Background and previous work
The ageing process involves a decline in the immune competence, which is characterized by functional and phenotypic alterations of cells of the immune system but also by changes in the macro- and microenvironment. This immunosenescence process in the elderly population is accompanied by an increased susceptibility to infections and cancer, a reduced efficacy of vaccination and an increased frequency of autoimmunity. The most prominent effects involve changes in T-cell function and phenotype concomitant with the presence of low-grade inflammation. Both immune responses and ageing processes can be enforced by the potentiation of NF-B transactivation efficiency, whose regulation involves acetylation and deacetylation of the subunit p65 by histone acetyltransferase p300 and deacetylase SIRT1, respectively. In previous work, we have characterised the differential protein expression pattern in T cells in response to oxidative stress and targeted therapies using proteome-based technologies. In addition, immunomonitoring of peripheral blood mononuclear cells and of tumour infiltrating lymphocytes from patients with renal cell carcinoma revealed a reduced frequency and activity of effector T cells in the elderly when compared to younger renal cell carcinoma patients.
Reversible acetylation on the ε-amino group of lysines has emerged as an important PTM with a crucial role in regulating the biology of its target proteins. This modification is the target of many sirtuins, NAD+-dependent deacetylases that control key physiologic processes involved in the health and lifespan of mammals. Therefore, the aim of this study is to characterise the acetylome of T cell subpopulations during ageing and to identify agerelated changes due to modulation of sirtuins or other deacetylase activities.
The global protein acetylation status will be analysed by immunoblotting, subcellular
fractionation (extranuclear/nuclear) and pull-down assays with PTM-specific antibodies.
Tryptic digestion of proteins isolated from different subcellular localisation of CD4+ and
CD8+T cells from young and old healthy donors, purification with an anti-acetyl-lysine
antibody and subsequent analysis via multidimensional liquid chromatography-tandem
mass spectrometry (LC/LC-MS/MS) will shed light on the age-related changes within the lysine acetylation pattern, which is responsible for reduction of the cognate function of CD4+ (helper) and CD8+ (cytotoxicity) T cells. In long term, these analyses will be extended to young and old tumour patients. We will collaborate with a number of projectsin particular regarding mass spectrometrical analysis (SP2, SP3, SP6, SP7, SP8 and SP12) or signal transduction pathways and their modulation in T cells (SP13).