Rev. Bras. Reumatol. vol.56 número1

w w w . r e u m a t o l o g i a . c o m . b r

REVISTA

BRASILEIRA

DE

REUMATOLOGIA

Original

article

Th17

cells

and

CD4

+

multifunctional

T

cells

in

patients

with

systemic

lupus

erythematosus

Júlio

Antônio

Pereira

Araújo

_,

_Danilo

_Mesquita

_Jr

_,

_Wilson

_de

_Melo

_Cruvinel

_,

Karina

Inácio

Salmazi

_,

_Esper

_Georges

_Kallás

_,

_Luis

_Eduardo

_Coelho

_Andrade

a_{DepartmentofRheumatology,UniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil}

b_{DepartmentofBiomedicine,PontifíciaUniversidadeCatólicadeGoiás(PUC-GO),Goiânia,GO,Brazil}

c_{DepartmentofClinicalImmunologyandAllergy,UniversidadedeSãoPaulo(USP),SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4April2014 Accepted22August2015

Availableonline19November2015

Keywords:

Systemiclupuserythematosus Tlymphocytes

Th17

MultifunctionalTcells

a

b

s

t

r

a

c

t

Introduction/Objective:RecentevidencesuggeststhatabnormalitiesinvolvingTh17

lympho-cytesareassociatedwiththepathophysiologyofsystemiclupuserythematosus(SLE).In addition,multifunctionalTcells(MFT),i.e.,thoseproducingmultiplecytokines simulta-neously,arepresentintheinflammatorymilieuandmaybeimplicatedintheautoimmune processobservedinSLE.Inthepresentstudy,weaimedtocharacterizethefunctionalstatus ofCD4+_{TcellsinSLEbysimultaneouslydeterminingtheconcentrationofIL-2,}

IFN-␥and

IL-17inlymphocyteculturesunderexogenousandself-antigenicstimuli.

Patientsandmethods:Eighteenpatientswithactivedisease,18withinactivedisease,and14

healthycontrolshadfunctionalstatusofCD4+_{Tcellsanalyzed.}

Results:WefoundthatSLEpatientspresentedadecreasednumberoftotalCD4+_cells,an

increasednumberofactivatedTcells,andanincreasedfrequencyofTh17cellscompared tohealthycontrols(HC).MFTcellshadincreasedfrequencyinSLEpatientsandtherewasan increasedfrequencyoftri-functionalMFTinpatientswithactiveSLEcomparedwiththose withinactiveSLE.Interestingly,MTFcellsproducedlargeramountsofIFN␥than mono-functionalTcellsinpatientsandcontrols.

Conclusion:TakentogetherthesedataindicatetheparticipationofrecentlyactivatedTh17

cellsandMTFcellsintheSLEpathophysiology.

©2015ElsevierEditoraLtda.Allrightsreserved.

Linfócitos

Th17

e

linfócitos

T

CD4

_{multifuncionais}

_em

_pacientes

com

lúpus

eritematoso

sistêmico

Palavras-chave:

Lúpuseritematososistêmico LinfócitosT

r

e

s

u

m

o

Introduc¸ão/Objetivo:Evidênciasrecentessugeremqueanormalidadesqueenvolvemos

lin-fócitosTh17estãoassociadasàfisiopatologiadolúpuseritematososistêmico(LES).Além disso, os linfócitos T multifuncionais (LTM), ouseja, aqueles que produzem múltiplas

∗

Correspondingauthor.

E-mail:luis.andrade@unifesp.br(L.E.C.Andrade).

http://dx.doi.org/10.1016/j.rbre.2015.10.003

Th17

LinfócitosTmultifuncionais

citocinassimultaneamente,estãopresentesnomeioinflamatórioepodemestarimplicados noprocessoautoimuneobservadonoLES.Nopresenteestudo,objetiva-secaracterizaro estadofuncionaldoslinfócitosTCD4+_{noLESedeterminarsimultaneamenteaconcentrac¸ão}

deIL-2,IFN-␥eIL-17emculturasdelinfócitossobestímulosexógenoseautoantigênicos.

Pacientesemétodos:Dezoitopacientescomdoenc¸aativa,18comdoenc¸ainativae14controles

saudáveisforamsubmetidosàanálisedoestadofuncionaldoslinfócitosTCD4+_.

