Bacalb,d, Paulo Vidal Campreghera,b, Nelson Hamerschlaka,b, Gustavo Pessini Amarante-Mendesc,f

w w w . r b h h . o r g

Hematology,

Transfusion

and

Cell

Therapy

Original

article

ZAP-70

expression

is

associated

with

increased

CD4

central

memory

T

cells

in

chronic

lymphocytic

leukemia:

cross-sectional

study

Rodolfo

Patussi

Correia

_,

_Flávia

_Amoroso

_Matos

_e

_Silva

_,

_Nydia

_Strachman

_Bacal

_,

Paulo

Vidal

Campregher

_,

_Nelson

_Hamerschlak

_,

Gustavo

Pessini

Amarante-Mendes

a_{InstitutoIsraelitadeEnsinoePesquisa,SãoPaulo,SP,Brazil} b_{HospitalIsraelitaAlbertEinstein,SãoPaulo,SP,Brazil}

c_{InstitutodeCiênciasBiomédicas,UniversidadedeSãoPaulo(ICBUSP),SãoPaulo,SP,Brazil} d_{CentrodeHematologiadeSãoPaulo(CHSP),SãoPaulo,SP,Brazil}

f_{InstitutoNacionaldeCiênciaeTecnologia(INCT),Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8March2017 Accepted28March2018 Availableonline11June2018

Keywords:

Chroniclymphocyticleukemia ZAP-70protein-tyrosinekinase MemoryTcells

a

b

s

t

r

a

c

t

Background:AlthoughchroniclymphocyticleukemiaisbasicallyaBcelldisease,its patho-physiologyandevolutionarethoughttobesignificantlyinfluencedbyTcells,astheseare probablythemostimportantinteractionpartnerofneoplasticBcells,participatingintheir expansion,differentiationandsurvival.ChroniclymphocyticleukemiaBcellsmayalsodrive functionalandphenotypicchangesofnon-malignantTcells.Therearefewdataaboutthe associationbetweenmemoryTcellsandprognosis,especiallyrelatedtoZAP-70,acommon reliablesurrogateofthegoldstandardchroniclymphocyticleukemiaprognosticmarkers.

Objective:Theaimofthisstudy wastoinvestigatewhethertheexpressionofZAP-70in chroniclymphocyticleukemiapatientsisassociatedwithabnormalpatternsofthe distri-butionofnaïveandmemoryTcellsrelatedtocrosstalkbetweenthesecells.

Methods:In this cross-sectional, controlled study, patients with chronic lymphocytic leukemiawerecomparedwithhealthyblooddonorsregardingtheexpressionofZAP-70 andthedistributionofnaïveandmemoryTcellsubsetsinperipheralbloodasmeasuredby flowcytometry.

Results:ZAP-70positivepatientspresentedanincreasedfrequencyandabsolutenumberof centralmemoryCD4+_{Tcells,butnotCD8}+_{Tcells,comparedtoZAP-70negativepatients}

andage-matchedapparentlyhealthydonors.

∗ _{Correspondingauthorat:LaboratoryofClinicalPathology,FlowCytometryDivision,HospitalIsraelitaAlbertEinstein,Av.AlbertEinstein,}

627,Morumbi,SãoPaulo,SP,CEP:05652-900,Brazil. E-mailaddress:rodolfoptc@gmail.com(R.P.Correia).

https://doi.org/10.1016/j.htct.2018.03.008

Conclusions: BecausecentralmemoryCD4+_{Tcellsarelocatedinlymphnodesandexpress}

CD40L,weconsiderthatmalignantZAP-70-positiveBcellsmayreceivebeneficialsignals fromcentralmemoryCD4+_{Tcellsastheyaccumulate,whichcouldcontributetomore}

aggressivedisease.

