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Abstracts & Posters

Afternoon Poster Session 1: Posters 1 - 70

Evening Poster Session 2:Posters 71 - 142

SoutheasternImmunologySymposium

June 8-9, 2019

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Presenter Dillon Patterson 1Title IRF4regulatestheproliferativeresponseduringBcelldifferentiationin

vivoEmail dillon.patterson@emory.eduCo-Authors Madeline J. Price, Tian Mi, Qiang Zhang, Sakeenah L. Hicks, Christpher

D. Scharer, and Jeremy M. BossAffiliation EmoryUniversity

CelldivisionisanessentialcomponentofBcelldifferentiationtoaplasmacell(PC).Divisioncoupled changes in the expression of transcription factors that coordinate the PC program are known; however, little is known regarding the timing/extent of reprogramming by these factors as the cells are dividing in vivo. Furthermore, how/when these factors coordinate cell divisionduringdifferentiationhasremainedunexplored.Toaddressthis,weassessedthecelldivisionkineticsofwild-type(WT)BcellsrespondingtoLPSinvivo. Interestingly,wefoundthatWTBcellsmustundergo8divisionsbeforedifferentiating intoPCs.Computa-tionalmodelingofdivisionratesdefinedaproliferativeburstbetween48-60hoursafterLPSinjection that is characterized by a rapid increase in the rate of cell division. In contrast, Inter-feronRegulatoryFactor4-deficient(IRF4-/-)Bcellsdividedbutstalledatdivisions2-6,failingto undergo the proliferative burst. To assess the timing/scope of IRF4-dependent reprogram-ming,WTandIRF4-/-respondingBcellsatdivisions0,1,and3-6weresortedforATAC-andRNA-seq.RNA-seqdatarevealedhundredsofdifferentiallyexpressedgenes(DEGs)whenIRF4 was deleted, a subset of which included Myc target genes. ATAC-seq data also ex-posedhundredsofdifferentiallyaccessibleregions,themajorityofwhichcontainedaknownIRF4bindingmotifandmappedtoacorrespondingDEG.Interestingly,IRF4-/-Bcellspro-gressivelybecamemoredivergentastheydividedwhencomparedtoWTBcellsinthesamedivision.Together,thesedatacreatearoadmapdefiningtheroleofIRF4throughoutBcelldifferentiationandrevealacriticalroleforIRF4inmaintainingtheproliferativeresponse.

Presenter: Srilatha Edupuganti, MD 2Title: Decreased Humoral Immune Responses to Mumps in Young Adults Im-

munizedwithMMR(Mumps,MeaslesandRubella)Vaccine inChild-hood

Email: sedupug@emory.eduCo-authors: AtaUrRasheedMohammed,CaroleJ.Hickman,MarciaMcGrew,Sun

Bae Sowers,SaraMercader,AmyHopkins,VickieGrimes,Tian-weiYu, JensWrammert,Mark J.Mulligan,WilliamJ.Bellini,PaulA.Rota,WalterA.Orenstein,RafiAhmed

Affiliation: HopeClinicoftheEmoryVaccineCenter,DepartmentofMedicine,Divi-sionof InfectiousDiseases,EmoryUniversitySchoolofMedicine,At-lanta,Georgia,USA

Inthepastdecade,multiplemumpsoutbreakshaveoccurredintheUS,primarilyinclose-contact, high-density settings such as colleges, with a high attack rate among young adults manyofwhomhadtherecommended2dosesofmumps-measles-rubella(MMR)vaccine.Waninghumoralimmunityandcirculationofdivergentwild-typemumpsstrainshavebeenproposed as contributing factors to mumps resurgence. To determine mumps immunity in healthycollegestudents,werecruited71students18to23yearsofageandconfirmedre-ceipt of MMR vaccination by review of immunization records. A one-time blood sample was assayed forantibodies, IgGavidity,andmemoryBcells (MBC) tomumps,measles,andrubella. Mumps plaque reduction neutralizing antibody titers were also determined. Antibod-iesdeterminedbyIgGELISAtomumps,measles,andrubellaweredetectedin93%,93%and100%ofparticipants, respectively.Theconcentrationof IgGper indexstandard ratio(ISR)wassignificantlylowerformumpscomparedtorubella.Inparticipantswhohadsuf-ficientIgGlevels(n=57/71),highaviditywasdetectedtomumpsEndersstrain.Themeanfrequencyofcirculatingmumps-specificMBCwassignificantlylowerthanformeaslesandrubella(p<.001).In7/71participants(10%)mumps-specificMBCwereundetectable,3/71hadundetectablemeaslesMBC,andallparticipantshaddetectablerubella-specificMBC.Geometricmeanneutralizingantibodytiters(GMT)tothepredominantcirculatingwild-typemumpsstrain(genotypeG)was6-foldlowerthantheGMTsagainstthegenotypeAJeryl

