Switching and loss of cellular cytokine producing capacity characterize in vivo viral infection and

25 Pages·2017·4.1 MB·English

RESEARCHARTICLE
Switching and loss of cellular cytokine
producing capacity characterize in vivo viral
infection and malignant transformation in
human T- lymphotropic virus type 1 infection
HuseiniKagdi1*,MariaAntoniettaDemontis1,JuanCarlosRamos2,GrahamP.Taylor1
1 SectionofVirology,DepartmentofMedicine,ImperialCollegeLondon,London,UnitedKingdom,
a1111111111 2 DepartmentofHematology/Oncology,UniversityofMiamiSchoolofMedicine,Miami,Florida,United
a1111111111 StatesofAmerica
a1111111111
a1111111111 *[email protected]
a1111111111
Abstract
AdultT-cellleukaemia/lymphoma(ATL)arisesfromchronicnon-malignanthumanTlym-
OPENACCESS photropicvirustype-1(HTLV-1)infectionwhichischaracterizedbyhighplasmapro-inflam-
Citation:KagdiH,DemontisMA,RamosJC,Taylor matorycytokineswhereasATLischaracterizedbyhighplasmaanti-inflammatory(IL-10)
GP(2018)Switchingandlossofcellularcytokine concentrations.ThepoorprognosisofATLispartlyascribedtodisease-associatedimmune
producingcapacitycharacterizeinvivoviral
suppression.ATLcellshaveaCD4+CCR4+CD26-CD7-immunophenotypebutinfected
infectionandmalignanttransformationinhuman
T-lymphotropicvirustype1infection.PLoS cellswiththisimmunophenotype(‘ATL-like’cells)arealsopresentinnon-malignantHTLV-1
Pathog14(2):e1006861.https://doi.org/10.1371/ infection.Wehypothesizedthat‘ATL-like’andATLcellshavedistinctcytokineproducing
journal.ppat.1006861
capacityandaswitchinthecytokinesproducedoccursduringleukemogenesis.Seventeen
Editor:DanielC.Douek,VaccineResearchCenter, asymptomaticcarriers(ACs),28patientswithHTLV-1-associatedmyelopathy(HAM)and
UNITEDSTATES
28withATLwerestudied.PlasmaIL-10concentrationandtheabsolutefrequencyofIL-10-
Received:September7,2017 producingCD4+TcellsweresignificantlyhigherinpatientswithATLcomparedtoAC.IL-
Accepted:January8,2018 10-producingATLcellsweresignificantlymorefrequentthan‘ATL-like’cells.Thecytokine-
producingcellswereonlyasmallfractionofATLcells.Clonalityanalysisrevealedthateven
Published:February14,2018
inpatientswithATLtheATLcellswerecomposednotonlyofasingledominantclone(puta-
Copyright:©2018Kagdietal.Thisisanopen
tiveATLcells)butalsotensofnon-dominantinfectedclones(‘ATL-like’cells).Thefre-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which quencyofcytokine-producingcellsshowedastronginversecorrelationwiththerelative
permitsunrestricteduse,distribution,and abundanceofthelargestcloneinATLcellssuggestingthattheputativeATLcellswerecyto-
reproductioninanymedium,providedtheoriginal
kinenon-producingandthatthe‘ATL-like’cellsweretheprimarycytokineproducers.These
authorandsourcearecredited.
findingswereconfirmedbyRNAseqwithcytokinemRNAexpressioninATLcellsinpatients
DataAvailabilityStatement:Allrelevantdataare
withATL(confirmedtobecomposedofbothputativeATLand‘ATL-like’cellsbyTCRanaly-
withinthepaperanditsSupportingInformation
sis)significantlylowercomparedto‘ATL-like’cellsinpatientswithnon-malignantHTLV-1
files.
infection(confirmedtobecomposedofhundredsofnon-dominantclonesbyTCRanalysis).
Funding:Thestudyfundingwasprovidedby
Asignificantinversecorrelationbetweentherelativeabundanceofthelargestcloneand
Bloodwisecharity(https://bloodwise.org.uk,grant
number:13060).Thefundershadnoroleinstudy cytokinemRNAexpressionwasalsoconfirmed.Finally,‘ATL-like’cellsproducedlesspro-
design,datacollectionandanalysis,decisionto andmoreanti-inflammatorycytokinesthannon‘ATL-like’CD4+cells(whicharepredomi-
publish,orpreparationofthemanuscript.
nantlyHTLVuninfected).Insummary,HTLV-1infectionofCD4+Tcellsisassociatedwitha
Competinginterests:Theauthorshavedeclared changeincytokineproducingcapacityanddominantmalignantclonalgrowthisassociated
thatnocompetinginterestsexist.
