Deciphering the tcr repertoire to solve the covid-19 mystery lucas gutierrez & john beckford & houda

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TrendsinPharmacologicalSciences

DecipheringtheTCRRepertoiretoSolvethe COVID-19Mystery

Thesevereacuterespiratorysyndromecoronavirus2(SARS-CoV-2)hasinfected severalmillionsandkilledmorethanquarterofamillionworldwidetodate.Importantquestionshaveremainedunanswered:whysomepatientsdevelopseveredisease,whileothersdonot;andwhatrolesdogeneticvariabilitiesplayin theindividualimmuneresponsetothisviralinfection.Here,wediscussthecriticalroleTcellsplayintheorchestrationoftheantiviralresponseunderlyingthe pathogenesisofthedisease,COVID-19.Wehighlightthescienti ficrationalefor comprehensiveandlongitudinalTCRanalysesinCOVID-19andreasonthatanalyzingTCRrepertoireinCOVID-19patientswouldrevealimportant findingsthat mayexplaintheoutcomedisparityobservedinthesepatients.Finally,weprovideaframeworkdescribingthediffere ntstrategies,theadvantages,andthe challengesinvolvedinobtainingusefulTCRrepertoiredatatoadvanceour fightagainstCOVID-19.

TheCOVID-19ImmuneConundrum

SinceitsemergenceinDecemberof2019,the severeacuterespiratorysyndromecoronavirus2(SARS-CoV-2) (see Glossary)hasinfectedmorethan7millionpeopleandkilledmore thanaquartermillionpeopleworldwide.IntheUS,atleast2millionindividualsareconfirmedpositiveforthe coronavirusdisease2019(COVID-19) andthedeathtollexceeds100000people. TheclinicalmanifestationsofpatientswithSARS-CoV-2infectionrangefromasymptomaticto mildorsevererespiratorysymptomaticdiseasewithhighviralload[1,2].Thecurrentcoronavirus infectsallages,yettheseverityofthediseaseisdifferentamongtheagegroups.Approximately 15%oftheconfirmedcasesaresevere,ofwhomthemajorityareeitherover65yearsofageor patientswithunderlyingmedicalconditions[3].

Aswithanyvirus,theadaptiveimmuneresponseplaysacentralroleinclearingSARS-CoV-2infectionsanddirectlyinfluencespatients’ clinicaloutcome.Aproperandspecificadaptiveimmune responsecansuccessfullyeliminatethevirus,whichlikelyoccursintheasymptomaticandmild cases,wherehavinganappropri ategeneticbackground[e.g., humanleukocyteantigen (HLA)]thatelicitsspecificantiviralimmunity,orboostingtheimmuneresponseswiththerapeutic strategies,maysupportthedevelopmentofaprotectiveimmuneresponse.However,insevere cases,animpairedimmuneresponseleadstot heviralinfectionrunninguncheckedandthe highlyproliferativeviruscausingdestructionof theinfectedtissues,particularlyinthelungs, wheretheexpressionof angiotensin-convertingenzyme2 (ACE2),thereceptorrequiredby thevirustoenterthecells,ishigh[4,5].Thetissuedamageinducesinnateimmuneandinflammatoryresponseinthelungs,mediatedbyproinflammatory macrophages andgranulocytes,often causingthelife-threateningacuterespiratoryfailureseenintheseverecases[1,6].

SeveralimmunecharacteristicshaveemergedfromearlyWuhancasesandwerefurtherconfi rmedincasesinItalyandinNewYorkhospitals,showingthatpatientswithsevereinfections

Highlights

TheCOVID-19pandemicrevealed vulnerabilityinelderlypopulationand underscoredtheroleoftheaging immunesysteminthe fightagainst emerginginfections.

Tcellsarecrucialmediatorsofantiviral adaptiveimmunityanddelineatingthe roleoftheTCRrepertoireinthecontext ofSARS-CoV-2meritsresearchefforts.

TheTCRrepertoiredynamics,composition,anddiversityareinfluencedby severalintrinsicandextrinsicfactors suchasaging,HLAgeneticvariabilities, viralinfection,etc.

ThearchitectureoftheTCRrepertoire largelycontributestotheperformance oftheadaptiveimmuneresponse againstSARS-CoV-2.

Advancesinsequencingtechnologies andsingle-cellimmuneprofilingcanbe leveragedtomonitoradaptiveimmune responsesinCOVID-19patientsand guidefutureSARS-CoV-2immunotherapyandbiomarkerdevelopment.

1DepartmentofClinicalPharmacy, SchoolofPharmacy,Universityof SouthernCalifornia,LosAngeles,CA 90089-9121,USA

*Correspondence: alachkar@usc.edu (H.Alachkar).

TrendsinPharmacologicalSciences,Month2020,Vol.xx,No.xx https://doi.org/10.1016/j.tips.2020.06.001 1 ©2020ElsevierLtd.Allrightsreserved.

TrendsinPharmacologicalSciences

exhibitincreasedseruminterleukin-6(IL-6),tumornecrosisfactor-α (TNF-α),andc-reactiveprotein.Inaddition,patientswithseverediseasedisplayhighlevelsofneutrophilscombinedwitha signi fi cantdecreaseintotal lymphocytes [ 7 ].Theseproin fl ammatoryimmunefeatureswere alsocorrelatedwithdiseaseseverityanddeath[8–10].Recentstudyhascharacterizedbronchoalveolarlavage fl uidimmunecellsfrompatientswithva ryingseverityofCOVID-19andfrom healthypeoplebyusingsingle-cellRNAsequencing.Thebronchoalveolarlavage fluidfrompatientswithsevereCOVID-9exhibitedhighproin fl ammatorymonocyte-derivedmacrophages, whilemoderatecaseswerecharacterizedbythepresenceofhighlyclonallyexpanded CD8+ T cells [11].Yet,manyimportantquestionsremainedunanswered:whysomepatientsdevelopseveredisease,whileothersdonot;andhowgeneticvariabilitieswithintheimmunesystemimpact individualantiviralimmuneresponse.

TheRoleofTCellsintheImmuneResponseagainstSARS-CoV-2 Respiratoryviruseshaveevolvedinmanywaystoevadeorneutralize innateimmunity;therefore, adaptiveimmunity ,whichiscomprisedofBand Tcells ,mustalsobecomeactivated forproperresponse.Bcellsarederivedfrom thebonemarrowandproduceantibodiesthat bindtovirusparticlesinthebloodandmucosalsurfaces,preventingthespreadoftheinfection (humoralmediatedantiviralresponse).Thus,thereisanimmenseresearchinterestintheidentifi cationandtheuseoftheantibodiesdevelopedagainsttheSARS-CoV-2asabiomarkerfor thedevelopmentofindividualimmunityorfortherapeuticpurposesasintheconvalescentplasma therapy[12,13].Therearealsosubstantialongoingeffortsplacedtodevelopserologicalteststo accuratelymeasurethelevelsofantiviralantibodiesinanindividual[14].Thisshouldhelptheepidemiologicalassessmentofthescaleoftheviralspreadandallowcountriestoplanforending lockdownsandthereturntonormallife.However,currentchallengeswiththesensitivityand specificityforsomeoftheseassaysarestilllimitingtheirwideuse.

