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Function in Patients with Active Systemic
Deficient CD4+CD25high T Regulatory Cell
Xavier Valencia, Cheryl Yarboro, Gabor Illei and Peter E.
2007; 178:2579-2588; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 1, 2013
Deficient CD4?CD25highT Regulatory Cell Function in
Patients with Active Systemic Lupus Erythematosus1
Xavier Valencia,2* Cheryl Yarboro,†Gabor Illei,†‡and Peter E. Lipsky*
CD4?CD25?T regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoim-
munity. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear
components. We hypothesized that altered function of CD4?CD25highTregs might play a role in the breakdown of immunologic
self-tolerance in patients with SLE. In this study, we report a significant decrease in the suppressive function of CD4?CD25high
Tregs from peripheral blood of patients with active SLE as compared with normal donors and patients with inactive SLE.
Notably, CD4?CD25highTregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein
and poorly suppressed the proliferation and cytokine secretion of CD4?effector T cells in vitro. In contrast, the expression
of FoxP3 mRNA and protein and in vitro suppression of the proliferation of CD4?effector T cells by Tregs isolated from
inactive SLE patients, was comparable to that of normal individuals. In vitro activation of CD4?CD25highTregs from
patients with active SLE increased FoxP3 mRNA and protein expression and restored their suppressive function. These data
are the first to demonstrate a reversible defect in CD4?CD25highTreg function in patients with active SLE, and suggest that
strategies to enhance the function of these cells might benefit patients with this autoimmune disease.
Immunology, 2007, 178: 2579–2588.
self-reactive T lymphocytes in the thymus at an early stage of
development (1, 2). Several mechanisms of peripheral tolerance
have also been described, including T cell anergy and ignorance.
In addition, studies in the murine system initially provided strong
evidence for the existence of a unique CD4?CD25?population of
naturally occurring regulatory/suppressor T cells that actively pre-
vent both the activation and the effector function of autoreactive T
cells that have escaped other mechanisms of tolerance (3–5). Re-
moval of this population from normal rodents leads to the spon-
taneous development of various autoimmune diseases both organ
specific as well as systemic. Recent studies have revealed their
presence in human peripheral blood, where they constitute up to
5% of the CD4?T cells (6, 7). These cells require cell-to-cell
contact to exert their suppressive effect in vitro. Whether or not a
soluble factor is involved depends on the experimental system
used (8, 9). Notably, the generation of CD4?CD25?T regulatory
cells (Tregs)3in the immune system is developmentally and ge-
netically controlled, as recent studies have demonstrated that the
The Journal of
he ability of the immune system to discriminate between
self and nonself is controlled by central and peripheral
tolerance mechanisms. The former involves deletion of
transcription factor FoxP3 is essential for their thymic develop-
ment (10) and is sufficient to activate a program of suppressor
function in peripheral CD4?CD25?T cells by ectopic expres-
sion (11). Genetic defects that primarily affect the development
or function of CD4?CD25?Tregs can be a primary cause of
autoimmune and other inflammatory disorders in humans (12).
However, regulation of the suppressive activity of Tregs is
more complex because in vitro activation of CD4?CD25?T
cells results in transient expression of FoxP3 but no regulatory
Systemic lupus erythematosus (SLE), the prototypical systemic
autoimmune disease, is characterized by a wide spectrum of clin-
ical manifestations and abundant production of autoantibodies to
nuclear Ags, cell surface molecules, and serum proteins (14, 15).
In SLE, it is well recognized that B cells are hyperactive and pro-
duce a variety of autoantibodies, resulting in the formation of im-
mune complexes, that play a central role in the effector phase of the
disease. Furthermore, it has also become evident that SLE T cells
participate in the attack on target cells or tissues through overpro-
duction of proinflammatory cytokines or an increase in cell-to-cell
adhesion, ultimately leading to the apoptosis of the target cells
(16). One possibility to explain the emergence of autoimmunity in
diseases such as SLE could relate to deficient function of Tregs.
The deficiency in Treg function could result in increased helper T
cell activity or directly in enhanced B cell activity, both of which
have been shown to be regulated by Tregs in normal subjects (17, 18).