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006861 February14,2018 1/25
CytokineprofileinHTLV-1infectionandATL
withlossofcytokineproducingcapacity.Non-dominantcloneswith‘ATL-like’cellscontrib-
utetoplasmacytokineprofileinpatientswithnon-malignantHTLV-1infectionandarealso
presentinpatientwithATL.
Authorsummary
HumanT-celllymphotropicvirustype-1(HTLV-1)infectionofCD4+Tcellsisassociated
withachangeintheircytokineproducingcapacityandisresponsibleforthedifferent
plasmacytokineprofilesinpatientswithadultT-cellleukaemia/Lymphoma(ATL)and
non-malignantHTLV-1infection.Dominantmalignantclonalgrowthoftheinfected
CD4+Tcellsisassociatedwithlossofcytokineproducingcapacity.ACs,patientswith
HAMandpatientswithATLhaveacommoncytokineclusterwithpositivecorrelations
betweenpro-(TNFαandIL-6)andanti-(IL-10)inflammatorycytokines.PlasmaIL-10
washigherintheHAMandATLstatescomparedtoACwhilsttherewasnodifferencein
pro-inflammatorycytokines.PatientswithHAMhaveraisedplasmaconcentrationsof
IFNγ,IL-10andIL-17suggestingacomplexinteractionbetweenthesecytokineinHAM
whichwasnotseeninATL.AggressiveATLisassociatedwithraisedplasmaconcentra-
tionsofpro-andanti-inflammatorycytokinescomparedtoindolentATL.Thiscytokine
profiledidnotprecedeorpredictaggressiveATL.The‘ATL-like’infectedcellsinACs
andinpatientswithHAMhavelowerpro-andhigheranti-inflammatorycytokinesecre-
tionthannon-‘ATL-like’cellswhicharepredominantlyHTLV-1uninfected.Putative
ATLcellshavelittleornocytokineproducingcapacity.‘ATL-like’infectedcellsfrom
non-dominantinfectedcloneswerepresentnotonlyinpatientswithnon-malignant
HTLV-1infectionbutalsoATL.‘ATL-like’cellshavecytokineproducingcapacityand
contributetoplasmacytokineprofileinpatientswithnon-malignantHTLV-1infection
andpossiblyalsoinATL.
Introduction
HumanT-lymphotropicvirustype-1(HTLV-1)isacomplexdeltaretrovirusinfectinganesti-
mated10millionindividualsworldwide[1].Inthemajority,infectionleadstoachronic
asymptomaticcarrierstate(AC)but2%to6%developadultT-cellleukaemia/lymphoma
(ATL)andanother3%inflammatorydisorderse.g.HTLV-1-associatedmyelopathy(HAM).
ThediagnosisofATLisbasedonclinicalfeatures,morphology(lymphocyteswithcharacteris-
tic‘flowercell’morphology),immunophenotyping(CD3+,CD4+,CCR4+,CD25+,CD26-
andCD7-)anddemonstrationofdominantHTLV-1infectedclones[2,3].ATLisclassified
intofoursubtypes:smouldering,chronic,acuteandlymphoma.Smoulderingandchronic
ATLaresuggestedtohaveanindolentcoursewhileacuteandlymphomaanaggressivecourse
[3].Survivalwithchemotherapyispoor[4–7]duetoprimarychemo-refractorydisease,or
earlyrelapseoropportunisticinfections.[8–11].
PatientswithATLhavehighplasmaconcentrationsofanti-inflammatorycytokinese.g.
interleukin(IL)-10[12,13].IL-10issecretedbyregulatoryCD4+Tcells[14].CD4+Tcellsin
patientswithATLhavebeenshowntohavehighIL-10expression[13,15].ATLcellsexpress
regulatoryTcell-associatedmarkersCD25andFOXP3[16–19].Thishasledtotheassumption
thatATLcellsarearegulatoryTcellcounterpartandmediateanimmunosuppressiveclinical
statebysecretingIL-10.IncontrastHAMischaracterizedbyorgandamageduetoimmune
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CytokineprofileinHTLV-1infectionandATL
activation,andhighconcentrationsofplasmapro-inflammatorycytokinese.g.interferonγ
(IFNγ)[20].HTLV-1infectedcellssecreteIFNγ[21,22]anddirectlycontributetotheplasma
cytokineprofileinHAM.Thesefindingssuggestadistinctcytokineproducingcapacityof
HTLV-1infectedcellsinkeepingwithclinicalstate.
