Compromised CD4+ CD25

high regulatory T-cell function in patients

with relapsing-remitting multiple sclerosis is correlated with areduced frequency of FOXP3-positive cells and reduced FOXP3

expression at the single-cell level

Introduction

CD4+ CD25+ regulatory T cells are a subpopulation of

suppressor T cells that play an important role in down-

modulating the activation and effector function of poten-

tial auto-aggressive T cells.1,2 Loss of Tregs in animal

models has demonstrated their importance in maintaining

self tolerance, as these animals develop a variety of auto-

immune manifestations. The interleukin (IL)-2 receptor

a-chain (CD25), a marker commonly used to quantify

Tregs, is not sufficiently specific as it is also expressed by

activated T cells. Measurement of FOXP3 expression on T

cells isolated directly ex vivo allows a more accurate inves-

tigation of the Treg frequency in healthy individuals and

patients with immune disorders. The gene encoding the

transcription factor FOXP3 was discovered as a master

gene in the development of Tregs of mice.3,4 Loss-

of-function mutations in the FOXP3 gene lead to the

development of a severe lymphoproliferative disease and

autoimmune manifestations in mice (scurfy model) and

in patients with the immuno-dysregulatory, polyendocrino-

pathy, enteritis X-linked (IPEX) syndrome.5

We and others have reported that Tregs are functionally

impaired in patients with relapsing-remitting (RR)

multiple sclerosis (MS), an inflammatory disease of the

central nervous system (CNS).6–8 In contrast, patients with

Koen Venken,1 Niels Hellings,1

Marielle Thewissen,1 Veerle

Somers,1 Karen Hensen,2 Jean-Luc

Rummens,2 Robert Medaer,1

Raymond Hupperts3 and Piet

Stinissen1

1Hasselt University, Biomedisch Onder-

zoeksinstituut and Transnationale Universiteit

Limburg, School of Life Sciences, Diepenbeek,

Belgium, 2Clinical Laboratory of Experimental

Hematology, Virga Jesse Hospital, Hasselt,

Belgium, and 3Department of Neurology,

University Hospital Maastricht, Maastricht,

the Netherlands

doi:10.1111/j.1365-2567.2007.02690.x

Received 31 May 2007; revised 3 July 2007;

accepted 5 July 2007.

Correspondence: Prof. Dr Piet Stinissen,

Hasselt University, Biomedisch

Onderzoeksinstituut (BIOMED), Agoralaan

(building A), 3590 Diepenbeek, Belgium.

Email: piet.stinissen@uhasselt.be

Senior author: Piet Stinissen

Summary

CD4+ CD25high regulatory T cells (Tregs) of patients with relapsing-remit-

ting (RR) multiple sclerosis (MS), in contrast to those of patients with

secondary progressive (SP) MS, show a reduced suppressive function. In

this study, we analysed forkhead box P3 (FOXP3) at the single-cell level

in MS patients and controls (healthy individuals and patients with other

neurological diseases) by means of intracellular flow cytometry. Our data

revealed a reduced number of peripheral blood CD4+ CD25high FOXP3+

T cells and lower FOXP3 protein expression per cell in RR-MS patients

than in SP-MS patients and control individuals, which was correlated with

the suppressive capacity of Tregs in these patients. Interestingly, inter-

feron (IFN)-b-treated RR-MS patients showed restored numbers of

FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25high

FOXP3+ Tregs in RR-MS patients, as compared with controls and SP-MS

patients, expressed CD103 and CD49d, adhesion molecules involved in

T-cell recruitment towards inflamed tissues. This was consistent with a

significantly increased number of CD27+ CD25high CD4+ T cells in the

cerebrospinal fluid (CSF), as compared with peripheral blood, in RR-MS

patients. Taken together, these data show aberrant FOXP3 expression at

the single-cell level correlated with Treg dysfunction in RR-MS patients.

Our results also suggest that Tregs accumulate in the CSF of RR-MS

patients, in an attempt to down-regulate local inflammation in the central

nervous system.

Keywords: autoimmunity; multiple sclerosis; tolerance; regulatory T cells;

FOXP3

Abbreviations: CSF, cerebrospinal fluid; FOXP3, forkhead box P3; GITR, glucocorticoid-induced tumour necrosis factor receptorfamily-related gene; HC, healthy control; RR-MS, relapsing-remitting multiple sclerosis; SP-MS, secondary progressive multiplesclerosis; Treg, CD4+ CD25high regulatory T cell; VLA-4, very late antigen-4.

� 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89 79

I M M U N O L O G Y O R I G I N A L A R T I C L E

secondary progressive MS (SP-MS) have a normal Treg

function, suggesting a possible restoration of Treg function

in the later disease stages.8 In the early disease phase of

MS, it is thought that myelin-reactive T cells, potentially

activated in the periphery by molecular mimicry, are reac-

tivated in the CNS and start an immune cascade, attracting

other immune cells. This local inflammation can eventu-

ally lead to damage to the myelin sheath, oligodendrocytes

and axons, resulting in the typical neurological symptoms

seen in MS patients.9,10 One hypothesis is that pathogenic

myelin-reactive T cells in MS patients escape from periph-

eral tolerance as a result of disturbed immunoregulation.

Therefore, it is important to further characterize the

observed regulatory T-cell dysfunction in RR-MS patients.

In addition to in vitro suppressive activity, FOXP3 expres-

sion has also been evaluated in MS patients. FOXP3 levels

were measured either by reverse transcription–polymerase

chain reaction (RT-PCR) (mRNA) or by western blotting

(protein), and these reports demonstrated lower FOXP3

expression in the CD4+ CD25+ T-cell populations of

RR-MS patients as compared with healthy controls (HCs)

and SP-MS patients.8,11,12 However, as these methods

investigate FOXP3 expression in bulk CD25+ populations,

it is still unclear whether the reduced FOXP3 mRNA

expression in RR-MS patients is a reflection of a reduced

number of FOXP3+ CD25+ CD4+ Tregs or decreased

FOXP3 expression at the Treg cell level.