©2018Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Chronic lymphocytic leukemia (CLL) is the most common lymphoproliferativeneoplasminWesterncountries;itis char-acterized by an accumulation of mature CD5+ _{B cells in}

hematopoietic tissuedue to the imbalance in the rates of proliferationandapoptosis.Ithasbeenreportedthatgenetic predisposition, mutations, familial history, environmental factorsandclonalevolutioninducedbyantigensand/or auto-antigensareelementsrelatedtotheetiologyofthedisease.1–3

The clinical course of CLL is heterogeneous and varies betweenindividuals.1,4 _{Several clinical and laboratory data}

areconsidered prognosticmarkers,includingRaiand Binet staging,lymphocytecountdoublingtime,cytogenetics, differ-entgeneticmutations,zeta-chain-associatedproteinkinase 70(ZAP-70)expressionandtheIgheavychainV-IIIregionVH26

(IgVH)genemutationalstatus.3,5–8 _{Ofthese,ZAP-70}

expres-sion, detected by flow cytometry, is commonly used as a reliablesurrogateforgoldstandardprognosticmarkers.8

AlthoughCLLisbasicallyaBcelldisease,ithasbeen pro-posedthatitspathophysiologyandevolutionaresignificantly influencedbyTcellsastheyparticipateintheirexpansion, differentiationandsurvival,whichmayalsoinfluenceT lym-phocytefunctionandphenotype,9,10 _{withtheaccumulation}

ofmemoryTcellsinCLLpatients.11,12_{Howevertherearefew}

dataabouttheassociationbetweenmemoryTcellsandthe prognosisofCLL,especiallyrelatedtoZAP-70.

Thus,thisstudy analyzedtheperipheralTcell compart-mentofCLLpatients,toevaluatewhetherZAP-70expression isassociatedwithanabnormaldistributionofnaïveand mem-oryTcellsrelatedtothecrosstalkbetweenthesecells,and consequentlytotheprognosisofthedisease.

Methods

Studydesign,ethicsandsetting

Thiscontrolledcross-sectionalstudycomparedCLLpatients withhealthyblooddonorsregardingthedistributionofnaïve andmemoryT-cellsubsetsinperipheralblood.Thestudywas conductedintworeferralcentersforhematologyand oncol-ogyin Brazil, one private and the other a public teaching institution.

Allhealthyblooddonorsandpatientssignedinformed con-sentformstherebyagreeingtoparticipateinthisstudy.The reviewboardsofbothinstitutionsreviewedandapprovedthe studyprotocol.

Healthydonorsandpatients

Thecasegroupofthisstudywerealladultpatientsadmitted forthediagnosisofCLL fromSeptember2011toSeptember 2012. The control groupcomprised blood donorsfrom the sameinstitutionsduringthesameperiod.

Avolumeofapproximately10mLofperipheralblood(PB) was collected from all participantsintubes with anticoag-ulantheparinorethylenediaminetetraaceticacid(EDTA)for flowcytometry.Inthebloodbanks,thesamplewascollected aftervoluntarydonations.

Flowcytometryassay

Theflowcytometryassay usedinthis studyforthe evalu-ation ofthe ZAP-70expressionwasperformed intheFlow CytometryLaboratoryoftheClinicalPathologyLaboratoryof Hospital Israelita Albert Einstein.Thetechnical procedures andflowcytometrysettingsfollowedstandardoperating pro-cedures(SOP)previouslyvalidatedinthelaboratoryandthe qualitywasmonitoredbyinternalandexternalquality con-trolsaspublishedbytheAmericanCollegeofPathology(CAP) and UnitedKingdomNational ExternalQualityAssessment Service(UKNEQAS).

TheexpressionofZAP-70inB-cellCLLpatientswasalways evaluatedinparalleltoasampleofperipheralbloodfroma healthydonorinordertovalidatetheintracytoplasmic reac-tionandtheZAP-70antigenicexpressioninT-cells(positive) andBcells(negative).

All fluorochrome-conjugated antibodies were validated beforeuseaccordingtothereactionspecificityandthebest volumecapableofprovidingtheantigen–antibodybinding sat-uration(titration).

CharacterizationofnaïveandmemoryTcells

Peripheral blood mononuclear cells (PBMC) from healthy donors and CLL patients were obtainedand purified using Ficoll-Paque PLUS(GEHealthcareBio-SciencesAB, Sweden) anddensitygradientcentrifugation.13

Thecellconcentrationandviabilitywereassessedusingan opticalmicroscope,Neubauerchamberandtrypanblue.An aliquotof1×106cellswerestainedwithanti-CD3,anti-CD4,