Tcells,however,matureinthethymus,and CD8+ Tcells killvirus-infectedcells(cell-mediated antiviralresponse)through Tcellreceptor(TCR)-mediatedrecognitionofviral antigens.Similar tothehumoralmediatedresponse,Tcellsarealsoessentialforantiviraldefense.Here,wefocus ontheroleofTcell(cell-mediated)responsesandthe TCRrepertoire ,astheyareoften overlookedinearlyviralstudies.Tcellsplayacrucialroleintheimmuneresponsetoviralinfectionsduetotheirabilitytoselectivelyeliminatevirus-infectedcells[15].CD8+ Tcellsrecognize viralantigensthroughtheirpresentationby majorhistocompatibilityclassI(MHCI) molecules onvirallyinfectedcellsaswellasonuninfected dendriticcells andmacrophagesinthelymph nodes(aprocessknownascross-presentation)(Figure1)[16].OnceTcellsbecomeactivated throughantigenrecognition,theyundergoaprocesstermed ‘clonalexpansion’,inwhichthe activatedTcellrapidlyproliferatestogeneratealargenumberofTcel lswithidenticalTCRs andthus,identicalantigenrecognition[17].Toeliminatevirus-infectedcells,expandedCD8+ T cellclones,withTCRsspecificforviralantigens,directlylysethembyperforin/granzymerelease, induceapoptosisthroughtumornecrosisfactor-relatedapoptosis-inducingligand(TRAIL)orFas ligand,andsecreteproin fl ammatorymediatorssuchas interferon-γ (IFN-γ) [18](Figure1).T cellsarecrucialmediatorsofantiviraladaptiveimmunityanddelineatingtheroleoftheTCRin thecontextofSARS-CoV-2shouldgarnervastinterest.

TheEvolutionoftheTCRRepertoireduringViralInfection

ThespecificityofTcellstowardviralantigenspresentedbyallnucleatedcellsonMHCsisdeterminedbytheirTCR[19–21].Roughly95%oftheTCRsoncirculatingTcellsarealpha(α)andbeta (β)subunitheterodimers.TCRspecificityforanygivenantigenisshapedthrough V(D)Jrecombination (rearrangement)[22].Duringrecombination,VandJ(TCRalpha)orV,D,andJ(TCR beta)genesegmentsarerandomlyselectedfromanarrayofVandJexons[23].Theyundergo

Glossary

Adaptiveimmunity: immunitythat occursinresponsetospecificantigens. Activationofthisimmunityisdependent onthepresentationofantigensbythe innateimmunesystem.Itismediatedby immunoglobulinsandTcells.

Angiotensin-convertingenzyme2 (ACE2): acarboxypeptidasethatis mainlyexpressedinvascularendothelial cellsandtherenaltubularepithelium.

ACE2bindstoSARS-CoV-2spike proteinandpromotesinternalizationof thevirusintothecells.

Antigens: moleculethatiscapableof stimulatinganimmuneresponse, specificallyactivatingTcellsorBcells.

CD8+ Tcells: TcellsthatexpressCD8 ontheircellsurfaceareselectedinthe thymustorecognizeandrespondto foreignpeptidesthatarepresentedin thegrooveofMHCclassImolecules. AlsoknownascytotoxicTcells.

Clonalexpansion: theprocessin whichasmallnumberofprecursorcells thatrecognizeaspecificantigen proliferateintoexpandedclones, differentiate,andacquirevariouseffector andmemoryphenotypes,which promoteeffectiveimmuneresponses.

Clonotypes: termusedtodescribea uniquenucleotidesequencethatarises duringthegenerearrangementprocess fortheTorBcellreceptor.

Complementarity-determiningregion3(CDR3): antigenbindingregion withinTCRsthatareencodedbythe regionspanningtheVandJforthe alphasubunitortheV,D,andJ junctionsforthebetasubunitofthe TCR.ThediversityoftheCDR3loops issigni fi cantlyincreasedbecauseof theadditionandlossofnucleotides duringthejoiningprocess.TheCDR3 regiondeterminesantigenspeci fi city ofTCRs.

Coronavirusdisease(COVID-19): infectiousdiseasecausedbySARSCoV-2virusandistheofficialnamegiven bytheWorldHealthOrganizationtothe diseasecausedbythisnewlyidentified coronavirus.

Cytomegalovirus(CMV): a betaherpesvirus,representsthemajor infectiouscauseofbirthdefects,aswell asanimportantpathogenfor immunocompromisedindividuals.The viralnucleocapsidcontainingalinear double-strandedDNAof230kbis surroundedbyaproteinaceous tegumentthatisenclosedbyalipid bilayer.Establisheslifelonglatent

TrendsinPharmacologicalSciences

randomadditionanddeletionofnucleotidestoformthe fi nalmatureTCRgene[24](Figure2). Thisprocesscangenerateupto10 15 uniqueTCRs[ 25 , 26 ].Theantigenspeci fi cityofeach TCRliesinits complementarity-determiningregion3(CDR3),whichislocatedattheV(D)J junction.Asaresult,eachuniqueTcellcloneisidentifiedbyitsuniqueCDR3regionand,therefore,uniqueantigenspecificity.TheTCRrecombinationprocessgeneratesadiverseTCRrepertoirecapableofrecognizingawiderangeofantigensandprovidingpotentantiviralimmunity[27]. ThediversityandthecompositionoftheTCRrepertoirechangesinresponsetoacuteand chronicinfection,aging,cancer,andmanyotherexternalandinternalforces,makingtherepertoirehighlydynamic.ExposuretoantigentriggersmassiveexpansionofTcellscapableofrecognizingtheparticularantigen(antigen-speci fi cTcells),leadingtoskewingoftheTCRrepertoire (TCRbias)tofavorantigen-specificTcells[28–30].

EvidenceforTCRbiasinantiviralimmunityisdemonstratedformanyviralinfections,suchasthe Epstein-Barrvirus(EBV) and influenza.TCRanalysisofperipheralbloodlymphocytesfrom eitherEBV-positiveorinfluenza-positiveindividualshasrevealedseveralTcell clonotypes with recurrentTCRmotifssharedbyindividuals[31–33].Inaddition,theexpansionandmaintenance ofTCRsduringchronic cytomegalovirus(CMV) infectiondemonstratesthedynamicandevolvingnatureoftheTCRrepertoireinresponsetoviralinfection[34].Therefore,itstandstoreason thatthepresenceofabiasexistsintheTCRrepertoireasaresultoftheSARS-CoV-2infection.

InterindividualVariabilityintheTCRRepertoireInfluencingTheirImmune Response

ThecharacteristicsoftheTCRrepertoire,suchasdiversity,composition,anddynamics,arecontrolledbybothgeneticandepigeneticfactors,leadingtointerindividualvariability.HerewediscusstwofactorsthatwebelievearerelevanttoCOVID-19andtheobservedimmuneresponse disparitiesamongpatients.

AgingoftheTCRRepertoire

ItiswellknownthatTCRrepertoirediversitydeclineswithaging.ThisdecreaseinTCRdiversityas weagehasalsobeendemonstratedinvariousstudiesusing high-throughputsequencing [TCRsequencing(TCR-seq)] technology.OnestudyestimatedtheTCRβ diversityin naïve Tcell repertoireswas60–120millionforindividualsinthe firsttwodecadesoflifeanddeclined to8–57millioninindividualsover70yearsold[35].TheevidenceofagingTCRrepertoireisdemonstratedintheantiviralresponseagainstthehumaninfluenzaAvirus[39,40].ThismaybeofparticularinterestinthecontextofCOVID-19,consideringthatthemortalityofelderlypatientswith COVID-19ishigherthanthatofyoungandmiddle-agedpatients[3,36–38].Whethertheaged andlessdiverseTCRrepertoireimpactstheabilitytogenerateasufficientlyrobustTcellresponse againstSARS-CoV-2inolderpatientsremainstobestudied.

GeneticVariabilitiesintheHLA

GeneticdifferencesintheHLAgenesarewellknowntoinfluencethecompositionofTCRrepertoirebyaffectingintra-andextrathymicclonalselection,partiallyaccountingforindividualvariationsintheimmuneresponsetopathogens[ 41 – 43 ].ThesegeneticdifferencesintheHLA genesdirectlyaffectthebindingaffinityofMHCI/IImoleculestoantigensand,consequently,influenceTcellrecognitionofpathogen-derivedantigens[44–47].Onestudyconductedacomprehensive insilico analysisofviralpeptide–MHCclassIbindingaffinityacrossoverahundredHLA -A,-B,and-CgenotypesforallSARS-CoV-2peptides.ItshowedthatHLA-B*46:01hadthe fewestpredictedbindingpeptidesforSARS-CoV-2,suggestinghighervulnerabilitytoCOVID19amongindividualswiththisallele[48].ThesameallelewaspreviouslyassociatedwithsusceptibilitytoSARS[49].Incontrast,HLA-B*15:03wasfoundtohavethegreatestcapacitytopresent

infectionandisreactivatedby immunosuppression.

