IL10 inversely correlates with the percentage of CD8(+) cells in MDS patients

Contents

lists

available

at

SciVerse

ScienceDirect

Leukemia

Research

j o

u r

n a l

h o m e p

a g e :

w w w . e l s e v i e r . c o m / l o c a t e / l e u k r e s

IL10

inversely

correlates

with

the

percentage

of

CD8

+

_cells

_in

_MDS

_patients

Matheus

Rodrigues

Lopes

a

_{, Fabiola}

_Traina

a

_{, Paula}

_de

_Melo

_Campos

a

_{, João}

_Kleber

_Novais

_Pereira

a

_,

João

Agostinho

Machado-Neto

a

_,

_Helymar

_da

_Costa

_Machado

b

_,

_Simone

_Cristina

_Olenscki

_Gilli

a

_,

Sara

Teresinha

Olalla

Saad

a

_,

_Patricia

_Favaro

a

,

c

,

∗

a_{HaematologyandHemotherapyCenter-UniversityofCampinas/Hemocentro-Unicamp,InstitutoNacionaldeCiênciaeTecnologiadoSangue,Campinas,SãoPaulo,Brazil} b_{MedicalSciencesSchool,StateUniversityofCampinas,SãoPaulo,Brazil}

c_{DepartmentofBiologicalSciences,FederalUniversityofSãoPaulo,Diadema,SãoPaulo,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18October2012

Receivedinrevisedform4January2013 Accepted25January2013

Available online 28 February 2013

Keywords:

MyelodysplasticSyndrome IL10

CD8+_cells

ImmuneSystem

a

b

s

t

r

a

c

t

Therole oftheimmune systeminmyelodysplasticsyndrome(MDS)progressionhasbeenwidely accepted,althoughmechanismsunderlyingthisimmunedysfunctionarenotclear.CD4+_andCD8+ lym-phocyteprofilesintheperipheralbloodofMDSpatientswereevaluatedandcorrelatedwithclinical characteristics,theexpressionofFOXP3andtheanti-inflammatorycytokinesIL10,TGFˇ1andCTLA4.IL10 expressioninverselycorrelatedwiththepercentageofCD8+_{cellsandwashigherinhigh-riskMDS.Our} findingsprovidefurtherevidencefortheroleofTcell-mediatedIL10productioninMDSandstrengthen theideaofdistinctcytokineprofilesinlowandhigh-riskMDS.

© 2013 Elsevier Ltd.

1.

Introduction

Myelodysplastic

syndrome

(MDS)

is

characterized

by

an

increased

programmed

cell

death

of

bone

marrow

(BM)

cells,

both

clonal

and

nonclonal

precursors,

which

contributes

to

ineffective

hematopoiesis

and

peripheral

cytopenias

[1–5]

.

Although

the

com-plex

pathogenesis

of

MDS

remains

poorly

defined,

several

studies

indicate

a

role

for

the

immune

system

in

the

progression

of

early

MDS

to

the

advanced

stage

[6,7]

.

Low-risk

MDS

is

characterized

by

excessive

apoptosis

in

the

BM

and

by

an

autoimmune

disease-like

profile;

whereas

advanced

MDS

is

distinguished

by

immune

evasion,

lower

apoptosis

and

secondary

DNA

damage,

facilitating

the

progress

into

acute

leukemia

[7,8]

.

Immunosuppressive

and

immunomodulatory

therapeutics

have

presented

favorable

results,

such

as

abrogation

of

transfusion

dependence

for

a

subset

of

the

patients

[8–12]

.

Although

regulatory

(Tregs)

and

cytotoxic

T

cells

are

reported

to

be

modulated

during

the

course

of

MDS

[13]

,

the

exact

mechanism

by

which

these

cells

contribute

to

MDS

progression

is

not

yet

clear.

Low

numbers

of

Tregs

in

low-risk

MDS

are

associated

with

T

cell

∗Correspondingauthorat:DepartmentofBiologicalSciences,FederalUniversity ofSãoPaulo,Diadema,SãoPaulo,Brazil,FederalUniversityofSãoPaulo,Diadema campus,RuaSãoNicolau,210,09913-030–Diadema,SP,Brazil.

