t cell activation and the hla locus associate with latent … · protein hla-g, were predictive of...

1

TcellactivationandtheHLAlocusassociatewithlatentinfectionsofhumanAfricantrypanosomiasisPaul Capewell1, Bruno Bucheton5,6,8, Caroline Clucas1, Hamidou Ilboudo5,6,8, AnneliCooper1, Taylor-Anne Gorman1, Kerry O’Neill3, Agapitos Patakas4, Andrew Platt4, HeliVaikkinen1,WilliamWeir1,MamadouCamara5,PaulGarside4,VincentJamonneau6,7,8,andAnnetteMacLeod1,8

1WellcomeTrustCentreforaMolecularParasitology,CollegeofMedical,VeterinaryandLifeSciences,SirHenryWellcomeBuildingforComparativeMedicalSciences,Glasgow,UnitedKingdom.3InstituteofBiodiversity,AnimalHealthandComparativeMedicine,CollegeofMedical,Veterinary and Life Sciences, Graham Kerr Building, University of Glasgow, Glasgow,UnitedKingdom.4InstituteofInfection,ImmunologyandInflammation,CollegeofMedical,VeterinaryandLife Sciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow,UnitedKingdom.5ProgrammeNational de LutteContre laTrypanosomoseHumaineAfricaine, Conakry,Guinea.6InstitutdeRecherchepourleDéveloppement,UnitéMixtedeRechercheIRD-CIRAD177INTERTRYP,CampusInternationaldeBaillarguet,Montpellier,France.7InstitutPierreRichet,Unitéderecherche“Trypanosomoses”,Bouaké,Côted’Ivoire.8TrypanoGENandH3AfricaConsortium.

was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 5, 2017. . https://doi.org/10.1101/184762doi: bioRxiv preprint

https://doi.org/10.1101/184762

2

Abstract

Infectionsbymanypathogenscanresultinawiderangeofphenotypes,fromsevereto

mild, or evenasymptomatic.Understanding thegeneticbasisof thesephenotypes can

lead to better tools to treat patients or detect reservoirs. To identify human genetic

factors that contribute to symptoms diversity, we examined the range of disease

severities causedby theparasiteT.b. gambiense, theprimary causeofhumanAfrican

trypanosomiasis (HAT). We analyzed the transcriptomes of immune cells from both

symptomaticHAT cases and individualswith latent infections.Our analysis identified

several genes and pathways that associated with the latent phenotype, primarily

suggestingincreasedTandBcellactivationinHATpatientsrelativetolatentinfections.

We also used these transcriptome data to conduct an exome-wide single nucleotide

polymorphism (SNP) association study. This suggested that SNPs in thehumanmajor

histocompatibilitylocus(HLA)associatewithseverity,supportingthetranscriptiondata

and suggesting that T cell activation is a determining factor in outcome. Finally, to

establish if T cell activation controls disease severity, we blocked co-stimulatory

dependentTcellactivationinananimalmodelforHAT.ThisshowedthatreducingTcell

activationduringtrypanosomeinfectionimprovessymptomsandreducesparasitemia.

Ourdatahasusedacombinationoftranscriptome-wideanalysisandaninvivomodelto

reveal that T cell activation and the HLA locus associate with the development of

symptoms during HAT. This may open new avenues for the development of new

therapeuticsandprognostics.


https://doi.org/10.1101/184762

3

Introduction

Infectious diseases often display a range of severities determined by variation in the

pathogen, the host, or both (Riggs et al. 2007; Fakhar et al. 2013; Perron et al. 2008;

Lindblade et al. 2014; Hentges et al. 1992). One such medically and economically

important disease is human African trypanosomiasis (HAT), primarily caused by the

parasite Trypanosoma brucei gambiense (T. b. gambiense). Latent infections of T. b.

gambiensehaverecentlybeenidentified,contradictingmorethanacenturyofdogmathat

thediseaseisinvariablyfatalifuntreated(Sudarshietal.2014;Ilboudoetal.2011;2014;

Jamonneau et al. 2012). Individuals with latent infections appear able to tolerate the

parasite without obvious symptoms for a prolonged period, with one recent case

persisting for29yearswithoutsymptoms(Sudarshietal.2014).Latent infectionsare

primarily identifiedusingserologicalmethodsdue to lowobservableparasitemia.The

differencesindiseaseseverityfoundduringT.b.gambienseinfectionsarelikelytobedue

tohostfactors,asthereislittlegeneticvariationinthisclonalparasite(Weiretal.2016;

Kaboréetal.2011).Thisprovidesanopportunitytoexaminehost-derivedfactorsthat

resultindifferentialdiseaseseverityandmayprovideinsightintolatentinfectionswith

T.b.gambienseandpossiblyotherpathogens.

Duetotherecentconfirmationthat latent infectionsofT.b.gambienseexist, therehas

beenlimitedinvestigationintopotentialfactorsthatassociatewiththephenotype.This

work,primarily inaGuineanHATfocus,hasdemonstratedthatelevatedIL-8andIL-6

levels associate with latent T. b. gambiense infections. In contrast high levels of

immunosuppressivemolecules,suchasIL-10andthematernalhumanleukocyteantigen

proteinHLA-G,werepredictiveofdiseasedevelopmentinserologicalpositivesuspects

(Ilboudoetal.2014;Gineauetal.2016).Contrastingassociationshavealsobeenreported

withAPOL1 alleles thatpredisposepatients to renaldisease (Cooper et al. 2017).The

APOL1G1allelewasfoundathigherfrequenciesinlatentinfections,whiletheG2allele


https://doi.org/10.1101/184762

4

associatedwithan increasedriskofHAT.However,agenome-widescreenhasnotyet

been applied to highlight novel genetic aspects involved in latent T. b. gambiense

infections.While variations in disease severity caused by African trypanosomes have

been extensively studied in mice and cattle, most of these studies used the animal

trypanosomespeciesT.congolense,T.vivax,andT.b.bruceiratherthanT.b.gambiense

(Magezetal.2006;MansfieldandPaulnock2005;Namangalaetal.2009;Shietal.2003;

2006;Tabeletal.2008).Thisworkhasrevealedthattheimmuneenvironmentiscentral

todiseaseoutcomeinanimals.Keyfeaturescurrentlyelucidatedincludetheinvolvement

of IFNγ and IFNγ-activatedmacrophages in controlling early infection (Kaushik et al.

1999), the subsequent reduction in the IFNγ-mediated inflammatory environment

(Biswas and Mantovani 2014), and the roles that elevated IL-10 and IL-4 play in

mediatingthisshiftintheimmuneenvironment(Inoueetal.1999;Mertensetal.1999).

To more fully understand the immune components involved in disease severity, we

comparedtheperipheralbloodmononuclearcell(PBMC)transcriptomesofage-matched

groupsofT.b.gambienseHATcases, individualswith latent infections,anduninfected

controls.Latentinfectionsweredefinedasindividualswhowereserologicallypositivefor

at least two-years without the development of neurological symptoms or visible

parasitaemiausingboththecardagglutinationtestfortrypanosomiasis(CATT)andthe

highlyT. b. gambiense specific trypanolysis test (Jamonneauet al. 2010; Ilboudoet al.

