© 2005 Hindawi Publishing Corporation
Mediators of Inflammation • 2005:3 (2005) 160–166 • PII: S0962935105501261 • DOI: 10.1155/MI.2005.160
Inflammatory Process of CD8+CD28−T Cells
in Induced Sputum From Asthmatic Patients
Agnes Hamzaoui,1,2Nawel Chaouch,1,2Hedia Gra¨ ıri,1,2Jamel Ammar,1,2and Kamel Hamzaoui2
1Department of Pediatric and Respiratory Diseases, Abderahmane Mami Hospital, Pavillon B, Ariana 2080, Tunisia
2Homeostasis and Cell Dysfunction Unit Research 99/UR/08-40, Medicine University of Tunis,
Secretary of State of Scientific Research and Technology, 1007 Tunis, Tunisia
Received 26 January 2005; accepted 22 February 2005
Previously unreported CD8+CD28−and CD8+CD28+T-cell subsets occur in healthy individuals and expand in patients suffering
from autoimmune disease. Here we studied, for the first time, the expression of CD8+CD28+, CD8+CD28−, and CD8+CD56+sub-
populations in induced sputum from asthmatics. Using sputum samples, purified CD8+T cells were stained for surface antigen
CD28, CD56, FITC-conjugated anti-perforin, and anti-IFN-γ. Cytotoxic activity was evaluated in a chromium releasing test. In-
duced sputum CD8+CD28−T cells were found to be more expanded and expressed low levels of IFN-γ in severe asthmatics than
mild asthma and age-matched healthy controls. The predominance of CD8+CD28−T cells can be in part explained by the expan-
sion of CD8+CD56+. CD8+CD28−T cells from severe asthmatics produced high intracytoplasmic perforin and exerted a potent
cytotoxic activity. Considering their phenotyping and functional properties, the CD8+CD28−T-cell subset may constitute an in-
termediate phenotype in the process of CD8+T-cell differentiation of effector-type cells in severe asthmatics. Functional studies
showed that CD8+CD28−T cells had cytotoxic function.
Asthma is one of the most common chronic diseases,
characterized by inflammation of the airways, with in-
filtration of lymphocytes, eosinophils, macrophages, and
mast cells . A critical role in the pathophysiology of
asthma has been attributed to the T helper 2 lymphocytes
(TH2) that promote IgE production and eosinophils acti-
vation by release of cytokines, such as interleukin (IL)-4,
IL-5, IL-10, and IL-13. However, the exact mechanisms
that cause this Th2 polarization remain to be elucidated
[2, 3]. CD4+T cells and their proinflammatory cytokines
role of CD8+cells were often overlooked. Severe asthma
involves different pathogenic pathways than mild form.
Pathogenic pathways in severe asthma are considered to
differ from those discussed during mild forms of asthma
as the inflammatory process resists to steroids, and no
usual anti-asthma treatment is efficient. We postulated
that CD8 cells, particularly cytotoxic T cells, are involved
in severe asthma. Human CD8+T cells comprise cells
that are in different states of differentiation and under the
control of complex homeostatic process. The CD8 T-cell
Correspondence and reprint requests to Kamel Hamza-
oui, Homeostasis and Cell Dysfunction Unit Research
99/UR/08-40, Medicine University of Tunis, 1007 Tunis,
subset can be classified according to CD28 surface marker
expression as either CD28+or CD28−, with different bi-
ological properties. The CD8+CD28+is predominant in
healthy individuals, whereas CD8+CD28−cells increase
during inflammation  and autoimmune disease model
[6, 7]. Human CD8+T lymphocytes are largely homoge-
nous at birth and essentially all express the CD28 recep-
tor, but healthy adults accumulate CD8+CD28−T cells.
Moreover, CD8+CD28−T cells are increased in numbers
latency, or other chronic immunostimulative conditions
[8, 9]. The origin of CD28−cells has been controversial,
but recent data show that they derive from their CD28+
counterparts . CD8+CD28−T cells are efficient pro-
ducers of a variety of cytokines [11, 12]; they express per-
forin and exert potent cytotoxic activity [9, 12, 13, 14].
Links between TH2 cells in asthma and NKT cells, a pop-
ulation of NK cells that express conventional T-cell recep-
tor particularly CD8 , were reported [15, 16]. These data
indicate new levels of complexity of interactions between
during an asthmatic response.
