Telomerase downregulation in memory CD8+ T cells by cytokine and surface inhibitory receptor signalling

Old individuals are very susceptible to infections. This suggests that an immune defect occurs during ageing. However, the nature of this defect at the molecular level is not known. One of the key protective cells of the immune system is the cytotoxic T lymphocyte that is a very effective safeguard of our health in youth. However, these cells lose vigour as we age and this may explain the increased incidence of infection. To mount an immune response, there is a massive recruitment drive in order to mobilize the immune army. Recruitment increases the number of cytotoxic combatants that are essentail to clear the infection. This increase occurs as the cytotoxic cell is induced to divide and proliferate. Our previous data has shown that the cytotoxic cells from old individuals do not divide as well as those from young subjects because the activity of a crucial enzyme within these cells known as telomerase is lost. This enzyme confers unlimited proliferative capacity to cells. The studies that we propose here are to investigate the nature of the telomerase defect in old cytotoxic T cells, with a view to determine if thes changes are reversible. This has obvious implications for the improvement of health in old subjects. The studies that we hae proposed are based on our previous data that has been generated through BBSRC funding and is a logical follow-on of this work.

Ageing is associated with an accumulation of highly differentiated CD8+ T cells as defined by the loss of expression of the co-stimulatory receptors CD28 and CD27 and a lack of telomerase activity. Firstly we will investigate the mechanism of telomerase downregulation in highly differentiated CD8+ T cells. Akt is essential for the function of telomerase and our preliminary data suggests there is defective phosphorylation. We will purify CD8+CD28-27- T cells using the VARIOMACS and determine whether Akt is functional by analysing well known downstream targets using western blot analysis. This will indicate whether the lack of Akt phosphorylation is specific to hTERT function. Further analysis will also assess upstream regulators of Akt such as PDK-1. In addition, we will investigate the ability of the cytokines IL-2 and IL-15 to modulate survival, proliferation and telomerase induction of CD8+CD28-27- T cells. We have shown that IFN-a inhibits telomerase activity of CD8+ T cells. We will investigate whether this is through the transcriptional regulation of hTERT, Akt or PDK-1 or their phosphorylation status. We will also investigate the change in transcriptional profiles of genes associated with intracellular signalling pathways and telomerase activity before and after IFN-a treatment to enable a global assessment of telomerase-associated gene regulation. We will also analyse early and late differentiated CD8+ T cells with these methods. Finally we will explore the role that the surface molecule PD-1 has in the downregulation of telomerase activity. PD-1 is a negative regulator of T cell activation and is associated with 'exhausted' T cells. This molecule is known to inhibit the phosphorylation of Akt raising the possibility that it may directly induce telomerase downregulation. We will block PD-1 signalling in vitro in highly differentiated CD8+CD28-27- T cells and see if we can reverse the functional defects oberved.