2011).Thisrevealedseveraldifferentiallyexpressedgenesandpathwaysassociatedwith

diseaseseverity.Wealsousedexomedatafromthescreentoidentifysinglenucleotide

polymorphisms (SNPs) that associatewith the latent infection phenotype. These data

revealedthatTcellactivationandthemajorhistocompatibility(MHC)/humanleukocyte

antigen(HLA)locusassociatewithdifferentdiseaseoutcomesinhumans.Todetermine

ifvariationinTcellactivationisresponsibleforvariationindiseaseseverity,weblocked

co-stimulationdependentTcellactivation inamousemodel fortrypanosomiasis.This


https://doi.org/10.1101/184762

5

resultedinreducedsymptoms(assessedbybodyweight)andareductioninparasitemia.

TheseresultsprovidedexperimentalvalidationoftheassociationwithTcellactivation

identified in the human transcriptomic study, and reveals a central role for T cell

activationindeterminingdiseaseseverityintrypanosomiasis.

Results

TranscriptomeandPathwayAnalysis

Toidentifygeneticdeterminantsthatunderliediseaseseverityorlatentinfections,total

mRNAwaspreparedfromperipheralbloodmononuclearcells(PBMCs)collectedfrom

individuals and sequenced using Illumina HiSeq. Transcripts with at least a 1.25-fold

difference inexpressionbetweenHATpatientsand latent infectiongroupsanda false

discoveryrate(FDR)-adjustedpvalue<0.05,wereconsideredasdifferentiallyexpressed

inthisstudy(SupplementalTableS1).ThetranscriptionalprofilesfortheHATandlatent

infectiongroupswerefoundtobedistinctandcouldbeseparatedusingtwo-dimensional

principalcomponentanalysis(PCA)(Figure1).Additionally,latentindividualsclustered

morecloselywithuninfectedcontrols,suggestingthatthelatentphenotypeisduetoa

lackofresponsetoinfection.Wefound205PBMCtranscripts(»0.5%)thatsignificantly

differedinabundance(bothrelativelyhigherandlower)betweenHATcasesandlatent

infections, consistingof61 transcripts found ingreater abundance in individualswith

latentinfectionsand144thatwereelevatedinHATcases.Therewasnoenrichmentfor

specificgeneontological(GO)terms,althoughtranscriptsgreaterinabundanceinlatent

infections included regulators of immune response, such as components of the T cell

receptor(TCR)complex(TRAV14,TRAV27),IL-4inducedprotein1(IL4I1),andthegene

fortheco-stimulatoryTcellprotein,CD26.CD8betachaintranscriptwasalsofoundtobe

elevatedinlatentinfectionsrelativetoHATpatients.Transcriptsgreaterinabundancein

activediseasepredominantlyassociatewithBcells(primarilyimmunoglobinsandCD38).

ThismayreflectthelargeBcellexpansionthathasbeenpreviouslyobservedinclinical

trypanosomiasis(Lejonetal.2014).


https://doi.org/10.1101/184762

6

Figure1.Principlecomponentanalysis(PCA).APCAdescribingthevariabilitypresent

intheexpressionprofilesofclinicalcases,(blue)latentinfections(red),anduninfected

controls(green).Linesjoineachsample(circle)tothecentroid(square)oftherespective

group. The two dimensions describe 26% of the variability present in the transcript

profilesofthesamples.

Thebiologicalfunctionsandpathwaysthatmaydifferduetothedifferenttranscriptional

profilesfoundinactivediseaseandlatentinfectionswereinvestigatedusingIngenuity

Pathway Analysis and a database of experimentally-verified protein functions. This

indicatedthat102functionalpathwaysdifferbetweentheHATcasesandindividualswith

latentinfections,althoughthelikelydirectionofregulationbetweengroups(thosewitha

z-score<2.0or2.0>)couldonlybepredictedforonepathway,Ca2+mobilization(z-score

2.158,p=0.0007).ThiswouldsuggestincreasedlymphocyteactivationinHATpatients,

relativetolatentcases.Othersignificantpathways(p<0.01)suggestinvolvementofthe

binding,activationandproliferationofBandTlymphocytes.IngenuityPathwayAnalysis

specifically predicted that activation of lymphocytes (p<0.0001), activation of T cells

(p=0.0004), co-stimulation of T lymphocytes (p=0.0013), and Ca2+ flux (p=0.0004)


https://doi.org/10.1101/184762

7

(indicative of lymphocyte activation) were all significantly different between HAT

patients and latent cases (SupplementalTableS2). Interestingly,pathwaysassociating

withanimmuneresponseintheskinandjoints,includingatopicdermatitis(p=0.008),

psoriatic arthritis (p=0.006), and inactivation of mast cells (p=0.005), were also

implicated.Theskinhasrecentlybeenshowntobeanoverlookedanatomicalreservoir

forT.b.gambiense(Capewelletal.2016).

FurtherIngenuityPathwayAnalysisidentified19predictedupstreameffectorsthatcould

explain the expression profile differences between HAT cases and latent infections,

includingsixtranscriptionalregulators(GATA2,SP1,GATA1,SATB1,CREM,andIZF1)(

SupplementalTableS3).Ofthese,onlytheSATB1pathwayhadasignificantz-scorethat

waspredicted tobe inhibited in latent cases relative toHATpatients (z score=-2.093,

p=0.008).Theactivityoftheotherfivetranscriptionfactorscouldnotbepredictedfrom

ourdata.Otherupstreamputativeeffectorsthatmayexplaintheexpressiondifferences

between HAT and latent infections include several immune regulators, such as CD40

(p=0.001),IL-4(p=0.002),CXCL12(p=0.004),andIL-21(p=0.040).Thesepathwaysare

each associated with CD4+ T cell activation and predicted to be less active in latent

individuals,relativetoHATcases(SupplementalTableS3).CD40,IL-4,andIL-21arealso

potentmediators of B cell proliferation and immunoglobulin release (Eto et al. 2011;

Spriggsetal.1992).Supportingapriorstudy(Gineauetal.2016),HLA-G(p=0.004)was

also an upstream predicted effector for the observed differences in transcriptional

profiles, although the predicted direction was unable to be determined by Ingenuity

PathwayAnalysis.Insummary,thesedataindicatethatindividualswithlatentinfections

demonstrateareducedBandTcellresponse,relativetopatientsdiagnosedwithactive

disease.