Resultados:Encontrou-sequeospacientescomLESapresentaramumadiminuic¸ãona

quan-tidadetotaldecélulasCD4+_{,um aumentonaquantidadedelinfócitosT ativadoseum}

aumentonafrequênciadelinfócitosTh17emcomparac¸ãocomcontrolessaudáveis(HC). AscélulasLTMtinhafrequênciaaumentadaempacientescomLESehouveumaumentona frequênciadeLTMtrifuncionaisempacientescomLESativoemcomparac¸ãocomaqueles comLESinativo.Curiosamente,ascélulasMTFproduziramquantidadesmaioresdeIFN-␥ doqueoslinfócitosTmonofuncionaisempacientesecontroles.

Conclusão: Analisadosemconjunto,essesdadosindicamaparticipac¸ãodoslinfócitosTh17

recentementeativadosecélulasMTFnafisiopatologiadoLES.

©2015ElsevierEditoraLtda.Todososdireitosreservados.

Introduction

Effector CD4+ _{T cells have been initially categorized into}

two subsets based onthe cytokines theyproduce.1–3 _Th17 cells are part of this new T cell scenario and display an activatedCD4+ _{T cell phenotype characterizedby the}

pro-duction of high amounts of IL-17.4–6 _{Th17 cells seem to} playa crucial role in the development of awide range of autoimmuneandchronicinflammatorydisorders.7–9 _Infact, ithasbeensuggestedthatinappropriateregulationofTh17 cells may be a key event in the pathogenesis of rheuma-toid arthritis and systemic lupus erythematosus (SLE).10–14 Several studies reported significantly higher serum levels ofIL-17andhigherfrequencyofIL-17-producingperipheral blood mononuclear cells (PBMC) in SLE patients as com-paredtonormal individuals.13,15–18 _{Itwas alsoshown that} Th17 response is correlated with disease activity in SLE patients.17

Anovel strategyforthe evaluationof Tcell functional-ity is based on the simultaneous determination ofseveral cytokinesexpressedbysubtypes ofcells.According tothis approach T cells that produce multiple cytokines simulta-neouslyaretermedmultifunctionalTcells.Multifunctional T cell responseshave been documented in HIV-119 _{and in} the immune response to vaccination against Hepatitis B virus20 _{and HIV.}19,20 _{This novel approach has been largely} possible due to technological advances in flow cytometry that nowadays allows for the simultaneous detection of numerousfunctional,phenotypic,andlineagemarkersonT cells.21

Inthepresentstudy,wesoughttofurthercharacterizethe functionalstatusofCD4+_{TcellsinSLEpatientswithactive}

andinactivediseasesbysimultaneouslydeterminingthe con-centrationofseveralcytokinesinlymphocyteculturesunder different antigenic stimuli. Wealso quantified the propor-tionofTh17andmultifunctionalTcellsandcorrelatedthem withSLEDAI(SystemicLupusErythematosusDiseaseActivity Index),andwiththefrequencyofactivatedTcellsandTREG

cells.

Materials

and

methods

Thirty-six adultpatients (33women and3 men,aged40± 7.2years)withadiagnosisofSLEbasedontheAmerican Col-legeofRheumatologycriteria22_{wereconsecutivelyenrolledin} thestudyafterprovidinginformedconsent.Allpatientswere referredfromtheoutpatientclinicattheDivisionof Rheuma-tology of the Universidade Federal de São Paulo (Federal UniversityofSãoPaulo).Patientsweredividedintotwogroups basedontheirSLEDiseaseActivityIndex(SLEDAI)score.23,24 TheactiveSLEgroup(A-SLE)comprised18patients(SLEDAI score≥6;17womenand1man,aged36.7±10.2years)and theinactiveSLEgroup(I-SLE)comprised18patients(SLEDAI score=0;16womenand2men,aged39.2±13.9years).Table1

depicts the demographic and clinical characteristics of all patientsenrolledinthestudy.Asacontrol,14healthy labora-toryworkerswereenrolledinthestudy(13womenand1man, aged33.9±10.4years)aftergivinginformedconsent.Patients and healthycontrolsunderwentastructuredquestionnaire anddonated60mLofvenousblood.Thestudyprotocolwas reviewed and approvedby the institution’s research ethics committee.

