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.