SNPAssociation


https://doi.org/10.1101/184762

8

To identify a potential genomic basis for the differences in disease outcome, single

nucleotidepolymorphisms(SNPs)wereidentifiedfromtheexomedata.Intotal,39,897

highqualitySNPswere identifiedandtheirpotentialassociationswithHATand latent

infectionswere examined (figure2). This highlighted482 SNPs that differedbetween

individualswithanactiveorlatentinfectionusingafalsediscoveryrate(FDR)-adjusted

significancevalueofp<0.05 (SupplementalTableS4). fastSTRUCTUREanalysisof the

marginal likelihoods for various values of K indicated that themost likely number of

populationswas1(marginallikelihood=-0.45),suggestingtherewasnounderlyingsub-

structuringthatwouldaffectsubsequentanalysis.Themostsignificantlatentinfection-

associating SNPwas found on exon 5 of the class Imajor histocompatibility complex

(MHC)gene,HLA-A(adjustedp=0.00089).Thispolymorphismcausesanonsynonymous

changeat the junctionof theHLA-Aα3CD8-bindingregionandmembrane-addressing

domains.Themajority(71%)ofindividualswithlatentinfectionspossessthisSNPallele

compared to 31% of HAT cases, equating to an estimated odds ratio of 5.231. The

statisticalpowertodetectaneffectofthissizewithα=0.05is0.6forastudyofthissize.

FouradditionalsignificantlyassociatingSNPswerealsofoundintheHLA-Aα3domain

andtheneighboringHLA-Bgene.Asecondlocusofhighassociationwasfoundinaregion

containingseveraltripartitemotif-containing(TRIM)genesthatareinvolvedinretroviral

defense(Figure2)(Nisoleetal.2005).ExaminingthedistributionofSNPsonagene-by-

genebasisrevealednoassociationbetweenindividualTRIMgenesanddiseaseoutcome,

but there was a significant association with HLA-A (adjusted nominal p=0.02597)

(SupplementalTableS5).


https://doi.org/10.1101/184762

9

Figure2.ThesegmentedmeanoddsratiosoftheassociationbetweenSNPsandthe

latentinfectionphenotype.ThemeansoddratioofgeneticassociationwitheachSNP

was calculated using PLINK. Each presented segment includes a genomic region

encompassing two neighboring SNPs,with themean value determined from the odds

ratios of the two SNPs. The highest peak of association lieswithin theHLA region on

chromosome6.

The impact of blocking co-stimulation dependent T cell activation on disease

outcomeinananimalmodeloftrypanosomiasis

OurdatafromhumaninfectionsindicatethatTcellactivationinindividualswithlatent

infections is lower than those experiencing active disease, implying a role for

trypanosome-inducedTcellactivationindiseaseoutcomeintrypanosomiasis.Inorder

to test thishypothesis,weuseda specific inhibitorof co-stimulationdependentT cell

activation(CTLA4-Ig)inthemousemodelofsymptomaticdisease(Linsleyetal.1992).

Balb/c mice were either treated with CTLA4-Ig or an isotype control, administered

intraperitoneallythedaybeforeparasiteinoculationandeveryotherdayafterinfection

for the length of the experiment. Mice were infected with the trypanosome genome

referencestrain,TREU927,andtheexpressionofTcellactivationmarkers,parasitemia,

and weight loss (an indicator of animal health) were measured and compared to

uninfected control mice. CTLA4-Ig significantly reduced the expression of activation-

associatedimmunecheckpointproteins(ICOS,PD1,LAG3)intheCD8+andCD4+Tcell

populationsduringinfection(SupplementalFigureS1).Trypanosomeinfectionstypically


https://doi.org/10.1101/184762

10

followasinusoidalpatternthroughdiseaseprogression,althoughwefoundthatreducing

Tcellactivation ledtoareductioninparasitemiaafterthe initialpeakofparasitaemia

(GLM, F statistic 12.96, p<0.001) and an associating recovery in animal condition

(assessed using body weight) (ANOVA, p=0.025) (Figure 3). This suggests that co-

stimulationdependentTcellactivationpromotessurvivaloftheparasiteinthehostand

interferingwiththisbiologicalpathwaycaninducealessseverediseasephenotypeinan

animalmodel,mirroringthehumanlatentinfectionphenotype.

Figure3.Theeffectsofreducingco-stimulationdependentTcellactivationduring

trypanosomeinfection.(a)CTLA4-Igtreatmentsignificantlyreducesparasitemiaafter

thefirstpeakofparasitemiaintrypanosomeBALB/cmiceinfectedwithT.bruceistrain

TREU 927 (red) versusmice administered an isotype control (blue) (GLM, F statistic

12.96,p<0.001).Parasitemiafromdifferentmicearealignedtothefirstpeaktoaccount

forvariationparasitemiacurves(0=firstpeakofparasitemia).Datarepresentsfifteen

miceineachgroupwitha95%confidenceinterval.(b)Meanweightchangefromday0

offivemicethatwereeitheruninfected(blue),infectedwithT.bruceistrainTREU927and

administeredanisotypecontrol(green)orinfectedwithT.bruceistrainTREU927with

regularadministrationsofCTLA4-Ig(red).Inallexperiments,CTLA4-Igwasadministered

intraperitoneallythedaybeforeinoculationandeveryotherdayafter,forthelengthof

theexperiment.


https://doi.org/10.1101/184762

11

Discussion

LatentinfectionsofT.b.gambiensehaveonlyrecentlybeenidentifiedandtherehasbeen

little investigation into thegenetic factors thatmayplaya role indeterminingdisease

outcomeinsuchindividuals(Sudarshietal.2014;Ilboudoetal.2011;2014;Jamonneau

etal.2012).Theselatentinfectionslikelyrepresentanoverlookedandhiddenreservoir

thatwillhamperongoingcontroleffortsforAfricantrypanosomiasis.Interestingly,our

study found that pathways involved in a skin immune response significantly differed

betweenactiveandlatentcases.IthasrecentlybeendemonstratedthatextravascularT.

b. gambiense skin-dwelling parasites can form an overlooked anatomical reservoir in

experimentally infected mice and humans living in HAT endemic areas. Individuals

harboring latent infections may therefore form an overlooked anatomical reservoir,

despiteanabsenceofbloodparasitesandHATspecificsymptoms,suchasneurological

signs (Capewell et al. 2016). This suggests a putative link between the host immune

responseandprogression toa latentphenotype that requires furtherexamination.To

understand the underlying mechanisms that may contribute to the diversity in

trypanosomiasisdiseaseseverity,ourstudyfirstexaminedthetranscriptomicdifferences

betweenlatentindividualsandclinicalcases.Thereweresignificantdifferencesbetween

thetranscriptionprofilesoflatentinfectedandactivecases.Furtherexaminationusing

IngenuityPathwayAnalysishighlightedactivationandproliferationof lymphocytesas

significantlydifferentpathways,withasuggestionthroughtheywerelessactiveinlatent

infectionsrelativetoHATcasesbasedonCa2+mobilization.Wenextusedexome-datato

identifySNPsthatassociatewiththelatentinfectionphenotype,albeitwithlowpower

duetoalimitedsamplesize.ThissuggestedanassociationwiththeHLAlocus,aregion

directlyinvolvedinTcellactivation,withthelatentandclinicalphenotypes.Interestingly,

no association was foundwith genes encoding proteins known to be involved in the

human infectivity of African trypanosomes (APOL1, HPR (Vanhollebeke et al. 2008;

Vanhammeetal.2003)).Indeed,APOL1alleleshavepreviouslybeenfoundtoassociate


https://doi.org/10.1101/184762

12

withthedevelopmentofalatentphenotypeinthispopulation(Cooperetal.2017).This

discrepancymayaresultofusingapopulationcomprisedof immunecells thatdonot

expresstheseproteinsorsimplyduetopopulationsize.