Tel.:+551140493300;fax:+551140493300.

E-mailaddresses:patricia.favaro@unifesp.br,favaropb@gmail.com(P.Favaro).

cytotoxicity

of

BM

precursor

cells,

whereas

higher

frequencies

of

Tregs

in

high-risk

MDS

result

in

a

suppression

of

immune

response

[8,14,15]

.

In

an

attempt

to

better

understand

the

role

of

the

immune

sys-tem

in

MDS,

we

evaluated

CD4

+

_and

_CD8

+

_lymphocyte

_profiles

in

the

peripheral

blood

of

MDS

patients.

These

data

were

corre-lated

with

clinical

characteristics,

the

expression

of

FOXP3

and

the

anti-inflammatory

cytokines,

IL10,

TGF

ˇ

1

and

CTLA4

.

2. Materialsandmethods

2.1. Patientsandhealthydonors

Peripheralbloodsamples,collectedfrom49patientswithMDSand29unrelated, random,andhealthyindividuals(medianage=39,range,28–60),wereanalyzed.All patientsthatattendedtheclinicbetween2010and2011,withaconfirmeddiagnosis ofMDSanduntreatedatthetimeofthestudywereincluded.Allhealthycontrols andpatientsprovidedinformedwrittenconsentandthestudywasapprovedby theethicscommitteeoftheUniversityofCampinas.Patients’characteristicsare describedinTable1.

2.2. Peripheralbloodanalyses

HematologicalvaluesweredeterminedwithaCELL-DYNSapphireautomated hematologyanalyzer(AbbottDiagnostics,Illinois,USA).Peripheralblood mononu-clearcells(PBMC)wereisolatedbyFicoll-Hypaquegradientcentrifugation(Sigma, StLouis,MO).PBMCwerestainedwiththeconjugatedmonoclonalantibodies; allophycocyanin(APC)anti-CD3,fluoresceinisothiocyanate(FITC)anti-CD8,and phycoerythrin (PE)anti-CD4. AnFSC/SSCgate was createdaroundthe viable lymphocytepopulationforfurtheranalysisofCD3+_cells,CD3+_CD4+_andCD3+_CD8+

subsets. Dataacquisition wasperformedusinga FACScaliburFlowCytometer

0145-2126© 2013 Elsevier Ltd.

http://dx.doi.org/10.1016/j.leukres.2013.01.019

Open access under the Elsevier OA license.

Table1

Clinicalcharacteristicsofpatients.

Characteristics Value

Agey,median(range) 67(27–89)

Sex,n(%)

Male/female 24(49)/25(51)

WHOclassification,n(%)

RCUD 09(19)

RCMD 23(47)

RARS 8(16)

RAEB1 7(14)

RAEB2 2(4)

RiskstratificationbyWHOa_,n(%)

Low-risk 40(82)

High-risk 9(18)

Cytogeneticriskgroup,n(%)

Good 41(84)

Intermediate 3(6)

Poor 2(4)

Nogrowth 3(6)

Peripheralbloodcounts,median(range)

Hemoglobin,(g/dL) 10.5(5.5–15.6)

Whitebloodcellcount,(×109/L) 3.58(0.86–9.8)

Neutrophilscount,(×109/L) 1.63(0.16–6.51)

Plateletcount,(×109/L) 158(0.7–648)

Numberofcytopenia,(%)

0 6(12)

1 20(41)

2 20(41)

3 3(6)

Bonemarrowblasts,%,median(range) 1.5(0–14)

RCUDindicatesrefractorycytopeniawithunilineagedysplasia;RCMD,refractory cytopeniawithmultilineagedysplasia;RARS,refractoryanemiawithring siderob-lasts;RAEB-1/2,refractoryanemiawithexcessblasts-1/2.

a_{Low-riskincludesRCUD,RARS,RCMD.High-riskincludesRAEB-1/2.}

(BectonDickinson,FranklinLakes,NJ)andanalyseswerecarriedoutusingCellQuest andBDFACSDivasoftware(BectonDickinson,FranklinLakes,NJ).TheCD3+_Tcells

fromPBMCweresortedusinganti-CD3monoclonalantibodyandMACS®_Magnetic

Cellsortingtechnique(MiltenyiBiotec,BergischGladbach,Germany).

