Rozprawy doktorskie na temat „MDSC”

L’oncologie actuelle est encore confrontée à la résistance et à la progression rapide des cancers. Les mécanismes de résistance intrinsèque développés par les cellules tumorales peuvent compromettre l’efficacité des chimiothérapies et des immunothérapies. Il est maintenant admis que l’état de la réponse immunitaire de l’hôte détermine en partie l’issue thérapeutique des patients. L’objectif de notre équipe de recherche est donc de caractériser cette réponse et d’étudier l’impact des thérapies conventionnelles sur celle-ci dans le but d’identifier les mécanismes liés à un échappement futur de la tumeur. Dans ce contexte, nous avons montré qu’une chimiothérapie (5-FU, oxaliplatine, anti-VEGF (« Vascular Endothelium Growth Factor » : FOLFOX-bevacizumab) provoque chez certains patients une chute des gMDSC (cellules myéloïdes immunosuppressives granulocytaires) périphériques qui est associée à une meilleure réponse thérapeutique. Comme chez la souris, cet effet sur les gMDSC provoque néanmoins une élévation des Th17, une population pro-angiogénique, qui limite l’efficacité de la chimiothérapie. La suite de notre travail a eu pour objectif de tester l’effet « anti-Th17 » de l’activation de l’histone désacétylase SIRT1. SIRT1 est une enzyme capable de perturber l’acétylation de STAT3, un facteur essentiel à la différenciation des Th17. Nous avons montré que l’utilisation d’agonistes pharmacologiques de SIRT1 (resvératrol, SRT1720, metformine) inhibe la polarisation des Th17 par la désacétylation de STAT3 et que cet effet permet de limiter la croissance tumorale dans un modèle de cancer colique et de mélanome chez la souris (B16F10, CT26). Nous avons validé ce concept chez l’homme, ce qui suggère qu’il est possible de cibler les Th17 par cette stratégie en complément de la chimiothérapie. Le dernier volet de ce travail est consacré à la comparaison du profil immunologique périphérique de volontaires sains à celui d’une cohorte prospective de cancers bronchiques non à petites cellules. Cette étude nous a permis de mettre en lumière les altérations immunitaires induites par la tumeur et de lier ces altérations à la réponse au nivolumab (anti-PD-1). Un premier modèle prédictif de réponse a pu être généré grâce aux données d’un panel d’analyse des cellules myéloïdes. Ce modèle révèle une fois encore que les cellules gMDSC ont un rôle prédictif défavorable, alors que les populations présentatrices d’antigènes (cellules dendritiques et monocytes) exprimant PD-L1 ont un bon rôle prédictif. Les données présentées dans cette partie sont préliminaires et devront être confirmées avec la cohorte de validation qui est en cours d’inclusion. L’ensemble de ce travail a permis de montrer qu’il est essentiel de cibler spécifiquement les cellules myéloïdes immunosuppressives et les Th17 pour favoriser l’efficacité des chimiothérapies et de l’immunothérapie dans le cancer
Actual oncology is still facing resistance and rapid progression of cancer. Intrinsic resistance mechanisms developed by tumor cells determine chemotherapy and immunotherapy efficacy. It is now recognized that the host immune response status is in part implicated in the therapeutic outcome of patients. The aim of our research team is to characterize this response and to study the impact of therapies in order to identify the mechanisms associated with future exhaust of the tumor. In this context, we have shown that chemotherapy (5-FU, oxaliplatin, anti-VEGF: FOLFOX-bevacizumab) in some patients causes a drop in devices gMDSC (granulocytic myeloid derived suppressive cells) that is associated with better therapeutic response. Nevertheless, as in mice, this effect on gMDSC causes an elevation of Th17, a pro-angiogenic population, which limits the effectiveness of chemotherapy. The result of our work was aimed to test the effect "anti-Th17" activating SIRT1 deacetylase histone. SIRT1 is an enzyme capable of disrupting the acetylation of STAT3, a key factor in the differentiation of Th17. We have shown that by using pharmacological agonists SIRT1 (resveratrol, SRT1720, metformin) inhibits Th17 polarization by deacetylation of STAT3 and that this effect can limit tumor growth in colorectal and melanoma murine models (B16F10, CT26). We validated this concept in humans, suggesting that it is possible to target Th17 cells by this strategy in addition to chemotherapy. The final component of this work is devoted to the comparison of peripheral immunological profile of healthy volunteers to a prospective cohort of non-small cell lung cancer. This study has allowed us to highlight the immune alterations induced by the tumor and to link these changes in response to nivolumab (anti-PD-1). A first response predictive model could be generated using data from a panel analysis of myeloid cells. This model proves once again that gMDSC have a negative predictive role, while antigen presenting (dendritic cells and monocytes) expressing PD-L1 has a good predictive role. Data presented in this section are preliminary and must be confirmed with the validation cohort that is currently included. All of this work has shown that it is essential to specifically target immunosuppressive myeloid cells and Th17 to promote the efficacy of chemotherapy and immunotherapy in cancer

SUMMARY Tumor can activate a complex network of negative control of the immune response, inducing immunological tolerance. Antitumor chemotherapy causes immunosuppression but also favours activation of immune effectors by either triggering immunogenic cancer cell death or removing immunosoppressive constraints established in the host by growing tumors. In this work we demonstrated that the antimetabolite 5-fluoruracil can reduce for a long time the number of myeloid derived suppressor cells (MDSCs) residing in the spleen. This chemothrapeutic drug does not affect directly cancer cells but perturb a biological niche shared by central memory CD8+ T cells and a population of tumor-induced, actively proliferating, immunosuppressive myeloid cells. Depletion of this niche by surgical removal completely abrogates tumor-induced tolerance. Moreover, we began to characterize the effects of ATP on the suppressive function of MDSCs; in particularly we focused on the ATP effects mediated by P2 purinergic receptors. The preliminary data that we obtained in this study open a new prospective on a different set of metabolites that can determine the functional properties of MDSCs. This is of particular interest in cancer therapies since the extra-cellular concentration of ATP at the tumor site is abnormally elevated.
RIASSUNTO Le cellule tumorali sono in grado di modulare la reattività del sistema immunitario mediante un insieme di processi che regolano negativamente la risposta immunitaria. Questo processo è meglio noto come tolleranza immunologica. La chemioterapia antitumorale causa immunosoppressione ma favorisce anche l’attivazione di cellule effettrici del sistema immunitario sia, promuovendo la morte immunogenica delle cellule tumorali, che riducendo una componente cellulare fondamentale nella deregolazione della risposta immune indotta dal tumore. In questo lavoro abbiamo mostrato come il farmaco antimetabolita 5-fluorouracile induca un riduzione della popolazione mieloide soppressoria residente nella milza, attraverso una duratura azione che non influenza direttamente le cellule tumorali e che dipende integralmente dalla perturbazione della nicchia biologica condivisa dalle cellule CD8+ T central memory (TCM) e da un popolazione di cellule mieloidi attivate dal tumore, altamente proliferanti e dotate di una potente azione immunosoppressiva. L’eliminazione di questa nicchia mediante rimozione chirurgica della milza abroga completamente la tolleranza immunitaria indotta dal tumore. Inoltre, allo scopo di studiare i complessi meccanismi molecolari responsabili della funzione soppressoria delle MDSC, abbiamo caratterizzato la risposta purinergica P2-mediata indotta da ATP nelle cellule mieloidi, gettando le basi per una futura e approfondita indagine degli effetti mediati da questo messaggero extracellulare presente in elevate concentrazioni nel microambiente tumorale.

Les Myeloid-Derived Suppressor Cells (MDSC) sont une population hétérogène de cellules myéloïdes immatures, regroupées en deux sous-populations : les monocytiques-MDSC (M-MDSC) et les polymorphonucléaires-MDSC (PMN-MDSC). Ces cellules ont des capacités immunosuppressives et peuvent exprimer le ligand PD-L1 induisant l’anergie des lymphocytes T qui expriment le marqueur PD-1. Au cours du sepsis, divers bouleversements immunologiques surviennent, et la fonction majeure des MDSC est probablement de réguler l’hyper-inflammation en participant à l’état d’immunodépression rencontré chez les patients. Ceux-ci ont alors un risque de développer des infections secondaires, et de réactiver des virus jusque-là en latence. Notre étude a pour objectifs de mettre en évidence l’origine des MDSC dans le sepsis, et d’approfondir leurs rôles dans l’état d’immunosuppression, notamment dans la réactivation du Torque Teno Virus (TTV). Nos résultats montrent tant ex vivo qu’in vitro, que dans le sepsis, les MDSC sont produites par la moelle osseuse, sous l’influence du G-CSF et de l’IL-6. Ces cellules exprimant PD-L1, sont augmentées dans le sang très tôt dans le sepsis et persistes au cours de l’hospitalisation. L’augmentation de la charge virale du TTV est observée dans le sang périphérique des patients, mais n’est pas corrélée à la fréquence des MDSC. Ces résultats suggèrent que lors d’un sepsis, l’orage cytokinique stimule la production de MDSC exprimant PD-L1 par la moelle osseuse, qui une fois en périphérie, vont participer à l’immunosuppression générale
Myeloid-Derived Suppressor Cells (MDSC) are a heterogeneous population of immature myeloid cell, and are regrouped in two subsets: the monocytic-MDSC (M-MDSC) and the polymorphonuclear-MDSC (PMN-MDSC). These cells have immunosuppressive capacities and mainly act on T cells. MDSC can express the ligand PD-L1 and induce PD-1 expressing-T cells exhaustion. During sepsis, several immunological changes occur, and MDSC probably downregulate the hyper-inflammatory state, contributing to the immunosuppression phase encountered in patients after a sepsis. Immunocompromised patients can develop secondary infections, and reactivate latent virus. The aims of our study were to highlight the origin of MDSC in sepsis, and to explore their roles in the immunosuppression state, especially in the Torque Teno Virus (TTV) reactivation. Our results show, both ex vivo and in vitro, that in sepsis, MDSC originate from bone marrow are induced by G-CSF and IL-6. These PD-L1 expressing-cells are increased in peripheral blood very early in sepsis, and persist during hospitalization. These MDSC are able to inhibit T cells in vitro. The increase of TTV viral load is observed in peripheral blood of patients but is not correlated with MDSC frequencies. These results suggest that during sepsis, the cytokine storm boosts PD-L1 expressing MDSC’s production by bone marrow, which contribute in peripheral blood to the immunosuppression