The SNPs putatively identified in our study may lead to HLA alleles with different

propensities for immune presentation, affecting T cell activation, or may perturb the

bindingoftranscriptionfactorsthatcontrolHLAtranscription(SupplementalTableS3).

For example, the SATB1 pathwaywas predicted to have reduced expression in latent

individualsrelativetoHATpatients(z-score=-2.093,p=0.008)(SupplementalTableS3).

Thisproteinmediates thedevelopmentofTcellsbyreorderingthechromatinofMHC

(HLA)genesandaffectingsubsequenttranslation(Kumaretal.2007).Polymorphismsin

theHLAlocusmaythereforeaffectchromatinremodelingbySATB1andsubsequentHLA

expressionandactivity.TheHLA/MHClocushasbeenshowntobeinvolvedinseveral

diseaseswithlatentinfections,mostnotablyduringHIVinfection(Hentgesetal.1992),

butalsoparasiticdiseasesrelatedtoT.brucei,includingLeishmania(Quinnelletal.2003)

andT.cruzi(Cruz-Roblesetal.2004).Similarly,ourworksuggeststhatHLA-typingmay

have a role in understanding the latent infection phenotype associated with T. b.

gambiense infections and provide a prognostic tool to predict disease severity. MHC

alleles have not been shown to associate with susceptibility in mouse models for

trypanosomiasis(LevineandMansfield1981),althoughanassociationhasbeenshown

in cattle (Karimuribo et al. 2011). HLA-G has also been previously been shown to be

predictiveofdiseasedevelopmentinserologicalpositivesuspects(Ilboudoetal.2014;

Gineauetal.2016).Whilemakingsubsequentexperimentalanalysiswithanimalmodels

difficult,thisfurtherhighlightstheimportanceoffieldstudiesandhypothesis-generating

researchtounderstandnovelcharacteristics.


https://doi.org/10.1101/184762

13

Although our genetic analysis has suggested that T cell activation is involved in

determiningdiseaseoutcome,itwasambiguousifthereducedlevelsobservedinlatent

infections relative to HAT patients was a cause or simply the result of low blood

parasitemia. We therefore utilized experimental infections in a mouse model for

trypanosomiasisusingaspecificinhibitorofco-stimulationdependentTcellactivation.

ThisconfirmedthatreducedTcellactivationduringtrypanosomeinfectioncanleadto

lowerparasitemiaand improvedhostcondition.This isan importantvalidationof the

hypothesis that T cell activation plays a central role in determining disease severity,

whichemergedfromthetranscriptomeandexomestudy.

OurdatasuggeststhatdecreasedTcellactivationassociateswithalatentphenotype,both

inthefieldandinanexperimentalmodel,itisunclearfromourdatahowTcellactivation

leadstohigherparasitemiaandgreatersymptomseverity.Apossiblecontributorisasub-

populationof IFNγ-secretingCD8+Tcells thathasbeenshown tobeactivatedduring

trypanosomeinfection(Olssonetal.1991).WhileIFNγandaTh1immuneenvironment

isassociatedwithcontrolofparasitaemiaearlyininfection(HertzandMansfield1999;

Shietal.2003;Hertzetal.1998;Shietal.2006),elevatedorsustainedIFNγproduction

canleadtomoreseveresymptomsafterthefirstpeakofparasitemia(Namangalaetal.

2001; Baetselier et al. 2001). The importance of a switch from a Th1 to Th2 immune

environmenthasalsobeenshowntobecriticalinmiceinfectedwithT.b.gambienseand

animals thatmaintainhigh levelsof IFNγdevelopmoreseveresymptoms(Inoueetal.

1999).Lackofexpansion in the IFNγ-secretingCD8+Tcellpopulation,either through

geneticpolymorphismsorexperimentallyviaCTLA4-Ig,mayleadtoreducedIFNγactivity

and an immune environment better able to control the parasite over the long-term.

Interestingly, the most significantly associating SNPs from our study were found

specifically in a class IHLA gene involved in CD8+ T cell presentation. Several other

factors have been shown to modify the severity of symptoms during trypanosome


https://doi.org/10.1101/184762

14

infection,suchasthecytokineIL-27thatamelioratespathogenicitycausedbyactivated

CD4+Tcells(Liuetal.2015).ItisthereforelikelythatmanyTcellassociatingfactorsact

in concert to generate an asymptomatic or latent phenotype and further studies are

required.Nevertheless,wehypothesizethatdifferentialTcellactivationcontributesto

therangeofdiseaseseverityphenotypesobservedintrypanosomeinfections,withlatent

infections associating with a relatively reduced T cell activation and clinical cases

associatingwithelevatedTcellactivation.

Finally,we have demonstrated that trypanosome-induced T cell activationmay be an

appropriate target for intervention to reduce symptoms and improve outcome.While

directlyreducingTcellactivationintheanimalmodelreducedparasitemiaandimproved

animalcondition,elicitingsuchaprofoundreductioninTcellactivityisnotdesirablein

humansduetothevarietyofinfectionspatientsarelikelytoencounterinsub-Saharan

Africa.Instead,targetingtheparasitemoleculesthatinduceTcellactivationmayserveas

aneffectivealternative.Priorworkhasshownthatepitopesfoundonparasitevariable

surfaceglycoprotein(VSG)canelicitaTcellresponse(Mansfield1994)andvaccination

withVSGGPI-anchorinananimalmodelcanleadtoreducedpathogenicity(Magezetal.

2002). Additionally, T. brucei possesses a protein, termed trypanosome lymphocyte

triggeringfactor(TLTF),thatcandirectlyinduceactivationinIFNγ-releasingCD8+Tcells

(Vaidya et al. 1997). This protein has also been demonstrated to be present in T. b.

gambiense(Bakhietetal.1996).Interestingly,TLTFispredictedtointeractdirectlywith

CD8 and polymorphisms in CD8 or class 1 HLA allelesmay therefore impact activity

(Olssonetal.1993).AntibodiesraisedagainstTLTFsuppressthereleaseofIFNγby in

vitroculturedTcells,althoughthetherapeuticpotentialoftheseantibodieshasnotbeen

assessed (Hamadien et al. 1999). By targeting the parasite factors that elicit T cell

activation and the expansion of the IFNγ-secreting CD8+ T cell population, itmay be

possible to elicit a protective immune environment that mirrors the latent infection


https://doi.org/10.1101/184762

15

phenotype. Our field and animal model data suggests that this will allow patients to

controlinfection,improvingoutcome.