Peripheralbloodmononuclearcells(PBMC)wereisolated bydensity-gradientsedimentationoverFicoll–Paque (Pharma-cia Biotech,Uppsala,Sweden) andwashed twiceinHank’s balanced salt solution (Gibco,Grand Island, NY). For cryo-preservation,cellswereslowlyfrozenin90%offetalbovine serum(FBS;HyCloneLaboratories,Logan,UT),andstoredin liquidnitrogen.Atthetimeoftheassay,PBMCwererapidly thawedina37◦_{Cwaterbathandwashedinpre-heatedRPMI}

1640supplementedwith10%fetalcalfserum,100U/mL peni-cillin, 100␮g/mLstreptomycin and 20mM glutamine (R10). Cellswere counted,checkedforviability,and suspendedin R10ataconcentrationof1×106_{viablecells/mL.}

Thawed PBMC were incubated in 96-well plates

Table1–Demographiccharacteristicsofcontrolsandpatientswithsystemiclupuserythematosus.

Controls(n=14) A-SLE(n=18) I-SLE(n=18) pValue

Age 33.9±10.4 36.7±10.2 39.2±13.9 0.624

Female(%) 92.8% 94.4% 88.9% 0.987

SD,standarddeviation;A-SLE,activesystemiclupuserythematosus;I-SLE,inactivesystemiclupuserythematosus;SLEDAI,systemiclupus

erythematosusdiseaseactivityindex;ns,notsignificant.

1500gfor5min,suspendedinMacsbuffer and transferred

into V-bottom 96-wellplates (Nunc,Roskilde,Denmark) in

100␮L of staining buffer [phosphate-buffered saline (PBS)

supplementedwith0.1%sodiumazide(Sigma)and1%FBS,

pH 7.4] with the panel of surface monoclonal antibodies

(CD4–PerCP, CD3–APC-CY7 and CD69–PE-Cy7). Cells were

incubatedat4◦_{Cinthedarkfor30min,washedtwicewith}

PBS and then suspended in 100␮L of fixation buffer [1%

paraformaldehyde(Polysciences,Warrington,PA)inPBS,pH

7.4].Forintracellularstaining,cellsalreadylabeledforsurface

markerswereincubatedwith100␮Lof4%fixationbufferand

washedwithpermeabilizationbuffer(PBSsupplementedwith

0.1%sodium azide,1%FBSand0.1%saponin;Sigma). Each

samplewassuspendedin100␮Lofpermeabilizationbuffer,

incubatedfor15minatroomtemperatureinthedark,washed

with100␮Lofstainingbufferandincubatedfor30minat4◦C

inthedarkwitheithernoantibody(unstainedtube)orwith

a pool of anti-IL-2–PE, anti-IFN-␥–APC, and anti-IL-17–FITC

monoclonal antibodies in 50␮L of staining buffer.25 Cells

werewashedwith200␮Lofstainingbufferandsuspendedin 100␮Loffreshlyprepared1%paraformaldehyde(PFA)pH7.4. TREG cells and effector CD25+ T cells were identified

according to a previously established protocol.26 _Briefly,

PBMCs were washed in PBS and 0.5×106 _{viable cells}

wereincubatedwithfluoresceinisothiocyanate(FITC)-labeled anti-CD127,allophycocyanin-Cy3(APC-Cy3)-labeledanti-CD3, PerCP-labeled anti-CD4, and phycoerythrin (PE)-Cy7-labeled anti-CD25 (Becton Dickinson, San Jose, CA, USA) antibod-ies, according to the manufacturer’s instructions. After 30minofincubation at4◦_{Cinthedark,cells werewashed}

withMacsbuffer, fixedandpermeabilizedwithFoxP3 fixa-tion/permeabilizationbuffer(eBioscience,SanDiego,CA,USA) andthenprocessedforFoxP3stainingusingtheFoxP3 stain-ingkitandAPC-labeledanti-FoxP3(eBioscience)accordingto themanufacturer’sinstructions.

Samples were processedon a FACSCanto flow cytometer, usingFACSDIVAsoftware(BDBiosciences),andtheacquired data were analyzed with FLOWJO software (Tree Star, San Carlo, CA). Fluorescence voltages were determined using matchedunstainedcells.Compensationwascarriedoutusing CompBeads(BDBiosciences)singlestainedwithCD3–PerCP,

CD4–FITC,CD8–APC–CY7,CD4–PE–CY7,CD3–PEorCD3–APC,

respectively. Samples were acquired until at least 500,000 eventsinalivecellgatewereobtained.

Data were reported as median and interquartile range (IQR). Comparisons among groups were carried out using the Kruskall–Wallisnon-parametric test, followed by inter-group comparisons by the Dunnet test. Correlations were

performed using the Spearman’s non-parametric method.

Statisticalinferencelevelwasestablishedat5%(p<0.05).