2.3. Quantitativepolymerasechainreaction

SortedCD3+_{cellsweresubmittedtoRNAextraction.QuantitativePCR(q-PCR)}

wasperformedinanABI7500SequenceDetectorSystem(AppliedBiosystems, Fos-terCity,CA)withspecificprimersforFOXP3,IL10,TGFˇ1,CTLA4,andHPRT(sequences uponrequest).Therelativegeneexpressionwascalculatedusingtheequation, 2−CT_[16].

2.4. Statisticalanalysis

Theage-adjustedmultivariatelinearregressionanalysiswasusedinorderto studytheinfluenceofbothageanddiseaseonallparametersevaluatedinperipheral blood[15,17].Themodelincludedage,group(patientsvscontrols)andan inter-actionterm(agevsdiseasestatus)asindependentvariables.Theinteractionterm

betweenageanddiseasestatuswasdroppedfromthefinalmodelwhennot statisti-callysignificant(P>0.05).Two-tailedSpearman’scorrelationcoefficient,univariate andstepwisemultivariatemodelswerealsoused.Numericvariableswithout nor-maldistributionweretransformedintoranksforanalysis.Atwo-sidedP<0.05was consideredasstatisticallysignificant.

3.

Results

3.1.

Distinct

profiles

of

peripheral

blood

lymphocytes

exist

in

MDS

We

observed

a

significant

decrease

in

lymphocyte

count

in

the

MDS

group

compared

to

the

control

group

after

adjusting

for

age

(

P

=

0.002,

Fig.

1

A).

This

statistical

difference

remained

after

we

classified

the

patients

into

subgroups,

according

to

WHO,

but

the

decreased

lymphocyte

count

was

more

pronounced

in

the

high-risk

MDS

(

P

<

0.001,

Fig.

1

B).

Lymphopenia

(<1.1

×

10

9

cells/L)

was

found

in

13

(26.5%)

of

49

MDS

patients.

Age-adjusted

percentages

of

CD3

+

_cells

_were

_{significantly}

higher

in

the

MDS

group

(

P

=

0.004,

Fig.

2

A).

Analyses

of

the

CD3

+

cell

subsets

presented

no

statistical

differences

for

CD3

+

_CD4

+

_cells

(

Fig.

2

C),

but

showed

a

strong

trend

toward

an

increased

percent-age

of

CD3

+

_CD8

+

_cells

_in

_the

_MDS

_individuals

₍

_P

₌

_0.05,

_Fig.

₂

_E).

Comparisons

between

the

subgroups

of

the

disease

showed

higher

CD3

+

_frequencies

_in

_the

_high-risk,

_compared

_to

_the

_low-risk

_MDS

(

P

=

0.02,

Fig.

2

B),

followed

by

higher

CD3

+

_CD4

+

_frequencies

_in

_the

high-risk

compared

to

the

control

group

(

P

=

0.02,

Fig.

2

D).

The

fre-quency

of

CD3

+

_CD8

+

_cells

_was

_{significantly}

_higher

_in

_the

_low-risk

MDS,

when

compared

with

the

control

group

(

P

=

0.04,

Fig.

2

F).

There

was

no

statistical

difference

between

the

MDS

and

the

control

groups

with

regard

to

the

CD4:CD8

ratios

(

Fig.

2

G);

however

comparison

among

MDS

patients

revealed

a

significantly

higher

CD4:CD8

ratio

in

the

high-risk,

compared

to

the

low-risk

groups

(

P

=

0.03,

Fig.

2

H).

