Unique Immunological Profile In Patients With COVID-19

Abstract

The relationship between SARS-CoV-2 and host immunity is unknown. We show here that patients with COVID-19 had an altered immune phenotype, with an expansion of adaptive FceRIg neg NK cells, and inflammatory CD14 ⁺ CD16 ⁺ monocytes. T cells were reduced and overexpressed the Tim-3 exhaustion molecule. Low frequencies of CD8 T cells and NKG2A ⁺ NK cells, and expansion of mature CD57 ⁺ NK cells were associated with poor prognosis. These findings unveil a unique immunological profile in COVID-19 patients.

Full text

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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.
UniqueImmunologicalProfileInPatientsWithCOVID-
19
CURRENTSTATUS:UND ERREVI EW
StefaniaVarchetta
FondazioneIRCCSPoliclinicoSanMatteo
DalilaMele
FondazioneIRCCSPoliclinicoSanMatteo
BarbaraOliviero
FondazioneIRCCSPoliclinicoSanMatteo
StefaniaMantovani
FondazioneIRCCSPoliclinicoSanMatteo
SerenaLudovisi
FondazioneIRCCSPoliclinicoSanMatteo
AntonellaCerino
FondazioneIRCCSPoliclinicoSanMatteo
MarcoVecchia
FondazioneIRCCSPoliclinicoSanMatteo
SilviaRoda
FondazioneIRCCSPoliclinicoSanMatteo
MicheleSachs
FondazioneIRCCSPoliclinicoSanMatteo
RaffaeleBruno
FondazioneIRCCSPoliclinicoSanMatteo
MarioU.Mondelli
FondazioneIRCCSPoliclinicosanMatteo
mario.mondelli@unipv.itCorrespondingAuthor

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DOI:
10.21203/rs.3.rs-23953/v1
SUBJECTAREAS
Immunology InfectiousDiseases
KEYWORDS
SARS-CoV-2,hostimmunity,alteredimmunephenotypeinpatients

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Abstract
TherelationshipbetweenSARS-CoV-2andhostimmunityisunknown.Weshowherethatpatients
withCOVID-19hadanalteredimmunephenotype,withanexpansionofadaptiveFceRIgnegNKcells,
andinflammatoryCD14+CD16+monocytes.TcellswerereducedandoverexpressedtheTim-3
exhaustionmolecule.LowfrequenciesofCD8TcellsandNKG2A+NKcells,andexpansionofmature
CD57+NKcellswereassociatedwithpoorprognosis.Thesefindingsunveilauniqueimmunological
profileinCOVID-19patients.
Introduction,ResultsAndDiscussion
SevereAcuteRespiratorySyndromeCoronavirus-2(SARS-CoV-2)isresponsibleforapandemicthus
farresponsiblefornearly1millioncasesofCoronavirusDisease-19(COVID-19)withacase/fatality
rateof4.5%[1].Theinfectionusuallycausesmildsymptoms,butmayberesponsibleforsevere
interstitialpneumonia,myocarditis,acutekidneyinjury,acuterespiratorydistresssyndrome(ARDS),
multiorganfailureanddeath[2].Laboratorytestsindicatethatpatientswithsevereprogressionof
COVID-19showsignsofsecondaryhaemophagocyticlymphohistiocytosis(sHLH),a
hyperinflammatorysyndromecharacterisedbyapotentiallyfatalcytokinestormwithmultiorgan
failure,whichmaybetriggeredbyviralinfections[3].AkintosHLH,COVID-19ischaracterizedby
lymphopenia,andincreasedserumferritin,D-dimer,C-reactiveprotein(CRP),andlactic-
dehydrogenase(LDH),whicharealsoconsideredpredictorsofpooroutcome[4].
Moreover,severalserumcytokineconcentrationsareincreasedduringCOVID-19,supportingthe
hypothesisthatvirallydrivenhyperinflammationplaysakeypathogeneticrole[2].
Despiteclearevidenceofongoingoverexuberantinflammation,therearenosystematicstudies
addressingphenotypicandfunctionalalterationsofinnateandadaptiveimmunecells,thatarelikely
exposedtoavarietyofstimuliinCOVID-19patientsatpresentation.Thelackofacomprehensive
immunologicalanalysispromptedustoassessthephenotypicandfunctionalstatusofNKcells,γδT
cells,monocytesandCD4andCD8Tcellsinpatientspresentingwithclinicallymoderatetosevere
interstitialpneumoniaemerginginthesettingofCOVID-19.Patientclinicaldetailsandlaboratory
findings,aswellasperipheralbloodmononuclearcells(PBMC)flowcytometricanalysisarereported