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Lynnvaccinestrain ( (GMT35vs217,P<0.0001).Themajorityof theparticipants (80%)received their second MMR vaccine >10 years prior to study participation. Lower mumps IgGISRbuthighavidity,lowerGMTstocirculatingmumpswildtypestrainandnodetectablemumps-specificMBCin10%ofpreviousMMRvaccinerecipientsunderscorestheneedforcomprehensive characterization of B and T cell immune responses to mumps vaccine. Strat-egies are needed to improve the quality and durability of vaccine-induced immunity

Presenter GiuseppeA.Sautto 3Title A computationally optimized broadly reactive antigen elicits unique

broadlyneutralizingantibodiestargetingtheinfluenzavirushemaggluti-nin receptor-binding site

E-mail gasautto@uga.edu Co-authors GregA.Kirchenbaum,JeffreyW.Ecker,RodrigoB.Abreu,SpencerR.

Pierce, Ted M. RossAffiliation CenterforVaccinesandImmunology,UniversityofGeorgia,Athens,

GA,USA

Thedevelopmentofbroadlyprotectiveinfluenzavaccineswillrepresentthemaincounter-measuretoovercomeinfluenzavirusspreadandimprovethecoverageofferedbythecur-rent standard of care vaccine.A computationally optimized broadly reactive influenza virus hemagglutinin (HA) antigen(COBRA), named P1, elicits a broadly reactive and neutralizing antibody (Ab) responseagainst multiple seasonal and pandemic H1N1 strains.InordertounderstandthemechanismofCOBRAP1-conferredbreadthofresponse,wefirstcharacterizedthebreadthoftheAbresponseattheB-celllevel.Specifically,thereactivityofsecretedAbsfromBcellsofBALB/cmiceimmunizedwithCOBRAP1,seasonalorpan-demic vaccine strains was assessed against a panel of H1N1 recombinant HA. Interestingly, whileantibodysecretingcells(ASC)fromCOBRAP1-immunizedmiceexhibitedabroaderrecognition, those from seasonal or pandemic immunized animals showed a predominant homologous response.Inordertodissecttheantibodyresponse,apanelofCOBRAP1-specificB-cellhybridomaswas generated. Subsequently, the corresponding purified monoclonal Abs (mAbs) wereassessedforthebreadthofHAbinding,hemagglutinationinhibition(HAI)andneutralizingactivity against a panel of H1N1 viruses. Collectively, bothmAbs fromCOBRAP1- andseasonal or pandemic H1N1 HA antigens recognized head or stem HA epitopes. However, onlyCOBRAP1-elicitedAbsexhibitedabroadspectrumofbindingandfunctionalactivities,spanningfromstrain-specifictobroadlyreactiveandneutralizingmAbs,whileseasonal-orpandemic strains-elicited mAbs displayed a narrower spectrum of activity. Importantly,allCOBRAP1-elicitedmAbshaduniqueCDRH3sequences, suggestingdif-ferent antigen-driven somatic mutations. Finally, epitope mapping studies revealed that COBRAP1-elicitedbroadly reactiveandneutralizingmAbs recognizeduniqueconservedepitopes within the HA receptor binding site. Collectively, thesestudiesshed lighton themechanismofbreadthconferredbyCOBRAP1antigenandleverageitsoptimizationforthedevelopmentofamoreeffectiveinfluenzavaccine.

Presenter: Brianna Swartwout1,3 4Title: Lactobacillus reuteri induced IgA production is strain-dependent and

bears consequences for neonatesEmail: bkswart@vt.eduCo-authors: LeeG2,3, Mu Q3*,HardinK4, Reilly CM5, Luo XM3

Affiliation: 1 Translational Biology, Medicine, Health Graduate Program, VirginiaTech,Roanoke,VA.2 VirginiaTech-CarilionMedicalSchool,Roanoke,VA. 3Department of Biomedical Sciences and Pathobiology, Virginia-MarylandCollege-VeterinaryMedicine,VirginiaTech,Blacksburg,VA.4DepartmentofBiochemistry,CollegeofScience,VirginiaTech,Blacks-burg,VA.5EdwardViaCollege-OsteopathicMedicine,Blacksburg,VA.

