Reversing senescence and exhaustion signalling pathways in primary human T lymphocytes during ageing

The potency of the immune system should not be undrestimated. Without effective immunity our bodies would turn into rotting carcasses within a few days. However, such a potent defense against invading microorganisms has to be effectively controlled as there would be dire consequences if our immune systems turns against ourselves. There are two processes that control immunity the first is a process called exhaustion, where the over activation of white blood cells known as T lymphocytes leads to loss of their functional activity. This is achieved through binding of surface proteins on the T cell surface that inhibit the production of toxins that kill microorganisms properly and also prevent their proliferation. Exhaustion is therefore regulated by the signalling through cell surface inhibitory receptors. Secondly, the repeated stimulation of T lymphocytes leads to the loss of their replicative capacity and this is due to the loss of specialized structures at the end of chromosomes that are made of DNA and are known as telomeres. Excessive loss of telomeres initiates signalling pathways that sense DNA damage that turn off the capacity of the cell to proliferate. This process is known as senescence and unlike exhaustion, is initiated by signals from damaged DNA in the nucleus.

As we get older (>70 years), we become more susceptible to infections, even those to which we were immune in our youth. This indicates that the immune system becomes too fragile to be fully effective in preventing the lifelong assault on our bodies by micro-organisms. However the reasons why the T lymphocytes (white cells) isolated from old individuals are dysfunctional is not known. It is well recognized that the T lymphocytes from older humans have characteristics of senescent cells however it is not know if they may also be exhausted. We have recently found that in young individuals (<40 years), we can block proteins in the cell that regulate senescence or proteins on the cell surface that regulate exhaustion to increase their function. The key question is whether we can boost the function of T lymphocytes from older humans by blocking senescence pathways, exhaustion pathways or both. This is an important goal given the demographic shift of humans towards an older age with defective immunity that leads to increases of infection and malignancy.

The main aims therefore are:
1) To investigate the expression of senescence and exhaustion control elements in the T lymphocytes of older humans.
2) To determine whether active mediators called cytokines that are increased in the blood of older humans can turn on senescence or exhaustion pathways.
3) To investigate if we can block senescence or exhaustion in T cells from older humans using specific inhibitors to boost their function and capacity to proliferate (we have shown recently that this can be done in young subjects).
4) To investigate whether blocking senescence or exhaustion signalling can increase the functional activity of T cells of older humans that are specific for cytomegalovirus, that appear senescent, or varicella zoster virus that causes shingles, the incidence of which is increased in older humans.

All this work will be performed on leucocytes that are obtained directly from the blood of older humans and therefore has directly relevance for the ability to boost the defective immunity that occurs during ageing in humans.

This study involves the use of freshly isolated leucocyte populations from the peripheral blood of older humans (>70 years). We have specific recruitment programmes for these individuals in place and two research nurses who perform the blood taking. Using cell surface markers, we either investgate specific T cells within mixed populations of leucocytes by flow cytometry or isolate specific T cell subsets by FACS sorting. The functional analyses that will be performed include proliferative assays involving staining with the cell cycle marker ki67, cell death by incorporating propidium iodide and annexin V staining, cytokine production by intracellular cytokine staining and telomerase activity using the telomere repeat amplification protocol (TRAP). We will also introduce specific signalling proteins into primary human T cells by transducing them with VSV vectors to examine directly the role of specific signalling components on functional activity.

Our laboratory specializes in the investigation of small populations of T cells. We will investigate varicella zoster specific CD4+ and CD8+ T cell populations by 16 hour stimulation of PBMC with VZV antigens in the presence of brefeldin A, followed by intracellular cytokine staining with IFN-gamma. In individuals who are HLA-DR15 positive, we have access to a class II tetramer and donors who are HLA-A2 we will use class I tetramer both directed against immediate-early 1 protein. We also have MHC class 1 tetramers to immunodominat epitopes of the CMV pp65 protein.

We will block senescence signalling using the specific p38 inhibitor BIRB 796, that blocks all isoforms of this kinase. We will block PD-1 signalling by blocking its ligands using a combination of anti-PDL-1 and anti-PDL-2 antibodies.

Immune function declines as we age resulting in an increased susceptibility to new infections and re-activation of latent pathogens to which we were once immune. This suggests that lifelong antigenic pressures drive specific T lymphocytes to a functional and/or replicative end-stage, when they are no longer able to support effective immunity. Key ambitions therefore are to define mechanisms associated with human T cell senescence and exhaustion, which are regulated by different triggering events and signalling pathways and to manipulate these to improve the immunity of older humans. The Akbar group is expert at and specializes in the study of human immunity directly ex vivo. The proposed work does not involve an artificial expermental cellular system or animal models but instead involves the direct study of leucocytes from old humans. Therefore the results obtained are of direct relevance to human immune ageing. To ensure that the wider scientific community benefits from this research we will disseminate our findings through the usual literature and conference route. Also, a recent publication by the Akbar group that arose from BBSRC funding, was highlighted by the BBC Online Science (http://news.bbc.co.uk/1/hi/health/8226129.stm). This work was on defective immunity in the skin in ageing and highlights our ability to disseminate significant findings to a wider audience. The data that will be generated on the effect of p38 blockade on restoring function of T cells , will provide a platform for discussions with Pharmceutical companies such as Pfizer, Roche, GSK and Bristol Meyer Squibb, that currently have ongoing clinical trials or licenced treatments for diseases including rheumatoid arthritis, psoriasis, COPD, atherosclerosis etc. using p38 inhibitors. Our findings may identify mechanisms by which to interrupt the senescence and/or exhaustion programmes in human T cells, which in turn would provide opportunities for boosting immune responses of older individuals. Very few (if any) current interventions are directed towards the non-disease functional declines of old age.

This work has a strong international profile. Prof Akbar has received a BBSRC-NIA Trans-Atlantic Collaborative award to align research in mechanisms of T cell ageing in human and murine experimental systems. The partnering group in the USA is headed by Dr. Janko Nikolic-Zugich at the University of Arizona. This collaboration adds value to the current application and the interchange of ideas, reagents between both groups has already been extensive. Prof Akbar is also investigating ageing-associated mechanisms in T cells with Dr. Jim Kreuger and Dr. Elias Coutavas at the Rockefeller University in New York and the travel and subsistence for this is provided by a BBSRC USA Partnertship grant. Thus extensive BBSRC support to the Akbar group has culminated in the strategic and productive engagement with international partners in ageing research.

Thus this project meets the BBSRC strategic priority of "Ageing research: lifelong health and wellbeing". In addition, initial development of routes to intervene to abrogate the deleterious side effects of immunosenescence in older people 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. This application therefore has the potential to have economic and social impact in the future, that is a BBSRC policy priority. The applicant has already been active in promoting this particular agenda and will continue to drive it forward through scientific policy and public fora.