Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

No description available.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.

Cancer, historically considered a genetic disease, is currently acknowledged to affect the whole body. Our immune system is one key player that can elicit a response against malignant cells but can also promote tumorigenesis. Tumors avoid immune recognition by creating a suppressive microenvironment and inducing tolerance. T-cells are regarded a major effector cell type in tumor immunotherapy. An important ”switch” needed for T-cell activation involves so-called costimulatory and coinhibitory receptors. In this thesis, experimental tumor models were used to investigate the potential of immunomodulatory antibodies to stimulate immune cells and subsequently eliminate tumors. First, systemic antibody blockade of two negative checkpoint regulators (CTLA-4 and PD-1) present on T-cells was evaluated in combination with local CpG therapy or standard BCG treatment. Indeed, this combinatorial therapy with CpG augmented anti-tumor effects with increased levels of tumor-directed T-cells and reduced tumor-infiltrating Tregs. Secondly, as these immunomodulatory antibodies elicit severe side effects in patients, a local low-dose delivery regimen was explored as an alternative to systemic bolus treatment. Our results demonstrated that an approximately seven times lower dose of aCTLA-4, compared to systemic delivery, could eradicate both primary and distant tumors. CD40-expressing APCs are another potential target in antibody-mediated cancer therapy. CD40-stimulated dendritic cells (DCs) have the capability to activate tumor-directed T-cells to kill tumor cells. We next sought to investigate agonistic CD40 antibody efficacy and in vivo biodistribution when delivered locally compared to the equivalent systemic dose. Anti-tumor effects were dependent on CD8+ T-cells, host CD40 expression and the presence of tumor antigen at the injection site. CD40 antibodies were cleared from the circulation and accumulated in lymphoid organs, where, upon repeated aCD40 dosing, target APC populations increased in numbers and upregulated their surface CD40 expression. Lastly, CD40 agonist antibodies were mixed with nanoparticles to enhance their stimulatory properties. B-cells demonstrated increased proliferative capacity and DCs became more activated when exposed to the cocktail. Further, this combination reduced serum levels of pro-inflammatory cytokines compared to plain antibodies.       The results herein advocate further exploratory studies of the delivery of monoclonal antibodies at the tumor site in order to improve anti-tumor effects and reduce toxicity.

