Altered susceptibility to HIV-1 infection has been observed in multiple cohort studies around the world, with a small proportion of HIV-Exposed, Seronegative (HESN) individuals remaining uninfected despite repeated exposure. Previous work has shown association of three polymorphisms in interferon regulatory factor 1 (IRF1) with decreased susceptibility to HIV-1 infection and a reduced likelihood of seroconversion. Peripheral blood mononuclear cells (PBMCs) from patients with protective IRF1 haplotype exhibited significantly lower basal IRF1 expression, reduced responsiveness to IFNγ stimulation and significantly lower HIV-1 LTR transcription during the initial stages of infection. The goal of this thesis was to characterize the effect of three IRF1 polymorphisms associated with HIV-resistant phenotype on: (1) IRF1 gene transcription, alternative splicing of IRF1 mRNA and IRF1 mRNA/protein stability (2) plasma and cervical lavage (CVL) cytokine/chemokine expression and (3) HIV pathogenesis and disease progression. Furthermore since differences in hormone expression are known to regulate IRF1 function we set out to determine if differences in plasma hormone levels contribute to the natural resistance against HIV-1 infection in the Majengo HESN cohort. Polymorphisms in the IRF1 gene do not directly affect IRF1 transcription but instead act as intronic splicing regulators. PBMCs from individuals with protective IRF1 haplotype were associated with increased inclusion of exons 7/8 and decreased protein stability when compared to cells from individuals with nonprotective haplotype. Individuals with protective IRF1 haplotype also exhibited significantly higher plasma IL15, IFNγ and IL6 expression and significantly higher CVL IL15, IFNγ, IL2 and sIL2Rα expression (with univariate analysis only) compared to those with nonprotective IRF1 haplotype. Additionally, individuals with protective IRF1 haplotype expressed significantly lower levels of plasma prolactin when compared to individuals with nonprotective haplotype. IRF1 polymorphisms were found to not be associated with HIV disease progression, suggesting that the protective effect of IRF1 polymorphisms is limited to the early stages, prior to establishment of HIV-1 infection. Furthermore, independent of IRF1 genotypes, significantly lower plasma prolactin, estrogen, progesterone and cortisol levels were observed in HESN women suggesting that hormonal regulation may be one of the main factors regulating natural resistance to HIV-1 infection in the Majengo HESN cohort.

In neurodegenerative diseases, the CNS becomes inflamed through activation of pathways, including the NF-B pathway. Some of the therapies for those diseases target neuroinflammatory pathways. Here, we explore the mechanisms for the upregulation of a subset of genes following a restimulation of the NF-B pathway. We discover that this upregulation occurs independent of IRF1 expression and type 1 interferon signaling. A knockdown of IRF1 using siRNA and an inhibition of JAK proteins using inhibitor AG490 both had no effect on priming. A secreted factor was found to upregulate the expression of both this subset of genes and genes encoding pro-inflammatory cytokines induced by NF-B activation. We also explored the role of IRF1 in a mouse model of multiple sclerosis. We found that the deletion of IRF1 from oligodendrocytes diminished EAE severity. A deletion of IRF1 from myeloid cells within mice did not diminish EAE severity, however showed a promising decrease in the expression of certain inflammatory genes. Thus, IRF1 plays a critical role in fine-tuning inflammatory responses in the brain.

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The aim of this thesis was to establish the therapeutic value of the IFITM1 monoclonal antibodies and to design and develop therapeutically valuable recombinant monoclonal antibodies so as to study the implication of these novel antibodies in cancer therapy. Cancer metastasis is one of the main interests that has given rise to the design and development of innovative strategies for cancer therapeutics. The Interferon Induced Transmembrane Protein 1(IFITM1), a notable member of the IFITM family of proteins has been identified as one of the most up-regulated trans-membrane proteins in metastatic breast cancer and cervical adenocarcinoma. This interferon-regulated protein is also involved in cell migration, invasion in glioma and squamous cancers. This PhD aimed to study IFITM1 as a pro-invasive cancer target by the use of IFITM1 monoclonal antibodies that were raised against the extracellular domain of the human IFITM1 gene. The epitope mapping of IFITM1 revealed the binding activity of the IFITM1 monoclonal antibody. This gave the opportunity to design and generate to new IFITM1-specific molecular tools, in the form of recombinant IFITM1 targeted murine scFv antibody, IFITM1-CPG2 yeast fusion protein antibody for potential application in ADEPT as well as a Mouse-Human Chimeric IFITM1 antibody secreting mammalian cell line. The immunohistochemical staining of IFITM1 in tissue micro array from breast, colon and oeosphegal cancer has revealed that the majority of these cancers produce this protein. However, IFITM1 is over produced in cervical cancer indicating it’s selective over expression in cervical cells. This PhD endeavored to investigate the expression of IFITM1 at a translational and transcriptional level and to study the clinical significance of IFITM1 in cervical cancer. The antibody dependent cell mediated cytotoxic activity of the chimeric IFITM1 antibody was found to be cytotoxic to SiHa cells in vitro. In the future these molecular tools could be used to regulate and further characterize the activity of this transmembrane protein antibody. In an effort to better understand the mechanisms that regulate the activity and the over production of the IFITM1 gene and its interacting proteins, a proteomic screen of cervical cancer cells was carried out using data-independent SWATH-MS on an AB SCIEX TripleTOF™ mass spectrometer. This Mass Spec analysis provided us with a host of IFITM1 biomarkers and revealed that the IFITM1 gene and its binding proteins also cross link with the IRF1 pathway. The data presented in this thesis, demonstrates that the IFITM1 gene can be targeted to either stimulate or inhibit IFITIM1 signaling to engage IFITM1 as a potential pro-invasive extracellular receptor as a target in antibody cancer therapy. In summary, this thesis aimed to confirm the activity and the binding specificity of the IFITM1 antibody. Additionally, this thesis demonstrated a promising application of the recombinant antibody in the ADEPT technology. Characterization of IFITM1 mAb effector functions indicated that the antibody was cytotoxic to cervical cancer cells. This highlights an important element in the immune suppressive tumour microenvironment. And finally, this thesis also provides the basis for the production of recombinant mouse human chimeric antibodies that are a part of a new group of immunotherapeutic molecules paving the way for cancer therapeutics.