Dendriticcells: professionalantigenpresentingcells,uniquelyabletoinduce naïveTcellactivationandeffector differentiation.

Epstein-Barrvirus(EBV): ahuman herpesvirus4withalargedoublestrandedDNAgenomethatbelongsto the γ-herpesviridaesubfamily.EBVwas the firsttumorvirusidentifiedinhumans. Thevirusisprimarilyassociatedwith lymphomasandepithelialcellcancers andisthecausativeagentof mononucleosisafterinfection. High-throughputsequencing: technologycapableofsequencing multipleDNAmoleculesinparallelto enablemillionsofDNAmoleculestobe sequencedatonce.Thisismuch quickerandcheaperthanthepreviously usedSangersequencingand,assuch, revolutionizedthestudyofgenomics andmolecularbiology.

Humanleukocyteantigen(HLA): geneclusterthatencodesthemajor histocompatibilitycomplexproteins, whichpresentantigenpeptidestothe hostimmunesystem.Theseareamong themostpolymorphicgenesinthe humangenome.

Influenza: aviruscommonlyknown asthe ‘ fl u’ thatattacksthe respiratorysystem.In fl uenzaviruses aremembersofthefamily Orthomyxoviridae.Thisfamily representsenvelopedviruses,the genomeofwhichconsistsof segmentednegative-sensesinglestrandRNAsegments.Thereare fourgeneraofthisfamily:typesA, B,C,and Thogotovirus ,ofwhich, however,onlygeneraAandBare clinicallyrelevantforhumans.

Innateimmunity: animmunological subsystemthatcomprisesthecellsand mechanismsthatprovidethe firstlineof defensefrominfectioninanonspecific manner.Itdetectsandrespondsto pathogenassociatedmolecularpatterns (PAMPS)thatarenotnaturallyoccurring withintheentireorganism.

Interferon-γ (IFN-γ): cytokinethathas sincebeencharacterizedasa homodimericglycoproteinwith pleiotropicimmunologicfunctions.IFN-γ isprimarilysecretedbyactivatedTcells andnaturalkillercellsandcanpromote macrophageactivation,mediateantiviral andantibacterialimmunity,enhance antigenpresentation,orchestrate activationoftheinnateimmunesystem, coordinatelymphocyte–endothelium

TrendsinPharmacologicalSciences

highlyconservedSARS-CoV-2peptidesthataresharedamongcommonhumancoronaviruses, suggestingapotentialcross-protectiveTcellimmunity[48].TheseobservationsraisetheimportantquestionofwhichallelesconfersusceptibilityorresistancetotheSARS-CoV-2infection.

UnravelingtheTCRRepertoiretoDecodetheDynamicofSARS-CoV-2Immune Response

AprotectiveTcell-drivenimmuneresponsetounpredictedantigensrequirestheimmunesystem togenerateadiverseTCRrepertoirethatisreadyforanynovelpathogen/antigenchallenge,such asSARS-CoV-2.Therefore,thesizeandthediversityoftheTCRrepertoirearemajordeterminantsoftheimmuneresponsetoagivenantigen[50].TheTCRrepertoirecanbefurtherdivided intonaiveandmemory.ThenaiveTCRrepertoi rerepresentstherepertoireofantigeninexperiencedTcells.Thispopulationisshapedby thymopoiesis ,whichintroducesnewT cellclonesand,therefore,newantigenspeci fi cities,todiversifytheTCRrepertoire[ 51 ]. ThymopoiesiscangenerateSAR-CoV-2reactiveTcellswheretheyhavenotexistedpreviously. ThememoryTCRrepertoirerepresentstherepertoireofantigen-experiencedTcells.Itisshaped byanindividual’shistoryofantigenexposure,wherebythoseTcellsthathaveparticipatedinimmuneresponsespersistlongterm. MemoryTcells,thoughtheyhavebeenselectedforbydifferentantigens,suchasthosegeneratedagainstpriorcoronaviruses,maystillgenerate responsesagainstnovelvirusessuchastheSARS-CoV-2[52].Indeed,arecentstudyreported thatTcellreactivitytoSARS-CoV-2epitopesisalsodetectedinnonexposedindividuals,suggestingcrossreactiveTcellrecognitionbetweencirculating ‘commoncold’ coronavirusesand SARS-CoV-2[53].

Thedevelopmentoffasterandcheapersequencingtechnologies,augmentedbytheadvancesin computationaltools,supportthefeasibilityofusingTCRanalysesnotonlytotrackSARS-CoV-2speci fi cTcellexpansionpost-COVID-19infectio norinthecourseoftreatingpatientswith COVID-19,butalsotoestablishcertainfeaturesoftheTCRrepertoirearchitectureaspredictive biomarkersforpatients’ clinicaloutcome.Arecentstudydemonstratedthefeasibilityandvalue oftheseanalysesinpatientswithCOVID-19.Intenpatientswitheitherearlyrecoverystageor laterecoverystageCOVID-19and fivehealthydonors,TCR-seqanalysisshowedthatTcellexpansionwaslowerintheearlyrecoverygroupthaninthehealthycontrolgroup.Also,thenaïveor centralmemoryTcellsshowedlittleclonalexpansion,whileeffectormemoryTcells,terminaleffectorCD8+ Tcells,andproliferatingTcellsshowedhigherexpansionlevels.Thestudyfoundthat themosthighlyexpandedcloneintheearlyrecoveredgroupwasTRAV8-6-TRAJ45:TRAV7-8TRBJ2-1[54].Thus,acomprehensivecharacterizationofthedynamicsandcompositionofthe TCRrepertoirestoSARS-CoV-2infectioncanlargelycontributetotheevolvingunderstanding ofthefunctionalandmechanisticinvolvementoftheadaptiveimmunecellresponseandpotentiallyguidethedesignofeffectivetreatment.HerewesummarizesomeofthemostrelevantclinicalapplicationsofobtainingcomprehensiveTCRrepertoiredatainthesettingofCOVID-19.

TCR-BasedBiomarkerstoEvaluateHerdImmunityandAdvancetheDevelopmentofCOVID-19 PreventiveVaccines

Recenteffortshavefocusedonidentifyingsharedmotifsincoresequencestopredictconserved residuesdrivingessentialelementsofTCRrecognition[22,55].Aswithanyotherviralinfection, theabilityofindividuals’ TCRrepertoiretoeffectivelyclearSARS-CoV-2infectionishighlydependentonitscomposition.Epitope-specificTcellshavebeenidentifiedinastudyusingsamplesobtainedfromindividualspreviouslyinfectedwithSARS-CoV[56 ].Similarly,anotherstudy demonstratedthepresenceofepitope-speci fi c publicTcells inbothhealthyandin fl uenzainfectedpatients[32].ThesestudiesshowacorrelationbetweenthepresenceofspecificTcell clonesandantiviralimmunity.Beyondidentifyingspecificclones,TCRβ profilinghasdemonstrated

interaction,regulateTh1/Th2balance, andcontrolcellularproliferationand apoptosis.

Lymphocyte: whitebloodcellsthat haveimportantimmunefunctions.The mainpopulationsoflymphocytesareB cells,Tcells,andnaturalkiller(NK)cells. Macrophage: myeloidcellsthathave importantinnateimmunefunctions. Macrophagesresideintissuesand respondtoinfectionbyproducing inflammatorymediatorsandengulfing bacteria.