Le CTLA4 permet de maintenir la tolérance du soi et prévient le développement d’auto-immunités. Contenu au sein de vésicules intra-cytoplasmiques des lymphocytes T au repos, le CTLA4 est exprimé à la membrane plasmique suite à l’activation du TCR, on le qualifie de rétrocontrôle inhibiteur du système immunitaire (ICB). L’anticorps bloquant le CTLA4, l’ipilimumab induit un contrôle immunitaire à long terme chez une fraction de patients atteints de mélanomes métastatiques. Deux études cliniques de phase III ont conduit à son autorisation de mise sur le marché dans le traitement du mélanome métastatique par la FDA et l’EMA en 2011. Cependant le blocage du CTLA4 est souvent associé au développement d’effets indésirables liés à l’immunité, irAEs, majoritairement au niveau de la peau et de l’intestin, deux sites colonisés par la flore microbienne. Afin de continuer le développement des ICB et des combinaisons de traitements, de nombreux efforts visent à découpler l’efficacité anti-tumorale de la toxicité associée à l’anti-CTLA4. Bien que la stimulation du système immunitaire soit responsable des effets thérapeutiques de l’anti-CTLA4, aucun biomarqueur immunologique d’efficacité n’a été décrit. Dans notre première étude nous avons étudié le mécanisme d’action de l’anti-CTLA4 et nous avons décrit un rôle de l’IL-2 et de ses récepteurs dans l’activité anti-tumorale de l’anticorps. Nous avons également décrit la fraction soluble du récepteur à l’IL-2, le sCD25 comme un biomarqueur potentiel de résistance au traitement. Une concentration élevée de sCD25 dans le sérum des patients atteints de mélanome prédit la résistance à l’ipilimumab. Dans notre second projet, nous avons révélé le rôle du microbiote intestinale et particulièrement de bactéries Gram négatives, des Bacteroides, dans l’efficacité anti-tumorale de l’anti-CTLA4. L’absence d’efficacité du blocage du CTLA4 chez les animaux dépourvus de flore intestinale peut être rétablie par l’administration de Bacteroides fragilis, ou bien de DC, ou encore de lymphocytes T spécifiques de B. fragilis, sans déclencher de colites. Ces travaux suggèrent de nouvelles stratégies thérapeutiques pour espérer améliorer la balance bénéfice / toxicité / coût de l’ipilimumab. / CTLA4, cytotoxic T lymphocyte antigen-4, which is present in the intracytoplasmic vesicles of resting T cells, is upregulated at the surface of activated T cells where it maintains self-tolerance and prevents autoimmunity. The CTLA4-blocking antibody, ipilimumab, induces immune-mediated long term control of metastatic melanoma in a fraction of patients, leading to its approval by the US Food and Drug Administration (FDA) and the European Medical Agency (EMA) in 2011 for the treatment of advanced metastatic melanoma. However, blockade of CTLA4 by ipilimumab often results in immune-related adverse events (irAEs) at sites that are exposed to commensal flora, namely the gut and the skin. Uncoupling efficacy from toxicity is a challenge for the development of immune checkpoint blockers and therapeutic combinations. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, relevant immune biomarkers that predict treatment efficiency remain elusive. Firstly, we unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides an immunologically relevant biomarker, elevated serum sCD25, which predicts resistance to CTLA-4 blockade in patients with melanoma. Secondly, we show that the antitumor effects of CTLA4 blockade depend upon intestinal Gram-negative bacteria, mostly Bacteroides species. These bacteria accumulate at the bottom of the intestinal crypts and elicit an IL-12-dependent Th1 immune response specific for distinct Bacteroides species, both in tumor bearing mice and in cancer patients. CTLA4 blockade lost its anticancer efficacy in antibiotic-treated or germ-free mice. This defect could be overcome by oral administration of Bacteroides fragilis (Bf), immunization with Bf polysaccharides, or adoptive transfer of Bf-specific T cells, all of which in the absence of colitis. Our study unravels the key role of Bacteroides in the immunostimulatory effects of CTLA4 blockade, suggesting novel strategies for safely broadening its clinical use

No description available.

