One of the most effective immunological interventions for human disease prevention is the administration of anti-red blood cell (RBC) IgG, more specifically, anti-D IgG, for prevention of hemolytic disease of the fetus and newborn (HDN), a serious and potentially fatal condition caused by the maternal immune response against the Rhesus (Rh) blood group system D antigen on fetal RBC. Despite its widespread clinical use, the mechanism of the suppressive anti-RBC IgG effect is not fully understood. In a murine model of immunity to foreign RBCs, transfusion of mice with IgG-opsonized RBCs strongly attenuated the antibody response compared to transfusion of untreated RBCs. This model was used to study the anti-RBC IgG effect on the host immune response. Contrary to the predominant theories of the anti-D effect, here it is shown that IgG-mediated RBC clearance is not sufficient for the attenuation of antibody responses. IgG-opsonized RBCs internalized by the mononuclear phagocytic cells could stimulate T and B cell responses against RBC antigens. This thesis also shows that the adaptive tolerance at the T or B cell level is not the reason for the attenuation of the antibody response. Instead, IgG selectively prevented the appearance of antigen-primed RBC-specific B cells and, surprisingly, induced the host B cell response against the IgG in complex with RBCs. These results suggest that the inability of RBC-specific B cells to recognize and present RBC-specific epitopes may explain the inhibitory IgG effect.

The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases.       The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology. Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model. By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice. To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.

<p>Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.</p><p> Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.</p> / Dissertation

Des peptides antigéniques sont présentés à la surface des cellules cancéreuses ou infectées par un virus pour être reconnus par des cellules du système immunitaire. Cette reconnaissance déclenche alors une réponse immunitaire spécifique contre les cellules présentatrices ciblées. L'objectif de ce projet est de stimuler cette réponse immunitaire afin qu'elle soit un moyen thérapeutique pour détruire spécifiquement les cellules cancéreuses ou infectées. Toutefois, les peptides antigéniques ne peuvent pas être administrés tels quels, du fait de leur pauvre stabilité en milieu biologique. Il est donc nécessaire de les modifier afin qu’ils soient plus résistants. Le défi de ce projet est de synthétiser des peptidomimes bio-résistants, capables de reproduire la même réponse immunitaire que celle du peptide antigénique naturel. / Abstract

<p>Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell-mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune-modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and preclinically characterizing their mechanisms of action and in vivo therapeutic efficacy.</p><p>CD8+ cytotoxic T cells have potent antitumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo-expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was upregulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired antitumor potential of patient CD8+ T cells. We determined that tumor-derived TGF-&#946; directly suppresses CD8+ T cell immune function by elevating miR-23a and downregulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF&#946;-mediated tumor immune-evasion pathway.</p><p>Having established that miR-23a-inhibition can enhance the quality and functional-resilience of anti-tumor CD8+ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers &#8210; such as TGF&#946; &#8210; within the tumor microenvironment. Since TGF&#946; poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune-competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wildtype and miR-23a-deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumor-specific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wildtype EGFRvIII-CAR T cells were vulnerable to functional suppression by TGF&#946;, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGF&#946;. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23a-deficient EGFRvIII-CAR T cells for GBM immunotherapy. </p><p>Lastly, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immune-suppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro: subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory IFN&#947; and IL-17 production by fully-differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.</p> / Dissertation

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. .

When breast tumor cells encounter stress due to cancer therapies, they may enter a dormant state, escaping from treatment-induced apoptosis. Dormant cells may eventually regain proliferative capabilities and cause recurrent metastatic disease, which is the leading cause of mortality in breast cancer patients. We sought to determine if a high dose of radiation therapy (RT) or combined chemo-immunotherapy, with and without the blockade of autophagy by chloroquine (CQ), could overcome treatment-induced tumor dormancy or relapse. We found that autophagy contributes in part to treatment-induced tumor dormancy. We also found that three therapeutic strategies were successful in inhibiting or preventing tumor relapse. These include: 18Gy/day RT, chemotherapy combined with the blockade of autophagy, and combined chemo-immunotherapy. Follow-up studies are needed to determine the feasibility of preventing tumor relapse by prolonging tumor dormancy versus eliminating dormant tumor cells.

