Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasia.

Abstract

Oncogenic human papillomavirus (HPV)-infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (T(regs)) might be involved in the failure of the immune system to control the development of HPV-induced cancer. We investigated frequencies, phenotype and activity of T(regs) in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4(+)/CD25(high) T cell frequencies in peripheral blood and CD4(+) T cell fraction. These CD4(+)/CD25(high) T cells represent T(regs) as demonstrated by their low proliferation rate, low interferon (IFN)-gamma/interleukin (IL)-10 ratio, high expression of CD45RO, GITR, CTLA-4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16(+) cervical cancer patients, in-vitro depletion of CD25(+) T cells resulted in increased IFN-gamma T cell responses against HPV16 E6- and E7 peptides. Thus, increased frequencies of T(regs) in cervical cancer patients may indeed suppress HPV-specific immunity. Longitudinal analysis of CD4(+)/CD25(high) T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of T(regs) in cervical cancer. These results imply that T(regs) may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by T(regs) will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia.

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Frequencies and role of regulatory T cells in patients with
(pre)malignant cervical neoplasia
J. Visser,*†H. W. Nijman,*
B.-N. Hoogenboom,*†P. Jager,†
D. van Baarle,‡E. Schuuring,§
W. Abdulahad,¶F. Miedema,‡
A. G. van der Zee,* and T. Daemen†
*Department of Gynecology, University Medical
Center Groningen, University of Groningen,
Groningen, the Netherlands,†Department of
Medical Microbiology, Molecular Virology
Section, University Medical Center Groningen,
University of Groningen, Groningen, the
Netherlands,‡Department of Immunology,
University Medical Center Utrecht, Utrecht, the
Netherlands,§Department of Pathology,
University Medical Center Groningen, University
of Groningen, Groningen, the Netherlands, and
¶Department of Clinical Immunology, University
Medical Center Groningen, University of
Groningen, Groningen, the Netherlands
Summary
Oncogenic human papillomavirus (HPV)-infection is crucial for developing
cervical cancer and its precursor lesions [cervical intraepithelial neoplasia
(CIN)]. Regulatory T cells (Tregs) might be involved in the failure of the
immunesystemtocontrolthedevelopmentof HPV-inducedcancer.Weinves-
tigated frequencies, phenotype and activity of Tregs in patients with cervical
neoplasia. CIN and cervical cancer patients showed increased CD4+/CD25high
T cell frequencies in peripheral blood and CD4+T cell fraction. These CD4+/
CD25highTcellsrepresentTregsasdemonstratedbytheirlowproliferationrate,
low interferon (IFN)-g/interleukin (IL)-10 ratio,high expression of CD45RO,
GITR,CTLA-4,forkhead box P3 (FoxP3) and low CD45RA expression.More-
over, in HPV16+cervical cancer patients, in-vitro depletion of CD25+T cells
resulted in increased IFN-g T cell responses against HPV16 E6- and E7
peptides. Thus, increased frequencies of Tregsin cervical cancer patients may
indeed suppress HPV-specific immunity. Longitudinal analysis of CD4+/
CD25highT cell frequencies in patients showed a modest decline 1 year after
curative surgery or chemoradiation. This study demonstrates increased fre-
quencies and suppressive activity of Tregs in cervical cancer. These results
imply that Tregs may suppress the immune control of cervical neoplasia and
furthermore that suppression of immunity by Tregswill be another hurdle to
overcome in therapeutic immunization strategies against cervical neoplasia.
Keywords: CD4+T cell, CD8+T cell, cervical cancer, HPV, regulatory T cells
Accepted for publication 18 June 2007
Correspondence and current affiliation: Dr
J. T. J. Visser, University Medical Center
Groningen, University of Groningen,
Department of Cell Biology, Section
Immunology, 3215 11th Floor, A. Deusinglaan
1, 9713 AV, Groningen, the Netherlands.
E-mail: j.t.j.visser@med.umcg.nl
Introduction
Natural
co-expression of CD4 and CD25, play an important role in
immune homeostasis [1–4]. In animal tumour models,
elevated frequencies of Tregs have been demonstrated and
Tregdepletion increased the anti-tumour immune responses
[5,6]. These observations led to the hypothesis that cancer
patients have an enlarged population of Tregs inhibiting
tumour-specific T cell responses [1–3]. A recent study
showed that CD4+/CD25+Tregs control the induction of
antigen-specific T-helper responses in cancer patients [7].
Intratumoral Tregs have been demonstrated in ovarian
cancer patients [8] and an enlarged population of Tregs in
peripheral blood of patients with different types of cancer
[9–13].
regulatoryTcells(Tregs),characterizedby
Infection with oncogenic human papillomavirus (HPV) is
involved in cervical carcinogenesis; HPV DNA can be
detected in ? 99% of all cervical cancers [14,15]. Most
women infected with oncogenic HPV types clear the infec-
tion and do not develop (pre)malignant cervical neoplasia.
The importance of the immune system in HPV clearance is
demonstrated by observations that immunocompromised
women fail more often to clear HPV infections and have an
increased risk of developing cervical cancer [16].