Recently, a number of reports have indicated that Tregs

can be divided into different subsets based on the expres-

sion of adhesion molecules.13–16 Adhesion molecules play

an important role in the trafficking of Tregs (and conven-

tional T cells) between the lymphoid and blood circula-

tion systems and also in the retention of T cells at sites of

inflammation and/or infection. For example, L-selection

(CD62L) is a lymph node homing receptor, whereas inte-

grin aE+ (CD103), integrin a4+ [CD49d, or very late anti-

gen-4 (VLA-4)] and the hyalorunate receptor (CD44) are

involved in T-cell migration into inflamed tissues.14,17–20

A report by Soilu-Hanninen et al. showed increased

expression of CD44 and CD49d in the total T-cell popu-

lation during MS relapses,21 but until now no informa-

tion has been available about the expression of adhesion

molecules on Tregs in patients with MS. Whether Tregs

migrate into the CNS of MS patients is not clear, but

results from the animal model, experimental autoimmune

encephalomyelitis (EAE), have demonstrated an accumu-

lation of Tregs in CNS lesions.22,23

To explore the reduced FOXP3 expression in MS

patients, we measured FOXP3 at the single-cell level in the

peripheral blood mononuclear cells (PBMC) of a large

panel of MS patients and controls by means of flow cytome-

try. In addition, we analysed the migration of human Tregs

towards the CNS by measuring the expression of adhesion

molecules on blood circulating Tregs, and we compared

Treg frequencies in paired cerebrospinal fluid (CSF) and

peripheral blood samples of MS patients and controls.

Materials and methods

Blood and CSF samples

Peripheral blood samples were obtained from 70 patients

with clinically definite MS, 18 patients with other neuro-

logical diseases (ONDs), 10 patients with rheumatoid

arthritis (RA), five patients with systemic lupus erythema-

tosis (SLE) and 40 HCs. The characteristics of patients

and controls are listed in Table 1. All MS patients fulfilled

the McDonald criteria,24 and were diagnosed as having

either RR-MS (n ¼ 55) or SP-MS (n ¼ 15). All RR-MS

patients were in clinical remission at the time of blood

Table 1. Characteristics of study subjects

Subjects No.

Gender1

(M/F)

Age (years)

[mean (range)]

Untreated/

treated2

EDSS

[median (range)]

MS patients

RR-MS 55 14/41 41.9 (18–59) 40/15 2.0 (0–4)

SP-MS 15 4/11 55.5 (45–69) 10/5 6.5 (4–9)

Control subjects

ONDs 18 8/10 47.7 (27–74) 18/0 NA

RA 10 3/7 67.0 (55–85) 5/5 NA

SLE 5 1/4 36.2 (20–52) 5/0 NA

HCs 40 12/28 38.9 (20–58) 40/0 NA

1Gender: male (M)/female (F).2Immunomodulatory treatment within 3 months of blood sampling. Fifteen RR-MS patients

and three SP-MS patients were treated with interferon-b; two SP-MS patients were treated

with methotrexate; RA patients were treated with disease-modifying antirheumatic drugs.

Untreated: no immunomodulatory drugs.

EDSS, expanded disability status score; HCs, healthy controls; MS, multiple sclerosis; NA, not

applicable; ONDs, other neurological diseases; RA, rheumatoid arthritis; RR-MS, relapsing-

remitting MS; SLE, systemic lupus erythematosus; SP-MS, secondary progressive MS.

80 � 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89

K. Venken et al.

collection. Fifty MS patients were untreated (no immuno-

modulatory drugs), whereas the other 20 MS pati-

ents were under treatment with interferon (IFN)-b1a

(RebifTM; Serono, Geneva, Switzerland) or methotrexate

at the time of the blood samplings. Other patients were

diagnosed following criteria described elsewhere.25–27 The

OND group consisted of 16 patients with non-inflamma-

tory neurological diseases (NINDs) and two patients with

other inflammatory neurological diseases (OINDs).

Paired CSF samples were collected by lumbar puncture

from a subgroup of MS (n ¼ 12; nine RR-MS and three

SP-MS patients, all untreated) and OND (n ¼ 11)

patients. The CSF was put immediately one ice and han-

dled within 1 hr for flow cytometric analysis. CSF samples

contaminated with red blood cells were excluded from

further analyses. This study was approved by the local

Medical Ethical Committee and informed consent was

obtained from all study subjects.

Flow cytometric analysis

Mononuclear cells were separated from whole blood

(PBMC) by means of Ficoll density gradient centrifuga-

tion (Histopaque; Sigma Diagnostics, St Louis, MO). CSF

cells were collected by centrifugation for 12 min at 250 g.

For surface marker analyses, PBMC or CSF cells were

incubated for 30 min at 4� with fluorescein isothiocya-

nate (FITC)-, phycoerythrin (PE)- or peridinin chloro-

phyll protein (PerCP)-conjugated monoclonal antibodies

(mAbs). The following mAbs were used: anti-human CD3

(clone SK7), CD4 (clone SK3), CD8 (clone SK1), CD25

(clone 2A3) (all from BD Biosciences, Erembodegem, Bel-

gium), CD27 (clone 9F4), CD44 (clone NKI-P2), CD49d

(clone BU49), CD62L (clone SK11), CD103 (clone 2G5)

(all from Immunotools, Friesoythe, Germany) and the

glucocorticoid-induced tumour necrosis factor (TNF)

receptor family-related gene (GITR; clone 110416; R&D

Systems, Abingdon, UK). For intracellular staining of

FOXP3 and/or cytotoxic T-lymphocyte-associated anti-

gen-4 (CTLA-4), cell surface staining was first completed

and cells were subsequently fixed and permeabilized

according to the FOXP3 staining buffer set protocol

(eBioscience, San Diego, CA) before anti-human FOXP3

(clone PCH101; eBioscience) and/or anti-human CTLA-4

(clone BNI3; Immunotech, Marseille, France) was added

for 30 min at 4�. Samples were analysed on a FACSCali-

bur flow cytometer (BD Biosciences). The percentage of

positive cells and the mean fluorescence intensity (MFI)

arbitrary units (AU) for a specific marker were calculated

using CELLQUEST software (BD Biosciences).

