Дисертації з теми "Suppressive myeloid cells"

Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network that develops during cancer. MDSC down-regulate immune surveillance and antitumor immunity and facilitate tumor growth. The ability of MDSC to suppress T cell responses has been documented; however the mechanisms regulating this suppression remain to be understood. This work proposes a biological dichotomy of MDSC regulated by the tumor microenvironment. In peripheral lymphoid organs MDSC cause T-cell non-responsiveness that is antigen-specific. These MDSC have increased expression of NOX2, enabling them to produce large amounts of reactive oxygen species. Since the transcription factor STAT3 is substantially activated in MDSC, its potential role in upregulation of NOX2 expression was investigated. Over-expression of a constitutively active form of STAT3 increases expression of NOX2 subunits, whereas attenuation of STAT3 activity leads to decreased expression of NOX2. The significance of NOX2 in ROS generation is demonstrated in mice devoid of NOX2 function; NOX2- deficient MDSC are unable to inhibit antigen-induced activation of T cells. In contrast, MDSC within the tumor microenvironment have a diminished potential to generate ROS but acquire expression of arginase and inducible nitric oxide synthase, enzymes plicated in T cell non-responsiveness. Upregulation of these enzymes results in MDSC ability to inhibit lymphocyte response in absence of antigen presentation. The tumor microenvironment also promotes the differentiation of MDSC to tumor associated macrophages. Hypoxia is an exclusive feature to the tumor microenvironment and we investigated its involvement in the properties of MDSC at the tumor site. Exposure of spleen MDSC to hypoxia converts MDSC to non-specific suppressors and induces a preferential differentiation to macrophages. Stabilization of HIF-1!, a transcription factor activated by hypoxia, induces similar changes in MDCS as hypoxic exposure. Finally, ablation of HIF-1! prevents MDSC from acquiring factors that enable the suppression of T cells in absence of antigen. These findings help to expand our understanding of the biology of MDSC and suggest a regulatory pathway of myeloid cell function exclusive to the tumor microenvironment. They may also open new opportunities for therapeutic regulation as we now should take into consideration how systemic location affects the function of MDSC.

The CD3ζ chain is indispensable for coupling antigen recognition to T cell response. During HIV infection, a down-modulation of CD3ζ was found on T cells, contributing to T cell anergy. It has been shown that circulating myeloid derived suppressor cells (MDSC) are elevated in HIV+ patients, and correlate with disease progression. In this work, we studied the correlation between MDSC frequency and T cell CD3ζ expression. Moreover, we investigated the mechanisms of CD3ζ decrease exploited by MDSC. CD3ζ expression and MDSC frequency were evaluated by flow cytometry on PBMC from 105 HIV+ patients. We found that granulocytic-MDSC (Gr-MDSC) were expanded in HIV+ patients compared to healthy donors; in particular, a higher Gr-MDSC frequency was observed in patients with a CD4 T cell count below 400 cells/μl. We found an inverse correlation between the percentage of Gr-MDSC and CD3ζ level. Moreover, in vitro Gr-MDSC depletion induced the up-regulation of CD3ζ in T cells, restoring the functionality of αβ, but not γδ T cells. The in vitro effect of isolated Gr-MDSC on CD3ζ expression was found cell contact-dependent, and was not mediated by previously described molecules. CD3ζ downmodulation corresponds to the decrease of its mRNA induced by silencing the transcription factor ELF-1.

Myeloid-derived suppressor cells (MDSCs) numbers increase significantly in sepsis and are associated with high mortality rates. These myeloid cell precursors promote immunosuppression, especially in the late (post sepsis) stage. However, the mechanisms that underlie MDSC expansion and programming are not completely understood. To investigate these mechanisms, we used a cecal-ligation and puncture (CLP) mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive phase. Previous studies in our laboratory showed that microRNA (miR)-21 and miR-181b elevate levels of the transcription factor nuclear factor 1 (NFI-A) that promotes MDSC expansion. We report here that miR-21 and miR-181b regulate NFI-A expression via a post-transcriptional regulatory mechanism by recruiting RNA-binding proteins HuR and Ago1 to stabilize NFI-A mRNA, thus increasing its protein levels. Studies in our laboratory also showed that inflammatory mediator S100A9 accumulates in the nucleus in Gr1+CD11b+ myeloid precursors in the later phases of sepsis and is necessary for their expansion and programming into immunosuppressive MDSCs. We demonstrate here that nuclear S100A9 associates with specific transcription factors that activate miR-21 and miR-181b expressions. In our final manuscript, we uncover another layer of the mechanisms of MDSC expansion and programming. We found that long non-coding RNA (lncRNA) Hotairm1 binds to and recruits S100A9 to the nucleus to program Gr1+CD11b+ myeloid precursors into MDSCs in the later phases of sepsis. Together, our results reveal three regulatory layers involving NFI-A, S100A9 and Hotairm1 in the pathway leading to MDSCs development in sepsis and suggest that therapeutically targeting these molecular switches might improve sepsis survival.

