Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive...

ORIGINAL ARTICLE

Patients with oral squamous cell carcinoma are characterizedby increased frequency of suppressive regulatory T cellsin the blood and tumor microenvironment

Thaıs Helena Gasparoto • Tatiana Salles de Souza Malaspina • Luciana Benevides •

Edgard Jose Franco de Melo Jr • Maria Renata Sales Nogueira Costa • Jose Humberto Damante •

Maura Rosane Valerio Ikoma • Gustavo Pompermaier Garlet • Karen Angelica Cavassani •

Joao Santana da Silva • Ana Paula Campanelli

Received: 23 August 2009 / Accepted: 23 November 2009

� Springer-Verlag 2009

Abstract Oral squamous cell carcinoma (OSCC) is a

cancerous lesion with high incidence worldwide. The

immunoregulatory events leading to OSCC persistence

remain to be elucidated. Our hypothesis is that regulatory T

cells (Tregs) are important to obstruct antitumor immune

responses in patients with OSCC. In the present study, we

investigated the frequency, phenotype, and activity of

Tregs from blood and lesions of patients with OSCC. Our

data showed that [80% of CD4?CD25? T cells isolated

from PBMC and tumor sites express FoxP3. Also, these

cells express surface Treg markers, such as GITR,

CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Puri-

fied CD4?CD25? T cells exhibited stronger suppressive

activity inhibiting allogeneic T-cell proliferation and IFN-cproduction when compared with CD4?CD25? T cells

isolated from healthy individuals. Interestingly, approxi-

mately 25% of CD4?CD25- T cells of PBMC from

patients also expressed FoxP3 and, although these cells

weakly suppress allogeneic T cells proliferative response,

they inhibited IFN-c and induced IL-10 and TGF-bsecretion in these co-cultures. Thus, our data show that

Treg cells are present in OSCC lesions and PBMC, and

these cells appear to suppress immune responses both

systemically and in the tumor microenvironment.

Keywords T regulatory cells � OSCC �Immunosuppression

Introduction

Treg cells (CD4?CD25?FoxP3?) represent 2–4% of the

total peripheral CD4? T cells in healthy humans, and these

cells are the key for the maintenance of peripheral self-

tolerance. Although the mechanism of action is not com-

pletely understood, these cells regulate CD4 and CD8 T

cells, NK, dendritic cell, and macrophage [1, 2] activity

during immune responses against pathogens, self-antigens,

and tumors [3, 4]. The involvement of Tregs in tumor

progression has been extensively investigated [5] and these

cells are increased in peripheral blood of patients with

tumors, lung, head and neck, prostate, and breast cancers

[4–11]. Although these reports have suggested a direct

correlation between Treg cells and the suppression of the

immune response, the presence and characteristics of Treg

T. H. Gasparoto � T. S. de Souza Malaspina �G. P. Garlet � A. P. Campanelli (&)

Department of Biological Sciences, Bauru Dental School,

University of Sao Paulo, Al. Octavio Pinheiro Brisolla,

9-75, CEP 17012-901, Bauru, SP, Brazil

e-mail: [email protected]

T. H. Gasparoto

e-mail: [email protected]

J. H. Damante

Department of Stomatology, Bauru Dental School,

University of Sao Paulo, Bauru, SP, Brazil

E. J. F. de Melo Jr � M. R. S. N. Costa

Lauro de Souza Lima Institute, Bauru, SP, Brazil

M. R. V. Ikoma

Amaral Carvalho Hospital, Jau, SP, Brazil

K. A. Cavassani

Departament of Pathology, Medical School,

University of Michigan, Ann Arbor, MI, USA

L. Benevides � J. S. da Silva

Department of Biochemistry and Immunology,

School of Medicine of Ribeirao Preto,

University of Sao Paulo, Ribeirao Preto, SP, Brazil

123

Cancer Immunol Immunother

DOI 10.1007/s00262-009-0803-7

cells at the site of tumor lesions from patients with squa-

mous cell carcinoma has not been fully described.