We

also

correlated

CD3

+

_CD4

+

_and

_CD3

+

_CD8

+

_cell

_frequencies

with

clinical

data

(age,

sex,

hemoglobin,

leukocyte,

granulocyte,

platelet,

number

of

cytopenias,

percentage

of

blasts

in

BM,

and

karyotype

risk

group).

Univariate

analysis

demonstrated

that

advanced

age

correlated

with

a

decreased

percentage

of

CD3

+

_CD8

+

cells

(Beta

=

−

0.28;

P

=

0.037;

R

2

=

0.11),

which

was

confirmed

by

multivariate

analysis

(Beta

=

−

0.30;

P

=

0.024;

R

2

=

0.14).

There

was

no

significant

correlation

between

CD3

+

_CD4

+

_cell

_frequency

_and

the

clinical

parameters

studied.

3.2.

IL10

inversely

correlates

with

the

percentage

of

CD8

+

_cells

and

presents

higher

expression

in

high-risk

MDS

To

better

understand

the

regulation

of

the

anti-inflammatory

cytokines

IL10,

TGF

ˇ

1,

and

CTLA4

,

as

well

as

FOXP3

,

in

MDS,

the

A

MDS (n=49) Control (n=29)

B

Low-riskMDS (n=40)

Control(n=29)

High-riskMDS (n=09)

A

b

s

olute

ly

mph

o

c

y

te

c

ount

(x10

9

/L)

A

b

s

olute

lymph

o

c

y

te

c

ount

(x10

9

/L)

Age (years

)

20

40

60

80

10

0

0

1

2

3

4

Age (years

)

20

40

60

80

10

0

0

1

2

3

4

P

=0.00

2

Controlx Low-risk

P

=0.00

4

Controlx High-risk

P

<0.00

1

37 (2013) 541–546 543

20

40

60

80

10

0

0

20

40

60

A

B

C

D

E

F

MDS (n=38) Control(n=29)

MDS (n=40) Control(n=29)

MDS (n=40) Control(n=29) Low-riskMDS (n=31) Control(n=29)

High-riskMDS (n=09)

Low-riskMDS (n=30)

Control(n=29)

High-riskMDS (n=08)

%C

D

3

+

CD

4

+

Low-riskMDS (n=31)

Control(n=29)

High-riskMDS(n=09)

G

MDS (n=40) Control(n=29)

Low-riskMDS (n=31)

Control(n=29)

High-riskMDS (n=09)

%

CD

3

+

Age (yea

rs)

%

CD

3

+

Age (yea

rs)

Age (yea

rs)

%C

D

3

+

CD

4

+

Age (yea

rs)

% C

D

3

+

CD

8

+

Age (yea

rs)

% C

D

3

+

CD

8

+

Age (yea

rs)

log

(C

D4:

CD

8)

Age (years)

log

(C

D4:

CD

8)

Age (years)

20

40

60

80

10

0

20

40

60

80

100

20

40

60

80

10

0

20

40

60

80

100

20

40

60

80

10

0

20

40

60

80

20

40

60

80

10

0

20

40

60

80

20

40

60

80

10

0

0

20

40

60

20

40

60

80

10

0

-0,5

0,0

0,5

1,0

1,5

20

40

60

80

10

0

-0,5

0,0

0,5

1,0

1,5

P

=0.00

4

Controle x Low-risk

P

=0.00

9

Controlx High-risk

P

<0.00

1

Low-riskx High-risk

P

=0.02

P

=0.20

Controlx High-risk

P

=0.02

P

=0.05

Controlx Low-risk

P

=0.04

P

=0.65

Low-riskx High-risk

P

=0.03

H

Fig.2. CD3+_,CD3+_CD4+_andCD3+_CD8+_{TcellprofilesinMDSandcontrolgroups.Multivariateregressionanalysiswasperformedwith%CD3}+_(A–B),%CD3+_CD4+_(C–D),

%CD3+_CD8+_{(E–F),andlog-transformedratioofCD4:CD8(G–H),asthedependentvariables,andageanddiseasestatusasindependentvariables.ThePvalueandthenumber}