Results

DecreasedfrequencyofCD4+_{Tcellsandincreased}

frequencyofnewlyactivatedcellsinSLE

TherelativefrequencyofCD4+ _{cellsovertotalCD3}+_cellsin

PBMCculturesstimulatedwithHEp-2cellextractwas signif-icantlylowerinsamplesfrompatientswithA-SLE(18.4±8.9) and I-SLE (17.3±7.6) as compared to controls (24.0±8.2) (Fig.1A).Likewise,PBMCculturesstimulatedwithPMA/Io pre-sentedlowerrelativefrequencyofCD4+_{cellsovertotalCD3}+

cellsinsamplesfrompatientswithA-SLE(16.6±8.4)and I-SLE(19.6±7.6) ascomparedtocontrols(25.3±6.4) (Fig.1B). The same wasobserved fornon-stimulated PBMCcultures (data not shown). In contrast, when we evaluated newly activatedcells,characterizedbytheexpressionoftheCD69 molecule,higherrelativefrequencyofCD4+_CD69+ _cellsover

totalCD4+_{cellswasobservedinpatientswithA-SLE(5.1±6.6)}

andI-SLE(4.9±5.2)whencomparedtocontrols(2.8±1.9)in culturesstimulatedwithHEp-2cell extract(Fig.1C)aswell aswithPMA/Io:A-SLE(6.6±8.3),I-SLE(5.9±5.5),andcontrols (3.5±2.0)(Fig.1D).Again,asimilarbehaviorwasobservedin non-stimulatedPBMCcultures(datanotshown).

MultifunctionalprofileofCD4+_TcellsinSLE

Next, we investigated the functional profile of CD4+ _cells

after auto-antigen-specific(HEp-2 extract)and non-specific (PMA/Io)stimuli.Thiswas accomplishedbysimultaneously determiningintracellularIL-2,IL-17andINF-␥byflow cytom-etryafterinvitrostimulationwithHEp-2extractandPMA/Io, respectively.Fig.2Ashowsthegatingstrategyandthe multi-parametriccharacterizationofmono-,bi-,andtri-functional responsepatternsinarepresentativepatientwithactiveSLE. The frequencyofthese Tcell functional subsets was then exploredinhealthycontrolsandinpatientswithactiveand inactiveSLE.

50

*

A

B

C

D

40 35 30 25 20 15 10 5 0

0 4 5

2

30

20

10

0 20

15

10

5

0

50

**

**

**

**

***

40 35 30 25 20 15 10 5 0

% CD4

+ T cells

Control I-SLE A-SLE

Control I-SLE A-SLE

Control I-SLE A-SLE

Control I-SLE A-SLE

% of CD4

+ CD69 + T cells

% of CD4

+ CD69 + T cells

% CD4

+ T cells

Fig.1–RelativefrequencyofCD4+TcellsinpatientswithactiveSLE,inactiveSLEandcontrols(A)andrelativefrequencyof CD4+CD69+TcellsinpatientswithactiveSLE,inactiveSLEandcontrolsinstimulationwithHEp-2(A,C)andstimulated withPMA/Io(B,D).Thebarsrepresentthemedianforeachgroup.*p<0.05;**p<0.01;***p<0.001.

frequencyinsamplesfromSLEpatientsandcontrols. How-ever,samplesfrompatientswithA-SLEandI-SLEpresented higherfrequencyofIL-17producingcellsincultures stimu-latedwithPMA/IobutnotinthosestimulatedwithHEp-2cell extract(Fig.2DandTable2).Inaddition,bi-functionalcells producingIL-17andINF-␥showedastrongtrendforhigher frequencyinPMA-stimulatedculturesfromA-SLEandI-SLE patientsascomparedwiththosefromnormalcontrols(Fig.2D andTable2).AscanbeappreciatedinFig. 2D,therewas a broadvariationinthefrequencyofmono-functionalcellsin stimulatedculturesfromSLEpatients,withseveralpatients presentingveryhighfrequencyofcellsexpressingIL-17and INF-␥.