The E6 and E7 oncoproteins of HPV play a crucial role in
the transformation and maintenance of the malignant phe-
notype [15]. Several reports showed impaired cellular
immunity against the HPV16 E6 and/or E7 oncoproteins in
cervical cancer patients [17–20]. It has been suggested that
impaired cellular immunity against these oncoproteins is
responsible for the failure to eradicate HPV infections,
Clinical and Experimental Immunology
ORIGINAL ARTICLE doi:10.1111/j.1365-2249.2007.03468.x
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leading subsequently to (pre)malignant cervical neoplasia
[18]. In other studies, significant cellular immune responses
against HPV16 E6 and/or E7 were demonstrated in cervical
intraepithelial neoplasia (CIN) and cervical cancer patients
[21–23].However,theseresponsesseemunabletoclearHPV
infections [21–23]. Interestingly, we observed that (subopti-
mal) T cell responses against HPV in CIN and cervical
cancer patients can be enhanced by invasive surgical
procedures [21].
CD4+/CD25+Tregs have been found in tumour-draining
lymph nodes of cervical cancer patients [24,25]. Immuno-
histochemistry also revealed the presence of CD25+Tregs in
infiltrate associated with CIN lesions [26].However,in these
studiesfrequenciesandphenotypesof TregsinCINorcervical
cancer patients were not compared to healthy controls;
nor was suppression of (HPV-specific) cellular immune
responses by Tregsdetermined.
To elucidate the role of Tregsin developing (pre)malignant
cervical neoplasia, we evaluated frequencies, phenotype and
suppressive activity of CD4+/CD25+T cells in peripheral
blood of patients with cervical cancer or CIN and healthy
controls. Longitudinal analysis of Treg frequencies was per-
formed during and following therapy.We also investigated if
the previously observed suboptimal T cell responses against
HPV can be ascribed to increased frequencies/activities
of Tregs.
Materials and methods
Ethical approval
The study was approved by the local medical ethical
committee of the University Medical Center Groningen
(UMCG). Written informed consent was obtained from
all patients.
Patients
CIN and cervical cancer patients (Table 1) were recruited
from the out-patient clinic of the gynecology department at
the UMCG,as described previously [21].In the Netherlands,
cervical smears are classified according a modified Papani-
colaou system in which borderline dyskaryosis corresponds
well with the Bethesda classification of atypical squamous
cells of undetermined significance, mild dyskaryosis with
low-grade squamous intraepithelial lesions and moderate
and severe dyskaryosis and carcinoma in situ with high-
gradesquamousintraepitheliallesions[27].Patientsreferred
with cervical carcinoma were staged according FigO criteria
[28].In general,patients with FigO stages Ib/IIa were treated
by radical surgery and patients with stages IIb–IV were
treated with chemoradiation. Radiotherapy consisted of
50 Gy in 25 fractions, five fractions a week, combined with
Table 1. Patient characteristics.
CIN patientsCervical cancer patients
Patient
no.
Age
(years)DiagnosisGrade
HPV16
status
Patient
no.
Age
(years)Diagnosis Stage
HPV16
status
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
26
28
27
50
45
27
41
48
34
34
33
45
40
38
40
40
35
30
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
CIN
I
I
I
I
I
I
I
I
II
III
III
III
III
III
III
III
III
III
neg
neg
neg
?
?
neg
pos
?
pos
neg
pos
pos
?
neg
pos
pos
?
?
1
2
3
4
5
6
7
8
9
37
30
71
36
33
40
75
60
35
45
36
33
26
54
43
70
43
46
36
48
51
41
45
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
IA1
IA1
IB1
IB1
IB
IB1
IB1
IB1
IB1
IB1
IB1
IB2
IBII
IIA
IIB
IIB
IIB
IIB
IIB
IIB
IIB
IIB
IIB
pos
pos
pos
pos
neg
neg
pos
pos
pos
neg
neg
pos
pos
pos
pos
neg
pos
pos
pos
pos
pos
pos
neg
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Avg 35 ? 8Avg 45 ? 13
CIN, cervical intraepithelial neoplasia; SCC, squamous cell carcinoma; HPV, human papillomavirus.
J. Visser et al.
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two fractions of brachytherapy if indicated. In addition to
radiotherapy, patients received 40 mg/m2of cisplatin per
week for 6 weeks.
Female healthy volunteers (age 39 ? 11 years) were
recruited from the departments of gynecology and medical
microbiology of the UMCG.
Isolation of cell subsets
Heparinized blood (50 ml) was obtained and peripheral
blood mononuclear cells (PBMC) were isolated with a
Ficoll-density gradient. PBMC were cryopreserved using
standardized conditions enabling batchwise analysis at a
later time.
Using fluorescent activated cell-sorting, thawed PBMC of
healthy controls and patients with (pre)malignant cervical
neoplasia were separated into CD4+/CD25negT cells, CD4+/
CD25lowT cells and CD4+/CD25highT cells. PBMC were
stained with aCD4-APC (IQ Products, Groningen, the
Netherlands) and aCD25-fluorescein isothiocyanate (FITC)
antibodies (BD Biosciences, San Diego, CA, USA). Cells of
interest were isolated with a Dako-Cytomation MoFlo High-
Speed Sorter (Glostrup, Denmark), using gate-settings as
described previously [13,29].
