Clinical Immunology (2008) 127, 151–157

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

CD4+CD25+ regulatory T cells (TREG) in Systemic Lupus

Erythematosus (SLE) patients: The possible influence
of treatment with corticosteroids
N.A. Azab a,⁎, I.H. Bassyouni a , Y. Emad a , G.A. Abd El-Wahab b ,
G. Hamdy c , M.A. Mashahit d
a
Rheumatology and Rehabilitation Department, Faculty of Medicine, Cairo University, Egypt
b
Clinical Pathology Department, National Cancer Institute, Cairo University, Egypt
c
Internal Medicine Department, Faculty of Medicine, Cairo University, Egypt
d
Internal Medicine Department, Faculty of Medicine, Fayoum University, Egypt

Received 29 August 2007; accepted with revision 14 December 2007

Available online 4 March 2008

KEYWORDS Abstract Systemic Lupus Erythematosus (SLE) is a chronic, systemic autoimmune disease
Regulatory T cells (TREG); characterized by loss of tolerance to self-antigens. Regulatory Tcells (TREG) are those CD4+ Tcells
Systemic Lupus that constitutively express high levels of CD25 and exhibit powerful suppressive properties. The
Erythematosus (SLE); aim of this work was to quantify CD4+CD25+ (TREG) cells and the Mean Fluorescence Index (MFI) of
CD4+CD25+T cells; TREG in the peripheral blood of patients with SLE and to correlate these findings with their disease
Corticosteroids activity scores and drug therapy. This study included 24 SLE patients with various disease activity
scores (SLEDAI) and 24 healthy age and sex matched controls. Flow cytometry was used to
examine the frequency of CD4+CD25+ T cells and the MFI of CD4+CD25+high T cells (TREG). CD4+
CD25+ T cells % and MFI of CD4+CD25+high T cells were higher in SLE patients than controls (p
value = 0.62 and = 0.037 respectively) and both CD4+CD25+ Tcell % and the MFI of CD4+CD25+high T
cells showed highly significant correlation with SLEDAI scores (both with a p value b 0.001) and
were higher in patients taking glucocorticoids than those not on glucocorticoids (p = 0.023, 0.048
respectively). We conclude that the increase in TREG cells in our patients may be due to
corticosteroid treatment.
© 2008 Elsevier Inc. All rights reserved.

Abbreviations: SLE, Systemic Lupus Erythematosus; TREG, regulatory
T cells; TNF, tumor necrosis factor; CTLA-4, cytotoxic T lymphocytes-
associated antigen; SLEDAI, SLE disease activity index; PBMCS,
peripheral blood mononuclear cells; MFI, mean fluorescence index.
⁎ Corresponding author. Fax: +20 002 02 25329113.
E-mail address: nfakharany70@yahoo.com (N.A. Azab).
Introduction
Growing evidence suggests that naturally acquired immunolo-
gical self-tolerance, in addition to clonal deletion, anergy and
ignorance, are accounted for by a population of CD4 (+) T cells
called natural regulatory T (TREG) cells, which actively suppress
the activation and expansion of self-reactive T cells. Those are

1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2007.12.010
152 N.A. Azab et al.