CurrentAffiliation: DepartmentofMedicine,DivisionofImmunologyandRheumatology,StanfordUniversitySchoolofMedicine,CA.

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IgA is the antibody responsible for protecting against foreign invaders at mucosal sites. At 50mg/kgproducedperday, it is themostabundantantibody in thebody.Antibodydiver-sificationduringearlydevelopment isstrictlyregulated,thustherepertoireofendogenousIgA antibodies in neonates are immature, have a lower frequency of somatic mutation, and havea loweraffinity forantigen.Themechanismofendogenousneonatal IgAproductionis still unknown. Mice studies show that early endogenous IgA production can be induced by nursing immunocompetent pups on immunocompromised dams.We hypothesize thatthe microbiota transferred through milk may play a critical role in instigating this increase in endogenous IgA production. Comparing milk from competent and compromised mice shows an increase in the species Lactobacillus reuteri. Evidence exists that L. reuteri can induce IgAproduction,butourdatasuggestthatthisfeatureisstrain-dependent.Outof4strainsanalyzed(CF48-3A,6475,100-23,and2010),CF48-3AmostpotentlyinducedIgAproduc-tion in spleen and intestinal lamina propria cultures. in vitro B-cell cultures showed that in-activatedbacteriaaloneweresufficient to induce increasedIgAproduction,andCF48-3Awas consistently more potent than non-stimulatory strains. This phenomenon was found to correlatewithincreasedproliferationofplasmablasts.Therefore,specificstrainsofL. reuteri, and other stimulatory bacteria, may directly perpetuate the presence of IgA positive B-cells in the neonatal gut by inducing proliferation of antibody secreting cells.

Presenter JingjingRen 5Title Selective histone deacetylase 6 inhibition normalize B cell activation

and germinal center formation in a model of systemic lupus erythemato-sus

Email renjj@vt.eduCo-Authors MichelleDCatalina,KristinEden,XiaofengLiao,KaitlinRead,XinLuo,

Ryan P McMillian, Hatt Hulver, Matthew Jarpe, Prathyusha Bachali, Am-rieC.Grammer,PeterE.Lipsky,ChristopherM.Reilly

Affiliation VirginiaTech,AMPELBioSolution,Edwardviacollageofosteopathicmedicine

Autoantibodyproductionbyplasmacells(PCs)playsapivotalrole inthepathogenesisofsystemic lupus erythematosus (SLE). Themolecular pathways bywhichB cells becomepathogenic PC secreting autoantibodies in SLE are incompletely characterized. Histone de-actylase6(HDAC6)isauniquecytoplasmicHDACthatmodifiestheinteractionofanumbertubulin-associatedproteins;inhibitionofHDAChasbeenshowntobebeneficialinmurinemodelsofSLE,butthedownstreampathwaysaccountingforthetherapeuticbenefithavenotbeen clearly delineated. In the current study, we sought to determine whether selective his-tonedeacetylase(HDAC)6inhibitionwouldabrogateabnormalBcellactivationinSLE.Wetreated20-week-oldNZB/WlupusmicewiththeselectiveHDAC6inhibitor,ACY-738,forfourweeks beginning at 20 weeks-of age. After only 4 weeks of treatment, manifestation of lupus nephritis(LN)weregreatlyreducedintheseanimals.WethenusedRNAseqtodeterminethe genomic signatures of splenocytes from treated and untreated mice and applied compu-tational cellular and pathway analysis to reveal multiple signaling events associated with B cellactivationanddifferentiationinSLEthatweremodulatedbyHDAC6inhibition.PCde-velopmentwasabrogatedandgerminalcenter(GC)formationwasgreatlyreduced.WhentheHDAC6inhibitortreatedlupusmousegenesignatureswerecomparedtohumanlupuspatientgenesignatures,theresultsshowednumerousimmuneandinflammatorypathwaysincreasedinactivehumanlupusweresignificantlydecreasedintheHDAC6inhibitortreatedanimals. Pathway analysis suggested alterations in cellular metabolism might contribute to thenormalizationof lupusmousespleengenomicsignatures,and thiswasconfirmedbydirectmeasurementoftheimpactoftheHDAC6inhibitoronmetabolicactivitiesofmurinespleencells.Takentogether,thesestudiesshowHDAC6inhibitiondecreasesBcellactiva-tionsignalingpathwaysandreducesPCdifferentiationinLNandsuggestthatacriticaleventmight be modulation of cellular metabolism.