The ability of the immune system to avoid self-reactivity and autoimmune diseases is achieved by central and peripheral tolerance. Immune tolerance is susteined by different population, one of which is represented by myeloid-derived suppressor cells (MDSC). MDSC are a heterogeneous population of cells consisting of myeloid progenitor cells and immature myeloid cells that are able to suppress both adaptative and innate immunity. In pathological conditions, such as cancer, various infectious diseases and autoimmune diseases, the release of soluble factors induce the increase of myelopoiesis and a partial block of the maturation of myeloid cells. These soluble factors are also believed to be involved in the mobilization and activation of MDSC. MDSC was found in tumour-bearing mice but also in cancer patients. While the phenotype of murine MDSC is well defined by specific markers, like Gr-1, CD11b e IL4Rα, the immunophenotype of human MDSC is still not well defined. In recent years, both in tumour-bearing mice and in cancer patients, MDSC was identified with granulocytes or monocytes features. In this work we demonstrated that human MDSC can be induced in vitro by cytokine treatment of myeloid progenitors. Our data show that the combination of G-CSF and GM-CSF induce the accumulation of immature myeloid cells (BM-MDSC) with features resembling those we identified in the blood of cancer patients. We also demonstrated that BM-MDSC are able to suppress T cells proliferation and that this suppression is accompanied by the downregulation of CD3ζ and CD3ε chains and by the reduction of IFN-γ secreted by activated lymphocyte. Since BM-MDSCs consist of a heterogeneous population, we focused our research to identify the subpopulation of BM-MDSC characterized by the suppressive activity. We demonstrated that human MDSC are immature cells blocked in a particular stage of differentiation. This cells, identifided by the phenotype CD16-/CD11blow/-, showed morphological features of ex-vivo-bone marrow isolated promyelocytes but, unlike these, they are smaller, less grainy, and are able to suppress T lymphocyte proliferation also by CD3ε downregulation. We also observed that the suppressive subpopulation is composed by other two fractions that distinguish each other by a different emission in the red wavelenght, a different grain and a different ability to suppress T lymphocyte proliferation. In the second section of this work we focused in the evaluation of chemotherapy on the accumulation and the functionality of MDSC, one of the main population involved in tumor immunosuppression and in the failure of immunotherapy. Our data showed that, in analogy to the results obtained in mice, 5-fluorouracil is able to remove the suppressive effect of BM-MDSCs on T lymphocyte proliferation. These observation allow us to suggest the use of chemotherapy like adiuvant in immunotherapy approaches.
Il sistema immunitario è in grado di bloccare il riconoscimento degli antigeni self e quindi lo sviluppo di risposte autoimmunitarie mediante il mantenimento della tolleranza immunitaria. Tale fenomeno viene sostenuto dall’azione di diverse popolazioni cellulari tolerogeniche, una delle quali è rappresentata cellule soppressorie di derivazione mieloide (MDSC). Le MDSC sono una popolazione molto eterogenea composta da cellule mieloidi immature caratterizzate dalla capacità di sopprimere sia la risposta immunitaria innata che quella adattativa. È stato ipotizzato che in condizioni patologiche, quali infezioni, malattie autoimmunitarie e neoplasie, il rilascio di diversi fattori di crescita induca l’aumento della mielopoiesi ed il blocco maturativo delle cellule mieloidi che si accumulano in uno stato di immaturità. Tali fattori solubili sono inoltre ritenuti coinvolti nella mobilizzazione e nell’attivazione delle MDSC. Le MDSC sono state identificate sia nel modello murino che in pazienti affetti da tumore. Mentre il fenotipo delle MDSC murine è facilmente identificabile mediante l’uso di marcatori specifici (Gr-1, CD11b e IL4Rα), le caratteristiche immunofenotipiche delle MDSC umane non sono ancora state definite. Nonostante questo, sia nel modello murino che nei pazienti affetti da tumore, le MDSC si sono dimostrate avere caratteristiche talvolta granulocitarie e talvolta monocitarie. In questo lavoro abbiamo dimostrato che è possibile indurre, in vitro, la generazione di MDSC umane a partire da precursori midollari coltivati in presenza di diversi fattori solubili, tra i quali il G-CSF ed il GM-CSF. Il nostro studio ha dimostrato che l’uso combinato di G-CSF e di GM-CSF permette di indurre l’accumulo di cellule mieloidi immature (BM-MDSC) con caratteristiche simili a quelle da noi identificate nel sangue di pazienti affetti da tumore. Le BM-MDSC sono in grado di inibire la proliferazione di linfociti T attivati sia con mitogeni che con allo-antigeni. Inoltre abbiamo dimostrato che la soppressione mediata dalle BM-MDSC è associata sia alla diminuzione dell’espressione delle catene ζ ed ε del CD3 che alla riduzione della produzione di IFN-γ secreto dagli stessi linfociti T. Considerando l’elevata eterogeneità delle BM-MDSC, in questo lavoro abbiamo cercato di identificare quali sottopopolazioni di BM-MDSC fossero responsabili dall’attività soppressoria. Mediante esperimenti di sorting cellulare abbiamo dimostrato, che le BM-MDSC sono cellule immature ascrivibili ad un preciso stadio di differenziazione. Queste cellule, definite dal fenotipo CD16-/CD11blow/-, presentano infatti caratteristiche morfologiche simili ai promielociti isolati dal midollo ex-vivo ma, a differenza di queste, sono caratterizzate da una minore granulosità, da maggiori dimensioni e dalla capacità si sopprimere la proliferazione linfocitaria accompagnata anche dalla diminuzione dell’espressione della catena ε del CD3 espressa sulla superficie dei linfociti T. Abbiamo inoltre evidenziato l’eterogeneità di questa frazione dimostrando che, al suo interno, si possono identificare altre due sottopopolazioni caratterizzate da una diversa fluorescenza nella lunghezza d’onda del rosso, da una diversa granulosità e da una diversa attività soppressoria. Nella seconda parte del lavoro ci siamo concentrati sulla valutazione degli effetti della chemioterapia sull’accumulo e sulla funzionalità delle MDSC, una delle principali popolazioni cellulari coinvolte nell’immunosoppressione associata ai tumori e responsabili dell’inefficacia delle terapie immunoterapiche. I dati da noi ottenuti dimostrano che, come osservato nel modello murino, l’aggiunta del 5-fluorouracile a basse dosi è in grado di indurre l’eliminazione dell’attività soppressoria delle BM-MDSC nei confronti dei linfociti T. Queste osservazioni permettono di suggerire il possibile utilizzo di alcuni chemioterapici come adiuvanti nei trattamenti immunoterapici, in quanto in grado di eliminare una delle popolazioni coinvolte nell’immunosoppressione associata ai tumori.

Le système immunitaire joue un double rôle dans le cancer : il peut non seulement supprimer la croissance tumorale en détruisant les cellules cancéreuses, mais aussi promouvoir la progression de la tumeur en sélectionnant les cellules tumorales ou en créant un microenvironnement tumoral immunosuppresseur. Notre idée principale est de développer des stratégies pour mieux comprendre l'immunologie du cancer colique.Au cours de ma thèse, je me suis tout d'abord intéressée à une population du système immunitaire : les MDSC (Myeloïd Derived Suppressor Cells). Nous avons exploré des stratégies pour réduire le nombre de ces cellules au cours de la croissance tumorale. Nous avons pu découvrir que de petites doses de 5 fluorouracil sont capables d'induire spécifiquement une mort par apoptose des cellules myéloïdes suppressives. Nous avons ainsi caractérisé un effet immunologique positif nouveau du 5-fluorouracil. Cet effet immunologique contribue à l'effet antitumoral du 5-fluorouracil chez la souris. Dans une deuxième partie nous avons étudié le rôle pronostic des infiltrats immunitaires dans une série de patients présentant un cancer du côlon avec des métastases hépatiques. Nous avons étudié le rôle pronostic des infiltrats en cellules CD8, CD45R0 et Foxp3. Nous avons mis en évidence que la présence d'un fort infiltrat en cellules CD45RO et Foxp3 est un facteur de bon pronostic. L'association des 2 marqueurs permet de définir 3 groupes pronostics et ainsi d'individualiser un groupe de mauvais pronostic ne bénéficiant probablement pas de la chirurgie hépatique.

Mast cells respond to a variety of signals, are associated with both increased inflammation and regulation of the immune response, and are able to interact with a variety of hematopoietic and non-hematopoietic cells. The majority of the work that highlights mast cell pleiotropic abilities has been completed in murine models. Though these models have significantly advanced our understanding of what mast cells can do, they cannot inform us as to what mast cells actually do in human beings. The goal of this dissertation is to assess fully mature, primary human mast cell function beyond the well-defined type 1 hypersensitivity function and place mature human mast cells in the context of interactions with other immune cells. The first project addresses the ability of IFNγ, a historically Th1 associated cytokine, to dramatically alter mast cell phenotype. In particular, IFNγ stimulation allows mast cells to act as antigen presenting cells to CD4+ T cells. The second project describes and addresses the T cell suppressive function of myeloid derived suppressor cells in Mastocytosis, a disease of clonal mast cells.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that expands during cancer, inflammation and infection and are potent inhibitors of T-cell-mediated antitumor immunity. MDSC accumulate in the blood, lymph nodes and bone marrow and at tumor sites in most patients and experimental animals with cancer and inhibit both adaptative and innate immunity. Expansion, mobilization and activaction of MDSC is driven by tumors-secreted growth factors, and by a profound alteration of myelopoiesis. In cancer patients the nature of MDSC is still poorly defined since evidence exists for both monocytic and granulocytic features. In the present study we evaluated the phenotype and the suppressive activity of leukocyte subsets freshly isolated from the blood of melanoma and colon cancer patients. Our results indicate that cells with characteristics of MDSC can be found in both mononuclear and polymorphonuclear fraction, and that a useful marker for their identification is the alpha chain of IL4R. Subsequently, we defined growth factors able to generate MDSC in vitro from human bone marrow precursors and to use these cells to characterize better the biology and phenotype of human MDSC. We demonstrated that combinations of some cytokine, such as G-CSF, GM-CSF and IL-6 induce the expansion of bone-marrow immature myeloid populations (BM-MDSC) expressing IL4Ra, with phenotype and inhibitory activity comparable to patients' MDSC. Functional assays revealed that only cytokine-treated bone marrow cells are able to suppress lymphocyte proliferation, while ex-vivo isolated cells and untreated bone marrow cells do not interfere significantly with T lymphocyte proliferation. BM-MDSC suppress activation of both alloactivated and mitogen activated T lymphocytes. We further examined BM-MDSC mechanisms of suppression, and we demonstrated that these cells are able to suppress lymphocyte proliferation by decreasing lymphocyte CD3z chain expression and that suppression requires cell-to-cell contact. Immunoregulatory activity of BM-MDSC is dependent on C/EBPb transcription factor, a key component of the emergency myelopoiesis, because the knock down of C/EBPb leads to a marked decrease in the immunosuppressive activity of BM-MDSC. Since BM-derived MDSC consist of a heterogeneous myeloid population, we separated myeloid fractions with immunomagnetic sorting and we demonstrated that the Lineage negative fraction of BM-MDSC contains the main immunosuppressive activity by inducing a decrease of lymphocyte proliferation and of CD3z expression.
Le cellule soppressorie di derivazione mieloide (MDSC) costituiscono una popolazione molto eterogenea che viene espansa in alcune condizioni patologiche, come le neoplasie, le infiammazioni e le infezioni ed ha la capacità di inibire potentemente l'immunità antitumorale mediata dai linfociti T. Le MDSC si accumulano nel sangue, nei linfonodi, nel midollo osseo e nel microambiente tumorale, in pazienti ed in modelli animali portatori di tumore e inibiscono sia la risposta immunitaria innata, che quella adattativa. L'espansione, la mobilizzazione e l'attivazione delle MDSC è indotta da fattori di crescita rilasciati della cellule tumorali e da un'estesa alterazione della mielopoiesi. In pazienti portatori di tumore la caratterizzazione fenotipica e funzionale delle MDSC non è ancora completamente definita, ma esistono evidenze sia sulla natura granulocitaria che su quella monocitaria di tale popolazione. In questo lavoro di tesi abbiamo valutato il fenotipo e l'attività soppressoria di popolazioni leucocitarie isolate dal sangue periferico di pazienti affetti da tumore del colon e melanoma. I nostri risultati indicano che cellule con caratteristiche delle MDSC possono essere trovate sia nella frazione monocitaria che in quella granulocitaria e che un marcatore utile per la loro identificazione è la catena alpha del recettore dell'interleuchina 4 (IL4Ra). Nella seconda parte di questo lavoro abbiamo definito i fattori di crescita necessari per l'induzione in vitro di MDSC da progenitori mieloidi midollari, ed abbiamo usato tali cellule per caratterizzare la biologia ed il fenotipo delle MDSC. Abbiamo dimostrato che le combinazioni di alcune citochine, come G-CSF, GM-CSF e IL-6 inducono l'espansione di popolazioni mieloidi immature midollari (BM-MDSC) che esprimono IL4Ra e dotate di caratteristiche fenotipiche e funzionali simili a quelle delle MDSC espanse nei pazienti. I saggi funzionali hanno mostrato che solo le cellule trattate con le citochine sono in grado di inibire la proliferazione linfocitaria, mentre le cellule isolate ex-vivo o le colture mantenute in vitro senza l'aggiunta dei fattori di crescita, non interferiscono in modo significativo con la proliferazione linfocitaria. Le MDSC derivate da midollo (BM-MDSC) sopprimono sia la proliferazione dei linfociti attivati da alloantigeni, che la proliferazione di linfociti attivati da mitogeni. Abbiamo quindi analizzato i meccanismi di soppressione delle BM-MDSC ed abbiamo dimostrato che tali cellule sono in grado di sopprimere la proliferazione linfocitaria inducendo una diminuzione dell'espressione della catena z del CD3 e che la soppressione mediata dalle BM-MDSC richiede il contatto cellula-cellula. L'attività immunoregolatrice delle BM-MDSC dipende dal fattore di trascrizione C/EBPb, un componente chiave della granulopoiesi di emergenza, dal momento che la diminuzione della proteina induce una marcata inibizione dell'attività soppressoria delle BM-MDSC. Infine abbiamo separato alcune sottopopolazioni mieloidi presenti nelle BM-MDSC, poichè queste sono rappresentate da una popolazione mieloide eterogenea, ed abbiamo dimostrato che la frazione cellulare Lineage- è responsabile della maggiore attività immunosoppressoria inducendo una diminuzione della proliferazione linfocitaria e dell'espressione del CD3z di superficie.

