Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

In dieser Arbeit wurde die Zytolyse, die durch HIV-spezifische zytotoxische T-Lymphozyten (CTL) bewirkt wird, untersucht. Grundsätzlich erscheinen in der Abwehr viraler Infekte sowohl CD4+ wie CD8+ CTL. In chronisch HIV-Infizierten sind in der Regel lediglich CD8+ CTL zu finden. HIV-spezifische CD4+ CTL können aber zum Beispiel in Impfstudien aus Probandenblut isoliert werden. Sie erwiesen sich als zytolytisch aktiv und waren außerdem, wie in dieser Arbeit erstmals gezeigt werden konnte, auch an einer Fusions-vermittelten Lyse beteiligt. Diese ist Kalzium-unabhängig, auch mit heterologen Zellen möglich und die Expression des HIV-gp120 an der Zelloberfläche der Zielzellen ist essentiell, während die Präsentation von gp120 Epitopen nicht ausreicht. Die Konsequenz dieser Lyseform ist die apoptotische Zerstörung aller beteiligten Zellen. CD4+ HIV-spezifische CTL können über diesen Lysemechanismus bereits in der akuten Phase der Infektion einer negativen Selektion unterliegen, was ein Grund für das Fehlen der HIV-spezifischen CD4+ CTL in chronisch Infizierten darstellen kann. HIV-spezifische CD8+ CTL konnten aus einem HIV-positiven Patienten isoliert werden. Insgesamt wurden fünf Klone näher charakterisiert. Zwei erkannten jeweils ein Epitop aus unterschiedlichen Bereichen des extrazellulären Anteils des Glykoproteins, einer einen Bereich des p17-Anteils und zwei dasselbe Epitop im p24-Anteil des gag-Proteins. Dieses Epitop, plaziert innerhalb der Aminosäuren 71 bis 85, konnte hier zum erstenmal für die Präsentation mit HLA B51 beschrieben werden. In der spezifisch induzierten Lyse wiesen alle CD8+ CTL-Klone zwei Lysemechanismen auf, einen Perforin-vermittelten und einen CD95-vermittelten Anteil. Die CD95-vermittelte Lyse war stets prozentual geringer, und zusätzlich, sowohl Klon- wie Zielzell-spezifisch, unterschiedlich stark ausgeprägt . In HIV-infizierten primären Zellen ließ sich kein CD95-Anteil signifikant nachweisen, was darauf hinweist, daß dieser Lysemechanismus wahrscheinlich keine Rolle bei der Zerstörung infizierter Zellen im peripheren Blut spielt. In anderen Geweben kann sich die Situation unterschiedlich darstellen. Desweiteren konnte in dieser Arbeit nachgewiesen werden, daß das Vakzinia-Virus B13R-Genprodukt die CD95-vermittelte Apoptose hemmt. Daher muß bei der Untersuchung apoptotischer Vorgänge, die Wahl der eingesetzten Vektoren genauestens bedacht werden. / In this work the HIV-specific cytotoxic T-lymphocyte (CTL) mediated cytolysis was investigated. Normally CD4+ and CD8+ CTL are generated to defend viral infections. In chronically infected HIV-patients generally only CD8+ CTL are detectable. But HIV-specific CD4+ CTL can be isolated fram blood of HIV-vaccine candidates, for example. These CTL were cytolytic active and, as shown in this work for the first time, were also involved in fusion-mediated lyses. This lyses is independent of extracellular calcium, works with heterologous cells and the expression of HIV-gp 120 at the cell surface is essential. A presentation of gp 120 epitopes by HLA is not sufficient. In consequence this form of lysis is destructive in an apoptotic manner for all participating cells. CD4+ HIV-specific CTL could be negative selected by this mechanism in the early stage of infection. This could be a reason for the absence of HIV-specific CD4+ CTL in chronically infected patients. HIV-specific CD8+ CTL were isolated from one patient. Five were characterized in more detail. Two of them recognized an epitope in different parts of the extracellular region of the glycoprotein, one discerned the p17-protein and two the same epitope of the p24-part of the gag-protein. This epitope, localized between amino acids 71 to 85, is here reported for the fist time for presentation with HLA B51. In specific induced lyses all CD8+ CTL-clones presented two lyses-mechanisms, a perforin-mediated and a CD95-mediated portion. The CD95-mediated lyses always showed lower percentages and differed dependant on the CTL-clone and the target-cells in its markedness. For HIV-infected primary cells no significant CD95-mediated lyses was detectable, leading to that there is no role for this lyses mechanism in destruction of infected cells in peripheral blood. In other tissues the situation can differ. In this work, furthermore the inhibition of CD95-mediated apoptosis by the Vaccinia Virus B13R gene product was proven. Therefore, the vectors used in investigations of apoptosis has to be chosen carefully.