Therefore, in this study we asked whether there are
phenotypic differences in the induced sputum CD8 cell
types, including CD56 cells in asthmatic patients.
The aim of this study was to quantify the
CD8+CD28−, CD8+CD56+cells in mild and severe
asthmatics in comparison with a healthy control group,
and to specify the level of their cytotoxic activity.
2005:3 (2005) CD8+CD28−T Cells in Asthmatics161
Table 1. Subject’s characteristics. Values are expressed as mean (SEM). (†) denotes significant differences (P < .05) compared to the
same subset of mild asthmatic patients and healthy controls. Successful sputum induction was achieved in all patients.
Total cell count × (106/g sputum)
Squamous-cell contamination (%)
PATIENTS AND METHODS
Induced sputum samples were collected from 15 suc-
cessive patients with asthma (8 patients in severe asthma)
(Table 1). All were outpatients in steady state, regularly
followed by an asthma specialist. The severity of the dis-
ease was classified according to Gina recommendations
. The samples were obtained on the fixed day of the
visit. A precise history of the patient was previously ob-
tained with functional respiratory tests. The following
cases were excluded: acute exacerbation of asthma, con-
comitant respiratory infection, other pulmonary diseases,
Ten induced sputum samples from healthy subjects
(females, with a mean age of mean age 32.4 years; range
25–42 years), who had normal pulmonary radiographs
and showed no clinical signs of respiratory diseases, acted
as controls. Informed consent was obtained from all the
patients. The study was approved by our Ethics Commit-
Sputum induction and processing were realized fol-
lowing the method recently reported . Briefly, after
the inhalation of salbutamol (2 × 200µg), subjects were
asked to inhale sterile, pyrogen-free, hypertonic saline in
increasing concentrations for a duration of 10 minutes.
The hypertonic saline was nebulized via an ultrasonic
nebulizer. Subjects were encouraged to cough throughout
the procedure. Most patients were able to expectorate an
In order to reduce salivary contamination, plugs were
selected and transferred into an Eppendorf tube. A freshly
prepared 10% solution of dithiothreitol (1mL) (DTT)
was added. The tube was vortex mixed and the sputum
was incubated for 5 minutes at room temperature, filtered
through 52µm nylon gauze to remove debris and mucus,
and subsequently centrifuged at 450xg for 10 minutes.
The cell pellet was resuspended in phosphate-buffered
saline (PBS) in a volume equal to the sputum plus DTT
solution volume. Total cell counting was carried out in a
hemocytometer and the cell concentration was adjusted
In order to enrich CD8+T cells, CD4+T cells were de-
pleted from induced sputum cells using magnetic beads
(Dynal AS, Norway). Induced sputum samples were
treated with beads directly conjugated to a monoclonal
antibody (mAb) against CD4 (according to the manufac-
turers’s recommendations) and then exposed to magnetic
field. The unbound cells consistently contained less than
2% CD4+cells compared to 30%–40% CD4+cells in the
Specific staining of the respective cell surface
anti-CD8, phycoerythrin-conjugated anti-CD28, and
Anti-CD56 Cy-Chrome (Cy) (BD Biosciences, Franklin
Lakes, NJ) was also used. For intracellular detection of
perforin or IFN-γ, cells were stained for their surface
antigens, CD28 and CD8, with FITC-conjugated anti-
perforin, and for IFN-γ or control immunoglobulin,
respectively. Monoclonal antibodies were provided from
Becton Dickinson (San Diego, Calif).
conjugated anti-CD8, phycoerythrin-conjugated anti-
anti-CD8 or Cy-Chrome (Cy) anti-CD56 monoclonal
antibodies, respectively (Becton Dickinson). Cells were
simultaneously stained for 30 minutes at 4◦C and, after
washing with phosphate-buffered saline, fixed with
4% paraformaldehyde in phosphate-buffered saline for
60 minutes. For intracellular detection of perforin or
162 Agnes Hamzaoui et al2005:3 (2005)
Table 2. CD28 and CD56 expression on CD8+T lymphocytes obtained from healthy controls and patients suffering from mild and
severeasthma.Valuesareexpressedasmean ±SD.(†)denotessignificantdifferences(P < .01)comparedtothesamesubsetofhealthy
controls. (††) means significantly different than severe asthmatics.