Hepatitis C is the most common chronic blood-borne infection in the United States. Research has focused on contributing factors to the development and progression of liver disease, but few studies have considered nicotine use as a potential prognostic factor with CHC. Research has commonly found that CHC patients report with a diminished quality of life. Several factors have been proposed to account for a decrease in QOL; however, the mechanisms underlying the impairment in QOL have not yet been elicited. 76 CHC patients completed self-report measures on a variety of psychosocial variables and biochemical data for determining the patient's liver disease severity was obtained. The findings revealed strong support for the deleterious effects of smoking cigarettes on liver disease symptomatology and its progression. Smokers endorsed experiencing significantly more severe symptoms of fatigue, poor appetite, and headaches. The CHC smokers tended to present with higher scores on the Aspartate Arninotransferase to platelet ratio index (APRI). The smokers' mean score is above the cut-off value of 1.50 that indicates a .88 predictive value for the presence of hepatic fibrosis. The level of cigarette consumption could also be a factor in the progression of liver disease. Individuals smoking more than one pack per day tended to report more severe symptoms of fatigue and a poorer appetite. Heavy smokers presented with an APRI mean score above the cut-off value of 2.00 that indicates a .93 negative predictive value for the presence of cirrhosis below the cut-off value.General active coping moderated the relationship between liver disease severity and QOL. The results revealed that patients using more avoidant coping reported lower levels of QOL on the physical and mental component of the SF-36. Tobacco use moderated the relationship between liver disease severity and QOL. Interestingly, smokers reported a higher level of QOL compared to nonsmokers when experiencing more severe liver disease. CHC patients with higher levels of psychological distress reported lower QOL on both physical and mental functioning. Individuals smoking marijuana also tended to report lower levels of QOL on mental functioning. Information garnered from this study is aimed to help slow the progression of advanced liver disease in CHC patients in addition to improving their QOL.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells at various stages of differentiation. These cells are broadly characterized by the simultaneous expression of the surface markers CD11b and Gr1 and have been found to accumulate in large numbers in response to many different tumors in both mice and humans, including HER2/neu+ breast cancers. The adoptive immunotherapy of cancers has been a promising field, yet the clinical efficacy of adoptive immunotherapies targeted against human breast cancers and many other cancers has been extremely limited. Given the influx of MDSC in tumor-bearing individuals, we hypothesized that these cells were the reason for the failure of adoptively transferred T cells to effectively reject primary tumors. Using either monoclonal antibodies or the chemotherapeutic drug, gemcitabine, we aimed to eliminate MDSC cells in vivo to determine if adoptively transferred T cells would be more effective in the absence of these cells. We further aimed to characterize the mechanism of T cell suppression by MDSC and the tumor-derived soluble factor(s) responsible for their accumulation. We have found that the elimination of MDSC in vivo does result in significant tumor inhibition when adoptively transferred T cells are administered. Furthermore, the use of gemcitabine in conjunction with adoptively transferred T cells resulted in complete tumor rejection in 100% of mice and was accompanied by large antibody titers against HER2/neu as well as strong recall responses characterized by IFN-g release and subsequent rejection of further tumor challenges. We report herein that suppression by MDSC is contact dependent and affects the proliferation of both CD4+ and CD8+ T cells. The accumulation of MDSC in tumor-bearing mice can be entirely attributed to tumor-derived soluble factors, with GM-CSF specifically causing the generation and maintenance of these cells. Our findings suggest that the adoptive immunotherapy of breast carcinomas in a clinical setting should be combined with the use of gemcitabine, and that the use of GM-CSF as an adjuvant in cancer vaccines should be carefully re-evaluated as this cytokine may result in increased MDSC accumulation in vivo.