20

40

60

80

10

0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

Age (years)

A

MDS (n=49) Control(n=28)

B

lo

g

(r

e

la

ti

ve

ex

p

re

ss

io

n

FO

X

P

3

/HP

R

T

)

Low-riskMDS (n=40) Control(n=28)

High-riskMDS (n=09)

lo

g

(r

e

la

ti

ve

ex

p

re

ss

io

n

FO

X

P

3

/HP

R

T

)

20

40

60

80

10

0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

Age (years)

Low-riskMDS (n=40) Control(n=29)

High-riskMDS (n=09)

MDS (n=49) Control(n=29)

F

_{MDS (n=49) Control(n=29)}

MDS (n=49) Control(n=29)

E

lo

g

(r

e

la

ti

ve

expr

essi

on

IL

1

0

/H

P

R

T

)

lo

g

(r

e

la

ti

ve

expr

essi

on

CT

LA

4/

HPRT

)

G

Age (years)

lo

g

(r

e

la

ti

ve

expr

essi

on

IL

1

0

/H

P

R

T

)

Age (years)

Age (years)

lo

g

(r

e

la

ti

ve

expr

essi

on

TGF

ββββ

1/

HPRT

)

Age (years)

20

40

60

80

10

0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

20

40

60

80

10

0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

20

40

60

80

10

0

-5

-4

-3

-2

-1

0

1

20

40

60

80

10

0

-5

-4

-3

-2

-1

0

1

C

D

0

10

20

30

40

50

-0.5

0.0

0.5

1.0

1.5

%CD3+CD8+

log (

re

la

ti

v

e

e

x

pr

e

s

s

ion

IL

1

0

/HP

R

T

)

P

=0.02

r=-0.36

P

=0.06

Controlx Low-risk

P

=0.02

High-riskx Low-risk

P

=0.01

P

=0.00

9

Con

_P

trol

_=0.00

x Low-risk

₆

Fig.3.FOXP3,IL10,TGFˇ1andCTLA4expressionsintheperipheralbloodCD3+_{cellsofMDSandcontrolgroups.Multivariateregressionanalysiswasperformedwiththe}

37 (2013) 541–546 545

transcript

expressions

of

these

genes

were

analyzed

in

periph-eral

CD3

+

_cells.

_We

_observed

_a

_decrease

_in

_FOXP3

_expression

_in

the

MDS

group,

when

compared

with

the

control

group

after

adjusting

for

age

(

P

=

0.009,

Fig.

3

A).

Similar

statistical

differences

remained

when

low-risk

MDS

was

compared

with

the

control

group

(

P

=

0.006,

Fig.

3

B).

IL10,

TGF

ˇ

1

,

and

CTLA4

correlated

posi-tively

with

FOXP3

expression,

according

to

Spearman’s

correlation

(

P

=

0.008,

r

=

0.37;

P

=

0.04,

r

=

0.28;

P

=

0.01,

r

=

0.35,

respectively).

Secondly,

there

was

a

trend

toward

a

lower

IL10

expression

in

the

MDS

group,

when

compared

with

the

control

group

(

P

=

0.06,

Fig.

3

C),

probably

due

to

the

lower

IL10

expression

observed

in

the

low-risk

MDS,

when

compared

to

the

control

group

(

P

=

0.02,

Fig.

3

D).

Additionally,

higher

levels

of

IL10

transcripts

in

the

high-risk,

compared

to

the

low-risk

MDS,

were

observed

(

P

=

0.01,

Fig.

3

D).

No

significant

differences

in

TGF

ˇ

1

and

CTLA4

expressions

were

observed

(

Fig.

3

E-F).

IL10

transcripts

inversely

correlated

with

CD3

+

_CD8

+

_frequency

_in

_MDS

_patients

_(Spearman

_r

₌

−

0.36;

P

=

0.02,

Fig.

3

G);

however,

there

were

no

correlations

between

the

cytokines

analyzed

and

CD3

+

_CD4

+

_frequency.

4.

