Mechanism for CD8+ T cell recognition and removal of senescent tissue cells during ageing

The prevailing view is that the repeated stimulation of the immune system by microbes throughout life exhausts and ages particular types of white blood cells. This may be why immunity decreases during ageing. It was therefore surprising to note that instead of being decrepit, these old cells actually become very good at responding to infections in a promiscuous fashion and not by using their usually very specific microbe recognition apparatus. Therefore, during ageing, white blood cells adapt to respond more broadly to microbes rather than lose their function totally. Our preliminary experiments identify a group of proteins known as sestrins that regulate this exchange of functions. We will first explore the roles of the sestrins further, to understand the breadth of functional changes in human white blood cells that they can regulate. We will also complement these studies by studying white blood cells for old genetically altered mice that do not express the sestrins.

The accumulation of old tissue during ageing reduces organ function. Exciting new studies in the mouse have shown that the removal of these old tissue cells improves the function of many different organs and the mouse behaves more like a young animal. Therefore, the removal of old immune cells in tissues may be a strategy for rejuvenation of organ function leading to increased well-being during ageing. White blood cells can directly recognize and eliminate old cells in tissues and exploiting this interaction may improve age-associated organ dysfunction. White blood cells that have more promiscuous immune activity in older people can also recognise and clear old tissue cells. Therefore, the sestrins, that regulate the acquisition of this activity, may be indirectly involved in the removal of old cells in the body. We will explore further the interactions between white blood cells and old tissue cells to identify new ways by which they may interact together.

One unanswered question is why do old tissue cells accumulate in organs of aged people if the immune system can recognize and clear them? We think that old tissue cells can hide from white blood cells that are looking to kill them. This evasion strategy involves the induction of inhibitory receptors on the senescent cells in organs. We have identified one such inhibitory receptor, HLA-E and we will now investigate if others are also involved. Understanding the nature of this evasion mechanism could lead to the identification of new ways to enable the immune system to work more efficiently to clear senescent cells and improve organ function in older humans that would lead to an increase in health and well-being.

The final question is whether interactions between white blood cells and old tissue cells happen in real life and not just in a test tube. We have developed methods for investigating immunity in human skin. We take small skin samples from young and old individuals and look at the multiple different populations of immune and non-immune cells in the same tissue sample. We will also inject the skin with an immune stimulus and investigate the multitude of interactions between different cell types at different times after the initiation of the immune response. We will identify the different types of interaction between immune cell and senescent tissue cells in the skin of young and old subjects. This will provide a roadmap to how sestrins and NK receptors develop on T cells during an immune response and potentially identify which cellular interactions in the skin can be targeted to boost immunity during ageing.

Leukocyte populations will be isolated from peripheral blood of young (<35 yrs) and older humans (>65 yrs). We will analyse leukocyte populations and signalling events by multi-parameter flow cytometry, Western Blot or by confocal microscopy. We will isolate specific subsets by Fluorescence- or Magnetic-activated cell sorting. We will inhibit the the sestrins, NK receptors and/or ligands in primary human T cells by antibody blocking and by lentiviral transduction of vectors containing inhibitory shRNA.

We will investigate sestrin k/o mice to determine the role of sestrins on NK receptor expression in vivo. We will investigate if T cells that express NKR can kill NK targets in vivo using old wild type or sestrin k/o mice. We will deplete NK cells from wt and sestrin k/o mice using NK1.1 then inject NKG2D-ligand positive and negative cells (Rae 1+ or Rae 1-) labelled with high or low concentrations of CFSE in to the mice and check ratio of specific killing using a calcein release assay.

We have cryopreserved skin samples taken at 24, 48, 72 hours and 7 days after varicella zoster virus (VZV) antigen injection from healthy young and old volunteers. We have a minimum of 5 separate samples at each time point for these subjects. This will enable us to map the cellular interactions between leukocytes and senescent stromal cells during an human immune response in vivo using multiplexed histology. We will also probe other published gene expression datasets of human antigen-specific CD8+ T cells isolated at different times after induction of an immune response to determine the kinetics of NK receptor and sestrin expression in vivo. We previously probed one dataset taken after yellow fever virus vaccination (Akondy et al Nature 552, 362-367, 2017). This confirmed that both sestrins and NK receptors were expressed by CD8+ T cells in the circulation after antigen-specific activation. We will now investigate the expression of these molecules on CD8+ T cells in the skin.