72.7% ± 8.5%
9.9% ± 5.4%†
IFN-γ, cells were stimulated with 25ng/mL phorbol
12-myristate 13-acetate and 1µg/mL ionomycin in the
presence of 10µg/mL brefeldin A for 4 hours (Sigma,
St Louis, Mo). Cells were then stained for their surface
antigens CD28 and CD8 and, after permeabilization, with
FITC-conjugated anti-perforin, anti-IFN-γ, or control
immunoglobulin, respectively. Fixed cells were analyzed
on a FACSCalibur flow cytometry (Becton Dickinson).
Data were analyzed using WinMDI software.
The cytotoxic activity of CD8 T-cell subsets was eval-
uated in an anti-CD3-redirected cytotoxicity assay .
Briefly 5 × 105Fc-receptor-bearing P815 target cells were
labeled with 50µCi of Na
Cells were then washed three times and incubated for 30
minutes at 4◦C in the presence or the absence of 2µg of
anti-CD3 mAb. Gated and sorted CD28−cells were incu-
bated for 4 hours at 37◦C with 5 × 103P815 target cells
at E:T ratios ranging between 5:1 and 25:1. Supernatants
were then collected and counted. Specific cytotoxicity was
calculated as follows: cpm of experimental release − cpm
of spontaneous release / cpm of maximum release − cpm
of spontaneous release × 100. The SE of the mean per-
centage lysis never exceeded 7%.
51CrO4for 2 hours at 37◦C.
The Mann-Whitney test, the two-sided Pearson test,
and regression analysis by ANOVA were performed using
the SPSS program, version 10.0 (Chicago, Ill). Bonferroni
nificance was defined as P < .05.
The clinical characteristics and sputum cell counts
were as shown in Table 1. There were significant dif-
ferences in the total cell count of the sputum cells be-
tween patients with mild asthma and patients with se-
vere asthma when compared to healthy controls. Sig-
nificant differences were observed in the percentages of
eosinophils and lymphocytes between severe asthmatics
and mild asthmatics (P < .001).
The presence of CD8+T lymphocytes was evalu-
ated in sputum samples from asthmatics and healthy
0 10 2030 4050 60
Figure 1. Relationship between CD8+CD28−and CD8+CD56+
cells in induced sputum cells of severe asthmatics. Significant
positive correlation (r = 0.748, P = .0034) was observed be-
tween CD8+CD28−cells and CD8+CD56+cells by Pearson’s
individuals after staining with a CD28 mAbs, which re-
vealed two subsets of CD8+T lymphocytes: CD8+CD28+
and CD8+CD28−subsets, as shown in Table 2. In healthy
individuals, the CD8+CD28+subset prevailed over the
CD8+CD28−subset. In severe asthmatics, we observed
a significant increase of CD8+CD28−cells, compared
to healthy subjects and mild asthmatics. This increase
of CD8+CD28−cells in severe asthma is paralleled by
a decrease of the CD8+CD28+subset. In mild asthma,
CD8+CD28−and CD8+CD28+subsets population were
expressed at similar levels.
The proportions of CD8+CD56+cells in the in-
duced sputum cells of patients with severe asthma were
higher compared to mild asthma and healthy con-
trols (Table 2). In terms of the relationship between
CD8+CD28−and CD8+CD56+subsets, analyzed by Pear-
son’s method, CD8+CD28−cells were correlated signif-
icantly with CD8+CD56+cells (r = 0.748, P = .0034)
For functional characterization of CD8+CD28−spu-
tum T cells, the frequency of perforin and IFN-γ pos-
itive cells were studied. In healthy controls, perforin
production was more frequent in CD8+CD28−T cells
(36.6% ± 8.3%) than in CD8+CD28+T-cell counterparts
2005:3 (2005) CD8+CD28−T Cells in Asthmatics163
Perforin-producing cells (%)
P < .001
P < .001
Interferon-γ-producing cells (%)
P < .001
P < .001
Figure 2. Intracellular production of perforin in CD8+CD28−
and CD8+CD28+cells. Induced sputum cells of asthmatics
and healthy controls (HC) were stimulated with phorbol 12-
myristate 13-acetate and ionomycin in the presence of brefeldin
A. Cells were stained with fluorescence-marked monoclonal an-
tibodies (mAbs) directed against CD8, CD28, and perforin and
counted by flow cytometry. The Mann-Whitney test was used to
perforin-CD28−T cells in induced sputum from severe, mild
asthmatics, and healthy controls.