Discussion

There

is

a

clear

involvement

of

multiple

myeloid

cell

lineages

in

the

MDS

clone,

which

results

in

the

loss

of

the

capacity

of

dif-ferentiation

and

apoptosis

in

the

bone

marrow,

with

consequent

peripheral

pancytopenia

in

patients

[18,19]

.

Conversely,

several

studies

have

shown

that,

in

most

cases

of

MDS,

the

lymphocytes

are

not

involved

in

the

malignant

clone

[20–22]

.

In

our

study,

there

was

a

significant

decrease

in

the

absolute

lymphocyte

counts

in

the

peripheral

blood

of

MDS

patients,

which

is

in

accordance

with

the

literature

[21,23,24]

and

supports

the

immunological

abnor-malities

that

have

been

extensively

described

in

MDS

patients.

Lymphopenia

in

MDS

has

been

suggested

to

occur

due

to

a

decrease

in

the

T

cell

numbers,

as

a

consequence

of

T

cell

apoptosis

in

the

peripheral

blood

of

MDS

patients

[23,25]

;

as

well

as

a

consequence

of

CD19

+

_cell

_apoptosis

_in

_both

_the

_peripheral

_blood

_and

_bone

mar-row

of

MDS

patients

[21,26]

.

Additionally,

a

decreased

NKT

cell

number,

already

described

in

MDS

patients

[27]

,

could

contribute

to

the

lower

absolute

lymphocyte

count.

Further

studies

addressing

the

absolute

values

of

each

cell

type

in

the

peripheral

blood

of

MDS

patients

could

clarify

which

specific

cells

are

involved

in

the

lower

absolute

lymphocyte

counts.

Although

our

findings

show

that

only

26.5%

of

patients

with

lymphopenia,

the

most

prominent

decrease

in

lymphocyte

counts

was

in

the

high-risk

group,

which

is

in

agree-ment

with

the

preview

reports

[21,28]

.

Data

presented

herein

show

that

the

increase

in

CD3

+

_cell

per-centage

in

MDS

may

be

a

reflection

of

CD8

+

_frequency

_in

_the

low-risk

group

and

CD4

+

_frequency

_in

_the

_high-risk

_group.

_The

increased

CD8

+

_frequency

_in

_the

_low-risk

_MDS

_is

_in

_agreement

_with

other

studies

[29–35]

and

supports

the

contribution

of

CD8

+

_cells

_to

the

apoptosis

of

hematopoietic

progenitors,

since

the

early

stages

of

this

disease

are

characterized

by

an

increased

apoptotic

activity

[7]

.

In

contrast

to

previous

reports

demonstrating

no

significant

dif-ferences

in

the

CD4

+

_cell

_frequency

_between

_patients

_and

_controls

[36,37]

,

we

showed

a

higher

CD4

+

_cell

_frequency

_in

_high-risk

_MDS,

with

a

consequently

increased

CD4:CD8

ratio

in

this

subgroup.

In

fact,

studies

of

T

cell

subsets

in

MDS

have

been

contradictory;

a

decreased

CD4:CD8

ratio

in

MDS

patients

has

been

reported

[15,38]

,

while

other

studies

have

shown

an

increased

CD4:CD8

ratio

in

intermediate

and

high-risk

MDS

[39]

.

It

has

been

shown

that

the

inversion

of

the

CD4:CD8

ratio

is

associated

with

the

response

to

immunosuppressive

therapy

(IST)

and

is

inversely

correlated

with

the

proliferative

T-cell

index

before

IST

in

these

patients

[15]

.

Taken

together,

our

results

suggest

the

contribution

of

T

CD4

+

_cells

_to

_the

pathophysiology

of

the

disease.

The

majority

of

CD4

+

_Treg

_cells

_present

_specific

_FOXP3

expres-sion,

a

transcription

factor,

which

is

important

for

the

development

and

function

of

these

cells

[40]

;

however,

a

population

of

CD8

+

_FOXP3

+

_T

_cells

_has

_been

_described

_in

_several

_autoimmune

diseases,

after

allergen

exposure

and

allogenic

transplantation

[41–46]

.