Flow cytometry
PBMC were stained with aCD25-FITC (BD Biosciences),
aCD152-PE (CTLA4; BD Biosciences), anti-glucocorticoid-
induced tumour necrosis factor (TNF) receptor family-
related gene (GITR)-phycoerythrin (PE) (R&D Systems,
Minneapolis, OK, USA), aCD45RO-PE (IQ Products),
aCD4-antigen-presenting cell (APC) (IQ Products), anti-
forkhead box P3 (FoxP3)-PE (eBioscience) and isotype con-
trols to determine the immunophenotype of the different
CD25Tcellsubsets.Flowcytometrywasperformedandcells
were measured with a fluorescence activated cell sorter
(FACS)Calibur (BD Biosciences). Cells were analysed using
CellQuest software (BD Biosciences).
Cell cultures, cytokine- and proliferation assays
Isolated CD4+/CD25 T cell subsets were cultured at a density
of 2·5 ¥ 104cells/well in 96-well round-bottomed plates
(Nunc,Rochester,NY,USA).Cells were cultured in a volume
of 200 ml RPMI-1640 (Gibco, Breda, the Netherlands)
supplemented with 10% fetal calf serum (FCS) (BioWhit-
taker,Verviers,Belgium),penicillin/streptomycinand50 mM
b-mercaptoethanol.
Cells were stimulated with 0·75 mg/ml aCD3/1 mg/ml
aCD28 (Sanquin Research, Amsterdam, the Netherlands).
Culture supernatants were harvested after 3 days and cell
proliferation was measured by overnight [3H]-thymidine
incorporation (1 mCi/well; Amersham, Bucks, UK). Labelled
cells were harvested and [3H]-thymidine incorporation
measured with a liquid scintillation counter (Canberra-
Packard, Meriden, CT, USA).
Cytokines were measured in culture supernatants using
commercially available enzyme-linked immunosorbent
assay (ELISA) kits (Sanquin Research).
Expansion and detection of interferon
(IFN)-g-producing HPV16E6- and E7-specific T cells
For expansion and detection of HPV16 E6- and E7-specific
T cells, we adapted an assay developed previously for detec-
tion of Epstein–Barr virus-specific CD4+and CD8+T cells
[30,31]. HPV16 E6- or E7-specific T cells were stimulated
using 15-mer peptides with an 11-aa overlap spanning the
complete sequence of HPV16 E6 (37 peptides) or E7 (22
peptides) protein. Peptides were synthesized by Mimotopes/
Perbio Sciences. Purity (> 90%) and sequences were verified
by high performance liquid chromatography (HPLC)/mass-
spectrometry.Peptidesweredissolvedindimethylsulphoxide
(DMSO)andpooled(finalconcentrationof 1 mg/mlof each
peptide).Stimulations with peptide pools and medium were
performed in the presence of co-stimulation (2 mg/ml
aCD28). As a negative control, cells were stimulated with
medium and co-stimulation only.As a positive control, cells
were stimulated with 10 ng/ml phorbol myristate acetate
(PMA)/2 mg/ml ionomycin.
To expand HPV16 E6- or E7-specific T cells, total PBMC
or CD25+cell-depleted PBMC were cultured for 12 days in
the presence of E6 or E7 peptide pools. Culture medium
consisted of RPMI-1640 (Gibco) supplemented with
penicillin/streptomycin,50 mMb-mercaptoethanoland10%
human pool serum (Sigma, Zwijndrecht, the Netherlands;
complete medium). Cells were cultured at 2 ¥ 105PBMC/
well in 100 ml complete medium in 96-well round-bottomed
plates at 37°C and 5% CO2. The peptide pool (at 2 mg/ml of
each peptide) was added on days 0 and 6. Interleukin (IL)-2
(10 U/ml)wasaddedondays3,6and9.Onday12,cellswere
pooled, washed in RPMI-1640 and incubated overnight in
complete medium.
On day 13,the number of IFN-producing cells were deter-
mined by intracellular cytokine staining; 106cells were
stimulated in 500 ml complete medium for 6 h with HPV16
E6 or E7 peptide pools (at 2 mg/ml of each peptide) and
aCD28(2 mg/ml;SanquinResearch)asco-stimulation.After
1 h, brefeldin-A (Golgiplug; BD Biosciences) was added at a
dilution of 1 : 1000 to allow cytokine accumulation in the
cytosol. After stimulation, cells were washed in phosphate-
buffered saline (PBS) + 0·5% bovine serum albumin (BSA),
permeabilized (FACS permeabilizing solution; BD Bio-
sciences), washed again and stained with aCD3-PE/Cy5
(SanquinResearch),aCD4-APC(IQProducts),aCD8-FITC
(Sanquin Research) and anti-IFN-PE (IQ Products). Cells
were washed again, fixed (Cellfix; BD Biosciences) and
2 ¥ 105events were acquired on a FACSCalibur and data
analysed using CellQuest software.