Selon une étude précédente, une limitation à l'efficacité anticancéreuse du 5-Fluorouracile (5-FU) repose sur la sécrétion d'IL-1β par des cellules myéloïdes immunosuppressives (MDSC). La libération d'IL-1β mature provient de l'activation de NLRP3 induite par le 5- FU et de l’augmentation de l’activité de la caspase-1 dans les MDSC, qui favorise la reprise de la croissance tumorale chez des souris traitées avec 5-FU. L'acide docosahexaénoïque (DHA) appartient à la famille des acides gras oméga-3 et possède des propriétés anticancéreuses et anti-inflammatoires qui pourraient améliorer la chimiothérapie à base de 5-FU. Dans ces travaux, nous démontrons que le DHA inhibe la sécrétion d'IL 1β induite par le 5 FU dans une lignée cellulaire de MDSC (MSC-2). Chez des souris porteuses de tumeurs traitées par 5 FU, nous avons montré qu'un régime alimentaire enrichi en DHA réduit la concentration d'IL 1β circulante et la récidive tumorale après une injection de 5 FU. Le traitement par 5 FU conduit à l'activation de JNK dans les MDSC et l'inhibiteur de JNK SP600125 diminue la sécrétion d’IL-1β. De plus, le DHA est capable de contrecarrer l'activation de JNK induite par 5-FU dans les MDSC, entraînant la chute de la libération de l’IL 1β. De plus, nous avons montré que la supplémentation en DHA dans les MDSC exposées au 5 FU diminuait l’activité de la caspase-1 ainsi que la modification des interactions entre NLRP3 et la caspase-1, ASC ou β-arrestine-2. De manière inattendue, la régulation de l'activité de la caspase-1 par le DHA était indépendante de JNK, ce qui suggère que le DHA pourrait contrôler la sécrétion de l’IL 1β par le biais de l'inflammasome NLRP3 et de la voie JNK. Enfin, nous avons trouvé une corrélation négative entre la teneur en DHA dans le plasma et l'induction du niveau d'IL 1β ou de la caspase-1 dans le sang de patients traités par chimiothérapie à base de 5-FU.L’ensemble de ces données fournissent de nouvelles informations sur la régulation de la sécrétion de l’IL-1β par le DHA et son bénéfice potentiel dans la chimiothérapie à base de 5-FU
A limitation to 5-Fluorouracil (5-FU) anti-cancer efficacy relies on the secretion of IL-1β by myeloid-derived suppressor cells (MDSC) according to a previous pre-clinical report. The release of mature IL-1β originates from 5 FU mediated NLRP3 activation with increased caspase-1 activity in MDSC and sustains tumor growth recovery in 5 FU treated mice. Docosahexaenoic acid (DHA) belongs to omega-3 fatty acid family and harbors both anti cancer and anti inflammatory properties which might could improve 5 FU chemotherapy. Here, we demonstrate that DHA inhibits 5 FU induced IL 1β secretion produced by a MDSC cell line (MSC-2). In tumor-bearing mice treated with 5 FU, we showed that a DHA enriched diet reduces circulating IL 1β concentration and tumor recurrence after 5 FU injection. 5 FU treatment led to JNK activation in MDSC and JNK inhibitor SP600125 decreased IL 1β secretion. Moreover, DHA was able to counteract 5 FU mediated JNK activation in MDSC leading to the drop of IL 1β release. In addition, we showed that DHA supplementation in 5 FU exposed MDSC decreases caspase-1 activity along with a modification of the interactions between NLRP3 and caspase-1, ASC or β arrestin-2. Unexpectedly, the regulation of caspase-1 activity by DHA was independent of JNK which suggests that DHA could control IL 1β secretion through both NLRP3 inflammasome and JNK pathway. Interestingly, we found a negative correlation between DHA content in plasma and the induction of circulating IL 1β level or caspase-1 activity in patients treated with 5 FU based chemotherapy.Together, these data provide new insights on the regulation of IL 1β secretion by DHA and its potential benefit in 5-FU based chemotherapy

The interactions between the immune system and the tumor cells occur through complex events that lead to tumor eradication or immune evasion by cancer. Recently, the prognostic role of Myeloid derived suppressor cells (MDSC) accumulation has been documented for some hematological malignancies where they correlates with disease progression and persistence of minimal residual disease. We first evaluated the change of MDSC frequency in hematological patients during therapy founding a significant correlation between the number of persistent monocytic-MDSC and major molecular response (MMR) value in chronic myeloid leukemia patients treated with dasatinib. Moreover, our data demonstrated that tumor cells, through the release of soluble factors and exosomes, are able to expand monocytic-MDSC, creating an immunotolerant environment that results in T cell anergy and facilitates tumor growth. In addition, cancer cells are also able to promote immune dysfunction in MSC with their consequent commitment, via TLR4 signaling, toward an activated status promoting immune escape through the polarization of neutrophils in immunosuppressive cells.

Myeloid derived suppressor cells (MDSCs) are heterogeneous population of immature cells at various stages of differentiation, characterized by the presence of CD11b and Gr1 in mice. They are major contributors of the tumor-induced immune suppression against the tumors. So far, various strategies have been introduced to overcome the endogenous MDSCs. Most of these approaches rely on the elimination of MDSCs and it is not clear whether tumor-reactive T cells may be differentiated towards phenotypes that are refractory to MDSCc. Our laboratory has previously shown that high affinity T cells derived from tumor-sensitized wild-type FVB mice and expanded ex vivo with the alternating common gamma chain cytokine formulation (initiation of culture with IL-7 + IL-15 followed by one day pulse with IL-2 and continuation of culture with IL-7 + IL-15) can successfully induce tumor regression in FVBN202 transgenic mouse model of breast carcinoma upon adoptive immunotherapy (AIT), only when combined with the depletion of endogenous MDSCs. In this study we have introduced a novel formulation of the sequential common gamma chain cytokines (initiation of culture with IL-7 + IL-15 followed by the expansion with IL-2 until 6 days) for the ex vivo expansion of the autologous and tumor-sensitized low affinity T cells derived from FVBN202 mice and further used for AIT. This novel formulation induced differentiation of tumor-reactive CD8+ T cells mainly towards effector and effector/memory phenotypes that were refractory to MDSCs in vitro and in vivo. AIT by using these T cells induced rejection of primary neu positive tumors and generated long-term memory responses against the recall tumor challenge. Importantly, these T cells also resulted in the inhibition of neu antigen negative relapsed tumor cells. Our findings in the present study provide a platform for AIT of breast cancer patients. .

Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play an essential role in the immunosuppressive networks that contribute to tumor immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross-regulation between MDSC and Treg remain incompletely defined. The current work evaluates the influence of MDSC, expanded in two mouse cancer models, on immunosuppressive Treg. We demonstrate that tumor-induced MDSC endowed with the potential of suppressing conventional T lymphocytes surprisingly impair TGF-β1-mediated generation of induced Treg (iTreg) from naïve CD4⁺ T lymphocytes. Suppression of iTreg generation by MDSC occurs early in the differentiation process, and is cell contact dependent. This inhibition of FoxP3-expressing T lymphocyte differentiation by MDSC does not depend on arginase 1, cystine/cysteine depletion, iNOS/NO, or PD-1/PD-L1 signaling. These findings therefore indicate that MDSC from tumor-bearing hosts have the heretofore unreported ability to restrict some immunosuppressive Treg subpopulations.