In summary,our studysuggests thatgenes involved in regulatingT-cell activationare

importantindetermininghumanT.b.gambienseinfectionoutcomes.Thisbuildsonprior

work demonstrating an associationwith APOL1 using the same patient samples. Our

studyisalsoanexampleofsuccessfullyapplyingtranscriptomeandgenetichypothesis-

generatingresearchtoidentifylociandpathwaysassociatingwiththedevelopmentofa

latentasymptomaticphenotypeinahumandisease. Importantly,wehaveverifiedthis

predicted association using an animal model to clearly demonstrate the relevance of

identifiedfactors.

Methods

StudyDesign

ParticipantswereidentifiedduringsurveysorganizedbytheGuineanNationalControl

Programme(NCP)between2007and2011fromthreefociinGuinea(Dubreka,Boffa,and

Forecariah)(Camara et al. 2005) according to theWHOandNational Control Program

policies(Ilboudoetal.2011).Foreachparticipant,500ulofplasmawerecollectedand

twomlofbloodweretakenonPAXgenBloodRNAtubes(PreAnalytix).Allsampleswere

frozeninthefieldat-20°Candwerethenstoredat-80°CatCIRDES(CentreInternational

de Recherche-Développement sur l’Elevage en zone Subhumide) until use.. For each

individual,theplasmawasusedtoperformtheimmunetrypanolysistest(TL)todetect

surfaceantigensspecificforT.b.gambiense(LiTat1.3,LiTat1.5)(Jamonneauetal2010).

Ofthetotalcohort,anage-matchedselectionof14clinicalcases(characterizedasCard

Agglutination Test for Trypanosomiasis (CATT) positive and trypanosomeswere also

detectedusingaminiAnionExchangeCentrifugationTechnique(mAECT), followedby

microscopyand/orexaminationofcervicallymphnodeaspiratesbymicroscopywhen


https://doi.org/10.1101/184762

16

adenopathieswerepresent),14 latent infections(CATTplasmatitre1/8orhigher,TL

positive,notrypanosomesdetectedbymeanofmAECTand/orexaminationofcervical

lymphnodeaspirateduringatwo-yearfollow-up),and7uninfectedcontrols(negativeto

theCATT,negativetothemAECTandnegativetoTL)wereusedinthisstudy(meanage

37years±17).RNAswereextractedfrombloodcollectedonPAXgenBloodRNAtubes

using the PAXgen® Blood RNA kit (PreAnalytiX) according to manufacturer’s

instruction. Twoof theHATpatientsweredetermined tobe relatedafter sequencing,

addingpowertothesubsequentPLINKanalysis.

Ethicsapprovalandconsenttoparticipate

All sampleswere collectedaspartofmedical surveyandepidemiological surveillance

activities supervised by the Guinea National Control Program. All participants were

informedof theobjectivesof the study in theirown languageandsignedan informed

consent form.Thisstudyispartof twolargerprojects.Thefirst isaimedat improving

diagnosis for which approval was obtained from the WHO (Research Ethics Review

Committee), Institut de Recherche pour le Développement (Comité Consultatif de

Déontologie et d'Ethique), and the University of Glasgow (MVLS Research Ethics

Committee)ethicalcommittees.ThesecondstudyisTrypanoGEN,partoftheH3Africa

consortium, aimed at identifying the underlying genetic determinants of human

susceptibility/resistance to trypanosome infections (Ilboudo et al. 2017), and ethical

approvalwassoughtfromallsubjectsundertheH3Africaconsortiumguidelines.

TranscriptomeAnalysis

Illumina paired-end sequencing libraries were prepared from PBMC RNA using an

IlluminaTruSeqstrandedmRNAkit.Fragment lengthranged from100-500bp,witha

median length of 150 bp. Libraries were sequenced by standard procedure on the

Illumina HiSeq platform to yield paired-sequence reads 75 bp in length. Reads were


https://doi.org/10.1101/184762

17

aligned to the complete human cDNA library (GRCh38.79) using bowtie2-2.2.5

(LangmeadandSalzberg2012).TranscriptcountswereextractedusingacustomPERL

scriptanddifferentialexpressionandsignificancecalculatedusingDeSeq2v2.11(Loveet

al.2014) forR.Themeanalignmentratepersample forpairedreads to thereference

genomewas87.94%.

PathwayAnalysis

Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) Software

346717M (Qiagen) with a dataset of differentially expressed genes (adjusted p

value<0.1),correspondingtoapproximately1,000transcripts.Thedatasetwasanalyzed

usingtheIPAcoreanalysistooltoidentifybothdirectandindirectrelationships,usingall

datasourcesandtissuetypesbutlimitedtoexperimentallyobservedrelationshipsamong

humanpatientdata.

SNPAssociationAnalysis

Illumina reads from the transcriptome analysis were aligned to the human genome

(GRCh38.79)usingBowtie2-2.2.5.Theaveragereaddepthforcodingregionswas22.12.

AsequencepileupwascreatedusingSAMtools1.2andsinglenucleotidepolymorphisms

(SNPs)calledfortheentirepopulationusingBCFtools1.2.SNPswerefurtherfilteredto

createafinalSNPdatasetcontainingbinarySNPsofatleastBCFtoolsquality20.Genes

and genomic features containing polymorphic loci were annotated using SnpEff4.1G.

Associating SNPs were identified using PLINK1.7 with the standard association test,

adaptivepermutationandadjustmentforfalsediscoverytogenerateempiricalp-values.

fastSTRUCTUREwasusedtoestablishanyunderlyingstructureinthegenomicdatafor

valuesofKupto10(Rajetal.2014).Posthocstatisticalpowerwascalculatedusingthe

formula:


https://doi.org/10.1101/184762

18

𝑁" = {𝑧"&'(∗ 𝑝 ∗ 𝑞 ∗ (1 +

1𝑘) + 𝑧&1 ∗ 𝑝" ∗ 𝑞" + (

𝑝( ∗ 𝑞(𝑘

)}(/Δ(

𝑞" = 1 − 𝑝"𝑞( = 1 − 𝑝(

𝑝 =𝑝" + 𝑘𝑝(1 + 𝐾

𝑞 = 1 − 𝑝

p1,p2=proportionsofaSNPinthelatentandHATgroups

Δ=|p2-p1|=absolutedifferencebetweenthetwoproportions

n1,n2=samplesizes

α=probabilityoftypeIerror(0.05)

β=probabilityoftypeIIerror

z=criticalZvalueforagivenαorβ

k=ratioofsamplesizeforgroup#2togroup#1 Eq1.

Sets of SNPs within gene-coding regions were grouped and additional set-based

association tests were performed using a maximum of 10,000 permutations to find

identifygeneassociations.