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Enzyme-linked immunospot (ELISPOT) to
determine HPV16 E6- and E7-specific T cell
responses
Cryopreserved PBMC were thawed and CD25+T cells were
depleted using anti-CD25 Microbeads (Miltenyi-Biotec,
Bergisch-Gladbach,Germany) according the manufacturer’s
instructions. This procedure leads to complete removal of
CD4+/CD25highT cell fractions. Unseparated PBMC and
CD25-depleted were seeded at a density of 1·5 ¥ 106cells/
well in a 24-well plate (Corning Life-Sciences,Schiphol-Rijk,
theNetherlands)in1·5 mlcompletemediuminthepresence
or absence of HPV16 E6 or E7 peptide-pools (10 mg/ml of
each peptide).
After 4 days of incubation, PBMC were harvested,
washed and seeded in quadruplicate at a density of 1 ¥ 105
cells/well in a coated ELISPOT-plate (Nunc, Silent-screen,
Rochester, NY, USA) with an IFN-g catching antibody
(IFN-g ELISPOT assay; Sanquin Research). The assay was
performed further according the manufacturer’s instruc-
tions (Sanquin Research). ELISPOT plates were analysed
with a fully automated imaging device (A.EL.VIS, Hanover,
Germany). The background in medium control wells was
below 10 spots/1 ¥ 105cells. Specific spots were calculated
using criteria similar to other studies [17–19,21]. Specific
responses were calculated by subtracting the mean number
of spots ?2 s.d. of medium control wells from the mean
number of spots of experimental wells. Response against
HPV16 E6 or E7 peptide-pools were considered positive
when the number of specific spots was ?10/1 ¥ 105cells
and the response in the experimental wells was at least two
times the background levels. As a positive control, PBMC
were stimulated with a memory recall mix (MRM; kind gift
of Dr S. van der Burg, LUMC, the Netherlands), consisting
of a mixture of Tetanus toxoid (0·75 LF/ml), Mycobacterium
tuberculosis sonicate (2·5 mg/ml) and Candida albicans
(0·005%).
Establishing HPV16 status
HPV16 status was established by HPV16-specific poly-
merase chain reaction (PCR) on DNA isolated from cervical
scrapings obtained at the patient’s initial visit. The scraped
cells were suspended in 5 ml PBS and kept on ice. The
HPV16-specific PCR was performed as described previously
[21]. HPV16-primers [product 152 base pairs (bp)] were:
sense: TGCTAGTGCTTATGCAGCAGCAA,
ATTTACTGCAACATTGGTAC.
anti-sense:
Statistical analysis
Differences between groups were determined using the
Mann-Whitney U-test and c2test. Significance was deter-
mined as P < 0·05.
Results
Frequencies of CD4+/CD25+T cells in peripheral blood
of healthy controls, patients with CIN and cervical
cancer
CD4+/CD25+and CD4+/CD25highT cells (Fig. 1a,d) were
examined in patients with CIN, cervical cancer and healthy
controls, using gate-settings as described previously [13,29].
As shown in Fig. 1, CD4+/CD25+(Fig. 1b,c) and CD4+/
CD25highT cells (Fig. 1e,f) were significantly increased in
both total PBMC (Fig. 1b,e) and CD4+T cell fraction
(Fig. 1c,f) of patients with CIN and cervical cancer com-
pared to healthy controls.
Immune phenotype and FoxP3 expression of
CD4+/CD25 T cell subsets
CD4+/CD25highT cells of representative CIN patients, cervi-
cal cancer patients and healthy controls were characterized
further for the expression of CD45RO, GITR, CTLA-4 and
CD45RA. The three first molecules are expressed relatively
highly and CD45RA is expressed relatively low on Tregs
[1–3,9–13,29]. CD4+/CD25highT cells of patients and con-
trols expressed increased levels of CD45RO, CTLA-4 and
GITR compared to CD4+/CD25negT cells (data not shown).
Also, as the CD4+/CD25highT cells expressed much lower
CD45RA levels than CD4+/CD25negT cells (data not shown),
theCD4+/CD25highTcellsof CINandcervicalcancerpatients
phenotypically resemble Tregs.
The transcription factor FoxP3 is considered to be a spe-
cific marker for Tregs[1,32,33].Therefore,we analysed FoxP3
expression in the CD4+/CD25 T cell subsets (Fig. 2, upper
panel).In both healthy controls and cervical cancer patients,
themajorityof CD4+/CD25highTcellsexpressedFoxP3,while
CD4+/CD25negT cells and CD4+/CD25lowT cells expressed
FoxP3 at very low and moderate levels, respectively (Fig. 2,
lower panel).
Cytokine profiles of CD4+/CD25negand CD4+/CD25high
T cell subsets
In general, Tregs are characterized by low proliferation, low
IFN-g production and robust IL-10 production [1–3,9–
13,29]. CD4/CD25negand CD4+/CD25highT cells were iso-
lated by cell-sorting (Fig. 3a) to study their functionality
in vitro.
