Vol.1, No.3, 57-64 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/oji/
Open Journal of Immunology
Increased expression of regulatory T cell-associated
markers in recent-onset diabetic children
Mikael Pihl1*, Mikael Chéram y1, Jenny Mjösberg2, Johnny Ludvigsson1, Rosaura Casas1
1Division of Pediatrics and Diabetes Research Center, Department of Clinical and Experimental Medicine, Linköping University,
Linköping, Sweden; *Corresponding Author : mikael.pihl@liu.se
2Division of Clinical Immunology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
Received16 June 2011; revised 16 September 2011; accepted 14 October 2011.
CD4+CD25hi T cells are thought to be crucial for
the maintenance of immunological tolerance to
self antigens. In this study, we investigated the
frequencies of these cells in the early stage of
type 1 diabetes, as well as in a setting of possi-
ble pre-diabetic autoimmunity. Hence, the ex-
pression of FOXP3, CTLA-4, and CD27 in CD4+
CD25hi T cells was analyzed using flow cytome-
try in 14 patients with recent onset type 1 dia-
betes, in 9 at-risk individuals, and 9 healthy in-
dividua ls with no known risk for ty pe 1 dia betes.
Our results show there were no differences in
the frequency of CD4+CD25hi cells between gro-
ups. However, compared to controls, recent-
onset type 1 diabetic patients had higher expre-
ssion of FOXP3, CTLA-4, and CD27 in CD4+
CD25hi cells from peripheral blood. The median
fluorescence intensity of FOXP3 was signifi-
cantly higher in CD4+CD25hi cells from patients
with type 1 diabetes t han from control s. Further -
more, a positive correlation between the fre-
quency of FOXP3+ cells and the median fluore-
scence intensity of FOXP3 was observed among
patients with type 1 diabetes. These data sug-
gest that the frequency of CD4+CD25hi FOXP3+ T
cells in the periphery is not decreased but
rather increased at onset of type 1 diabetes.
Thus, functional deficiencies rather than re-
duced numbers of CD4+CD25hi cells could con-
tribute to the development of type 1 diabetes.
Keywords: Regulatory T cells; Type 1 Diabetes;
There is little doubt today that a regulatory su bset of T
cells necessary for peripheral tolerance exists, and that
absence of these cells causes autoimmunity in a variety
of experimental settings [1]. Naturally occurring regula-
tory T cells (Treg) are CD4+ T cells which constitutively
express the Interleukin (IL)-2 receptor α chain (CD25),
the transcription factor FOXP3 and Cytotoxic T lym-
phocyte associated antigen 4 (CTLA-4, CD152), and are
normally produced in the thymus [2]. Expression of the
transcription factor FOXP3 is vital to the development
and function of Treg [3-4] and has therefore been used to
delineate regulatory T cell populations. However, activa-
tion of human T cells induces transient expression of
FOXP3 in non-regulatory T cells without conferring a
regulatory phenotype to the affected cells [5-9]. Some
report this phenomenon to be a consequence of T cell
receptor (TCR) stimulation [7], while others have postu-
lated that TCR stimulation does not produce FOXP3
expression at either gene or protein level [10]. Tran et al
recently found that high levels of FOXP3 could be in-
duced in CD4+CD25 T cells by TCR stimulation, only
in the presence of transforming growth factor (TGF)-β.
FOXP3 expression induced in this way was maintained
for weeks in the presence of IL-2 [9]. CTLA-4 is a cos-
timulatory molecule with potent suppressive function
constitutively expressed on Treg but also on activated
effector T cells [11,12]. In addition to its negative
co-stimulatory effect, CTLA-4 up-regulates Indolamine
2,3-dioxygenase in dendritic cells [13], resulting in ca-
tabolism of tryptophan to kynurine, which has potent
local immunosuppressive effects [14]. Expression of the
transmembrane costimulatory receptor CD27 was used
to define Treg in inflamed synovia in conjunction with
CD25 [15]. Other evidence suggests that Treg expressing
CD27 are more suppressive than CD27-negative coun-
terparts [16].