Infectionandstrains

Fortheanimalmodeloftrypanosomiasis,6-8weekoldfemaleBALB/cmicewerehoused

inaspecificpathogen-freeenvironmentandwereusedinaccordancewithHMGHome

Office regulations. Bloodstream forms of the genome strain Trypanosoma brucei

TREU927wereusedforallassays(Gibson2012).Micewereinoculatedintraperitoneally

with 104 parasites. Parasitemia was assayed on each subsequent day using phase

microscopy(Lumsden1963).

Flowcytometry


https://doi.org/10.1101/184762

19

SinglecellsfrombloodandspleenswereincubatedwithFcblockfor10minutesbefore

staining for 40minuteswith fluorochrome-conjugated antibodies raised against CD8a

(APC anti-CD8a Biolegend #100712), CD4 (APC anti-CD4 Biolegend #100408), and

several immune checkpoint proteins to assess activation (AlexaFluor488 anti-ICOS

Biolegend#313514,PerCP5.5anti-PD1Biolegend#109120,andPEanti-LAG3Biolegend

#125208). Cells were washed, fixed with 4% paraformaldehyde, and analyzed on a

MACSquantFlowCytometer(Miltenyi).Subsequentpopulationgatingandanalysiswas

performedusingFlowJosoftware.

Invivotreatments

CTLA4-Ig was provided by Bristol-Myers Squibb and administered by intraperitoneal

injectionatadoseof10mg/kgeverysecondday.Theinitialdosewasadministeredatday

-1relativetoinoculation.HumanIgG1wasusedasanisotypecontrolinthesestudies.

DataAccess

TheRNASeqdatasetsassociatedwiththestudyareavailableintheEBIrepositoryunder

theprimaryaccessionnumberPRJEB21742.

Acknowledgements

TheauthorswouldliketothankProf.EileenDevaneyforadviceinwritingthemanuscript

and to staff at Glasgow Polyomics for their support sequencing the samples. The

sequencing and analysis undertaken in the project was funded by a Wellcome Trust

Institutional Strategic Support Fund Catalyst Grant (097821/Z/11/B) and a Tenovus

ScotlandProjectGrant(S14/18)awardedtoPCandaWellcomeTrustSeniorFellowship

awarded to AM (095201/Z/10/Z). Funding for the Wellcome Centre for Molecular

Parasitology isprovidedby theWellcomeTrust (085349).The fundershadno role in


https://doi.org/10.1101/184762

20

studydesign,datacollectionandinterpretation,orthedecisiontosubmittheworkfor

publication.

References

BaetselierPD,NamangalaB,NoelW,BrysL,PaysE,BeschinA.2001.AlternativeversusclassicalmacrophageactivationduringexperimentalAfricantrypanosomosis.InternationalJournalforParasitology31:575–587.

BakhietM,BüscherP,HarrisRA,KristenssonK,WigzellH,OlssonT.1996.DifferenttrypanozoanspeciespossessCD8dependentlymphocytetriggeringfactor-likeactivity.ImmunologyLetters50:71–80.

BiswasSK,MantovaniA.2014.Macrophages:BiologyandRoleinthePathologyofDiseases.eds.S.K.BiswasandA.Mantovani.Springer,NewYork,NY.

CamaraM,KabaD,KagbaDounoM,SanonJR,OuendenoFF,SolanoP.2005.[HumanAfricantrypanosomiasisinthemangroveforestinGuinea:epidemiologicalandclinicalfeaturesintwoadjacentoutbreakareas].MedTrop(Mars)65:155–161.

CapewellP,Cren-TravailléC,MarchesiF,JohnstonP,ClucasC,BensonRA,GormanT-A,Calvo-AlvarezE,CrouzolsA,JouvionG,etal.2016.Theskinisasignificantbutoverlookedanatomicalreservoirforvector-borneAfricantrypanosomes.eLifeSciences5:e17716.

CooperA,IlboudoH,AlibuVP,RavelS,EnyaruJ.2017.APOL1renalriskvariantshavecontrastingresistanceandsusceptibilityassociationswithAfricantrypanosomiasisrenalriskvariantshavecontrastingresistanceand….eLifeSciences.

Cruz-RoblesD,ReyesPA,Monteón-PadillaVM,Ortiz-MuñizAR,Vargas-AlarcónG.2004.MHCclassIandclassIIgenesinMexicanpatientswithChagasdisease.HumImmunol65:60–65.

EtoD,LaoC,DiToroD,BarnettB,EscobarTC,KageyamaR,YusufI,CrottyS.2011.IL-21andIL-6arecriticalfordifferentaspectsofBcellimmunityandredundantlyinduceoptimalfollicularhelperCD4Tcell(Tfh)differentiation.ed.L.N.F.Poh.PLoSONE6:e17739.

FakharM,MotazedianMH,HatamGR,AsgariQ,KalantariM,MohebaliM.2013.AsymptomatichumancarriersofLeishmaniainfantum:possiblereservoirsforMediterraneanvisceralleishmaniasisinsouthernIran.AnnalsofTropicalMedicine&Parasitology102:577–583.

GibsonW.2012.TheoriginsofthetrypanosomegenomestrainsTrypanosomabruceibruceiTREU927,T.b.gambienseDAL972,T.vivaxY486andT.congolenseIL3000.ParasitesVectors5:71.

GineauL,CourtinD,CamaraM,IlboudoH,JamonneauV,DiasFC,TokplonouL,MiletJ,MendonçaPB,CastelliEC,etal.2016.HumanLeukocyteAntigen-G:APromisingPrognosticMarkerofDiseaseProgressiontoImprovetheControlofHumanAfricanTrypanosomiasis.ClinInfectDis63:1189–1197.


https://doi.org/10.1101/184762

21

HamadienM,LyckeN,BakhietM.1999.Inductionofthetrypanosomelymphocyte-triggeringfactor(TLTF)andneutralizingantibodiestotheTLTFinexperimentalafricantrypanosomiasis.Immunology.

HentgesF,HoffmannA,OliveiradeAraujoF,HemmerR.1992.ProlongedclinicallyasymptomaticevolutionafterHIV-1infectionismarkedbytheabsenceofcomplementC4nullallelesattheMHC.ClinExpImmunol88:237–242.

HertzCJ,FilutowiczH,MansfieldJM.1998.ResistancetotheAfricantrypanosomesisIFN-gammadependent.JImmunol161:6775–6783.

HertzCJ,MansfieldJM.1999.IFN-γ-DependentNitricOxideProductionIsNotLinkedtoResistanceinExperimentalAfricanTrypanosomiasis.CellularImmunology192:24–32.

IlboudoH,Bras-GonçalvesR,CamaraM,FloriL,CamaraO,SakandeH,LenoM,PetitdidierE,JamonneauV,BuchetonB.2014.Unravellinghumantrypanotolerance:IL8isassociatedwithinfectioncontrolwhereasIL10andTNFαareassociatedwithsubsequentdiseasedevelopment.ed.M.Boelaert.PLoSPathogens10:e1004469.