ATLarisesdenovoinACandpatientswithHTLV-1-associatedinflammationsuchas
HAM,hereafterreferredtoasnon-malignantHTLV-1infection.HTLV-1-infectedcellshavea
CD4+CCR4+CD26-immunophenotype;thelossofCD7expressionbythesecellsdifferenti-
atesATLfromnon-malignantHTLV-1infection[23–27].Weandothershaveshownthat
‘ATL-like’HTLV-1-infected(CD4+CCR4+CD26-CD7-)cellsarepresentinpatientswith
non-malignantHTLVinfection.WehypothesizethatthecytokineproducingcapacityofATL
and‘ATL-like’cellsaredistinctfromeachotherandaredirectlyresponsiblefortherespective
plasmacytokineprofileinATLandnon-malignantHTLV-1infection,andthatthechangein
cytokineproducingcapacityreflectsmalignanttransformationfromnon-malignantHTLV-1
infectiontoATL.Pro-andanti-inflammatorycytokineweremeasuredinplasmaandcells
(intracellularstainingandgeneexpressionbymRNAsequencing)inpatientswithfourdiffer-
entHTLVdiagnoses:AC;HAM;indolentATL;aggressiveATL.ClonalityanalysisofATLand
‘ATL-like’cellswasperformedandtherelationshipbetweenplasma,cellularcytokine,clonal-
ityandclinicalstatewasstudied.
Materialandmethods
Patientsandcells
ThepatientcohortisbasedattheNationalCentreforHumanRetrovirology(NCHR)atSt
Mary’sHospital,Paddington,London,UKandtheUniversityofMiamiSchoolofMedicine,
Miami,USA.DiagnosisofHTLV-1infection,HAMandATLwasmadeaccordingtoWorld
HealthOrganizationcriteria.
Ethicsstatement
Patients’samplesarecollectedandstoredinaCommunicableDiseasesResearchTissueBank
approvedbytheUKNationalResearchEthicsService(references09/H0606/106and15/SC/
0089).SamplesatUniversityofMiamiSchoolofMedicinesarecollectedunderIRB-approved
study"StudyofBlood,TissueandBodyFluidsofViralAssociatedMalignancies"(EPROST
No.20030608).SamplesarestoredunderlicenseinaccordancewiththeHumanTissuesAct
2004.SampleswerecollectedpriortoanysystemictherapyinpatientwithATLorimmune
modulatorytreatmentinpatientswithHAM.Baselinedemographicandclinicalcharacteris-
ticsofthestudypopulationareshowninS1Table.
Peripheralbloodmononuclearcells(PBMCs)andplasmawereseparatedfromfreshwhole
bloodbydensity-gradientcentrifugationonHistopaque-1077(Sigma-Aldrich,StLouis,USA).
Plasmawasstoredat-80˚C.PBMCswereharvestedfromtheinterface,washedinphosphate
buffersaline(PBS,Sigma-Aldrich),cryopreservedin10%dimethylsulphoxide(Sigma-
Aldrich)and90%heatinactivatedfoetalcalfserum(FCS)(Gibco,Carlsbad,USA),andstored
inliquidnitrogenuntiluse.
Electrochemiluminescenceassay
Plasmacytokineconcentrationsofthefollowingninecytokines/chemokinesweremeasured
usingsensitiveandspecificV_PLEXimmunoassaysaccordingtothemanufacturer’sprotocol
(MesoScaleDiscovery,Gaithersburg,USA):Humanpro-inflammatorycytokines:interferon
gamma(IFNγ),interleukin(IL)-2,IL-6,IL-7,IL-17α,tumournecrosisfactor-alpha(TNFα);
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CytokineprofileinHTLV-1infectionandATL
anti-inflammatorycytokine:IL-10andchemokines:macrophage-derivedchemokine(MDC)
alsoknownasCCL22andC-X-Cmotifchemokine10(CXCL-10)alsoknownasIFNγ-
inducedprotein10(IP-10),.
Forsampleswithdetectableconcentrationsbelowthelimitsofquantificationmissingval-
ueswerereplacedwith99%ofthelowestdetectableconcentration.