105 105

CD3+CD4+ 32,07_TCM

8,47 TCM

TEMRA 10,24

TEMRA 43,28 TEM

22,97

TEM 14,75

34,72

Naïve

33,50

Naïve

CD3+CD4+

CD3+CD8+

CD3+CD8+

105

105

105

105

101 ₁₀1

101

101

102 102

150K

150K

SSC

100K

100K 50K

FSC

CD4

CD45RA CD8

50K

CD3 _CD62L

200K

200K 250K

250K

102

102

102

103 ₁₀3

103

103

103

104

105

101

102

103

104

104

104

105

101 ₁₀2 ₁₀3 ₁₀4 ₁₀1 ₁₀2 ₁₀3 ₁₀4 105

104

104

Figure1–CharacterizationofnaïveandmemoryTcellsinperipheralbloodofhealthydonorsandchroniclymphocyte leukemia(CLL)patientsbyflowcytometry.Thelymphocyteregionwasevaluatedconsideringtheforwardandsidescatter ofcells.CD4+_andCD8+_{TcellswererespectivelydeterminedbydoublepositivityofCD3}+_CD4+_andCD3+_CD8+_,andthe

CD45RAandCD62LexpressionpatternswereusedtoidentitynaïveTcells(CD45RA+_CD62L+_{),centralmemoryTcells}

(CD45RA−_CD62L+_{),effectormemoryTcells(CD45RA}−_CD62L−_{)andterminaleffectormemoryTcells(CD45RA}+_CD62L−_).For

standardization,50,000eventswereacquiredintheCD3+_{T-cellpopulation.Thisstrategywasrepresentativeforhealthy}

donorsandCLLpatients.

Afterincubationandwashing,sampleswereanalyzedbyflow cytometryinaBDFACSCantoIIapparatus(Becton,Dickinson, SanJose,CA,USA)usingtheFlowJosoftware–(TreeStar,Inc.). CD4+ _{and CD8}+ _{T cells were respectively determined}

by double positivity of CD3+_CD4+ _{and CD3}+_CD8+_.

More-over,CD45RAandCD62L expressionswere usedtoidentify naïve T cells (CD45RA+_CD62L+_{), central memory T cells}

(TCM; CD45RA−CD62L+), effector memory T cells (TEM;

CD45RA−_CD62L−_{) and terminal effector memory T cells}

(TEMRA;CD45RA+CD62L−)(Figure1).Thisstrategywasusedfor

bothhealthydonorsandCLLpatients.

ZAP-70expressioninCLLpatients

For the ZAP-70flow cytometry assay, 5×106 PB cells from

healthy donors and CLL patients were stained with anti-CD3andanti-CD19mAbsandconjugatedwithphycoerythrin cyanine (PE-Cy5) and PE, respectively (Beckman Coulter). For cytoplasmic staining, cells were fixed, permeabilized

and stained withanti-ZAP-70 mAbconjugated with Alexa-Fluor488(clone1E7.2,Invitrogen).Sampleswereanalyzedina CytomicsFC500apparatus(BeckmanCoulter–Figure2).

Thecut-offforZAP-70positivitywasbasedonpublished data,whichrecommendspositivitywhenmorethan20%of neoplasticBlymphocytesexpressZAP-70.8

Tostandardizethetechnique,theZAP-70expressionwas alwaysanalyzedinthesamplesofhealthydonorsinparallel toCLLpatients.Furthermore,theisotypecontrolfor Alexa-Fluor488wasusedasanegativecontrolofthereaction,while theexpressionofZAP-70onnormalTlymphocyteswas con-sideredthepositivecontrol.14

Statisticalanalysis

A

[Ungated] SSC / FSC

[Lymphocytes] CD19PE / CD3PE-Cy5

[CD3+ T cells] IgG1 AF488 / CD3PC5

[CD3+ T cells]

ZAP70 AF488 / CD3PE-Cy5

ZAP70+ : 1,47% ZAP70+ : 0,14%

ZAP70+ : 44,19% ZAP70+ : 99,53%

ZAP70 AF488 ZAP70 AF488 [CD3+B cells] IgG1 AF488 / CD19PE

[CD19+ B cells] ZAP70 AF488 / CD19PE

1000

1000

SSC Lymphocytes

CD3+ T cells

CD19+ B cells

CD19PE

IgG1 AF488 IgG1 AF488

CD3PE-Cy5

CD3PE-Cy5

CD3PC5 CD19PE

CD19PE

FSC

800

800 600

600 400

400 200

200 0

0

103

103

103 102

102

102 101

101

101 100

100

103

102

101

100

103

102

101

100 103

102

101

100

100

103

102

101

100 ₁₀0 ₁₀1 ₁₀2 ₁₀3

103

102

101

100

103

102

101

100

B

C

E

F

D

Figure2–DetectionofZAP-70expressioninhealthydonorsandchroniclymphocyteleukemia(CLL)patients.Analiquotof 5×105peripheralbloodcellswerestainedwithmonoclonalantibodies(CD19PE,CD3PE-Cy5,intracellularmouseIgG1