Presenter: RodrigoB.Abreu 6Title: Longitudinal Assessment of Memory B cell and Plasmablast Reactivity

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AgainstInfluenzaAinSubjectsofVaryingAgeEmail: rbabreu@uga.eduCo-Authors: GregA.Kirchenbaum1, Emily F. Clutter1 and Ted M Ross1,2

Affiliation: 1Center forVaccinesand Immunology, 2Department of Infectious Dis-eases,UniversityofGeorgia,Athens,GA,USA

Influenzaisahighlycontagiousviralrespiratorydiseasethataffectsmillionsworldwideeachyear.AnnualvaccinationisrecommendedbytheWorldHealthOrganizationwiththegoaltoreduceinfluenzaseverityandlimittransmissionthroughelicitationofantibodiestargetingthehemagglutinin(HA)glycoprotein.Theantibodyresponseelicitedbycurrentseasonalin-fluenzavaccinesispredominantlystrain-specific;however,continuousantigenicdriftbycir-culatinginfluenzavirusisolatesfacilitatesescapefrompre-existingantibodiesandrequiresfrequent reformulation of seasonal influenza vaccine. Furthermore, pre-existing influenzaimmune responses can greatly impact the serological antibody response to vaccination. However, it remains unclear how B cell memory is shaped by annual vaccination over the course of multiple seasons, especially in high risk elderly populations. Here, we systemati-callyprofiledtheBcellresponseinyoungadult(18-34yearsold)andelderly(65+yearold)vaccinerecipientsthatreceivedannualsplitinactivatedinfluenzavaccinationfor3consecu-tiveseasons.Specifically,wequantifiedchangesinfrequencyofmemoryBcellandplasma-blastinperipheralbloodprior,7and21daysaftervaccinationover3consecutiveseasons.Additionally,wetrackedthefrequencyofvaccine-elicitedH1andH3-reactivememoryBcells(Bmem)byflowcytometryusingtetramericrHAprobesablatedforsialicacidbindingactivity.Moreover,toassesstheimpactofsequential influenzavaccinationontheBmem repertoire, we in vitro-differentiateddonorPBMCscollectedover3consecutiveyearsandevaluatedthe breath of Bmem-derived antibodies against a panel of rHA by ELISA. In summary, we show that vaccine elicited Bmem and plasmablast expansion is impaired in elderly subjects compared to young adults and that the memory B-cell repertoire is transiently reshaped by annualTIVvaccination.Collectively,thesestudieswillshedinvaluableinsightintohowpre-existingimmunityshapesthememoryBcellresponsetorecurrentinfluenzavaccination.

Presenter Byron Au-Yeung 7Title BasalTCRsignalingdrivesthefunctionalheterogeneityofnaïveCD4+

T cellsEmail byron.au-yeung@emory.eduCo-authors WendyZinzow-Kramer,ArthurWeiss,andByronAu-YeungAffiliation EmoryUniversity

Prior to their activation with peptides derived from foreign proteins presented by MHC Class II (pMHC), naïveCD4+T cells experienceweakTCRsignals in response to self-pMHC.These weak “basal” TCR signals in response to self-antigens do not result in IL-2 production orcelldivision,thoughtheymayinfluenceresponsestoforeignantigens.Weaimedtode-termine whether relatively strong basal TCR signaling introduces a bias toward more robust activationtoforeignantigenstimulation.AssessinghowthesebasalTCRsignalsinfluenceT cell function upon foreign antigen stimulation requires a method for visualizing the relative strengthofbasalTCRsignalinginsinglecells.Wefoundthatthecombinationofatransgenicreporter of antigen receptor signaling (Nur77-GFP) and staining for the surface receptorLy6CenablesvisualizationofabroadrangeofbasalTCRsignaling.CellsexperiencingtheweakestbasalTCRsignalsareGFPlowLy6C+,whereascellsexperiencingthestrongestbasalTCRsignalsareGFPhighLy6C–.ThoughTCRspecificitycanskewthedistributionofNur77-GFPandLy6Cexpression,wefindthatthereisheterogeneityinthenaïveCD4+Tcellpopulation,eveniftheyexpressidenticaltransgenicTCRs,suchastheOT2orANDTCR. Furthermore, adoptive transfer studies indicate that expression of a given amount of Nur77-GFPandLy6Cisrelativelystable.Subpopulationsofcellsexperiencingweak,strong,orintermediatebasalTCRsignalsweresortedandstimulatedwithanti-CD3antibodiesorcognate peptide and antigen presenting cells. Cells experiencing weak basal TCR signaling generate more sustained IL-2 responses and proliferate to a greater extent than cells experi-encingmoderatebasalTCRsignalstrength.TheGFPhighLy6C–cellsconsistentlymountedattenuated IL-2 and proliferative responses, and expressed proteins associated with anergy, suchasGrailandCbl-b.WeproposethatthispopulationofGFPhighLy6C–naïveCD4+