Bi-functionalIL-2+

+CD4+TcellsproducemoreINF thanmono-functionalINF-+CD4+Tcells

Nextweaskediftheamountofcytokinesproducedby mul-tifunctionalandmono-functionalTcellswasequivalent.To evaluatethisparameterweassessedthemeanfluorescence intensity(MFI)foreachofthethreecytokinesevaluated.As canbeseeninexperimentswithPMA/Io-stimulatedsamples from controls, IFN-␥MFI in bi-functional IL-2+/INF-␥+ cells wassignificantlyhigherthaninotherTcellsubpopulations, eventhosethatproduceonlyINF-␥(Fig.3AandB).This fea-turewasequallyobservedinsamplesfromSLEpatientsand controls(Fig.3C–E).Thisphenomenonwasnotobservedin thosecultureswithoutstimulationorstimulatedwithHEp-2 cellsextracts(datanotshown).Thisbehaviorwasobserved inall36 SLEpatients and14 healthyvolunteers examined.

SuchabehaviorwasnotobservedforIL-17andIL-2inCD4+

T cells in any ofthe three groupsof individuals (datanot shown).

CorrelationbetweenthefrequencyofTh17cellsandnewly activatedcellsinpatientswithinactiveSLE

andwithCD4+_{TeffectorcellsinactiveSLE}

There was apositive correlation betweenthe frequencyof Th17cellsandrecentlyactivatedcellsinPBMCsamplesfrom patients with I-SLE innon-stimulated cultures(Fig. 4A) or under stimulus with HEp-2 cells extract (Fig. 4B), but not whencellswerestimulatedwithPMA/Io(Fig.4C).Therewas also a positive correlation between the frequency of Th17 lymphocytes and CD4+_CD25+_Foxp3∅ _{cells in samples from}

patients with active SLE either without or under different stimuli(Fig.4D–F).Incontrolsamples,therewasalsoapositive correlationbetweenthefrequencyofTh17andCD25+_Foxp3∅

cells when stimulated withHEp-2 cell extract and in non-stimulatedcultures,butnotinthosestimulatedwithPMA/Io (datanotshown).Therewasnosuchcorrelationincultures ofsamplesfrompatientswithinactiveSLE(datanotshown). Therewasnocorrelationbetweentherelativefrequencyof Th17cellsandTreg cells(CD25highCD127∅Foxp3+)insamples

IL-17

IL-2 IL-17 IFN-γ

% of total CD4+ _{T cells that produce}

cytokines

Boolean gate % of total CD4+ _{T cells that produce}

cytokines

% of total CD4

+ T cells that produce cytokines

A-SLE

Aa

B

Ab

I-SLE

Control

A-SLE

A-SLE I-SLE

I-SLE

4%

2% 2% p < 0.5 3%

86%

86% 86%

88%

82% 85%

4% 4%

Control

Control HEp-2

PMA/lo

A-SLE I-SLE Control 0 5 10

40

30

20

10

0

15 20 _{0 10 20 30 40}

*** *

***

* *

**

A

B

D

C

3 functions

3 functions 2 functions

2 functions

1 function

1 function

SSC _CD4

CD3

CD4 FSC

R1

R3 R4 R5

R2

IL-2

IL-17 + + +

+ + –

– + +

+ – +

+ + +

+ + –

+ – +

– + +

+ – –

– + +

– – + – + + – + – + –

– + – + +

– – + +

– – + +

– – + +

– – + +

– –

–

– +

–

+ – IL-2

INF-γ

INF-γ

IL-2

Hep-2

PMA/lo

IL-17 IFN-γ

Fig.2–(A)Multi-parameterstrategyofanalysistoidentifyTh17cellsandpolyfunctionalcellsinPBMCofHCstimulated withPMA/Io.Initiallywasestablishedagatedinlymphocytepopulation(R1),thenonlythepopulationCD3+_CD4+_was selected(R2).FromthispopulationwereobtainedcellsexpressingIL-2(R3)IL-17(R4)andINF-␥(R5).Relativefrequencyof

CD4+_{Tcellsthatproducecytokines(IL-2,IL-17or}

INF-␥)bystimulationwithHEp-2(B)andPMA/Io(C).Thebarsrepresent

thestandarderrorforeachgroup.(D)DiagramillustratingthebehavioroffunctionalsubsetsofCD4+_{TcellsfromA-SLE,} I-SLEandcontrols.TheboxplotgraphrepresentstherelativefrequencyofeachCD4+_{Tfunctionalsubsetinthethree} groupsunderthetwostimulusconditions(extractofHEp-2andPMA/Io,respectively).Theresponsepatternsaregrouped

andcolor-codedbynumberofpositivefunctionsandsummarizedinpiechartformwhereeachpieslicerepresentsthe

meanproportionofthetotalCD4+_{Tcellresponsecontributedbyresponsepatternsthathaveallthree(green)orany} combinationoftwo(red)orone(blue)ofthemeasuredfunctions.*p<0.05;**p<0.01;***p<0.001.