produced in the normal thymus as functionally mature cells – capacity – most of their markers become up-regulated and
hence they are called natural regulatory Tcells – and seed into have therefore limited capacity to identify TREG [6]. However,
the periphery, creating a distinct subpopulation of CD4 (+) only cells expressing the highest levels of CD25 (termed
T lymphocytes with immunosuppressive properties [1]. Other CD25+high) demonstrate potent regulatory function [5,24]. So,
regulatory cells have been described including Tr1 and Th3 gating of CD4+CD25+high T cells is usually preferred to define
regulatory Tcells [2,3] and CD8+ regulatory Tcells [4]. The CD4+ TREG and the most suppressor function purportedly attributed
CD25+ TREG cells are currently the best defined and character- to TREG comprises the bright population (CD4+CD25+high) [5].
ized among the various subgroups of regulatory T cells [1]. Phenotypic identification and quantification and suppressor
Within the population of CD4+ CD25+ T cells from healthy functional studies of the CD4+CD25+ regulatory Tcell population
volunteers, there exists a minor subset of in-vivo activated have been investigated in several autoimmune diseases, with
anergic regulatory Tcells (TREG) which mediate contact-dependent, conflicting results in most studies [25,26]. In contrast to strong
cytokine-independent suppression of T cells activation [5,6]. suspicions, few data yet exist as to whether the uncontrolled T
Tregulatory cells that are positive for CD4 and CD25 surface and B cell activation and pathogenesis in SLE can be attributed
expression have generated the highest level of interest amongst to a deficiency in CD4+CD25+ regulatory T cells.
immunologists. It is well documented that the breakdown of This study was carried out aiming to quantify CD4+CD25+
mechanisms assuring the recognition of self and non-self T cells population and CD4+CD25+high (TREG) T lymphocytes in
antigens is a hallmark of autoimmune diseases, which adds SLE patients and to elucidate if there is a possible influence
to the importance to those natural regulatory Tcells (TREG) [7]. of disease activity scores and drug therapy.
Systemic Lupus Erythematosus (SLE) is a chronic inflamma-
tory autoimmune disease. In those patients, there is loss of Patients and methods
tolerance to self-antigens with polyclonal activation of B lym-
phocytes, production of different auto-antibodies, and an Patients
altered function of Tcells [8]. The CD4+CD25+ Tregulatory cells
not only play a major role in the maintenance of self-tolerance
This study was carried out in the Rheumatology and Rehabilita-
and the control of various autoimmune diseases [9,10] but they
tion Department and internal medicine department in Kasr
are also involved in the regulation of Tcell homeostasis [11,12]
Elaini hospitals, Cairo University. Participants in the study
and in the modulation of immune responses to allergens, cancer
included twenty-four consecutive SLE patients and twenty-
cells, and pathogens [13,14]. Those findings have opened new
four age and sex matched volunteers.
prospects in immunotherapeutic interventions for several
SLE patients were diagnosed according to the revised
diseases. Such T regulatory cells represent a master player in
Criteria of the American College of Rheumatology (ACR) for
the immune system, and their manipulation could be used in
SLE [27]. They were 20 females (83.3%) and 4 males (16.7%),
new therapeutics. Recently, Scherer et al. [15] successfully
their ages ranged from 16 to 45 years old with a mean±SD
treated a case of scleroderma with anti-CD25 monoclonal
(24.46±6.48 years).The control group comprised 24 healthy
antibody and Nieuwhof et al. [16] reported successful treat-
volunteers, 21 females (87.5%) and 3 males (12.5%), their ages
ment of cerebral vasculitis with anti CD25 monoclonal antibody.
ranged from 17 to 44 years old with a mean±SD (25.67±
T regulatory cells (TREG) can be classified into two major
7.56 years). Information with regards to clinical manifestations
categories; thymus-derived natural TREG cells and those
of patients was obtained (age, disease duration, malar rash,
induced in the periphery [3]. There are some signals that
discoid lesions, photosensitivity, oral ulcers, arthritis, and
facilitate peripheral TREG formation which may involve co-
renal, neurological and hematological disorder). In addition, at
stimulatory molecules such as cytotoxic T lymphocytes-as-
the time of sampling, SLE participants were asked precise
sociated antigen (CTLA-4) and cytokines such as IL2, IL10,
questions regarding the treatment received during the last
TNF-α and TGF-B.[1,17]. TREG lymphocytes are characte-
3 months. On the day of sampling, laboratory investigations as
rized by the expression of CD4 and by high levels of the
CBC, ESR, liver and kidney functions and complete urine
α chain of IL-2 receptor (CD25) as well as by their lack of
analysis were done. Lupus disease activities were assessed
responsiveness to antigenic stimulation [18,19].
using SLE disease activity index (SLEDAI) score which defines
FOXP3, which is the transcription factor whose expression
SLE activity according to 24 clinical and laboratory parameter
confers the regulatory phenotype on both murine and human
[28] The results of renal biopsy grading from patients with
Tcells, is now thought to represent the most specific TREG cell
nephritis were retrospectively obtained from the patients'
marker [20]. However, its intracellular expression doesn't yet
medical records and used for correlations.
allow live sorting of cells for functional assays [21]. Also,
The protocol for this research conforms to the provisions
Valencia et al. [22] on their work on TREG reported that the
of the World Medical Association's Declaration of Helsinki.
regulation of the suppressive activity of TREG is more complex
Informed Consent was obtained from all participants prior to
because in vitro activation of CD4+CD25+ T cells results in
the study. Approval from our institutional ethics committee
transient expression of FOXP3 but no regulatory activity.
was also obtained.
However, FOXP3 has been shown to be essential for both the
development and function of regulatory T cells [20]. Other
surface markers have been reported to be expressed on Methods
human CD4+CD25+high T cells, including cytotoxic T lympho-
cytes — associated antigen (CTLA-4), CD 69 legend, gluco- Flow cytometry
corticoid-induced tumor necrosis factor receptor (GITR),
membrane bound TGF-β and α4β7/α4β1 integrin [23]. Upon It was applied for evaluation of the level of CD4+CD25+ % Tcells
activation of T cells – independent of their regulatory and their subtype CD4+CD25+high Tcells in the peripheral blood
Possible influence of treatment with corticosteroids in Systemic Lupus Erythematosus patients 153