One of the mechanisms used by cancer to evade the immune response is the expansion of myeloid-derived suppressor cells (MDSCs), a population of immature myeloid cells able to inhibit immune responses in cancer patients and experimental animals with neoplasia. The role of MDSCs in promoting tumor growth and metastasis has gained importance over the years, highlighting the need to find specific target of intervention that could be used in the treatment of cancer patients. The aim of the present work was to analyze the signaling pathways active in MDSCs, using an in vitro model of MDSC generation developed by our group. Our previous studies revealed that the phenotype and suppressive ability of MDSCs were influenced by the presence of activated T cells, thus suggesting the existence of an interplay between the two populations. We therefore focused our attention on soluble molecules and surface markers mediating the interaction. We demonstrated that IL-10 release is increased in the culture between MDSCs and activated T cells and that this cytokine leads to the activation of STAT3 both in myeloid and lymphoid cells. One of the targets of STAT3 is B7-H1, a molecule that can deliver an inhibitory signal to T cell, interacting with its receptor PD-1. We therefore analyzed the expression of B7-H1 on MDSCs and we found that it is up-regulated in the presence of activated T cells through a STAT3-dependent signaling. By analyzing the fate of suppressed T cells, we observed that they express at higher level two markers of T cell exhaustion, PD-1 and LAG-3. LAG-3 is a negative co-stimulatory receptor on T lymphocytes and the natural ligand of HLA class II, whose expression we found up-regulated in MDSCs after culture with activated T cells. These results thus suggest that the interplay between MDSCs and activated T cells could be mediated by the couples of receptor/ligand PD-1/B7-H1 and LAG-3/HLA class II, leading to T cell exhaustion.<br>Uno dei meccanismi utilizzati dalle cellule tumorali per evadere la risposta del sistema immunitario è costituito dall’espansione delle cellule soppressorie di derivazione mieloide (MDSC), una popolazione di cellule mieloidi immature capaci di inibire le risposte immunitarie nei pazienti con tumore e in modelli murini con neoplasie. Il ruolo delle MDSC nel promuovere la crescita tumorale e la metastatizzazione ha acquisito sempre maggiore importanza negli ultimi anni, evidenziando la necessità di trovare specifiche vie di segnalazione attive in queste cellule che possano diventare bersaglio di interventi terapeutici mirati nel trattamento dei pazienti con tumore. Lo scopo di questo lavoro è stato quello di analizzare le vie di segnalazione attive nelle MDSC, utilizzando un modello sviluppato dal nostro gruppo per la generazione in vitro delle MDSC umane. I nostri studi precedenti hanno rivelato che il fenotipo e la capacità soppressoria delle MDSC sono fortemente influenzati dalla presenza dei linfociti T attivati, suggerendo l’esistenza di un’interazione tra le due popolazioni. Abbiamo quindi focalizzato la nostra attenzione su molecole solubili e marcatori di superficie che potrebbero essere coinvolti nell’interazione tra MDSC e cellule T attivate. Abbiamo dimostrato che il rilascio di IL-10 è aumentato nelle colture tra MDSC e cellule T attivate e che questa citochina porta all’attivazione di STAT3 sia nelle cellule mieloidi che in quelle linfoidi. Uno dei bersagli di STAT3 è B7-H1, una molecola che può fornire un segnale inibitorio alla cellula T, interagendo con il suo recettore PD-1. Abbiamo pertanto analizzato l’espressione di B7-H1 sulle MDSC e abbiamo notato che essa è aumentata in presenza delle cellule T attivate, mediante una via di segnalazione dipendente dall’attivazione di STAT3. Analizzando il destino dei linfociti T soppressi, abbiamo osservato che essi esprimono ad alto livello due marcatori di “exhaustion” delle cellule T, che sono PD-1 e LAG-3. E’ noto che LAG-3 è un recettore co-stimolatorio negativo sui linfociti T ed è il ligando naturale di molecole HLA di classe II, che sono significativamente aumentate nelle MDSC dopo coltura con le cellule T attivate. Questi risultati pertanto suggeriscono che l’interazione tra le MDSC e le cellule T attivate potrebbe essere mediata dalle coppie di recettore/ligando PD-1/B7-H1 e LAG-3/HLA di classe II, portando ad “exhaustion” delle cellule T.

