Microbiology & Immunology - Theses
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ItemMolecular mechanisms regulating CD8+ T cell differentiation following external cues( 2018)Upon activation, naive CD8 T cells undergo a program of proliferation and differentiation that results in the acquisition of effector functions. Optimal T cell activation requires the integration of multiple signals including cross-linking of the T cell receptor (signal 1), co-stimulation (signal 2) and soluble factors such as cytokines (signal 3). Once a CD8 T cell has received these three signals they differentiate into an effector cell, which are able to control infection by directly killing the infected cell. Once the infection is cleared, these effector cells contract by controlled cell death and a long-lived population of memory cells remain. These potent memory cells are the defining feature of adaptive immunity as they offer protection for the life of the host due to their unique capabilities to survive in the absence of antigen and respond rapidly to secondary challenge. Therefore, effective CD8 T cell memory is the goal of cell-mediated vaccination strategies. While it is well established that CD4 help is required for CD8 T cell memory formation, it is unclear when during CD8 differentiation this help is required. Further, the effect that CD4 help has on the transcriptional profiles of CD8 T cells and the molecular pathways they use during the generation and maintenance of memory CD8 T cells remains elusive. Using a mouse model of Influenza A virus infection, where priming occurs in the presence or absence of CD4 T cell help, we have pinpointed that help is required during the initial priming of CD8 T cells, and not during memory maintenance or recall. Genome wide RNA sequencing analysis of the transcriptional signatures between resting helped and unhelped memory CD8 T cells revealed surprisingly few differentially expressed genes. However, upon reactivation, helped memory CD8 T cells exhibited greater transcriptional up-regulation than their unhelped counterparts and utilization of alternate molecular pathways. Intriguing metabolism defects combined with similarities to an ‘exhausted phenotype’ suggest that help is required to defer a cell away from terminal differentiation, towards a memory cell. Further, our analysis revealed that CD4 T cell help during initial priming is essential for establishing a memory cell pool with enhanced transcriptional potential. Thus, CD4 T cell-dependent programming likely underpins rapid responsiveness, a key characteristic of memory CD8 T cells. Each stage of T cell differentiation; naive, effector and memory, are characterized by distinct transcriptional and functional profiles. However, the molecular mechanisms regulating the acquisition of these profiles remain poorly defined. Further, contention remains around the pathway of differentiation towards memory cell status. This thesis has compared the transcriptional profiles of each of these cell stages, aiming to identify any previously unappreciated genes, gene networks or TFs that may be vital during the differentiation of memory CD8 T cells. These transcriptional profiles were first compared globally, which highlighted the similarities between each of the cell stages. Based on transcriptional profiles of gene expression across each of the cell stages, two key genes were identified, Dmrta1 and Zbtb32. These were then validated and assessed to determine if they translate from mice models into human studies. The data shown in this thesis suggests each of these genes may be molecular signatures used to identify memory cells. Each gene should be further evaluated, but alone have validated the method of data mining and comparison to identify previously undescribed genes as having a role in cell differentiation. Finally, using mathematical modelling of in vitro activated cells combined with bioinformatic analysis of ATACSeq, this thesis also has explored the role of signal three on chromatin remodelling during CD8 T cell activation. Our analysis has identified that each cytokine has a slightly different impact on the degree of chromatin accessibility, and a combination of signal 3 cytokines resulting in the highest level of chromatin accessibility and subsequent activation, proliferation and downstream acquisition of effector function. We suggest signal 3 itself is an under-appreciated cascade of regulation during the activation of CD8 T cells by directly controlling chromatin structure. Taken together, this data suggests that each signal received by a CD8 T cell during activation is gearing it towards a particular fate. It can be speculated from this data that the default pathway after activation is towards terminal differentiation, that is, to become an effector cell, destined to die by controlled death after the infection is cleared. It is the cues from the environment that skew the cell away from this fate in a healthy environment, such as CD4 T cell help and signal three. Importantly, in chronic disease states such as HIV, or LCMV in mice, this balance is tilted and results in a mass of activated cells and exhaustion. Therefore, the work contained in this thesis demonstrates the importance of each factor during activation and their effect on not only the functional capacity of CD8 T cells, but also their fate.