Cellularcytokineassay
ThawedcryopreservedPBMCswereincubatedineithercompletemedia(CM)comprising
10%heatinactivatedFCSinRPMIwithL-glutamineplus1%Penicillinonly,CMwithphyto-
hemaggulutinin(PHA,finalconcentrationof5μg/mL,Sigma)orCMwith2%cellactivation
cocktail(containingphorbol12-myristate13-acetate(PMA)andionomycin,(BioLegend,San
Diego,USA))at37˚C,in5%CO ataconcentrationof106cells/50μLfor6hours.BrefeldinA
2
(BioLegend)wasaddedforthelastfivehours.Allwashesweredonebysuspendingcellsin
PBScontaining1%FCSfollowedbycentrifugationat600gfor5minutestwice.PBMCswere
washedthriceattheendofincubationandstainedsequentiallywithnearinfraredfixablevia-
bilitystainfollowedbyflurochromeconjugatedmonoclonalantibodiesagainstcellsurface
markers(CD3,CD4,CD7,CD8andCCR4)for30minutesatroomtemperature(RT).The
cellswerethenfixedusingfixationbufferfromFoxP3/TranscriptionFactorStainingBuffer
Set(eBioscience,SanDiego,USA).Thefixedcellswerewashedwithpermeablizationbuffer
followedbyincubationwithfluorochrome-conjugatedmonoclonalantibodiesagainstIL-6,
IL-10,TNFαandIFNγfor15minutesatRT.PBMCswerethenwashedtwiceandstoredat
4˚CinPBS1%FCSovernightuntilanalysisonBectonDickinsonFortessaII.Aminimumof
20,000eventswererecordedforanalysis.CompensationwascomputedusingBDFACSdiva
softwareusingsinglestainingofCompebeads(eBioscience)andcheckedmanually.Fluores-
cenceminusonecontrol(antibodycocktailcontainingallantibodiesexceptone)wasusedfor
gating.DatawereanalysedbyFlowjosoftware.
Magneticcellsorting
Magneticcellsortingwasperformingbynegativeselection.ThawedPBMCswereincubated
withprimarybiotinylatedantibodies(CD7,CD26,CD8,CD14,CD15,CD16,CD19,CD36,
CD56,CD123,TCRγ/δ,CD235a[MiltenyiBiotecLtd.,UnitedKingdomandBiolegend,
USA])at4˚Cfor10minand107cells/100μl.Allwashesweredonebysuspendingcellsin1%
BSA(Sigma)followedbycentrifugationat600gfor5minutestwice.LabelledPBMCswere
washedandincubatedwith30%dilutionofstreptavidinconjugatedmicrobeadsatafinalcon-
centrationof107cells/100μL.Microbead-conjugatedPBMCswerewashedandmagnetically
sortedtwiceonLSColumns(MiltenyiBiotecLtd)accordingtothemanufacturer’sdirections.
Theflow-throughcontainingCD3+CD4+CD7-CD26-cells,ofwhich99%wereCCR4+,was
washedtwice.Ofthesecells,104cellswereusedtocheckforpuritybyflowcytometryandthe
remainderwereusedforHTLV-1proviralloadquantificationandclonalityanalysis.
HTLV-1proviralload(PVL)quantification
GenomicDNAwasextractedfromPBMCsandsortedCD4+TcellsubsetsusingQIAamp
DNAminikit(Qiagen,Hilden,Germany).Theproviralloadwasdeterminedbyreal-time
PCRaspreviouslydescribed[28].
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CytokineprofileinHTLV-1infectionandATL
LigationmediatedPCRfollowedbyHTS
Clonalityanalysiswasperformedusinglinker-mediated(LM)-PCR,high-throughputsequenc-
inganalysisofHTLV-1integrationsitesaccordingtothemethodpreviouslydescribed[29].
Randomfragmentsof1μggenomicDNA(100ngofsampleDNAmixedwith900ngofunin-
fectedDNAfromJurkatcells)weregeneratedbysonicationandligatedtocustom,partially
double-stranded,DNAadaptors.NestedPCRusingspecificprimerswasperformedtoselec-
tivelyamplifyadaptor-ligatedDNAfragmentsabuttingtheHTLV-13’LTR.ThePCRprod-
uctsfromeachsamplehadtwounique8bpmultiplexingbarcodesandwerepooledfor
sequencing.Paired-end150-basereadsweregeneratedonanIlluminaMiSeq.Thereadswere
de-multiplexedusingtheMiSeqreporter.ThelinkerandprimersequencesarelistedinS2
Table.