Multiple myeloma (MM) is an essentially incurable malignancy associated with profound cellular and soluble immune deficiencies. Despite recent progresses in the treatment of MM, the prognosis remains frequently poor due to the difficulty in targeting MM progenitor cells, which are responsible for disease relapse. Immunotherapy, and in particular the employment of Natural Killer (NK) cells, offers the potential to target and eliminate MM cells within the bone marrow stromal sanctuaries, where they appear to be better protected against conventional therapeutic interventions. However, these strategies have so far provided limited clinical benefit, possibly reflecting the various escape mechanisms employed by MM cells to avoid immune recognition. The work presented in this thesis aims to further elucidate the mechanisms underlying MM-induced inhibition of NK cells and to investigate the therapeutic potential of immunomodulatory strategies to reverse this inhibition. Initially, the effect of co-culturing MM cell lines and healthy donor (HD) peripheral blood mononuclear cells (PBMCs) on NK cell phenotype and function was analysed. The results demonstrate that MM cell lines are able to impair NK cell cytolytic activity. This inhibition, which correlates with the downregulation of activating receptors such as NKG2D, NKp30, and DNAM-1, was shown to be the product of direct and contact-dependent interactions between MM and NK cells, without the need for other peripheral blood components. Importantly, the analysis of NK cells isolated from MM patients show that they display the same suppressed phenotype and activity as healthy donor NK cells co-cultured in the presence of MM cells lines, thereby suggesting that the detected suppression of NK cell activation by MM cells is a function of MM cells rather than any inherent defect in NK cells isolated from MM patients. In the light of this immunosuppressive effect, MM cells genetically-modified with a self-inactivating lentiviral vector encoding CD80 (B7.1) and IL-2 were tested for their ability to enhance and recover NK cell functional competence. Our results show that the in vitro co-culture of healthy donor or MM patient PBMCs with CD80/IL-2-modified MM cells is able to expand NK and T cell numbers, and to induce a significant increase in the fraction of NK cells expressing activating receptors such as NKp44, NKG2D, NKp30, and CD69, when compared to unmodified MM cells. More importantly for potential therapeutic applications, stimulated NK cells from healthy donors show increased cytolytic activity. These data suggest that the stimulation of PBMCs with CD80/IL-2-modified MM cells may be able to overcome the immune suppressive functions of unmodified MM cells and to stimulate NK and T cell mediated responses. Therefore, vaccination with CD80/IL-2-modified MM cells may represent a potential strategy for NK cell recovery and stimulation and, possibly, for the induction of a broad ranging immunological responses against multiple myeloma cells.

Le sepsis est à l’origine d’une dysfonction immunitaire prolongée responsable d’infections nosocomiales et d’une mortalité tardive élevée. Sa physiologie complexe demeure mal connue et il n’existe aucun traitement spécifique en dehors de l’antibiothérapie et des thérapeutiques de suppléance d’organes. Nous nous sommes intéressés au rôle des cellules myéloïdes dans cette dysfonction immunitaire. Nous avons pu montrer qu’il existe chez les patients atteints de sepsis une augmentation du nombre de cellules suppressives d’origine myéloïde monocytaires (M-MDSC) CD14+HLA-DRlow/- et granulocytaires (G-MDSC) identifiées comme des granulocytes de faible densité CD14-CD15+. Ces cellules sont responsables d’une activité Indoléamine 2,3-dioxygénase (IDO) et arginase 1, et leur déplétion permet de restaurer la prolifération des lymphocytes T in vitro. L’augmentation précoce des G-MDSC prédit la survenue ultérieure d’infections nosocomiales. De même, l’augmentation de l’activité IDO et de l’arginase 1 plasmatique sont associées à un mauvais pronostic. Au total, nous avons pu démontrer que les cellules myéloïdes acquièrent un phénotype suppresseur en partie responsable de l’immunodépression acquise et du pronostic péjoratif chez les patients septiques. Afin de restaurer les capacités immunitaires des patients, les MDSC pourraient devenir une future cible thérapeutique<br>Sepsis results in a sustained immune dysfunction responsible for poor prognosis and nosocomial infections. Sepsis physiology remains poorly understood and no treatment exists currently, excepted from antibiotherapy and life-support techniques. We asked if myeloid cells could play a role in this sustained immune dysfunction. We demonstrated that Peripheral CD14+HLA-DRlow/- monocytic-myeloid-derived suppressor cells (MDSCs) and CD14-CD15+ low-density granulocytes identified as granulocytic- (G-)MDSCs were increased in septic patients. In vitro, arginase and IDO activities relied on MDSCs and depletion of both subsets restored T-cell proliferation. The initial proportion of G-MDSC predicted occurrence of nosocomial infections. Similarly, high plasma Indoleamine 2,3-dioxygenase (IDO) activity and arginase 1 level were associated with poor outcome. Altogether, our results demonstrate that myeloid cells acquire suppressive functions during sepsis, partially responsible for the sustained immune dysfunction and poor outcome. MDSCs may become a future therapeutic target to restore the immune capacities of septic patients