Tumor cells release cytokines that convert a conven-

tional T-cell to Tregs. This can occur via a direct effect on

the T cells or indirectly via effects on accessory cells, such

as dendritic cells and macrophages [8, 12]. The migration

of Tregs to the tumor environment is mediated by che-

moattractants (i.e. CCL22) [13] resulting in the suppression

of immune effector cells via IL-10 and TGF-b1 in the

absence of cell-to-cell contact [14]. Importantly, there is

evidence that naıve peripheral CD4?CD25negFoxP3neg T

cells convert extrathymically into FoxP3? regulatory T

cells under certain conditions, including those associated

with the tumor environment [15], via the activation of the

T-cell receptor (TCR) in the presence of TGF-b [16, 17].

Based on the importance of Tregs in the regulation of

tumor immune response, we explored the presence and

characteristics of CD4?CD25? and CD4?CD25- T cells in

the peripheral blood and tumor from patients with oral

squamous cell carcinoma (OSCC). The results showed that

more than 80% of CD4?CD25? T cells found in the tumor

site and peripheral blood of these patients expressed

FoxP3, the classical phenotype, and similar function with

the natural Treg cells. More importantly, higher frequency

of CTLA-4 GITR, TGF-b, and FoxP3? T cells were not

restricted to CD25? expression in OSCC samples from

blood and tumor sites, indicating the possible conversion of

Tregs or a peculiar phenotype of Tregs in the tumor

environment. Moreover, increased TGF-b, IL-10, and

lower IFN-c levels were found in the tissues of OSCC

lesions compared with tissue samples from healthy indi-

viduals. Altogether, these data suggest the accumulation of

CD41CD251FoxP3? T cells in OSCC sites might regulate

the effector function of T cells, which impairs tumor

immunosurveillance.

Materials and methods

Patients with OSCC and healthy volunteers

We used tumor samples and peripheral blood mononuclear

cells (PBMCs) from 12 patients with a diagnosis of OSCC.

Eight tissue samples were derived from lip tumors and one

from tongue tumor and PBMCs were obtained from 9

patients with OSCC (age ranged 41–96 years, mean

age = 58.42 ± 2.25 years old), as well as 10 age-matched

healthy volunteers (7 men and 3 women; age ranged

27–74 years). All patients had active disease at the time of

phlebotomy and surgery. Tumors were classified as well,

moderately, or poorly differentiated according to the WHO

classification of histological differentiation grade. All

patients presented undergone surgical resection of their

tumors with a curative intent, alone or combined with

radiotherapy and chemotherapy. All subjects signed an

informed consent releasing the use of specimens (tissues

and blood) for research purposes approved by Bauru Dental

School of University of Sao Paulo.

Media and reagents

All human cells were grown in RPMI 1640 (Invitrogen

Life Technologies) supplemented with 10% heat-inacti-

vated fetal calf serum (FCS, GIBCO), 100 U/ml penicillin,

100 lg/ml streptomycin, 2 mM L-glutamine, 10 mM

HEPES, 0.1 mM nonessential amino acids, and 1 mM

sodium pyruvate (all from Sigma-Aldrich). PHA and

PE-conjugated streptavidin were purchased from Invitro-

gen Life Technologies.

Abs and flow cytometry analysis

For immunostaining, PerCP, PE-, and FITC-conjugated

Abs against CD3 (UCHT 1), PerCP-CD4 (RPAT4), PerCP-

CD8 (RPA-T8), FITC-CD14 (M5E2), PE-CD19 (HIB 19),

FITC-CD25 (M-A251), PE-CD45RO (UCHL 1), PE-

CD152 (BNI3.1), PE-CD103 (Ber-ACT8), PE-CD69

(FN50), PE-CD25 (M-A251), PE-CCR4 (1G1), FITC-

FoxP3 (PCH101, eBiosciences, San Diego, USA), and the

respective mouse and rat isotype controls were used (BD

Biosciences). PE-conjugated mice mAb anti-human GITR

(110416), and biotinylated anti-TGF-b1 (LAP, 27240)

were purchased from R&D Systems. The Abs used for

intracellular cytokine staining were PE-conjugated anti-IL-10

(JES3-19F1) and biotinylated anti-TGF-b (4492) both from

R&D Systems. The cell acquisition was performed on a

FACSort flow cytometer using CellQuest software (BD

Biosciences). Unconjugated anti-CD3 (UCHT1) and anti-

CD28 (CD28.2) (BD Biosciences) were used for polyclonal

activation.