Murine models that lack CD4?CD25?Tregs develop a systemic au-
toimmune disease, characterized by gastritis, oophoritis, arthritis, and
thyroiditis (5). Interestingly, some animal models lacking Treg also
develop glomerulonephritis and increased titers of anti-dsDNA (5,
19), which are hallmarks of SLE.
Initial studies in SLE suggested there was a decrease in circu-
lating CD4?CD25?T cells in patients with active disease (20, 21),
and more recently it was claimed that Treg from active SLE were
decreased in number during disease flares but displayed normal in
vitro suppressive function (22). Therefore, the potential role of
Tregs in SLE remains to be fully delineated.
*Autoimmunity Branch,†Office of the Clinical Director National Institute of Arthritis
and Musculoskeletal and Skin Diseases/National Institutes of Health, and‡Gene Ther-
apy and Therapeutics Branch, National Institute of Dental and Craniofacial Research/
National Institutes of Health, Bethesda, MD 20892
Received for publication June 13, 2006. Accepted for publication November
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Intramural Research Program, National Institute of
Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health.
2Address correspondence and reprint requests to Dr. Xavier Valencia, National
Institutes of Health, 10 Center Drive, Room 6D44, Bethesda, MD 20892. E-mail
3Abbreviations used in this paper: Treg, T regulatory cell; SLE, systemic lupus ery-
thematosus; SLEDAI, SLE disease activity index; GITR, glucocorticoid-induced
tumor factor receptor.
The Journal of Immunology
by guest on June 1, 2013
We have previously reported a reliable system to assess human
Treg function in vitro. Together with flow cytometric analysis of
cell surface phenotype and determination of FoxP3 expression (7),
this has provided an objective means to assess the presence and
function of Tregs in human autoimmune diseases. We, therefore,
used these approaches to compare the frequency and function of
CD4?CD25highTreg from a group of SLE patients and with those
from age-matched healthy control subjects. In this study, we found
that CD4?CD25highTregs from active but not inactive SLE pa-
tients manifest deficient in vitro suppressive activity. Importantly,
this defect is associated with a decrease in FoxP3 mRNA and
protein that can be restored after in vitro stimulation. A reversible
defect in Treg function may contribute to flares of disease activity
in patients with SLE.
Materials and Methods
We enrolled 25 patients who were 18 years or older and fulfilled the Amer-
ican College of Rheumatology criteria for the classification of SLE (23,
24), and 40 healthy donors between the ages of 23 and 69 years with no
history of autoimmune disease. Disease activity was scored based on the
SLE disease activity index (SLEDAI) (25), with one group comprising
patients with inactive disease (SLEDAI ?3; n ? 8) and another group with
active SLE (SLEDAI ?3; n ? 17), with or without immunosuppressive
treatment. We excluded patients with a history of infection within 3 wk and
comorbidities, such as diabetes mellitus. Informed consent was provided
according to the declaration of Helsinki. The study was approved by the
Institutional Review Board of the National Institute of Arthritis and Mus-
culoskeletal and Skin Diseases/National Institute of Diabetes and Digestive
and Kidney Diseases, National Institutes of Health. Data from some normal
controls (n ? 20) have been previously reported (7).
Cell culture reagents
X-VIVO 20 medium (BioWhitaker) supplemented with 1% heat-inacti-
vated normal human serum (BioWhitaker), 20 ?g/ml gentamicin, 1 ?g/ml
Fungizone, and 2 mM glutamine (all obtained from Invitrogen Life Tech-
nologies) was used for T cell cultures. FBS was obtained from HyClone.
