The integration of human T cell senescence and function at the molecular level

Immunity decreases during ageing, resulting in the increased susceptibility of older individuals to both infections and cancer. The powerhouse of immunity is the T lymphocyte, that recognizes and combats malignant and infected cells. However these cells have to expand in number, akin to the mobilization of an army, to combat the infectious or cancerous challenge. This mobilization takes time and during this lag period, protection is offered by another population of leukocytes in the front line known as natural killer (NK) cells, that are less specialized in the recognition of the invaders but highly efficient in killing them. Efficient immunity therefore, requires that the 'beserker' NK cells that are involved in the first line of defense and the specialized T cells that are mobilized later fulfill their role at different phases of the war.

In earlier studies we showed that T cells get older progressively as a result of repeated immune challenges and in older humans, immune protection is mediated by the equivalent of "Dad's Army" soldiers that do not function as well as younger leukocytes. The changes in old T lymphocyte population includes the decrease in their ability to proliferate and increase in numbers after immune activation, this expansion is essential for increasing the number of T cells that can seek and destroy the invaders. We showed that the decreased activity in old T cells was due to an active signaling process mediated by p38 MAPkinase and that by blocking the activity of this molecule, we could enhance proliferative activity. p38 is one of a family of distinct MAP kinases, others include JNK. Our preliminary results indicate that the T cells in old individuals also express JNK spontaneously, without the requirement of any additional activation and that these molecules may also regulate the function of T cells. Thus while we showed that p38 was a brake on lymphocyte function, our recent preliminary observations show that JNK is an additional brake, that may be engaged simultaneously. Both brakes work to inhibit different processes. Furthermore, both brakes are engaged by the energy sensor molecule AMPK that is activated by low nutrient availability or the process of ageing in the cells.

We will investigate whether blocking JNK, using novel reagents that we have developed in the laboratory can restore additional functions to those that are regulated by p38 in these old T cells. Furthermore, we will determine whether either or both p38 and/or JNK MAP kinases regulate the NK-like function of old T cells.

Another novel part of this investigation is the use of a human experimental system where we induce and antigen specific response by injecting chickenpox virus proteins into the skin to induce a memory immune response that we can harvest cells from. This is a totally unique model that has been developed and refined in the Akbar lab over 18 years. All the appropriate safety and ethical approvals are in place to do this. This will enable the study of how quickly leukocytes age in the skin during an immune response and the role of AMPK and the MAP kinases in this process. We will also be able to determine when T cells start to develop NK characteristics in real life, after immune stimulation in vivo.

These studies will identify changes that occur in the early and late phases of the immune response and how ageing affects this. More importantly, we will determine whether it is possible to enhance the activity of old T cells by removing the MAP kinase brakes that will provide proof of principle of whether we can enhance the activity of these cells during ageing.

We will use of freshly isolated T cell populations fromperipheral blood of young (<35 years) and older humans (>65 years). We will also isolate T cells from the site of recall antigen challenge in the skin of humans using suction blister technology that we developed. We have specific recruitment programmes for this and the ethical approval is in place for these studies. This project involves analysis of human leukocyte populations by multi-parameter flow cytometry. Using cell surface marker panels we will investigate T cells within mixed populations of leucocytes by flow cytometry or isolate these populations of cells by cell sorting. We will also separate these cells using magnetic bead technology. In addition, we utilize Western Blot to confirm flow cytometric data on expression of phosphorylated and non-phosphorylated signaling molecules. We will analyze the interaction between different molecules by co-immunoprecipitation followed by Western Blot analysis. All these techniques are currently in use in my laboratory.

The proliferative assays of isolated T and NK cells include staining with the cell cycle marker Ki67, dye dilution or trittiated thymidine uptake assays. Cell death is measured by propidium iodide and annexin V staining. Cytokine production is measured by intracellular cytokine staining and telomerase activity using the telomere repeat amplification protocol (TRAP). We will measure cytotoxic activity by CD107a staining that indicates cytotoxic deganulation of these cells and also by a caspase 3 activation cytotoxic assay.

We will transduce primary T cells with lentiviral vectors (pseudotyped with glycoprorein G of vesicular somatitis virus) containing short hairpin interfering RNA specific for AMPK, p38, JNK or ERK kinases to block the activity of these specific signaling components on functional activity.

The UK is shifting in demography towards a more aged society. Ill health in old individuals is often associated with the increase in the incidence of infections that is related to impaired immunity. This study will provide new information on the nature and the possibility for manipulation of defective T lymphocyte responses during human ageing.

Scientific excellence:
Two recent paradigm shifting papers were published by the Akbar group in 2014 in Nature Immunology (Lanna A et al 2014) and the Journal of Clinical Investigation (Henson SM et al 2014) show that cell metabolism and cell senescence are linked by the same signaling pathways and that this pathway can be manipulated to boost the function of human T lymphocytes. This has considerable implications for immune enhancement during ageing. These papers have been highlighted on the BBSRC Business Magazine (http://www.bbsrc.ac.uk/news/health/2014/140826-pr-are-you-as-old-as-what-you-eat.aspx), and in the Daily Express (Change in diet can slow rate you age at Daily Express 25 Aug 2014, p4. and in the Science Daily (http://www.sciencedaily.com/releases/2014/08/140824152345.htm). In addition, the UCL Media Department produced a video podcast that highlights this work that has been posted on Youtube (http://youtu.be/oQ-unC7D9i4) that has been featured on the BBSRC, MRC and also the UCL website. This media coverage highlights the scientific impact of our studies and the current proposal builds on the investigations cited above.

Pharmaceutical Companies:
1. We previously showed that blocking of p38 signaling either directly or indirectly, by targeting upstream molecules such as AMPK, can enhance the proliferation of T cells. We will now test the hypothesis that AMPK integrates p38, JNK and ERK activation in T and that the inhibition of AMPK or the MAP kinases can enhance their functions. It is of note that p38 inhibitors that have been through phase II and phase III drug trials have been developed by many different pharmaceutical companies and our investigations may build a case for drug repurposing. Akbar is PI on a MRC Experimental Medicine Grand Challenge (MICA) Grant that uses a GSK p38 inhibitor to inhibit inflammation during cutaneous recall antigen challenge in older subjects.

Health Care Policy: If this study identifies ways to improve the immune response in elderly subjects, this may ultimately lead to significant changes in our ability to immunize the UK population, maintaining their health during ageing.

The Ageing Public: We aim to establish a close dialogue with the older community who are the key stakeholders in this work. In July 2014 and December 2014, my research group hosted public engagement events aimed specifically at older adults. There were ~50 attendees at each meeting. The aim of the event was to contact and engage with older adults from within the local community and UCL to inform past, present and potential blood volunteers of the important research that has and is taking place on ageing immunity in my group. The intention was to make our research understandable and accessible to all. In addition, I have given talks to publicize our work to lay audiences at the Cheltenham Science Festival in 2013 and the University of the Third Age in 2013 and 2015. I was interviewed for a programme on BBC Radio 4 hosted by Ms. Vivienne Parry that discussed ageing and immunity (http://www.bbc.co.uk/programmes/b012qtw0). This publicity underscores the impact of our work.