MajorhistocompatibilityclassI (MHCI): proteinfoundonthesurfaceof allnucleatedcellsthatfunctionsin antigenpresentationtoCD8+ Tcells. MemoryTcells: aCD8Tcellthathas respondedtocognateantigen(Ag)and persistslong-term.Thesecellscan populateperipherallyandarepoisedto immediatelyproliferate,execute cytotoxicfunctions,andsecreteeffector cytokinesuponAgre-encounterand existindifferentmetabolic, transcriptional,andepigeneticstates. NaïveTcell: aTcellpoolthatis generallyconsideredtobeafairly quiescent,homogeneouspoolof antigen-inexperiencedcells.Inresponse toencounteringandinteractingwith cognateantigensintheperiphery,naive Tcellswillproliferateanddifferentiate intodifferenttypesofeffectorand memoryTcells,whichcanmigrateto differenttissuesforlocalantigenpatrol. PublicTcells: referstoTcellsthat carryTCRsequencesthatoccurin multipleunrelatedindividuals,oftenin responsetothesameantigen.

Severeacuterespiratorysyndrome coronavirus2(SARS-CoV-2): severe acuterespiratorysyndrome(SARS)-like coronavirusthatbelongstothefamily Coronaviridaeandthesubfamily Coronavirinae.Itusestheangiotensinconvertingenzyme2(ACE2)receptorto enterintothehumancells.Itisthe causativeagentofCOVID-19.

Tcell: whitebloodcellsthatare importantforadaptiveimmunity.They haveuniquecellsurfacereceptorsthat aregeneratedbyrandomlyassorting genes.ThesereceptorsallowTcellsto senseandrespondtodiversetypesof infection.

Tcellreceptor(TCR): proteinthat recognizessmallproteinfragmentsthat aredisplayedbytheMHCmoleculeon thesurfaceofnucleatedcells.EachTcell expressesauniqueTCRthatis generatedbyrandomlyrecombined genes.ThisensuresthatTcellscan

TrendsinPharmacologicalSciences

convergentTCRβ repertoireevolutioninlargecohortsofunrelatedindividualsinfectedwithCMVor vaccinatedagainstyellowfever[57,58].AdditionalworkhasdemonstratedthatinformationobtainedfrompublicTCRsinsmallpoxvaccinatedmicecanbeusedtoidentifysmallpoxvaccinated micewith N99%accuracy[59].Also,ithasbeendemonstratedthatinfluenzaAinfectioninducesa polyclonalTcellresponseinpatients,byTCRspredominantlyutilizingTRBV19gene[60].

Collectively,thesestudiessuggestthattheTCRrepertoirechangesinresponsetoinfectionor vaccinationinapotentiallypredictablemannerandcanthereforebeleveragedtoidentifyclones againstSARS-CoV-2.Thus,TCRrepertoireprofilinginCOVID-19patientsafterrecoverymayuncoverSARS-CoV-2reactiveclonesand/orgeneralTCRrepertoirefeaturesthatdifferentiatepatientswithpriorinfectionfromthosethathaveneverbeeninfected(Figure3A).TCRrepertoire pro fi lingcombinedwithcomputationalstrategiesmayalsoenabletheidenti fi cationofSARSCoV-2-specificTCRclonesandgeneralTCRrepertoiresignaturesthatmayserveasabiomarker forherdimmunityatthepopulationlevel(Figure3B).

TCR-BasedImmunotherapyforCOVID-19

Adoptivetransferof TCRengineeredTcell(TCR-T) hasbeenappliedtotreatviralinfections suchashepatitisBandC[ 61 , 62 ].Thiswasalsodemonstratedinpreclinicalmodelsforprior coronavirusessuchasSARS;engineeredSARS-CoV-speci ficTCR-TcellsfromnormalindividualshavedemonstratedantiviralactivitythatwascomparablewiththatofnaturalSARS-CoVspeci fi cmemoryCD8+ Tcells[63].TheabilitytoidentifypotentialTCRclonesthatarespeci fi c toSARS-CoV-2antigenscanbeleveragedinthedevelopmentofhighlyspecificandadvanced formsofimmunotherapybasedonTCRstructuralandsequenceinformation.Identi fi cationof publicclonesthroughTCRrepertoirepro filingininfectedandvaccinatedindividualsmayguide thedesignoffutureTCR-Timmunotherapiesa gainstSARS-CoV-2.SARS-CoV-2reactiveT cellscanbeexpanded exvivo andinfuseddirectlyintoanindividualtoshortenrecoverytime andincreasethemagnitudeofresponse(Figure4).

TCRRepertoireChangesasaMeasureofClinicalOutcome

TCRrepertoirecompositionanddiversityaremajor determinantsofdiseaseoutcomesfollowingviral infection.MultiplestudieshaveprovidedclearevidenceindicatingahighlydiverseTCRrepertoireprovidesprotectionagainstawidearrayofantigens[22,64,65].ItispossiblethatahighlydiverseTCR repertoirecanallowfortherightamountofavidity,affinity,andoverallfunction[66,67].Similarstudies canbeperformedtotrackchangesintheTCRrepertoireinSARS-CoV-2positiveindividuals,beginningathospitalization,topreciselyidentifychangesintheTCRrepertoireandcompositionthroughoutdiseaseprogression.ByexaminingtheTCRrepertoirecompositionanddiversityinalargepatient population,beginningatearlystagesofinfection,wemaybeabletounderstandhowthesetwoparameterschangeinassociationwithimmuneresponseandoutcomesinCOVID-19patients.

StrategiesforTCRRepertoireProfilinginPatientswithCOVID-19 BiologicalSamples

Theselectionofproperbiologicalsamplesanddetermininganappropriatecollectionscheduleis crucialtopreparetheinputforTCRanalysis.Sputumsamples,whilefeasible,maynotoffersufficientdepthoftheTCRrepertoiresequencing,asthenumberofCD8+ Tcellsinsputum,relative tootherbiologicalmaterials,islow[68].Bronchoalveolarlavageandlungtissuesamplesmaybe anattractiveandinformativechoicebutacquiringsuchsamplesisdifficultforavarietyofreasons, particularlypatientwillingnessandworkersafety[69].Inaddition,theseproceduresareinvasive toperformonmildcasesandotherwisehealthyindividualsforresearchpurposes.Peripheral bloodsamplesareeasytocollectandarethemostfeasible,particularlytoexaminethedynamics oftheTCRrepertoire,asmultiplecollectionscouldbeperformed,startingfromdiagnosisand

respond(atthepopulationlevel)to almostanyinfection.

TCRbias: referstotheoverrepresentationofTCRgenesorclonesin theTCRrepertoire.ThebiasinTCR usagecanbecategorizedbasedonthe diversityinaspecificTcellpopulation. TCRbiascanbeinfluencedbymany factors,suchasthymicselectionand antigen-drivenselection,whichcan narrowtheavailableTcellrepertoireover time.

TCRengineeredTcell(TCR-T): T cellsengineeredbyviralvectorsto expressthe TCR genewithdefined specificity.Thesecellscanbemodified totargetcancercellsorbacterial/virusinfectedcells.

TCRrepertoire: thesumofallTCRsby theTcellsofoneindividualgeneratedby somaticDNArearrangements.

TCRsequencing(TCR-seq): highthroughputsequencingtechnologies usedintheexaminationofantigen receptorrepertoiresatsingle-nucleotide and,morerecently,single-cellresolution. Thymopoiesis: processthatturns thymocytesintomatureTcells accordingtoselectionprocesses.This selectionprocessisvitallyimportantin shapingthepopulationofthymocytes intoaperipheralpoolofTcellsthatare abletorespondtoforeignpathogens butremaintoleranttowardsthebody’s ownantigens.

V(D)Jrecombination: theprocessby whichTcellsrandomlyassemble differentgenesegments,knownas variable(V),diversity(D),andjoining (J)genes,inordertogenerateunique receptors(knownasT-cellreceptors) thatcancollectivelyrecognizemany differenttypesofmolecule.