Table 1 SLE patients' characteristics and disease pipette and washed twice with cold Phosphate buffer saline
manifestations (PBS) (Sigma cat No.k8211).The number of cells was adjusted to
1×106 cells/ml, then the cells were prepared into three tubes:
Total SLE patients N= 24
Female/male n (%) 20/4 (83.3/16.7%) Two test tubes:
Age (mean ± SD) 24.46 ± 6.48 A. Tube containing 100 μl of cell suspension + 10 μl
Disease duration from each of the following monoclonal antibodies
Range 1 −12 years (CD4 and CD25)
(mean ± SD) 3.2 ± 2.48 years B. Tube containing 100 μl of cells suspension + 10 μl
Malar rash n (%) 13 (54.2) from CD8 monoclonal antibodies
Arthritis n (%) 19 (79.2) Control tube: containing 100 μl of cells suspension and
Renal disorders n (%) 16 (66.7) 10 μl of the matched isotypic control.
Neurological disorder n (%) 5 (20.8)
Cardiac affection n (%) 4 (16.7) Then the three tubes were left for 30 min in the dark. The
Chest affection n (%) 1 (4.2) stained cells were then washed twice by PBS and the cell
Ocular disease n (%) 1 (4.2) pellets were suspended in 0.5 ml of sheath fluid and
Hematological disorder n (%) 6 (25) cytometric analysis was performed by Partec-III from Dako
ESR (mean ± SD) 64.83 ± 40.91 cytomation.
Presence of anti-ds DNA n (%) 19 (79.2) Results were expressed as the percentage of cells showing
SLEDAI ⁎ scores positive expression in PBMCS or/and as mean fluorescence
Range 4–40 index (MFI), to detect the level of expression of CD25 on CD4+
Mean ± SD 17.42 ± 10.51 T cells, defined as the ratio between the mean fluorescence
Coticosteroids intensity of the cells incubated with CD4/CD25 monoclonal
No. of patients on Coticosteroids (%) 18 (75) antibodies, and the mean fluorescence intensity of cells incu-
Dose (mg/day) (mean ± SD) 17.71 ± 15.32 bated with the isotypic control. For quantification of MFI of
Antimalarials n (%) 6 (25) CD4+CD25+high (TREG) cells, the gate for CD25+ was deliber-
Cyclophosphamide n (%) 12(50) ately set high for isolation of TREG cells (i.e. top 2% of CD4+
⁎SLE disease activity index. T cells with the brightest CD25 expression) [25].

