Increased expression of regulatory T cell-associated markers in recent-onset diabetic children

doi:10.4236/oji.2011.13007

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Vol.1, No.3, 57-64 (2011)

doi:10.4236/oji.2011.13007

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.

ABSTRACT

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;

Autoantibodies

1. INTRODUCTION

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

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. MATERIALS AND METHODS

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

At-risk individuals IAA GADIA-2 IAA GAD IA-2 IAAGADIA-2 DQB1DQB2 DQA1 DQA2DRB

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

5959

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

Zealand).

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

dyes.

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.

3. RESULTS

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

(a)

(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

6161

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)

(c)

(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

blood.

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.

4. DISCUSSION

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

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.

5. ACKNOWLEDGEMENTS

This project was supported by the Swedish Child Diabetes Foun-

dation and the Medical Research Council of Southeast Sweden

(FORSS-8847).

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