The CD4+/CD25highsubset showed a low proliferation rate
(Fig. 3b), high levels of IL-10 production (Fig. 3c) and low
IFN-g production (Fig. 3d). The CD4+/CD25negsubset, on
theotherhand,showedtheoppositeprofile.AlthoughCD4+/
CD25highand CD4+/CD25negcells of cervical cancer patients,
on average, produced lower cytokine levels, the cytokine
profile of these T cell subsets was similar compared to these
subsets in healthy controls and patients with CIN.
J. Visser et al.
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Effect of CD25+T cell depletion on HPV16
E6/E7-specific and general T cell responses
Using IFN-g ELISPOT analysis,we determined the influence
of CD4+/CD25+T cells on HPV16 E6- and E7-specific T cell
responses in patients with cervical cancer.
In vitro, depletion of CD25+T cells enhanced IFN-g T cell
responses against HPV16 E6 and/or E7 peptide-pools in
50% (five of 10) of the HPV16-DNA+cervical cancer
patients (Table 2; P = 0·05 compared to HPV16negcervical
cancer patients and P = 0·07 compared to the controls). In
HPV16negcervical cancer patients no significant responses
against HPV16 E6 and E7 peptide-pools were observed, and
in only one healthy control CD25+T cell depletion enhanced
the HPV16 E6-specific T cell response (Table 2).
In a number of HPV16-DNA+, HPV16-DNAnegand
control donors, depletion of CD25+T cells enhanced MRM-
specific T cell responses (Table 2). In one patient (patient 7)
and three controls (controls 3, 5, 10) depletion of CD25+T
cells caused a decrease of MRM-specific T cell responses.
This is probably due to depletion of CD25+activated
memory T cells.
Using standard IFN-g ELISPOT analysis the total T cell
response is measured,not discriminating between CD4+and
CD8+T cell responses. As Tregscontrol both CD4+and CD8+
T cell responses [1–4], we determined if depletion of CD25+
T cells enhances HPV-specific T cell responses of both cell
subsets.
All cervical cancer patients thus analysed displayed low,
yet detectable CD4+T cell responses against HPV16 E6
and/or E7 peptide-pools (Table 3). As observed by others
[19–22], we also found higher CD4+and CD8+T cell
responses against the E6 peptide pool compared to the E7
peptide pool (Table 3). Upon depletion of CD25+cells, the
responsewasenhancedintheCD4+Tcellfractioninthreeof
four patients. Patients 2, 4 and 20 showed CD8 responses
against the HPV16 E6 and/or E7 peptide pools. However, in
only patient 2 was the E6-specific response enhanced after
CD25+celldepletion.BackgroundIFN-gproductionwasnot
enhancedsignificantlybyCD25+celldepletion.Althoughthe
results for the HPV16 E6- and E7-specific CD8+responses
are inconclusive, these results suggest that CD25+T cells can
at least suppress the HPV16 E6- and/or E7-specific CD4+T
cell responses.
100
100
101
102
APC
CD4-APC
103
104
6
7
9
101
102
FITC
CD25-FITC
Controls
n = 23
p = 0·043
p = 0·012
p < 0·001
CD4+/CD25+ T cell levels
0
2
4
6
8
% of PBMC
10
12
14
16
CIN I
n = 8
CIN II/III
n = 10
CxCa
n = 23
(a)(b)
CD25+
103
104
100
100
101
102
APC
CD4-APC
103
104
67
9
101
102
FITC
CD25-FITC
(d)
CD25+
103
104
Controls
(n = 23)
p < 0·01
p = 0·01
p < 0·001
CD4+/CD25high T cell levels
0
0·5
1·5
2·5
1
2
3
% of PBMC
CIN I
(n = 8)
CIN II/III
(n = 10)
CxCa
(n = 23)
(e)
Controls
n = 23
p = 0·21
p = 0·01
p < 0·001
CD4+/CD25+ T cell levels
0
5
10
20
15
30
25
35
% of CD4
CIN I
n = 8
CIN II/III
n = 10
CxCa
n = 23
(c)
Controls
(n = 23)
p = 0·038
p = 0·015
p < 0·001
CD4+/CD25high T cell levels
0
2
4
6
8
10
% of CD4
CIN I
(n = 8)
CIN II/III
(n = 10)
CxCa
(n = 23)
(f)
Fig. 1. (a, d) Gate-settings for calculating CD4+/CD25+and CD4+/CD25highT cell frequencies. Markers to establish CD4+/CD25+T cell frequencies
are set on the isotype control. (b, c) CD4+/CD25+T cell frequencies in total peripheral blood mononuclear cells (PBMC) (b) and CD4+T cell
population (c). (e, f) CD4+/CD25highT cell frequencies in total PBMC and CD4+T cell population, respectively. Analysis was performed with PBMC
collected before invasive therapy. Differences between groups were analysed using the Mann–Whitney U-test.