All cytokines used in this study were recombinant human proteins. Final
concentrations were as follows: 100 ng/ml GM-CSF, IL-4 and 2 ng/ml
TGF?1 (R&D Systems), and 100 U/ml IL-2 (National Cancer Institute,
For immunostaining, mouse PE-, FITC-, and CyChrome-conjugated mAbs
against human CD3 (UCHT 1), CD4 (RPA-T4), CD8 (RPA-T8), CD14
(M5E2), CD25 (M-A251), CD45RA (HI 100), CD45RO (UCHL 1),
CD62L (DREG-56), CD80 (L307.4), CD83 (HB15e), CD86 (FUN-1),
CD122 (MIK-?2), CD127 (hIL7R-M21), CD152 (BNI3.1), HLA-DR
(G46-6), CCR4 (1G1), and corresponding mouse isotype controls (all ob-
tained from BD Pharmingen), glucocorticoid-induced tumor factor
receptor (GITR)-FITC (110416), TNFRI-FITC (16803), and TNFRII-
allophycocyanin (22235.311) (obtained from R&D Systems), and
CD25-PE (Beckman Coulter) were used. Cells were stained with
FoxP3-allophycocyanin (PCH101; eBioscience) and FoxP3-AlexaF488
(150D; Biolegend) according to the manufacturer’s instructions for fix-
ation and permeabilization, after the cells were stained for surface ex-
pression of CD4 and CD25 with CD25-PE and CD4-CyChrome. Anti-
CD3 (64.1; Ref. 26) was used for polyclonal activation of T cells.
T cells were stimulated with plate-bound anti-CD3 mAb 64.1 (1 ?g/well).
Cytokine analysis was conducted after a 72-h incubation by analysis of
supernatants with commercially available ELISA kits for human IFN-?
anti-CD25 PE, and the population was sorted into CD4?CD25?and CD4?CD25highsubsets as indicated in Materials and Methods. Staining with the
isotype-matched control mAb is indicated by the horizontal bracket. Numbers indicate the percentages of cells in each gate, and those in parentheses show
the mean fluorescence intensity of staining. Data are representative of results from ?10 different experiments.
Phenotype of CD4?CD25highTregs. Purified CD4?T cells from healthy donors and active SLE were stained with anti-CD4 CyChrome and
2580SLE AND CD4?CD25highTregs
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(BD Pharmingen), according to the manufacturer’s instructions or by the
cytometric bead array kit (BD Biosciences).
CD4?T cells were enriched from PBMC by negative selection using the
AutoMACS (Miltenyi Biotec). Enriched CD4?T cells were stained with
anti-CD4-CyChrome and PE-conjugated anti-CD25 (15 ?g/108cells) for
20 min at 4°C. CD4?CD25?T cells and CD4?CD25highTregs were pu-
rified using a MoFlo high-speed cell sorter (DakoCytomation) to a purity
of ?98%. In some experiments, CD4?CD25?and CD4?CD25highTregs
were stimulated in vitro before analysis. This was accomplished by cul-
turing them for 3 days in microtiter plates coated with anti-CD3 mAb 64.1
(1 ?g/well) in medium containing 100 U/ml IL-2.
TNF preincubation experiments
Purified CD4?CD25?and CD4?CD25highT cells were incubated over-
night with TNF at 50 ng/ml in medium supplemented with 1% NHS and
100 U/ml IL-2. Afterward, cells were washed extensively and used in the
assays of Treg function.
Flow cytometric analysis
Single-cell suspensions were prepared and stained for 20 min at 4°C with
optimal dilutions of each mAb. Expression of cell surface markers was
assessed using the flow cytometer (FACSCalibur; BD Biosciences), and
data were analyzed using FlowJo software (Tree Star).
To assess proliferation, 5 ? 104sorted cells were incubated in X-VIVO-20
medium with 10% FBS in 96-well U-bottom plates coated with anti-CD3
(64.1) at 1 ?g/well. For assessment of regulatory properties, 5 ? 104CD4?
CD25?T cells were cultured with plate-bound anti-CD3 in 96-well U-
bottom plates. Purified autologous CD4?CD25highTregs were added, usu-
ally at a 1:1 ratio if not indicated differently. After 3–4 days of culture, 100
?l of supernatant was removed from each well and used for cytokine de-
tection and 1 ?Ci of [3H]thymidine (37 KBq/well) was added for an ad-
ditional 16 h to each well. [3H]Thymidine incorporation was measured
using a liquid scintillation counter.
