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

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.

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.

The goal of this study is to identify whether human CD8+ T cells specific for persistent viruses that have the best antigen binding avidity are lost through repeated lifelong re-stimulation, leaving less avid cells with lower functionality, to combat these organisms during ageing. Further an inherent characteristic of the immune system is the capacity for immunosurveillance. Neither T cell avidity nor the migratory properties of T cells in older humans have been investigated in detail previously. A third novel element in this proposal is the investigation of the effect of induced experimental stress on the blood donor on leukocyte mobilization during ageing. All three key points will provide important new information on why older humans may be more susceptible to infections and malignancy. 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. To ensure that the wider scientific community benefits from this research we will disseminate our findings through the usual literature and conference route. We will make these tools available to the academic community through the usual collaborative arrangements. The results we will generate on the effects of psychological stress on the mobilization of leukocytes will provide a better understanding of the mechanisms underlying the common anecdotal observation that stress reduces immunity. In particular, we will obtain data on whether older humans have increased susceptibility to this phenomenon. We envisage that our route to allowing older people to benefit from our research will come via longer-term engagement with pharma and drug development to identify mediators to reduce stress and/or increase virus-specific T cell mobilization.

This study involves the use of unique reagents that will be provided by Prof. Andy Sewell in Cardiff. The tetramers his lab will produce will be used to explore the quality of virus specific T cells during ageing. Prof Sewell has had extensive BBSRC funding but does not work on ageing per se. This project will bring his extensive expertise on dissecting the nature of the T cell receptor and investigating clonal evolution into the ageing research arena.

Very few current interventions are directed at investigating the non-disease-related functional decline during old age such as transient stress. Thus this project meets the BBSRC strategic priority of "Ageing research: lifelong health and well-being". 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. Prof Akbar has been invited to give talks about his research to older audiences at the University of the Third Age, a seminar series organized for retired doctors at the Royal Free Hospital. He has also been interviewed by the Daily Telegraph and also Radio 4 about immunity and ageing. He has agreed to give 2 talks this year at the Cheltenham Science Festival on aging and immunity. Therefore by disseminating the results on ageing research from his group by public engagement, this application has the potential to have economic and social impact in the future, that is a BBSRC policy priority.