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inSupplementaryInformation,PatientsandMethods.
ThefrequencyofNKcellswassignificantlyhigherinCOVID-19patientscomparedtohealthycontrols,
beingsignificantlyenrichedinmature(CD56dimCD57+)NKcells(Fig.1a).Interestingly,therewasa
relativeexpansionofCD57+/FcεRIγnegadaptiveNKcellscomparedwithnon-COVID-19disease
controlsandhealthycontrols(Fig.1a)suggestingaSARS-CoV-2-relatedexpansionofthispopulation,
whereastheproportionofCD56brightNKcellswasreduced.AnincreaseinCD16+NKcellswasalso
evidentcomparedwithhealthycontrols(Fig1a).Notably,thefrequencyofCXCR6-expressingNKcells
waslowinCOVID-19patients(Fig.1a),mostlikelysincethesecellshometothelungswherethey
concentrate,theirligandCXCL16beingproducedinlargeamountsbyalveolarmacrophages[5].
AdditionalchangesinNKcellsincludedsignificantreductionsinthefrequenciesofSiglec-7,DNAM-1,
NKG2D,NKp30(Fig.1a),thelatterbeingparticularlyevidentintheadaptivesubset(Fig.1c).
Importantly,thefrequencyofPD-1positiveNKcellswassignificantlyhigherintheadaptivecompared
withconventionalNKcellsinpatientswithCOVID-19(Fig.1c).NochangeswerenotedinbulkNKcell
expressionofNKG2C,NKG2A,GITR,TRAIL,CD69,PD-1,TIGIT.ThetrendnotedforTIM-3wasnot
statisticallysignificant(Suppl.Fig.1a).Ofnote,althoughnosignificantchangesindegranulation
activityorIFNγproductionwereobservedusingK562astargetcells(Suppl.Fig.1b),therewasan
increasedabilityofNKcellstoexertantibody-dependentcell-mediatedcytotoxicity(ADCC),a
functionexquisitelyperformedbyadaptiveNKcells[6](Fig.1d).TheproportionsofCD56bright,
NKG2AandNKp46positiveNKcellsweresignificantlylowerandtheproportionofmatureCD57+cells
significantlyhigherinpatientswhosuccumbedcomparedwiththosewhosurvived(Fig.2a).The
relativefrequenciesoftotalCD3+,CD4+andCD8+Tcellsweresignificantlylowerthanhealthy
controls,althoughnoapparentdifferenceswerenotedwithdiseasecontrols(Fig.2b).Patientswho
diedshowedasignificantlylowerfrequencyofCD8Tcellscomparedwiththosewhosurvived(Fig.2a).
Moreover,bothCD4andCD8TcellsfromCOVID-19patientsoverexpressedTim-3comparedwith
healthycontrols,suggestingapanT-cellexhaustionprofile(Fig.2c).Nodifferenceswerefoundin
CD45RO,HLA-DR,GITRexpressionorTregpopulationfrequency(Suppl.Fig.2).Importantly,therewas