AF488andintracellularZAP-70AF488)andanalyzedbyflowcytometry.(A)Firstagateinthelymphocyteregionwas createdaccordingtoforwardandsidescattertobetteridentifythespecificpopulations.(B)Followingthegatestrategy, CD19+_BcellsandCD3+_{Tcellswerecharacterizedinthelymphocytegate.Thedefinitionofnegativefluorescencebymouse}

IgG1AF488(C)inCD3+_{Tcellsand(D)inCD19}+_{Bcells.(E)ZAP-70positiveexpressioninnormalCD3}+_{Tcellsand(F)in}

neoplasticCD19+_{Bcells.TheZAP-70expressioninnormalTcellsofCLLpatientswasusedastheinternalcontrolof}

stainingreaction.Atotalof50,000eventswasacquiredforallcounts.

usingthepairedStudentt-testandthecomparisonbetween thecellsubpopulationswasperformedusingrepeated mea-sures analysis ofvariance (ANOVA) followed byBonferroni multiplecomparison.Comparisonbetweengroups(healthy and case) and cell types or subtypes were achieved using two-wayANOVAfollowedbyBonferronimultiplecomparison whennecessary.Statisticaldifferenceswereconsidered sig-nificantwhenp-valueswere<0.05.

Results

In the study period, 21 CLL patients and 43 controls were enrolled.Inthecontrolgroup,17patientswereover40years old. Table 1 summarizes the characteristics ofthe control groupandtheclinicalandlaboratorydataoftheCLLpatients aresummarizedinTable2.

DistributionprofileofnaïveandmemoryTcells

Asexpected,thefrequencyandtheabsolutenumberofCD4+

TcellswerehigherthanthoseofCD8+_{TcellsintheControl}

Group(Figure3).TheTcell compartmentwasalso charac-terizedandthe CD4and CD8subpopulationshaddifferent distributions according to memory T cell subsets. Central memoryTcells(TCM)werepredominantintheCD4+ Tcell

Table1–Characteristicsofhealthydonors.

Characteristic Total Over40-yearolds

Patients–n(%) 43(100) 17(100)

Sex–n(%)

Male 26(60.5) 9(52.9)

Female 17(39.5) 8(47.1)

Age(years)

Median 36 55

Range 19–72 41–72

compartment,whereas TCM werefewerofthe CD8+ T

lym-phocyteswithTEMandTEMRAbeingdistributedequallyamong

CD8+_{Tlymphocytes(Figure3CandD).}

CharacterizationofTcellcompartmentinchronic lymphocyticleukemiapatients

TheanalysisoftheTcellcompartmentinCLLpatientsshowed that,duetotheincreasednumbersofneoplasticB lympho-cytesinPB,thefrequenciesofCD4+_andCD8+_Tlymphocytes

C

D

B

A

CD4+ _{T cells CD8}+ _{T cells}

T cells T cells

CD4

TCM TEM TCM TEM

TEMRA TEMRA

CD4

CD8 CD8

0 20 40

60 _***

***

** ***

0 20 40 60

0 20 40 60 80

1500

1000

0 500

F

requency (%) Cells/mm

3

F

requency (%) Frequency (%)

Figure3–DistributionprofileofTcellsinhealthydonors.(A)Frequencyand(B)absolutenumberofperipheralbloodCD4+

andCD8+_{Tcellsfromhealthyindividualsasassessedbyflowcytometry.Frequencyofmemory(C)CD4}+_and(D)CD8+_Tcell

subpopulationsaccordingtotheCD45andCD62Lexpression[centralmemoryTcells(TCM–CD45RA−CD62L+),effector

memoryTcells(TEM–CD45RA−CD62L−)andterminaleffectormemoryTcells(TEMRA–CD45RA+CD62L−)].Thevalueswere

statisticallysignificantwithp-value<0.001(***)andp-value=0.003(**).