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cells adapts to strong basal TCR signals by initiating negative regulation that attenuates their response to foreign antigen stimulation.

Presenter: PeytonVanWinkle 8Title: Characterizing the role of Jagunal homolog 1 protein in neutrophil func-

tionEmail: pev18@uab.eduCo-Authors: PeytonVanWinkle,TomaszNawara,HollyThomas,EunjooLee,Da-

mianKuna,PiotrStasiak,ZdenekHel,JohnParantandElizabethSztulAffiliation: UniversityofAlabamaatBirminghamThehighlyconservedendoplasmic reticulumproteinJagunal (JAGN1)wasfirst identifiedasarequirementforDrosophilamelanogasteroocytedevelopment.Subsequently,JAGN1mutationswerecorrelatedwithSevereCongenitalNeutropenia (SCN) inhumanpatients.SCN is characterized by arrested granulocytic maturation in the promyelocyte stage. Neutro-philsdeficientinJAGN1havealteredgranules,diminishedfungal-killingcapacity,andaber-rantglycosylationofproteins.Overall,individualswithmutationsinJAGN1aresusceptibleto bacterial and fungal infections due to decreased number of circulating neutrophils and impaired release of granule content by the remaining cells. Despite its physiological impor-tance,thecellularroleofJAGN1ispoorlyunderstood.Ourgoalistouseazebrafishmodel,alongwithmammalian cells in culture, to define themolecular functions of JAGN1. Thezebrafish(Daniorerio)offersauniqueadvantageforneutrophilstudiesastheyaretranslu-cent, and neutrophil migration, degranulation and pathogen killing can be visualized in intact tissuesinrealtime.ZebrafishhavetwohomologsofhumanJAGN1-JAGN1aandJAGN1b,an additional advantage since each may have a distinct function, providing additional infor-mationaboutthemolecularmechanismofJAGN1action.WehavegeneratedhomozygousJAGN1a-/-andJAGN1b-/-knockoutfish,andarenowpoisedtoelucidatetheroleoftheseproteinsbymonitoringneutrophildifferentiationandbyvisualizingneutrophilmigrationandbehaviorduringwoundhealinginintactanimals.AhumanHL-60myeloidleukemiacelllinecapableofdifferentiationintoneutrophil-likecellswillbeusedtoanalyzegranuleformationintheabsenceofJAGN1,definethestepofsecretionrequiringJAGN1function,andidentifyingJAGN1interactors

Presenter Zerick Dunbar1,2,3 9Title DISSECTIONOFNATURALKILLERCELLANTI-TUMORRECRUIT-

MENT,MATURATIONANDEFFECTORFUNCTIONEmail zdunbar18@email.mmc.eduCo-Authors Anil Shanker2,3,4,5Affiliation 1Department of Microbiology, Immunology and Physiology,School of

Medicine, Meharry Medical College, Nashville, TN 2Department of Bio-chemistry, Cancer Biology, Neuroscience & Pharmacology, School of Medicine, Meharry Medical College, Nashville, TN 3SchoolofGraduateStudies and Research, Meharry Medical College, Nashville, TN 4Host-TumorInteractionsResearchProgram,Vanderbilt-IngramComprehen-siveCancerCenter,VanderbiltUniversitySchoolofMedicine,Nashville,TN, 5Vanderbilt Institute for Infection, Immunology and Inflammation,VanderbiltUniversitySchoolofMedicine,Nashville,TN