Treg suppression assay

CD4+ T cells were pre-enriched from whole blood by

means of the RosetteSepTM CD4+ T-cell Enrichment

Cocktail (StemCell Technologies, Grenoble, France) as

described previously.8 Next, cells were incubated for

30 min with CD25-PE- and CD4-PerCP-labelled antibod-

ies (BD Biosciences) at 4�. CD4+ CD25high T cells were

sorted as the top 2% of CD4+ T cells (showing the bright-

est expression of CD25) using a FACSariaTM (BD Bio-

sciences). In parallel, CD4+ CD25– T cells were isolated as

responder T cells. The purity of fluorescence-activated cell

sorter (FACS)-sorted cell fractions was routinely 97–99%

for each T-cell fraction. For assessment of Treg suppressive

capacity, Tregs and 5,6-carboxy fluorescein diacetate succi-

nimidyl ester (CFSE) (Invitrogen, Merelbeke, Belgium)-

labelled CD4+ CD25– T cells were cocultured at different

ratios, as described previously.8,28 Cell cultures were stim-

ulated with 2 lg/ml anti-CD3 Ab (clone 2G3). After

5 days, cells were harvested and the CFSE signal of gated

CD4+ T cells was analysed by flow cytometry. The sup-

pressive capacity of Tregs towards responder T cells in

coculture was expressed as the relative inhibition of the

percentage of CFSElow responder cells.

Statistical analyses

All statistical analyses were performed using PRISM soft-

ware version 4�0 (Graphpad, San Diego, CA). For com-

parisons of Treg parameters between patients and

controls, Student’s t-tests were applied. CD27+

CD25high CD4+ T-cell frequencies in paired CSF and

blood samples of patients and controls were compared

by paired Student’s t-tests. Correlations between para-

meters were evaluated using Spearman’s correlation tests.

Differences were considered significant when P < 0�05.

Regarding the IFN-b-treated patients, a treatment respon-

der was defined as any patient free of relapse during the

12-month period of IFN-b therapy preceding the sample

collection.

Results

Analysis of FOXP3 expression at the single-cell levelin MS patients and controls

FOXP3 expression was measured in freshly isolated

PBMC from 70 patients with MS, 40 HCs and 18 patients

with ONDs by means of flow cytometry. FOXP3 was pre-

dominantly expressed by CD4+ T cells (range 1�7–10�9%)

and was also expressed by a small fraction of CD8+

T cells (0�1–1�6%), but was not expressed by B cells

or monocytes for both patients and controls (Fig. 1 and

data not shown). A significantly reduced frequency of

FOXP3+ CD25+ CD4+ T cells was observed in untreated

RR-MS patients (3�5 ± 1�0% of CD4+ T cells) as com-

pared with HCs (5�4 ± 0�3%; P < 0�01) and patients with

ONDs (5�4 ± 0�4%; p < 0�01) (Figs 1 and 2a). Interest-

ingly, SP-MS patients showed a normal frequency of

� 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89 81

Single-cell analysis of FOXP3 in patients with MS

FOXP3+ CD25+ CD4+ T cells (4�8 ± 0�4%; P > 0�05 as

compared with HCs and patients with ONDs; P < 0�05 as

compared with RR-MS patients; Figs 1 and 2a). A

reduced number of FOXP3+ cells in the blood of RR-MS

patients was also observed in the total CD4+ T-cell popu-

lation (Fig. 1 and data not shown).

As expected, the FOXP3 expression correlated with the

CD25 expression of CD4+ T cells, i.e. the CD25high CD4+

T cells showed the brightest expression of FOXP3. A frac-

tion of CD25int CD4+ T cells also expressed FOXP3,

whereas CD25– CD4+ T cells were mostly FOXP3-negative

(Fig. 1). We therefore further analysed the Treg frequency

in these different CD25 subpopulations of MS patients

and controls. A reduced proportion of FOXP3+ cells in the

CD25high CD4+ T-cell population (defined as the top 2%

of CD25+ cells) in RR-MS patients (53�1 ± 3�3%) was seen

as compared with HCs (85�0 ± 1�6%; P < 0�01), patients

with ONDs (82�0 ± 3�5; P < 0�01) and SP-MS patients

(71�0 ± 3�1%; P < 0�05) (Figs 1 and 2b). In contrast, no

differences in FOXP3 expression were detected between,

respectively, CD25int or CD25– CD4+ T cells of patients

and controls (Figs 1 and 2b).

For five RR-MS patients and three HCs, we assessed

the Treg frequency at three different time-points. The

number of FOXP3+ CD4+ T cells in MS patients and con-

trols did not change significantly in a 1-year period

(Fig. 2c).

To obtain information about FOXP3 expression levels

per cell, the FOXP3 MFI of CD4+ CD25high FOXP3+ T

cells was determined in MS patients and controls. A

decreased level of FOXP3 was seen in RR-MS patients

(MFI: 28�7 ± 3�4) as compared with HCs (41�4 ± 2�6;

P < 0�05) and OND patients (44�4 ± 2�5; P < 0�01)

(Fig. 2d). There was a trend towards lower FOXP3

expression in SP-MS patients, but this difference was not

statistically significant (29�7 ± 4�1; P ¼ 0�07 compared

with HCs and patients with ONDs) (Fig. 2d). No differ-

ences were seen in FOXP3 frequency or MFI between

OIND and NIND patients (data not shown). Overall,

there was a positive correlation between the percentage of

FOXP3+ cells within the CD4+ CD25high population and

the FOXP3 MFI of CD4+ CD25high FOXP3+ T cells

(Fig. 2e; total population: r ¼ 0�59, P < 0�0001; HCs:

r ¼ 0�32, P < 0�05; RR-MS patients: r ¼ 0�47, P < 0�01;

SP-MS patients: r ¼ 0�82, P < 0�001; OND patients: r ¼0�50, P < 0�05).

To evaluate whether changes in FOXP3 expression were

also observed in patients with other autoimmune diseases,

we measured intracellular FOXP3 expression in a small

number of patients with RA (n ¼ 10) and SLE (n ¼ 5).

Our preliminary data showed no significant differences in

FOXP3 frequency (FOXP3+ CD25+ CD4+ T cells: RA

patients: 5�0 ± 0�7%; SLE patients: 6�7 ± 2�0%) or expres-

sion level (MFI: RA patients: 39�9 ± 2�9; SLE patients:

47�1 ± 5�2) between these patient groups and HCs

(Figs 2a and d). Remarkably, SLE patients consisted of

two groups on the basis of high and low numbers of

Tregs, which was in agreement with a recent report.29

In conclusion, RR-MS patients showed a reduced

number of blood circulating CD4+ CD25high FOXP3+

regulatory T cells and decreased FOXP3 expression level

per cell.