Defects in the function of Tregs have been hypothe-
sized to be involved in the pathogenesis of numerous
autoimmune diseases, including type 1 diabetes [17]. In
mice, islet antigen specific FOXP3 transduced T cells
were able to suppress recent onset type 1 diabetes [18].
M. Pihl et al. / Open Journal of Immunology 1 (2011) 57-6 4
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/oji/
However, studies of human Tregs in type 1 diabetes have
produced contradictory results. Kukreja et al. reported
reduced numbers of CD4+CD25hi cells [19], while Lind-
ley et al. and Brusko et al. could find no change in
CD4+CD25hi frequency between healthy and diabetic
individuals [20,21]. A meta-analysis comparing these
findings suggested that the lack of consensus between
studies is a consequence of differently matched diabetic
and control groups. The authors advocated further stud-
ies of phenotypical markers associated with Treg, in-
cluding FOXP3 [22].
A conundrum of type 1 diabetes is the lack of good
indicators before disease onset, since it is of interest to
study individuals at risk of developing the disease. Thus,
we are interested in describing T cell populations in
at-risk individuals, with possible autoimmune activity
prior to diagnosis of type 1 diabetes. Most people pro-
gressing to type 1 diabetes produce autoantibodies to one
or more islet autoantig ens, most commonly against insu-
lin, glutamic acid decarboxylase (GAD), and the tyro-
sine phosphatase like protein IA-2 [23]. Therefore, a
group of healthy children with autoantibodies was se-
lected as a risk population in this study. To our knowl-
edge, no previous studies have examined the expression
of FOXP3 and CTLA-4 in autoantibody-positive chil-
dren, nor in children with recent onset T1D with as
closely age-matched healthy controls as in the present
study. This is important as the immune system changes
with age from childhood, through puberty until adult-
hood, and as the autoimmune process leading to type 1
diabetes is more rapid and aggressive in children than in
higher age groups.
We hypothesized that patients with type 1 diabetes as
well as healthy individuals expressing high levels of
autoantibodies against islet antigens would have a de-
creased proportion of Treg compared to healthy indi-
viduals without autoantibodies. Therefore we analyzed
the expression of proteins related to Treg function to
determine their frequency in type 1 diabetes patients,
healthy subjects and healthy subjects at r isk of type 1 dia-
betes. Here, we report an increased expression of FOXP3
and CTLA-4 on CD4+CD25+ cel ls in patients with typ e 1
diabetes and subjects at risk of ty pe 1 diabetes.
2.1. Stud y Population
All the participants and their parents received written
information on the study, and consent was obtained ac-
cording the Declaration of Helsinki. Via proxy consent
was obtained for participating children, and presumed
consent was obtained from paren ts upon completing and
submitting questionnaries on entering the study.
The study was approved by the Regional Ethics Com-
mittee for Human Research “Regionala etikprövnings-
nämnden i Linköping, Avdelningen för prövning av
medicinsk forskning”, Linköping University Hospital,
Sweden (Dnr 03-092).
Venous blood samples were collected from 14 patients
with recent onset type 1 diabetes with a median age of
10 years (range 3 - 17 years, SEM 1,123) and a diabetes
duration of three months. Nine 8-year-old healthy chil-
dren with autoantibodies against Insulin, GAD65, or
IA-2 in the 95th percentile or high er at 5 and/or 2 .5 years
of age were included as a risk popu lation. Some, but not
all, at-risk children exhibited an antibody response to
more than one autoantig en (Table 1). And some, but not
all, were positive for autoantibodies on several occasions
a few years apart prior to sampling. Nine healthy chil-
dren, 8 years old, with no known type 1 diabetes-asso-
ciated HLA-genotypes, allergy, or autoimmune disease
were included as reference. All at-risk and control sub-
jects were participating in the ABIS study (All Babies in
Southeast Sweden).
Table 1. Presence of autoantibodies in at-risk children.