La LMMC est une pathologie clonale de la cellule souche hématopoïétique dont les caractéristiques la classent parmi les syndromes myélodysplasiques/myéloprolifératifs (SMD/SMP). L’invalidation conditionnelle de Tif1γ au niveau hématopoïétique chez la Souris (Tif1γΔ/Δ) est responsable du développement d’un SMD/SMP mimant la LMMC humaine lorsque la souris atteint l’âge de 6 mois, faisant de Tif1γ un gène suppresseur de tumeur. Par ailleurs, malgré une monocytose, la population macrophagique péritonéale de ces souris est diminuée.Les objectifs de mon travail étaient de caractériser chez les souris malades la population myéloïde et d’étudier la différenciation macrophagique. Nous avons identifié chez les souris homozygotes Tif1γΔ/Δ une population morphologiquement immature, associant des caractéristiques granulocytaires et monocytaires. Les propriétés phénotypiques et moléculaires de cette population évoquent celles des cellules myéloïdes suppressives granulocytaires de type PMN-MDSC, faisant de Tif1γ un régulateur négatif de son développement. En sus, la différenciation in vitro des cellules myéloïdes médullaires en macrophages sous l’effet du CSF-1 est altérée. La baisse d’expression du CSF-1R n’explique pas à elle seule ces altérations puisque celle des cellules dendritiques est également perturbée sans modification de l’expression du GM-CSFR. Nous émettons l’hypothèse que l’augmentation d’expression de S100A8 et S100A9 chez les souris malades induit le développement des progéniteurs myéloïdes en cellules proches des PMN-MDSC au détriment des différenciations dendritique et macrophagique. En conclusion, Tif1γ est un régulateur majeur de la myélopoïèse<br>Chronic myelomonocytic leukemia (CMML) is a hematologic stem cell disease whose characteristics correspond to myelodysplastic/myeloproliferative syndroms (MDS/MPS). Hematopoietic conditional deletion of Tif1γ in mice leads to the development of a MDS/MPS, mimiking human CMML, when age is comprised between 6 to 10 months, defining Tif1γ as a tumour suppressor gene. Moreover, peritoneal macrophage population in these mice is decreased despite a monocytosis.The aims of my work were first to characterize in sick mice the myeloid population, and second to study macrophage differentiation. The myeloid population in Tif1γΔ/Δ mice is morphologically immature, with granulocytic and monocytic features. We demonstrated that phenotypic and molecular characteristics of this population are close to those observed in PMN-MDSC (polymorphonuclear myeloid-derived suppressor cells), suggesting that Tif1γ is a negative regulator gene of this myeloid subset. Furthermore, we showed that in vitro macrophage differentiation of myeloid progenitors upon CSF-1 treatment is altered. Decreased expression of CSF1-R (CSF-1 receptor) does not totally explain this alteration since dendritic cell differentiation is also abnormal, without alteration in GM-CSFR expression. Therefore, we hypothesize that S100A8 and S100A9 hyperexpression in Tif1γΔ/Δ mice is able to promote PMN-MDSC-like differentiation at the expense of macrophage and dendritic differentiations. In conclusion, Tif1γ is a major myelopoiesis regulator gene

L'échappement des cellules tumorales au processus d'immunosurveillance semble être une condition nécessaire au développement tumoral dans les modèles précliniques, comme chez l'homme. Les mécanismes par lesquels la tumeur parvient à médier une immunosubvertion sont multiples et font intervenir la plupart des cellules du système immunitaire, au sein desquelles, les cellules immunorégulatrices telles les cellules myéloides suppressives (MDSCs) ou les lymphocytes T régulateurs (Tregs, exprimant le facteur de transcription Foxp3), semblent jouer un rôle prépondérant. Les résultats présentés dans ce travail visent à mieux comprendre les rôles fonctionnels et pronostics des cellules myéloïdes suppressives et des Tregs dans le cancer, avec une attention plus particulière sur la façon dont ces cellules peuvent être modulées par la chimiothérapie. Concernant les MDSCs, nos travaux ont permis de mieux comprendre les mécanismes moléculaires présidant à leur accumulation d'une part, et d'autre part à l'acquisition de leur propriétés immunosuppressives, à travers une voie de signalisation impliquant les exosomes d'origine tumorale. Cette découverte, et la propriété d'une molécule d'usage thérapeutique courant, l'amiloride, de diminuer la production d'exosomes, y compris par les cellules tumorales, offrent une nouvelle possibilité de ciblage pharmacologique des MDSCs. Par ailleurs, l'étude des effets cytotoxiques sur les MDSCs de plusieurs molécules de chimiothérapie nous a permis de montrer que le 5-fluorouracile, probablement en raison d'un faible niveau d'expression de sa cible, la thymidilate synthase, dans les MDSCs, possédait une capacité sélective à éliminer ces cellules. Nos travaux d'immunohistochimie conduits sur des prélèvements tumoraux issus de patientes porteuses de cancers du sein localisés traitées par chimiothérapie néoadjuvante ont quand à eux permis de démontrer que la chimiothérapie néoadjuvante s'accompagne de modifications qualitatives de l'infiltration tumorale à la fois en lymphocytes T CD8+ et en lymphocytes T régulateurs Foxp3+. L'existence, après chimiothérapie néoadjuvante, d'une balance favorable de la réponse immunitaire, associant forte infiltration en CD8+ et faible infiltration en Foxp3+ s'accompagne d'une augmentation significative des marqueurs de cytotoxicité à médiation cellulaire, et est significativement corrélée à une éradication complète des cellules tumorales. Cette signature immunologique favorable se traduit également à long terme par une meilleure survie sans récidive et une meilleure survie globale, indépendamment du type de chimiothérapie reçue, de l'obtention ou non d'une réponse complète histologique, et du sous type moléculaire de cancer du sein. La combinaison de cette information immunologique avec la connaissance de la taille du résidu tumoral après traitement permet de considérablement affiner le pronostic des patientes. Enfin, nos travaux préliminaires semblent montrer que l'expression de Foxp3 dans les cellules cancéreuses de tumeurs du sein HER2+++ constitue un facteur de bon pronostic. Ces résultats illustrent donc l'importance non pas seulement des caractéristiques tumorales, mais aussi des caractéristiques de l'hôte, en particulier de la réponse immunitaire qu'il est capable de susciter, et de l'influence de la chimiothérapie sur cette dernière.