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aclearrelativeexpansionofCD14/CD16doublepositivemonocytes,aphenotypeassociatedwithan
inflammatoryprofile(Fig.2d)[7].Therewerenostatisticallysignificantchangesinthefrequencyof
γδTcells(Fig.2e).Negativecorrelationswerefoundbetweenlaboratoryindicatorsofsevereor
progressivedisease.Thus,NKcellsexpressingtheactivatingreceptorsNKp30andNKp46,aswellas
CD45RO+andTim-3+CD4Tcells,correlatednegativelywithLDH(Fig.2f).Anegativecorrelationwas
alsopresentbetweenNKp46+NKcellsandCRP(Fig.2g).
InformationonPBMCphenotypeandfunctionarevirtuallylackinginpatientswithCOVID-19.Herewe
hadtheopportunitytoevaluatepatientsadmittedtohospitalbecauseofmoderatetosevereCOVID-
19interstitialpneumoniaandcomparedthemtoasmallgroupofSARS-CoV-2negativepneumonia
andhealthyWeshowedthatpatientswithCOVID-19hadarelativeexpansionofmatureadaptiveNK
cellsendowedwithADCCfunction,whichwasincreasedinthissettinginlinewithfindingsinother
viralinfections,particularlycytomegalovirus[6].Otherphenotypicfeatureswerecompatiblewitha
dysfunctionalNKcellphenotype,namelythereducedfrequenciesofSiglec-7-,NKG2D-andNKp30-
expressingcells[8,9].Arecentstudyaddressedthekineticsandbreadthofimmuneresponses
associatedwithclinicalresolutionofCOVID-19inasinglepatientwithrelativelymilddisease[10].
Antibody-secretingcellsappearedatthetimeofviralclearancetogetherwithfollicularhelperTcells
andactivatedCD4andCD8Tcells.Incontrast,inourpatientswithmoderatetosevereinterstitial
pneumonia,someofwhomsadlysuccumbed,Tim-3positiveexhaustedCD4andCD8Tcellslargely
prevailedatpresentationandlowerfrequenciesofCD8+Tcellswerelinkedtopoorprognosis.A
recentstudyfoundlowerfrequenciesofCD8TcellsandNKcellswitharelativeenrichmentofNKG2A-
expressingcellswhichreturnedtonormalafterclinicalrecovery,suggestingrescueofimpairedTand
NKcellfunction[11].Interestingly,althoughnodifferenceinthefrequencyofNKG2A-expressingNK
cellswasfoundbetweenpatientsandcontrolsinthepresentstudy,NKG2A+NKcellswerelowerin
patientswhodidnotsurvive,suggestingthatlossofthisinhibitoryreceptorsomehowunleashedNK
cellsinpatientswithfataloutcome.
OurstudyprovidesimportantnovelinsightsintothepathogeneticmechanismsofCOVID-19,