IlboudoH,JamonneauV,CamaraM,CamaraO,DamaE,LenoM,OuendenoF,CourtinF,SakandeH,SanonR,etal.2011.DiversityofresponsetoTrypanosomabruceigambienseinfectionsintheForecariahmangrovefocus(Guinea):perspectivesforabettercontrolofsleepingsickness.MicrobesInfect13:943–952.

IlboudoH,NoyesH,MulindwaJ,KimudaMP,KoffiM,KaboréJW,AhoutyB,NgoyiDM,FatakiO,SimoG,etal.2017.IntroducingtheTrypanoGENbiobank:AvaluableresourcefortheeliminationofhumanAfricantrypanosomiasised.A.Caccone.PLoSNeglectedTropicalDiseases11:e0005438.

InoueN,InoueM,KurikiK,YamaguchiH,NagasawaH,MikamiT,FujisakiK,SuzukiN,HirumiH.1999.Interleukin4isacrucialcytokineincontrollingTrypanosomabruceigambienseinfectioninmice.VeterinaryParasitology86:173–184.

JamonneauV,BuchetonB,KaboréJ,IlboudoH,CamaraO,CourtinF,SolanoP,KabaD,KambireR,LingueK,etal.2010.RevisitingtheImmuneTrypanolysisTesttoOptimiseEpidemiologicalSurveillanceandControlofSleepingSicknessinWestAfricaed.D.K.Masiga.PLoSNeglectedTropicalDiseases4:e917.

JamonneauV,IlboudoH,KaboréJ,KabaD,KoffiM,SolanoP,GarciaA,CourtinD,LaveissiereC,LingueK,etal.2012.UntreatedhumaninfectionsbyTrypanosomabruceigambiensearenot100%fatal.ed.J.M.Ndung'u.PLoSNeglectedTropicalDiseases6:e1691.

KaboréJ,KoffiM,BuchetonB,MacleodA,DuffyC,IlboudoH,CamaraM,DeMeeûsT,BelemAMG,JamonneauV.2011.FirstevidencethatparasiteinfectingapparentaparasitemicserologicalsuspectsinhumanAfricantrypanosomiasisareTrypanosomabruceigambienseandaresimilartothosefoundinpatients.InfectGenetEvol11:1250–1255.

KarimuriboED,MorrisonLJ,BlackA,TurnerCMR,KambarageDM,BallingallKT.2011.AnalysisofhostgeneticfactorsinfluencingAfricantrypanosomespeciesinfectioninacohortofTanzanianBosindicuscattle.VeterinaryParasitology179:35–42.


https://doi.org/10.1101/184762

22

KaushikRS,UzonnaJE,GordonJR,TabelH.1999.InnateresistancetoTrypanosomacongolenseinfections:differentialproductionofnitricoxidebymacrophagesfromsusceptibleBALB/candresistantC57Bl/6mice.ExperimentalParasitology92:131–143.

KumarPP,BischofO,PurbeyPK,NotaniD,UrlaubH,DejeanA,GalandeS.2007.FunctionalinteractionbetweenPMLandSATB1regulateschromatin-looparchitectureandtranscriptionoftheMHCclassIlocus.NatCellBiol9:45–56.

LangmeadB,SalzbergSL.2012.Fastgapped-readalignmentwithBowtie2.NatureMethods9:357–359.

LejonV,MumbaNgoyiD,KestensL,BoelL,BarbéB,KandeBetuV,vanGriensvenJ,BottieauE,MuyembeTamfumJ-J,JacobsJ,etal.2014.Gambiensehumanafricantrypanosomiasisandimmunologicalmemory:effectonphenotypiclymphocyteprofilesandhumoralimmunity.ed.W.A.Petri.PLoSPathogens10:e1003947.

LevineRF,MansfieldJM.1981.GeneticsofresistancetoAfricantrypanosomes:roleoftheH-2locusindeterminingresistancetoinfectionwithTrypanosomarhodesiense.InfectionandImmunity34:513–518.

LindbladeKA,SteinhardtL,SamuelsA,KachurSP,SlutskerL.2014.Thesilentthreat:asymptomaticparasitemiaandmalariatransmission.ExpertReviewofAnti-infectiveTherapy11:623–639.

LinsleyPS,WallacePM,JohnsonJ,GibsonMG,GreeneJL,LedbetterJA,SinghC,TepperMA.1992.ImmunosuppressioninvivobyasolubleformoftheCTLA-4Tcellactivationmolecule.Science257:792–795.

LiuG,XuJ,WuH,SunD,ZhangX,ZhuX,MagezS,ShiM.2015.IL-27SignalingIsCrucialforSurvivalofMiceInfectedwithAfricanTrypanosomesviaPreventingLethalEffectsofCD4+TCellsandIFN-γ.ed.J.E.Uzonna.PLoSPathogens11:e1005065.

LoveMI,HuberW,AndersS.2014.ModeratedestimationoffoldchangeanddispersionforRNA-seqdatawithDESeq2.GenomeBiol.

LumsdenWH.1963.Quantitativemethodsinthestudyoftrypanosomesandtheirapplications:Withspecialreferencetodiagnosis.BullWorldHealthOrgan28:745–752.

MagezS,RadwanskaM,DrennanM,FickL,BaralTN,BrombacherF,BaetselierPD.2006.Interferon-γandNitricOxideinCombinationwithAntibodiesAreKeyProtectiveHostImmuneFactorsduringTrypanosomacongolenseTc13Infections.JInfectDis193:1575–1583.

MagezS,StijlemansB,BaralT,DeBaetselierP.2002.VSG-GPIanchorsofAfricantrypanosomes:theirroleinmacrophageactivationandinductionofinfection-associatedimmunopathology.MicrobesandInfection4:999–1006.

MansfieldJM.1994.T-cellresponsestothetrypanosomevariantsurfaceglycoprotein:Anewparadigm?ParasitolToday(RegulEd)10:267–270.

MansfieldJM,PaulnockDM.2005.RegulationofinnateandacquiredimmunityinAfricantrypanosomiasis.ParasiteImmunol27:361–371.


https://doi.org/10.1101/184762

23

MertensB,TaylorK,MuriukiC,RocchiM.1999.CytokinemRNAprofilesintrypanotolerantandtrypanosusceptiblecattleinfectedwiththeprotozoanparasiteTrypanosomacongolense:protectiveroleforinterleukin-4?JInterferonCytokineRes19:59–65.

NamangalaB,DeBaetselierP,BeschinA.2009.Bothtype-Iandtype-IIresponsescontributetomurinetrypanotolerance.JVetMedSci71:313–318.

NamangalaB,NölW,DeBaetselierP,BrysL,BeschinA.2001.RelativeContributionofInterferon-γandInterleukin-10toResistancetoMurineAfricanTrypanosomosis.JInfectDis183:1794–1800.

NisoleS,StoyeJP,SaïbA.2005.TRIMfamilyproteins:retroviralrestrictionandantiviraldefence.NatRevMicro3:799–808.