Treg FOXP3 expression is correlated with in vitroCD4+ CD25high T-cell-mediated suppression

To evaluate whether FOXP3 expression was correlated

with the in vitro suppressive capacity of Tregs, FACS-

sorted CD4+ CD25high T cells (Tregs) and CD4+ CD25– T

cells (Tresp) of 10 HCs, 10 RR-MS patients (untreated)

and 10 SP-MS patients were set up in a coculture assay28,

next to the flow cytometric FOXP3 analysis of the

CD4+ CD25high T cells. First, we could confirm previous

observations6,8 of reduced Treg function in RR-MS

patients and normal suppression in SP-MS patients

(Fig. 3). Furthermore, a clear positive correlation was

observed between CD4+ CD25high T-cell-mediated sup-

pression (at a Tresp:Treg ratio of 1 : 1) and FOXP3

expression levels (MFI) (Fig. 3; Spearman’s r ¼ 0�55;

HC

PBMC

47·8

48·8

46·6 4·2

FOXP3FITC

CD

4 P

erC

P

100 101 102 103 104 100 10

1 10

2 10

3 10

4

CD

4 P

erC

P

100 101 102 103 104 100 10

1 10

2 10

3 10

4

CD

4 P

erC

P

100 101 102 103 104 100

0 10

20

30

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nts

40 5

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70

101 102 103 104 100

0 10

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101 102 103 104 100

0 5

10

15

Cou

nts

101 102 103 104

100 101 102 103 104 100

0 20

Cou

nts

40 6

0 80

100

0 20

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nts

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100

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nts

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nts

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0 5

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nts

101 102 103 104

100

0 5

10

15

Cou

nts

101 102 103 104

100 10

1 10

2 10

3 10

4

FOXP3FITC FOXP3FITC FOXP3FITC

1·3% 7·3% 88·5%

2·3 0·4% 8·7% 66·2%

5·5 0·9%

M1 M1 M1

M1 M1 M1

M1 M1 M1

10·9% 94·1%

CD4+CD25– CD4+CD25int CD4+CD25high

RR-MS

SP-MS

Figure 1. Intracellular forkhead box P3 (FOXP3) expression in peri-

pheral blood mononuclear cells (PBMC) isolated from healthy

controls (HCs) and multiple sclerosis (MS) patients. PBMC of HCs

and MS patients were surface-stained with anti-CD4 and anti-CD25

antibodies. After permeabilization, cells were labelled with anti-

human FOXP3 antibody. Cells were analysed by means of flow

cytometry. The CD25 signal was subdivided into negative, intermedi-

ate (int) and high levels. The CD25high CD4+ T-cell population was

defined as the top 2% of CD4+ T cells showing the brightest CD25

expression. The figure shows costainings of FOXP3 and CD4 (dot

plots) or FOXP3 expression of CD4+ CD25–, CD4+ CD25int and

CD4+ CD25high T cells (histograms). The cut-offs for positive and

negative signals were set based on isotype-matched control antibody

staining. Numbers in each histogram indicate the percentage of

FOXP3-positive cells. Data are shown for one HC, one relapsing-

remitting multiple sclerosis (RR-MS) patient and one secondary pro-

gressive multiple sclerosis (SP-MS) patient, and are illustrative for all

study subjects, as indicated in Figure 2.

82 � 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89

K. Venken et al.

P < 0�01). In addition, we also observed a correlation

between the CD4+ CD25high T-cell suppressive capacity

and the number of FOXP3+ cells within this population

(Fig. 3; Spearman’s r ¼ 0�44; P < 0�05).

These results indicate that a combination of a reduced

number of FOXP3+ CD25high CD4+ T cells and reduced

FOXP3 expression per cell in RR-MS patients contribute

to the functional Treg defect in these patients.

Influence of IFN-b treatment on FOXP3 expression

A panel of 15 RR-MS patients treated with IFN-b were

also tested for FOXP3 expression. Interestingly, these

patients showed significantly higher frequencies of CD4+

CD25+ FOXP3+ T cells (5�3 ± 1�2%) than untreated

RR-MS patients (3�5 ± 1�0%; P < 0�01; Fig. 2a). This

higher number of CD4+ CD25+ FOXP3+ T cells was

00

20

MF

I FO

XP

3

40

60

0t = 0

% F

OX

P3+

CD

25+ T

cel

ls%

FO

XP

3+C

D25

+C

D4+

T c

ells

t = 6 m t = 12 m

MS1MS2MS3MS4MS5HC1HC2HC3

HC

MF

I FO

XP

3 (A

U)

0

20

40

60

80*

**

**

**

**

**

* HCONDUntreated RR-MSIFN-β-treated RR-MSSP-MS

0

20

% F

OX

P3

40

60

80

100

****

*

OND Untreated

RR-MS

IFN-β SP-MS

CD25high CD25int CD25–

RA SLE

HC

0

3

6

9

12

15(a) (b)

(c)

(e)

(d)

OND Untreated

RR-MS

IFN-β SP-MS RA SLE

2

4

6

8

10

25 50 75

P < 0·001r < 0·59

100

HCRR-MSSP-MSOND

% FOXP3

Figure 2. Frequency of CD4+ CD25+ FOXP3+ T cells and forkhead box P3 (FOXP3) expression at the cellular level in multiple sclerosis (MS)

patients and control subjects. Peripheral blood mononuclear cells (PBMC) of 40 healthy controls (HCs), 18 patients with other neurological dis-

eases (ONDs), 55 patients with relapsing-remitting multiple sclerosis (RR-MS) (40 untreated and 15 IFN-b-treated), 15 patients with secondary

progressive multiple sclerosis (SP-MS), 10 patients with rheumatoid arthritis (RA) and five patients with systemic lupus erythematosis (SLE) were

stained for FOXP3, CD25 and CD4 as described in Fig. 1. (a) The percentage of FOXP3+ CD25+ CD4+ T cells relative to the total number of

CD4+ T cells. (b) Percentage of FOXP3+ cells within the CD4+ CD25–, CD4+ CD25int and CD4+ CD25high T-cell populations. (c) For five

untreated RR-MS patients and three HCs, FOXP3+ CD25+ CD4+ T-cell frequencies were measured at different time-points (0, 6 and 12 months

after initial measurement). (d) Mean fluorescence intensity (MFI) of FOXP3 from CD4+ CD25high FOXP3+ T cells in patients and controls. Indi-

cated values (shown as box and whiskers) represent MFI signals obtained from FOXP3 stainings. MFI values of cells stained by isotype-matched

control antibody (background MFI) were subtracted from FOXP3 MFI values. *P < 0�05; **P < 0�01 as compared with the indicated study popu-

lations. (e) FOXP3 MFI of CD4+ CD25high FOXP3+ T cells was plotted against percentages of FOXP3+ cells within the CD4+ CD25high T-cell

populations of 55 MS patients, 18 OND patients and 40 HCs. r and P values were calculated using Spearman’s correlation test.