TheinsilicoanalysiswasconductedinshellandRenvironment.Read1andRead2were
alignedtoreferencehuman(hg38,UCSC)andHTLV-1genomesusingBowtie2[30].The
numberofuniqueintegrationsites(clones)andtherelativeandabsoluteabundanceofeach
uniqueintegrationsite(clonalsize)werecalculatedaspreviouslydescribed[29].
RNAsequencing
RNAwasextractedfromMACS-sortedcellsusingQiagenAllprepDNA/RNAcolumn-based
extractionkits(Qiagen)asperthemanufacturer’sinstructions.RNA-seqwasperformedon
DNase-treatedsamplesusingTruSeqStrandedmRNALibraryPrepKit(Illumina,United
States)asperthemanufacturer’sinstructions.AllsortedcellshadthehighestRNAquality
(>100ngRNAandRNAqualityscore(cid:21)8).Allsequencingwasperformedusing50nucleo-
tidepaired-endreadsonanIlluminaHiSeq4000instrumentattheImperialbiomedical
researchcentre(BRC)genomicsfacility,London,UnitedKingdom.
AnalysisofT-cellreceptorclonality. MiXCRsoftware[31]withstandardsettingswas
usedtoidentifyTCRalphaandbetaCDR3-containingreadspresentintheRNAsequencing
data,generatingalistofCDR3s(individualclones)andtheirrelativeabundances(clonal
fraction=readsofindividualclone/readsofallclones).Duetoallelicexclusion,eachTcell
cloneshouldonlyexpressonebetachain[32],thereforethemostabundantbetaclonotype(if
present,otherwisealpha)wasusedtodefinetherelativeabundanceoftheclonalTcellineach
subject.
Mappingandidentificationofdifferentiallyexpressedgenes. Beforereadmapping,
cleanreadswereobtainedbyremovingreadsthatcontainedadapterorpoly-N,andlowqual-
ityreadsfromrawdatabyusingTrimmomatic,aflexiblereadtrimmingtools(Version0.36,
[33]).Atthesametime,Q20,Q30,andGCcontentsofthecleandatawerecalculatedusing
FastQC(BabrahamBioinformatics).Allthedownstreamanalyseswerebasedonthehighqual-
itycleandata.Thenormalizedanddifferentialgeneexpressionwasperformedusingthenew
Tuxedopipeline[34].Thecleanreadswerealignedtothehumangenome(version:GRCH38)
usingtheHISAT2program(V2-2.0.1).WeappliedStringtie(Version0.36)andBallgownalgo-
rithmstoidentifythesignificantlydifferentiallyexpressedgenes(p<0.05).Cytokine,chemo-
kineandtheirreceptorsgeneexpressionwasextractedfromthedifferentialgeneexpression
data.Hierarchicalclusteringwasperformedtogenerateanoverviewofthecharacteristicsof
thecytokinegeneexpressionprofiles,basedonvaluesofsignificantlydifferentiallyexpressed
transcripts.
Statistics
StatisticalanalysiswasperformedusingGraphpadPrismsoftware.Thesignificanceofdiffer-
enceincontinuousvariablesbetweenmultiplepatientgroupswasdeterminedbyaKruskal-
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006861 February14,2018 5/25
CytokineprofileinHTLV-1infectionandATL
WallistestwithDunnpost-testanalysis.Thesignificanceofdifferenceincontinuousvariables
betweentwocellsubsetswasdeterminedusingaWilcoxonsigned-ranktest.Thesignificance
ofdifferenceincontingencyvariableswasdeterminedbyachi-squaredtest.Differenceswere
consideredstatisticallysignificantifp<0.05.Thecorrelationbetweentwocontinuousvari-
ableswasdeterminedbyanon-parametricSpearmantest.TheSpearmancorrelationwascon-
sideredsignificantforp<0.05andshowingatrendifthepvaluewasbetween0.05and0.1.
Aclassificationtreewasconstructedtoidentifythehierarchicalorganisationofplasma
cytokineconcentrationinACs,patientswithHAMandpatientswithATL.Theclassification
treewasproducedusingaRecursivePartitioningandRegressionTrees(RPART)analysisinR.
ThenetworkanalysiswasperformedusingNodeXL.Intheanalysis,absoluteCD3+,
CD4+andCD8+cellcountsandPBMCPVLwereusedascellularvariables,andplasmacyto-
kineconcentrationswereusedascytokinevariables.Thecellularandcytokinevariableswere
usedasedgesofthenetworkwhiletheSpearmancorrelatesbetweennodeswereusedasverti-
ces.VerticeswithatleastasignificanttrendonSpearmancorrelation(p<0.1)wereincluded.