L'analyse de la réponse lymphocytaire T représente une exploration de plus en plus importante pour la compréhension de la physiopathologie de maladies et le suivi de protocoles d'immunothérapie. La mise en évidence de la production de cytokines par ces cellules permet d'identifier les sous populations de lymphocytes T (LT) et d'analyser leur fonctionnalité et leur état d'activation. L'Elispot est une technique déjà largement utilisée dans le suivi de la réponse vaccinale pour la détection de cellules sécrétrices de cytokines. Bien que la séroprotection soit le critère reconnu pour déterminer l'efficacité de la vaccination antigrippale, un rôle important de la réponse lymphocytaire T a été démontré dans le contrôle de l'infection. Nos travaux ont permis de mettre en évidence que l'utilisation de vaccins totaux pour le suivi de la réponse lymphocytaire T, par l'introduction de leurs excipients, peut induire un biais dans la détection des cytokines. Le thimérosal, conservateur utilisé dans certains vaccins, provoque in vitro lors de la détection d'interféron (IFN) par Elispot l'apparition de petits spots que nous avons associés a l'activation abortive précoce des LT. Leur taille permet cependant d'éliminer automatiquement du comptage ces spots non spécifiques. Contrairement à l'Elispot dont elle est dérivée, la technique Fluorospot permet l'analyse simultanée de plusieurs cytokines et est donc plus adaptée à la détection des profils de sécrétion et de la fréquence des LT polyfonctionnels, associés à un bon pronostic dans plusieurs pathologies. Nos résultats ont permis la validation technique de ce test pour la détection des couples IFN/interleukine (IL)-10 et IFN/IL-2. Grâce au développement au laboratoire d'une lignée produisant de l'IFN et l'IL-10, nous avons montré que le Fluorospot avait une sensibilité supérieure à la cytométrie intracellulaire pour la détection de l'IFN et de l'IL-10. Il était déjà connu que l'IL-10 était difficilement détectable par cytométrie, renforçant l'intérêt du Fluorospot pour détecter les cellules Tr1. L'utilisation du Fluorospot lors du suivi de la réponse vaccinale antigrippale a également permis de démontrer une équivalence de sensibilité avec l'Elispot. Au cours du suivi de ce protocole anti-grippal, nous avons montré à l'aide du Fluorospot que la réponse LT antigrippale était surtout caractérisée par une forte production d'IL-2 et que la détection isolée d'IFN a une faible sensibilité pour la mesure des réponses LT anti-grippales. Il s'agit de la première utilisation de ce test pour le suivi d'un protocole vaccinal démontrant sa robustesse et sa faisabilité dans un contexte clinique. Nos résultats offrent donc une validation complète de la technique Fluorospot, aussi bien du point de vue technique que clinique, ouvrant des perspectives d'utilisation pour le suivi de futurs protocoles. / The detection of cellular response via its different T cell subpopulations was proved to be crucial to better understand mechanisms of pathologies and monitor protocols of immunotherapy. Unicellular cytokine measurement not only allows the assessment of their inherent role on immunological response, but also the detection of T lymphocytes (LT) secretion profile which gives information on subpopulations involved, their activation state and their functionality. Detection of the cytokine secreting cells frequency is achievable with Elispot. This test is widely used in clinical trials to monitor vaccine response to diseases such as influenza. Although seroprotection is the recognized parameter to assess anti-influenza vaccine efficiency, cellular response has been proved to play an important role in infection control. Our results highlighted that the use of the whole vaccine to monitor T cell response may induce bias in cytokine detection. Indeed, their excipients can be considered as contaminants, like Thimerosal which is used as a preservative in some vaccines. We demonstrated that it induced detection of small spots in interferon (IFN) Elispot. We proved that these spots were associated with in vitro early abortive T cell activation. However, their size enabled us to remove these unspecific spots from bona fide spots.Fluorospot test is suited for multiplex cytokine analysis. Thus it is more adapted than Elispot to detect cytokine secretion profiles and polyfunctional T cells frequency. It is worth noting that polyfunctionnal Tcells are associated with a better clinical outcome in several pathologies. Our results led to technical validation of this test for detection of two cytokines couples, IFN/interleukin (IL)-10 and IFN/IL-2. Fluorospot showed a better sensitivity than intracellular staining cytometry (ICS) in detection of IFN and IL-10 produced by a cell line transfected with the cDNA encoding these cytokines. Moreover, Fluorospot demonstrated a similar sensitivity than Elispot when used to monitor the immunological response to an anti-influenza vaccine. Furthermore, using this technique, we showed that anti-influenza T cell producing IL-2 were the dominant population of T cells. Isolated IFN producing T cells were less sensitive than the detection of IL-2 to detect specific T cell response against influenza. Therefore our results provided a full validation of Fluorospot test, for the technical part as well as for the clinical part. These conclusions open perspectives of using this method to monitor protocols in a near future.