mononuclear cells (PBMCS) in patients and controls groups
using Partec-III flow cytometry.
Monoclonal antibodies and staining procedure
Phycoerythrin-labeled (PE) monoclonal anti-CD25 (clone Act-1)
and antiCD8 (104), fluourescein isothiocyanate-labeled (FITC)
monoclonal anti-CD4 (clone MT310) as well as isotype and
fluorochrome-matched control antibodies (IgG FITC-IgG2 PE)
were used for setting fluorescence markers around negative
population and detecting non-antigen specific antibody bind-
ing. Anti CD4 and anti CD25 were purchased from Dako
cytomation (Danemark) and anti CD8 were from IQ product
(Granigen).
Heparinized whole blood was collected and immediately
peripheral blood mononuclear cells (PBMNCs) were separated
using Ficoll hypaque gradient (Density 1.077 — Bichrome AG.
Berlin (cat No. 6113)), then the separation was done by centri-
fugation. The mononuclear cells were collected by using Pasteur
Statistical analysis
Data were coded and summarized using SPSS (statistical
package for Social Sciences) version 12.0 for Windows [29].
Quantitative variables were described using mean ± standard
deviation and categorical data by using frequency and
percentage. Nonparametric Mann-Whitney U test compared
independent groups and Kruskal–Wallis test compared more
than two groups. Spearman's rank correlation test was used
as a measure of association of quantitative variables. P value
b 0.05 was considered statistically significant.
Results
This study comprised 24 SLE patients; their general char-
acteristics, disease parameters as well as their drug therapy
are shown in Table 1.
To determine the size of the TREG population in patients
with SLE and controls, we analyzed the percentage of gated
CD4+ T lymphocytes expressing CD25, which was higher in

