The functional and migratory characteristics of low avidity virus-specific T cells during ageing

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We will investigate freshly isolated leucocyte populations from the blood of young (<40 years) and older humans (>70) as well as lymph node and bone marrow samples paired with blood from healthy individuals. We will either investigate specific T cells within mixed populations of leukocytes by flow cytometry or isolate specific T cell subsets by FACS sorting. Proliferative assays involving staining with the cell cycle marker ki67, cell death by incorporating propidium iodide and annexin V staining, cytotoxicity by degranulation and cytokine production by intracellular cytokine staining will be performed. Fluorochrome labelled MHC class I tetrameric complexes (tetramers) will be used to recognize specific T cells for viral antigens such as cytomegalovirus and Epstein-Barr virus. Furthermore, we will use tetramers that have been engineered to have an altered alpha3 domain in the conserved binding site for CD8 so that only T cells with receptors that bind very avidly to the viral peptide will bind the mutated tetramer. We will use conventional (binds low and high avidity T cells) and mutated tetramers (only binds high avidity cells) containing the same viral peptide to test virus specific cells in the same individual. The clonal diversity of low and high avidity T cells will be compared in freshly isolated T cells and following repeated antigen stimulations by TCR sequence analysis. We will investigate telomere length by fluorescence in situ hybridization coupled to flow cytometry (flow-FISH). We have developed extended our multi-parameter flow-FISH technology to enable the detection of surface and cytoplasmic markers together with telomeres in the same tetramer positive virus-specific T cell. We will use time-lapse microscopy as part of an existing collaboration with Prof. John Greenwood to investigate the migration of T cells across endothelial monolayers. We will also induce mild stress in volunteers using a defined speaking task that was used in Dr. Riddell's PhD.

This project directly addresses the BBSRC strategic priority of "Ageing research: lifelong health and well-being". To date, T-cell immunity in older humans has been considered to be the same as that in younger people. Emerging evidence suggests that this may not be the case and that immunosenescence in the T-cell compartment might explain why older people are more likely to succumb to infections and malignancies.

It is well established that T-cell populations specific for persistent viral infections accumulate over the human life course. Recent experiments in the laboratory of Professor Arne Akbar at UCL indicate that the T-cell populations for these viruses have a lower overall avidity for their cognate antigen in older people than in the young (i.e. responses are weaker in the elderly). This study will examine whether the cells with the greatest sensitivity for antigen are lost during aging as a result of repeated lifelong re-stimulation thereby leaving only less avid cells with lower functionality, to combat these organisms in old age.

The Cardiff group has produced unique reagents that stain T-cells that only have a high overall avidity for cognate antigen. If funding is forthcoming this powerful collaboration will combine the Cardiff expertise in T-cells, TCRs, T-cell antigens and the clonotypic analysis with the world-leading expertise in immune aging in the laboratory of Professor Arne Akbar. This partnership will enable the first examination of T-cell avidity and migration over the human life course. We will further study the effects of psychological stress on the mobilization of leukocytes to provide a better understanding of the mechanisms underlying the common anecdotal observation that stress reduces immunity.

The biggest impact of this work will be to show that the sensitivity of important parts of our immune system decreases with age. Such immunosenescence has not been properly considered to date. We anticipate that the hard molecular proof of senescence in the T-cell compartment over time will result in a shift in how we view an older immune system and thereby lead to further studies in this increasingly important area.

The immediate beneficiaries of this work will be the wider biomedical community of researchers who have an interest in T cell biology and its role in human health and disease. We also expect that our work will be of benefit to the elderly by showing that this grouping needs to be considered distinctly when it comes to vaccination etc. thereby directly affecting the nation's health and increasing the quality of life.

Very few (if any) current interventions are directed towards the non-disease functional declines of old age such as transient stress. In addition, initial development of routes to identify the deleterious side-effects of immunosenescence in the elderly would provide a concrete "worked example" through which to engage with the public and policy makers on the topic of research for a better old age.