� 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89 83

Single-cell analysis of FOXP3 in patients with MS

consistent with a significantly higher number of FOXP3+

cells in the CD4+ CD25high population of the IFN-b-trea-

ted patients (Fig. 2b). The mean FOXP3 expression levels

(MFI: 40�6 ± 8�1) of IFN-b-treated patients were also

higher than those of untreated patients (MFI: 28�7 ± 3�4),

although the difference was not statistically significant

(P > 0�05; Fig. 2d). We next divided patients into IFN-bresponders (n ¼ 11) and non-responders (n ¼ 4), based

on clinical data (see ‘Materials and methods’), to see

if there was a correlation with treatment responsive-

ness. FOXP3 MFI values and the percentage of

CD4+ CD25+ FOXP3+ T cells were slightly higher in

responders (MFI: 44�3 ± 7�2; frequency: 5�4 ± 0�3%)

than in non-responders (MFI: 40�1 ± 3�3; frequency:

4�8 ± 0�7%), although no significant differences were

found. However, the difference in FOXP3 MFI between

IFN-b responders and untreated RR-MS patients was sig-

nificant (P < 0�05).

In conclusion, IFN-b treatment in vivo could poten-

tially compensate for the reduced Treg numbers in

RR-MS patients.

Phenotypic analysis of Tregs in MS patients andcontrols

It has recently been shown that CD25high Tregs represent

a heterogeneous population which can be divided into

different Treg subsets based on the expression of adhesion

molecules, which regulate T cell trafficking.13,14 Therefore,

we determined the expression of L-selectin (CD62L), the

hyalorunate receptor (CD44), integrin aE+ (CD103), inte-

grin a4+ (CD49d) and Treg function-associated molecules

(GITR and CTLA-4) on CD4+ CD25high T cells and their

counterparts CD4+ CD25– T cells from MS patients (15

untreated and 10 IFN-b-treated RR-MS patients and 12

SP-MS patients) and control subjects (20 HCs and 15

patients with OND) by means of flow cytometry. An

overview of these results is shown in Fig. 4 and Table S1

(Supplementary material). GITR, CTLA-4 (intracellular)

and CD103 were mainly expressed by CD4+ CD25high

T cells in both MS patients and controls. All CD4+

CD25high and CD4+ CD25– T cells expressed CD44

(100% for all individuals tested; data not shown) and

were CD62Lhigh, whereas high expression levels of CD49d

(CD49dhigh) were found in a smaller subset of both cell

types in patients and controls (Supplementary material,

Table S1). In a comparison of the expression profiles of

CD4+ CD25high T cells of MS patients and controls, we

observed a significantly higher number of cells positive

for CD103 and CD49dhigh in RR-MS patients (untreated

and IFN-b-treated) as compared with HCs and patients

with OND. Also, significantly more CD4+ CD25– T cells

of RR-MS patients were CD103+ compared with controls,

although the number of CD4+ CD25– CD103+ T cells in

these patients was still very small. No significant correla-

tion (P > 0�05) was observed between the percentage of

CD103+ or CD49dhigh cells and the percentage of FOXP3+

cells or FOXP3 MFI within the CD4+ CD25high T-cell

population of MS patients (data not shown). Regarding

the CTLA-4 and GITR expression of peripheral CD4+

CD25high T cells, we observed no differences between MS

patients and controls, which was in agreement with our

previous observations.8

Remarkably, whereas no differences in CD44 expression

levels or numbers of CD62Lhigh CD4+ CD25high T cells of

Suppression (%)

0

25

50

75

% F

OX

P3

100

0

20

40

MF

I FO

XP

3 (A

U)

% FOXP3+ FOXP3 MFI

60

0 25 50 75 100

P < 0·05r = 0·44

P < 0·01r = 0·52

Suppression (%)0 25 50 75 100

Figure 3. The suppressive capacity of CD4+ CD25high T cells in func-

tion of forkhead box P3 (FOXP3) expression. The suppressive capacity

of CD4+ CD25high T cells from 10 patients with relapsing-remitting

multiple sclerosis (RR-MS; .), 10 patients with secondary progressive

multiple sclerosis (SP-MS; r) and 10 healthy controls (HCs; s) were

plotted against the percentage of CD4+ CD25high T cells positive for

FOXP3, and FOXP3 mean fluorescence intensity (MFI). Suppression

values represent percentages of inhibition of proliferation (5,6-carboxy

fluorescein diacetate succinimidyl ester (CFSE)) of CD4+ CD25– T

cells (Tresp) by CD4+ CD25high T cells (Tregs) as measured in cocul-

ture experiments at a 1 : 1 (Tresp:Treg) ratio. r and P values were

calculated using Spearman’s correlation tests.

CD62L CD44

%%

100**

*

**

***

**

****

****

*

**

806040200

400

300

200MF

I

100

0

50403020100

%

20

15

10

5

0CD25high CD25– CD25high CD25–

HC

ONDRR-MS,untreated

SP-MS

RR-MS,IFN-β treated

CD25high CD25– CD25high CD25–

CD49d CD103

Figure 4. Expression of adhesion molecules on regulatory T cells

(Tregs) of multiple sclerosis (MS) patients and controls. CD4+

CD25high and CD4+ CD25– T cells within the peripheral blood

mononuclear cell (PBMC) population of 20 healthy controls (HCs),

15 patients with other neurological diseases (ONDs), 15 untreated

and 10 interferon (IFN)-b-treated relapsing-remitting multiple

sclerosis (RR-MS) patients and 12 secondary progressive multiple

sclerosis (SP-MS) patients were analysed for expression of the indi-

cated markers [percentage positive cells or mean fluorescence

intensity (MFI)] by means of flow cytometry. *P < 0�05; **P < 0�01

compared with the corresponding population.