Isolation of leukocytes

Peripheral blood was harvested with heparin (50 U/ml)

from healthy subjects and OSCC patients. PBMC were

isolated using Ficoll-Hypaque (Pharmacia Biotech) density

gradient centrifugation, washed, counted, and labeled with

specific Abs for phenotypic analysis in flow cytometer or

for purification of T-cell subpopulations. To characterize

the leukocytes present in the tumor site, the tumor samples

from patients were collected and incubated 1 h at 37�C in

RPMI 1640 medium, containing 50 lg/ml collagenase CI

enzyme blend (Boehringer Ingelheim Chemicals). Next,

the tissues were dissociated, for 4 min, in the presence of

RPMI 1640 containing 10% serum and 0.05% DNase

(Sigma-Aldrich) using a Medimachine (BD Biosciences),


123

according to the manufacturer’s instructions. The tissue

homogenates were filtered using a 30 lm cell strainer

(Falcon; BD Biosciences). The leukocyte’s viability was

evaluated by Trypan blue exclusion and used for cell

activation or immunolabeling assays.

CD4?CD25? T cells separation

Cells were fractionated into CD4?CD25- and

CD4?CD25? populations using a Regulatory T-Cell Iso-

lation Kit (Miltenyi Biotec Ltd, Surrey, UK) and the

autoMACS separator (Miltenyi Biotec, Bergisch Gladbach,

Germany) according to the manufacturer’s instructions.

The isolation of these subpopulations is performed in a

two-step procedure. First, all non-CD4? T cells were

depleted with a cocktail of biotin-conjugated antibodies

and anti-biotin microbeads. Second, CD4?CD25? T cells

were positively selected using CD25 microbeads, leaving

CD4?CD25- T cells in the pre-enriched CD4? T-cell

population. A two-color flow cytometric analysis was

performed to determine the relative proportions of

CD4?CD25low and CD4?CD25high cells within the bead-

isolated CD4?CD25- and CD4?CD25? T-cell popula-

tions. The levels of contamination (OSCC cells) were less

than 1%. The purity of CD4?CD25? T-cell populations

(after magnetic selection) was [90% and determined by

flow cytometry.

T-cell stimulation

Due to the low yield of pure cells, the CD4?CD25? cells

were cultured in 96-well tissue-culture U-bottom plates

(Corning) and primarily activated with anti-CD3 mAb

(0.5 lg/ml) in the presence of anti-CD28 (1 lg/ml) and

PHA (1 lg/ml). rhIL-2 (10 ng/ml) was added at days 2, 5,

and 7 after primary stimulation. At day 10, the cells were

harvested and used in a secondary anti-CD3 and PHA

stimulation with identical conditions. At day 15, after

secondary stimulation, cells were harvested and co-culture

assays were performed as described below.

Co-cultures and proliferation assays

To verify the ability of CD4?CD25? T cells isolated from

tumor samples from patients and controls PBMCs suppress

allogeneic T-cell proliferation as described previously by

Campanelli et al. [18] with some modifications. Briefly,

magnetically sorted CD4?CD25? T cells were co-cultured

with CFSE labeled-PBMC (1 9 105/well) from normal

donors at ratio 1:10, in 96-well U-bottom plates, in the

presence of PHA (1 lg/ml), at 37�C and 5% CO2. On day

4, the cells were harvested and the proliferative response of

allogeneic T cells was assessed by measuring the CFSE

dilution using flow cytometry. T cells proliferation was

characterized by sequential halving of CFSE fluorescence,

generating equally spaced peaks on a logarithmic scale.

The data represent the percentage of inhibition calculated

on the PHA-induced proliferation of allogeneic T cells

cultured with PHA in the absence of CD4?CD25? T cells.

Intracellular cytokines and FoxP3 detection

The intracellular detection of IL-10, TGF-b, and FoxP3 in

leukocytes obtained from lesions and blood of patients, as

well as control individuals was performed using Cytofix/

Cytoperm and Perm/Wash buffer from BD Biosciences,

according to the manufacturer’s instructions. First, the cells

were labeled with Abs of cell surface, such as FITC or PE-

conjugated anti-CD25 and PerCP-conjugated anti-CD4.

