The regulation of CD8+ T cells by nutrient availability
The aim of this project was to explore how nutrient availability control CD8+ T lymphocytes. The focus was the regulation and function of autophagy as CD8 T cells encounter amino acid deprived conditions. The study also examined the role of the AMP dependent kinase in CD8+ T cell responses to metabolic stress. To study autophagy a novel fluorescent autophagy reporter mouse model was used that allowed for quantitative analysis of autophagic flux at the single cell level in T lymphocytes. Using this fluorescent reporter naïve T cells were shown to have high levels of basal autophagy, and this was required to maintain cell survival and expression of the surface molecule L-selectin. Immune activation of CD8+ T cells resulted in downregulation of autophagy. The downregulation of autophagy was dependent on the availability of extracellular glucose and amino acids. We established that T cells switch off autophagy when they respond to immune activation and increase amino acid transport: they revert to autophagy when their capacity for amino acid transport is low. Effector CD8+ cytotoxic T cells generated in vitro displayed low levels of basal autophagy. This was increased upon removal of key cytokines or amino acids. It is generally considered that the amino acid sensing pathway that controls autophagy is mediated by the serine/threonine mTORC1. The current data found a modest role for mTORC1 as a regulator of autophagy in naive T cells and no role for mTORc1 in CTL. These data highlight that the regulatory processes that determine autophagy are not universal.To examine the role of autophagy in CD8+ cytotoxic T cells we used an inhibitor of VPS34, a phosphoinositide 3-kinase (PI3K) class III isoform that controls T cell autophagy. A key strategy was to use mass spectrometry to explore how Vps34 signalling pathways control the proteome restructuring that occurs in amino acid deprived CD8+ cytotoxic T cells to gain insights about how autophagy controls T cell function. Quantitative analysis of >6000 proteins identified ~1300 proteins which are degraded by VPS34 dependent autophagy. These included cytolytic effector molecules, nutrient transporters, regulators of protein translation, cell survival and cell cycle progression. The experiments also identified 96 proteins whose expression requires autophagy to be maintained under conditions of amino acid deprivation. These data provide a new understanding how T cell phenotypes are selectively shaped by autophagy and give insights that explain how autophagy controls T cell survival and the transition of effector T cells to memory cells. At the beginning of this PhD project there was widely held belief that a key regulator of autophagy is AMPK activated in response to glucose deprivation (Hardie, 2011). The present data found no evidence that glucose deprivation or AMPK regulated autophagy in cytotoxic T cells again highlighting that some of the well characterized autophagy pathways are not universal to T cells. We did however find experimentally that AMPK acts intrinsically to restrain T cell anti tumor effector function in a mouse tumour model system. The use of mass spectrometry to explore the impact of glucose deprivation on the CTL proteome also gave some new understanding of how glucose deprivation modifies the functional capacity of CTL by regulating the expression of cytolytic effector molecules, key transcription factors, cytokine receptors and the CTL protein synthesis machinery. Overall this project has been able to highlight how the ability of CD8+ T cells to react to metabolic stress through proteomic remodelling can impact CD8+ T cell effector function, survival and proliferative capacity.
Immunology, Autophagy, Metabolism, T cells
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