TrendsinPharmacologicalSciences

Trends in Pharmacological Sciences Figure1.TCell-MediatedResponsetoSevereAcuteRespiratorySyndromeCoronavirus2(SARS-CoV-2)Infection. Infectionoccursinthepatientsafter respiratorytractexposure.Subsequently,virusentryoccursviabindingtoangiotensin-convertingenzyme2(ACE2)onthesurfaceofvariouscelltypesandbeginsviral replication.Antigen-presentingcellssuchasdendriticcellsendocytosetheSARS-CoV-2virusanddegradethemthroughaprocesscalledantigenprocessing.These antigenfragmentsarethenpresentedbyproteinstermed ‘ MHCclassmolecules’,onthecellsurfaceandallowrecognitionbyaTcell.IfaCD8 + Tcelliscapableof binding,itwillundergoclonalexpansionanddirectlytargetinfectedce llsthrougheitherperforin/granzymes,FASligand/tumornecrosisfactor -relatedapoptosisinducingligand(TRAIL)pathways,orsecretionofproin flammatorymediators.IftheCD4+ Tcelliscapableofbinding,itcanactivateBcellsthatrecognizetheantigen bycausingthemtoclonallyproliferateandsecreteantibodiestotargettheSARS-CoV-2virus.Abbreviations:BCR,Bcellreceptor;MHC,majorhistocompatibility complex;TCR,Tcellreceptor.

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Trends in Pharmacological Sciences

Figure2.SchematicDiagramoftheV(D)JSomaticRecombinationtoGenerateTCellReceptor(TCR). TheTCRis composedofthealpha(α)andbeta(β)subunit.DuringTcelldevelopment,thelocithatencodeTcellreceptor α and β-chains arerearranged.Variable(V),diversity(D),joining(J),andconstant(C)genesegmentsarerepresentedasblue,red,green,and purplerectangles,respectively.Diversityforthe β chainis firstcreatedviarecombinationofDandJsegments,followedbytheV segments,and fi nallyoneoftwoCsegments.Diversityforthe α chainiscreatedviarecombinationofVandJsegments, followedbytheoneCsegment.ThisprocessalsoinvolvesthedeletionandinsertionofnucleotidesattheV–D,D–Jjunctions forthe β chainandV–Jjunctionsforthe α chain,thusaddingmorepotentialdiversitytotheTCRrepertoire.

thenweeklytillrecoveryorevenafterwardtotrackmemoryTcellclones.SARS-CoV-2reactiveT cellswouldtransientlyappear/persistinbloodastheymigratefromlymphnodestotissuesites viaperipheralbloodcirculationandthuscouldbecapturedinperipheralbloodsamples.However,itispossiblethattheirfrequencyint hebloodmaybelow.Itisalsopossiblethatthe lymphocytopeniaobservedinCOVID-19patients(particularlyinseverecases)couldrequirea largervolumeofbloodtoobtainasufficientnumberofTcellsforcomprehensiveTCRrepertoire pro fi ling.Anotherdrawbacktousingperipheralbloodsamplesisthattheywouldnotcapture SARS-CoV-2-speci fi cTcellexpansionaswellassamplesfrominfectedtissuessuchaslung sampleswould,asSARS-CoV-2Tcellswouldtransientlypersistinperipheralbloodcirculation. Ultimately,sampleselectiona ndcollectionschedulewillvar ybasedonstudyobjectivesand

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Trends in Pharmacological Sciences

Figure3.WorkflowforIdentifyingPublicTCellReceptors(TCRs)AssociatedwithCoronavirusDisease2019 (COVID-19)toDetermineHerdImmunity. (A)PeripheralbloodsamplescollectedfromCOVID-19infectedandhealthy uninfectedindividualswillbetheinputfortheTCRrepertoireanalyses.SequencesandmotifanalyseswillidentifysharedTCR clonesthatareuniquetosevereacuterespiratorysyndromecoronavirus2(SARS-CoV-2)epitopes.(B)Predictionalgorithm generatedin(A)shouldenabletheuseofTCRsequencinganalysisofpatientwithunknowninfectionstatustodetermine SARS-CoV-2exposurehistoryinanindividual.Thisinformationcanbevaluableinthe fightagainstCOVID-19asitcanbe appliedtoobservethedegreeofherdimmunityinaparticularpopulation.

theTCRprofilinganalysisplatform,whichwouldaffectsamplecollectionandstorage(cells,RNA, orDNA).

SequencingPlatforms

EffortstocharacterizetheTCRrepertoireinhealthanddiseasehaveincreaseddramaticallyinrecentyearsduetoadvancesinsequencingtechnologies.TCR-seqhasprovidedimportantnew insightsintothehumanTCRrepertoire,suchasthemoreaccurateestimationoftherepertoire sizeandthepresenceofTCRsthataresharedb ymultipleindividuals.TCR-seqisdifferent fromstandardDNA/RNA-seqinthatlibrarypreparationisachievedthroughselectiveamplifi cationofTCRgeneproducts,allowingthecaptureoftheTCRrepertoireatloweroverall financial, reagent,anddatastoragecosts.DifferentmethodshavebeendevelopedtoanalyzetheTCRrepertoirevianextgenerationsequencingusinggenomicDNA(gDNA)andRNAasinput.Multiplex

8 TrendsinPharmacologicalSciences,Month2020,Vol.xx,No.xx

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Trends in Pharmacological Sciences

Figure4.TCellReceptor(TCR)RepertoirePro fi lingWork fl owinSevereAcuteRespiratorySyndromeCoronavirus2(SARS-CoV-2)Studiesand Applications. (A)RNA/DNAcanbeisolatedfromperipheralbloodcollectedfromSARS-CoV-2infected/vaccinatedindividuals.(B)PeripheralbloodRNA/DNAwillbe usedasaninputforTCRsequencinglibrarypreparation.(C)TCRsequencingwillbedoneusingIlluminaorothersequencingplatformsandresultingda taanalyzed usingpopularbioinformatictoolstocharacterizetheTCRrepertoireofSARS-CoV-2infected/vaccinatedindividuals.(D)Theinformationobtainedcanbeusedto advanceimmunotherapydevelopment,characterizeTCRrepertoiresinspecificpopulations,andmonitorherdimmunity.

PCR-,targetenrichment-,and5′-RACE(RNAonly)-basedapproachesarethemostwidelyused TCR-seqstrategies[71].MultiplexPCR-basedmethodsinvolvetheuseofaprimermixthatannealstoeveryVandJexonandtheconstantregion.However,preferentialamplificationofhighly abundantgeneproductscanoccurwhenusingmultiplexPCR,contributingtoinaccuraciesinreportedclonefrequencies.Additionally,intronicsequencesandincompleteVDJrecombination productsmaycontributetodatanoisewhenusinggDNAsamples.

TargetenrichmentstrategiesutilizeprobestocaptureTCRgDNA/RNAsequencesafterinitiallibrarypreparationusingcompa tiblekits(suchastheIllumi naTruSeqkitortheAgilent SureSelectXTkit)followedbyrelativelyfewcyclesofmultiplexPCRampli fi cation[72, 73 ].This methodreducestheextentofampli fi cationbiasseeninstandardmultiplexPCRbutismore labor/timeintensiveanddoesnotresolvetheissuesassociatedwithgDNAinput.The5′-RACE methodwithanRNAinputinvolvesthereversetranscriptionofTCRtranscriptsfollowedbythe additionofaknownsequenceatthe5′ endofeachcDNAmolecule.This5′ sequenceisleveraged alongwiththehighlyconservedTCR α and β constantregionduringnestedPCR(separate primersneededtoamplifyeither α or β transcripts)toachieveequallyef fi cientampli fi cationof

TrendsinPharmacologicalSciences

allTCRtranscripts,regardlessofabundance[74 ,75].Whilethe5′-RACEapproachavoidsthe PCRbiasseenintheothertwomethods,itrequirestheuseofsequencingplatformscapable oflongreads,suchastheIlluminaMiseq,asfragmentsizescanexceed600bp.