Tregsand cervical neoplasia
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Longitudinal follow-up of CD4+CD25highT cell
frequencies in patients with cervical cancer
Longitudinal analyses of CD4+/CD25highT cell frequencies
were performed in four healthy controls and 10 cervical
cancer patients during and following therapy. In healthy
controls the CD4+/CD25highT cell population appeared very
stable (Fig. 4a). In two patients with cervical cancer who
underwent curative surgery only, with no sign of recurrence
for more than 2 years, CD4+/CD25highT cell frequencies
showed a modest decline following curative surgery
(Fig. 4b,c). CD4+/CD25highT cell frequencies showed strong
fluctuation in patients treated with chemoradiation (RCT;
Fig. 4d–k). In six patients the CD4+/CD25highT cell
frequencies increased after radiochemotherapy (Figs 4d–h,
k); in two patients these frequencies remained stable
(Fig. 4i,j). After completion of chemoradiation, CD4+/
CD25highT cell frequencies did not fall below the levels as
measured before chemoradiation in all eight patients.
Discussion
In this study we provide evidence that patients with CIN and
cervical cancer have increased CD4+/CD25highTreg frequen-
cies in their peripheral blood compared to healthy controls.
Our data show that these CD4+/CD25highT cells, but not
CD4+/CD25lowT cells, of CIN and cervical cancer patients
display all Treg characteristics. The CD4+/CD25highT cells
Fig. 2. Forkhead box P3 (FoxP3) expression
was measured by flowcytometry in CD4+/CD25
subsets of a healthy control and three cervical
cancer patients with peripheral blood
mononuclear cells collected before invasive
therapy. The upper panel shows gate-settings
for CD4+/CD25neg, CD4+/CD25lowand
CD4+/CD25highcells. Within the T cell fractions
FoxP3 expression was analysed (lower panel).
CD25-FITC
I
I = CD25neg
II = CD25low
II
III
III = CD25high
CD25neg
4·1%
3·5%
3·6%
4%
16%
10%
17%
20%
76%
66%
57%
60%
Health control
Cervical cancer
patient 1
Cervical cancer
patient 2
Cervical cancer
patient 3
CD4-APC
FOXP3-PE
CD25lowCD25high
100
100
101
102
103
104
67
9
8
101
102
FITC
103
104
APC
CD4-APC
J. Visser et al.
6
© 2007 British Society for Immunology, Clinical and Experimental Immunology

Page 7

express high levels of CD45RO, GITR and CTLA-4. Further-
more, these CD4+/CD25highT cells produce high levels of
IL-10, low levels of IFN-g and have a low rate of
proliferation. Moreover, this subset is highly positive for the
Treg-specific marker FoxP3.
It has been shown that in vitro CD25+T cell depletion
enhanced virus-specific CD4+and/or CD8+T cell responses
[33]. Moreover, in melanoma patients, CD4+/CD25+Tregs
control tumour-specific CD4+T cell responses [7].We there-
fore hypothesized that increased Treg numbers in cervical
cancer patients might be responsible for impaired cellular
immunity against HPV. Indeed, in vitro, depletion of CD25+
T cells, enhanced HPV16 E6- and/or E7-specific T cell
responses in 50% of the HPV16-DNAposcervical cancer
patients.This enhancement did not occur in HPV16-DNAneg
cervical cancer patients, while only one healthy control
showed an enhanced E6-specific T cell response, probably
reflecting T cell memory of previous HPV-infection(s). Our
results indicate that suppression of HPV16 E6/E7-specific T
cell responses by CD25+cells was at the level of CD4+and
possibly also CD8+T cells. The increased IFN-g responses
could not be explained by enrichment of responder T cells
after CD25+cell depletion, because relative changes of CD4+
T and CD8+T cells remained below 4% and 2%,respectively
(data not shown).
CD25+T cell depletion also enhanced T cell responses
against MRM in a considerable number of cervical cancer
patients and healthy controls. In one patient (patient 7) and
three controls (controls 3, 5 and 10), CD25+T cell depletion
resultedinadecreaseof theMRM-specificTcellresponse.In
view of the fact that activated memory T cells express
increased levels of CD25, this decrease might be due to
depletion of activated memory T cells.
Thesimultaneousincreaseof HPV16andMRM-specificT
cellresponsesinHPV16+cervicalcancerpatientsafterCD25+
T cell depletion indicate that suppression of cell-mediated
immunity by Tregs in these patients is not antigen-specific.
However,our results imply that increased frequencies of Tregs
incervicalcancerpatientsmightsuppresstheimmunological
control of cervical neoplasia. Insufficient cell numbers were
available to characterize further the suppression of general
immune responses by Tregsin cervical cancer patients.
CD25high
(a)
(c)
(d)
(b)
Proliferation
IL-10 production
IFN-y production
CD25-FITC
0
0
0
1000
2000
3000
100
200
300
400
500
pg/ml
500
DPS1000
1500
Controls (n = 7) CIN (n = 11) CxCa (n = 12)
Controls (n = 7) CIN (n = 11)CxCa (n = 12) Controls (n = 7) CIN (n = 11)CxCa (n = 12)
CD4-APC
100
100
101
102
APC
103
104
67
9
8
101
102
FITC
103
104
Fig. 3. (a) Gate-settings used for sorting the different CD4+/CD25 T cell populations. White bars represent the CD25negT cells and the black bars
the CD25highT cells. Cell-sorting experiments were performed with peripheral blood mononuclear cells (PBMC) collected before invasive therapy of
seven representative controls, 11 representative patients with cervical intraepithelial neoplasia and 12 representative patients with cervical cancer.