Aab at 1 year of age Aab at 2.5 years of ageAab at 5 years of age Genotype where available
3932 98th 02 0501 05
8107 95th
8772 95th 03020602 0401
11,124 98th 03020602 0405
14,903 98th 02 0301 0201 05
17,450 95th99th 99th 0602
18,034 90th 95th90th 03010302 03 05 0401
19,032 99th 99th 98th>5.5 RA U03010302 03 05 0401
23,735 98th>10 RA U02 0302 03 05 0401
M. Pihl et al. / Open Journal of Immunology 1 (2011) 57-6 4
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/oji/
2.2. Flow Cytometry
Peripheral Blood Mononuclear Cells (PBMC) were iso-
lated from blood samples by Ficoll (Pharmacia Biotech,
Sollentuna, Sweden) gradient centrifugation within 24 h
of collection. Cells at interface were harvested and wa-
shed three times in RPMI 1640 (Gibco, Auckland, New
PBMC were washed with Phosphate Buffered Saline
(PBS)(Medicago AB, Uppsala, Sweden) containing 0.1%
Bovine Serum Albumin (BSA)(Sigma-Aldrich, St Louis,
MO, USA). Approximately 2 × 106 cells were used in
each FACS tube for staining of Treg-like cells. In addi-
tion, ~105 cells were used to set compensation and as
isotype and unstained controls. Cells were aliquoted
(200 µl per tube) along with appropriate antibodies, pe-
ridin chlorophyll (PerCP) anti-CD4 (BD Biosciences,
San Jose, CA, USA, clone SK3), fluorescein isothiocy-
anate (FITC) anti-CD27 (BD Pharmingen, M-T271), and
Allophycocyanin (APC) anti-CD25 (BD Biosciences,
2A3). Cells were stained for 30 minutes at 4˚C, washed
with PBS 0.1%BSA, and then fixed and permeabilized
with the eBioscience intracellular staining kit (eBio-
science, San Diego, CA, USA). Finally, cells were stained
intracellularly with FITC- or Phycoerythrin (PE)- con-
jugated anti-FOXP3 (eBioscience, PCH101) and FITC-
or PE-conjugated anti-CTLA-4 (R&D Systems, Min-
neapolis, MN, USA, clone 48815 and BD Biosciences,
BNI3, respectively) as above. FITC- conjugated CTLA-4
antibody was combined with PE-con- jugated FOXP3
antibody, and FITC-conjugated FOXP3 with PE-conju-
gated CTLA-4. Though cells were stained with FITC-
and PE-conjugated anti-FOXP3, only the samples
stained with PE-conjugated antibody were used when
analyzing FOXP3 expression alone. PCH101 has been
shown to bind both isoforms of FOXP3 [8]. Stained cells
were kept in the dark at 4˚C until analysis or were ana-
lyzed immediately. The following isotype control anti-
bodies were used: FITC-conjugated mouse IgG1, PE-
conjugated mouse IgG2a, PerCP-labeled mouse IgG1,
and APC-labeled mouse IgG1. Unstained cells were used
to estimate autofluorescence. Cells stained with single
antibodies were used to compensate spectrally adjacent
Samples were acquired on a four-color BD FAC-
SCalibur flow cytometer. The cytometer was calibrated
daily using BD Calibrite 3 beads, with added APC beads
(BD Biosciences). Compensation was set manually and
gates were set subjectively (Figures 1(a)-(c)). Analysis
was performed using Cellquest Pro software (BD Biosci-
ences). All analyses were performed in a blinded manner,
the evaluator did not know the identity of the sample.
CD4+CD25hi cells were defined by first gating on small
lymphocytes by forward and side scatter, and then on
CD4 and high CD25 expression [24]. The CD25hi gate
was ad justed to cont ain CD4+ cells that expressed higher
levels of CD25 than the discrete population of CD4
cells [25]. This gate contained approximately 2% of
small lymphocytes, and 2% - 6% of CD4+ cells. Ap-
proximately 5 × 105 small lymphocytes were collected
from each tube, while approximately 104 cells were ac-
quired from tubes with unstained cells, cells stained with
isotype contro ls, and tubes used to set compensation.
2.3. Statistical Analysis
Some, but not all, of our material was normally dis-
tributed according to the D’Agostino & Pearson omni-
bus normality test. We decided to use non-parametric
tests based partly on this and because of the small size of
our population. Analysis of variance between groups was
performed for each parameter using the Kruskal-Wallis
test, followed by Dunn’s multiple comparison test. Se-
lected groups were compared using the Mann-Whitney
U-test; two-tailed P-values were obtained throughout.