WhilebulkTCR-seq(TCR-seqofaheterogeneousTcellpopulation)viatheearlier-discussed strategiescanprovideinformationabouttheevolution,thedynamic,andthearchitectureofthe TCRrepertoireduringthecourseofSARS-CoV-2infections,itcannotbeusedtoidentifythe exactTCR α/β pairsthatparticipateinantiviralresponses(onlydrawinferencesbasedonclone frequencychanges)[52,57 ].Additionally,bulkTCR-seqwilllikelycapturebystanderactivated andexpandedTcells,contributingtodatanoise.Single-cellTCR-seqintandemwithsinglecell fl owcytometrysortingisanapproachto preciselyidentifySARS-CoV-2-speci fi cTcells. Single-cellTCRanalysiscanbelimitedbytheirdepthandtheirabilitytocaptureminorclones.Regardless,theseapproachesgeneratemillionsofTCRsequencesinoneexperimentandenable thecharacterizationoftheTCRrepertoireinCOVID-19patients[73,76].

ComputationalTools

BioinformatictoolshavebeendevelopedtoenablestreamlinedandstandardizedTCR-seqdata analysisandhavebeenusedbyourgroupandothersextensively.MiXCRi isaUNIX-basedsoftwareusedtoidentifyandbuildTCRsequencesusingrawsequencingdata[77].MiXCRusesa built-inalignerandreferencelibraryofV,D,J,andCsegmentsequencestoefficientlyandaccuratelycallV,D,J,andCregionsfrommillionsofreadsandthengroupssimilarreadstogetherto builda finalTCRsequence.TheassembledTCRsequencesalongwithV,D,andJannotations arewrittenintoatext-formatted filethatcanbeusedbyanalysistoolssuchasVDJToolsii tocharacterizeTCR-seqdataandidentifychangesintheTCRrepertoirethatmayoccurduringdisease progressioninCOVID-19patients[78].

ConcludingRemarks

RecentadvancesinTCR-seqhaveprovidedevidenceforacloserelationshipbetweenTCRdiversityandimmuneresponsesagainstviralantigens.Underhomeostaticconditions,theTCR repertoireislargelydiverseandpolyclonal.However,inthecontextofviralinfection,preferential selectionofTcellclonescancontributetothenarrowingofanantigen-selectedTCRrepertoire. TowhatextentSARS-CoV-2influencestheTCRrepertoireremainstobeassessed.Inaddition, whatimpacttheCOVID-19resultinglymphocytopeniahasontheTCRrepertoirearchitecture,diversity,andcomposition,andhowcomorbiditiessuchasdiabetes,obesity,andhypertension whichareimplicatedinCOVID-19severitymayinfluencetheTCRrepertoire,arequestionsthat warrantattention(see OutstandingQuestions).

StudyingtheTCRrepertoiremayalsorevealpotentialpublicTCRs,whichmayallowustotrackinfectionaswellasunderstandimmunityagainstthisinfection.Inlinewithouropinion,severalefforts areonthewaytoperformTCRanalysisinCOVID-19patients.Arecentstudyhasreportedalongitudinalhigh-throughputTCR-seqtotrackchangesintheTcellrepertoirefollowingtwomild casesofCOVID-19infection[79].Inaddition,clinicaltrials(clinicaltrialnumbersiii:NCT04379466 andNCT04362865)havebeeninitiatedtoexaminetheimmunerepertoireinCOVID-19patients. TheNCT04362865trial,sponsoredbytheNationalInstituteofHealth,isparticularlyinteresting asitaimstoinvestigateboththeBandTcellrepertoireandimmuneresponseinpatientswith acuteandresolvedCOVID-19infection.WeshouldemphasizethattheBcellimmunoglobulinrepertoireanalysisisalsoasignificantandanurgentlyneededtask.Indeed,arecentstudyhasapplied asingle-cellimmuneprofi lingapproachthatcombinessingle-celltranscriptomicanalysiswith single-cellTCRandBcellreceptor(BCR)sequencingtechnologiestoexaminethecellularcontext oftheadaptiveimmuneresponseandimmunerepertoiresofTandBcellsinbloodsamplesof

OutstandingQuestions

HowdoesSARS-CoV-2in fluencethe TCRrepertoire?

Howandwhichcharacteristicsofthe TCRrepertoiremayexplainthedisproportionalriskofCOVID-19inolder population?

Whatimpactdoesthelymphocytopenia inCOVID-19haveontheTCRrepertoire architecture,diversity,andcomposition?

HowdoesgeneticdiversityintheHLAs contributetotheSARS-CoV-2-specific TCRrepertoireand,potentially,theimmuneresponse?

Howmightfactorssuchascomorbidities influencetheTCRrepertoireinCOVID19patients?

TrendsinPharmacologicalSciences

COVID-19patients[54].Weforeseethatotherplatformscanalsoprovidevaluabledata,suchas CyTOFmasscytometryandthe10×Genomicsiv immunepro fi lingplatformthatutilizesDNAbarcodedpeptide-MHC(pMHC)multimersandantibodiesconjugatedtobarcodestodissect theimmunerepertoireinthesettingofCOVID-19.Theseanalysesshouldrevealclonality,diversity, antigenspecificity,andcellularcontext;andpair α and β chainTCRsequencesfromindividualT cells.ItalsoallowsforpairingheavyandlightchainimmunoglobulinsequencesfromindividualB cells.Thesedata,combinedwithsophisticatedcomputationalmethods,willenabletheidentificationofthepairedTCR α and β chainsequenceswithTCR-pMHCspecifi citycorrespondingto SARS-CoV-2.Moreimportantlyitwillsimultaneouslymeasurecellsurfaceproteinexpression withgeneexpressionanduncovermeaningfuldetailsrelatedtothemechanismscontributingto thepathologyofthisinfectionandthepatient’sclinicaloutcome[57].Together,thesetechnologies canbeleveragedtomonitoradaptiveimmuneresponsesinCOVID-19patientsandguidefuture SARS-CoV-2immunotherapydevelopment.WhichcharacteristicsoftheTCRrepertoiremayexplainthedisproportionalriskofCOVID-19inolderpopulationandhowgeneticdiversityinthe HLAscontributetotheSARS-CoV-2-specificTCRrepertoireandpotentiallytheimmuneresponse aresomeofmanyoutstandingquestionstobetackled(see OutstandingQuestions).

Acknowledgments

WeacknowledgetheUniversityofSouthernCalifornia,SchoolofPharmacySeedFund,andTheNorrisCancerCenterpilot fundingthatsupportHAresearcheffort.All figuresweremadewithBioRender(https://biorender.com/).

Resources

ihttps://mixcr.readthedocs.io/en/master/ iihttps://vdjtools-doc.readthedocs.io/en/master/ iiihttps://clinicaltrials.gov/ ivwww.10xgenomics.com/products/single-cell-immune-profiling/

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“There’s bunth, of course,” said Hastings, “but perhaps he doesn’t count.” He lit a cigarette.

“Never heard of such a word.”

“No? He—or it if you prefer it—is a jolly little beast, usually to be found in the tropics under a leaf, and is something between a marmoset and a beetle.” Hastings threw away the match with which he had lighted his cigarette and Marcus picked it up; he hated matches thrown about.

“A what?” asked Watkins, his pocket-book ready and pencil poised.

“It’s no time for joking, Hastings,” said Marcus. “A storm in this part of the world can get up in a moment: we’re in for one now, unless I’m very much mistaken and we may be here for hours. What can have happened?”

