La plupart des chimiothérapies sont connues pour exercer une immunosuppression en plus de leur effet cytotoxique, car elles ciblent sans distinction les cellules à prolifération rapide telles que les cellules tumorales ainsi que les cellules du système immunitaire. Cependant, la radiothérapie ainsi que certaines chimiothérapies comme les anthracyclines ou l’oxaliplatine ont montré une propriété immunostimulante, grâce à l’induction d’une mort cellulaire dite immunogène. Cette mort cellulaire se caractérise par la libération de signaux de danger par la cellule mourante qui vont activer le système immunitaire. Tout d’abord, l’exposition de la calréticuline à la surface de la cellule va être un signal de phagocytose pour les cellules dendritiques. Les cellules succombant à la mort cellulaire libèrent aussi HMGB1 qui en se liant à TLR4 permet un apprêtement et une présentation des antigènes tumoraux efficace. Ensuite, la libération d’ATP qui agit sur les récepteurs P2RX7 permet l’activation de l’inflammasomme NLRP3 et conduit à la sécrétion d’IL-1β indispensable pour l’activation des lymphocytes T CD8+ sécrétant de l’IFN-γ. L’autophagie est un processus de dégradation permettant de limiter l’instabilité génomique et l’initiation de cancers. L’autophagie, peut être induite après un stress du réticulum endoplasmique, qui est nécessaire à l’exposition de la calréticuline lors de la mort cellulaire immunogène. Nous avons donc cherché à évaluer l’importance de l’autophagie après traitement aux chimiothérapies immunogènes. Nous avons montré que l’autophagie est requise pour induire la libération d’ATP lors de la mort cellulaire immunogène. De plus, nous avons montré que l’ATP libéré par les cellules mourantes après traitement aux chimiothérapies immunogènes permet le recrutement de cellules de type monocyte inflammatoire (CD11b+Ly6ChighLy6G-) ainsi que de leurs précurseurs. En outre, l’ATP est un facteur important dans la différenciation de ces cellules en cellules dendritiques inflammatoires. Les cellules CD11b+Ly6ChighLy6G- ont montré une grande capacité à présenter les antigènes tumoraux aux lymphocytes T CD8+ permettant leur activation. Les cellules déficientes pour l’autophagie n’ont quant à elles pas permit le recrutement de cellules dendritiques dans les tumeurs ni l’activation de lymphocytes T CD8+. Ces travaux ont permit de montrer l’importance de l’autophagie pour mettre en place une réponse immunitaire anti-tumorale spécifique lors du traitement avec des chimiothérapies immunogènes. De plus, nous avons montré que l’ATP est impliqué dans le recrutement et la différenciation de cellules avec un phénotype de monocytes inflammatoires. L’ensemble de ces résultats apporte de nouveaux éléments dans la caractérisation du processus de mort cellulaire immunogène. / Most of the cytotoxic agents used in cancer therapy are known to be immunosuppressive, because of their unspecific targeting of rapidly dividing cells, like tumor cells, but also cells of the immune system. However, radiotherapy, as well as some chemotherapeutic agents such as anthracyclines or oxaliplatine were reported to have immunostimulatory effects, thanks to their capacity to induce immunogenic tumor cell death. This type of cell death is characterized by the release of danger signals from the dying tumor cell, which will activate the immune system. As a first event, exposure of calreticulin from the dying tumor cell will act as an « eat-me » signal for dendritic cells. Once released, the nuclear protein HMGB1 will bind to TLR4, facilitating antigen processing and presentation. The dying tumor cells will also release ATP, which acts on P2X7 receptors and activates NLRP3 inflammasomme, leading to IL-1 release, necessary for IFN-- producing CD8+ T cells activation. Autophagy is a degradation process limiting genomic instability and cancer initiation. It can be induced after a stress of the endoplasmic reticulum (ER). ER-stress is also involved in calreticulin exposure during immunogenic cell death, so we aimed at understanding the role of autophagy in immunogenic cell death. We found that autophagy is required for the release of ATP after treatment with immunogenic chemotherapies. Moreover, we showed that ATP released from the dying cells is necessary for the recruitment of immune cells with inflammatory monocyte phenotype (CD11b+Ly6ChighLy6G-), as well as their precursors. ATP was also important for the differentiation of these inflammatory monocytes into dendritic cells. These CD11b+Ly6ChighLy6G- cells were efficient in presenting the tumor antigens to CD8+ T cells, and to induce a tumor-specific immune response. However, autophagy-deficient cells were not able to recuit dendritic cells or to induce CD8+ T cells activation. These studies showed the importance of autophagy in tumor-specific immune response, after treatment with immunogenic chemotherapies. We also reported that ATP is involved in the recruitment and differentiation of cells with inflammatory monocytes phenotype. Altogether, these results give new insights in the concept of immunogenic cell death.