Résumé :Notre équipe a développé une thérapie cellulaire originale dérivant de la lignée monocytaire. Cette sous-population de cellules humaines suppressives d’origine myéloide, appelée Human Monocyte-derived Suppressor Cells (HuMoSC, cellules CD33+), est capable d’inhiber la prolifération des lymphocytes T effecteurs et d’induire des CD4 et CD8 Treg. De plus, les HuMoSC préviennent l’apparition de la maladie du greffon contre l’hôte (GvHD).Dans un premier temps, nous avons montré qu’un environnement inflammatoire ou la présence d’immunosuppresseurs ne diminuaient pas la capacité des HuMoSC à inhiber la prolifération lymphocytaire et à favoriser l’induction de CD4 et CD8 Treg. Enfin, nous avons montré que l’effet graft-versus-leukemia (GvL) est préservé en présence des HuMoSC. Toutes ces données confirment l’intérêt des HuMoSC dans la prévention de la GvHD.Cependant, en raison d’un faible rendement de génération des HuMoSC et d’un problème de disponibilité de billes de tri CD33+ GMP, nous avons aussi modifié notre protocole pour isoler les cellules CD14+, appelées CD14-HuMoSC. Ainsi, dans un second temps, nous nous sommes intéressés aux propriétés des surnageants des HuMoSC et des CD14- HuMoSC. Ces modifications du protocole ont permis d’obtenir un grand nombre de cellules CD14-HuMoSC et de grandes quantités de surnageant produit en conditions GMP. Nous avons montré que les deux surnageants diminuaient l’activation et la prolifération des LT, diminuaient la réponse Th1 au profit de la réponse Th2, favorisaient l’induction des Treg et diminuaient la capacité des cellules dendritiques à induire la prolifération des LT. In vivo, les surnageants préviennent le développement de la GvHD dans un modèle murin de GvHD xénogénique. Enfin, pour montrer que ces deux surnageants seront efficace chez les patients, nous avons montré qu’un environnement inflammatoire ou que la présence d’immunosuppresseurs n’altéraient pas l’effet immunosuppressif des surnageants. Ces résultats confirment leur intérêt thérapeutique. L’étude proteomique de ces deux surnageants a permis d’identifier des protéines immunosuppressives qui pourraient être responsables de leurs capacités immunosuppressives.En conclusion, les HuMoSC et les surnageants des cellules dérivées des HuMoSC représentent un arsenal thérapeutique prometteur dans la prévention de la GvHD mais aussi dans les maladies inflammatoires<br>Abstract :Our team has developed an original cell therapy derived from monocytes. This sub-population of human suppressor cells of myeloid origin, called Human Monocyte-Derived Suppressor Cells (HuMoSC, CD33+ cells) is able to inhibit effector T cell proliferation and to induce CD4 and CD8 Treg. It has been demonstrated that HuMoSC prevent from graft-versus-host disease (GvHD).In a first time, we showed that an inflammatory environment or the presence of immunosuppressive drugs did not decrease HuMoSC abilities to inhibit T cell proliferation and to promote CD4 and CD8 Treg induction. Finally, we showed that graft-versus-leukemia (GvL) effect is preserved in presence of HuMoSC. Taken together, those data confirm the interest of HuMoSC in GvHD prevention.Nevertheless, due to a low yield of HuMoSC generation with this protocol and problem with avaibility of CD33 GMP beads, we also modified our protocol to isolate CD14+ cells, called CD14-HuMoSC. This is why in a second time, we took interest in HuMoSC and CD14-HuMoSC supernatant properties. These protocol modifications allow us to obtain large number of CD14-HuMoSC cells and large quantities of supernatant produced under GMP conditions. We showed that both supernatants decrease T cell activation and proliferation, decrease Th1 response in favor of Th2 response, promote Treg induction and decrease capacity of dendritic cells to induce T cell proliferation. In vivo, supernatants prevent from GvHD in a murine model of xenogenic GvHD. Finally, in order to assess that these supernatants will be efficient in patient, we showed that an inflammatory environment or presence of immunosuppressive drugs did not alter both supernatant immunosuppressive effects. These results confirm their therapeutic interest. Proteomic analysis allowed us to identify immunosuppressive proteins which could be responsible for supernatants immunosuppressive capacities.In conclusion, HuMoSC and supernatant derived from HuMoSC represent a promising therapeutic arsenal for GvHD prevention but also in inflammatory diseases