(5.76% ± 2.6%) (Figure 2a). Perforin production was
ics (59.2.5% ± 8.5%) (Figure 3) compared to mild asth-
matics (39.8 ±12.3%) and healthy controls. CD8+CD28+
T cells from mild, severe asthmatics and healthy controls
expressed similar perforin levels.
In severe asthmatics, the frequency of CD8+CD28−
T cells producing IFN-γ is lower than what is ob-
served in healthy controls and mild asthmatics (P <
.001) (Figure 2b). Mild asthmatics and healthy control
CD8+CD28−T cells and CD8+CD28+T cells expressed
similar IFN-γ levels. However, severe asthmatics ex-
pressed a significantly decreased percentage of cells pos-
itive for intracytoplasmic IFN-γ.
Relative cell number
a patient with severe asthma. Cells were permeabilized and
stained for intracellular perforin. The histogram shows the in-
tracellular protein after staining with anti-perforin-PE (——),
and after staining with isotype-matched PE-labeled mouse IgG
mabs, used as the negative control (------). Results are the mean
of at least two independent experiments.
CD8+CD28+and CD8+CD28−T cells from 5 severe
investigated for their cytotoxic activity against the mouse
cell line P815 as a nonspecific target in a CD3-restricted
cytotoxicity assay (Figure 4).
CD8+CD28−T cells from severe asthmatics exhibited
efficient cytotoxic responses (30% ± 4%, 20% ± 6%, and
17% ± 5%) at different E:T ratios (25:1, 10:1, and 5:1),
els (12%±6%, 10%±2%, and 5%±3%) at the same E:T
ratios (P < .001). In healthy controls and in mild asth-
matics, CD8+CD28−T cells, cytotoxicity was low (healthy
vere asthmatics CD8+CD28−T cells (P < .001), at all E:T
ratios (25:1, 10:1, and 5:1). In healthy controls and mild
Our data clearly show the presence of two CD8+T-
cell subsets in induced sputum from both healthy con-
trols and asthmatic patients, and particularly the enlarge-
ment of the CD8+CD28−T-cell subset in severe asthma;
they expressed low IFN-γ production. Intracytoplasmic
perforin is highly increased in CD8+CD28−T cells from
severe asthmatics. This increased perforin expression is
164Agnes Hamzaoui et al 2005:3 (2005)
Effector : target cell ratio
% of lysis
CD8+CD28−, healthy controls
CD8+CD28+, healthy controls
CD8+CD28−, severe asthmatics
CD8+CD28+, severe asthmatics
CD8+CD28−, mild asthmatics
CD8+CD28+, mild asthmatics
Figure 4. Cytotoxic activity of CD8+T-cell subpopulations. Pu-
rified CD8+T-cell subsets were directly analyzed for cytotoxicity
antibody in a 4-hour51Cr release assay, in 4 healthy controls, 5
mild and 5 severe asthmatics. Results are the mean of indepen-
associated with high CD8+CD28−cytotoxic activity in se-
vere asthmatics. According to these results, we can spec-
ulate that effector cytotoxic function of CD8+T cells re-
side in the subpopulation lacking CD28 expression. Pre-
vious studies have shown that most healthy elderly hu-
mans harbor clonal CD8+T-cell expansions in their pe-
ripheral blood . Chamberlain et al  reported that
loss of CD28 expression marks functional differentia-
tion to cytotoxic memory cells . Some studies have
also shown that polyclonal CD8+CD28−T cells found
in the peripheral blood have significantly shorter telom-
eres than CD8+CD28+population, suggesting replicative
senescence , with more resistance to apoptosis 
and increased expression of BCL2 . Patients with
acute exacerbation of asthma were characterized by in-
creased expression of BCL2 proto-oncogene in induced
sputum lymphocytes from asthmatics .
are increased in various infectious diseases , in pa-
tients with autoimmune diseases , and in animal au-
tion during an asthma exacerbation is more reminiscent
of an antiviral response than an eosinophil-predominant
response to allergen. This implies an independent role of
ing glucocorticoid efficacy in the treatment of this disease
An imbalance of T-cell subsets in asthma with a pre-
dominance of TH2 type cells has been characterized .