Anissueinthefightagainstcanceristhegrowingdiscrepancybetweenresourcesbeingap-plied to treat cancer and stagnant patient survival rates. In part to help decrease this discrep-ancy by improving patient outcomes, research ongoing into immunotherapy uses one’s own immune cells to combat cancer cells. Cytotoxic innate lymphoid cells, conventionally known asnaturalkiller(NK)cells,playmajorrolesincancerimmunitylargelyduetotheirnaturalcy-tolytic and immune regulatory functions. However, due to the extensive heterogeneity seen amongNKcells,theirfunctionshaveyettobefullyharnessed.Theobjectiveofthisresearchproject is toelucidateNKcellmaturation, recruitmentandeffector function in thecontextofsolidtumormicroenvironments.Usingmulti-colorflowcytometryandfunctionalassays,wewill characterizeNKcellsover timeatdifferent tumorstages, localizedversusdistantmetastatictumors.WehypothesizethattumorinfiltratingNKcellsdisplayauniquegenetic

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andfunctionalprofilewithahigherexpressionofdevelopmentandrecruitmentmarkerssuchas ID2, NCAM1andNotch receptors,aswellas increasedeffector functioncompared tonon-tumorinfiltratingNKcells.Here,weshowtheheterogeneitythatexistsamongNKcellsfromdifferentlocations,aswellasNKcellspecificclusterofdifferentiationmarkersandgeneexpressionperspectivesbasedonflowcytometry,RNA,andbioinformaticsanalyses.WewillalsoexplorepotentialmechanismsthatcanbealteredforenhancingNKcellanti-tumorfunc-tionsuchastreatmentwiththeFDAapproveddrugBortezomib.Understandingandsubse-quentlymanipulatingNKcellsinthetumormicroenvironmentcouldprovetobeanovelclini-cal immunotherapy tool for patients and mitigate the resources-results cancer discrepancy.

ThisworkwassupportedbyfundstoAShankerviathefollowingNIHgrants:U54CA163069,U54MD007593,SC1CA182843andR01CA175370;andsupported, inpart,by theNIHRISEgrantR25GM059994.

Presenter Ichiro Misumi 10Title TcellresponsestoHepatitisAVirusinamousemodelEmail imisumi@email.unc.eduCo-authors JosephMitchell,MakaylaLund,StanLemon,andJasonWhitmireAffiliation UniversityofNorthCarolinaatChapelHill,DepartmentofGenetics

Hepatotropicvirusesaremajorcausesofhumandisease.HepatitisAvirus(HAV)istrans-mittedfrompersontopersonthroughtheoral-fecalrouteandcausesacutehepatitis.Glob-ally,thereare~1.4millioncasesofhepatitisAeveryyear.IntheUS,sporadichepatitisAoutbreakshavebeenoccurringmorefrequently.Morethan15,000casesand140deathswerereportedinthelastthreeyears(CDCreport,2019).HAVinfectioncanbepreventedby a vaccine that induces strong humoral immunity. However, the contribution of T cells to viruscontrolandtheunderlyingmechanismsofpathogenesishavebeendifficulttostudyduetothelackoftractablesmallanimalmodels.OurgrouprecentlyshowedthatIfnar1-/- mice arepermissiveforHAVinfectionanddevelopcriticalfeaturesoftypeAhepatitis,includingacuteliverinjuryandinflammatoryimmuneresponses,thoughdeclininglevelofviralRNAcouldbefoundformonthsafterinfection.Usingthismodelandmouse-passagedHAV,wemappedCD8+andCD4+TcellepitopesandtrackedTcellresponsesovertimeafterinfec-tion.MurineTcellresponsestoHAVemergedonlyafterseveralweeksofinfection,similartoresponsesininfectednon-humanprimates.HAV-specificCD4+andCD8+Tcellsweremostabundant in the liver,coincidentwithdeclines in fecalvirussheddingand liverHAVRNA. Moreover, T cell depletion resulted in an increase in infection and ALT release in both Ifnar1-/- and the chimeric mice. Finally, T cells accumulated PD1 expression over time, and interference with the PD1-PDL1 pathway improved immune control. Collectively, these re-sultsdemonstratethatTcellsprotectagainstHAVinfectionanddonotcausepathogenesis.Further, this study reveals that mice with defects in interferon responses can serve as a modelforinvestigatingHAV-specificTcellresponses.