Autoimmune diseases, unwanted overshooting immune responses against self antigens, are due to an imbalance in immunity and tolerance. Although negatively impacting cancer prognosis, myeloid derived suppressor cells (MDSC), with their potent suppressive capabilities, might be applicable in a more beneficial light when applied in to autoimmunity. As previous shown MDSC have protective roles in Experimental Autoimmune Encephalomyelitis (EAE) (Zhu et al., 2007), the established inducible mouse model for the autoimmune disease multiple sclerosis (MS). This decrease in disease severity indicates in vitro generated immature myeloid cells (IMC) from bone marrow (BM) as precursors of MDSC are promising candidates for cellular therapy. Important to any cellular therapy by adoptive transfer, the major questions regarding IMC efficacy was addressed within the thesis. This thesis attempts to elucidate how IMC operate in EAE. This thesis defines the factors within the autoimmune microenvironment that lead to the activation of MDSC, where IMC home once delivered in vivo, and the protective mechanisms BMIMC employ. To emulate BM cells when they first enter circulation through the blood, IMC were injected intravenously (i.v.). IMC are protective with no regard to the various routes delivered (i.v., i.p.). They protect to a lesser extent when pre-activated before injection. IMC suppress by causing a delay and/or by decreasing the severity of the disease via a mechanism yet determined. To understand the migration pattern of IMC after i.v. injection, in vivo kinetics experiments employing bioluminescence imaging were performed. This techinique allows for whole in vivo mouse imaging daily, allowing the tracking of cell migration over days within a single mouse. During steady-state, BMIMC circulate and appear to accumulate in the spleen by day 4 after injection, whereas they alternatively home to inflammatory sites (immunization site), draining lymph nodes, and the spleen within mice with low grade EAE. Visualization of CMDiI-labelled BMIMC by fluorescence microscopy could locate IMC injected cells outside the white pulp, as they were colocalizing in the regions stained with CD169 or outside, but not within the follicles of spleens on day 4. Consistant with these findings, the attempt to analyze the phenotype of these cells by flow cytometry was problematic as these cells seem to adhere strongly to collagen also indicating the cells are located in the collagenous area of the marginal zone and the red pulp.To determine factors influencing MDSC activation, we utilized different stimuli through a high throughput method detecting release of nitric oxide (NO). Extracts from yeast, fungi, and bacteria were observed to activate MDSC to produce nitric oxide. Surprisingly, material mimicking viral DNA (CpG) and RNA (poly I:C), and several self glycolipids, could not activate the MDSC to produce NO. Upon attempts to understand synergistic effects between microbial pathogens and host cytokines, IFNg was determined to boost the signal of pathogen stimuli, whereas IL17, another cytokine which causes pathology during EAE, and IFNb, a drug used in therapy to treat MS, did not cause any additional effects. Activation of MDSC was determined by the microbial pathogens components LPS, curdlan, and zymosan, to induce upregulation of B7H1 on the cell surface. MDSC did not increase any co-stimulatory markers, such as CD40, CD80, CD86, CD70, or the co-inhibitory marker, PDL2. On day 1 after EAE induction, endogenous MDSC populations when stimulated showed an increase in B7H1 expression and a downregulation of CD80. After further analysis, these cells were concluded to be mostly granulocytic cells (Ly6G+). As the B7H1 ligand PD1 is upregulated in chronic diseases and correlates to an exhausted phenotype, the PD1 : B7H1 interaction was a good candidate for the mechanism our cells may employ for their suppressive capacity. To investigate this interaction, fixed BM-IMC deficient in B7H1 were incubated with restimulated memory T cells. IMC deficient in B7H1 resulted in a significant loss of T cell suppression, as compared to the wildtype control BMIMC. To assess this interaction in vivo, we injected wildtype (WT) and B7H1-/- IMC into mice followed by induction of EAE to assess whether B7H1 mediated this suppression. The lack of B7H1 did not alter their suppressive capacity under these conditions, contrary to other findings which have described this interaction to be important in their suppressive capacity when administered post EAE induction (Ioannou et al., 2012). Interestingly, EAE mice pre-treated with IMC had similar amounts of cytokine production in the CNS after restimulation. Spleens from IMC injected mice had increased amounts of Arg-1 suggesting suppression is via oxidation or recruitment by soluble mediators may lead to this protection. We speculate this may inhibit T cell reactivation in the CNS<br>Autoimmunerkrankungen, unerwünschte, überschießende Immunantworten gegen Selbstantigene, resultieren aus