In this project two novel murine autoimmune models were to be established in an attempt to further investigate the nervous system disorders of Multiple Sclerosis and Guillain Barré Syndrome. Previous experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN) models have demonstrated that T cells play a major role in these diseases. Which roles CD4 and CD8 T cells specifically have in the initiation, propagation and termination of an autoimmune nervous system disorder remains controversial. To this end two transgenic mice specifically expressing the neo-antigen (Ag) ovalbumin (OVA) in either the central nervous system (CNS) or peripheral nervous system (PNS) were to be generated. The myelin basic protein (MBP) is a major component of the myelin sheath both within the CNS and the PNS. Therefore the MBP promoter was employed for its distinct regulatory elements to facilitate exclusive CNS or PNS OVA expression. The adoptive transfer of OVA specific MHCI restricted (OT-I) and MHCII restricted (OT-II) TCR Tg T cells extended the OVA Tg mouse model by allowing potentially encephalitogenic T cells to be tracked in vivo. Specificity for the target Ag should enable the dynamic role of antigen specific T cells in neuroinflammatory diseases to be revealed in more detail. / Im Rahmen der vorliegenden Arbeit wurden zwei neue Mausmodelle für Autoimmunerkrankungen etabliert, um weitere Fortschritte bei der Aufklärung der zellulären und molekularen Interaktionen bei den Erkrankungen des Nervensystems Multiple Sklerose und Guillain Barré Syndrom zu erzielen. In früheren Experimenten mit EAE (experimentelle autoimmune Enzephalomyelitis) und EAN (experimentelle autoimmune Neuritis) konnte bereits gezeigt werden, dass T-Zellen eine Hauptrolle bei diesen Erkrankungen spielen, wobei jedoch die Bedeutung von CD4 bzw. CD8 T-Zellen im Einzelnen noch nicht aufgeklärt ist. Zu diesem Zwecke sollten zwei transgene (Tg) Mauslinien generiert werden, die speziell entweder im peripheren (PNS) oder im zentralen (ZNS) Nervensystem das Zielantigen OVA exprimieren. MBP ist eine Hauptkomponente der Myelinscheide sowohl im ZNS als auch im PNS. Daher kam der Myelin Basic Protein (MBP) Promoter zum Einsatz, dessen unterschiedliche regulatorischen Elemente eine Expression von intaktem OVA ausschließlich im ZNS bzw. ausschließlich im PNS steuern können. Eine Erweiterung dieser OVA tg Mausmodelle stellte der adoptive Transfer von OVA spezifischen MHCI-restringierten OTI und MHCII-restringierten OTII T-Zellen dar, da es so möglich wurde, potentiell enzephalitogene T-Zellen in vivo zu verfolgen. Dadurch sollte ebenfalls eine detailliertere Darstellung der dynamischen Rolle von antigenspezifischen T-Zellen bei neuroinflammatorischen Erkrankungen ermöglicht werden.

Selection of the T cell repertoire in the thymus is governed by the need to create a repertoire of peripheral T cells that can respond to any foreign antigen in the context of self-major histocompatibility complex (MHC), while enforcing central tolerance to self-antigens. Perturbations in signalling molecules, that reduce the affinity of thymic selection, can lead to the production of a peripheral repertoire with increased autoimmunity, as has been shown for mutations in the Zap-70 kinase. Upstream of Zap-70 is Lck, the most proximal tyrosine kinase required for T cell receptor (TCR) triggering upon TCR engagement by peptide:MHC. In order to study how Lck influences T cell activation, a transgenic mouse model (LckVA), in which Lck is expressed constitutively from a T cell specific transgene and mice have very low expression of Lck (~5% of WT) in both the thymus and periphery, was used. It has been shown that Lck is critical for successful T cell development, yet the results of this thesis show that even 5% of WT levels of Lck are sufficient for selection of thymic T cells on both polyclonal and F5 TCR transgenic backgrounds. Previous studies utilising mice expressing an inducible Lck transgene, which also had reduced Lck expression in the periphery, showed Lck to be critical in determining the activation threshold of T cells. In contrast, peripheral T cells in LckVA mice had similar activation thresholds to wild type T cells, as measured by in vitro upregulation of early activation markers. Further analysis of LckVA peripheral T cells revealed differential influences of low expression of Lck on downstream signalling pathways upon TCR engagement. For example, ERK signalling was impaired, while calcium flux and proliferation were enhanced in LckVA T cells. Finally, LckVA T cells were altered in their ability to differentiate, showing enhanced production of cytokines and retaining the capacity to form memory cells.