Presenter Bhalchandra Mirlekar 11Title IL-35+ B cells establish immunosuppressive network in pancreatic

ductal adenocarcinomaE-mail rmirlekar@med.unc.eduCo-Authors YuliyaPylayeva-GuptaAffiliation Department of Genetics, University of North Carolina School

of Medicine, Chapel Hill, North Carolina, 27599 and The Line-berger Comprehensive Cancer Center, University of NorthCarolina School of Medicine, Chapel Hill, North Carolina.

Pancreaticcanceristhe3rd leading cause of cancer-related death worldwide with a dismal 5-yearsurvivalrateof7percent.Unfortunately,recenteffortsgearedtowardstreatmentofpancreatic cancer with immunotherapy have not seen success. A major barrier for immu-notherapeutic approaches in pancreatic cancer is marked immunosuppression within the pancreatic tumor milieu. Mechanisms driving immunosuppression in pancreatic cancer are notwellknownandunderstandingthesemechanismsmayhelpimproveefficacyofcurrent

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immunotherapy.Ouranalysisrevealsonesuchmechanism:aspecializedsubsetofBcells,termed regulatory B cells, which promote pancreatic tumorigenesis through production of the immune-suppressivecytokineIL-35.Here,wesetouttoelucidatethecellularandmolecularmechanismsofBcellderivedIL-35 in the initiationandprogressionofpancreaticcancer.OurresultsdemonstratethatIL-35productionbyBcells,notbyTcellsfunctionsinthetumormicroenvironment and is essential for suppression of anti-tumor T cell responses. Impor-tantly, while PDAC is typically resistant to anti-PD-1 immunotherapy, we demonstrate robust synergisticreductionintumorgrowthwhenBcellspecificIL-35deficiencyiscombinedwithanti-PD-1treatment.Furthermore,weassessedtheeffectofanti-IL-35blockadeincombina-tion with immune checkpoint blockade therapy as a pharmacological approach to provide additional invigoration forTcellmediatedkillingof cancercells.Collectively,our findingswillrevealapotentialnovelfunctionofBcellderivedIL-35inthepathogenesisofpancreaticcancer and could provide therapeutic insight for patient treatment.

Presenter: AnnaKania 12Title: H3K27me3-specificdemethylases restrict themagnitudeofBcelldif-

ferentiationEmail: akania@emory.eduCo-authors: Madeline M. Price, Christopher D. Scharer, Robert R. Haines, Lou-Ella

Alexander-George,andJeremyM.BossAffiliation: DepartmentofMicrobiologyandImmunology,EmoryUniversity,Atlanta

GA

Bcell terminaldifferentiation intoantibodysecretingplasmacells (PCs)mustbeproperlyregulated to ensure robust humoral immune responses against foreign but not self-antigens. Whiletheroleoftranscriptionfactorsinthisprocessiswell-established,thereisalsoagrow-ingappreciationthatepigeneticmechanismsalsoregulateBcelldifferentiation.ThehistonemodificationH3lysine27trimethylation(H3K27me3)isassociatedwitharepressivechro-matinstate resulting ingenesilencingand ishighlydynamicduringBcell differentiation.EZH2,theenzymethatdepositsH3K27me3,isrequiredforproperBcelldevelopment,aswell as germinal center, and PC formation. However, the role of active demethylation of H3K27me3bythetwodemethylasesUTXandJMJD3inBcellsisstilltobeelucidated.Todetermine the requirements for these enzymes in B lymphocytes, we crossed Utxfl/flJmjd3fl/flmice onto the Cd19Cre/+background (dKOmice).InoculationofCd19Cre/+(CreCtrl)anddKOmicewithLPS,aTcell independentantigen,ledtoasignificantincreaseinCD138+PCsinUTX-andJMJD3-deficientmice.Thisincreasewasattributedtoaltereddifferentiationofmarginalzone(MZ)butnotfollicularBcells(FoB)inresponsetoLPS.RNA-seqanalysisofPCsgeneratedfromMZandFoBofCreCtrlanddKOmicerevealedenhancedexpressionofgenesassociatedwithoxidativephosphorylationmetabolismindKOPCs.TodeterminetheroleofUTXandJMJD3inTcelldependentBcellresponses,weinfectedCreCtrlanddKOmicewithPR8influenzavirus.Fourteendayspostinfection,weobservedanincreaseinGCBcellsandskewingintheGCdark/lightzonepolarityindKOmice.Takentogether,thesedataplaceH3K27me3demethylasesascriticalenzymesthatregulatetheepigeneticdynamics of B cell fate and act to restrict the expansion of B cells following T cell dependent and independent stimuli.