B

A

CLL patients Healthy donors

Cells/mm

3

F

requency (%)

CD4 CD8 CD4 CD8

0 20 40

60 ***

***

1500

1000

0 500

Figure4–AbsolutenumberofCD4andCD8Tcellsisnotchangedinchroniclymphocyteleukemia(CLL)patients.(A) FrequencyofperipheralbloodCD4+_andCD8+_{TcellsinCLLpatientscomparedtohealthydonors.Thevalueswere}

statisticallysignificantwithp-value<0.001(***).(B)AbsolutenumberofperipheralbloodCD4+_andCD8+_{TcellsfromCLL}

patientscomparedtothehealthydonors.

DistributionpatternofnaïveandmemoryTcellsin chroniclymphocyticleukemiapatients

Nodifferencesinthefrequencyandtheabsolutenumberof naïve,TCM, TEM and TEMRA were observedinthe CD4+ and

CD8+_{Tcellpopulations(Figure5).However,ZAP-70positive}

patients had significant differences in the frequencies of naïveandabsolutenumber ofTCM intheCD4+ Tcell

com-partment.Therewasno differenceinthe frequencyorthe

absolutenumberofCD8+_{TcellsinCLLpatientscomparedto}

age-matchedapparentlyhealthyindividuals(Figure6).

Age-dependentdifferencesinthedistributionofnaïveand memoryTcells

Cells/mm

3

CD4+_{T cells}

CD8+_{T cells} CD8+_{T cells}

CD4+_{T cells}

B

A

D

C

CLL patients Healthy donors (group 2)

CLL patients Healthy donors (group 2)

Cells/mm

3

F

requency (%)

F

requency (%)

0

50

50 100 150 200

10

0 0

20 30 40

0

20 200

40 400

60 600

80 800

TEMRA TEM TCM Naiv

e

TEMRA TEM TCM Naiv

e

TEMRA TEM TCM Naiv

e

TEMRA TEM TCM Naiv

e

Figure5–NaïveandmemoryTcelldistributionsinchroniclymphocyteleukemia(CLL)patients.PeripheralbloodTcell subsetsinCLLpatientsandage-matchedhealthydonors(Group2)weredeterminedbyflowcytometry.(A)Frequencyand (B)absolutenumberofCD4+_{naïveandmemoryTcellssubsets.(C)Frequencyand(D)absolutenumberofCD8}+_naïveand

memoryTcellssubsets.TCM:centralmemoryTcells;TEM:effectormemoryTcells;TEMRA:terminaleffectormemoryTcells.

CD4+_{T cells CD4}+_{T cells}

A

D

C

B

CD8+T cells CD8+T cells

Cells/mm

3

Cells/mm

3

F

requency (%)

F

requency (%)

CLL ZAP-70 positive CLL ZAP-70 negative Healthy donors (group 2)

CLL ZAP-70 positive CLL ZAP-70 negative Healthy donors (group 2) 0

20 40 60 80

0 500 1000 1500

***

***

50 100 150 200

0 50

10

0 20 30 40

TEMRA TEM TCM Naiv

e

TEMRA TEM TCM Naiv

e

TEMRA TEM TCM Naiv

e

TEMRA TEM TCM Naiv

e

Figure6–NaïveandmemoryCD4+_{TcelldistributionsarerelatedtotheZAP-70expressioninchroniclymphocyteleukemia}

(CLL)patients.(A–C)Frequencyand(B–D)absolutenumberofCD4+_andCD8+_{naïveandmemoryTcellssubsetsinCLL}

patientswithandwithoutZAP-70expressionincomparisontoage-matchedhealthydonors.Differencesinthefrequencies ofnaïveandabsolutenumberofcentralmemoryTcellsintheCD4+_{Tcellcompartmentwerestatisticallysignificantwith}

p-value<0.001andp-value=0.008,respectively.TCM:centralmemoryTcells;TEM:effectormemoryTcells;TEMRA:terminal

p=0,769 r=0,002

p=0,963 r=0,000

p=0,0004 r=0,263

p=0,2776 r=0,028 p=0,0337 r=0,105

p=0,0465 r=0,093 p=0,473 r=0,126 p=0,948 r=0,000

Age (years) Age (years)