Discussion

Thepurposeofthepresentstudywastoevaluatetheimmune systemofpatientswithSLE,regardingeffectorpathwaysof CD4+_{TcellsproducingIL-17(Th17)andmultifunctionalCD4}+

Tcells.Therefore, we stimulatedPBMC cultureswith HEp-2cellextract,whichexpressesmostself-antigensofclinical importanceinSLE,includingSm/RNP,SS-A/Ro,andSS-B/La polypeptides,aswell aschromatin antigens.Thisstimulus allowedustoevaluate,amongthepoolofCD4+_Tcells,those

withself-reactivecapacity,comparedtonon-stimulatedcells andwithcells stimulated withPMA/Io(polyclonal stimula-tion).TheseexperimentsshowedthatPBMCfromSLEpatients didrespondtostimulationwithauto-antigens.Furthermore, weobservedthateveninhealthyindividuals,therewereCD4+

TcellssensitivetostimulationwithHEp-2cell-derived self-antigens, but in smaller proportions compared topatients withSLE.

Inthis context,weobservedthat culturesfrom patients withactiveSLEpresentedlowerrelativefrequencyofCD4+_T

cells,aspreviouslyreportedbyWoutersetal.27_Ontheother hand,samplesfrompatientswithactiveandinactiveSLEhad anincreasedfrequencyofnewlyactivatedTcells(CD4+_CD69+_).

ThereductioninCD4+_{cellrelativefrequencymayberelated}

totheimmunosuppressant therapyeffects,sinceseveralof the studied patientswere under immunosuppressant ther-apy.Incontrast,theincreasedfrequencyofCD4+_CD69+_Tcells

possiblycorrespondstothe fractionofrecentlyactivatedT cellsthatmaycontributetothemaintenanceofexacerbated autoimmuneresponseanddiseaseactivitydespitetreatment. TherewasanincreasedrelativefrequencyofCD4+_Tcells

T able 2 – Rela ti v e fr equenc y of cells pr oducing cytokines in rela tion to total CD4+ T cells in cultur es stim ula ted with HEp-2 cell extr act or PMA/Io. Subpopulations Stim ulus HEp-2 cell e xtr act PMA/Io Contr ols A-SLE I-SLE p Contr ols A-SLE I-SLE p IL-2 + 0.70% (0.32–1.03) 0.99% (0.75–1.54) 1.07% (0.79–1.61) 0.245 1.09% (0.89–1.69) 1.65% (1.41–2.20) 1.90% (1.62–2.44) 0.388 IL-17 + 2.47% (0.73–5.88) 4.37% (0.63–20.90) 3.71% (0.73–25.80) 0.132 3.60% (1.09–9.66) 6.25% (1.83–25.62) 7.20% (2.51–33.20) 0.010 INF-␥ + 1.67% (0.35–6.69) 3.61% (2.46–5.55) 3.16% (2.27–9.61) 0.653 4.96% (3.56–6.37) 6.25% (5.10–8.19) 11.47% (10.58–17.92) 0.156 IL-2 +lL17 + INF-␥ + 0.17% (0.05–0.54) 0.42% (0.27–1.08) 0.48% (0.30–0.80) 0.074 0.26% (0.11–0.52) 0.47% (0.32–1.13) 0.54% (0.36–0.86) 0.526 IL-2 +lL17 + 0.27% (0.09–0.75) 0.47% (0.023–1.28) 0.49% (0.27–0.91) 0.179 0.33% (0.15–0.80) 0.71% (0.47–1.52) 0.85% (0.63–1.27) 0.422 IL-2 + INF-␥ + 0.12% (0.07–0.20) 0.16% (0.11–0.21) 0.14% (0.08–0.37) 0.279 0.54% (0.48–0.61) 0.60% (0.54–0.64) 0.94% (0.88–1.17) 0.944 IL-17 + INF-␥ + 0.17% (0.09–0.77) 0.41% (0.20–2.02) 0.94% (0.76–1.50) 0.166 0.23% (0.16–0.83) 0.66% (0.45–2.27) 1.19% (1.01–1.75) 0.087 Bold v alue is highlight significant p v alue .

takeoneffector function.Itispossiblethat thisextendsto inflammatorysitesoftargetorgans,therebycontributingto theperpetuationofinflammationatthesesites.