SUMMARY OF THE STUDY The World Health Organization (WHO) estimates that approximately 3% of the global population is chronically infected with Hepatitis C Virus (HCV) and that approximately 3-4 million new cases of hepatitis C occur each year worldwide. While African countries have the highest prevalence of HCV infection (up to 26%) in the world, however, HCV infection represents a global health challenge from which no country, rich or poor, is spared. The acute phase of HCV infection is asymptomatic in the majority of infected individuals (75-80%), and except few cases of acute hepatitis C followed by viral clearance, in approximately 80% of patients the virus establishes a chronic infection and, among these patients, about 20% develop cirrhosis with possible degeneration in hepatocellular carcinoma (HCC) in 1-5% of cases. At present, although numerous candidates have been pursued, there is no vaccine available to prevent HCV infection and, even if antiviral drugs are the choice of treatment, HCV infection indeed represents a major health problem worldwide. New generation of highly effective interferon-free, direct acting antivirals (DAAs) therapies have been recently introduced in the clinical practice promising to cure HCV and to overcome the issues related to interferon-based therapies. DAAs have revolutionized the care of HCV-infected individuals due to their dramatically high cure rate, above 90%. Nonetheless, recent reports describe the presence of occult HCV infection in some patients, and the occurrence and recurrence of HCC, despite sustained virological response (SVR) after treatment with DAAs (Koutsoudakis G.et al., 2017; Elamarsy S. et al., 2017; Vukotic R. et al., 2017; Reig M. et al., 2016). In addition, the emergence of drug resistance and suboptimal activity of DAA-based therapies against different HCV genotypes have been observed, causing treatment failure and hampering the control of HCV spread globally (Pawlotsky J.M.et al., 2016; Gimeno-Ballester V. et al., 2017). For all these reasons, and considering also that a previous HCV cleared infection does not ensure prevention from re-infection, at present it is unclear if HCV eradication worldwide will be achieved with DAAs therapies alone or with the combination with immunotherapies. Mechanisms regulating viral clearance or establishment of chronic infection and disease progression have been clarified only partially and several questions are still open (Manns M.P. et al., 2017). During HCV chronic infection, HCV-specific CD8+ T lymphocytes are present in the liver, but these cells are not able to control the replication of HCV, because they have lost their antiviral effector functions, such as cytokine production, proliferation and cytolytic activity. Recent hypothesis is that the loss of function of T lymphocytes may be due to both the liver microenvironment and other cell populations such as CD4+ regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs) exerting inhibitory functions and favoring viral escape and disease progression. MDSCs have been well described in multiple severe human diseases such as cancer, autoimmune diseases, and infections but little is known on their role in HCV infection. A hallmark feature of persistent HCV infection is chronic immune activation and dysfunction of several types of immune cells, including naïve and memory CD4+ and CD8+ T cells, which have been linked to perturbation of anti-viral and anti-tumoral immune responses. Besides, HCV may exert direct effects on B and T lymphocytes and accelerate T cell immune senescence, as the presence and replication of HCV in these cells has been reported, contributing to viral persistence and impairing overall immune responses and vaccination against other infectious agents. Therefore, the global dysregulation of the immune system caused by HCV infection, in addition to affect HCV clearance itself, may be deleterious in terms of response to other infectious agents and tumor onset. In this context, how DAA treatments influences immune responses and immune activation, and whether effective inhibition of HCV replication by DAAs restores defective innate and adaptive immune responses in HCV chronically infected patients are unclear and require further investigation. Recent evidence indicate that, in patients with SVR after interferon-free DAA treatments, HCV clearance was associated with improved blood HCV specific immunity (Spaan M. et al., 2016; Serti E. et al., 2015; Larrubia J.R. et al., 2015; Burchill M.A. et al., 2015). However, contradictory results have been reported for MDSCs and other recent studies indicate that DAA-induced HCV clearance does not completely restore the altered cytokines profile in T lymphocytes and CD4+ Treg cells frequency and activation status (Hengst J. et al., 2016; Langhans B. et al., 2017), implying that HCV cure does not lead to complete immune reconstitution and that regulatory cells may play a role in progression of liver disease even long-term after HCV cure. This issue is of crucial interest in the development of strategies aimed at eradicating HCV infection. Indeed, the incomplete reconstitution of HCV-specific and non-specific immune responses even after DAAs treatment may lead to the occult HCV infection and the development of HCC despite SVR (Koutsoudakis G. et al., 2017; Elamarsy S. et al., 2017; Vukotic R. et al., 2017; Reig M. et al., 2016). To gain further insights into the activity of DAAs on the immune dysfunction, the main objective of this study is to evaluate the capacity of DAA treatments of restoring immune functions, focusing on features of cellular responses known to be affected by HCV infection and/or to be crucial for the effectiveness of adaptive immune responses, such as: 1) the evaluation of the presence, frequency and function of suppressive regulatory cells, including MDSCs. I have focused my attention on M-MDSCs as other reports already showed an increase of this monocytical population in patients infected by HCV, while the effects of HCV antiviral DAA-based treatments on frequency and phenotypes of these cells remain unknown; 2) the phenotype of different CD4+ and CD8+ T cell subpopulations, including evaluation of chronic immune activation, exhaustion, and differentiation, and the presence of Treg, that in other contexts have been shown to be affected by chronic immune activation (Maue A.C. et al., 2009; Papagno L. et al., 2004; Sforza F. et al., 2014); and 3) some metabolic properties of different CD4+ and CD8+ T cell subpopulations. T cell metabolism drives lymphocyte functionality, and may be affected by chronic infections (Dimeloe S. et al., 2016). However, no data are available for HCV infection. Finally, since in the last years several studies demonstrated the regulatory role of microRNAs (miRNAs) in gene expression and their implication in HCV replication and in MDSCs expansion, I have also analysed the expression profile of miRNA-122, miRNA-196b, miRNA-21 and miRNA-29a (known to play a role in HCV replication and in the expansion of myeloid progenitors) as possible biological markers in peripheral blood of selected HCV infected patients under different therapies or untreated. For the purpose of this study I have enrolled a total of 262 HCV-chronically infected patients, grouped in: 1) untreated (n=75); 2) during different pharmacological therapies (n=70) (IFN-based n=10, and IFN-free n=60); 3) with cleared infection after the end of pharmacological therapy (n=115) (IFN-based n=38, and IFN-free n=77); 4) patients who have spontaneously cleared HCV infection (n=2) and 5) healthy controls (n=47). The main results of the study demonstrates that M-MDSCs are deeply altered by HCV infection both quantitatively and qualitatively, and that this is part of a more general phenomenon of HCV-induced immune dysregulation involving also CD4+ and CD8+ T cell subsets. In addition, the results indicate that DAA-based therapy only partially, and slowly, restores these phenomena.
RIASSUNTO L'Organizzazione mondiale della sanità (OMS) stima che circa il 3% della popolazione mondiale sia cronicamente infettata dal virus dell'epatite C (HCV) e che ogni anno nel mondo si verifichino circa 3-4 milioni di nuovi casi di epatite C. Mentre i paesi africani hanno la più alta prevalenza di infezione da HCV (fino al 26%) nel mondo, tuttavia, l'infezione da HCV rappresenta una sfida sanitaria globale dalla quale nessun paese, ricco o povero, è risparmiato. La fase acuta dell'infezione da HCV è asintomatica nella maggior parte dei soggetti infetti (75-80%) e, tranne alcuni casi di epatite C acuta seguita da clearance virale, in circa l'80% dei pazienti il virus determina un'infezione cronica e, tra questi pazienti, circa il 20% sviluppa cirrosi con possibile degenerazione nell’epatocarcinoma (HCC) nell'1-5% dei casi. Allo stato attuale, sebbene siano stati perseguiti numerosi candidati, non esiste un vaccino disponibile per prevenire l'infezione e, anche se i farmaci antivirali rappresentano la miglior scelta, l'infezione da HCV rappresenta davvero un grave problema in tutto il mondo. La nuova generazione di terapie anti-antivirali ad azione diretta, senza interferone (DAAs) è stata recentemente introdotta nella pratica clinica che promette di curare l'HCV e di superare i problemi relativi alle terapie basate sull'interferone. I DAAs hanno rivoluzionato la cura degli individui con infezione da HCV a causa del loro tasso di guarigione estremamente alto, superiore al 90%. Nondimeno, rapporti recenti descrivono la presenza di infezione occulta da HCV in alcuni pazienti e l'insorgenza e la recidiva di HCC, nonostante la risposta virologica sostenuta (SVR) dopo il trattamento con DAA (Koutsoudakis G.et al., 2017; Elamarsy S. et al. , 2017; Vukotic R. et al., 2017; Reig M. et al., 2016). Inoltre, è stata osservata l'insorgenza della resistenza ai farmaci e dell'attività sub-ottimale delle terapie basate su DAA contro diversi genotipi dell'HCV, causando fallimento del trattamento e ostacolando il controllo dell'HCV diffuso a livello globale (Pawlotsky JMet al., 2016; Gimeno-Ballester V. et al., 2017). Per tutti questi motivi, e considerando anche che una precedente guarigione dall'HCV non garantisce la prevenzione dalla re-infezione, al momento non è chiaro se l'eradicazione dell'HCV a livello mondiale sarà raggiunta con le terapie DAAs da sole o con l'associazione con le immunoterapie. Meccanismi che regolano la clearance virale o l'instaurarsi di infezioni croniche e la progressione della malattia sono stati chiariti solo in parte e molte domande sono ancora aperte (Manns M.P. et al., 2017). Durante l'infezione cronica da HCV, i linfociti T CD8 + HCV specifici sono presenti nel fegato, ma queste cellule non sono in grado di controllare la replicazione dell'HCV, poiché hanno perso le loro funzioni antivirali, come la produzione di citochine, la proliferazione e l'attività citolitica. L'ipotesi recente è che la perdita di funzione dei linfociti T possa essere dovuta sia al microambiente epatico che ad altre popolazioni cellulari come le cellule T regolatorie CD4 + (Tregs) o le cellule soppressorie derivate da mieloidi (MDSCs) che esercitano funzioni inibitorie e favoriscono la fuga e la malattia virali progressione. Le MDSCs sono state ben descritte in molte malattie umane gravi come il cancro, le malattie autoimmuni e le infezioni, ma si sa poco sul loro ruolo nell'infezione da HCV. Una caratteristica distintiva dell'infezione persistente da HCV, è l'attivazione e la disfunzione immunitaria cronica di diversi tipi di cellule immunitarie, comprese le cellule naïve e CD4 + e CD8 + di memoria, che sono state collegate alla perturbazione delle risposte immunitarie anti-virali e anti-tumorali. Inoltre, l'HCV può esercitare effetti diretti sui linfociti B e T e accelerare la senescenza immunitaria delle cellule T, poiché è stata segnalata la presenza e la replicazione del virus in queste cellule, contribuendo alla persistenza virale e compromettendo le risposte immunitarie e la vaccinazione contro altri agenti infettivi. Pertanto, la disregolazione del sistema immunitario causata dall'infezione da HCV, oltre a influire sulla stessa eradicazione del virus, può essere deleterio in termini di risposta ad altri agenti infettivi e insorgenza di tumore. In questo contesto, sono andata a valutare se i trattamenti con DAA influenzano le risposte immunitarie e l'attivazione immunitaria; e se l'effettiva inibizione della replicazione dell'HCV da parte dei DAA ripristina le risposte immunitarie innate e adattive nei pazienti con infezione cronica da HCV. Recenti lavori indicano che, in pazienti con SVR dopo trattamenti con DAA, la clearance dell'HCV era associata a una migliore immunità specifica per HCV (Spaan M. et al., 2016; Serti E. et al., 2015; Larrubia JR et al. , 2015; Burchill MA et al., 2015). Tuttavia, sono stati riportati risultati contraddittori per MDSCs. Altri recenti studi indicano che la clearance dell'HCV indotta da DAA non ripristina completamente il profilo alterato delle citochine nei linfociti T e nella frequenza delle cellule CD4+ Treg e nello stato di attivazione (Hengst J. et al., 2016; Langhans B. et al., 2017), il che implica che la cura dell'HCV non porta a un completo ripristino immunitario e che le cellule regolatrici possono svolgere un ruolo nella progressione della malattia epatica anche a lungo termine dopo la cura. Questo problema è di fondamentale interesse nello sviluppo di strategie volte a eradicare l'infezione da HCV. In effetti, la ricostituzione incompleta delle risposte immunitarie specifiche e non specifiche dell'HCV anche dopo il trattamento con DAA può portare a un'infezione occulta da HCV e allo sviluppo di HCC nonostante SVR (Koutsoudakis G. et al., 2017; Elamarsy S. Per ottenere ulteriori informazioni sull'attività dei DAA nella disfunzione immunitaria, l'obiettivo principale di questo studio è valutare la capacità dei trattamenti DAA di ripristinare le funzioni immunitarie, concentrandosi sulle caratteristiche delle risposte cellulari note per essere affette da infezione da HCV e/o essere cruciali per l'efficacia delle risposte immunitarie adattive, come ad esempio: 1) la valutazione della presenza, della frequenza e della funzione delle cellule regolatorie soppressive, comprese le MDSCs. Ho focalizzato la mia attenzione su M-MDSCs poiché altri studi mostravano già un aumento di questa popolazione monocitica in pazienti infetti da HCV, mentre gli effetti dei trattamenti antivirali basati su DAA su antivirali su frequenza e fenotipi di queste cellule rimangono sconosciuti; 2) il fenotipo di diverse sottopopolazioni di cellule T CD4+ e CD8+, compresa la valutazione dell'attivazione cronica, dell'exhaustion, della differenziazione e la presenza di Treg. (Maue AC et al ., 2009; Papagno L. et al., 2004; Sforza F. et al., 2014); e 3) alcune proprietà metaboliche di diverse sottopopolazioni di cellule T CD4+ e CD8+. Il metabolismo delle cellule T guida la funzionalità dei linfociti e può essere influenzato da infezioni croniche (Dimeloe S. et al., 2016). Tuttavia, non sono disponibili dati per l'infezione da HCV.et al., 2017; Vukotic R. et al., 2017; Reig M. et al., 2016). Infine, poiché negli ultimi anni diversi studi hanno dimostrato il ruolo regolatore dei microRNA (miRNA) nell'espressione genica e la loro implicazione nella replicazione dell'HCV e nell'espansione dei MDSC, ho anche analizzato il profilo di espressione di miRNA-122, miRNA-196b, miRNA- 21 e miRNA-29a (noto per svolgere un ruolo nella replicazione dell'HCV e nell'espansione dei progenitori mieloidi) come possibili marcatori biologici nel sangue periferico di pazienti con infezione da HCV selezionati sottoposti a terapie diverse o non trattati. Ai fini di questo studio, ho arruolato un totale di 262 pazienti con infezione cronica da HCV, raggruppati in: 1) non trattati (n = 75); 2) durante diverse terapie farmacologiche (n = 70) (n = 10 basato su IFN e n = 60 senza IFN); 3) con infezione risolta dopo terapia farmacologica (n = 115) (basato su IFN n = 38 e senza IFN n = 77); 4) pazienti che hanno spontaneamente eliminato l'infezione da HCV (n = 2) e 5) controlli sani (n = 47). I principali risultati dello studio dimostrano che la frequenza delle M-MDSCs è profondamente alterata dall'infezione sia quantitativamente che qualitativamente e che questo, fa parte di un fenomeno più generale della disregolazione immunitaria indotta da HCV che coinvolge anche i sottogruppi di cellule T CD4+ e CD8+. Inoltre, i risultati indicano che la terapia basata su DAA solo parzialmente, e lentamente, ripristina questa situazione di perturbazione immunitaria.