OlssonT,BakhietM,EdlundC,HöjebergB,vanderMeidePH,KristenssonK.1991.BidirectionalactivatingsignalsbetweenTrypanosomabruceiandCD8+Tcells:Atrypanosome-releasedfactortriggersinterferon-γproductionthatstimulatesparasitegrowth.EurJImmunol21:2447–2454.

OlssonT,BakhietM,HöjebergB,LjungdahlÅ,EdlundC,AnderssonG,EkreHP,Fung-LeungWP,MakT,WigzellH.1993.CD8iscriticallyinvolvedinlymphocyteactivationbyaT.bruceibrucei-releasedmolecule.Cell72:715–727.

PerronGG,QuessyS,BellG.2008.AReservoirofDrug-ResistantPathogenicBacteriainAsymptomaticHostsed.D.A.Carter.PLoSONE3:e3749.

QuinnellRJ,KennedyLJ,BarnesA,CourtenayO,DyeC,GarcezLM,ShawM-A,CarterSD,ThomsonW,OllierWER.2003.SusceptibilitytovisceralleishmaniasisinthedomesticdogisassociatedwithMHCclassIIpolymorphism.Immunogenetics55:23–28.

RajA,StephensM,PritchardJK.2014.fastSTRUCTURE:VariationalInferenceofPopulationStructureinLargeSNPDataSets.Genetics197:573–589.

RiggsMM,SethiAK,ZabarskyTF,EcksteinEC,JumpRLP,DonskeyCJ.2007.AsymptomaticcarriersareapotentialsourcefortransmissionofepidemicandnonepidemicClostridiumdifficilestrainsamonglong-termcarefacilityresidents.ClinInfectDis45:992–998.

ShiM,PanW,TabelH.2003.ExperimentalAfricantrypanosomiasis:IFN-γmediatesearlymortality.EurJImmunol33:108–118.

ShiM,WeiG,PanW,TabelH.2006.ExperimentalAfricantrypanosomiasis:asubsetofpathogenic,IFN-γ-producing,MHCclassII-restrictedCD4+Tcellsmediatesearlymortalityinhighlysusceptible….JImmunol.

SpriggsMK,ArmitageRJ,StrockbineL,CliffordKN,MacduffBM,SatoTA,MaliszewskiCR,FanslowWC.1992.RecombinanthumanCD40ligandstimulatesBcellproliferationandimmunoglobulinEsecretion.JExpMed176:1543–1550.

SudarshiD,LawrenceS,PickrellWO,EligarV,WaltersR,QuaderiS,WalkerA,CapewellP,ClucasC,VincentA,etal.2014.HumanAfricantrypanosomiasispresentingatleast29yearsafterinfection--whatcanthisteachusaboutthepathogenesisandcontrolofthisneglectedtropicaldisease?ed.C.Franco-Paredes.PLoSNeglected


https://doi.org/10.1101/184762

24

TropicalDiseases8:e3349.

TabelH,WeiG,ShiM.2008.TcellsandimmunopathogenesisofexperimentalAfricantrypanosomiasis.ImmunolRev225:128–139.

VaidyaT,BakhietM,HillKL,OlssonT,KristenssonK,DonelsonJE.1997.ThegeneforaTlymphocytetriggeringfactorfromAfricantrypanosomes.JExpMed186:433–438.

VanhammeL,Paturiaux-HanocqF,PoelvoordeP,NolanDP,LinsL,VanDenAbbeeleJ,PaysA,TebabiP,VanXongH,JacquetA,etal.2003.ApolipoproteinL-Iisthetrypanosomelyticfactorofhumanserum.Nature422:83–87.

VanhollebekeB,DeMuylderG,NielsenMJ,PaysA,TebabiP,DieuM,RaesM,MoestrupSK,PaysE.2008.Ahaptoglobin-hemoglobinreceptorconveysinnateimmunitytoTrypanosomabruceiinhumans.Science320:677–681.

WeirW,CapewellP,FothB,ClucasC,PountainA,SteketeeP,VeitchN,KoffiM,DeMeeûsT,KaboréJ,etal.2016.Populationgenomicsrevealstheoriginandasexualevolutionofhumaninfectivetrypanosomes.eLifeSciences5:e11473.

SupplementalFileLegends

SupplementalTableS1.Transcriptsthatdiffersignificantlybetweensymptomatic

andlatentinfections.Transcriptssignificantlyupinlatentindividuals(orsignificantly

down insymptomaticcases),withanFDR-adjustedpvalueof<0.05determinedusing

DeSeq2.0,arehighlightedinblue.Transcriptssignificantlyupinsymptomaticcases(or

significantlydowninlatentindividuals)arehighlightedinred.

Supplemental Table S2. Disease or functional pathways that differ significantly

differbetweensymptomaticandlatentinfections.Pathwayslistedhavepvaluesof

<0.05determinedusingIngenuityPathwayAnalysissoftware.Inhibition(z-score<-2.0)

oractivation(z-score>2.0)ofapathwayinsymptomaticHATpatientsrelativetolatent

infectionsisindicated.Moleculescontributingtothepathwaypredictionarelisted.

SupplementalTableS3.Predictedupstreamregulatorsofthepathwaydifferences

observedbetweenlatentindividualsandsymptomaticcases.Regulatorslistedhave


https://doi.org/10.1101/184762

25

pvaluesof<0.05determinedusingIngenuityPathwayAnalysissoftware.Inhibition(z-

score<-2.0)oractivation(z-score>2.0)ofeachregulatorinlatentindividualsrelativeto

symptomaticcasesisindicated.

Supplemental Table S4. SNPs that significantly segregated between clinical and

latent infections. SNPs listed have an FDR-adjusted significance value of p<0.05,

assessedusingPLINK.Theallelesandproportionsofeachareshownforsymptomatic

cases and latent individuals. The odds ratio for the SNP to associate with the latent

phenotype,andtheresultingmutationtypeandgenearealsoshown.

Supplemental Table S5. SNPs that significantly segregated between clinical and

latentinfections,groupedbygene.SNPswereallocatedtogenesetsgroupsbasedon

theirpositionandanalyzedusingPLINK.Thisrevealedgenesthatsignificantlysegregated

betweenclinicalandlatentinfectionswithanominalsignificancevalueofp<0.05.The

numberofSNPSineachset,includingnon-significantandnon-significantSNPS(including

position)arelisted.

SupplementalFigureS1.ICOS,PD1,andLAG3expression(markersofactivation)

onCD4+Tcellsduringinfection.TheexpressionofICOS,PD1,andLAG3(markersof

activation)on CD4+T cells and ICOS and LAG3 on CD8+ T cells on days 5 and 11 of

experimentalTREU927trypanosomeinfectioninmice(n=5pergroup,pertimepoint),

assessed via flow cytometry. This demonstrates that CTLA4-Ig (ABA) reduces T cell

activationduringinfectionrelativetoanisotypecontrol(ISO).


https://doi.org/10.1101/184762

t cell activation and the hla locus associate with latent … · protein hla-g, were predictive of...

Documents