Statistical testing was carried out with GraphPad Prism
5.01 and SPSS 14. 0, b ot h f or W indows.
Expression of Tr eg associated markers in CD4+CD25hi
T cells is more frequent among at-risk and recent onset
type 1 diabetic children than among controls.
To determine the frequency of cells with a Treg phe-
notype, we compared the percentages of CD4+CD25hi
cells expressing FOXP3, CTLA-4, and CD27 deter-
mined by FACS. Lymphocytes were thus gated based on
forward and side scatter (Figure 1(a)), followed by CD4
and CD25 expression (Figure 1(b)). Quadrant lines de-
marcate the isotype controls. Gated cells were analyzed
for expression of FOXP3, CTLA-4, and CD27 (Figure
1(c)). Results are expressed as percentages of cells posi-
tively stained for each molecule. For clarity, no-risk in-
dividuals will be termed controls.
Approximately 2% small lymphocytes and 2% - 6%
CD4+ lymphocytes were gated as CD4+CD25hi. The
groups were not significantly different in their frequen-
cies of CD4+CD25hi cells (Figure 1(a)). The percentage
of FOXP3-expressing CD4+CD25hi cells was signifi-
cantly higher in diabetic children compared to controls
(p = 0.0061, Figure 1(b)), and tended to be higher in
diabe tic chi ldren c ompared to at-risk children (p = 0.0518,
Figure 1(b)). CD4+CD25hi cells from at-risk and diabetic
children more frequently expressed CTLA-4 than cells
from control children (p = 0.0078, p = 0.0006, Figure
1(c)). CD27 was also more frequently expressed in
CD4+CD25hi cells among diabetic children compared to
controls (p = 0.0092, Figure 1(d)). In addition, we ana-
lyzed the frequencies of FOXP3+CTLA-4+ CD4+CD25 hi
M. Pihl et al. / Open Journal of Immunology 1 (2011) 57-6 4
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/oji/
(b) (c) (d)
(e) (f) (g)
(a) shows the frequency of CD25high cells among CD4+ cells . The p ercen tages of CD4+CD2 5 hi cells expressing FOXP3, CTLA-4, and CD27 are depicted in (b),
(c), and (d), respectively. The percentage of CD4+CD25 hi cells coexpressing both FOXP3 and CTLA-4 is given in (e). (f) and (g) show percentages of
CD4+CD25hi cel ls that are CD27 +FOXP3+ and CD27+CTLA-4+, respectively. Ns = not significant. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Significance
was determined by Kruskal-Wallis test followed by Dunn’s post test. A clear trend of increasing Treg-associated molecules with increasing autoimmune activity
is discernable throughout. The manual setting of the lymphocyte gate is shown in H, with 5% of all events shown in the plot. In I, the manually set CD4+CD25hi
gate is depicted. Thirty-three percent of lymphocyte-gated events are shown, and the gate is set according to where the expression of CD25 in the CD4-negative
populat ion becomes s carce. J sh ows a typical plot of CD27 FITC (x -axis) and FOXP 3 PE (y-axi s); all coll ected event s in th e CD4+CD25hi gate are shown. All
plots show permeabilized cells. Isotype staining is compared to FOXP 3 stained cells in K.
Figure 1. Differential expression of FOXP3 and CTLA-4 among healthy, at risk and recent onset diabetic children.
cells. In concordance with separately determined FOXP3
and CTLA-4 levels, co-expression on single cells was
higher among children with type 1 diabetes than controls
(p = 0.0018, Figure 1(e)).