einem Ungleichgewicht von Immunität und Toleranz. Obwohl sie einen negativen Einfluss auf Tumorerkrankungen haben, könnten Myeloide Suppressorzellen (MDSC) durch ihre potenten immunsuppressiven Eigenschaften, in einem besseren Licht bei Anwendung gegen Autoimmunerkrankungen erscheinen. Wie zuvor gezeigt, können MDSC eine protektive Rolle bei der Experimentellen Autoimmunenzephalomyelitis (EAE) entfalten, dem etablierten induzierbaren Mausmodel für die Autoimmunerkrankung Multiple Sklerose (MS). Die Verminderung der Erkrankungssymptome deutet darauf hin, dass in vitro aus Knochenmark generierte unreife myeloide Zellen (IMC) als Vorläufer von MDSC viel versprechende Kandidaten für eine Zelltherapie darstellen. Da für jede Art der Zelltherapie die Effektivität der transferierten Zellen eine entscheidende Rolle spielt, sollte in dieser Arbeit die Funktionalität von IMC untersucht werden. Diese Dissertation erarbeitet wie IMC bei der EAE funktionieren. Die Arbeit versucht die Faktoren innerhalb der AutoimmunMikroumgebung zu definieren, welche zur MDSC Aktivierung führen, wohin applizierte IMC in vivo wandern und welche protektiven Mechanismen IMC anwenden. Um nachzubilden, wie BM Zellen bei ihrem Eintritt in das Blut sich in der Zirkulation verhalten, wurden IMC intravenös injiziert. Die injizierten gemischten IMC verhielten sich protektiv, unabhängig von der Art der Injektion (iv, ip). Sie sind jedoch weniger protektiv, wenn sie voraktiviert injiziert wurden. IMC supprimieren auf eine Weise, dass sie eine Verzögerung und/oder Verminderung der Erkrankungssymptome bewirken, wobei die dafür zugrunde liegenden Mechanismen noch nicht definiert sind. Um die Wanderungsmuster der BMIMC nach iv Injektion zu verstehen, wurden in vivo Kinetikexperimente mittels der Biolumineszenz-Darstellung durchgeführt. Diese Technik erlaubt eine tägliche Betrachtung der gesamten lebenden Maus, so dass die Zell-Wanderungsmuster über Tage in derselben Maus aufgezeichnet werden können. Unter homöostatischen Bedingungen zirkulieren IMC bis sie nach 4 Tagen in der Milz akkumulieren, wogegen sie alternativ zu Entzündungsherden wandern (Immunisierungssstelle), in Lymphknoten und Milz in Mäusen mit milden EAE Symptomen. Deren Lokalisierung konnte durch Fluoreszenzmikroskopie von CMDiI-markierten IMC in der roten Pulpa der Milz an Tag 4 lokalisiert werden. In Übereinstimmung mit diesem Befund, waren durchflusszytometrische Phänotyp-Analysen problematisch, da die Zellen fest an Kollagenfasern gebunden schienen, was als weiterer Hinweis auf ihre Kollagenbindung dienen kann. Um Faktoren zur Aktivierung zu bestimmen, wurden verschiedene MDSC Stimuli benutzt und deren Freisetzung von Stickstoffmonoxid (NO) mittels einer Hochdurchsatzmethode bestimmt. Es konnte nachgewiesen werden, dass Extrakte aus Hefen, Pilzen und Bakterien MDSC aktivieren und zur NO Produktion führen. Überraschenderweise konnten DNS (CpG) oder RNS-Bestandteile (Poly I:C) mit viralen Charakteristika oder verschiedenen Selbst-Glykolipide keine NO Freisetzung hervorrufen. Darüber hinaus konnte für das Zytokin IFNg eine wichtige verstärkende Rolle gezeigt werden, wobei ein anderes bei der EAE-Pathogenese beteiligte beteiligtes Zytokin, IL17, und auch IFNb, eine Substanz zur Therapie der MS, keinerlei Effekte zeigten. Untersuchungen nach MDSC-Aktivierung mit den mikrobiellen Komponenten LPS, Curdlan und Zymosan zeigten eine Hochregulation des B7H1 Moleküls auf der Zelloberfläche. Andere kostimulatorische Marker, wie CD40, CD80, CD86, CD70 oder der inhibitorische Marker PDL2 nahmen nicht zu. Einen Tag nach EAE-Induktion exprimierten auch die endogene MDSC Populationen nach Stimulation eine erhöhte B7H1 und eine erniedrigte CD80 Expression. Nach weiterer Analyse konnten diese Zellen überwiegend als granulozytär (Ly6G+) eingestuft werden. Da der B7H1-Ligand PD1 bei chronischen Erkrankungen hochreguliert wird, und mit einem verbrauchten Phänotyp korreliert, sollte die PD1:B7H1 Interaktion als guter Kandidat für den Suppressionsmechanismus untersucht werden. Fixierte B7H1-defiziente IMC wurden auf ihre Suppressorfunktion auf Gedächtnis-T-Zellen getestet. B7H1-defiziente IMC zeigten eine signifikant niedrigere Suppression, im Vergleich zu Wildtyp IMC. Um diese Interaktion in vivo zu untersuchen, wurden Wildtyp oder B7H1defiziente IMC in Mäuse injiziert und danach EAE induziert um auch hier eine B7H1-vermittelte Suppression nachzuweisen. Die Abwesenheit von B7H1 veränderte jedoch die suppressiven Eigenschaften unter diesen Bedingungen nicht, im Gegensatz zu anderen beschriebenen Befunden bei denen eine wichtige suppresive Rolle bei Injektion nach EAE-Induktion beschrieben wurde. Interessanterweise zeigten Mäuse, welche mit BMIMC vorbehandelt wurden, eine vergleichbare Zytokinfreisetzung im ZNS nach Restimulation. Milzen zeigten nach IMC Injektion auch erhöhte Mengen Arg-1 könnte dies auf eine Suppression durch oxidative Mediatoren hindeuten. Man kann also annehmen, dass so eine Reaktivierung der T Zellen im ZNS verhindert wird