Thenextstepwastoevaluatetherelativefrequencyofcells abletoproduceeach cytokineindividuallyandcellsableto takeamultifunctionalphenotypebyproducingmorethanone cytokine.Infact,wefoundanincreasedrelativefrequencyof CD4+ _{IL-17-producingTcells(Th17lymphocytes)in}

PMA/Io-stimulatedculturesfrompatientswithA-SLEandI-SLE.This findingcorroboratespreviousreportsintheliterature, show-inganincreasedfrequencyofTh17cellsandincreasedserum levelsofIL-17inSLEpatients.10,15,16,18,28–31_However,we can-not exclude the participation of Th1cells and the related cytokine INF-␥intheinflammatoryprocess ofSLE.Infact, our data showthat the frequency ofIFN-␥-producing cells was consistentlyhigherinsamplesfrom SLEpatientsthan incontrolsamples,underPMA/IoorHEp-2stimulus.

Tothebestofourknowledge,thisisthefirststudyassessing therelativefrequencyofmultifunctionalTcellsinSLE.This typeofanalysisallowsaflexibledefinitionoffunctional sub-typesofTcellsthatcontrastswiththetraditionalpolarized classificationofsubtypesdefinedbysurfacemarkers.Based onthisconcept weanalyzedthemultifunctionalactivityof CD4+_{Tcells.Therewasnodifferenceintherelativefrequency}

ofbi-functional(INF-␥+/IL-2+;IL-2+/IL-17+;IFN-␥+/IL-17+)and tri-functionalcells(INF-␥+IL-2+IL-17+)CD4+cellsinpatients withA-SLE,I-SLEandcontrols.However,whenweanalyzed thefrequencyoftri-functionalcellsrelativetoallcells produc-inganyoneoftheanalyzedcytokines,wedidfindasignificant differenceinculturesstimulatedwithPMA/Io,wheresamples frompatientswithA-SLEhadsignificantlyhigherrelative fre-quencyofmultifunctionalTcellsthanthosefromI-SLE.This findingsuggeststhatpatientswithA-SLEshowanincreased subpopulationofThelpercellsabletoproduceabroad spec-trumofpro-inflammatorycytokinesafterastrongstimulus, suchasPMA/Io.Thisincreasedsubpopulationmaycontribute totheimmunedisordersinSLEautoimmuneprocess.Another interestingfindingconcerningmultifunctionalTcellswasthe observationthatCD4+_{TcellsexpressingIL-2andINF-␥}

pro-ducedmoreIFN-␥thandidcells thatexpressedonlyIFN-␥. Thiswasequallyobservedinsamplesfrompatientsand con-trols.Ananalogous phenomenonhasbeenshown byBetts et al.,19 _{who observedan increasedproductionofINF-␥by} tri-functionalcellsinHIV-infectedpatients.

Therewasasignificantcorrelationbetweenthefrequency of Th17 cells and recently activated cells (CD4+_CD69+_{) in}

patients with I-SLE as well as a correlation between the frequency of Th17 cells and CD4+_CD25+_Foxp3∅ _{in patients}

with A-SLE. These resultssuggest that IL-17 productionis predominantly associatedwithrecentlyactivatedTcellsin inactive-stageSLEandwithregularactivatedeffectorTcellsin active-stageSLE.ThisconceptisillustratedinFig.5.Wefound nocorrelationbetweenSLEactivity,measuredbySLEDAI,and thefrequencyofIL17-producingTcellsinpatientswithA-SLE, asalsoobservedbyWongetal.(2000).However,other stud-iesshowedsignificantcorrelationbetweenSLEDAIandTh17 frequency.16,29

INF-γ+ 386 323 564 1150 CD4 Functionality

A

B

C

D

E

INF-γ MFI INF-γ MFI INF-γ MFI INF-γ MFI

CD4 Functionality CD4 Functionality CD4 Functionality

INF-γ+IL-2+IL-17+

INF-γ+ IL-2+IL-17+ INF-γ+ IL-17+ INF-γ+ IL-2+

INF-γ

INF -γ+IL-2+IL-17+C

INF -γ+IL-2+IL-17+C

INF -γ+IL-2+IL-17+I

INF -γ+IL-2+IL-17+A

INF -γ+IL-2+I

INF -γ+IL-2+A

INF -γ+IL-17+I

INF -γ+IL-17+A

INF+IINF+A

INF -γ+IL-2+IL-17+IINF

-γ+IL-2+C

INF -γ+IL-2+I

INF -γ+IL-17+C

INF -γ+IL-17+IINF

-γ+C

INF -γ+I

INF -γ+IL-2+IL-17+AINF

-γ+IL-2+C

INF -γ+IL-2+A

INF -γ+IL-17+C

IINF -γ+IL-17+A

INF +C

INF+A

INF -γ+IL-2+

INF

-γ+IL-17+ INF

+

100 80

60 40

% of cells

20 8000 _*** ** *** 6000 4000 2000 8000 6000 4000 2000 0 8000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 6000 4000 2000 0 0 0