Production of IFN-γ has been reported to be reduced in
patients with asthma , particularly in CD8+T cells
of asthmatics, and the proinflammatory activities of IFN-
γ may play an important role in the pathogenesis of child-
hood asthma and may suggest that asthma is not simply
a TH2-driven response. Our result reported a decreased
IFN-γ in severe asthma-producing CD8+CD28−T cells.
This suggests a real imbalance of TH1/TH2 cytokine-
producing cells in asthma, which linked asthma to a
chronic T-cell-mediated bronchial inflammation.
Because little is known about the physiologic role and
the putative cytotoxic functions in asthma, we studied
perforinproduction in CD8+CD28−Tcells.Perforinpro-
duction was found increased in CD8+CD28−T cells in se-
vere asthmatics. Increased perforin expression has been
reported by other authors in peripheral blood of asth-
matics  and in patients with COPD . Increased
perforin expression has been reported in several other
chronic inflammatory disorders with autoimmune phe-
nomena such as multiple sclerosis [33, 34], Takayasu ar-
teritis , or autoimmune thyroid disease , which is
localized to CD4+, CD8+, CD16+γδ T cells or NK cells.
Recently we have reported increased γδ T-cell expression
in induced sputum asthmatics, which mediated a potent
natural killer cytotoxic activity . CD8+CD28−T cells
have been reported to be effector cells, producing per-
forin, granzyme B, tumor necrosis factor-α, and IFN-γ
. CD28−T cells derive from their CD28+T-cell coun-
terparts [37, 38]. In our asthmatic patients, CD28−T
cells produced high levels of perforin-mediated cytotoxic-
ity, inversely correlated to IFN-γ production. We assumed
that the functional differentiation of CD8+T cells in se-
vere asthmatics into mature effector cells, with the disap-
pearance of CD28 makes them able to adhere to human
microvascular endothelial cells as reported by Fiorentuni
et al . CD8+CD28−T cells contain clonally expanded
cytotoxic T cells for unknown antigen specificity  and
ing their ability to elicit T helper cell activation and pro-
liferation . The role of CD8+CD28−T cells in asth-
matics is still unclear. Some recent publications have shed
light on some of the key processes controlling CD8+cells,
TH2 cytokines, and NKT cells . CD8+CD56+cells was
the predominant subtype in severe asthmatics. A signif-
icant correlation was found between CD8+CD28−cells
and CD8+CD56+cells in severe asthmatics. NKT cells
are heterogeneous T-cell populations that are character-
ized by the coexpression of TCRs and various NK re-
ceptors, including CD16, CD56, CD161, CD94, CD158a,
and CD158b . More investigations were needed to
define NKT cells in asthmatics. Umetsu et al  bear
on the driving roles of NKT cells and T cells in asthma.
In asthmatic patients, occurrence of CD8+CD28−T cells
seemed to be independent of prior viral infection, as our
patients were in steady state. A recent study highlights
the relationship between the functional activities of lym-
phocytes and their migration properties. Cells migrating
2005:3 (2005) CD8+CD28−T Cells in Asthmatics 165
to lymph nodes lack inflammatory and cytotoxic func-
tions, whereas cells migrating to peripheral tissues are en-
dowed with various effector functions . Inflamma-
tion is present in the lungs of asthmatics despite treat-
ment, but the inflammation in severe asthma may be dis-
tinct from the inflammation seen in mild asthma. These
findings are not without limitations, including overlap
among the groups and the unclear relationship to type
and severity of disease . The mechanisms associated
with the development of severe asthma are poorly under-
stood, but likely heterogenous. It was hypothesized that
flammatory groups based on the presence or absence of
eosinophils and that the inflammatory subtype would be
associated with distinct structural, physiologic, and clini-
cal characteristics .
The immunologic mechanisms reported are specific
with the development of severe asthma. Severe asthmatics
were characterized by a large expression of CD8+CD56+
cells and CD8+CD28−cells. Functional studies showed
that CD8+CD28−T cells had a cytotoxic function.
However, more work is required to describe the
relationship between: (i) CD8+CD28−T cells and
This work was supported by research grants of
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