84 � 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89

K. Venken et al.

untreated RR-MS and SP-MS patients could be detected

as compared with controls, IFN-b-treated RR-MS patients

showed a significantly lower number of CD4+ CD25high T

cells that were CD62Lhigh than untreated RR-MS patients

(P < 0�05) and controls (P < 0�05).

In the analysis of the same set of markers on FOXP3+

and FOXP3– CD4+ T cells in a small cohort of MS

patients and control subjects, the same significant differ-

ences were detected in percentages of CD103+ and

CD49dhigh cells within CD4+ FOXP3+ T cells of RR-MS

patients compared with control individuals (Table S1).

Taken together, the detection of a higher percentage of

CD103+ and VLA-4+ T cells in the Treg population in

RR-MS patients could reflect an increased migration capa-

city of Tregs towards inflammatory lesions in the CNS.

Treg numbers are increased in the CSF comparedwith peripheral blood in RR-MS patients

To evaluate a possible migration of Tregs towards the

CNS in MS patients, we quantified Treg numbers in

paired CSF and blood samples in MS patients (n ¼ 11;

eight RR-MS patients and three SP-MS patients, all

untreated) and patients with ONDs (n ¼ 12; 10 NIND

patients and two OIND patients). Interestingly, we could

detect CD4+ CD25+ FOXP3+ T cells in the cerebrospinal

fluid of RR-MS patients (n ¼ 2; Fig. 5a). However,

because of the low number of cells collected and the

requirements of the intracellular staining protocol, it was

not feasible to analyse all the CSF samples for FOXP3

expression. In an alternative approach, we focused on the

frequency of CD27+ CD25high CD4+ T cells, as a previous

study showed that Tregs and effector T cells could be dis-

tinguished near the site of inflammation in patients with

juvenile idiopathic arthritis (JIA) based on CD27 expres-

sion.30 In agreement with that report, we demonstrated

that the majority of CD4+ FOXP3+ T cells in both the

peripheral blood (data not shown) and CSF (Fig. 5a) of

MS patients was positive for CD27. To further verify our

approach, we compared percentages of CD27+ CD25high

and FOXP3+ CD25high CD4+ T cells (within the total

CD4+ T-cell population) in PBMC isolated from OND

(n ¼ 11) and RR-MS (n ¼ 12) patients. Percentages

of CD27+ CD25high and FOXP3+ CD25high CD4+ T cells

were strongly comparable in all individuals tested

(P < 0�0001; linear regression analysis r2 ¼ 0�92; Fig. 5b).

These results indicate that quantification of CD27+

CD25high CD4+ T cells accurately reflects the number

of FOXP3+ CD25high CD4+ T cells and therefore can be

used to measure CD25high Treg frequencies in CSF

samples.

Interestingly, we observed a significantly higher number

of CD27+ CD25high CD4+ T cells in the CSF of RR-MS

patients (3�0 ± 0�6%) compared with numbers in paired

blood samples (1�4 ± 0�2%; P < 0�01; Fig. 5c). For

patients with OND and SP-MS, the frequencies of

CD27+ CD25high CD4+ T cells in the CSF (OND patients:

2�2 ± 0�3%; SP-MS patients: 2�0 ± 0�5%) and in blood

6

(c)

(b)

P < 0·05

Blood CSF

OND

Blood CSF

RR-MS

Blood CSF

SP-MS

P < 0·01

5

4

3

% C

D4+

CD

27+C

D25

high

T c

ells

2

1

0

% F

OX

P3+

CD

25hi

gh

CD

4+ T

cel

ls

00

1

2

3

4

5

1 2% CD27+CD25high CD+ T cells

ONDRR-MS

r 2 = 0·92P < 0·0001

3 4 5F

OX

P3

CD27

CD

25

FOXP3

0·1 4·4

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

3·3

1·3

CSF(a)

Figure 5. Analysis of regulatory T cell (Treg) frequency in the cere-

brospinal fluid (CSF) of patients with multiple sclerosis (MS) and

patients with other neurological diseases (ONDs). (a) Flow cyto-

metric analysis of forkhead box P3 (FOXP3), CD25 and CD4 on

cells isolated from the CSF of relapsing-remitting multiple sclerosis

(RR-MS) patients. The dot plots shown are representative for

data obtained from two RR-MS patients. (b) Percentages of

CD27+ CD25high and FOXP3+ CD25high CD4+ T cells (within the

total CD4+ T-cell population) were determined by flow cytometric

analysis of peripheral blood mononuclear cells (PBMC) isolated from

11 OND and 12 RR-MS patients. The cut-off value for high staining

of CD25 was set at a constant value (102) to allow comparison

between the T-cell subsets. The percentages of CD27+ CD25high

and FOXP3+ CD25high CD4+ T cells were significantly correlated

(P < 0�0001) in the PBMC of the same individuals as measured by

linear regression analysis (r2 ¼ 0�92). (c) Frequency of CD4+

CD27+ CD25high T cells in paired peripheral blood and CSF samples

of 12 OND, eight RR-MS and three SP-MS patients.

� 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89 85

Single-cell analysis of FOXP3 in patients with MS

(OND patients: 1�4 ± 0�4%; SP-MS patients: 0�8 ± 0�3%)

were not significantly different (Fig. 5c). Numbers of

CD27+ CD25high CD4+ T cells in the peripheral blood

were lower in RR-MS patients than in OND patients

(Figs 5b and c), further supporting our initial observa-

tions of a reduced frequency of FOXP3+ T cells in RR-MS

patients.

Taken together, these results indicate that Tregs of

RR-MS patients, but not of SP-MS or OND patients,

accumulate in the CSF.

Discussion

The transcriptional repressor FOXP3 plays a key role in

the development and function of naturally occurring

CD4+ CD25+ Tregs. Analyses of FOXP3 mRNA and pro-

tein expression using, respectively, quantitative RT-PCR

or western blotting indicated reduced FOXP3 levels in the

CD25 Treg population of RR-MS patients but provided

no information about FOXP3 expression at the single-cell

level. This report is the first to demonstrate lower num-

bers of circulating CD4+ CD25high FOXP3+ T cells and

decreased FOXP3 expression per cell in RR-MS patients

compared with SP-MS patients and controls.