Proliferation (b) is expressed as dissociations per second (dps; mean ? s.e.m.), interleukin-10 (c) and interferon-g (d) production as pg/ml
(mean ? s.e.m.).
Tregsand cervical neoplasia
7
© 2007 British Society for Immunology, Clinical and Experimental Immunology

Page 8

Table 2. Effect of CD25+cell depletion on interferon (IFN)-g T cell responses against HPV16 E6 and/or E7 in cervical cancer patients and healthy
controls.
Cervical cancer patients
PBMCCD 25 depleted PBMC
E6 E7MRM E6E7MRM
HPV16 pos
Patient 1
Patient 2
Patient 3
Patient 7
Patient 8
Patient 12
Patient 14
Patient 15
Patient 19
Patient 21
9
0
0
0
3
3
0
1
0
2
4
3
0
1
3
38
31
9
0
0
0
1088
37
9
6
0
> 400
170
> 400
91
74
56
76
56
157
12
39
38
45
39
42
43
16
29
10
12
1
0
0
7
7
2
4
0
3
7
> 40014
> 400
HPV16 neg
Patient 5
Patient 6
Patient 10
Patient 11
Patient 16
0
0
0
0
1
0
1
0
0
1
> 400
0
0
9
0
0
0
4
0
0
1
> 400
6
0
95
4
102
28
167
38
Controls
Control 1
Control 2
Control 3
Control 4
Control 5
Control 6
Control 7
Control 8
Control 9
Control 10
0
1
0
0
0
0
0
0
0
0
1
1
1
0
0
1
77
65
170
0
0
0
0
0
0
0
X
0
3
2
0
0
0
0
X
0
0
112
80
51
85
120
118
159
5
170
43
110
49
78
10
2
0
0
X
79
269
21
> 400
6
HPV16 E6- and E7-specific T cell responses in patients with cervical cancer and controls as determined by interferon (IFN)-g enzyme-linked
immunospot. The table shows the number of spots per 1 ¥ 105cells. Shown are the responses in total peripheral blood mononuclear cells (PBMC) and
the CD25-depleted PBMC. Specific responses (? 10/1 ¥ 105cells) are indicated in bold type. Analyses were performed with PBMC collected before
invasive therapy. MRM: memory recall mix, E6: E6 peptide pool, E7: E7 peptide pool, X: insufficient cells available for analysis.
Table 3. Effect of CD25+cell depletion on antigen-specific interferon (IFN)-g responses against human papilloma virus (HPV)16 E6 and/or E7 in both
CD4+and CD8+T cell subsets of HPV16+cervical cancer patients.
Cervical cancer patients
PBMCCD25 depleted PBMC
Neg controlE6 pept E7 peptNeg control E6 peptE7 pept
% IFN-y + CD4+ T cells
Patient 2
Patient 4
Patient 20
Patient 22
%IFN-g + CD8+ T cells
Patient 2
Patient 4
Patient 20
Patient 22
0·9
2·3
0·4
0·8
3·4
7
2·3
7·1
3·6
2·9
0·6
1·7
1
2·8
0·4
1·2
7·5
9·2
4·5
7·8
4·8
n.a.
1·6
1·4
0·5
0·8
0·4
0·5
1·4
4·1
2·1
0·7
0·8
1·9
0·6
0·3
0·3
n.a.
0·7
0·3
6·3
n.a.
1·7
0·6
0·8
n.a.
1
0·3
Human papilloma virus (HPV)16 E6 and E7-specific interferon (IFN)-g T cell production was measured by intracellular cytokine staining after in
vitro expansion for 12 days with peripheral blood mononuclear cells (PBMC) or CD25-depleted PBMC of four HPV16 + cervical cancer patients in the
presence of HPV16 E6 or E7 peptide pools.The results are expressed as percentage IFN-g positive cells within the CD4+or CD8+T cell fraction.Positive
responses(atleasttwotimesthebackground)areindicatedinboldtype.AnalyseswereperformedwithPBMCobtainedbeforeinvasivetherapy.E6pept:
E6 peptide pool, E7 pept: E7 peptide pool, n.a. insufficient cells available for analysis.
J. Visser et al.
8
© 2007 British Society for Immunology, Clinical and Experimental Immunology

Page 9

Our results regarding suppression of HPV16 E6- and
E7-specific T cell responses by CD25+T cells shed new light
on observations of relatively low or impaired HPV-specific
cellular immunity in cervical cancer patients [17–23].In this
study we provide evidence that intrinsically these anti-HPV
responses exist, but are suppressed by Tregs. Therefore, the
observed (low) level of HPV-specific immunity in PBMC of
cervical cancer patients [17–23] is most probably an under-
estimation of the actual HPV-specific T cell numbers.
Malignant cervical tumour cells can produce large
amounts of transforming growth factor (TGF)-b [34].
Because TGF-b induces Tregdevelopment [35], this might be
one of the mechanisms leading to increased Treg levels in
cervical cancer patients. However, theoretically, it cannot be
ruled out that women developing CIN or cervical cancer
display intrinsically increased Tregnumbers. If the increased
Treg numbers are induced by the cervical tumour only,
tumour eradication might lead to a decline in Tregnumbers.