“We are stranded on a desert island, that’s quite clear,” said Hastings, and he remembered Diana’s words. What had she said about desert islands? She had rather harped on the subject. Was she playing a practical joke? It looked like it. If she had planned a ridiculous game he would play it with her. If she had meant to be funny he would laugh with her. He would enter into the spirit of any joke she chose to perpetrate—be it good, bad, or indifferent; and after all an island is an island, and a man is a boy, and what man is there, who is as much a boy as he ought to be, who can be on an island and not light a fire? or be in a wood and not look for birds’ nests? Being very much of a man he was very much of a boy Diana had spoken in fun of desert islands. Did she realize how deeply implanted in the heart of every real man is the longing for the primordial life? Not for long, perhaps, but to experience it, for once? Hastings had dreamed all his life of a desert island. He had cooked, he had built, he had slept, on a desert island. He had lain awake under the stars above it, slept, lulled by the wind that rocked it. He had risen with the sun that rose behind it, and had bathed in the noonday heat that scorched it. Did Diana know the lure of those dreams? He set out to explore and at the mouth of the cave he came upon the cairn of her building. He took the paper from under the top

stone and he read of the historical one match, with which she dared him to light a fire! It required, she said, the very particular skill of the experienced explorer to light a fire with the last match. “I am sure you can do it,” she wrote. Further she said she knew exactly how a desert island should be furnished—there should, by rights, of course, be a chest of drawers in the cave. She knew how it should be stocked: with what cunning the stores should be hidden; they should stand upon a rock in full view—but she realized how little time there was to spare, how soon Uncle Marcus would tire of a desert island; so she had placed all he should need in a box, and in that box he would find two halves of a cocoanut shell, in which to make soup, and other things, such as whiskey, that should help to keep Uncle Marcus warm and happy for a little while—and sardines that should sustain him. Hastings wished Diana had thought of a funnier joke, but he had vowed he would be amused. He would light the fire; that at least would be good fun, and he would stick faithfully to the one match. He would tell her he had made soup and boiled eggs. He would get Uncle Marcus to swear he had swallowed them; he—Miles —should not be the loser in the end: the joke should be his ... until such time as it should find its way home again to her!

He went out of the cave to gather driftwood. The storm had risen, he was caught by the scudding rain and whipped by the wind. He wished she would come. In a moment the rain was running down his neck and oozing over the tops of his shoes. The joke was beginning to pall, but he said to himself, and truly, that if it had been any one else’s it would have palled long before. He was going back to the cave when he saw at his feet a little bird that looked about to die. “Poor little beggar!” he said. “It’s a poor joke, isn’t it?” And lifting it up he examined it, promising to do what he could to save its funny little life. “You would, would you?” he asked, as the little bird pecked at his finger. “Wait a bit, my little friend, until you are sitting by a warm fire with a speck of whiskey inside you—eh? Come along!”

The joke was not such a bad one after all. A fire has been lighted for worse things than for the warming of a little half-dead bird. “You’re an ugly little beggar—yes, you are!” he said. “Now be quiet while I

light a fire with one match—one match, old man, is all that stands between you and death—yes, death!”

To the little bird it must have been an enormous giant that placed him so tenderly in a safe place, under the shadow of a great rock. The little bird watched with interest the arranging of that fire: gasped when the one match nearly went out: blinked when the flame fostered in the hollow of a gigantic hand flared up straight and strong —and yellow, the colour of its mother’s eyes—a colour warm, comforting, and kind. The giant put the flame to the driftwood; it caught here, went out there, blazed up here; the little bird squeaked. “Wait a bit, sonny—it’ll be all right. It’s no joke dying when you’re so young, is it? I had measles myself long ago—here—draw up to the fire, closer—that’s right—now for a little whiskey. Like it, old man? No, no more! True Scotsman that you are—later on, perhaps! Now go to sleep. I’ll go and see what the others are doing—squawk if you’re frightened—I shan’t be far off.”

It wasn’t such a bad joke after all. Bother the rain!

Meanwhile Marcus had taken all the photographs he could take and began to find Diana’s joke—if joke it were—a poor one—and a stupid one. He made no vow to be amused by it—no uncle would. Watkins, anxious to help, said the only thing he could do to while away the time was to recite and he cleared his throat.

“If you would shout instead, I should be very much obliged,” said Marcus; “some one might hear you.”

Shouting was not at all the same thing to Watkins because it was a thing he did with great difficulty. However, he must try and he should be very glad if some one should chance to hear him because his landlady never did, and she always put the blame on his voice. He asked St. Jermyn to shout too, and St. Jermyn shouted.

“You should be a fine singer,” said Watkins, clearing his throat again.

“Think so?” asked St. Jermyn, indifferent to praise.

“I do—indeed I do—don’t you?”

“Yes, I do, of course.”

“You? Oh, I see you are joking.”

“I never felt less like joking—d’you know what a storm here can mean?”

“Not particularly here,” said Mr. Watkins.

“I thought not. Shout!”

Watkins shouted. Marcus stood in the fine drizzling rain peering out to sea—and saw nothing.

Again St. Jermyn shouted.

“Be quiet!” said Watkins; his ears, attuned to the elements, he said, were the first to hear an answering call. It was the hoot of a steamer—at that moment of all sounds most blessed—even the voice of Diana must have been less sweet.

“It’s a yacht,” said Marcus.

“The Scotts, I expect,” said Ralph St. Jermyn; “I told them if they should be round here to look us up.” (What a comfortable thing it is to have cousins who have yachts! and other things, most desirable.) “They will find difficulty in getting a boat off—unless they can get under the lee of the island,” he added.

Until that moment when Mrs. Scott held out two hands to Marcus to greet him, he had never been able to excuse his want of judgment in having allowed himself to call her beautiful. He had always felt he had risked his reputation in so doing—his reputation as a judge of beauty. Now as he took her hands in his he found her of all women the most to be admired. There was something after all that was better than beauty of line, there was charm of expression, and she was, at this moment, perhaps, even beautiful: but he could not see because she wore a sou’wester well pulled down over her eyes, but he could imagine the kindness beaming from those eyes. The smile on her lips he could see, so that if he did not stand completely exonerated he at least must be largely excused—to a cold, wet man all women may seem beautiful.

“You dear moist things,” she said, “how did you get stranded here?”

A few minutes after they had left the island, and Marcus was being ministered to by Mrs. Scott, Miles Hastings came in search of him. He shouted; but there was no answer except the screeching of birds as they flew up in their thousands at his approach. There was no sign of man. Was this another joke?—this time a poor one—a very poor one? It had not been Diana’s fault that the day had turned out wet; but this was childish. He walked on and shouted again. There was still no answer. The waves hurled themselves against the rocks, making a noise like the booming of guns—old Watkins had said that. It was quite evident that by some miraculous chance the others had got off the island, and had forgotten him. He wondered that St. Jermyn had forgotten him: perhaps he had not—that was another way of looking at it!

There was nothing to be done so he went back to the cave, where he found that the little bird, at all events, had not forgotten him. “What shall we do?” he asked, and the little bird said nothing—how should he know when he had lived so short a time—and knew nothing as yet of the division of days? Breakfast, lunch, dinner, were to him as one. He would have no divisions between them—if he had the ordering of things.

There was one thing Hastings could always do, and that anywhere, no matter where—think of Diana! He pulled up the collar of his coat closer round his ears, sat down in the best shelter he could find, picked up the little bird, told it to be good, and proceeded to think of Diana. If she had forgotten him the situation was about as bad as it could be, but he was convinced something must have happened—a thousand things might have happened. The motor launch might have gone wrong—if it had, then she might be in danger. He sprang up to look. “All right, old chap, I won’t drop you.” He looked first one way, then another, and in neither direction could he see anything. A veil impenetrable hung between him and the mainland. The storm raged—more and more furiously. He knew it must spend itself in time, it was bound to. Diana at that moment was wondering what he was doing?

She could never have guessed that he was greatly exercised over the feeding of his young charge with bits of sardine—meant for Marcus. That done he was going to think about her. That he would think about her she might have guessed—but she could hardly have guessed how tenderly he was going to do it.

XVII

It’s a good joke that keeps out the rain, stays the wind, and makes the fire burn brightly.