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characterizedbyarapidexpansionofphenotypicallymatureNKcellspersistingathighfrequencyin
patientwithpoorprognosis.ThesimultaneouslyreducedfrequencyofCD4+andCD8+Tcells
expressingtheTim-3exhaustionmarkerunveilsamultifacetedbehaviorofthetwoarmsofimmunity
inthisclinicalsetting.Therelativeenrichmentofinflammatorymonocyteslendssupporttothe
hypothesisthatCOVID-19resemblesinparttothemacrophage-activationsyndromewhichisthought
tobecloselyrelatedtohemophagocyticlymphohistiocytosis(HLH)[12],anuncommonlife-
threateningdisorderofseverehyperinflammationcausedbyuncontrolledproliferationofactivated
lymphocytesandmacrophagesthatsecretehighlevelsofinflammatorycytokines.Ofnotepatterns
similartocytokinestormsyndromeshavebeendescribedforCOVID-19andSARS[2].
ItisdifficultatthisearlystagetopreciselyframeCOVID-19withinanimmunologicallycoherent
clinicalentity.Indeed,severalpeculiaritieshaveemergedthatcontributetotheuniquenessofits
immuneprofile.UnderstandingthedynamicsandthequalityofimmuneresponsestoSARS-CoV-2will
provideinvaluabletranslationalinformationtodesigneffectivetreatmentsforthispotentiallydeadly
disease.
Methods
Providedassupplementalfile.
Declarations
AuthorContributions:SVandDLdesignedandperformedexperimentsandcriticallycontributedto
draftingthemanuscript;BO,SM,ACperformedexperimentsandcriticallyreadthemanuscript;SL,
MV,SR,MS,RBrecruitedpatients,preparedthedatabaseandcriticallyreadthemanuscript,MUM
designedanddiscussedtheexperimentsandwrotethemanuscript.
CompetingInterests:none.
Ethics:Thestudyprotocolconformedtotheethicalguidelinesofthe1975DeclarationofHelsinkiand
wasapprovedbytheInstitutionalReviewBoardandEthicalCommitteeofFondazioneIRCCS
PoliclinicoSanMatteo(Protocolnumber20200033215).Allpatientsprovidedwrittenor,incasethey
wereunabletosign,verballywitnessedinformedconsentaspertheabovestudyprotocol.
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9.  V i t a le ,etal.AnHistoricalO v e r v i e w :  T h e D iscoveryofHowN K  C e l l s  C a n  K i l l  E nemies,
RecruitDefenseTro o p s ,  a n d  M ore.Front.Immuno l .  10,1415(2019).
10.  T h e v arajan,etal.Breadth  o f  c o n c o m it a ntimmunerespon s e s  p r i o r  t o  p atient
recovery:acaser e p o r t  o f  n o n - severeCOVID-19.Na t .  M e d .
https://doi.org/10.1 0 3 8 / s 4 1 5 9 1-020-0819-2(20 2 0 ) .
11.  Z h e n g,M.etal.Functiona l  e x h a u s t i o n  o fantivirallymphocyte s  i n  C O V I D - 1 9 patients.
Mol.Immunol.https : / / d o i . o r g / 1 0 .1 038/s41423-02 0 - 0 4 0 2 - 2  ( 2 020).
12.  F i s m an,N.Hemophago c y t i c  s y n d r o mesandinfection.E m e r g i n g  I n f e ct.Dis.6,601–8
(2000).

8
Covid19IrccsSanMatteoPaviaTaskForce
IDStaff
RaffaeleBruno,MarioUMondelli,EnricoBrunetti,AngelaDiMatteo,ElenaSeminari,LauraMaiocchi,
ValentinaZuccaro,LaylaPagnucco,BiancaMariani,SerenaLudovisi,RaffaellaLissandrin,AldoParisi,
PaoloSacchi,SavinoFAPatruno,GiuseppeMichelone,RobertoGulminetti,DomenicoZanaboni,
StefanoNovati,RenatoMaserati,PaoloOrsolini,MarcoVecchia.
IDResidents
MarcoSciarra,ErikaAsperges,MartaColaneri,AlessandroDiFilippo,MargheritaSambo,Simona
Biscarini,MatteoLupi,SilviaRoda,TeresaChiaraPieri,IlariaGallazzi,MicheleSachs,PietroValsecchi.
EmergencyCareUnit
ECUStaff:StefanoPerlini,ClaudiaAlfano,MarcoBonzano,FedericaBriganti,GiuseppeCrescenzi,
AnnaGiuliaFalchi,RobertaGuarnone,BarbaraGuglielmana,ElenaMaggi,IlariaMartino,Pietro
Pettenazza,SerenaPiolidiMarco,FedericaQuaglia,AnnaSabena,FrancescoSalinaro,Francesco
Speciale,IlariaZunino.
ECUResidents:MarziaDeLorenzo,GianmarcoSecco,LorenzoDimitry,GiovanniCappa,IgorMaisak,
BenedettaChiodi,MassimilianoSciarrini,BrunoBarcella,FlaviaResta,LucaMoroni,GiuliaVezzoni,
LorenzoScattaglia,ElisaBoscolo,CaterinaZattera,TassiMicheleFidel,CapozzaVincenzo,Damiano
Vignaroli,MarcoBazzini.
IntensiveCareUnit
GiorgioIotti,FrancescoMojoli,MirkoBelliato,LucianoPerotti,SilviaMongodi,GuidoTavazzi
PaediatricUnit
GianluigiMarseglia,AmeliaLicari,IlariaBrambilla
VirologyStaff
BarbariniDaniela,BrunoAntonella,CambieriPatrizia,CampaniniGiulia,ComolliGiuditta,Corbella
Marta,DaturiRossana,FurioneMilena,MarianiBianca,MaseratiRoberta,MonzilloEnza,Paolucci
Stefania,PareaMaurizio,PercivalleElena,PirallaAntonio,RovidaFrancesca,SarasiniAntonella,
ZavattoniMaurizio.