Dendritic cells (DCs) comprise a distinct lineage of potent antigen-presenting mononuclear phagocytes that serve as both mediators of innate immune responses and key facilitators of the adaptive immune response. DCs play both immunogenic and tolerogenic roles through their dual ability to elicit pathogen-specific T cell immunity as well as induce regulatory T cell (Treg) responses to promote tolerance in the steady state. The aim of the work presented here is to examine the normal regulatory mechanisms of DC development and function, starting with the dissection of mechanisms behind an aberrantly activated developmental pathway, followed by the exploration of new mechanisms governed by two candidate transcription factors. The first chapter of the thesis focuses on the growth factor receptor Flt3, an essential regulator of normal DC development in both mice and humans, and concurrently one of the most commonly mutated proteins found in acute myeloid leukemia (AML). We investigated the effect of its most common activating mutation in AML, the Flt3 internal tandem duplication (Flt3-ITD), and found that this mutation caused a significant cell-intrinsic expansion of all DC populations. This effect was associated with an expansion of Tregs and the ability to dampen self-reactivity, with an inability to control autoimmunity in the absence of Tregs. Thus, we describe a potential mechanism by which leukemia can modulate T cell responses and support Treg expansion indirectly through DCs, which may compromise immunosurveillance and promote leukemogenesis. The subsequent chapters explore the basic molecular mechanisms of DC development by using Flt3 expression as a guide to uncover new candidates involved in the DC transcriptional program. We show that Myc family transcription factor, Mycl1, is largely dispensable for DC development and function, contrary to recent published findings that propose a role in proliferation and T cell priming. On the other hand, we find that conditional deletion of our second candidate gene, an Ets family transcription factor, has diverse effects on DC development, monocyte homeostasis, and cytokine production. Overall, our studies highlight an unexpected molecular link between DC development and leukemogenesis, and elucidate novel mechanisms controlling DC differentiation and function.

A proposta do nosso estudo foi comparar o número de células apresentadoras de antígenos (CAAs) presentes em biópsias de gengivite (G) e de hiperplasia gengival induzida por ciclosporina (HGIC). Vinte e oito biópsias de pacientes com G e 14 biópsias de HGIC foram analisadas no epitélio de revestimento bucal (ERB), epitélio sulcular (ES) e no tecido conjuntivo (TC). O número de macrófagos (CD68+), de células de Langerhans (CD1a+) e de células dendríticas intersticiais (CDIs) (FXIIIA+) foi investigado por técnica imunoistoquímica. Células CD1a+/mm2 foram significantemente aumentadas na G quando comparada com HGIC, no TC, no ES e no ERB (p<0,05). Em contrapartida, o número de células CD68+ no TC e no ERB, e o número de células FXIIIA+/mm2 no TC foram aumentadas no grupo de HGIC (p<0,05). Ciclosporina A está relacionada com a diminuição de células de Langerhans. Podemos sugerir que este fato aumenta as infecções oportunistas, conseqüentemente, um maior número de macrófagos é necessário para combater os microorganismos. / The aim of this study was to compare the number of antigen-presenting cells (APCs) observed in biopsies of gingivitis (G), and in cyclosporine-induced gingival overgrowth (CIGO). Twenty eight biopsies from patients with G, and 14 with CIGO were analyzed in oral epithelium (OE), sulcular/junctional epithelium (SJE), and connective tissue (CT). The number of macrophages (CD68+), Langerhans’cells (CD1a+), and interstitial dendritic cells (FXIIIA+) was investigated by immunohistochemistry. CD1a+ cells/mm2 were significantly increased in G when compared with CIGO, in CT, in SJE, and in OE (p<0.05). In contrast, the number of CD68+ in CT, and in OE, and FXIIIA+ cells/mm2 in CT were increased in CIGO group (p<0.05). Cyclosporine is related with the diminution of Langerhans’ cells. We can suggest that this fact increase opportunistic infections, consequently, a greater number of macrophages is necessary in order to combat the microorganisms.