MDSCs (myeloid derived suppressor cells) are one of the most important immunoregulatory populations involved in the generation of a permissive environment allowing tumor escape, progression and spreading. In physiologic conditions MDSCs protect the organism from exacerbated immune responses in order to protect surrounding tissues from damage. During tumor growth, cancer cells produce TDSFs (tumor derived soluble factors), which induce MDSC accumulation and their acquisition of a persistent suppressive activity. Among the several mechanisms exploited by MDSCs to exert their suppressive function, L-arginine metabolism seems to be fundamental for the alteration of the tumor microenvironment and for the generation of T effector cell dysfunctions. This work highlights the in vivo requirement for ARG1 and NOS2 in the biology of MDSCs during tumor development. MDSCs are known for their phenotypic and functional complexity. It is clear that many factors can shape their identity and function: here we show, indeed that tumor microenvironment can “educate” them progressively. We illustrate that ARG1 and NOS2 are fundamental for MDSC-mediated T cell suppression in vivo but also for the expansion of the suppressive subsets of MDSCs. L-arginine metabolism mediated by these enzymes is crucial for tumor spreading and metastasis, as depletion of either enzyme produces a 50-75% reduction in the number of lung metastases. Finally, we show that the effects mediated by the novel drug AT38 are mediated by ARG1/NOS2 reduction and sufficient to improve ACT (adoptive cell therapy) therapeutic approaches. Data here produced represent a body of evidence that could be exploited for improving the efficiency of anti-cancer therapies, and identifies ARG1 and NOS2 as key targets for restoring T cell mediated tumor responses.
Le MDSC rappresentano un’importante popolazione immunoregolatoria coinvolta nella generazione di un microambiente tumorale permissivo, in grado di favorire l’instaurarsi, la crescita e la diffusione del tumore. Le MDSC sono fisiologicamente presenti nell’organismo dove, in caso di infezione o infiammazione si espandono transitoriamente allo scopo di limitare risposte immunitarie eccessive e quindi di evitare danni ai tessuti. Durante la crescita del tumore, le cellule tumorali secernono fattori solubili in grado di indurre l’accumulo delle MDSC e l’acquisizione del loro fenotipo soppressivo. Tra i diversi meccanismi utilizzati dalle MDSC per svolgere le loro funzioni, il metabolismo dell’aminoacido L-arginina sembra avere un ruolo fondamentale nell’alterazione del microambiente tumorale e nella generazione di linfociti T effettori incapaci di generare un’adeguata risposta anti-tumorale. In questo lavoro è stato studiato il coinvolgimento degli enzimi ARG1 e NOS2 nell’attività delle MDSC durante lo sviluppo tumorale. Le MDSC sono note per la loro complessità sia fenotipica che funzionale ed ormai è chiaro che queste caratteristiche possono essere plasmate da molti stimoli differenti. In questo lavoro, infatti, è stato dimostrato che durante la sua crescita il tumore è in grado di “educare” le MDSC. Abbiamo inoltre mostrato che ARG1 e NOS2 sono fondamentali non solo per la soppressione in vivo dei linfociti T ma anche per l’ espansione delle MDSC. Risulta inoltre evidente come il metabolismo di L-arginina mediato da ARG1 e NOS2 sia cruciale nella per la diffusione delle cellule tumorali e per il processo metastatico. Infatti la deplezione di anche uno solo di questi due enzimi provoca una riduzione del 50-75% nel numero di metastasi polmonari. Infine, abbiamo dimostrato come l’effetto mediato da un nuovo farmaco, AT38, sia dovuto, almeno in parte, alla riduzione di ARG1 e NOS2 e come questo sia sufficiente a migliorare l’efficacia di approcci terapeutici, quali il trasferimento adottivo. I dati riportati in questa tesi rappresentano quindi una solida evidenza del fatto che ARG1 e NOS2 possano rappresentare un rilevante bersaglio terapeutico in grado di aumentare l’efficacia delle terapie anti-tumorali.

ABSTRACT Although there is ample evidence linking chronic inflammation with cancer, the cellular mechanisms involved in early events leading to tumor development remain unclear. Myeloid cells are an intricate part of inflammation. They consist of mature cells represented by macrophages, dendritic cells and granulocytes and a population of Immature Myeloid Cells (IMC), which in healthy individuals are cells in transition to mature cells. There is a substantial expansion of IMC in cancer and many other pathological conditions which is associated with pathologic activation of these cells. As a result, these cells acquire the ability to suppress immune responses and are termed Myeloid-derived Suppressor Cells (MDSCs). Although the role of MDSC in immune suppression in cancer and tumor progression is well established, their contribution to tumor development is still uncertain. The fact that cells with MDSC phenotype and function are observed in chronic inflammation raised the possibility that these cells can contribute to initial stages of tumor development. To address this question, we used an experimental system where the number of IMC was regulated by the expression of S100A9 protein. In this project, we used two different models of chronic inflammation in S100A9 transgenic (S100A9tg) and S100A9 knock-out (S100A9KO) mice. In the first model, we created the conditions for topical accumulation of these cells in the skin in the absence of infection or tissue damage using S100A9tg mice. Accumulation of IMC in the skin resulted in a dramatic increase in the formation of skin tumors during epidermal carcinogenesis. Conversely, lack of myeloid cell accumulation in S100A9KO mice substantially reduced the formation of skin papillomas. The effect of IMC was not associated with immune suppression but with the recruitment of CD4+ T cells mediated by CCL4 chemokine released by activated IMC. Elimination of CD4+ T cells or blockade of CCL4 abrogated the increase in tumor formation caused by myeloid cells. Thus, this study implicates the accumulation of IMC as an initial step in facilitating of tumor formation, which can mediate the recruitment of CD4+ T cells via the release of CCL4 chemokine. In the second model, we used inflammation-associated lung cancer caused by the chemical lung carcinogen urethane in combination with exposure to cigarette smoke referred to throughout as CS. Exposure of mice to CS alone resulted in a significant accumulation of cells with typical MDSC phenotype in different organs; however, these cells lacked immune suppressive activity and could not be defined as bona fide MDSC. When CS was combined with the single dose of urethane, it led to the accumulation of immune suppressive cells. The expansion of MDSC followed the onset of lung tumors development. This suggests that MDSC in this model is not the preceding factor but rather a consequence of tumor formation. Further studies are necessary to determine the relevance of targeting these cells for cancer treatment and prevention.

Polymer/clay nanocomposites consisting of an epoxy resin matrix filled with organoclays have been investigated. The main objective of this study was to determine which combination of components led to the greatest enhancement in properties of the epoxy resin. Exfoliation of the clay was desired, as exfoliated nanocomposites are known to exhibit great improvements in mechanical properties [1]. The epoxy resins studied were di-functional DGEBA and tetra-functional TGDDM. The epoxy resin was cured with three different hardeners, these included: the high functionality amine hardener, TETA, and two anhydride hardeners, accelerated MTHPA and pure HHPA. The three organoclays used, contained alkylammonium cations and were also compared to the unmodified clay. Morphology was investigated by XRD and TEM, and the flexural properties of the resulting nanocomposites were studied. The effect that the addition of an organoclay has on the cure of the epoxy resin was investigated using MDSC. Both the temperatures required to cure the resin with, and without, the clay, and any changes in the total heat flow that occurred were studied. The Tg�++ of the cured nanocomposites was also measured using MDSC. The heat flow results indicated that the clays added to the epoxy resins act as a physical barrier, which prevents the resin from reaching full cure. In the higher functional resin, the addition of clay resulted in a significant decrease in the total heat flow, suggesting that a large amount of epoxy remains uncured, and, as a result, there should be a reduction in the amount of cross-linking. The lower cross-link density led to a significantly lower Tg and the mechanical properties were also poorer. The reactivity of the hardener towards the resin was found to have the greatest impact on the cured nanocomposite morphology. Intragallery polymerisation occurring at a faster rate than the extragallery polymerisation causes exfoliation. In order to achieve a balance that favours intragallery polymerisation, it was found that the curing reaction was required to be catalysed by the alkylammonium cation of the organoclay, and not catalysed by other means. The DGEBA cured with HHPA provided the largest layer expansion in the clay structure due to the alkylammonium cation catalysing the anhydride ring-opening reaction. The effect was not seen with TGDDM due to the tertiary amine in its structure. The accelerator within the MTHPA assisted extragallery polymerisation of the resin and the TETA cured readily without additional catalysis.