Single cells expressing both FOXP3 and CD27 were
common among CD4+CD25hi cells regardless of group,
as were CTLA-4+CD27+ cells. As the majority of CD4+
CD25hi cells expressed CD27, the frequencies of FOX
P3+CD27+ cells are essentially the same as those of
FOXP3 single positive cells. Hence, both FOXP3+
CD27+ and CTLA-4+CD27+ co-expression was signifi-
cantly higher in CD4+CD25hi cells from diabetic chil-
M. Pihl et al. / Open Journal of Immunology 1 (2011) 57-6 4
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/oji/
dren compared to controls (p = 0.0021, Figure 1(f), p =
0.0006, Figure 1(g)). Recent onset diabetic children had
a higher percentage of FOXP3+CD27+ coexpressing CD
4+CD25hi cells than at-risk children (p = 0.0033), wh er eas
at-risk children more frequently co-expressed CTLA-
4+CD27+ than no-risk controls (p = 0.0315). While the
at-risk population did not always differ significantly
from the control and diabetic groups, a clear trend is
discernable from the graphical presentation of our find-
ings, where the at-risk group has higher expression of
most analyzed markers than controls, and lower expres-
sion than recent onset type 1 diabetic patients. Even
though the most distinct variations in FOXP3 and CTL
A-4 expression were present within the CD4+ CD25hi
subset, the groups remain significantly different when
analyzing CD4+ cells in general (data not shown).
The FOXP3 Median Fluorescence Intensity (MFI) of
CD4+CD25hiFOXP3+ cells is higher among recent onset
Type 1 diabetic children than among controls.
The median fluorescence intensity for FOXP3 PE in
CD4+CD25hiFOXP3+ cells was higher in diabetic chil-
dren than in controls (p = 0.0061, Figure 2(a)). Intrigu-
ingly, CD4+CD25hiFOXP3+ cells had higher FOXP3
MFI than CD4+ cells among children with type 1 diabe-
tes (p = 0.0007, Figure 2(b)). The same pattern was evi-
dent in children with risk for type 1 diabetes (p <
0.0001). In contrast, no difference in the FOXP3 MFI
was detected between CD4+ cells and CD4+CD25hi-
FOXP3+ cells in the control group. Furthermore, there is
a distinct correlation between the frequency of CD4+
CD25hiFOXP3+ cells and the MFI of FOXP3 among
CD4+CD25hiFOX P3+ cells (Figure 2(c)). This correla-
tion is found exclu sively in the diabetic popu lation when
the groups are examined independently.
(a) (b)
(a) illustr ates the FOXP 3 MFI of CD4 +CD25hi cell s, whereas (b) sho ws FOXP3 MFI between the CD4 + and CD4+CD25hi populations in each group. (c) shows
the correlation of CD4+CD25hiF OXP3 + cell frequency and FOXP3 MFI in CD4+CD25hi cells in a mixed population and among healthy, at risk and diabetic
children separately. r and p values were calculated using Spearman’s correlation test.
Figure 2. Analysis of FOXP3 MFI in CD4+CD25hi cells.
CD27 cannot define CD4+CD25hi cells in peripheral
To explore whether CD27 could define CD4+CD25 hi
cells in peripheral blood, the expression of this receptor
was analyzed. CD27 was commonly expressed on more
than 95% of CD4+ cells. Further, CD27 expression did
not differ between the CD4+CD25hi and the CD4+ popu-
lation, regardless of the autoimmune state of the indi-
vidual, defined as diagnosed type 1 diabetes or the pres-
ence of autoantibodies.
In this study we found that the percentages of CD4+
CD25hi cells expressing FOXP3 and CTLA-4 was higher
in the peripheral blood of diabetic and at-risk children
compared to healthy individuals. It has been argued that
the age of the control population relative to the studied
M. Pihl et al. / Open Journal of Immunology 1 (2011) 57-6 4
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/oji/
population may affect the outcome of such comparisons
[21]. In the present study, both at-risk and control sub-
jects were 8-year-old children, and the recent-onset type
1 diabetic patients were only slightly older (median 10
years). Thus, the differences between at risk and control
groups cannot be explained by differences in age.
In agreement with most of the studies including adults
[20,21,26], we could not detect a difference in the fre-
quency of CD4+CD25 hi cells. One previous publication
on the subject has reported reduced frequencies of
CD4+CD25+ cells in children with type 1 diabetes, but
the control population was considerably older than the
diabetic population [19]. Brusko et al. found no changes
in the frequency of CD4 +CD25+ FOXP3 regulatory cells
in type 1 diabetics [27]. However, the samp les from first
degree relatives and healthy controls were from indi-
viduals considerably older than the diabetic children.