Following the staining of surface markers, the cells were

fixed, permeabilized, stained with PE-labeled anti-human

(h) IL-10, FITC-FoxP3 (BD Biosciences), or control iso-

type. To TGF-b detection, the leukocytes from tumors

were stained with mouse biotin-labeled anti-hTGF-b and

incubated with PE-streptavidin (from Invitrogen Life

Technologies) according to manufacturers’ protocols.

Samples were acquired on FACSort flow cytometer and

data were analyzed using CellQuest software (BD Biosci-

ences). The graphs show the absolute number of cells/

lesion and percentage of cells/lesion.

Cytokine assays

The supernatants of tumor samples were obtained by

disaggregation through treatment with RPMI 1640 med-

ium containing 0.25% collagenase (Worthington), and

frozen at -70�C until analysis. The total protein con-

centration was measured using Quick StartTM Bradford

Protein assay kit (Bio-Rad, CA, USA). IL-10, TGF-b, and

IFN-c were quantified in the samples by the quantitative

sandwich Enzyme-Linked Immunosorbent Assay (ELISA)

using commercial capture and biotinylated detection

antibodies (BD Pharmingen Corp., San Diego, CA) and

the respective human recombinant cytokines (diluted in

PBS) as standards, according to the manufacturer’s

instructions. The concentrations of each cytokine were

dosed as pg/ml and the results were normalized and

expressed as mg/protein. Healthy gingival tissues,

removed for orthodontic treatments, were used as cytokine

controls in this analysis.

Statistical analysis

Data obtained from flow cytometry and co-culture assays

were expressed as mean ± SEM. Statistical analysis was

performed using ANOVA followed by the Tukey’s


123

multiple comparison test (INSTAT Software; GraphPad).

All values were considered significant when p B 0.05.

Results

Phenotypic characterization of CD4?CD25? T cells

in PBMC from patients with OSCC

First, we analyzed the phenotype of circulating subpopu-

lations of lymphocytes (gate shown in Fig. 1a) in PBMC

from OSCC patients and healthy control subjects (Fig. 1a).

We found that the frequencies of B cells (CD191), CD4? T

cells, CD8? T cells, and CD41 T cells expressing CD251

in patients with OSCC were similar to those detected in

normal donors. Our data did not show any difference in the

amount of CD31CD41 T cells expressing the IL-2Ra-

chain (CD25) between patients and controls (Fig. 1a),

however, cells from OSCC patients had a higher percent-

age of GITR (27.2 ± 12.2%), CD45RO (90.2 ± 3.5%),

CD69 (14 ± 7%), FoxP3 (88.5 ± 6%), and IL-10

(77.8 ± 13.2%) than those from control individuals

(6.7 ± 3.5%, 75.5 ± 8.1%, 4.1 ± 3.1%, 24.9 ± 8.3%, and

5.7 ± 3.7%, respectively) (Fig. 1b). Also, when we ana-

lyzed the population of CD4? T cells that did not express

CD25, no differences were found in the expression of

CCR4, CD103, GITR, CTLA-4, CD45RO, C69, FoxP3

and IL-10 expression (Fig. 1b). These data indicate

CD4?CD25? T cells from PBMC patients with OSCC,

unlike from healthy controls, showed a regulatory

phenotype.

Functional characterization of CD4?CD25? T cells

of PBMC from patients with OSCC

We next asked whether CD41CD251 T cells, which[90%

express FoxP3, from OSCC patients had altered suppres-

sive properties compared with healthy controls. The addi-

tion of CD4?CD25? T cells from OSCC patients strongly

inhibited the proliferation of allogeneic T cells stimulated

with PHA compared with CD4?CD25? T cells purified

from healthy controls (suppression index = 53.7 ± 7.9%

Fig. 1 Characterization of

lymphocytes from control

subjects and OSCC patients.

a PBMC from both groups in

these analyses were gated on

lymphocytes via their forward

(FSC) and side scatter (SSC)

properties. The CD3?CD4?,

CD3?CD8?, CD4?CD25?, and

CD19? cells. b Gated TCD4?

lymphocytes expressing CD25

or not were characterized.