Polymer/clay nanocomposites consisting of an epoxy resin matrix filled with organoclays have been investigated. The main objective of this study was to determine which combination of components led to the greatest enhancement in properties of the epoxy resin. Exfoliation of the clay was desired, as exfoliated nanocomposites are known to exhibit great improvements in mechanical properties [1]. The epoxy resins studied were di-functional DGEBA and tetra-functional TGDDM. The epoxy resin was cured with three different hardeners, these included: the high functionality amine hardener, TETA, and two anhydride hardeners, accelerated MTHPA and pure HHPA. The three organoclays used, contained alkylammonium cations and were also compared to the unmodified clay. Morphology was investigated by XRD and TEM, and the flexural properties of the resulting nanocomposites were studied. The effect that the addition of an organoclay has on the cure of the epoxy resin was investigated using MDSC. Both the temperatures required to cure the resin with, and without, the clay, and any changes in the total heat flow that occurred were studied. The Tg++ of the cured nanocomposites was also measured using MDSC. The heat flow results indicated that the clays added to the epoxy resins act as a physical barrier, which prevents the resin from reaching full cure. In the higher functional resin, the addition of clay resulted in a significant decrease in the total heat flow, suggesting that a large amount of epoxy remains uncured, and, as a result, there should be a reduction in the amount of cross-linking. The lower cross-link density led to a significantly lower Tg and the mechanical properties were also poorer. The reactivity of the hardener towards the resin was found to have the greatest impact on the cured nanocomposite morphology. Intragallery polymerisation occurring at a faster rate than the extragallery polymerisation causes exfoliation. In order to achieve a balance that favours intragallery polymerisation, it was found that the curing reaction was required to be catalysed by the alkylammonium cation of the organoclay, and not catalysed by other means. The DGEBA cured with HHPA provided the largest layer expansion in the clay structure due to the alkylammonium cation catalysing the anhydride ring-opening reaction. The effect was not seen with TGDDM due to the tertiary amine in its structure. The accelerator within the MTHPA assisted extragallery polymerisation of the resin and the TETA cured readily without additional catalysis.

Mit der Entwicklung und Einführung ökologischer Bauweise im Neubau sowie neuen Baustoffsystemen in Sandwichbauweise wird es zunehmend erforderlich, neue effektive Befestigungsvarianten zu entwickeln, die eine dauerhafte Fixierung auch unter sicherheitstechnischen Bestimmungen sowie aus Garantie- bzw. haftungsrechtlichen Gründen ermöglichen. Die aus der Praxis bisher bekannten chemischen Befestigungssysteme (Zweikomponentenverbundmörtel, Verbundankerpatronen) weisen hinsichtlich der Applikation unter bautechnischen Bedingungen noch einige Nachteile auf. Dazu gehören vor allem längere Aushärtungszeiten zur Realisierung der abschließenden Verbundfestigkeit, Inhomogenitäten im Verbund, der Einsatz toxischer Verbindungen und eine Limitierung der Applikationsmöglichkeiten in horizontalen und Überkopf-Einsatzbereichen sowie Hohlkammersystemen. Alle zuvor genannten Punkte haben bis jetzt die Nutzung solcher Verbundwerkstoffe als universale Anwendungsmöglichkeit verhindert. Ein neues chemisches Befestigungssystem, welches aus Novolak gehärteten mit Hexamethylentetramin (Hexa) und anorganischen Füllstoff besteht, wurde für Applikationen in Beton entwickelt. Das Bindemittel härtet bei der Temperaturzuführung aus. Die unkatalysierte Befestigungsmasse zeigt bei einer Temperatur zwischen 150-300 °C eine hohe Reaktivität. Die Vorteile dieses Systems sind die unbegrenzte Lagerfähigkeit der vorgemischten härtbaren Masse sowie die Gewährleistung einer homogenen Netzwerkstruktur im gesamten Verbund und sie ist frei von giftigen und flüchtigen Substanzen. Auf den Einsatz toxischer Substanzen wurde verzichtet. In dieser Arbeit wurde die Gesamtkinetik der Reaktion während des Aushärtungsprozesses dieser Polymerkomposite untersucht. Die DSC- (nicht-isothermen, isothermen) und MDSC-Untersuchungen haben sich als ein sicheres Verfahren zur Qualitätskontrolle des Aushärtezustands der Befestigungssysteme herausgestellt. Parallel zur nicht-isothermischen und isothermischen DSC wurden Leitfähigkeitsmessungen durchgeführt, um den Endpunkt der Aushärtungsreaktion zu bestimmen
The development and introduction of ecological construction methods and the use of sandwich materials make it necessary to develop new fixing systems and technologies. Dealing with the application in concrete and other substrates commercial chemical fixing systems show some disadvantages up to date. Especially the rather long curing time in order to realize the final bond strength, inhomogenities in the composite, the partial use of toxic substances and application limits of such systems in horizontal direction as well as hollow section materials has so far prevented the use of such composites for all-purpose applications. A new chemical fixing system, which consists of hexamethylene tetramine (hexa) cured novolac and inorganic filler, was developed for application in concrete. It is applied by a thermo-curing procedure. The uncatalyzed curable mixture has a high reactivity at temperature between 150-300 °C. Compared with commercial chemical fixing systems, the premixed curable mass has many benefits. First it has a unique storage stability and second, it is free of toxic and volatile substances. Another important aspect is, it is self-foaming. In this study was investigated the overall kinetics of the reaction during the curing process of these polymer composites. An appropriate method for this experiment proved to be the DSC in isothermal and non-isothermal mode and MDSC. This turned out to be a safe quality control technique for these systems. Parallel to the non-isothermal and isothermal DSC conductivity measurements have been performed to determine the end point of the curing reaction

ADAM10 is a zinc-dependent metalloprotease. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. In this study, we examined the role of ADAM10 in the immune system. Here, we show that knocking out ADAM10 on the mature B2 cell causes a defect in the development of secondary lymphoid architecture that becomes more severe post-immunization. We also show that overexpression of ADAM10 leads to a defect in hematopoiesis, which eliminates B2 lymphocyte development. This defect additionally induces accumulation of myeloid derived suppressor cells, MDSCs. ADAM10Tg MDSCs function synonymous to tumor MDSCs. Of the two subpopulations of MDSCs, granulocytic MDSCs increase parasitic clearance in a model of N. brasiliensis. Monocytic MDSCs are more immunosuppressive in regards to tumor. Both subpopulations are dependent on the presence of mast cells for activity. It is thought that this relationship is mediated through histamine and IL-13. During N. brasiliensis infection, ADAM10Tg mice, lacking B2 B cells but having intact B1 B cells, makes increased IgE over WT mice. This production of IgE is thought to be produced by the B1 cells. Of the two types of B1 cells, B1a cells make the majority of the IgE. This IgE production is enhanced by MDSC accumulation and can be induced by MDSC adoptive transfer in a parasite-free mouse. Lastly, ADAM10 is the key sheddase for CD23 on B2 cells. When IgE is bound to its antigen to form an immune complex, IC, it binds CD23 and is internalized. After CD23 bound to IgE ICs is internalized, it is sorted into bexosomes. These bexosomes are transferred to dendritic cells which are responsible for presenting to T cells and inducing an increased antigen-specific immune response. Overall, ADAM10 is important for many aspects of the immune response.

De nombreuses études ont impliqué des TLR dans le développement et la progression tumorale. Précédemment, il a été démontré que les cellules tumorales expriment TLR7, un récepteur à ARNsb, et qu’une forte expression de TLR7 par les cellules tumorales de patients atteints de cancer du poumon est associée à un mauvais pronostic. Dans un modèle murin de cancer du poumon, cet effet pro-tumoral a été reproduit lors de l’injection d’agoniste de TLR7. Mes travaux de thèse ont eu pour objectif de déterminer les mécanismes impliqués dans les effets pro-tumoraux de TLR7. La stimulation de ce récepteur au niveau des cellules tumorales induit une production plus importante de CCL2 et GM-CSF, ainsi qu’un fort recrutement de MDSC au site de la tumeur. Ces MDSC, de par leurs propriétés immunosuppressives sont responsables de l’effet pro-tumoral à la suite de la stimulation de TLR7. Nous avons également mis évidence que la stimulation de TLR7 était pro-métastatique dans un modèle murin de cancer du poumon et que les MDSC étaient également impliquées dans cet effet. Ces effets pro-métastatiques associés au TLR7 ont été confirmés chez l’homme, grâce à l’étude de transcrits de molécules associées à l’invasion, l’angiogénèse, la transition épithélio-mésenchymateuse et les métastases. Enfin nous avons démontré la présence de ligand de TLR7 chez les patients atteints de cancer du poumon et démontré que l’injection intratumorale de virus respiratoires, IAV et RSV, a un effet pro-tumoral dans notre modèle de cancer du poumon. Ces virus respiratoires pourraient donc être à l’origine de la surexpression de TLR7 et du mauvais pronostic associé à ce récepteur chez les patients atteints de cancer du poumon. Ces recherches ont donc permis de mettre en évidence de nouveaux facteurs aggravants dans le cancer du poumon, dont les virus respiratoires, et de découvrir les mécanismes impliqués
Numerous studies have implicated some TLR in tumor development. Previously, we have demonstrated that lung tumor cells express TLR7, a receptor for ssRNA, and that high TLR7 expression confers to NSCLC patients bad clinical outcome. In mice models of lung cancer, we further demonstrated that the injection of TLR7 agonists led to a pro-tumoral effect.My thesis work has firstly demonstrated the mechanisms involved in the pro-tumoral effects of TLR7 in lung cancer: TLR7 stimulation on tumor cells induces a high production of CCL2 and GM -CSF, as well as a sharp MDSC recruitment within the tumor. These MDSC, by their immunosuppressive properties, are implicated in the pro-tumoral effect upon TLR7 stimulation. We also demonstrated that TLR7 stimulation was pro-metastatic in a mice model of lung cancer and that MDSC were also involved in this effect. These pro-metastatic effects associated with TLR7 have been confirmed in humans through the studies of transcripts and proteins involved in invasion, angiogenesis, Epithelial–mesenchymal transition and metastasis. Finally, we demonstrated that TLR7 ligands are present in tumor microenvironment of lung cancer patients and that intratumoral injection of respiratory viral infections such as IAV and RSV, have a pro-tumoral effect in lung cancer mice model. These respiratory viruses could therefore be at the origin of the overexpression of TLR7 and the poor clinical outcome associated with this receptor in lung cancer patients. This research has thus made possible to highlight new aggravating factors in lung cancer, including respiratory viruses, and to discover the mechanisms involved