Lawson et al. has also reported no difference in the per-
centages of CD4+CD25hi cells co-expressing FOXP3,
but in contrast to our study the patients had long-stand-
ing diabetes, and both patients and controls were adult
subjects [28].
It has been shown recently that the effector cells of
diabetic subjects are resistant to regulation via Treg, and
that this resistance is intrinsic to the effector population
[29]. Thus, the increased level of FOXP3 expression we
detect might be due to a resistance to Treg-mediated
suppression in effector T cells. Marwaha et al. recently
demonstrated that recent-onset type 1 diabetes patients
have a higher percentage of non-suppressive CD45RA-
CD25intFOXP3low cells that secrete IL-17 [30]. Since CD
45RA was not included as a marker in the present study,
it cannot be excluded that the increased percentage of
FOXP3-expressing cells presented here may represent a
population of non-suppressive cells.
Our study also showed higher frequencies of CD4+
CD25hi cells expressing intracellular CTLA-4 among
recent-onset type 1 diabetes children. This is in agree-
ment with a previous result from a study in type 1 dia-
betic adults [20]. In addition we observed that children
with risk for type 1 diabetes also had increased percent-
ages of CD4+CD25hi expressing CTLA-4. CTLA-4 is
constitutively expressed by Treg and has been linked to
Treg function in vitro [12,31,32]. However, sharply con-
trasting results indicate that CTLA-4 blockade does not
alter the ability of Treg to suppress proliferation of re-
sponder T cells [24]. FOXP3+ Treg capable of in vitro
suppression are present in CTLA-4 deficient mice,
which further questions the role of CTLA-4 in the me-
chanism of Treg suppression [33]. Our results indicate
that it is unlikely that a lack of CTLA-4 is a causative
factor in type 1 diabetes development in children.
CD27 has previously been suggested to define Treg in
combination with CD25 [15]. It has also been reported
that CD27 expr ession correlates with FOXP3 expression
in peripheral blood of patients with relapsing-remitting
multiple sclerosis [34]. In the present study, expression
of CD27 did not vary noticeably between CD4+ and
CD4+CD25hi cells. It has been shown that 80% of
CD4+CD25int cells from synovial fluid expressed CD27,
arguing against the use of CD27 as a marker to define
Treg, since it is unlikely that as many as 80% of
CD4+CD25int cells are Treg [35]. Thus, our results indi-
cate that CD27 is not suitable to define Treg in periph-
eral blood fro m children with type 1 diabetes.
The median fluorescence intensity of FOXP3 has been
shown to be higher in Treg than in effector T cells [8].
Thus, increased FOXP3 expression in children with type
1 diabetes might represent Treg and not be due to activa-
tion of effector T cells. Both diab etic and at-risk child ren
exhibited higher FOXP3 MFI in CD4+CD25hi cells
compared to CD4+, whereas controls did not. This could
represent a higher fraction of CD4+CD25hi cells with a
regulatory phenotype in the at-risk and diabetic groups
compared to controls. Finally, we found a correlation
between the frequency of FOXP3+ cells and FOXP3
MFI among patients with type 1 diabetes but not in
at-risk or healthy children. This is in agreement with a
previous study where the authors observed a correlation
between FOXP3+ cell frequency and FOXP3 MFI in
patients with multiple sclerosis [34]. They further dem-
onstrated that the suppressive capacity of CD4+CD
25hiFOXP3+ cells correlates with the MFI of FOXP3
in-vitro. Thus, it cannot be excluded that an abundant
population of functional Treg exists in the peripheral
blood of children with type 1 diabetes.
In conclusion, the result of our study prov ide eviden ce
of an altered frequency of cells with a regulatory T cell
phenotype in peripheral blood of children with type 1
diabetes and in children with risk for developing the
disease. It will be important to clarify whether the in-
creased frequency of CD4CD25hiFOXP3 cells is a fail-
ed attempt at controlling autoimmunity.
This project was supported by the Swedish Child Diabetes Foun-
dation and the Medical Research Council of Southeast Sweden
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