ThelayoutwasperformedusingHarel-KorenFastmultiscale.
Results
Plasmacytokineprofileinnon-malignantHTLV-1infectionandATL
TherelativeandabsolutefrequenciesofCD3+,CD4+TcellsandHTLV-1PVLinPBMCs
weresignificantlyhigherinpatientswithATLcomparedtoACsorpatientswithHAM
(Table1).PatientswithHAMhadsignificantlyhigherplasmaconcentrationsofIFNγ,
CXCL10,IL-2andIL-17(pro-inflammatorycytokines)comparedtoACsandpatientswith
ATL(Fig1A–1D).PatientswithATLandpatientswithHAMhadsignificantlyhigherplasma
concentrationsoftheanti-inflammatorycytokineIL-10comparedtoACs(Fig1E).Therewas
nosignificantdifferenceinplasmaconcentrationsofIL-6,IL-7,CCL22andTNFαbetween
thethreepatientgroups(Fig1F–1I).ThemedianplasmaconcentrationsofIL-6andTNFαin
allthreepatientgroupswereneartheupperlimitofthemanufacturer’snormalhumanrange
whilethoseofCCL22,IL-2andIL-7werenearthelowerlimit.
PatientswithaggressiveATLhadsignificantlyhigherTNFα,IL-6andIL-10plasmaconcen-
trationsthanthosewithindolentATL(Fig2A–2C).Therewasnosignificantdifferencein
plasmaconcentrationsofIFNγ,CXCL10,CCL22,IL-2andIL-7betweenATLsubtypesas
showninS1Fig.
Theplasmachemokineandcytokineconcentrations,offourpatientswithHAMwhodevel-
opeddenovoaggressiveATLandtwopatientswithindolentATLwhoprogressedtoaggressive
ATL,weremeasuredataggressiveATLdiagnosisandat3–12monthsearlier.PlasmaTNFα,
IL-6andIL-10concentrationsweresignificantlyhigheratdiagnosis,withmedianincreasesof
1.9-fold;3.7-foldand7.1-foldrespectively(Fig2D).Therewasalargevarianceinchangesof
plasmaIFNγconcentrations.
Insummary,patientswithHAMhavethehighestconcentrationsofpro-inflammatory
cytokineandACshadthelowestIL-10plasmaconcentrations.PatientswithaggressiveATL
hadhigherplasmaconcentrationsofTNFα,IL-6andIL-10comparedtopatientswithindolent
ATL.Thesehigherplasmaconcentrationsdidnotprecedemalignantprogression.
Networkandclassificationtreeanalysisofimmuneprofileinnon-
malignantHTLV-1infectionandATL
Networkanalysiswasperformedtobetterunderstandtheinteractionbetweencellularand
plasmaimmunemarkers.TheabsolutefrequencyofCD3+,CD4+TcellsandPVLwere
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CytokineprofileinHTLV-1infectionandATL
Table1. T-cellsubsetsandproviralloadinAC,HAMandATL.
Patientgroup Parameter CD3 CD4 CD8 PVL(copies
per100
Absolutecount Relativecount(% Absolutecount Relativecount(% Absolutecount Relativecount(%
PBMCS)
(10^6/uL) lymphocytes) (10^6/uL) lymphocytes) (10^6/uL) lymphocytes)
AC(n=17) Minimum 881 61.4 539 30 194 12 <0.1
25% 1139 67 860 45 289 15 0.7
Percentile
Median 1454 73 989 53 385 19 2.8
75% 1852 79 1260 58 518 27 18.9
Percentile
Maximum 2970 87 1563 66 1470 42 27.9
Mean 1458 73 1049 51 461 22 9.4
Std. 631 8 301 9 360 10 10.4
Deviation
Std.Error 158 2 78 2 90 2 2.5
HAM(n=28) Minimum 723 59 264 22 232 17 0.4
25% 1112 74 678 44 342 22 4.4
Percentile
Median 1347 79 864 50 508 25 6.9
75% 2057 84 1301 55 855 36 12.6
Percentile
Maximum 3768 95 2368 60 1495 73 28.8
Mean 1644 78 1012 48 633 30 9.0
Std. 789 9 522 10 371 13 7.2
Deviation
Std.Error 147 2 97 2 69 2 1.3
ATL(n=28) Minimum 574 38 334 22 160 1 2.0
25% 2345 81 1454 58 287 3 20.3
Percentile
Median 6494 93 5499 83 366 8 29.8
75% 11868 95 10600 91 531 16 64.6
Percentile
Maximum 91000 99 11612 94 1252 31 276.2
Mean 14534 85 5695 73 446 10 54.3
Std. 24674 16 4343 22 263 9 59.9
Deviation
Std.Error 5384 4 1024 5 62 2 11.8
Significanceof ACvs Ns Ns ns ns ns ns ns
difference? HAM
