incubation. The off-kinetics revealed a decrease of cell-bound rPra1 over time to the edge of detectability. The receptor of Pra1 on T cells has not been identified yet. The structurally and functionally comparable surface proteins Crry, CD59a and CD55 were tested in knockout mice for each of these proteins and could be excluded as possible receptors. After binding of secreted Pra1 to neutrophilic granulocytes these cells experience a decreased capacity to phagocytose pathogens. The binding of Pra1 to CD4+ T cells leads to a costimulation of T cells, which results in increased cell activation and proliferation. This costimulatory capacity of Pra1 can be augmented by adding 10 μM zinc chloride. During activation of naïve CD4+ T cells Pra1 reduces the secretion of IFN-γ. The reduction of IFN-γ-producing cells is not due to an influence of Pra1 during cell activation of naive CD4+ T cells to Th1 cells and is also not due to induction of apoptosis in IFN-γ-producing Th1 cells. The binding of Pra1 to ex-vivo isolated CD4+ T cells reduces the in vitro secretion of IFN-γ after stimulating these cells via their T cell receptor. Additionally, the secretion of IL-2 and TNF-α was reduced.