We

found

that

the

lower

expression

of

FOXP3

transcripts

in

the

peripheral

CD3

+

_cells

_of

_MDS

_patients

_was

_clearly

_due

_to

_the

lower

expression

of

this

gene

in

the

low-risk

group.

The

only

study

that

reports

on

CD8

+

_Treg

_cells

_in

_MDS,

_related

_no

_difference

_in

_the

number

of

CD8

+

_Treg

_cells

_between

_MDS

_groups,

_IPSS

_or

_disease

progression

[14]

.

Although

data

regarding

CD4

+

_Treg

_frequency

_in

the

low-risk

MDS

patients

are

uncertain,

mainly

due

to

the

different

flow

cytometry

strategies

used

[47]

,

our

data

for

FOXP3

expression

support

studies

that

report

that

the

number

of

CD4

+

_Tregs

_is

_lower

in

low-risk

MDS

[7,14]

.

Although

IL10

and

TGF

ˇ

1

are

secreted

by

many

cell

types,

the

production

of

IL10

and

TGF

ˇ

1,

as

well

as

the

expression

of

CTLA4,

are

indications

of

the

activation

of

Treg

cells

[40]

.

Our

results

in

CD3

+

_cells

_showed

_a

_significant

_positive

_correlation

_between

the

expression

of

these

regulatory

molecules

and

FOXP3

expres-sion,

indicating

that

these

transcripts

are

derived

from

Treg

cells.

IL10

and

FOXP3

expressions

were

lower

in

the

low-risk

group,

compared

to

the

control

group,

corroborating

the

hypothesis

of

down-regulated

Tregs

in

low-risk

MDS

[7]

.

A

significant

increase

in

IL10

expression

was

observed

in

high-risk,

compared

to

low-risk

MDS,

which

is

in

agreement

with

the

measurement

of

IL10

concentrations

in

serum,

described

by

Kor-dasti

et

al.

[48]

.

Interestingly,

we

observed

an

inverse

correlation

between

CD8

+

_cell

_frequency

_and

_IL10

_expression,

_supporting

_data

describing

the

recruitment

of

CD8

+

_cells

_in

_an

_inverse

_relationship

with

the

levels

of

Tregs

in

the

bone

marrow

of

MDS

patients

[49]

.

IL10

is

thought

to

contribute

to

the

immune

suppressive

milieu,

by

inhibition

of

antigen

presentation,

cytokine

expression

and

T

helper

cell

functions

[50,51]

,

all

features

of

the

immune

evasion

that

is

characteristic

of

high-risk

MDS

[7]

.

We

postulate

that

IL10,

secreted

by

Tregs,

may

have

a

role

in

the

prevention

of

MDS

clone

elimination,

with

a

consequent

role

in

MDS

progression.

In

conclusion,

our

findings

provide

further

evidence

for

Treg

deregulation

in

low-risk

MDS;

and

most

importantly,

add

new

insight

into

the

role

of

T

cell-mediated

IL10

production

in

MDS

and

strengthen

the

idea

of

distinct

cytokine

profiles

in

low

and

high-risk

MDS.

Funding

This

work

received

financial

support

from

the

Conselho

Nacional

de

Desenvolvimento

Científico

e

Tecnológico

(CNPq),

Fundac¸

ão

de

Amparo

à

Pesquisa

do

Estado

de

São

Paulo

(FAPESP).

The

Hema-tology

and

Hemotherapy

Center

–

UNICAMP

forms

part

of

the

National

Institute

of

Blood,

Brazil

(INCT

de

Sangue–CNPq/MCT).

Authors’

Contributions

Conflict

of

interest

Authors

have

no

conflicts

of

interest.

Acknowledgements

The

authors

would

like

to

thank

Nicola

Conran

and

Raquel

Susana

Foglio

for

English

review.

The

authors

would

also

like

to

thank

Tereza

Salles

for

her

invaluable

technical

assistance.

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