En raison de l'efficacité pour délivrer des antigènes directement dans le cytoplasme des CAPs in vivo, les vecteurs bactériens atténués et basés sur les propriétés du système de sécrétion de type 3 (SST3) attirent de plus en plus l'attention grâce à leur potentiel dans le développement des vaccins contre le cancer. Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les personnes immunodéprimées, les grands brûlés et les patients atteints de la mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence dont le SST3. Dans nos travaux précédents, le potentiel de souches atténuées de P. aeruginosa dans le domaine de la vaccination anti-tumorale a été démontré. Dans ce travail, nous avons optimisé des vecteurs vaccinaux basés sur le SST3 de P. aeruginosa pour des applications cliniques. Dans un premier temps, la performance de ces vecteurs bactériens a été améliorée en utilisant différents modèles de tumeurs murines. Ceci par : 1) l'ajout d'un épitope spécifique des lymphocytes CD4+ Th aux vecteurs; 2) l'application d'un modèle d'expression bi-antigénique aux vecteurs; 3) la construction de vecteurs induisant une réponse humorale. Dans un deuxième temps, la performance thérapeutique du vecteur bactérien a été optimisée par la modulation de la fréquence des injections et l'intervalle qui les sépare. Cette performance a été confirmée dans des modèles différents de tumeurs murines. Dans un troisième temps, un candidat qui pourrait être appliqué en clinique a été généré par l'adaptation d'un mutant (CHA-OAL) de P. aeruginosa totalement avirulent dans un milieu chimiquement défini. La très faible infectiosité de cette souche a été surmontée par en vaccinant à des emplacements multiples. Par la suite, le potentiel du vecteur bactérien dans l'immunothérapie humaine a été également évalué- dans un premiers temps-dans un modèle de souris humanisées (HHD). Enfin, nous avons observé qu'une immunité anti-vecteur pré-existante n'a pas d'effet sur l'efficacité de la vaccination par le vecteur bactérien. L'ensemble de nos résultats a mis en évidence le potentiel de nos vecteurs vivants et atténués de P. aeruginosa pour des applications dans des essais cliniques pertinents. / Due to the endowed effective ability to deliver antigen to cytoplasm of APCs in vivo, T3SS based attenuated bacterial vectors attracted more and more attention for their potential interest in cancer vaccine development. Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les personnes immunodéprimées, les grands brûlés et les patients atteints de la mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT). In our previous work, the potential of attenuated P. aeruginosa strains as the carriers for anti-tumor vaccination purpose has been reported. In this work, we would like to strengthen P. aeruginosa T3SS based vaccine vectors and direct the development of these bacterial vectors toward clinical applications. First, the performance of these bacterial vectors has been improved in different murine cancer models by: 1) adding one CD4+ Th epitope to vectors; 2) applying bi-antigen expression pattern to vectors; 3) constructing potential humoral response inducing vectors. Second, the therapeutic performance of bacterial vector has been optimized by modulating injection frequency and interval and then be confirmed in murine tumor models. Third, one clinically applicable candidate has been generated by adapting one totally avirulent P. aeruginosa mutant (CHA-OAL) in a chemically defined medium and the poor infectivity of this new strain has been overcome by vaccinations at multiple loci. Fourth, the potential of bacterial vector for human immunotherapy has been further evaluated in one first level humanized mice (HHD) model. Finally, we observed that the pre-existing anti-vector immunity didn't impair the vaccination efficiency of bacteria vector. Taken together, our results highlight the potentials of our live attenuated P. aeruginosa vectors for applications in relevant clinical trials.

Epithelial ovarian cancer is the sixth most common tumor disease among women and it is the leading cause of death from all types of gynecologic malignancies. The current standart of care consist of debulking surgery followed by platinum-taxane chemotherapy. Althought some patients benefit from the treatment, most eventually experience platinum-resistance and die from this disease. Immunotherapy based on application of immune checkpoint blockers represents a new treatment strategy in different cancer malignancies. However, emerging clinical data show only limited clinical efficacy of these agents in ovarian cancer patients with objective response rates of 10-15%. Therefore there is a strong need to identify a potential biomarkers, which allows to identify the group of patients, who will benefit the most from this costly treatment. The aim of my diploma thesis was to characterize the prognostic and predictive role of the immune checkpoints within the retrospective and prospective cohort of patients with high-grade serous ovarian cancer (HGSOC). Our study follows, that the expression of PD-L1 molecule and high frequencies of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor microenviroment is significantly correlated with a better prognosis of patients with HGSOC. Moreover, PD-L1 and PD-1...

Bladder carcinoma (BCa) is among the most common carcinomas in the Western world. Despite the availability of effective therapies, there is currently an urgent need to develop a stratification method, which would enable the accurate identification of patients responsive to therapy. In the theoretical part of my diploma project I describe the heterogeneity of BCa and the currently applied immunotherapeutic approaches. I specifically focused on the Bacillus Calmette-Guérin (BCG) vaccine instillation. For decades another use of BCG has been a prophylactic vaccination against tuberculosis (TB) infection. BCG serves as a model treatment because it is highly efficient when prescribed to the responsive patient. However, an effective stratification is yet to be developed for BCa and latent tuberculosis infection (LTBI) diagnosis and/or monitoring. In the experimental part of my project, I developed and tested a 10-parameter panel for T cell- specific activation test (TAT) applicable for a stratification of BCa patients as well as for the detection of LTBI. I tested the panel on positive controls using flow cytometry (FCM) method because it allows for detection and measurement of dozens of markers at a single cell level. It is easily applicable to available urine and blood samples obtained from BCa...