DOWN on the shore of the loch Diana waited, buffeted by the wind, drenched by the rain. Her hair loosened by the wind blew into her eyes. She pushed it back impatiently: she was waiting for the storm to clear. So soon as the clouds broke, and she saw light on the horizon, and the waters were stilled, she would send the launch for Uncle Marcus. That he must by this time be suffering from congestion of the lungs, she was gloomily certain. There was now no question as to which of the men she loved; she loved them all, in the sense, at least, that there was not one of them she wouldn’t marry to save him from drowning, or from pneumonia. Not one! She had only meant to be funny and she had not been funny. A poor joke was a crime in itself—nothing excused it. If it had been a good joke they would all have forgiven her, but what with the rain and the wind and the island—horrible at all times—she had sinned beyond forgiveness. Oh, to be with Aunt Elsie—the peace of it—the perfect peace! The memory of the sun-steeped garden, with the booming of bees—so different from the booming of waves—of the scent of the roses, was more than she could bear—and the deliciousness of Shan’t became an aching memory.

Pillar was watching, too, clad in oilskins and wearing a sou’wester. He made the agony still more acute, by carrying a rope. As Napoleon is pictured standing wrapt in melancholy, so stood Pillar— awaiting the worst.

Suddenly he stiffened and came towards her, as though, she thought, he were in London, about to announce a visitor. “Mr. St.

Jermyn, miss,” he said. She had been right, he was a butler again announcing a visitor, and never was visitor so welcome as this one.

“You!” she cried, as St. Jermyn came towards her across the sandy bay and took her hands in his.

“I am abjectly sorry,” she said, and withdrew them gently, explaining they were wet. Then she asked him if he had forgiven her, which was a dangerous question to ask.

“Yes ... now,” he said, with a still more dangerous emphasis.

“Why now?” she asked—a foolish question to ask. She might have guessed why.

“Don’t spoil it,” he said gently.

“I only meant it as a joke—leaving you on the island, I mean. And then, when I got back and was going to send the launch to fetch you, the storm had got up and the men said the sea was too rough.”

“Why did you leave us?”

“As a joke! I told you.” It was awful to have to go on explaining that the idiotic thing she had done was a joke.

“For no other reason?”

“I was tired of so many men, that was all!”

“You couldn’t put up with one, I suppose, who is very humble and doesn’t ask much?”

“No—at the moment I want you all—tell me, how did you get off the island?”

“The Scotts picked us up. They were out fishing or had been before the storm got up. There was a little difficulty in sending out the boat for us, but they managed it—they got under the lee of the island. Your uncle is anxious about you—he is afraid you are wet— you are!” St. Jermyn put his hand on her arm.

She moved away. “Not really wet,” she said; “a little damp, but what does it matter how wet I am now you are all back.”

“We are not all back. Hastings is still on the island.”

The wind and rain had stung Diana’s cheeks to a vivid colour and she wore a blue hood drawn over her hair and wore it as St. Jermyn had thought only an Irishwoman could wear it. He found her distractingly pretty.

“Still on the island? He must be frightfully wet,” she said.

“And very hungry,” he added.

“Oh—yes, perhaps—” Hunger she knew would not be the worst thing he had to bear. “But some one must fetch him.”

“Yes, some one must fetch him. We forgot him, which is a thing I should not have thought I could possibly have done, whatever the others might do. The storm may clear off at any moment. They do in these parts as suddenly as they come up.”

“But if it doesn’t clear up?”

St. Jermyn looked at her—then out to sea and back again to her.

“If you want him to be fetched I will fetch him—for you.”

“Why for me?”

“Because you want some one to go and I want to please you—is that reason enough?”

“Do you mean that if I didn’t want him fetched you wouldn’t go?”

“I mean that. I would let some one else go.”

“Why not let some one else go now, then? A man on an island must be fetched. Why should you make it a personal thing?”

“Because I want everything between you and me to be personal. I want my chance, that’s all. You say a man on an island must be fetched. Do you realize that for me to do it is an act of heroism? Supposing it would suit me that Hastings should stay forever on the island, what then? Suppose that in fetching him off the island I know I destroy my one chance of happiness, and I still fetch him—for your sake—what does that argue?”

“I can’t argue—I hate it. He is cold and wet—he is cold and wet because I behaved like an idiot—he must be fetched.”

“Many a time during the last few days I have wished to drown him.”

Diana said she didn’t believe him, but he assured her it was true. “Just as heartily as he has wished to drown me,” he added.

That Diana refused to believe.

“No? Well, then, his position is evidently more assured than I imagined it, and he is a more fortunate man than I am—he can afford to be magnanimous. Well, I am going to fetch him for your sake—you can’t rob me of that nobility of character—I shall fetch him in order to make you happy—just as I would do anything else in the world to make you happy. It will make you happy—I should like to be certain of that—before—”

Diana said of course it would make her happy that any one cold and miserable should be made warm and happy, “But I don’t want you to run any danger—that I wouldn’t face myself. I will come with you.”

“Will you?” The thought of an hour—or as long as he liked to make it—alone with Diana was a delirious thought—less delirious was the thought of the return journey with Hastings in the boat. Hastings with the glamour of martyrdom upon him would be invulnerable. St. Jermyn said he wasn’t sure that Hastings would like that, and Diana asked why? He would be glad to see her, she knew.

“Without me—yes, but with me? What do you think?”

“Why should he mind?” she asked; then added, “It will be safe for you to go, though?”

If it were not would she mind? he asked her.

She answered, of course, why not? If she had proved herself devoid of humour, it did not show she was heartless.

“I wish I understood you—perhaps if I did I should be even less happy than I am. It’s clearing, I’ll go; any message for him?”

She shook her head—then said: “My humble duty, perhaps—”

“I am glad you did not ask me to take him your love because I should have kept half for myself—but if you would trust it all to me— to give what I like of it to Hastings—”

Diana, interrupting him, said she was tired of jokes. So was he, he vowed: he was in deadly earnest. “By the way—if in earnest I asked you—‘Will you marry me?’ What should you say?”

“I should say—‘Is this a serious proposal?’”

“And if I said ‘Yes’—what should you say then?”

“I should say, it was too windy to hear—too wet to answer—too cold to marry.”

“Too cold—that’s it! Isn’t it? Why are you so cold?”

“The wind—I can’t hear—I told you!”

“I am very serious—it’s no joke—”

Diana looked at him. “Are you really serious?” she asked.

He said he was very serious.

“Then I, too, am very serious. I should say I was very sorry, but I couldn’t.”

“Would you say why?”

“I should say it had nothing to do with the weather—the wind or the rain—but just to do with my heart.”

“You would mean that seriously?”

“I should mean it very seriously.”

“Then I shall not propose.”

“It would be a careless thing to do.”

She went into the Lodge, very unhappy, very wet, and very much perturbed. She had a bath, dressed for dinner, and went downstairs to meet Uncle Marcus. Uncle Marcus was clean and dry and dressed for dinner. And he was very serious. Not at all a nice Uncle Marcus:

but Diana was quick to see the justice of this. There was no reason he should be nice.

“You were extremely foolish,” he said, “and Hastings is still on the island.” He didn’t look up from the “Scotsman” he was reading. Diana said Mr. St. Jermyn had gone to fetch him.

“Why St. Jermyn, when there are plenty of men about?”

“He went to please me,” said Diana, sitting down on the table—an attitude of hers Uncle Marcus particularly disliked.

“Because he is in love with you he goes to fetch another man who is also in love with you. You will find it difficult to choose between them. You are under an obligation to one and you have—”

“Captain Hastings would never retaliate.”

“Don’t ask me to help you, that’s all.”

“Aunt Elsie will help me.”

Uncle Marcus put down the “Scotsman.” Diana had taken his middle stump with a fast underhand ball—so he would have described it.

“My dear child,” he said, “don’t do anything rash—it’s all perfectly simple. What has happened to you has happened to most women— girls—I expect—attractive girls, I mean. You are, I am sure, in no way to blame....”

“Let me get on to the sofa, darling,” said Diana; “there, that’s right, now go on.”

“What was I saying?” resumed Uncle Marcus. “You are in no way to blame—”

“I am kissing my hand to you hard,” said Diana, from the depths of the sofa.

“Well, don’t interrupt me. I was saying, you are in no way to blame. Men must take their chances. It happens that two men are in love with you—two at least—both are excellent young men. It is perhaps difficult for you to choose between them, for in your inexperience you

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