A proposta do nosso estudo foi comparar o número de células apresentadoras de antígenos (CAAs) presentes em biópsias de gengivite (G) e de hiperplasia gengival induzida por ciclosporina (HGIC). Vinte e oito biópsias de pacientes com G e 14 biópsias de HGIC foram analisadas no epitélio de revestimento bucal (ERB), epitélio sulcular (ES) e no tecido conjuntivo (TC). O número de macrófagos (CD68+), de células de Langerhans (CD1a+) e de células dendríticas intersticiais (CDIs) (FXIIIA+) foi investigado por técnica imunoistoquímica. Células CD1a+/mm2 foram significantemente aumentadas na G quando comparada com HGIC, no TC, no ES e no ERB (p<0,05). Em contrapartida, o número de células CD68+ no TC e no ERB, e o número de células FXIIIA+/mm2 no TC foram aumentadas no grupo de HGIC (p<0,05). Ciclosporina A está relacionada com a diminuição de células de Langerhans. Podemos sugerir que este fato aumenta as infecções oportunistas, conseqüentemente, um maior número de macrófagos é necessário para combater os microorganismos. / The aim of this study was to compare the number of antigen-presenting cells (APCs) observed in biopsies of gingivitis (G), and in cyclosporine-induced gingival overgrowth (CIGO). Twenty eight biopsies from patients with G, and 14 with CIGO were analyzed in oral epithelium (OE), sulcular/junctional epithelium (SJE), and connective tissue (CT). The number of macrophages (CD68+), Langerhans’cells (CD1a+), and interstitial dendritic cells (FXIIIA+) was investigated by immunohistochemistry. CD1a+ cells/mm2 were significantly increased in G when compared with CIGO, in CT, in SJE, and in OE (p<0.05). In contrast, the number of CD68+ in CT, and in OE, and FXIIIA+ cells/mm2 in CT were increased in CIGO group (p<0.05). Cyclosporine is related with the diminution of Langerhans’ cells. We can suggest that this fact increase opportunistic infections, consequently, a greater number of macrophages is necessary in order to combat the microorganisms.

La macroautophagie est un processus catabolique, situé au carrefour entre l’homéostasie et le métabolisme cellulaire. Dans le système immunitaire, elle joue des rôles spécialisés, en contribuant notamment à la régulation de l’inflammation et la présentation antigènique. En utilisant deux modèles murins, nous avons pu démontrer que la macroautophagy est nécessaire à la lignée lymphocytaire B et contribue à l’élaboration d’une réponse humorale optimale. En effet, la macroautophagie contribue à la survie des cellules sécrétrices d’antigènes, notamment la population plasmocytaire, ainsi que des cellules à longue durée de vie, telles que les lymphocytes B mémoire. Nous avons également démontré qu’une forme d’autophagie non-canonique était nécessaire pour la présentation efficace d’antigènes particulaires reconnus par le récepteur des lymphocytes B. Dans ce contexte, la machinerie macroautophagique contribue à la polarisation du cytosquelette des lymphocytes B, afin de former une synapse immunologique, nécessaire au chargement efficace du complexe majeur d’histocompatibilité de classe II, et ainsi, à la présentation antigènique. A l’aide d’un troisième modèle de souris transgénique, nous avons caractérisé un rôle jusqu’alors inconnu de la macroautophagie dans le maintient de l’homéostasie des cellules de Langerhans. L’inhibition de la macroautophagie altère la survie de ces cellules, en les exposant à potentiel stress du réticulum endoplasmique, potentiellement non compensé. En somme, nous avons démontré que la macroautophagie était un acteur majeur au sein des cellules présentatrices d’antigènes. / Macroautophagy is a catabolic process at the crossroad between homeostasis and metabolism. In the immune system it also possesses specialized roles such as inflammation regulation and antigen presentation. Here we demonstrated in two mice models that macroautophagy is integral to B cell lineage for proper humoral responses. Indeed it insures the survival of secreting cells such as plasma cells and long living cells such as memory B cells. We also report that non-canonical autophagy is also needed for an efficient presentation of particulate antigen recognized by the B cell receptor. In this context it drives B cell cytoskeleton polarization to form an immune synapse necessary for the efficient loading of class two major histocompatibility complexes and the subsequent antigen presentation. Using a third mice model we unveiled a yet uncharacterized function of macroautophagy in Langerhans cells, a subset of epidermal dendritic cells, homeostasis. Macroautophagy inhibition impairs their survival by exposing them to a potentially uncompensated endoplasmic reticulum stress response. Altogether we demonstrated that macroautophagy is a major actor in several types of antigen presenting cells.