Transcriptional enhancers represent the primary basis for differential gene expression. These elements regulate cell type specificity, development, and evolution, with many human diseases resulting from altered enhancer activity. To date, a key gap in our knowledge is how enhancers select specific promoters for activation. To fill this gap, in this thesis, I first developed an Integrated Method for Predicting Enhancer Targets (IM-PET). Leveraging abundant “omics” data, I devised and characterized multiple genomic features for distinguishing true enhancer-promoter (EP) pairs from non-interacting pairs. I integrated these features into a probabilistic predictor for EP interactions. Multiple validation experiments demonstrated a significant improvement over extent state-of-the-art approaches. Systematic analyses of EP interactions across twelve human cell types reveals global features of EP interactions. Second, we used a well-established viral infection model to map the dynamic changes of enhancers and super-enhancers during the CD8+ T cell responses. Our analysis illustrated the complexity and dynamics of the underlying EP interactome during cell differentiation. Taking advantage of the predicted EP interactions, we constructed stage-specific transcriptional regulatory networks, which is critical for understanding the regulatory mechanism during CD8+ T cell differentiation. Third, recent progress in mapping technologies for chromatin interactions has led to a rapid increase in this type of interaction data. However, there is a lack of a comprehensive depository for chromatin interactions identified by all major technologies. To address this problem, we have developed the 4DGenome database through comprehensive literature curation of experimentally derived interactions. We envision a wide range of investigations will benefit from this carefully curated database.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection and hospitalization in infants and young children, but no vaccine is currently available. CD4 and CD8 T cells are critical for mediating viral clearance but also contribute to immunopathology following an acute RSV infection. However, few RSV-derived CD4 and CD8 T cell epitopes in the commonly used C57BL/6 mouse strain have been described. I utilized an overlapping peptide library spanning the entire RSV proteome and intracellular cytokine staining for interferon-gamma (IFN-γ) to identify novel CD4 and CD8 T cell epitopes in C57BL/6 mice. I discovered and characterized two novel CD4 T cell epitopes and three novel CD8 T cell epitopes located within multiple RSV proteins. Overall, the novel RSV-derived CD4 and CD8 T cell epitopes identified in C57BL/6 mice will aid in future studies of RSV-specific T cell responses. While CD8 T cells are important for viral clearance following an acute RSV infection, the contribution of memory CD8 T cells in providing protection against reinfection with RSV remains unclear. I used a prime-boost immunization approach to induce robust, systemic memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. I determined that high magnitude, systemic memory CD8 T cell responses efficiently reduced lung viral titers following RSV infection, but unexpectedly did so at the expense of severe and fatal immunopathology. The exacerbated disease was mediated by the rapid and excessive production of IFN-γ by memory CD8 T cells in the lung and airways. In contrast, I found that local immunization generated a large population of tissue-resident memory CD8 T cells in the lung that efficiently reduced lung viral titers in the absence of exacerbated disease. Additionally, I observed that pre-existing RSV-specific neutralizing antibodies prevented the immunopathology induced by high magnitude, systemic memory CD8 T cell responses following RSV infection. Prophylactic treatment with neutralizing antibodies against RSV efficiently restricted early virus replication, which resulted in a significant decrease in lung IFN-γ levels, memory CD8 T cell activation, and the frequency of IFN-γ producing CD8 T cells. Thus, my results demonstrate that high magnitude, systemic memory CD8 T cells induce lethal immunopathology following RSV infection, which can be prevented by pre-existing RSV-specific neutralizing antibodies. Overall, my results have important implications for the development of future RSV vaccines. The development of a live-attenuated vaccine for RSV has been prevented by the inability to properly balance attenuation with immunogenicity and efficacy. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. As the M protein is required for virion assembly following infection of a host cell, RSV M-null induces a single-cycle infection. I evaluated RSV M-null as a potential live-attenuated vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared to its recombinant wild-type control virus (RSV recWT). RSV M-null was sufficiently attenuated, as significantly reduced lung viral titers, weight loss, and pulmonary dysfunction were observed compared to mice infected with RSV recWT. Surprisingly, despite its attenuation, I found that RSV M-null infection induced effector T cell, germinal center B cell, serum antibody, and memory T cell responses of similar magnitude to that elicited by infection with RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, my results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.