Indiana University-Purdue University Indianapolis (IUPUI)<br>More than 200,000 American women are diagnosed with breast cancer each year. Although therapies effective in treating metastatic breast cancer currently exist, each year approximately 40,000 women die from this disease. Current evidence indicates that anti-cancer immune responses can be induced by vaccination in situ to the growth of metastasis and protect patients from the tumor recurrence. However, induction of anticancer immune responses may be limited in their efficacy due to immune suppression mechanisms induced by the developing cancer. Myeloid-derived suppressor cells are one population of immune regulators comprised of immature cells of myeloid origin with important roles in blocking immune activation and promoting tumor progression. Elimination or maturation of these cells has been found to promote enhanced anti-tumor effects and improve overall survival. This thesis identifies a new role for interleukin-12 as a modulator of myeloid-derived suppressor cell activity. Interleukin-12 was found to promote up-regulation of cell maturation markers on the surface of myeloid-derived suppressor cells with an accompanying decrease in factors responsible for conferring suppressive activity such as nitric oxide synthase 2 and arginase I. The alterations in myeloid-derived suppressor cells were observed following both in vitro and in vivo treatment with interleukin-12. Further analysis of the anti-tumor efficacy of interleukin-12 revealed that at least part of its suppression of tumor growth can be linked to reductions in myeloid-derived suppressor cell populations in the tumor microenvironment and an influx of active CD8+ T cells into the tumor microenvironment. The findings outlined in this thesis show that interleukin-12 alters the suppressive function of myeloid-derived suppressor cells leading to significant immune infiltration and activation resulting in increased overall survival and a reduction in metastasis.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Multiple myeloma (MM) is the most frequent cancer to involve the skeleton, with over 80% of myeloma patients developing lytic bone disease (MMBD). Importantly, MM-associated bone lesions rarely heal even when patients are in complete remission. Bone marrow stromal cells (BMSCs) isolated from MM patients have a distinct genetic profile and an impaired osteoblast (OB) differentiation capacity when compared to BMSCs from healthy donors. Utilizing an in vivo model of MMBD and patient samples, we showed that BMSCs from tumor-bearing bones failed to differentiate into OBs weeks after removal of MM cells. Both Runx2 and Osterix, the master transcription factors for OB differentiation, remained suppressed in these BMSCs. However, the molecular mechanisms for MM-induced long-term OB suppression are poorly understood. We characterized both Runx2 and Osterix promoters in murine pre-osteoblast MC4 cells by chromatin immunoprecipitation (ChIP). The transcriptional start sites (TSSs) of Runx2 and Osterix in untreated MC4 cells were co-occupied by transcriptionally active histone 3 lysine 4 tri-methylation (H3K4me3) and transcriptionally repressive histone 3 lysine 27 tri-methylation (H3K27me3), termed the “bivalent domain”. These bivalent domains became transcriptionally silent with increasing H3K27me3 levels when MC4 cells were co-cultured with MM cells or treated with TNF-α, an inflammatory cytokine increased in MM bone marrow microenvironment. The increasing H3K27me3 levels induced by MM cells or TNF-α were associated with the downregulation of the H3K27 demethylase JMJD3 in MC4 cells and murine BMSCs. Knockdown of JMJD3 in MC4 cells was sufficient to inhibit OB differentiation. Further, ectopic overexpression of JMJD3 in MC4 cells partially rescued the suppression of osteoblast differentiation induced by TNFa. We also found that pre-incubation of MC4 cells with the NF-kB inhibitor quinazoline (QNZ) before TNF-a treatment prevented the downregulation of JMJD3. In agreement with our in vitro findings, BMSCs from MM patients had persistently decreased JMJD3 expression compared to healthy BMSCs. Our findings together demonstrate that decreased JMJD3 expression in BMSCs contributes to the long-term OB suppression in MMBD by remodeling histone landscapes at the Runx2 and Osterix TSSs. Thus, developing strategies to restore JMJD3 expression in BMSCs should increase bone formation and possibly decrease tumor burden in MM.