Total RNA was isolated from sorted cells using the RNAeasy Mini kit
(Qiagen) according to the manufacturer’s instructions. RNA samples were
treated with DNase I to remove contaminating genomic DNA and reverse
transcribed with Superscript II (Invitrogen Life Technologies). FoxP3 ex-
pression was tested using Assays on Demand reagents from Applied Bio-
systems (Hs00203958m1). All reported mRNA levels were normalized
to the GAPDH mRNA level, where GAPDH ? 1.
The mean ? SEM thymidine uptake and mean ? SEM cytokine secretion
of triplicate cultures were calculated for each experimental condition. The
Mann-Whitney U test was used to evaluate possible differences in the
CD4?CD25highfunction following in vitro and TNF stimulation. Percent-
age of suppression was determined as 1 ? (cpm incorporated in the co-
culture/cpm of responder population alone) ? 100%. Correlations between
percentage of FoxP3?CD25highcells or percentage of suppression by CD4?
CD25highTregs and SLEDAI scores were assessed by nonparametric
Spearman correlation. All statistical tests were performed using StatView
software (SAS Institute).
CD4?CD25?T cells exhibit phenotypical differences compared
with CD4?CD25?T cells
CD4?CD25highTregs represented ?0.5–3 ? 1% (mean ? SEM)
of total CD4?T cells from healthy donors (n ? 40). Because it has
been demonstrated that the brightest 2% of the CD25?population
contains most of the Treg (7, 27), the CD25?brightest subset was
studied further. Because we analyzed only the brightest 2% of
CD4?CD25?T cells in both SLE and normal controls, assessment
of the comparative number of Tregs could not be undertaken. The
surface phenotype of CD4?CD25?and CD4?CD25highTreg sub-
sets among healthy volunteers and patients with active (SLEDAI
?3; n ? 17) SLE was characterized. As shown in Fig. 1, the
CD4?CD25highTreg subset from active SLE patients expressed
modestly higher but not significantly different levels of GITR
(20 ? 8% (mean ? SEM) vs 15 ? 5% (mean ? SEM) in normal
volunteers). An increased expression of TNFRII was also observed
by the freshly isolated CD4?CD25highTreg subset from active
SLE (30 ? 12%, mean ? SEM) compared with normal individuals
inactive SLE express reduced FoxP3. A, Freshly sorted
CD4?CD25highTregs and CD4?CD25?effector cells
were isolated from normal donors and patients with active
and inactive SLE, and their expression of FoxP3 was char-
acterized by intracellular staining. Data shown are repre-
sentative of three different experiments. The isotype stain-
ing control is shown by the dotted line, and the staining for
FoxP3 is illustrated in black. Numbers in each histogram
indicate the percentage of positive cells, and those in pa-
function and the data is shown in Fig. 3. B, CD127 ex-
CD25?effectors from the same donors as in A. The iso-
type staining control is shown in the dotted line and the
staining for CD127 in black. Numbers in each histogram
indicate the percentage of positive cells, and those in pa-
rentheses show the mean fluorescence intensity of stain-
ing. C, Freshly sorted CD25 very high CD4?T cells
(upper 0.6% of CD4?CD25?T cells) and CD4?CD25?
effector cells were isolated from patients with active SLE,
and their expression of FoxP3 was characterized by intra-
cellular staining. Data shown are representative of three
different experiments. The isotype staining control is
shown by the dotted line, and the staining for FoxP3 is
illustrated in black. Numbers in each histogram indicate
the percentage of positive cells, and those in parentheses
show the mean fluorescence intensity of staining.
CD4?CD25highTreg from active but not
2581The Journal of Immunology
by guest on June 1, 2013
(18 ? 5%, mean ? SEM; p ? 0.05), whereas patients with inac-
tive SLE had similar expression of TNFRII as normal donors (n ?
8; 25 ? 6%, mean ? SEM; p ? 0.32). In contrast, TNFRI
(CD120a) was practically undetectable in both groups (mean 1.3 ?