CD8 T cells play a critical role in the clearance of pathogenic bacteria, viruses, and protozoan parasites. Upon encountering their cognate antigen through either infection or vaccination, naïve CD8 T cells undergo robust proliferative expansion, which is followed by contraction and the formation of a memory population. Memory CD8 T cells are long-lived, and because they persist in increased numbers and possess enhanced functional abilities compared to naïve CD8 T cells, they are able to provide the host with increased protection following re-infection. Because of these properties, vaccines designed to elicit memory CD8 T cells have the potential to reduce health care burdens related to infection with pathogens including human immuno deficiency virus (HIV), malaria, influenza, and hepatitis virus. However, stimulating protective CD8 T cell responses against these pathogens through vaccination has proven challenging. Therefore, a better understanding of the properties of memory CD8 T cells generated following vaccination, and the characteristics of memory CD8 T cells best suited for providing protection against diverse pathogens is needed. While memory CD8 T cells can be maintained for as long as the life of the host, evidence suggests that their properties change with time after infection. Because CD8 T cell-mediated protection is based upon both the numbers and quality or functional abilities of memory cells present at the time of re-infection, changes in memory CD8 T cell function over time could impact their ability to provide protection upon re-infection. Therefore, a better understanding of how memory CD8 T cells change with time after infection is needed. As part of the studies presented in this thesis, I found that the phenotype and function of memory CD8 T cells including localization, interleukin (IL)-2 cytokine production, responsiveness to homeostatic cytokines, metabolic capabilities, and proliferation and secondary memory generation potential change with time after infection. Interestingly functional changes could not be completely explained by changes in subset composition that occur with time, as changes over time were also seen in defined CD62Lhi subsets. Importantly, functional changes of memory CD8 T cells that occurred with time led to an increased ability to provide protection against a chronic viral infection. These data improve our knowledge of the capabilities of memory CD8 T cells generated following infection, and suggests that the outcome of vaccination strategies designed to elicit protective memory CD8 T cells using single or prime-boost immunizations will depend upon the timing between antigen encounters. Following re-infection, memory CD8 T cells become activated and produce effector cytokines and cytolytic molecules that aid the host in clearing invading microbes. Activation can be triggered not only through cognate antigen recognition, but also by antigen-independent cytokine driven signals. However, our knowledge of how antigen-dependent and –independent signals contribute to CD8 T cell activation and protection following infection is incomplete. In the second part of my thesis, I show that the ability of memory CD8 T cells to become activated in response to inflammation decreases with time after infection, that antigen and inflammation act synergistically to induce activation of memory CD8 T cells, that the presence of cognate antigen enhances activation of memory CD8 T cells that contribute to clearance of infection, and that bystander memory CD8 T cell responses following unrelated bacterial infection do not provide the host with a protective benefit. Together, the data in this thesis further our understanding of memory CD8 T cells generated following infection and/or vaccination, and the properties of memory CD8 T cells important for providing protection upon re-infection with invading pathogens.

Antigen-specific CD8 T cells play a critical role in protecting the host from infection by intracellular pathogens including viruses, bacteria and parasites. During the course of an infection, antigen-specific CD8 T cells undergo proliferative expansion to increase in number, which is followed by contraction and generation of a stable pool of long-lived memory cells. Importantly, memory CD8 T cells provide enhanced resistance to re-infection by the same pathogen. Moreover, the number of memory CD8 T cells correlates strongly with the level of protection against re-infection. Therefore, vaccines designed to promote cellular immunity should logically focus on achieving sufficiently high number of these memory cells for protection. Most current vaccines have relied on inducing antibodies to protect the host by neutralizing pathogens or blocking pathogen entry into the cells. However, there is a recognized need to design vaccines that also stimulate a strong CD8 T cell component of the adaptive immune response in addition to antibodies. Importantly, inflammatory cytokines induced by infection or vaccination with adjuvant act directly or indirectly on CD8 T cells to modulate their expansion, contraction and acquisition of memory characteristics. Thus, an understanding of how inflammatory cytokines regulate CD8 T cell memory differentiation may help guide the strategies for rational vaccine design. My studies examine the roles of inflammatory cytokines in regulating CD8 T cell memory differentiation. Specifically, my studies investigate the timing of inflammatory cytokine exposure and the role of type I IFNs and IL-12 in regulating effector/memory CD8 T cell differentiation, and exploiting the cross-presentation pathway to rapidly generate protective CD8 T cell immunity. Specifically, my results indicate that (i) encounter with inflammatory cytokines during the rapid proliferative phase deflects CD8 T cell differentiation away from memory towards a sustained effector program, (ii) that direct signaling by either type I IFN or IL-12 to the responding CD8 T cells promotes maximal expansion, but neither of these cytokines is essential to regulate the effector/memory differentiation program, and (iii) cross-priming with both cell-associated antigen and antigen-coated, biodegradable microspheres, accelerates CD8 T cell memory development that can be exploited to rapidly generate protective CD8 T cell immunity.