Airway hyperresponsiveness (AHR) is one of the primary features of allergic airways disease. Despite continuous allergen exposure atopic asthmatics do not develop progressively worsening AHR. The mechanism(s) that limit AHR are unknown. Two valid candidates are regulatory T cells (Treg) and antigen presenting cells (APC). Dendritic cells (DC) are the main APC within the airways. Presentation of allergens to T cells can result in the differentiation and expansion of different subsets of T cells including effector Treg cells. The precise role of Treg and DC in the attenuation of allergen-induced AHR remains unknown. The general aim of this thesis is to investigate mechanisms to limit AHR in a murine model of atopic asthma. Specific aims are to: 1. develop a murine model of allergen-induced attenuation of AHR, 2. determine the potential role of regulatory T cells (Treg) in allergen-induced AHR attenuation, and 3. determine the potential role of airway dendritic cells (DC) in allergen-induced AHR attenuation. Balb/c mice were sensitised with intraperitoneal Ovalbumin (OVA) in aluminium hydroxide and challenged with a single, 3-weeks or 6-weeks of OVA aerosols. Aerosols were 1% OVA in sterile saline delivered for 30 minutes for three days per week. Animals were sacrificed 24 hours after the final aerosol for measurements of lung function and Methacholine (MCh) responsiveness (low-frequency forced oscillation technique), collection of bronchoalveolar lavage fluid (BALF) and serum. '...' In contrast, 6-weeks of OVA challenges decreased Treg numbers back to control levels. Adoptive transfer of 1x106 Treg taken from DLN of 3-week challenged mice attenuated AHR in single-OVA recipients (p<0.05). Furthermore, in vivo depletion of Treg in 3-week OVA challenged mice restored AHR (p<0.05 compared with control). Similar proportions of CD4+ T cells became activated following both aerosol regimes, however total numbers of airway CD4+ T cells were decreased (p<0.05), and OVA-specific CD4+ T cell proliferation in DLN was reduced (p<0.05) after 3-weeks versus one OVA aerosol. Analysis of antigen handling by airway APC populations showed antigen uptake (OVA-647) and processing (DQ-OVA) by macrophages and airway DC subsets to be down-regulated (p<0.05) after 3-weeks of OVA aerosols. In addition, adoptive transfer of Treg into single-OVA recipients did not affect antigen handling by airway APC populations. These data suggest that Treg are responsible for allergen-induced attenuation of AHR in vivo in established airways disease. AHR attenuation was associated with an altered function of airway DC, resulting in reduced antigen capture and processing, leading to limited clonal expansion of antigen-specific CD4+ T cells with limited production of Th2 cytokines. Furthermore, Treg were not directly responsible for the down-regulation of allergen capture in the airways. In conclusion, knowledge of the role of Treg and DC in attenuation of AHR could potentially result in improved and more directed therapies for the attenuation of AHR in atopic asthmatics.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 12, 2007) Vita. Includes bibliographical references.

Graduation date: 1994

In 1995 and 2000 a total of 29 886 non surgical ED visits at Uppsala University Hospital were registered. Presenting complaint, admittance to a ward, length of stay, in-hospital mortality, discharge diagnoses, 30-day and long-term mortality were registered. The presenting complaints were sorted into 33 presenting complaint groups (PCGs). For different PCGs there was different in-hospital fatality rate. Compared to the largest PCG, chest pain, the gender and age adjusted OR was 2.12 (95% CI 1.01 – 4.44) for the miscellaneous complaint group and 2.04 (95 % CI 1.35 – 3.08) for the stroke–like symptom group. Within a given PCG the in-hospital mortality could vary depending on discharge diagnoses. By relating PCG and long term mortality to the expected mortality in the population, the Standardized Mortality Ratio (SMR) could be calculated. The SMR was found to be highest in seizure 2.62 (95 % CI 2.13 – 3.22), intoxication 2.51 (95% CI 2.11-2.98) and symptoms of asthma 1.8 (1.65 – 2.06). For the same discharge diagnoses the long term mortality could differ considerably depending on PCG at ED arrival (p&lt;0.001). Between 1995 and 2000 there was a 30 % increase in ED visits at the non surgical ED. PCGs representing lesser severe conditions had increased. Demographic changes could account for 45 % of the increment and the remaining increase could be ascribed to change in visiting pattern. In the 2000 cohort 41.0 % of all visits were performed by re-visitors. The number of revisits and five-year mortality had an inversed u-shaped relationship were patients with three re-visits within the same year had an increased mortality compared to patients with more or less visits. Conclusion: It is possible to define presenting complaint groups (PCGs) that are robust and consistent over time and useful as a tool for epidemiological studies in the ED.

Diss. Stockholm : Karolinska institutet, 2007.