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ItemInvestigating the requirements for robust CD4+ T cell immunity to Herpes Simplex Virus.( 2016)CD4+ T cell responses are crucial for the control and clearance of many intracellular pathogens, including Herpes Simplex Virus type 1 (HSV-1), yet the requirements for the induction of optimal effector responses are unclear. To better understand the stimuli required for the generation of sustained T cell immunity, we compared transgenic HSV-1-specific CD4+ T cell responses following epicutaneous, intranasal and intravenous inoculation of mice with UV-inactivated (UV-HSV) and live HSV-1. This thesis describes these observations and demonstrates that despite comparable early proliferative responses, HSV-1-specific CD4+ T cells increased T-bet expression and accumulated in mice infected with live HSV, but not following inoculation with UV-HSV. Phenotypically, HSV-1-specific CD4+ T cells activated by UV-HSV in vivo lacked typical effector cell characteristics including CD25, a component of the high affinity IL-2 receptor, which promotes T cell expansion, survival and Th1 differentiation. This was found to occur despite the prolonged persistence of UV-HSV-derived antigen during early priming, a known requirement for CD4+ T cell differentiation. Dividing CD25-, CD4+ T cells elicited by UV-HSV resembled those that were adoptively transferred into mice at a late stage of an acute epicutaneous infection when virus in the skin had cleared and antigen levels had presumably waned. Congruent with this, infection with an attenuated mutant HSV-1, incapable of more than one replication cycle, elicited a proportion of CD25+ CD4+ T cells significantly greater than and less than that generated by UV-HSV and wildtype HSV-1, respectively. These findings suggest that HSV-1 antigen abundance during T cell priming is vital to generate a sustained CD4+ T cell response. To resolve whether these disparate priming patterns were due to differences in how dendritic cells (DC) stimulate CD4+ T cells in response to live and UV-HSV, DC were isolated from the lymph nodes draining the site of infection and assessed their ability to stimulate HSV-1-specific CD4+ T cells ex vivo. Intriguingly, although both CD8α DC and migratory dDC presented virus-derived antigen on MHC II molecules following live virus infection, presentation of UV-HSV-derived antigen was largely confined to the dDC with very little, if any contribution by the CD8α DC. The reduced contribution by CD8α DC in UV-HSV-inoculated mice also correlated with a lack of CD40 up-expression, a DC maturation marker, from this subset. Consistent with the view that epicutaneous UV-HSV inoculation failed to convert naïve HSV-1-specific CD4+ T cells into T-bet expressing effectors cells because CD8α DC did not participate in the priming, live virus infection of mice lacking CD8α DC resulted in similarly impaired HSV-1-specific CD4+ T cells priming characterized by reduced T-bet expression. Finally, supplementation of epicutaneous UV-HSV administration with targeted HSV-1 antigen to CD8α DC was found to profoundly augment T-bet expression in respondent CD4+ T cells. Collectively, these data implicate lymph node-resident CD8α DC as a source of critical Th1 differentiation cues and propose a functional rationale for recently reported DC:CD4+ T cell dynamics during CD4+ T cell induction in response to skin infection with this pathogen.
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ItemIncreasing the efficacy of subunit vaccines with synthetic adjuvants( 2016)The results of experiments in which two different vaccine delivery systems, demonstrating the safety, economy of dose and the ability to induce antibody and CD8+ T cell responses using model antigen and real world antigens are described in this thesis. A chitosan polymer-based depot system was shown to induce a long-lasting antibody-mediated immune response that was maintained for at least one year following inoculation with a single dose of vaccine. Furthermore, the vaccine was able to facilitate antigen and adjuvant dose-sparing effects. A cationically charged vaccine delivery system, R4Pam2Cys and its PEGylated form, R4Pam2Cys-PEG were also investigated. PEGylation of R4Pam2Cys was shown to reduce the size of vaccine particulates and enhanced the expansion of primary antigen-specific CD8+ T cells. The PEGylation of R4Pam2Cys provided a vaccine candidate which suppressed tumour growth and improved survival time. Finally, the efficacy of a whole inactivated influenza virus (WIV) preparation formulated with R4Pam2Cys was shown to induce influenza-specific antibody and also cross-protective CD8+ T cells which protected against homologues and heterologous influenza virus challenge. A dose-sparing effect was also observed using WIV which could be of great assistances during pandemic outbreaks when vaccines are often in short supply. The findings described in this work highlight the importance of the use of appropriate vaccine delivery systems to activate appropriate arms of the immune response.
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ItemAn investigation into how the TLR-2 agonist Pam2Cys stops influenza and its sequelae( 2015)Influenza continues to be a major public health concern with up to 1 billion people being infected annually. Mitigation of the disease and its symptoms are effected by the use of seasonal influenza vaccines i.e. vaccines against the currently circulating strains of influenza virus and a small number of antiviral drugs which are often used in concert. The development of antiviral resistance to the antiviral drugs and the need for vaccines to accommodate pathogen evolution, limit the effectiveness of these treatments and as a consequence focus has been directed on developing alternate treatments to combat influenza. Immunostimulants are a new class of antiviral agents that are currently being examined and function by activating the host’s own innate immune system. Because these agents target the host and not the pathogen, they are unlikely to be plagued with issues of resistant strains and can also be used to combat a broad range of pathogens including influenza strains yet to be encountered. When administered intranasally, the Toll-like receptor-2 (TLR-2) ligand S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys) has been shown to alter the pulmonary immune environment in a matter of hours in such a way that the host is protected from infection with influenza virus. This study has identified those components of the innate immune system which are elicited and/or activated by Pam2Cys and are responsible for the immediate protective effect. In the absence of an effective influenza vaccine, treatment with Pam2Cys also allows the induction of influenza-specific immunity on exposure to the virus providing the opportunity for population immunity to be achieved naturally. Furthermore, treatment with Pam2Cys reduces the morbidity and mortality associated with secondary bacterial infections which are a major contributor to the mortality associated with influenza. The work described here provides an opportunity for clinical development of Pam2Cys to combat the influenza virus and other respiratory pathogens