0 102 103 104 105

Fig.3–(A)Histogramrepresentingthemeanfluorescenceintensity(MFI)ofINF-␥afterstimulationwithPMA/Iointhe

differentfunctionalsubpopulationsofCD4+_{Tcellsfromarepresentativesampleofnormalcontrol.Thebluelinerepresents} theMFIofcellswithonefunction,thegreenlinethreefunctionscellsandrowsoforangeandredtwofunctionscellsto INF-␥+IL-17andINF-␥+IL-2,respectively.(B)GraphrepresentingthedifferencebetweentheMFIofIFN-␥betweendifferent

functionalsubpopulationsofCD4+_{TcellsincontrolsbystimulationwithPMA/Io.ComparisonoftheMFIofIFN-␥by} differenttypesofCD4+cellsafterstimulationwithPMA/IoamongthecontrolandA-SLE(C),controlandI-SLE(D)andI-SLE andA-SLE(E).**p<0.01;***p<0.001.

SLEandopensperspectivestothedevelopmentofalternative therapiesforthisdisease.Webelievethattheimmunological patternobservedinthepresentstudyoccursinSLEpatientsin general,butthereisconsiderableheterogeneityinthegroup.

The present study did not aim to investigate the associa-tionofthisphenomenonwiththediversemanifestationsof SLE. Thisis arelevant pointto beinvestigatedina future study speciallydesignedforthisaim. Itisconceivablethat

% of CD4+ _CD69+ _{T cells}

15 40 30 20 10 0 40 30 20 10 0 30 20 10 0 40 30 20 10 0 40 30 20 10 0 10 P=.0002

A

B

C

F

E

D

0 5 0

0 10 15

10 15

10 15 20 25

10 15 20 25 10 20 30

10 20 30

% of CD4+ _CD69+ _{T cells % of CD4}+ _CD69+ _{T cells}

% of Th17 cells

% of CD25

+ Foxp3

. T cells

% of CD25

+ Foxp3 . T cells

% of CD25

+ Foxp3

. T cells

% of Th17 cells

% of Th17 cells % of Th17 cells % of Th17 cells

% of Th17 cells

R=.7665 P=.006 R=.619 P=.100 R=.399 P=.010 R=.732 P=.006 R=.763 P=.023 R=.674

Fig.4–CorrelationamongtherelativefrequencyofTh17cellsandCD4+CD69+TcellsinculturesofPBMCofpatientswith I-SLEwithoutstimulation(A),culturestimulatedwithHEp-2cellsextract(B)andculturestimulatedwithPMA/Io(C)n=18.

CorrelationamongtherelativefrequencyofTh17cellsandCD4+CD25+Foxp3−inculturesofPBMCofpatientswithA-SLE

Th17 cells

Th17 cells CD69+

T cells

CD69+ T cells CD25+Foxp3φ

T cells

CD25+_Foxp3φ

T cells

Active SLE Inactive SLE

Fig.5–ProposedconceptionofcorrelationamongtherelativefrequencyofTh17cellsandCD4+_CD69+_{Tcellsinpatients} withI-SLEandcorrelationamongtherelativefrequencyofTh17cellsandCD4+_CD25+_Foxp3−_{inpatientswithA-SLE.}

ShowingthatthemajorityofcellsresponsibleforproductionofIL-17arethosenewlyactivatedinpatientswithI-SLE,while inpatientswithSLE-A,mostoftheIL-17producingcellsarethosechronicallyactivated.

personalizedcytokineblockingtherapyspecificallydesigned accordingtothepredominantprofileofmultifunctional effec-torTcellswillbeeffectiveinhelpingrestoretheimmunologic balanceineachpatient.IL-17-producingTlymphocytesseem toplayaprominentroleinSLEpathophysiologyandmay rep-resentapotentialtargetfortherapy.Infact,itispossiblethat cytokine-targetedandpersonalizedtherapymaycontributeto improvingthebalance ofeffectorimmuneresponse,

avoid-ing orminimizing the damagecausedbythe autoimmune

response.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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