Award details

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

Reference	BB/L005328/1
Principal Investigator / Supervisor	Professor Andrew Sewell
Co-Investigators / Co-Supervisors
Institution	Cardiff University
Department	School of Medicine
Funding type	Research
Value (£)	87,852
Status	Completed
Type	Research Grant
Start date	28/02/2014
End date	27/02/2016
Duration	24 months

Abstract

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.

Summary

Older humans are susceptible to infections and malignancy indicating that their immune systems become less effective. The continuous challenge by infectious agents, especially those such as viruses that persist in the body throughout life, may drive the reactive white cells towards exhaustion. Secondly, one of the main attributes of the immune system is the capacity of white cells to migrate from the blood to the tissues and back again, a phenomenon known as immunosurveillance. In this project we make use of reagents that enable us to investigate the quality of T cells as well as their capacity for migration in older humans, to determine if one or both of these essential functions are defective. We will also investigate the white cells in the blood and in the lymph nodes and in the blood and bone marrow in two different groups of individuals simultaneously. This is a unique opportunity as normally only the leukocytes in blood are studied in humans due to the difficulty of obtaining tissue samples. We have all the appropriate ethical approval to be able to perform these studies. This project will also utilize additional novel technology that we developed, to measure the telomeres, the equivalent of an ageing clock in white cells that react to viruses. The cells with long telomere have the potential to persist in the body while those with short telomeres (senescent) are close to running out of time and will be lost. Furthermore, by using time-lapse microscopy, we can visualize the capacity of white cells from old and young subjects to migrate across blood vessel cells that are grown in the laboratory. Collectively this will tell us about whether senescent cells, that have decreased function accumulate during ageing and whether these cells are defective in imunosurveillance. Stress is known to be bad for immunity, especially during ageing. The scientist for whom funding is being sought in this proposal, Dr. Natalie Riddell used a technique for generating mild stress in humans during her PhD studies. This involved asking volunteers to give a verbal account of a situation to a small audience of 3 people. Significantly, this mild stress in young subjects (<40 yrs) was able to induce the mobilization of white cells from tissues into the blood to the same extent as excercise. This method is therefore a surrogate assessment of capacity for immunosurveillance. What we will now investigate is the capacity of older subjects (>70 yrs) to mobilize white cells during this stress response and to determine the quality of the cells that are mobilized. These studies will provide new information on changes in the quality of immunity during ageing and also the ability of the immune system in older humans to respond to external influences. This is cross-disciplinary work that utilizes multiple technologies to understand the impact of ageing on human immunity.

Impact Summary

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.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Ageing, Immunology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/L005336/1 The functional and migratory characteristics of low avidity virus-specific T cells during ageing

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