The induction of type I interferon (IFN) and subsequent activation of interferon stimulated genes (ISGs) represent a first line of defense against viral infection. Typically type I IFN signaling leads to the phosphorylation of the STAT1 and STAT2 transcription factors (TFs) which then form a trimetric complex with IRF-9 and translocate to the nucleus to induce ISG expression. However, the results of this study showed that IFN-mediated upregulation of the ISG Oas1b, the product of which confers resistance to flavivirus induced disease, can be induced in a STAT1-independent manner. Since numerous ISGs have antiviral functions, many viruses have evolved strategies to disrupt the type I IFN-signaling pathway. In cases when STAT1 activation is blocked by a viral infection, STAT1-independent upregulation of ISGs provides an additional strategy for the cell to mount an effective antiviral response. Infection of mouse embryofibroblasts (MEFs) with West Nile virus (WNV) induced the production of IFN beta and STAT1 and STAT2 phosphorylation but blocked nuclear translocation and binding of these TFs to the promoters of the ISGs, Oas1a, Oas1a, Irf7 and Irf1. However, each of these antiviral ISGs was efficiently upregulated in infected cells and IRF-9 was shown to be crucial for the upregulation of Oas1a, Oas1b and Irf-7. IRF-3 or IRF-7 was needed to maintain the upregulation of these genes at later times of infection. In contrast, the upregulation of Irf1 by WNV infection did not depend on the tested IRFs but was reduced by inhibition of the p38 or NF-kappa B pathways. Although Irf1 mRNA was efficiently upregulated in WNV-infected cells IRF-1 protein synthesis was blocked. The precise mechanism of the IRF-1 translational suppression is not yet known, but the suppression was shown not to be due to increased proteasomal degradation of IRF-1 nor to alternative splicing of Irf1 mRNA. Preliminary results suggest miRNAs may play an indirect role in regulating IRF-1 translation. The results of this study expand knowledge about the strategies evolved by viruses to evade host cell antiviral responses and also provide valuable insights about alternative mechanisms utilized by the host cell to counteract viral infections.