9
VirologyTechnicalstaff
AdzasehounGuy,BellottiLaura,CabanoErmanna,CasaliGiuliana,DossenaLuca,FriscoGabriella,
GarbagnoliGabriella,GirelloAlessia,LandiniViviana,LucchelliClaudia,MaliardiValentina,Pezzaia
Simona,PremoliMarta.
VirologyResidents
BonettiAlice,CanevaGiacomo,CassanitiIrene,CorcioneAlfonso,DiMartinoRaffella,DiNapoli
Annapia,FerrariAlessandro,FerrariGuglielmo,FiorinaLoretta,GiardinaFederica,MercatoAlessandra,
NovazziFederica,RatanoGiacomo,RossiBeatrice,SciabicaIreneMaria,TallaritaMonica,Vecchio
NepitaEdoardo.
ResearchLaboratories,DivisionofInfectiousDiseasesandImmunology
AntonellaCerino,StefaniaVarchetta,BarbaraOliviero,StefaniaMantovani,DalilaMele.
PharmacyUnit
MonicaCalvi,MichelaTizzoni
HospitalManagement
CarloNicora,AntonioTriarico,VincenzoPetronella,CarloMarena,AlbaMuzzi,PaoloLago
DataUnit
FrancescoComandatore,GherardBatistiBissignandi,StefanoGaiarsa,MarcoRettani,ClaudioBandi
Figures

10

11
Figure1
NKcellcharacterizationinSARS-CoV-2infection.a)FrequencyofNKcellsandexpansionof
matureCD57+andadaptive(FcεRIγneg)NKcellsinCOVID-19patients.Reducedfrequency
ofCXCR6,Siglec-7,NKG2DandNKp30,andincreasedproportionofCD16+cells.b)Dotplot
showinggatingonCD57+FcεRIγnegadaptiveandCD57+FcεRIγposconventionalNKcells.c)
RepresentativedotplotsandgraphsshowingNKp30reductionandPD1increaseinadaptive
comparedwithconventionalNKcellsinCOVID-19patients.Representativedotplotsare
gatedontotalCD57+NKcells.CirclesindicateadaptiveNK;squares,conventionalNK.d)
IncreasedNKdegranulationandIFNγexpressioninCOVID-19patients.e)Representative
IFNγandCD107adotplotsinpatientsandcontrols.Fullredsymbolsindicatepatientswho
subsequentlydied.Middlebarsrepresentmedians.TheOneWayAnovatestwasusedto
comparethreegroups.*p<0.05,**p<0.01,***p<0.001.

12
Figure2

13
a)ExpansionofmatureCD57+NKcellsandreductionofCD56brightNKcells,NKG2A+and
NKp46+NKcellsandCD8Tcellsinpatientswhosurvivedandinthosewhosuccumbed.b)
FrequenciesoftotalCD3+,CD4+andCD8+TcellswerereducedinCOVID-19patients
comparedtoHD.c)Tim-3expressingCD4andCD8TcellswereincreasedinCOVID-19
patients.d)ExpansionofCD14+CD16+doublepositivemonocytesinCOVID-19patients
andrepresentativedotplots.e)NodifferenceswereobservedintheproportionofγδTcells.
f&g)CorrelationsofNKandCD4TreceptormoleculeswithLDHandCRP.Middlebars
representmedianvalues.TheMann-Whitneytestwasusedtocomparesurvivorsversus
deadpatients.TheOneWayAnovatestwasusedtocomparethreegroups.ThePearson
testwasusedtoexaminecorrelations.*p<0.05,**p<0.01.
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