In our study, FOXP3 expression of CD4+ CD25+ T

cells was measured on freshly isolated PBMC in a large

panel of patients and different controls by means of

flow cytometry. The observation of a lower number of

CD4+ CD25high FOXP3+ T cells and a reduced FOXP3

expression level in RR-MS patients compared with HCs

and SP-MS patients is consistent with our current and

previous functional analyses of Tregs in these patients.

We and others reported a reduced suppressor function of

CD4+ CD25high T cells isolated from RR-MS patients

and a normal Treg function in SP-MS patients.6–8 We

now extend these results by demonstrating a correlation

between the suppressive capacity of Tregs and FOXP3

expression at the cellular level. This is consistent with our

earlier report of an association between FOXP3 mRNA

levels and Treg suppression.8 Moreover, for the majority

of patients and controls, there was a positive correlation

between FOXP3+ cells within their CD4+ CD25high T-cell

population and FOXP3 levels. This means that the blood

circulating Tregs in some RR-MS patients, in addition to

being reduced in number, also expressed low levels of

FOXP3 per cell. These data further support a potential

Treg dysfunction in a subset of RR-MS patients as a

result of alterations in FOXP3 expression.

Several mechanisms may account for the reduced

FOXP3 expression and Treg frequency in RR-MS patients.

First, we and others showed that the number of

CD4+ CD25high T cells is not significantly different in MS

patients and HCs.6–8 Here, we found that the frequency

of FOXP3+ cells within the CD4+ CD25high T cells of

RR-MS patients was reduced, suggesting that a subgroup

of the CD25high Treg cells may have completely or partly

lost their FOXP3 expression. Oh and coworkers recently

reported that patients with human T-lymphotrophic virus

type 1 (HTLV-1)-associated myelopathy/tropical spastic

paraparesis (HAM/TSP), an inflammatory neurological

disease, have a reduced expression of FOXP3 in PBMC.31

Their data indicated that this reduction was mediated by

HTLV-1 tax proviral DNA load in these patients. Viral

infections have also been associated with MS pathology,

although an unequivocal role for a specific virus has not

been demonstrated to date.10

Alternatively, it could be that the reduced FOXP3

expression is associated with FOXP3 gene alterations

within our MS population. For type 1 diabetes, another

T-cell-mediated autoimmune disorder, a functional

FOXP3 polymorphism has been associated with disease

susceptibility in a Japanese population.32 In MS, no

FOXP3 polymorphism studies have yet been conducted.

Our preliminary data show that FOXP3 levels can be

up-regulated in the CD4+ T cells of RR-MS patients after

T-cell receptor stimulation (KV, unpublished data), indi-

cating that FOXP3 expression can be regulated in these

patients. Nevertheless, polymorphisms within the FOXP3

gene could have contributed to altered FOXP3 expression,

transcript stability, protein turnover or function.

In addition, suboptimal thymic development or periph-

eral homeostasis of Tregs could have accounted for the

lower Treg numbers. Indeed, several reports indicate an

age-inappropriate reduction in thymic output or altered

T-cell homeostasis in a subset of MS patients.33–35 A

detailed analysis of parameters related to Treg thy-

mic output in MS patients is necessary to investigate this

possibility.

It could also be that the balance between auto-aggres-

sive T cells and Tregs is disturbed in RR-MS patients,

resulting in more activated T cells in the CD25high CD4+

T-cell population. It has recently been shown in a mouse

model that decreased FOXP3 expression can cause defec-

tive Treg function and Treg conversion into effector T

cells, which would contribute to immune disorders

observed in these animals.36 Results obtained by Viglietta

et al. also support this possibility, as these investigators

showed a lower cloning efficiency of CD4+ CD25high T

cells with suppressor function in RR-MS patients com-

pared with HCs.6

Finally, the possibility cannot be excluded that Tregs

actively migrate to the site of inflammation in RR-MS

patients, causing a preferential shift towards the CNS

compartment. To investigate this possibility, we first mea-

sured the expression of a set of adhesion molecules

(CD62L, CD44, CD103 and CD49d) and subsequently

quantified Tregs in the CSF of MS patients and controls.

We found that a significantly higher percentage of

CD4+ CD25high FOXP3+ Tregs expressed CD103 and

VLA-4 in RR-MS than in HCs and SP-MS patients. In

86 � 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89

K. Venken et al.

mice, it has been demonstrated that CD103+ CD4+

T cells, regardless of their CD25 expression, represent a

subpopulation of effector/memory Tregs, which can

actively migrate to inflammatory lesions induced in these

animal models.14 In this regard, we should also draw

attention to our observation of higher numbers of

CD25– CD103+ T cells in RR-MS patients. In an EAE

study, an accumulation of CD103+ FOXP3+ T cells was

detected in CNS lesions.23 A similar role for CD103 on

human Tregs as that shown for animal-derived Tregs is

therefore possible.37 In addition, it is known that VLA-4

plays a central role in endothelial transmigration of

T cells into the inflamed CNS.18 Our observations there-

fore suggest that a number of peripheral Tregs in RR-MS

patients have an increased capacity to migrate into the

inflamed CNS lesions in RR-MS patients. Indeed, we

observed FOXP3+ CD4+ T cells in the CSF of MS patients

and OND. However, because of technical restrictions we

could not analyse FOXP3 expression for all CSF/blood

samples. Therefore, to allow accurate comparison of

Treg numbers among the different tissue samples and

subjects, we focused on the CD4+ T cells with high

expression of CD25 and CD27. The number of these

CD27+ CD25high CD4+ T cells corresponded to the fre-

quency of FOXP3+ CD25high CD4+ T cells, indicating that

this gating strategy excluded a possible contamination of

activated cells in our Treg quantification. We have to

mention that regulatory FOXP3+ CD25int CD4+ T cells

could be missed using this approach. Nevertheless, our

analysis showed that Tregs with a similar phenotype (i.e.