La surveillance immunologique des tumeurs repose sur la capacité des cellules effectrices du système immunitaire à détecter et à éliminer les cellules cancéreuses. Nonobstant ce constat, la régression complète et spontanée de cancers établis n’est observée que dans de très rares cas. L’échec de la résolution des cancers par le système immunitaire pourrait résulter de la conjonction de plusieurs facteurs : i) une réponse immune inadéquate liée au manque d’immunogénicité des tumeurs, ii) l’incompétence du système immunitaire consécutif à des immunodéficiences acquises ou induites et iii) la sélection de variants tumoraux résistants capables de déjouer la surveillance opérée par le système immunitaire ou de subvertir ses effets. Ainsi, le développement de stratégies visant à potentialiser les réponses antitumorales de l’hôte revêt un enjeu crucial en cancérologie.Au laboratoire, notre travail de recherche a pour objectif de mieux caractériser les liens entre réponse immunitaire et cancer. Mon travail de thèse vise précisément à comprendre les mécanismes moléculaires impliqués dans la différenciation des lymphocytes T CD4 et à déterminer le rôle de ces cellules dans l’immunité antitumorale. Au cours de ma thèse, nous nous sommes particulièrement attachés à explorer les mécanismes moléculaires qui sous tendent la différenciation des populations lymphocytaires Th17, Th9 et TFh pour mieux appréhender et moduler leurs fonctions effectrices afin d’optimiser les réponses antitumorales. Ces travaux s’inscrivent dans une démarche d’application potentielle en immunothérapie anticancéreuse, un domaine de recherches qui connaît actuellement des avancées spectaculaires.Nous avons tout d’abord étudié l’influence de l’acide docosahexaénoïque (DHA), un acide gras à longue chaîne de la série n 3, sur la différenciation des cellules Th17. Nous avons mis en évidence le mécanisme moléculaire responsable de l’inhibition directe de la polarisation cellulaire Th17 par le DHA. L’activation de PPARγ par le DHA induit l’expression de SOCS3 qui agit comme un répresseur intrinsèque de la différenciation Th17. Dans deux modèles de cancers murins, nous avons également montré que l’activité anticancéreuse du DHA était dépendante de sa capacité à inhiber la sécrétion d’IL 17 par les cellules T CD4 in vivo. Nous avons ainsi caractérisé l’un des mécanismes impliqués dans l’effet anticancéreux du DHA.Dans un deuxième travail, nous avons caractérisé les effets de l’interleukine 1β sur le programme moléculaire des cellules Th9. Nous avons montré que les cellules Th9 différenciées en présence d’IL-1β possédaient de puissantes propriétés anticancéreuses dépendantes de l’IL-21 reposant sur l’activation du facteur de transcription IRF1. Au niveau moléculaire, nous avons démontré que l’IL-1β induisait la phosphorylation de STAT1 elle même responsable de l’activation d’IRF1 qui est alors capable d’interagir sur les promoteurs de l’Il9 et de l’Il21 pour induire l’expression de ces gènes dans les cellules Th9.Le dernier projet porte sur la régulation transcriptionnelle du facteur de transcription IRF1 sur la réponse T folliculaire auxiliaire et cherche à en évaluer les retombées potentielles en immunothérapie anticancéreuse. Notre étude met en évidence l’activation précoce d’IRF1 dans la différenciation TFh et suggère que ce facteur de transcription semble initier le développement de ces cellules. Des approches de transfert adoptif révèlent que les TFh semblent posséder des propriétés anticancéreuses capables de limiter efficacement la croissance des tumeurs dans des modèles murins. Enfin, après caractérisation phénotypique nous montrons que les cellules TFh sont présentes dans des tumeurs mammaires chez l’Homme et validons la présence d’IRF1 dans ces lymphocytes. / Immune surveillance of tumors is based on the ability of effector cells of the immune system to detect and eliminate the cancer cells. Notwithstanding, the complete and spontaneous regression of established cancers was observed only in very few cases. The failure of cancer resolution by the immune system could result from the combination of several factors: i) inadequate immune response related to a low tumor immunogenicity, ii) incompetent immune system consecutively to induced or acquired immunodeficiencies and iii) the selection of resistant tumor variants able to thwart immune surveillance or subverting immune responses. Developing novel cancer immunotherapy strategies leading to potentiation of the host antitumor responses is thus a key challenge in oncology.We aim to better characterize the relationships between immune response and cancer. My work is precisely to understand the molecular mechanisms involved in CD4 T cell differentiation and to determine the role of these cells in antitumor immunity. I am particularly committed to explore the molecular mechanisms underlying the Th17, Th9 and TFh cell differentiations. The goal is to better understand and adjust their effector functions to optimize antitumor responses. This work is part of a potential application in cancer immunotherapy approach, an area that is experiencing dramatic advances and is likely to grow in the years ahead.We first studied the influence of the n 3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on Th17 cell differentiation. We unraveled the molecular mechanism responsible for the direct inhibition of Th17 cell polarization by DHA, explaining one way of DHA to exert its anticancer activity. TH17 cells induced in vitro displayed increased SOCS3 expression and diminished capacity to produce interleukin 17 following activation of PPARγ by DHA. In two different mouse cancer models, DHA prevented tumor outgrowth and angiogenesis in an IL 17 dependent manner. Altogether, our results uncover a novel molecular pathway by which PPARγ induced SOCS3 expression prevents IL 17 mediated cancer growth.Then, we characterized the effects of interleukin 1β (IL-1β) on Th9 cells molecular program. We found that the transcription factor IRF1 enhanced the effector functions of Th9 cells and dictated their anticancer properties. Under Th9 skewing conditions, IL-1β induced phosphorylation of the transcription factor STAT1 and subsequent expression of IRF1, which bound to Il9 and Il21 gene promoters and enhanced their secretion by Th9 cells. In addition, IL-1β induced Th9 cells exerted potent anticancer functions in an IRF1 and IL 21 dependent manner. Thus, our findings identify IRF1 as a target for controlling the function of Th9 cells.We are currently investigating the transcriptional regulation of IRF1 on follicular helper CD4 T (TFh) cell program. We address the question whether TFh cells could be beneficial in cancer immunotherapy. Our study highlights the early activation of IRF1 during the TFh cell polarization and suggests that IRF1 appears to initiate the development of these cells. Adoptive transfer approaches show that TFh lymphocytes seem to habor anticancer properties by limiting efficiently tumor outgrowth in mouse models of cancer. Finally, phenotypic characterization of TFh cells points out that they infiltrate human breast tumors and express IRF1.

Background & Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system. Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats. Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet. / Doctoral thesis