Age (years)

Age (years)

Age (years) Age (years)

Age (years) Age (years)

B

C

F

E

H

G

D

A

0 20

30 50

20

0 15

5

0

10

10 30 40

20

0 10 30 40

20 20 40

40 60

60 70 20 30 40 50 60 70

30 50

20 40 60 70 20 30 40 50 60 70

30 50

20 40 60 70 20 30 40 50 60 70

30 50

20 40 60 70 _{20 30 40 50 60 70}

80

0 20 40 60 80

0 20 40 60 80

0 20 40 60

0 20 40 60

CD4 T_cm

CD4 T_EM CD4 T_EMRA

CD8 T_CM

CD8 T_EM CD8 T_EMRA

CD4 naïve

CD8 naïve

F

requency (%)

F

requency (%)

F

requency (%)

F

requency (%)

F

requency (%)

F

requency (%)

F

requency (%)

F

requency (%)

Figure7–DistributionofnaïveandmemorysubtypesofCD8+_{Tcells,butnotCD4}+_{Tcells,isagedependent.Analysisof}

thefrequencydistributionof(A–D)CD4+_{and(E–F)CD8}+_{naïve,centralmemoryTcells(T}

CM),effectormemoryTcells(TEM)

andterminaleffectormemoryTcells(TEMRA)inhealthydonors(19–72yearsold).Linearregressionwasusedtoevaluatethe

Table2–Clinicalandlaboratorydataofchronic lymphocyteleukemiapatients.

Characteristic Value

n 21

Sex–n(%)

Male 16(76.2)

Female 5(23.8)

Age(years)

Median 64

Range 46–88

Leucocytes(×103_/L)

Median 24.46

Range 2.60–61.00

Lymphocytes(×103/L)–n(%)

Median 20,465.0(78.3)

Range 1.609(41.7)-53,741.0(92.0)

Hemoglobin(g/dL)

Median 13.6

Range 8.5–16.7

Hematocrit(fL)

Median 39.8

Range 25.6–54.2

Platelets(×109/L)

Median 205

Range 32,000–265,000

ZAP-70expression–n(%)

Negative 16(76.2)

Positive 5(23.8)

distributions.6 _{Therefore, thenext stepwastoruleout the}

possibilitythattheincreaseinTCMCD4+cellswasdueto

dif-ferencesintheagesofthepatients.Onanalyzingthehealthy group(19–72yearsold),nodifferenceswereobservedinthe naïveandmemoryCD4+_{Tcellswithage(Figure7),butinthe}

CD8+_{Tcellcompartment,thenaïveTcellsdiminishedand}

TCMandTEMRAcellsincreasedwithage(Figures7E–H).

Discussion

The involvement of T cells in the pathophysiology of hematologicmalignancies,especially inCLL, iswidely dis-cussed. The interaction of neoplastic B cells with their microenvironment, particularly CD4+ _{T lymphocytes and}

withextracellularcomponents,couldregulatetheexpansion, differentiation, and survivalofneoplastic Bcells,and pos-siblyinfluencetheTlymphocytegene expression,function andphenotype.9,10 _{Thisstudy showedthat ZAP-70positive}

patientspresentedincreasedTCM CD4+ Tcellscomparedto

ZAP-70negativepatientsandtoage-matchedhealthydonors (Figure5).

MemoryTcellsrepresentaheterogeneousgroupofcells characterizedbytheirphenotypicdiversity,proliferativeindex versuseffectorfunction,and migratorycapabilitytolymph nodesand/or toperipheraltissues.They areclassified into three major groups: TCM, TEM, and TEMRA. Briefly, TCM are

locatedinlymphnodesandhaveloweffectorfunctionwith

ahighproliferativeindex.Ontheotherhand,TEMandTEMRA

arecharacterizedbyhigheffectorfunctionintheliver,lung, and gutand withalowproliferativeindex. Together,these cellsprovideimmediateprotectioninperipheraltissuesand efficientsecondaryresponseinlymphnodes.15–18

Itiswellestablishedthatthesignalingandantigenic stim-ulationbyBcellreceptors(BCRs)iscrucialforsurvivaland growthofCLLcells,eventhoughitisadiseasewithalow pro-liferativeindex.19_{Inthiscontext,T}

CMCD4+cellscanmigrate

tothelymphnodesduetotheexpressionofCD62LandCCR7, and interactwithBcells byCD40L andCXCR5,that conse-quentlycouldcontributetostrongerBCRsignaling,expansion andevolutionofthedisease.18_{Thesedatacouldsupportthe}

findingthatZAP-70positiveCLLpatientshaveincreasedTCM

CD4+_Tcells.