TCD4?CD25? and

TCD4?CD25- cells were

analyzed for their expression of

GITR, CD103, CCR4, CTLA-4,

CD45RO, CD69, and

intracellular FoxP3 and IL-10

on freshly isolated PBMCs from

control (open square) and

patients (filled square) were

initially evaluated by flow

cytometry. The results are

expressed as the mean ± SEM

for patients (n = 9) or control

subjects (n = 10) tested

individually. *p \ 0.05,

**p \ 0.01, and ***p \ 0.001

when lymphocytes from

patients are compared with

controls


123

vs. 33.7 ± 5.7 %) (Fig. 2a). Figure 2b shows representa-

tive histograms of CFSE analysis of allogeneic PBMC

cultivated with or without CD41CD251 T cells from

patients and control individuals. In addition, culture of

allogeneic PBMCs in presence of CD41CD251 T cells

from patients evidenced high production of IL-10 (Fig. 3a),

but not affect IFN-c and TGF-b production (Fig. 3b, c).

Although, approximately 25% of CD41CD25- T cells

from patients expressed FoxP3 (Fig. 2b), these cells only

inhibited marginally the allogeneic PBMCs proliferation

mediated by PHA (suppression index: 13.3 ± 6.1, data not

shown), but these cells promoted significantly greater

amounts of TGF-b (Fig. 3b), and modestly inhibited IFN-clevels produced by allogeneic PBMCs (Fig. 3c). Therefore,

these results confirm that CD41CD251 T cells, but not

CD41CD25- T cells, from OSCC patients have potent

inhibitory consequences on T effector function and pro-

mote the generation of IL-10.

CD4?CD25? T cells present regulatory profile

and exert suppressive function in the OSCC lesions

We next analyzed the leukocytes infiltrate in tumor lesions

obtained from the OSCC patients. Our results showed that

Fig. 2 Functional

characterization of

CD4?CD25? T cells in patients

and controls. Magnetic

bead-sorted CD4?CD25? and

CD4?CD25- T cells were

purified from PBMC from

patients (n = 9, closed bars)

and control subjects (n = 10,

open bars) and tested for their

ability to suppress the

proliferation of allogeneic

PBMC (a). Allogeneic PBMCs

(1 9 105 cells/well) were

cultured with medium alone,

PHA, PHA plus CD4?CD25?,

or CD4?CD25- T cells

(1 9 104 cells/well) from

patients or control subjects.

Proliferation was determined

after 4 days of culture via CFSE

incorporation. b Representative

histograms of CFSE-PBMC

allogeneic proliferation

cultivated with PHA or PHA

plus CD4?CD25? cells

obtained from controls or OSCC

patients. The results are

expressed as the mean ± SEM

of stimulation index of

proliferation for patients tested

individually (n = 5). p \ 0.01

compared proliferation of

allogeneic T cells plus PHA

with or not CD4?CD25? T cells

from OSCC blood patients


123

6.7 ± 3.8 9 106 leukocytes were present in this infiltrate

(Fig. 4a) of which 35 ± 5.5% represents T cells (CD3?).

From these T cells, 23 ± 5.4% were CD4? and 18.6 ±

4.8% were CD8? (Fig. 4a). The OSCC lesions contained

approximately, 1 9 106 (15.4 ± 3.5%) CD4? T cells

co-expressing CD25 (ranged from 5.0 9 105 to 4.2 9 107

cells/lesion) (representative dot plot in Fig. 4b). From the

gated CD4?CD25? T cells (Fig. 4c) we found that these

cells expressed CTLA-4 (39.3 ± 7.1%), GITR (38.4 ± 7%),

CD103 (22.9 ± 6.7%), CD45RO (95.1 ± 2.2%), CD69

(35.2 ± 4.8%), LAP (15.6 ± 4.6%), and the chemokine

receptor CCR4 (50.6 ± 11.7 %) (Fig. 4b, black bars).

However,\10% of CD4?CD25- T cells express T regula-

tory surface markers (Fig. 4b). Intracellular FoxP3, IL-10,

and TGF-b were identified at high percentages of

CD4?CD25? T cells (Fig. 4b). Interestingly, although most

of the CD4?CD25- T cells did not express surface markers

typically found on Tregs, 49.2 ± 12% of CD4?CD25- T

cells expressed FoxP3 (Fig. 4b), indicating that these cells

might have regulatory functions.