However, we found only a modest decline of CD4+/CD25high
T cell frequencies in the year following curative surgery.
Patientstreatedwithchemoradiationshowedstrongfluctua-
tions but no decrease of their CD4+/CD25highT cell frequen-
cies during and following therapy. Chemoradiation causes
tissue damage and apoptosis which may lead to immune
activation. As a consequence the number of Tregs might be
up-regulatedtopreventimmunepathology[1–4].Itremains
Time in Days
healthy controls
cervical cancer patient 13 (stage IB/II)
cervical cancer patient 15 (stage IIB)
cervical cancer patient 19 (stage IIB)
cervical cancer patient 18 (stage IIB)
cervical cancer patient 22 (stage IIB)
cervical cancer patient 23 (stage IIB)
cervical cancer patient 17 (stage IIB)
cervical cancer patient 20 (stage IIB)
(a)
donor 1
EUA, biopsy
EUA, no
residual tumor
EUA,
start RCT
Biopsy
Biopsy
RCT
RCT
RCT
RCT
RCT
RCT
RCT
RCT
Lympedectomy,
start RCT
Wertheim,
high tumor load
At day 231 still high
tumor load,
palliative theapy
At day 625,
residual disease
0
2
4
6
% of CD4
8
10
donor 2
11
Time in Days
Time in Days
Time in Days
Time in Days
Time in Days
Time in Days
Time in Days
Time in Days
(d)
(g)(h) (i)
(j) (k)
0
4
8
12
% of CD4
% of CD4
% of CD4
% of CD4
% of CD4
% of CD4
% of CD4
% of CD4
16
1 26169186231
1
1
1
11
1
1
4088 152
80126
234
1760 24424164
EUA, Lletz
no residual tumor
EUA, Lletz
no residual tumor
365
2173136
130
Wertheim, no
residual tumor
Wertheim
>2 years no
recurrence
>2 years no
recurrence
>2 years no
recurrence
>2 years no
recurrence
>2 years no
recurrence
>2 years no
recurrence
154384
868
136260
250 951251 95171 150232 350
Time in Days
cervical cancer patient 1 (stage IA1)
(b)
(e) (f)
0
2
4
6
% of CD4
8
10
0
0
2
4
4
6
8
8
12
16
0
4
8
12
16
0
4
8
12
16
0
4
8
12
16
0
4
8
12
16
0
4
8
12
16
20
24
10
17 21330
donor 3 donor 4
LIetz
Vag. Hysterectomy,
no residual tumor
Time in Days
cervical cancer patient 35 (stage IB1)
(c)
0
2
4
6
% of CD4
8
10
1 1428298
LIetz
Wertheim, no
residual tumor
Fig. 4. The figure shows the longitudinal CD4+/CD25highT cell frequencies as a percentage of the CD4+T cell fraction in four healthy controls (a)
and 10 cervical cancer patients (c–k) during and following their therapy. Type of intervention/therapy and remarks about clinical status is
mentioned in the figure. EUA: examination under anaesthesia; RCT: radiotherapy combined with chemotherapy.
Tregsand cervical neoplasia
9
© 2007 British Society for Immunology, Clinical and Experimental Immunology

Page 10

an unanswered question as to whether Tregs are induced or
already present in patients with (pre)malignant cervical
neoplasia. Therefore, further research will be required to
elucidate fully the influence of therapy on Tregs in these
patients.
Because E6 and E7 transforming oncoproteins are crucial
for transformation and maintenance of the malignant phe-
notype,theyareidealcandidatesfortumour-specificcervical
cancer immunotherapy [36]. The results presented in this
study indicate that the presence of increased Treg numbers
is another hurdle to overcome for successful therapeutic
immunotherapy, especially when this therapy is given as an
adjuvant to chemoradiation.Therefore,immunization strat-
egies should elicit strong anti-tumour immune responses:
strong enough to overcome the immunosuppressive state of
the patient. Such strong responses have been described for
immunizations with a genetic vector derived from Semliki-
Forest virus (an alphavirus) expressing HPV16 E6/E7,which
couldbreakimmunetoleranceinHPV-transgenicmice[37].
Another approach could be (temporarily) bypassing sup-
pression of cellular immunity by Tregs during therapeutic
immunizations for treatment of (pre)malignant cervical
neoplasia. The clinical potential of enhancing anti-tumour
immune responses by Tregdepletion has been shown recently
in patients with metastatic renal carcinoma [38]. However,
interfering in Treg-controlled immune responses should be
conducted with extreme caution, because of the risk of
developing autoimmunity [39].
Acknowledgements
We thank Nening Nanlohy, Klaske 10 Hoor, Harry Klip and
Esther Nijhuis for their excellent assistance.T.Daemen,A.G.
van der Zee and F. Miedema were supported financially by
the Dutch Cancer Society, grant no. RUG 2001–2398; H. W.
Nijman was supported financially by the Dutch Cancer
Society, grant no. 2002–2678. J. Visser was supported finan-
cially by a grant from Maurits en Anna de Kock stichting.
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© 2007 British Society for Immunology, Clinical and Experimental Immunology