In addition to the modulation of BCR signaling, the cytokinesproducedbymemoryTcells,especiallyinterleukin (IL)-4andinterferon-gamma(IFN-␥),couldpromoteincreased

survivaloftheCLLclone.20,21 _{Recently,astudyshowedthat}

ZAP-70expressioninCLL isassociatedwithareductionof naïveCD4+_{Tcellsandincreasesinactivation/differentiation}

ofCD4+_{TcellstoamemoryprofilethatexpressesIL-4or}

IL-10.Inaddition,thesecytokinesmightfavorthegrowthand survivalofCLLcells.22

Furthermore,as neoplastic CLLBcells are inefficientas antigenpresentingcells,theevidencethatincreasedTCMCD4+

TcellscouldbeaCLLmechanismissupportedbythetheoryof thegenerationofmemoryTcells;aweakstimulationofnaïve Tcellsduringantigenpresentationdrivesthedifferentiation ofthesecellsintoTCMcells.23,24

ToratifythatthechangesdetectedintheCD4+_memoryT

cellcompartmentareaCLLintrinsicmechanismandnotan age-dependentevent,thefrequencyofthesecellswas evalu-atedinhealthydonorsstratifiedbyage.Indeed,eventsrelated toage,suchashomeostaticregulation,thymusinvolutionand antigenic stimulation were observed,6 _{but this mechanism}

onlyaffectedtheCD8+_{Tcellcompartment(Figure7).}

There-fore, ourfinding ofincreasedTCM CD4 cellsisaconsistent

resultanddirectlyassociatedwithCLL,inparticular, associ-atedtoZAP-70expressioninthisdisease.

This study has limitations in evaluating the real asso-ciation between the results with CLL prognosis, especially due to absent ofexperimental verification and correlation with other clinical and laboratory data. Still, we believe the increasein TCM CD4+ T cells might be a resultof the

crosstalk mechanism between CLL ZAP-70 positive B cells with normal CD4+ _{T cells, in which the neoplastic clone}

could obtain greater survivalandexpansion signalsdueto cytokinesandcostimulatorysignalsasdescribedabove. Inter-estingly, it is reported that murine models using animals deficientinBcellshaveimpairedgenerationofCD4+_memory

Tcells.15,25

These findings could be important to understand the pathophysiology,andconsequently,theycouldhelpdrive fur-therinvestigationsintheprognosisandtreatmentofCLL.

Conflicts

of

interest

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1. SwerdlowSH,CampoE,HarrisNL,JaffeES,PileriAS,SteinH, etal.WHOclassificationoftumoursofhaematopoieticand lymphoidtissues.4thed.Lyon:InternationalAgencyfor ResearchonCancer;2008.

2. InamdarKV,Bueso-RamosCE.Pathologyofchronic lymphocyticleukemia:anupdate.AnnDiagnPathol. 2007;11(5):363–89.

3. PuenteXS,PinyolM,QuesadaV,CondeL,Ordó ˜nezGR, VillamorN,etal.Whole-genomesequencingidentifies recurrentmutationsinchroniclymphocyticleukaemia. Nature.2011;475(7354):101–5.

4. RichesJC,RamsayAG,GribbenJG.Chroniclymphocytic leukemia:anupdateonbiologyandtreatment.CurrOncol Rep.2011;13(5):379–85.

5. RaiKR,SawitskyA,CronkiteEP,ChananaAD,LevyRN, PasternackBS.Clinicalstagingofchroniclymphocytic leukemia.Blood.1975;46(2):219–34.

6. BinetJL,AuquierA,DighieroG,ChastangC,PiguetH, GoasguenJ,etal.Anewprognosticclassificationofchronic lymphocyticleukemiaderivedfromamultivariatesurvival analysis.Cancer.1981;48(1):198–206.