Next, we determined TGF-b, IFN-c, and IL-10 levels in

lesions of OSCC patients. Our data clearly showed that

tumor samples contained elevated amounts of TGF-b and

IL-10 when compared with healthy gingival tissue from

Fig. 3 Cytokine levels from CD4?CD25? T cells of OSCC patients.

Allogeneic PBMC were co-cultured in the presence of magnetic bead-

sorted CD4?CD25? and CD4?CD25- T cells from PBMC from

patients (n = 9) or medium alone. IL-10 (a), TGF-b (b), and IFN-c(c) levels were analyzed in supernatants from cultures described

above. Results are expressed as the mean ± SEM from each patient

analyzed individually. *p \ 0.05 and **p \ 0.01 when compared

with results of allogeneic PBMC plus PHA

Fig. 4 Phenotypic characterization of leukocytes derived from tumor

lesions and cytokine profiles of OSCC lesions and control tissue.

Tumor sample and healthy gingival tissue of OSCC patients (n = 9)

and healthy control subjects (n = 4) were obtained. a The number of

leukocytes was determined. The frequency of tumor-derived leuko-

cytes that expressed CD3?CD4?, CD3?CD8?, CD4?CD25?,

CD8?CD25?, and CD19? were analyzed by flow cytometry.

Positives cells were determined over the appropriated isotype-

matched control. b CD4?CD25? (closed bars) and CD4?CD25-

(squared bars) gated T cells were analyzed for their surface

expression of CCR4, CTLA-4, GITR, CD103, CD45RO, CD69,

LAP, and intracellular expression of FoxP3, IL-10, and TGF-b.

c Cytokine profiles of OSCC lesions and control tissue was

determined by ELISA. The results are expressed as the mean ± SEM

from each patient analyzed individually. *p \ 0.05 and **p \ 0.01

when compared with controls


123

control individuals. In contrast, IFN-c level from OSCC

lesions was lower in comparison to the healthy gingival

tissue. These data establish that regulatory cytokines pre-

dominate in OSCC lesions and low levels of IFN-c, which

is essential for anti-tumor responses [19] (Fig. 4c).

Functional characterization of tumor infiltrating

CD4?CD25? T cells from OSCC patients

To verify the function of tumor infiltrating CD4?CD25?

T cells, we next assessed suppressive effects of these

cells in co-culture assays. Our results showed that

CD4?CD25? T cells isolated from tumor sites signifi-

cantly inhibited PHA activated-allogeneic PBMC prolif-

eration (SI = 74.5 ± 4.6) (Fig. 5a, c). These results

confirmed that infiltrating CD4?CD25? T lymphocytes

from patients presenting OSCC had a regulatory role.

Further, as we observed FoxP3 expression in the

CD4?CD25- T cells, we addressed whether these cells

had a suppressive role in the tumor sites. Although at

lower rate in comparison with CD4?CD25? T,

CD4?CD25- T cells from OSCC lesions were able to

reduce PHA activated-allogeneic PBMC proliferation

(Fig. 5a, d).

The production of cytokines is modulated

by CD4?CD25? T cells from the OSCC lesions

generating high levels of IL-10 and TGF-band inhibiting IFN-c

As we observed high levels of IL-10, TGF-b, and low

levels of IFN-c (Fig. 5) in the tumor environment, we

determined weather Treg isolated from tumor sites were

responsible for the regulation of cytokine production. In

co-culture assays, the addition of CD4?CD25? T cells

from OSCC lesions induced IL-10 and TGF-b at higher

levels when compared to allogeneic PBMC stimulated with

PHA (Fig. 6a, b). The regulatory properties of

CD4?CD25? T cells from OSCC lesions were confirmed

by the inhibition of IFN-c in these co-cultures (Fig. 6c).