Table 2 Comparison of CD4+CD25+T lymphocytes%, the Mean Fluorescence Index(MFI)of CD25 of CD4+CD25+high Tcells and MFI of
CD25 on total CD4+CD25+population of SLE patients and control group
CD4+CD25+ T cells% MFI ⁎⁎ of CD4+CD25+high MFI⁎⁎ of CD25 on total
(mean ± SD) T cells (mean ± SD) CD4+CD25+T cells (mean ± SD)
Total SLE patients n = 24 10.37 ± 4.44 71.70 ± 19.20 12.5 ± 3.82
Control group n = 24 7.78 ± 4.69 59.6 ± 16.8 10.08 ± 2.39
P value 0.62 0.037⁎ 0.067
⁎P value b0.05 significant.
⁎⁎Mean Fluorescence Index.
154
Figure 1 Correlation of SLEDAI scores with both CD4+CD25+ T
cell % and MFI of CD4+CD25+high T cells in patients not receiving
corticosteroids.
SLE patients than controls(p value = 0.062).Also, the level of
CD25 expression per cell, when expressed as Mean Fluores-
cence Index (MFI) was higher on CD4+CD25+high T cells (TREG)
in SLE patients group than controls (p value = 0.037) as in
Table 2.
Seeing that the SLE patients included in the study were
unselected, possible correlations between both the percen-
tages of CD4+CD25+ T cells and the MFI of CD4+CD25+high
T cells with some clinical manifestations of the disease were
considered. As for SLEDAI score, both CD4+CD25+ T cell % and
the MFI of CD4+CD25+high T cells showed highly significant
correlation (both with a p value b 0.001) as shown in Fig. 1
and Fig. 2.
However, neither CD4+CD25+ T lymphocytes % nor CD25
MFI of CD4+CD25+high T cells showed any significant correla-
tion with age or the duration of the disease (p value N 0.05).
Figure 2 Correlation of SLEDAI scores with both CD4+CD25+ T
cell % and MFI of CD4+CD25+high T cells in patients receiving
corticosteroids.
N.A. Azab et al.
Figure 3 Comparison of CD4+CD25+ T cell% between patients
receiving glucocorticoids and those not on glucocorticoids and
controls.
Similarly, we determined possible correlations with the
treatment received during the last 3 months before
sampling. No significant relation was detected with the use
of antimalarials or immunosuppressive drugs. However, a
significant higher level of CD4+CD25+ % T cells and a sig-
nificant higher level of MFI of CD4+CD25+high T cells (TREG)
was found in patients taking glucocorticoids compared with
non-glucocorticoids receiving patients and control group
(Kruskal–Wallis test p = 0.023,0.048 respectively) as in Figs. 3
and 4.
Correlation of both CD4/CD25+ T cell% and MFI of CD25
of CD4+CD25+high T cells, with corticosteroids dose showed
significant correlation with correlation coefficient (rho) =
0.814, 0.515 and p value b0.001, =0.010 respectively).
Figure 4 Comparison of MFI of CD25 on CD4+CD25+high T cells
between patients receiving glucocorticoids and those not on
glucocorticoids and controls.
Possible influence of treatment with corticosteroids in Systemic Lupus Erythematosus patients
Discussion
Accumulating evidence indicates an immune suppressive role
of the thymus-derived CD4+ T cells population constitutively
expressing a high level of CD25 (TREG) in autoimmune dis-
eases [30]. Despite abundant interest in CD4+CD25+ T cells as
they are involved in the pathogenesis of autoimmune
diseases; they have been investigated in several autoimmune
diseases, presenting conflicting results in most cases. A
higher proportion of functional CD4+CD25+ regulatory T cells
compared to controls was observed in the peripheral blood of
patients with primary Sjögren's syndrome [31] and in the
synovial fluid of rheumatoid arthritis patients [21]. However,
normal, elevated and diminished numbers of TREG cells have
been reported in the peripheral blood of patients with
rheumatoid arthritis or with other chronic rheumatic
diseases [21] Similarly, various studies in patients with type
I diabetes mellitus showed different results in the number of
CD4+CD25+ cells and in the level of CD25 expression [26].
Normal numbers of TREG cells with normal or diminished
suppressor function were reported in patients with multiple
sclerosis [32], autoimmune polyglandular syndrome [33]
and myasthenia gravis, whereas diminished amounts of the
CD4+CD25+ regulatory T cell population were reported in
autoimmune liver disease patients [25].
For SLE, however, data concerning TREG are limited. So we
performed this study to quantify the CD4+CD25+ Tcells mainly
CD4+CD25+high T cells in healthy subjects and in SLE patients.
The definition of human TREG is still under discussion and
no definite surface marker is currently available. The high
constitutive surface expression of the IL-2 receptor α chain
(CD25) is generally considered as a characteristic feature of
the majority of human TREG and regulatory activity is enriched
in CD4+ T cells expressing the highest levels of CD25, i.e.
the most bright population of CD25+ T cells (CD4+CD25+high
Tcells) [34]. Moreover, it has been previously shown that only
those cells expressing high levels of CD25 (CD25high) effi-
ciently suppress proliferative responses, thus being consid-
ered true regulatory T cells [1].
The mean ± SD of CD4+CD25+ T cells % in the peripheral
blood mononuclear cells (PBMCS) of control subjects was
7.78 ± 4.69%. Nearly similar results were reported by Liu
et al. [35], who reported that the mean ± SD of CD4+CD25+
T cells % of the controls group was 11.11% ± 4.58% when
expressed as percentage of the PBMCs. Also, the percentages
of CD4+CD25+ T cells are in line with the published “normal”
range of 5–10% mentioned by Todd et al. [36].
In the present study the levels of CD4+CD25+ % T cells
in SLE patients were higher than in the control group but it
was statistically insignificant. There was also a higher level
of MFI of total CD4+CD25+ T cells and MFI of CD4+CD25+high T