high CD25 and CD27 expression) were increased in the

CSF as compared with the peripheral blood in RR-MS

patients, but not in OND or SP-MS patients. This was in

agreement with recent observations by Feger et al., who

quantified CD25high CD45RO+ T cells in the CSF and

blood of MS patients.38 Functional analysis of CSF-

derived CD4+ CD25high T cells of MS patients would be

very informative. Unfortunately, because of the low

frequency of these cells in the CSF, it is not possible to

perform Treg/Tresp coculture experiments. It can be spec-

ulated that Tregs in the CSF of MS patients have a lower

suppressive capacity, as these Tregs could represent the

same population of cells (i.e. with lower FOXP3 expres-

sion levels) as those in the peripheral blood. However, it

is possible that the most potent Tregs are present within

the MS lesions, as has been shown in inflammatory

lesions in EAE animals22 and in the synovial fluid of

patients with RA.39 Korn et al.23 recently showed, in an

EAE model, that CNS-accumulated Tregs could inhibit

the proliferation of peripheral blood-derived naıve and

myelin oligodendrocyte glycoprotein (MOG)-reactive

T cells but not the proliferation of (inflamed) CNS

tissue-derived MOG-reactive T effector cells. Their data

underline a role for the local inflammatory cytokine envi-

ronment in determining the in situ outcome of the Treg

suppressive capacity. In our study, no correlation between

CD103 or VLA-4 expression and FOXP3 levels in

CD4+ CD25high T cells of MS patients was detected, sug-

gesting that CD103 and CD49d are not exclusively

expressed on Tregs with the strongest suppressive capac-

ity. The identification of surface markers related to the

in vitro suppressor function is needed to clarify this issue.

In the light of our own and other findings, we suggest

the following hypothesis concerning the role of Tregs in

MS pathology. In the early phase of the disease, a defect

in Treg function caused by either genetic alterations or

reduced thymic Treg output could contribute to subopti-

mal peripheral tolerance, leading to an escape of auto-

aggressive T cells. As a feedback mechanism, circulating

Tregs could up-regulate tissue retention molecules and

preferentially migrate to the inflamed CNS lesions.

Whether these Tregs are capable of influencing local

inflammation will depend on the functional capacity of

these Tregs, the in situ Treg/Teffector balance, the costim-

ulatory and cytokine environment (e.g. IL-6, TNF-a and

IL-17) and possibly the presence of other (induced) regu-

latory immune cells (e.g. CD8+ Tregs). The outcome of

this response could be associated with phases of relapse

and remission during the disease course. In the more pro-

gressive disease stage, neurodegenerative processes may

dominate inflammatory pathological features and periph-

eral T-cell-dependent inflammatory processes could play a

minor role,40,41 which may have led to a normalization of

the peripheral Treg/Teff balance. The detection of a nor-

mal Treg frequency in the peripheral blood and CSF of

SP-MS patients is consistent with this idea. Some SP-MS

patients showed relatively low Treg FOXP3 expression

(MFI) which was not correlated with their high Treg sup-

pressive capacity. However, this could have been partly

compensated by a high number of FOXP3+ cells within

the CD25high population in these patients. In addition,

the possibility cannot be excluded that other factors

besides FOXP3 [e.g. suppressive cytokines such as trans-

forming growth factor (TGF)-b] could also have contrib-

uted to the observed high Treg suppressive capacity in

these SP-MS patients.

A disturbance of FOXP3 expression and/or Treg func-

tion could be associated with a general susceptibility

for autoimmune disease development, although we must

emphasize that this represents only one of the many risk

factors for autoimmunity, as genetic background and

environmental factors also have to be taken into

account. Our preliminary observation of normal FOXP3

levels in RA patients and a subset of SLE patients,

in agreement with recent studies,29,42 supports this

conclusion.

From a therapeutic point of view, it would be challeng-

ing to restore Treg dysfunction in RR-MS patients in the

early disease stage. In this regard, it was interesting to

observe that RR-MS patients treated with IFN-b had

� 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89 87

Single-cell analysis of FOXP3 in patients with MS

increased FOXP3 levels compared with untreated patients.

In addition to other effects such as suppression of T-cell

proliferation or a shift in T helper type 1 (Th1) towards

a Th2 cytokine profile,43 our data suggest that IFN-btreatment may also affect regulatory T-cell function in

MS patients. Consistent with this observation, we8 and

others44 showed increased Treg suppression in RR-MS

patients treated with IFN-b. Whether there was a direct

correlation between a beneficial clinical outcome medi-

ated by IFN-b in MS patients and increased FOXP3 levels

has yet to be established. The exact mechanism by which

IFN-b increases Treg numbers is unknown. Our prelimin-

ary data demonstrate no induction of FOXP3 upon

short-term in vitro incubation of Tregs with IFN-b (KV,

unpublished data). It was recently shown that plasma-

cytoid dendritic cells in RR-MS patients, in contrast to

those in healthy donors, were impaired in the induction

of CD4+ FOXP3+ T cells.45 Therefore, it could be that

IFN-b has an indirect effect on Treg induction via modu-

lation of dendritic cells, as reported previously.46 Of note,

CD4+ CD25high T cells in IFN-b-treated RR-MS patients

in our study included a higher number of CD62L-nega-

tive cells. It has been demonstrated that anti-TNF-a ther-

apy in RA patients induces a distinct regulatory T-cell

population lacking CD62L expression.15 Whether the

CD62L– CD4+ CD25high FOXP3+ T cells in IFN-b-treated

RR-MS patients also represent a different subset of Tregs

should be investigated in future studies.

Taken together, the results obtained here show differ-

ences in numbers of circulating FOXP3+ CD4+ CD25+ T

cells in the peripheral blood of RR-MS patients and HCs.

In addition, lower FOXP3 expression at the cellular level

was observed, further supporting a Treg dysfunction in

RR-MS patients. Our results indicate that Tregs accumu-

late in the CSF of RR-MS patients, probably in an

attempt to down-regulate local inflammation in the CNS

of these patients.

Acknowledgements

This study was supported by research funding from the

Belgian Charchot Foundation and the Hasselt University

and Transnational University Limburg. The authors thank

Dr D. Van Hoof and Dr P. Van Paassen for providing

RA and SLE blood samples. We acknowledge A. Bogaers,

Bertine Timmermans and Riny Wieers for blood collec-

tions. We are grateful to Hanne Jongen for excellent tech-

nical assistance. We also thank all patients and control

subjects involved in this study for participation in blood

and CSF collections.

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Supplementary material

The following supplementary material is available for this

article online:

Table S1. Overview of the phenotype of regulatory and

non-regulatory T cells of healthy controls (HCs), patients

with other neurological diseases (ONDs) and multiple

sclerosis (MS) patients.

This material is available as part of the online article

from http://www.blackwell-synergy.com

� 2007 Blackwell Publishing Ltd, Immunology, 123, 79–89 89

Single-cell analysis of FOXP3 in patients with MS