Fig. 5 Functional characterization of CD4?CD25? T cells derived

from tumors of patients with OSCC. a CD4?CD25? T or

CD4?CD25- T cells (1 9 104 cells/well) isolated from tumors of

patients with OSCC (n = 9) were expanded with 0.5 lg/ml anti-CD3,

1 lg/ml anti-CD28, 1 lg/ml PHA, exogenous 10 ng/ml rhIL-2, and

tested for their ability to suppress the proliferation of allogeneic

PBMC. a Allogeneic PBMC (1 9 105 cells/well) were cultured with

medium alone, PHA, PHA plus CD4?CD25?, or CD4?CD25-

T cells. Proliferation was determined after 4 days of culture by flow

cytometry. b–d Representative histograms of PBMC allogeneic

proliferation-CFSE stained cultivated with PHA, PHA plus

CD4?CD25?, or CD4?CD25- T cells from OSCC lesions. The

results are expressed as the mean ± SEM of SI of proliferation for

patients (n = 9) tested individually. p \ 0.01 when compared

suppression by CD4?CD25- with CD4?CD25? T cells


123

These data demonstrate that CD4?CD25? T cells in OSCC

lesions present active regulatory/inhibitory profile.

Discussion

Previous studies have demonstrated that T regulatory cells

are present in higher numbers in patients suffering of many

diverse cancers, and these cells inhibit efficient immune

defenses against such neoplasias [12, 14]. In addition,

clinical studies have indicated the marked presence of

CD4?CD25? regulatory T cells as a measurement of

immune index, providing information regarding the general

status of the antitumor immune response and the antitumor

cytokine network in individual cancer patients [20, 21].

Accordingly, our results show that CD4?CD25? T cells

from peripheral blood of OSCC patients had regulatory

phenotype, with higher detection of GITR, CD69, FoxP3,

and IL-10 than controls. This finding held true despite the

fact that equal percentages of total CD4?CD25? T cells

were presented in patients and control volunteer peripheral

blood. In addition, peripheral blood purified CD4?CD25?

T cells from OSCC patients exhibited a marked inhibitory

effect on PHA-stimulated allogeneic PBMC, which was

statistically significant compared with that seen in

CD4?CD25? T cells from control group. These data con-

firm that OSCC patients have circulating Tregs, which are

able to suppress T lymphocyte proliferation to a greater

degree than healthy individuals. We speculate whether

these differences in Treg functions occur due to different

ratio between Tregs and effector CD4? T cells in patients

and controls or whether Tregs alter their phenotype in

squamous cell carcinoma. Therefore, we theorize that

Tregs in OSCC patients could adjust the phenotypic

appearance to increased regulatory function. We intend to

test this hypothesis in future experiments.

Moreover, we analyzed whether Tregs from OSCC

patients blood induced and/or altered IL-10, TGF-b, and

IFN-c production by allogeneic PBMC. Our data showed

that the co-culture of peripheral Treg and PHA-stimulated

allogeneic PBMC generated significantly higher levels of

IL-10 when compared with PHA-stimulated allogeneic

PBMC in the absence of Tregs. However, peripheral blood

circulating Tregs from OSCC patients did not alter IFN-cor TGF-b levels produced by PHA-stimulated allogeneic

PBMC, suggesting that IL-10 is an important factor in the

suppression of effector T cells by CD4?CD25?GITR?-

FoxP3? Tregs cells from patients [12]. Despite the fact that

FoxP3? Treg cells inhibit T-cell proliferation in a micro-

environment rich in IFN-c seems paradoxical, this effect

has been demonstrated previously [22]. Therefore, OSCC

patients have higher circulating T lymphocytes with potent

suppressor roles and such cells resemble of the natural

regulatory T cells [17]. On the other hand, higher per-

centage of circulating CD4?CD25- T cells from OSCC

patients expressed FoxP3 when compared with control

CD25- T cells. These cells also caused a significant

increase of IL-10, TGF-b and suppressed IFN-c secretion.