cells (TREG) in SLE patients than the control, with p value
(0.067 and 0.037) respectively. In agreement with our result
Minami et al. [37], reported that the percentage of CD4+
CD25+ T cells was higher in patients than controls, but it was
not statistically significant. Also Suárez et al. [38] found no
significant difference of CD4+CD25+ % T cells between SLE
patients and control group. On the other hand, different
results were found by others as Crispin et al. [39] who
reported a decreased percentage of CD4+CD25+ T cells and
TREG in 10 untreated SLE patients with active disease.
However, Lui et al. [35] found a normal percentage of CD4+
155
CD25+ T cells and of TREG among CD4+ lymphocytes, yet, with
a reduced level within the total PBMCs.
Discrepancies reported by different studies might be due,
at least in part, to technical difficulties in TREG cells pheno-
typic characterization. Since the IL-2 receptor α chain
(CD25) is transiently up-regulated in T cells after activation,
circulating CD4+CD25+ T cells make a heterogeneous popu-
lation. Also is probably due to differences in the selection of
patients as most of our cases were under glucocorticoid
treatment as well as being of different SLEDAI scores and this
was not the case in the previously mentioned studies.
In agreement with Suarez et al. [38], correlation tests in
our study didn't show any significant correlation of CD4+CD25+
% T cells, MFI of CD4+CD25+high (TREG) cells with age of the
patients or disease duration. However, Spearman correlation
tests revealed strong positive correlation between both CD4+
CD25+ % T cells, MFI of CD4+CD25+high (TREG) cells and SLEDAI
score for disease activity, (both with a p value b 0.001). This
was different from that reported by Liu et al. [35], who failed
to find a significant correlation between levels of CD4+CD25+
TREG cells and disease activity of SLE. However, the association
of high corticosteroid dose with more active disease in our
patients makes it difficult to reach a firm conclusion and
further studies are required to confirm these results to exclude
any bias due to contamination with effector activated T cells
expressing high level of CD25 in patients with a worse disease
course [6].
With regards to glucocorticoids treatment, the present
work showed that the group of patients treated with
corticosteroids, alone or with other immunosuppressive
drugs, had a significantly higher level of CD4+CD25+ % than
the group of patients not on corticosteroids treatment and
the control group with p value (0.023). Moreover there was a
significantly higher level of MFI of CD4+CD25+high (TREG) cells
of this group than both controls and SLE group not on
corticosteroids with p value = 0.048.
This is in consistence with the results of Suárez et al. [38],
who found that only patients under glucocorticoid ther-
apy during the last 3 months presented an elevated CD4+
CD25+high T cells, but patients under antimalarial or immu-
nosuppressive therapy were not significantly different from
normal subjects. So these results suggest that corticosteroids
may induce the expansion or generation of TREG. Some
authors support the role of corticosteroids in expansion of
TREG cells by the higher number of CD4+CD25+ % T cells in
peripheral blood of pregnant women [40,41] and in asth-
matic patients following systemic glucorticoids treatment
[42]. Also our result is in agreement with Fattorossi et al. [30]
who showed that the number of CD4+CD25+ and TREG in the
peripheral blood is significantly lower in untreated myasthe-
nia gravis patients, whereas it is normal or elevated in pa-
tients on corticosteroids.
Glucocorticoid-induced generation of TREG activity may
be due to over-expression and modulation of glucocorticoid-
induced TNF-receptor in regulatory T cells [43] or it may be
that the increase in CD25 expression after corticosteroids
therapy may reflect a general increase in the expression of
cytokines receptors which is a well recognized effect of
corticosteroids [44]. Also several studies have shown that
whereas corticosteroids suppress the expression of pro-
inflammatory cytokines, they induce TGF-β expression [45]
and this may be an important effect of oral corticosteroids as
156
TGF-β, induce the differentiation of TREG cells in humans.[1]
Also, steroids may induce selective apoptosis in CD4+CD25 T
cells as it has been previously reported [46]. So, it indirectly
increases the frequency of CD4+CD25+ T cells population.
However, the way in which glucocorticoids may increase TREG
cells population is still completely unknown and prospective
studies before and after treatment with corticosteroids are
needed to answer these questions.
In conclusion our results suggest the absence of quanti-
tative abnormalities in the population of the CD4+CD25+
regulatory T cells in non-glucocorticoid treated SLE patients.
However, patients users of glucocorticoids showed signifi-
cant higher MFI of CD4+CD25+high T cells (TREG) than controls
or non-steroid-treated patients, and the strong positive
correlations between MFI of CD4+CD25+high (TREG) cells in SLE
patients and disease activity scores as well as with
corticosteroid treatment necessitates further functional
studies to clarify the effect of corticosteroids on suppressor
activity of TREG. Finally, despite the conflicting reports in
TREG frequencies between SLE patients and control subjects,
the therapeutic potential of this cell population holds a great
promise for the future employment of effective treatment
modalities using TREG population.
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