7. DamleRN,WasilT,FaisF,GhiottoF,ValettoA,AllenSL,etal. IgVgenemutationstatusandCD38expressionasnovel prognosticindicatorsinchroniclymphocyticleukemia. Blood.1999;94(6):1840–7.

8. CrespoM,BoschF,VillamorN,BellosilloB,ColomerD, RozmanM,etal.ZAP-70expressionasasurrogatefor immunoglobulin-variable-regionmutationsinchronic lymphocyticleukemia.NEnglJMed.2003;348(18):1764–75.

9. CorreiaRP,MatosE,SilvaFA,BacalNS,CampregherPV, HamerschlakN,etal.InvolvementofmemoryT-cellsinthe pathophysiologyofchroniclymphocyticleukemia.RevBras HematolHemoter.2014;36(1):60–4.

10.ScrivenerS,GoddardRV,KaminskiER,PrenticeAG.Abnormal T-cellfunctioninB-cellchroniclymphocyticleukaemia.Leuk Lymphoma.2003;44(3):383–9.

11.TinhoferI,WeissL,GassnerF,RubenzerG,HollerC,GreilR. DifferenceintherelativedistributionofCD4+T-cellsubsets inB-CLLwithmutatedandunmutatedimmunoglobulin(Ig) VHgenes:implicationforthecourseofdisease.J

Immunother.2009;32(3):302–9.

12.HofbauerJP,HeyderC,DenkU,KocherT,HollerC,TrapinD, etal.DevelopmentofCLLintheTCL1transgenicmouse

modelisassociatedwithsevereskewingoftheT-cell compartmenthomologoustohumanCLL.Leukemia. 2011;25(9):1452–8.

13.BöyumA.Isolationofmononuclearcellsandgranulocytes fromhumanblood.Isolationofmonuclearcellsbyone centrifugation,andofgranulocytesbycombining centrifugationandsedimentationat1g.ScandJClinLab InvestSuppl.1968;97:77–89.

14.VillamorN.ZAP-70staininginchroniclymphocyticleukemia. CurrProtocCytom.2005;6:19.Chapter6:Unit619.

15.JamesonSC,MasopustD.DiversityinTcellmemory:an embarrassmentofriches.Immunity.2009;31(6):859–71.

16.PepperM,JenkinsMK.OriginsofCD4(+)effectorandcentral memoryTcells.NatImmunol.2011;12(6):467–71.

17.SallustoF,GeginatJ,LanzavecchiaA.Centralmemoryand effectormemoryTcellsubsets:function,generation,and maintenance.AnnuRevImmunol.2004;22:745–63.

18.SallustoF,LenigD,FörsterR,LippM,LanzavecchiaA.Two subsetsofmemoryTlymphocyteswithdistincthoming potentialsandeffectorfunctions.Nature.

1999;401(6754):708–12.

19.StevensonFK,Caligaris-CappioF.Chroniclymphocytic leukemia:revelationsfromtheB-cellreceptor.Blood. 2004;103(12):4389–95.

20.BuschleM,CampanaD,CardingSR,RichardC,HoffbrandAV, BrennerMK.Interferongammainhibitsapoptoticcelldeath inBcellchroniclymphocyticleukemia.JExpMed.

1993;177(1):213–8.

21.PanayiotidisP,GaneshaguruK,JabbarSA,HoffbrandAV. Interleukin-4inhibitsapoptoticcelldeathandlossofthe bcl-2proteininB-chroniclymphocyticleukaemiacellsin vitro.BrJHaematol.1993;85(3):439–45.

22.MonserratJ,SánchezMÁ,dePazR,DíazD,MurS,ReyesE, etal.Distinctivepatternsofnaive/memorysubset

distributionandcytokineexpressioninCD4Tlymphocytesin ZAP-70B-chroniclymphocyticpatients.CytomBClinCytom. 2014;86(1):32–43.

23.LanzavecchiaA,SallustoF.Understandingthegenerationand functionofmemoryTcellsubsets.CurrOpinImmunol. 2005;17(3):326–32.

24.DazziF,D’AndreaE,BiasiG,DeSilvestroG,GaidanoG,Schena M,etal.FailureofBcellsofchroniclymphocyticleukemiain presentingsolubleandalloantigens.ClinImmunol

Immunopathol.1995;75(1):26–32.