Les approches thérapeutiques de la DMD basées sur la transplantation de myoblastes se sont heurtées à un faible taux de survie cellulaire et une dispersion limitée des cellules. L'identification de cellules souches au sein de tissus adultes et la définition de leur potentiel myogénique ont ouvert de nouvelles perspectives. Dans un 1er temps, nous avons utilisé les propriétés d'adhérence des cellules dérivées du muscle afin d'isoler dans un modèle aviaire des cellules progénitrices résidantes du muscle distinctes des myoblastes, les LAC (late adherent cells). En utilisant la technique de préplating, nous avons montré, comme cela avait été démontré chez la souris, qu'une fraction marginale de cellules présente un défaut initial d'adhérence à une matrice collagénique et que celle-ci se compose de cellules immatures ou peu engagées dans le programme myogénique. De plus, nous avons démontré que ce défaut ne peut être attribué à la méthodologie employée et que ces cellules ne sont pas générées in vitro. Dans un 2nd temps, nous avons mis en évidence dans un modèle canin que les propriétés de quiescence, de forte capacité de prolifération, de faible capacité de fusion in vitro, de phénotype et de multipotence faisaient des LAC des cellules souches musculaires : les MDSC (Muscle Derived Stem Cells). Après injection intramusculaire chez le chien GRMD (Golden Retriever Muscular Dystrophy), modèle cliniquement relevant de la DMD, les MDSC participent à la formation de fibres musculaires, permettent une restauration de la dystrophine, et génèrent des cellules satellites. L'ensemble de ces caractéristiques positionne les MDSC comme des candidates intéressantes pour la thérapie de la DMD
Therapeutic approaches for Duchenne Muscular Dystrophy by myoblast transplantation have been hindered by poor survival rates and the limited spread of the injected cells. Stem cell identification in adult tissues and the definition of their myogenic potential have open new prospects. First, we used the muscle-derived cell’s adhesion properties in an avian model to isolate progenitor cells residing in skeletal muscle and that are distinct from myoblasts: the LAC (Late-Adherent Cells). Using the preplating technique, we showed that a marginal cell fraction displays an initial adhesion defect to collagen matrix and that it is composed of cells poorly committed in myogenic program and immature progenitor cells, as this has been previously described in mice model. Also, we demonstrated that this defect could not be attributed to the methodological approach and that the LAC are not generated in vitro by myoblasts. Second, we showed in canine model that the LAC are characterized by an initial quiescent status, a high in vitro proliferation rate as well as a low fusion ability, a phenotype and a multi-lineage differentiation potential that defined them as muscle stem cells: the MDSC (Muscle Derived Stem Cells). After intramuscular injection in dystrophic GRMD (Golden Retriever Muscular Dystrophy) dogs that represent clinically relevant animal model for DMD, we established that MDSC are able to participate in muscle fiber formation, to allow recovery of dystrophin expression and to generate satellite cells. Collectively, these results qualify MDSC as potential candidates for future cell therapy for DMD

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that a disintegrin and metalloproteinase 10 (ADAM10) is a critical regulator of Notch and that its deletion is embryonically lethal, we generated transgenic mice that overexpress ADAM10 at early stages of lymphoid and myeloid development (A10Tg). ADAM10 transgene expression alters hematopoiesis post-hematopoietic Lineage-Sca-1+c-kit+ (LSK) subset differentiation but prior to lineage commitment of progenitor populations. This results in delayed T cell development, abrogated B2 cell development, and dramatic expansion of functionally active myeloid derived suppressor cells (MDSCs) in A10Tg mice. Given ADAM10’s role in Notch signaling, we hypothesized that the observed hematopoietic alterations may be a consequence of perturbed Notch signaling. In fact, blockade of ADAM10 (S2) rescues B cell development and reduces myeloid cells in A10Tg LSKs. Inhibition of γ-secretase (S3) in wild type (WT) LSKs results in enhanced myelopoiesis, mimicking the phenotype of A10Tg mice. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical for hematopoietic cell-fate determination. Albeit arising in a tumor-free host, A10Tg MDSCs are functionally and phenotypically analogous to tumor-derived MDSCs. A10Tg MDSCs inhibit T cell activation in vitro, and inhibit adoptive immunotherapy (AIT) of metastatic melanoma in vivo, which can be reversed with MDSC depletion. Intriguingly, A10Tg mice are resistant to parasitic infection upon inoculation of Nippostrongylus brasiliensis. However, depletion of MDSCs abrogates this response, while adoptive transfer (AT) of MDSCs into WT mice increases their resistance. This polarized activity of MDSCs is heavily dependent upon interaction with mast cells (MCs). In fact, B16 melanoma cells metastasize more rapidly in WT mice infused with MDSCs when compared to MC-deficient mice (Kit Wsh/Wsh), with or without MDSC AT. Parallel to B16 progression, the ability of MDSCs to promote anti-Nb immunity is significantly diminished in MC-deficient (Kit Wsh/Wsh) mice even with MDSC AT. This augmentation of MDSC activity in the presence of MCs is further corroborated by in vitro co-culture assays that demonstrate a synergistic increase in cytokine production. Furthermore, MDSCs preferentially migrate to the liver in a MC-dependent manner. This interaction is mediated by MC-released histamine. In fact, MDSCs express histamine receptors (HR) and histamine induces MDSC survival, proliferation, and activation. We demonstrate that MDSC activity is abrogated with histamine blockade. Moreover, in humans, allergic patients present with an increase in MDSC population, and MDSCs purified from a stage I breast cancer patient exhibit increased survival in the presence of histamine. Taken together, our studies indicate that MCs and MC-released histamine are critical for the observed functional duality of MDSCs, ranging from immunosuppressive to immunosupportive, depending on the disease state.

L’augmentation de l’incidence et l’absence de ressources thérapeutiques pour les formes non opérables de patients atteints d’un cancer HPV+ est un enjeu important. La forte immunosurveillance au sein des tumeurs associées aux HPV ainsi que la présence d’antigènes viraux associée à l’oncogenèse de ces cancers devraient privilégier le développement de stratégies d’immunothérapies comme la vaccination anti tumorale ainsi que le transfert adoptif de TILs. Ces avancées technologiques incitent à mieux comprendre les réponses immunitaires dans ces pathologies et à développer des stratégies combinant les immunothérapies pour le traitement de l’ensemble des cancers liés aux HPV. Nous avons dans un premier temps recherché les antigènes de tumeurs associés au pronostique de patients atteints d’un cancer du canal anal. Les patients ont été traités par une combinaison de chimiothérapies dont son bénéfice thérapeutique a été démontré par notre équipe. Le monitoring des réponses T spécifiques dans le sang périphérique de ces patients a permis de montrer que la Télomérase est un antigène de tumeur associé au SCCA ; et que la présence de LT Th1 spécifiques de la Télomérase est un facteur prédictif de la survie sans progression à 12 mois. Nos données ont également mis en évidence l’influence des M-MDSC sur les réponses immunitaires T spécifiques de nos antigènes ainsi que sur la survie des patients atteints d’un SCCA. Aucun effet des Treg n’a été observé sur les réponses immunitaires et la survie dans cette maladie. Les M-MDSC sont un facteur pronostique de la réponse au traitement des patients atteints d’un SCCA traités par chimiothérapies. Le second travail de cette thèse a permis de valider la faisabilité d’isoler des TILs à partir de prélèvements biopsiques issus de patients atteints d’un cancer HPV+. Les réponses immunitaires T spécifiques des oncoprotéines d’HPV16 sont corrélées dans 50 et 60% des cas entre le sang périphérique et la tumeur. Le dernier objectif de ce travail de thèse a permis de démontrer l’immunogénicité de la protéine SALL4, un facteur de transcription associé à la pluripotence et à l’auto-renouvèlement des cellules souches embryonnaires. Nous avons généré une technologie d’analyse des réponses CD4 Th1 anti-SALL4 permettant le suivi des réponses immunes chez les patients atteints d’un adénocarcinome digestif. Nos travaux ont également permis de montrer une faible fréquence de LT spécifiques de SALL4 dans le sang périphérique de patients atteints d’un SCCA. Ces résultats suggèrent que la présence de réponses spécifiques de SALL4 dans le sang périphérique de patients atteints d’un SCCA pourrait être l’objet d’une immunosurveillance menant à une diminution de la fréquence des LT CD4 SALL4+. Il serait intéressant d’analyser la présence de LT spécifiques de cet antigène dans des formes précoces de cancers HPV+
The increased incidence and the lack of therapeutic resources for non-operable forms of HPV+ cancer patients constitutes a major challenge. High immunosurveillance in HPV-associated tumors and the presence of viral antigens associated with oncogenesis of these cancers should focus on the development of immunotherapy strategies such as anti-tumor vaccination and adoptive transfer of TILs. These technological advances encourage a better understanding of immune responses in these pathologies and aim to develop strategies combining immunotherapies for the treatment of all HPV-related cancers. We first looked for tumor antigens associated with the prognosis of patients with anal canal cancer. The patients were treated with a combination of chemotherapy whose therapeutic benefit was demonstrated by our team. Monitoring specific T-immune responses in the peripheral blood of these patients has shown that telomerase is a tumor antigen associated with SCCA; and moreover that the presence of Telomerase-specific LT Th1 is a predictor of progression-free survival at 12 months. Our data also highlighted the influence of M-MDSC on the T-specific immune responses of our antigens as well as on the survival of patients with SCCA. As a result, M-MDSCs are a prognostic factor in the response to treatment of patients with metastatic SCCA treated with chemotherapy. The second work of this thesis validated the feasibility of isolating TILs from biopsy samples from patients with HPV-associated cancer. The specific T immune responses of HPV16 oncoproteins are correlated in 50 and 60% of cases between peripheral blood and tumor. The final objective of this thesis work demonstrated the immunogenicity of the SALL4 protein, a transcription factor associated with pluripotency and self-renewal of embryonic stem cells. We have generated anti-SALL4 CD4 Th1 response analysis technology to monitor immune responses in patients with digestive adenocarcinoma. Our work has also shown a low frequency of SALL4-specific T responses in the peripheral blood of patients with SCCA. These results suggest that the presence of specific SALL4 responses in the peripheral blood of SCCA patients could be subject to immunomonitoring resulting in a decrease in the frequency of CD4 LT SALL4+. It would be interesting to analyze the presence of specific LT of this antigen in the early forms of HPV-related cancers

Le système immunitaire joue un double rôle dans le cancer: il peut non seulement supprimer la croissance tumorale en détruisant les cellules cancéreuses, mais aussi promouvoir la progression de la tumeur en sélectionnant les cellules tumorales ou en créant un microenvironnement tumoral immunosuppresseur. Au cours de ma thèse, je me suis intéressée à deux populations du système immunitaire : les MDSC (Myeloïd Derived Suppressor Cells) et les lymphocytes Th17. Dans ce travail, nous avons exploré les mécanismes impliqués dans l’activation et dans les fonctions suppressives de ces deux populations cellulaires au cours de la croissance tumorale. Tout d’abord, nous avons montré que les cellules tumorales activaient les MDSC en libérant des exosomes exprimant HSP72 à leur surface. HSP72 à la surface des exosomes permet l’activation de STAT3 via la voie de signalisation TLR2/Myd88 dans les MDSC et favorise leur fonction suppressive. Le facteur de transcription STAT3 joue aussi un rôle fondamental dans la différenciation des lymphocytes Th17. Le rôle des lymphocytes Th17 est controversé dans la croissance tumorale. Nous avons découvert que les lymphocytes Th17 exprimaient les ectonucléotidases CD39 et CD73 soutenant ainsi leur fonction suppressive. L'expression des ectonucléotidases est dépendante de l’activation de STAT3 par l’IL-6 et de l’inhibition de Gfi-1 par le TGFβ lors de leur différenciation
Immune system plays a dual role in cancer: it can not only suppress tumor growth by destroying cancer cells, but also promote tumor progression by selecting immunoresistant tumor cells or by establishing an immunosuppressive microenvironment.During my thesis, I focused on two populations of the immune system: MDSC (myeloid derived suppressor cells) and Th17 lymphocytes. In this work, we explored the mechanisms involved in the activation of suppressive functions of these two cell populations during tumor growth. First, we showed that tumor cells activated MDSC by releasing exosomes that express HSP72 on their surface. HSP72 on exosomes surface induces STAT3 activation via Myd88/TLR2 signaling pathway in MDSC and promotes their suppressive function. The transcription factor STAT3 also plays a fundamental role in Th17 cells differentiation. The role of Th17 cells is controversial in tumor growth. We reported that Th17 cells express CD39 and CD73 ectonucleotidases and thereby supporting their suppressive function. The expression of ectonucleotidases is dependent on the activation of STAT3 by IL-6 and inhibition of Gfi-1 by TGFβ during their differentiation

