Phenotype and number of regulatory T cells present in peripheral blood of COPD patients versus healthy controls

COPD is a leading cause of death worldwide and its morbidity and mortality are
still rising. Although the pathogenesis of the disease is still largely
unknown, smoking is widely accepted as the most important cause for development
of the disease. So far, no effective treatment is available to arrest the
accelerated lung function loss associated with COPD, nor is there a cure. To
find better treatment methods more insight is needed in the nature/origin of
the chronic inflammation that underlies the development of COPD.
The important role of neutrophils, macrophages and cytotoxic T cells is well
established in this respect, yet the role of CD4 T cells and B cells has only
recently (re)attracted attention. We detected the presence of lymphoid
follicles in lung tissue of COPD patients, consisting of B cells surrounded by
T cells. Recently, we have found the presence of Foxp3 positive T cells as a
component of these lymphoid follicles in COPD. Since Foxp3 is a distinctive
marker of regulatory T cells (Tregs), this finding suggests that Tregs are
involved in the inflammatory response in COPD.
Tregs have been subject of investigation in allergy and asthma. They are
important in controlling immunological tolerance and preventing auto-immune
responses by inhibiting T-cell responses. Dysfunction of Tregs can lead to
auto-immune diseases, allergy and chronic inflammatory diseases. However,
nothing is known so far about their contribution to the chronic inflammatory
response in COPD. The currently best described subset of Tregs is that of the
naturally occurring Tregs. Naturally occurring Tregs are generated in the
thymus and express CD4, CD25 and Foxp3. Recent studies show that, next to
direct inhibition by cell-cell contact, the inhibitory effects of Tregs are
mediated by heme oxygenase-1 (HO-1) expression and membrane bound TGFβ.
We hypothesise that a dysfunction of regulatory T cells underlies the
development of the (antigen driven) inflammatory response in COPD. This could
be due to a decreased presence of Tregs in COPD, or to an altered function of
Tregs. The latter may be due to a decreased HO-1 expression, as we have shown
in macrophages of COPD patients compared to those in healthy controls, and/or
an altered TGFβ regulation, a cytokine that plays a prime role in COPD.

Research questions:
Is the chronic inflammatory response in COPD caused by a dysfunction of
regulatory T cells?
1. Is the number of Tregs decreased in COPD?
2. Is the function of the Tregs impaired in COPD? And if so,
a. Is this associated with a decreased HO-1 expression?
b. Is this associated with an altered TGFβ regulation (e.g. by the signalling
of SMAD7)?

Experiments:
1. Treg numbers and HO-1,TGFβ, Smad 7 and Foxp3 expression in COPD patients
compared to controls
First it is important to investigate whether COPD patients have lower numbers
of Tregs than healthy individuals and whether COPD patients have Tregs with an
altered HO-1, TGFβ, Smad 7 or Foxp3 expression.
Numbers of Tregs (CD4, CD25, Foxp3) present in freshly isolated lymphocytes
from peripheral blood from COPD patients will be analysed using flowcytometry
and compared with age and smoking history matched healthy controls. In addition
the expression of HO-1, TGFβ and Smad 7 will be analysed in these cells.

2. Treg function in COPD

To study the function of Tregs in COPD patients the inhibitory capacity of the
Tregs will be investigated with proliferation assays and the cytokine
production will be measured.
CD4CD25high T cells will be freshly isolated from peripheral blood of COPD
patients and healthy individuals and will be co-cultured with stimulated
CD4CD25neg T cells (CD3/CD28). Proliferation of the CD4 T-cell population will
be analyzed by 3H-thymidine incorporation and the cytokine production of these
cells (IL-2, IL-10, and TGFβ) will be studied using ELISAs.

3. Involvement of TGFβ signalling

To investigate whether TGFβ signalling is involved in the suppressive effect of
Tregs and is altered in COPD, the expression of Smad 2, 3 and 7 will be
analysed.
CD4CD25high T cells will be freshly isolated from peripheral blood of COPD
patients and healthy individuals and will be co-cultured with stimulated CD4 T
cells (CD3/CD28). Intracellular expression of Smad 2, 3 and 7 will be analysed
with flowcytometry in CD4 T cells and CD4CD25 Tregs after co-culture and in the
single populations without co-culture.

Participants involved in this study are asked to visit the lung department of
the UMCG two or three times.
At the first visit there will be a short interview with the physician to check
their current health status, participants will perform spirometry to check for
their lung function, and a skin test to check for allergies. In addition, they
will be asked to fill in a Clinical COPD Questionnaire (CCQ). When a
reversibility test is not necessary participants will also perform a
methacholine provocation test to measure the hyperreactivity (PC20) and donate
20ml of peripheral blood for the flowcytometry experiments during the first
visit. Healthy controls do not need a reversibility test for inclusion in the
study and will perform a methacholine provocation test during the first visit.
COPD patients with a known FEV1 < 80% predicted and an FEV1/FVC < 70% measured
during the last 5 years also do not need a reversibility test, but when this
information is not present a reversibility test is required for inclusion and
the methacholine provocation test has to be performed at a second visit.
At the second or third visit, participants are asked to donate 80ml of
peripheral blood for experiments 2 and 3.

Risk for the participants in this study is are:
• Dyspnea during methacholine provocation test
• Irritated skin after skin test
• Haematoma from the donation of blood.
Participants have to stop using (inhaled) corticosteroids 6 weeks before visit
2 and 3, because this medication can influence the results of the study.