ACvsATL (cid:3)(cid:3)(cid:3) (cid:3)(cid:3)(cid:3) (cid:3) (cid:3)(cid:3)(cid:3) ns (cid:3) (cid:3)(cid:3)(cid:3)
HAMvs (cid:3)(cid:3)(cid:3) (cid:3)(cid:3)(cid:3) (cid:3)(cid:3)(cid:3) (cid:3)(cid:3)(cid:3) ns (cid:3)(cid:3)(cid:3) (cid:3)(cid:3)(cid:3)
ATL
(cid:3)denotesp<0.05,
(cid:3)(cid:3)(cid:3)denotesp<0.001,nsdenotesnotsignificant
ACdenotesasymptomaticcarriers,HAMdenotesHTLV-1-associatedmyelopathy,ATLdenotesadultT-cellleukaemia/lymphoma
https://doi.org/10.1371/journal.ppat.1006861.t001
significantlypositivelycorrelatedwitheachotherinpatientswithHAMorATL(Fig3Aand
3B).Therewerealsosignificantpositivecorrelationsbetweentheplasmaconcentrationsof
TNFα,IL-6andIL-10inallthreediagnosticgroups(Fig3A–3C).Theplasmaconcentrationof
IL-10correlatedsignificantlywithIFNγinpatientswithATLandtoalesserextentwithboth
IFNγandIL-17inpatientswithHAM.
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CytokineprofileinHTLV-1infectionandATL
Fig1.Plasmacytokineconcentrationinnon-ATLHTLV-1infectionandATL.A-I)Alignedcolumnplotsofplasmacytokine/chemokineconcentrationsin
patientswithnon-ATLHTLV-1infectionandATL.Thebarrepresentsmedianvalues.Thecontinuouslineandshadedareashowsmanufacturersupplied
medianandrangeinhealthyindividuals.Statisticalanalysis:Kruskal-WallistestwithDunnpost-test,95%confidenceinterval.(cid:3)denotesp<0.05,(cid:3)(cid:3)denotes
p<0.01,(cid:3)(cid:3)(cid:3)denotesp<0.001.
https://doi.org/10.1371/journal.ppat.1006861.g001
Aclassificationtreeanalysiswasperformedonplasmacytokineconcentrationstoidentify
thecytokineprofilewhichbestdifferentiatedAC,HAMandATLstates.AplasmaIL-10
concentration<0.16pg/mLidentifiedACwitha64.7%sensitivityand73%specificityas
showninFig3D.AplasmaIL-17concentration<1pg/mLoriftheIL-17concentrationwas
>1pg/mLanIL-10concentrationof>0.8pg/mLidentifiedATLwith78.5%sensitivityand
85%specificitywhilstanIL-17concentrationof(cid:21)1pg/mLwithanIL-10concentration
between0.16and0.8pg/mLidentifiedHAMwith82.1%sensitivityandaspecificityof71.8%.
Insummary,apositivecorrelationbetweentheplasmaconcentrationsofspecifiedpro-and
anti-inflammatorycytokineswaspresentinallthreeHTLV-1patientgroups.Together,the
plasmaconcentrationsofIL-10andIL-17discriminatedbetweentheclinicalstatesassociated
withHTLV-1infection.
Cellularcytokinestudiesinnon-malignantHTLV-1infectionandATL
Toidentifythecellularsourceofthecytokinesidentifiedinplasma,thecytokineproducing
capacityofmonocytes,CD4+andCD8+Tcellswasstudied.Intracellularcytokinestainingfor
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006861 February14,2018 8/25
CytokineprofileinHTLV-1infectionandATL
Fig2.PlasmacytokineconcentrationsinATL.A-C)Alignedcolumnplotsofplasmacytokine/chemokineconcentrationsinindolentandaggressiveATL.D)
BoxplotsoffoldchangeinsixpatientswithaggressiveATLatdiagnosisandat3–12monthspre-diagnosis.Thebarrepresentsmedianvalues.Statistical
analysis:Kruskal-WallistestwithDunnpost-test,95%confidenceintervalandWilcoxonsignedranktest.(cid:3)denotesp<0.05,(cid:3)(cid:3)denotesp<0.01,(cid:3)(cid:3)(cid:3)denotes
p<0.001.
https://doi.org/10.1371/journal.ppat.1006861.g002
TNFα,IFNγ,IL-6andIL-10wasperformedin10ACs,11patientswithHAMand10with
ATL(fourwithindolentandsixwithaggressiveATL).Thegatingstrategytodetectcytokine
producingcellsisshowninS2Fig.Therelativeandabsolutefrequenciesofcellssecretingeach
cytokineareshownaspercentagesandcellcountperlitre.