The severe disease associated with seasonal epidemics of influenza A virus (IAV), as well as pandemic outbreaks, have highlighted the necessity for novel, broadly cross-reactive vaccination and therapeutic strategies against IAV. Our studies have focused on the contribution of IAV-specific CD8 T cells to mediating protection following IAV vaccination and infection as IAV-specific CD8 T cells are required for clearance of IAV. Further, IAV-specific CD8 T cells are typically cross-protective as they are generally directed at highly conserved areas of IAV. Recently, influenza virus-like particles (VLPs) have been developed from recombinant baculoviruses containing influenza proteins hemagglutinin (HA) and/or neuraminidase (NA) on the surface and matrix (M1) in the VLP core. Influenza VLPs induce potent antibody responses and have been shown to provide protection from morbidity and mortality during lethal homo- and hetero-subtypic IAV challenge. This suggests that conserved, VLP-induced CD8 T cell responses may also contribute to the overall protective ability of VLPs. However, whether influenza VLPs can induce influenza-specific CD8 T cell responses and if these T cells are protective during IAV challenge remains unknown. Here, I demonstrate that a single, intranasal vaccination with VLPs containing HA and M1 leads to a significant increase in HA533-specific CD8 T cells in the lungs and lung-draining lymph nodes. Our results also indicate that HA533-specific CD8 T cells primed by influenza VLP vaccination are significantly increased in the lungs following lethal IAV challenge. These VLP-induced memory CD8 T cells are critical in providing protection from lethality following subsequent challenge infections, as depletion of CD8 T cells leads to increased mortality, even when total, but not VLP-induced memory, CD8 T cell numbers have been allowed to recover prior to lethal dose IAV challenge. In addition, my studies also importantly demonstrate that these VLP-induced, HA533-specific CD8 T cells aid in protection from high-dose, heterosubtypic IAV infections where CD8 T cell epitopes are conserved, but the targets of neutralizing antibodies have been destroyed. This dissertation further elucidates the requirements for the regulation of the IAV-specific CD8 T cell response in the periphery (i.e. lung) by pDC and CD8α+ DC. Our studies have previously demonstrated that pDC or CD8α+ DC must present viral antigen in the context of MHC class I along with trans-presentation of IL-15 to effector, IAV-specific CD8 T cells in the lungs to protect the T cells from apoptosis and allow generation of the full magnitude CD8 T cell response needed to clear IAV infection. Herein, I demonstrate that in addition to antigen presentation and IL-15, costimulatory molecules on the surface of pDC and CD8α+ DC are also required. However, the specific costimulatory molecules required depends upon both the mouse strain utilized for IAV infection as well as DC subset. In addition to costimulatory molecules, I also demonstrate that the requirement for pDC and CD8α+ DC to be infected differs in order for them to participate in this pulmonary rescue of the IAV-specific CD8 T cell response. While CD8α+ DC are able to efficiently cross-present exogenous antigen, pDC must be directly infected and utilize the endogenous, direct antigen presentation pathway to present viral antigen to IAV-specific CD8 T cells in the lungs during IAV infection. These data suggest there are distinct differences between pDC and CD8α+ DC in their mechanism of regulating the pulmonary IAV-specific CD8 T cell response, which had not been previously appreciated. Together, the results presented herein further detail the mechanism of regulation of effector IAV-specific CD8 T cells by DC as well as the contribution of IAV-specific CD8 T cells to a novel, IAV VLP vaccination strategy. These findings highlight the importance of IAV-specific CD8 T cells in mediating protection following IAV vaccination and infection.