However, these cells marginally inhibit PHA-stimulated

allogeneic PBMC proliferation. Again, an alternative

explanation would be possible differences in Treg func-

tions related to ratio between Tregs and effector CD4? T

cells. In addition, the tumor promotes generation of Tr1

cells which have the phenotype distinct from

CD4?CD25?FoxP3? natural Treg and mediate IL-10-

dependent immune suppression in a cell contact-indepen-

dent manner [12, 17]. Therefore, we speculate that

CD4?CD25- T cells in peripheral blood of OSCC patients

are Tr1 cells and may play a critical role in cancer

Fig. 6 Cytokine profiles of CD4?CD25? subsets in the lesions of

patients with OSCC. Magnetic bead-sorted CD4?CD25? T cells were

purified from lesions from patients (n = 9) and cultivated with

allogeneic PBMC. IL-10 (a), TGF-b (b), and IFN-c (c) levels were

analyzed in supernatants from cultures described above. Results are

expressed as the mean ± SEM from each patient analyzed individ-

ually. ***p \ 0.001 when compared with results of allogeneic PBMC

plus PHA


123

progression. Accordingly, it has been shown previously

that high production of IL-10 by CD4? T cells in squamous

cell carcinoma do not stem from natural Tregs [14]. Also,

Tregs expressing FoxP3 and IL-10 (named Tr1) are dif-

ferentiated in the periphery from a conventional T cells and

they have been described previously to be involved in

tumor escape mechanisms [11–14, 23]. High levels of

IL-10 are a strong indicative that soluble factors are involved

with the presence of Treg in the tumor microenvironment.

We will pursue these studies more fully in the future.

Functional characterization of Tregs has been varied and

this appears to depend on tumor localization [14, 21, 24,

25]. Although, OSCC lesions have been shown to present a

high number of infiltrating CD25?FoxP3? regulatory T

cells [26, 27], the specific inhibitor/regulatory role of these

cells was not previously investigated. Here, OSSC lesions

contained high numbers of TILs and a higher percentage of

CD4? CD25?GITR?FoxP3?IL-10? T cells. In contrast to

circulating Tregs, OSSC TILs expressed CTLA-4, CD69,

LAP, and intracellular TGF-b. Nevertheless, Tregs in

OSCC lesions were CD45RO?, which agrees with previous

studies showing that Tregs in adult individuals have this

phenotype [16, 17]. As expected, IL-10 and TGF-b were

strongly expressed in the tumor microenvironment.

Although many cell types might be producing such sup-

pressive factors, our results showed that the Tregs infil-

trating lesions might be the major source for purified

CD4?CD25? T cells which produced large amounts of

TGF-b and IL-10. For the first time, we show that these

cells from OSCC lesions also strongly inhibited PHA-

stimulated allogeneic PBMC proliferation and, unlike cir-

culating CD4?CD25? T cells, they induced high levels of

TGF-b and suppressed IFN-c secretion. Here, the tumor

microenvironment might facilitate Tregs infiltration, and

these cells perform their function through cytokine influ-

ence as well as via suppression of T-cell proliferation. This

regulatory T-cell-mediated immunosuppression may char-

acterize one of the tumor immune-evasion mechanisms

facilitating bad prognostic and relapse of the disease

[7, 11]. Also, tumors could actively prevent the induction

of tumor-associated antigen (TAA)-specific immunity

through induction of regulatory T-cell trafficking, differ-

entiation and expansion [12]. Accordingly, from our data,

OSCC patients seem to have a different Treg population

circulating in peripheral blood. Although these two Tregs

had diverse phenotypes and cytokine pattern, both were

able to suppress allogeneic proliferation effectively.

Tregs from OSCC lesions present a phenotype and

function consistent with natural Treg cells [17]. However,

we verified that TILs Tregs expressed also high level of

TGF-b and our data do not exclude other regulatory T cells

groups [17]. In general, marked presence of Tregs infil-

tration in OSCC lesions correlates with a negative

prognosis because of local immune suppression [28].

According to our results, the presence of Tregs may be, in

part, responsible for down-regulation of antitumor immune

responses as by inhibition of T-cell proliferation as by

high secretion of immunosuppressor cytokines. Further

studies are necessary to establish exact influence of Tregs

on activated T cells and their role in the regulation of

OSCC lesion. Understanding the role of Tregs infiltrating

OSCC lesion might contribute with novel therapeutic

interventions.

Acknowledgments We thank Dr. Cory M. Hogaboam for critical

reading of the manuscript. This study was supported by a grant (# 06/

04264-9) from The State of Sao Paulo Research Foundation

(FAPESP). The authors report no conflicts of interest related to this study.

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