0.6%; n ? 10). No differences were detected in the CD45RO ex-
pression on the CD4?CD25highTreg subset from normal individ-
uals (90 ? 5%, mean ? SEM) and active SLE patients (75 ? 8%,
mean ? SEM). Additionally, analysis of CD69 expression on the
Treg subset (mean 1.0 ? 0.7%; n ? 40) and in SLE (mean 1.2 ?
0.9%; n ? 20) confirmed that these cells were not simply contam-
inated with recently activated CD4?effector cells, because these
would be mainly CD69?. Finally, ?25% of CD4?CD25highTregs
in normal donors and patients with active SLE were HLA-DR?as
has been previously reported (27), indicating that CD4?CD25high
Tregs in SLE were not enriched in persistently activated T cells.
Our data also confirmed previous findings (28) of selective expres-
sion of CCR4 on CD4?CD25highTregs in normal donors (90 ?
7%, mean ? SEM; n ? 40) and SLE (85 ? 18%, mean ? SEM;
n ? 20) compared with CD4?CD25?from normal donors (30 ?
10%, mean ? SEM; n ? 40) and inactive SLE patients (20 ? 8%,
mean ? SEM; n ? 8). These phenotypic characteristics indicate
that the cells analyzed were authentic CD4?CD25highTregs in
both normal donors and SLE patients. However, CD4?CD25high
Tregs from patients with active SLE exhibited increased expres-
sion of GITR and TNFRII, which has been reported in patients
with rheumatoid arthritis and after exposure to TNF (7). By scatter
characteristics, CD4?CD25highTregs were not larger or more
complex than CD4?CD25?effector cells in either normal donors
or SLE patients (data not shown).
As shown in Fig. 2, CD4?CD25highTregs from normal donors
uniformly expressed high levels of FoxP3 protein by flow cytom-
etry (mean 85 ? 5%; n ? 40), whereas CD4?CD25highTregs from
subjects with active SLE expressed significantly less FoxP3 pro-
tein (mean 45 ? 10%, n ? 10; p ? 0.003). Notably, Tregs from
patients with inactive SLE expressed increased levels of FoxP3
(mean 64.4 ? 15%; n ? 8) that were not significantly different
than normal (p ? 0.20). Neither CD4?CD25?effectors from nor-
mal donors or SLE expressed this transcription factor that governs
Treg function (10, 11). Additionally, analysis of CD127 expres-
sion, which can be used to discriminate Tregs from effector T cells
(29), confirmed that Tregs from normal donors (4.5%), from in-
active lupus (8.1%) and active lupus (8.7%) contained few CD127-
expressing effector T cells as shown in Fig. 2B. Finally, to ensure
that results did not reflect activated effector cells contaminating the
CD25highpopulation, even more stringent sorting conditions were
used. In these experiments, only the brightest 0.6% of the CD4?
CD25?cells from patients with active SLE were sorted. In these
additional experiments, only a fraction of these CD25veryhigh
(5 ? 104/well) and CD4?CD25highTreg (5 ? 104/well) were cultured with plate-bound anti-CD3 (1 ?g/well) either alone or at a 1:1 ratio. After
72 h, [3H]thymidine incorporation was determined. Results are the mean ? SEM of 20 separate experiments using individual donors and patients
with active SLE. Also shown is the percentage of inhibition of proliferation in these 20 experiments.
CD4?CD25highT cells from patients with active SLE fail to suppress proliferation of CD4?CD25?T cells. CD4?CD25?responder
active SLE patients can be suppressed by
CD4?CD25highTregs from healthy con-
trols. CD4?CD25?responder (5 ? 104/
well) and CD4?CD25highTregs (5 ? 104/
well) were cultured with plate-bound anti-
CD3 (1 ?g/well) either alone or at a 1:1
ratio. After 72 h, [3H]thymidine incorpo-
ration was determined. CD4?CD25?ef-
fectors from active SLE patients were also
cocultured with CD4?CD25highTreg from
normal individuals. CD4?CD25?effectors
from normal individuals were also cocul-
tured with CD4?CD25highTreg from ac-
tive SLE patients. Results are the mean ?
SEM of three separate experiments.
CD4?effector cells from
2582 SLE AND CD4?CD25highTregs
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