Cancers induce ‘emergency’ hematopoiesis and expansion of myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), immunosuppressive and tumor-promoting cell populations, correlated with poor prognosis and resistance to chemo-immunotherapies. Molecular characterization of these cells offers new potential therapeutic opportunities. Previously, we showed that nuclear accumulation of p50 NF-kB transcription factor in TAM regulates expression of anti-inflammatory, pro-tumoral genes. Monocytic MDSC share myeloid progenitor and immunosuppressive properties with TAM. Here, we described how p50 NF-kB mediates the protumor functions of M-MDSC. Indeed, during cancer-related inflammation, chronic production of PGE2 induces nuclear p50 accumulation in M-MDSC, epigenetically reprogramming the response to IFNγ towards an immunosuppressive phenotype. Myeloid-specific deletion of p50 or antagonists of PGE2 receptors restore the antitumor inflammatory response to IFNγ of MMDSC. Recently, we identified that RORC1/RORγ drives cancer-related myelopoiesis. RORγ is a nuclear receptor co-activated by cholesterol-related molecules (eg. oxysterols). Of note, hypercholesterolemia predisposes to cancer and induces expansion of immature monocytes. Hence, we hypothesized an interplay between cholesterol metabolism and RORγ-driven myelopoiesis. Noteworthy, while tumor bearers (mice and humans) displayed increased levels of LDL-cholesterol, diet-induced hypercholesterolemia promotes metastasis and expansion of immunosuppressive M-MDSC and TAM. RORγ deficiency prevents hypercholesterolemia-induced myelopoiesis disabling metastasis formation. Collectively, we identified p50 NF-kB as key driver of immunosuppressive M-MDSC in response to tumor-derived PGE2, and we underlined that RORγ induces emergency myelopoiesis remarkably in dyslipidemic conditions. Hence, inhibitors of PGE2/p50 NF-kB and of cholesterol/RORγ axis could be useful in the improvement of anticancer therapy.

Tumor growth is associated with a profound alteration of myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). Analyzing the cytokines affecting myelo-monocytic differentiation produced by various experimental tumors, we found that GM-CSF, G-CSF and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by their ability to impair priming of IFN-gamma-producing CD8+ T cells upon in vivo adoptive transfer. Moreover, adoptive transfer of syngeneic, GM-CSF+IL-6-conditioned MDSCs to diabetic mice transplanted with allogeneic pancreatic islets resulted in long term allograft acceptance and correction of the diabetic status. Cytokines inducing MDSCs acted on a common molecular pathway. Immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on C/EBPbeta transcription factor, a key component of the emergency myelopoiesis triggered by stress and inflammation. Adoptive transfer of tumor antigen-specific CD8+ T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in myeloid compartment. These data unveil another link between inflammation and cancer and identify a novel molecular target to control tumor-induced immune suppression.
La crescita tumorale è associata ad una profonda alterazione della mielopoiesi, che porta al reclutamento di cellule immunosoppressive conosciute come cellule soppressorie di origine mieloide (Myeloid-derived suppressor cells, MDSCs). Analizzando le citochine prodotte da vari tumori sperimentali, e che influenzano la differenziazione mielo-monocitaria, abbiamo trovato che GM-CSF, G-CSF e IL-6 permettono la rapida generazione di MDSC dai precursori presenti nel midollo di topo. Le MDSC derivate in vitro da colture di midollo osseo con la combinazione di citochine GM-CSF+IL-6, possiedono la più forte attività tollerogenica, e sono in grado, dopo trasferimento adottivo in vivo, di limitare l’attivazione dei linfociti T CD8+ secernenti IFN-gamma. Inoltre il trasferimento adottivo di MDSC singeniche, ottenute impiegando le citochine GM-CSF ed IL-6, in topi resi diabetici e trapiantati con insule pancreatiche allogeniche permette l'accettazione del trapianto allogenico a lungo termine e la relativa modificazione dello stato diabetico. Le citochine che inducono MDSC agiscono attraverso una via molecolare comune. L'attività immunoregolatoria sia delle MDSC indotte dal tumore sia di quelle indotte in vitro dal midollo osseo risulta interamente dipendente dal fattore di trascrizione C/EBPbeta, un componente chiave della mielopoiesi di emergenza innescata da stress ed infiammazione. Il trasferimento adottivo di linfociti T CD8+ specifici verso antigeni del tumore garantisce la terapia di tumori stabilizzati solamente nei topi che mancano di C/EBPbeta nel comparto mieloide. Questi dati svelano un ulteriore legame tra infiammazione e cancro e identificano un nuovo bersaglio molecolare per controllare l’immunosoppressione indotta dal tumore.

Aim of this work was to phenotypically and functionally distinguish myeloid-derived suppressor cell (MDSC) subsets and study their cytokine- and microRNA-mediated regulation. In the first part of the work we silenced granulocyte-macrophage colony stimulating factor (GM-CSF) in 4T1 mammary carcinoma line by means of RNA interference (RNAi), highlighting the relevance of this factor in the preferential induction of suppressive myeloid subpopulations and in the control of their maturation and functional properties. By using this in vivo model, we were also able to show that GM-CSF released by cancer cells, in addition to promote myeloid recruitment to the tumor site, is critical for their invasive potential and the induction of neo-angiogenesis, factors that contribute to rapid tumor growth and that are indeed inhibited when the cytokine is silenced. We also developed an in vitro model, by culturing bone marrow (BM) cells with GM-CSF and other hematopoietic cytokines, that allowed us to easily examine the molecular mechanisms driving MDSC subset differentiation. In particular, we explored the role of some microRNA, found to be differentially expressed between non-suppressive myeloid cells and MDSCs, on phenotype and function of the different BM-MDSC subsets obtained in vitro. The microRNA we identified were able to limit the expansion of a monocyte/macrophage suppressive cell fraction and to inhibit the proliferation of BM cultures. From some recent evidences, these microRNA seems to regulate genes involved in myeloid differentiation and hematopoietic homeostasis maintenance.
Lo scopo di questa tesi è stato quello di distinguere sottopopolazioni di cellule mieloidi soppressorie (MDSC) con caratteristiche fenotipiche e funzionali differenti e studiarne la regolazione mediata da citochine o da microRNA (miRNA). Nella prima parte del lavoro è stato messo a punto il silenziamento genico, mediante RNA interference (RNAi), del gene per il granulocyte-macrophage colony stimulating factor (GM-CSF) nella linea tumorale 4T1, che ci ha permesso di evidenziare l’importanza di questo mediatore nell’indurre l’accumulo preferenziale di sottopopolazioni mieloidi con attività soppressiva e nel modularne sia la maturazione che l’attività. L’impiego di questo modello in vivo ci ha inoltre permesso di osservare come il rilascio di GM-CSF da parte del tumore sia cruciale, oltre che per il reclutamento dell’infiltrato mieloide, anche per l’induzione di neo-angiogenesi e per il potenziale invasivo della neoplasia, fattori che concorrono alla rapida crescita tumorale e risultano inibiti dal silenziamento della citochina. Grazie all’utilizzo di un sistema di coltura in vitro di cellule midollari con GM-CSF e altre citochine ematopoietiche, siamo poi stati in grado di studiare agevolmente alcuni aspetti molecolari alla base della maturazione delle diverse sottopopolazioni di MDSC. In particolare, abbiamo esaminato il ruolo di alcuni miRNA, differenzialmente espressi in vivo tra cellule mielodi non soppressive e cellule MDSC di topi portatori di tumori istologicamente differenti, sul differenziamento e la funzione delle varie frazioni cellulari di MDSC inducibili in vitro. I miRNA in esame sono in grado di inibire la formazione della frazione monocito/macrofagica soppressiva e di alterare la proliferazione delle colture midollari. Da recenti evidenze ottenute, questi miRNA sembrano inoltre regolare alcuni geni coinvolti nel differenziamento mieloide e nel mantenimento dell’omeostasi ematopoietica.

The research programme studied the cure reaction of a phenolic novolak resin and the effects of various additives and fillers on the reaction. The programme utilised the recently developed thermal analysis technique of temperature-modulated differential scanning calorimetry (TMDSC) performed in conjunction with other available thermal analysis techniques. TMDSC enables the signal for the heat of reaction to be separated from the underlying specific heat change in the resin. This meant that the reaction could be studied without interference from any physical changes in the resin. The manufacture of composite brake materials required the use of numerous additives and fillers to produce the desired properties. The influence of such additives on the cure rate and final properties of the resin was known to occur but had not previously been measured due to the difficulties presented by the presence of opaque additives. Some additives also underwent thermally induced physical changes in the temperature range of the cure. The final properties and the processing of new brake materials undergoing development often required trial and error adjustments to compensate for changes in cure rate. An understanding of the influence of additives would enable more rapid commercial development of brake materials through an improvement in the ability to predict both the properties of the product and the optimal processing parameters. Processing efficiency could also be improved through detailed knowledge of the kinetics. Moulding cycle times and post-baking times and temperatures were longer than necessary in order to ensure adequate cure at the end of each stage because of the lack of kinetic data. The cure of phenolic resin has been shown to be highly complicated with numerous alternate and competing reactions. For the manufacture of composite materials, knowledge of the kinetic parameters of individual reactions is not considered to be important; rather the overall kinetic parameters are required for prediction. Therefore the kinetic model parameters that best described the observed behaviour were chosen even though the model had no basis in the molecular interaction theory of reaction. Rather it served as a convenient tool for predictions. Characterisation of the resin proved to be difficult due to the presence of overlapping peaks, and volatile reaction products. TMDSC was successfully used to determine the reaction kinetics of the pure resin and the influence of certain additives on the reaction kinetics. The determination of the kinetic parameters using TMDSC agreed well with the traditional Differential Scanning Calorimetry isothermal and non-isothermal techniques. Both the Perkin-Elmer and TA Instruments were utilised for the research and were found to provide reasonably good agreement with each other. The capabilities and limitations of the individual instruments were critically examined, frequently beyond the manufacturers' specifications. TMDSC suffers from a limitation in the heating rate of the sample compared to DSC. However, it was observed that valuable information could still be obtained from TMDSC despite using heating rates that were higher than specified by manufacturers. Hot Stage Microscopy and thermogravimetry were additional experimental techniques used to aid in the characterisation of the resin. Some inhomogeneity of the resin was identified as well as differences in the behaviour of the cure between open (constant pressure) and closed (constant volume) environments were observed. A novel method of determining the orders of the cure reactions and their kinetic parameters was utilised. Reaction models for the overall cure reactions were postulated and tested by fitment to sections of experimental data in temperature regions which appeared to be free of interference from overlapping peaks. Once an individual peak was reasonably well modelled, adjacent overlapping peaks were able to be modelled both individually and in combinations by fitment to experimental data. The Solver function in Microsoft Excel was utilised to find the best fitting model parameters for the experimental data. The model parameters were able to be refined as overlapping peaks were progressively incorporated into the calculations. This method produced results that agreed well with the traditional method of analysing reaction peak temperatures at multiple scanning rates. Model fitment was shown to be of benefit where overlapping reactions occur. Various model scenarios could be tested and optimised to particular sections of experimental data. This enabled the researcher to easily identify areas of possible anomalies and postulate alternative scenarios. The accuracy of the postulated model was able to be determined by its successful fitment to experimental data from experiments run under different conditions.