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2011-2013 Daisy Sandhu and Milica Vukmanovic-Stejic
Project: The Basis For Decreased Responses To Recall Antigen Challenge In
The Skin During Ageing
Our group has developed a human model to allow us to study the memory T cell
response in the skin of healthy young and old donors, as well as patients with skin
diseases or cancers. Our earlier studies using this experimental system have already
demonstrated that old humans have decreased cutaneous responses to injected
recall antigens. Preliminary data indicates that there is defective recruitment of
memory T cells into the skin after antigen challenge and that this may be due to
defective macrophage secretion of TNF- which can also be related to increased
abundance of regulatory T lymphocytes.
Over the last 2 years we focused on investigating defective immune responses in old
individuals. We use Varicella zoster virus, which is responsible for chickenpox and
reactivation of which in later life causes shingles. This is a good model to understand
the key cellular and soluble mediators required for effective immunity in the skin.
We have been studying immune responses to VZV by injecting old and young
volunteers with VZV antigen into the skin of the forearm and subsequently sampling
the injected skin site by either taking a biopsy or raising a suction blister over it and
analysing the blister fluid. More recently we
have adopted a technique of digesting skin samples to isolate the cells of the
immune system for further analysis. As soon as we are confident that the technique
can produce reliable and reproducible results we will initially recruit healthy old and
young volunteers and eventually hope to enlist those with active skin diseases.
There were three main areas in the last year:
1.We focused on early stages of the response, as this is the time where the decision
to mount or not mount an effective immune response is made. Looking at these early
time points we have found that although blood vessel activation is impaired 24 hours
after injection of the VZV antigen in our older volunteers, this is not the case at 6
hours after injection when activation appears normal. Blood vessel activation is
essential for recruiting cells to mount an appropriate immune response. From this we
concluded that there were some missing signals in the old skin- most likely soluble
inflammatory mediators which were necessary for adequate activation of the
endothelium between 6 and 24hrs post challenge.
2. In collaboration with Prof Kreuger’s group at the Rockefeller University in New
York we have generated and analysed transcriptional profiles (mircroarray data) from
normal skin, and VZV or saline injected skin at 6hr and 72hr post injection. To do this
we have recruited 8 young and 8 old people to undergo skin testing with VZV skin
test and saline as control (representing non-specific response). Punch biopsies were
performed at the injection sites at 2 different time points and the tissue was used for
RNA preparation. This gave us materials to analyse gene expression at different time
points and to compare effects of Ag versus non specific injury, at different time points
and in different age groups. We have found that, in agreement with our data from
histological analysis, a strong inflammatory response is induced by VZV at 72 hrs in
young but not in old individuals. Interestingly, even at 6hrs the inflammatory response
to VZV was stronger in the young compared to old skin. However, old individuals
appear to respond more strongly to saline injection suggesting that they are more
prone to non-specific inflammation than the young. We are currently analysing data
from normal skin to understand if this increased inflammation is the result of saline
injection or if the old skin per se shows increased background inflammation. This
type of analysis gives insight into crucial molecules that are present/ absent during
the immune response and highlights those proteins of interest which we will now
scrutinise further.
3. Much of the blister work over the last year has concentrated on investigating the
relationship between varicella zoster virus (VZV) specific memory CD4+ T cells and
CD4+ Foxp3+ regulatory T cells (Tregs) that accumulate after intradermal challenge
with a VZV skin test antigen. Specifically we focused on identifing regulatory T cells
which are specific for the VZV virus. Although identifiable, they are present in very
low frequency. These VZV specific regulatory T cells may play a role in limiting
immune responses in the skin in older individuals as increased numbers of Foxp3+
Tregs in the skin of old individuals were associated with a significantly decreased
cutaneous reaction, confirming that these cells had inhibitory activity.
In progress/ future plans:
During this year we have also recruited 20 healthy old individuals to undergo
vaccination with Zostavax, standard vaccine used for prevention of shingles. In these
individuals we have collected samples pre and post vaccination in order to
investigate changes in cutaneous responses as a consequence of vaccination.
Samples are collected for transcriptional analysis (again both saline and VZV injected
skin, at 6 and 72 hours post injection from each individual) as well as histological
analysis of infiltrates at 3 and 7 days after VZV challenge. We hope to be able to
identify transcriptional changes that precede T cell infiltration in those individuals
which have been induced to respond well to VZV (majority of old individuals show
improved clinical response to VZV challenge following vaccination). This should
improve our understanding of defects which result in the reduced cutaneous
immunity in old individuals. These samples are currently being processed.