AlthoughtherewasnodifferenceintherelativefrequencyofIL-10+CD4+cells(Fig4E)
theirabsolutefrequencywashigherinpatientswithATLcomparedtoACandHAM(Fig4A).
TherelativeandabsolutefrequencyofIL-6+CD4+cellsdidnotdifferbydiseasestate(Fig
4B–4F).TheabsolutefrequenciesofTNFα+CD4+T-cellswerethesameineachgroup(Fig
4C)butTNF+cellsmadeupasignificantlylowerpercentageofallCD4+Tcellsinpatients
withATLcomparedtonon-malignantHTLV-1infection(Fig4G).Finally,althoughtheabso-
lutefrequencyofCD4+T-cellssecretingIFNγwasincreasedinATL(Fig4D),therelativefre-
quencyofthesecellswassignificantlylowerinpatientswithATLthaninACsandpatients
withHAM(Fig4H).InpatientswithATLthemedianrelativefrequenciesofTNFα,IFNγ,IL-
6andIL-10secretingCD4+cellswere5%,1.7%,0.6%and0.3%respectively.Thefrequencies
ofTNFα,IFNγ,IL-6andIL-10-secretingCD8+cellsandmonocytes(exceptIFNγ,whichis
notsecretedbymonocytes)didnotdifferbetweendiagnosticgroupsasshowninS3Fig.
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CytokineprofileinHTLV-1infectionandATL
Fig3.Networkandclassificationtreeanalysisofplasmacytokineconcentrationinnon-ATLHTLV-1infectionandATL.Networkanalysis
ofabsoluteT-cellsubsetcount,HTLV-1PVLandplasmacytokineconcentrationusingatleastsignificantSpearmancorrelationtrendsisshown
forpatientswithATL(A),HAM(B)andAC(C).Thegreenandredlinesdenotepositiveandnegativecorrelationsrespectively.Thecontinuous
andbrokenlinedenotestatisticallysignificantandtrendcorrelations.ThepruneclassificationtreetoclassifydiagnosisofAC,HAMandATL
onthebasisofIL-10andIL-17concentrationisshowninfigureD.Thepercentageshowsthedistributionofallpatientsintodifferentarms
whilstthethreedecimalnumbersshownspecificityofeachclassificationfordiagnosisofAC,ATLandHAMrespectively.
https://doi.org/10.1371/journal.ppat.1006861.g003
Insummary,althoughtheabsolutefrequencyofthecytokine-producingCD4+T-cellswas
greaterinpatientswithATLthesemakeuponlyaminorityoftheirCD4+Tcellssuggesting
thatATLcellsareingeneralnotsecretingthesecytokines.
CytokineproducingcapacityofATLand‘ATL-like’cells
CD4+Tcellsarethedominantreservoirofinfectedcellsinbothnon-malignantHTLV-1
infectionandATL[23,35].Theinfectedcellsarederivedfromthousandsofnon-dominant
clonesinnon-malignantHTLV-1infectionandfromadominantcloneonapolyclonalback-
groundofnon-dominantclonesinATL[29,36,37].TofurthercharacterisetheATLand
‘ATL-like’cells,theircytokineproducingcapabilitywasdetermined.ATLcellshaveaCD4
+CCR4+CD26-CD7-immunophenotypeand‘ATL-like’cellsarepresentinnon-malignant
HTLV-1infection.TheCD4+CCR4+CD7-immunophenotypewasusedtostudythecytokine
producingcapacityofATLcellsas>99%ofCD4+CCR4+CD7-cellswerealsoCD26-.
Therelative(Fig5A)andabsolutefrequencies(Fig5B)ofCD4+CCR4+CD7-Tcellswere
significantlyhigherinpatientswithATLcomparedtonon-malignantHTLV-1infectionwhilst
therewasnodifferenceintheabsolutefrequencyofnon-CCR4+CD7-CD4+Tcells(Fig5B).
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