Presenter RobertHaines 13Title LSD1 cooperates with non-canonical NF-κB signaling to regulate mar-

ginal zone B cell developmentEmail r.r.haines@emory.eduCo-authors Christopher D. Scharer, Jeremy M. BossAffiliation EmoryUniversity

MarginalzoneBcells(MZB)areamatureBcellsubsetthatrapidlyrespondtoblood-bornepathogens and are transcriptionally poised to differentiate. Although the transcriptionalchanges that occur throughout MZB development are known, the corresponding epigenetic changes and epigenetic modifying proteins that facilitate these changes are poorly under-stood.TheH3K4mono-anddi-methyldemethylaseLSD1,anepigeneticandtranscriptional

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modifierthatpromotesplasmablastformation,representsaprimecandidateforregulatingMZB development because of the high degree of overlap between MZB and plasmablast transcriptomes. B cell-conditional deletion of LSD1 resulted in a two-fold decrease in MZB whilefollicularBcells(FoB)andbonemarrowBcellpopulationswereminimallyaffected.LSD1 repressed genes in MZB that were normally upregulated in FoB and are involved in adhesion, migration, interferon response, transcriptional regulation, and signaling. MZB lacking LSD1 exhibited alterations in chromatin accessibility at transcription factor family motifs, including NF-κB.TheimportanceofNF-κBsignalingwasexaminedthroughanex vivo MZBdevelopmentassay,whichshowedthatbothLSD1-deficientandNF-κB-inhibited transitionalBcellsfailedtoundergofullMZBdevelopment.Geneexpressionandchromatinaccessibility analyses of in vivo- and ex vivo-generatedLSD1-deficientMZBindicatedthatLSD1 regulated the downstream target genes of non-canonical NF-κB signaling. Immuno-precipitation experiments demonstrated that LSD1 directly interacted with the non-canonical NF-κBtranscriptionfactorp52.Overall,thesedatarevealthatsplenicBcellepigenomesaredynamicallyregulatedandthattheepigeneticmodulationoftheNF-κBpathwaybyLSD1isan essential process during MZB development.

Presenter Lou-EllaGeorge-Alexander 14Title CUT&RUNmapshistonemodificationsinlymphocyteswithhigh-resolu-

tionEmail lou-ella.alexander@emory.eduCo-Authors Sakeenah Hicks, Tian Mi, Chris Scharer, Jeremy BossAffiliation EmoryUniversity

Thesystematicprofilingofepigenomesinmultiplecelltypesand(differentiation)stagesisessential for understanding developmental processes and disease states. For this purpose Chromatinimmunoprecipitation(ChIP)hasbeenacommonlyusedtechniquethathasun-dergonelittlechangesinceitsinception30yearsago.Thismethodallowsforpreservationoftheinvivopatternofprotein-DNAcomplexesthroughformaldehydefixation,fragmenta-tion of the genome and immunoprecipitation of targets. Together with the development of next-generation sequencing platforms, this method has led to base-pair resolution mapping of transcription factors. However, the use of formaldehyde is thought to promote epitope maskingandfalsepositivebindingsites.ThisfindingdrovethedevelopmentofNative-ChIPwithout crosslinking using ionic conditions to maintain electrostatic contacts, with sensitivity andspecificitytrade-offs.Theseuncertaintiesemphasizedtheneedformethodsusingdif-ferentprinciples,whichbroughtaboutCleavageUnderTarget&ReleaseUsingNuclease(CUT&RUN).Thispreviouslydescribedtechniquetethersthemicrococcalnucleaseenzymeto protein A through a chimeric fusion, allowing for antibody directed cleavage of the protein-DNA complex of interest into solution for subsequent sequencing. In this report we assessed the sequencing resolutionachievedwith useofCUT&RUNonmouse lymphocytes.WithsomemodificationstotheoriginalprotocolwewereabletoachievecleavageandreleaseofDNAfragmentsinmurineB-andT-cellsforvarioushistonemodifications.ThereleasedDNAfragments sizes were visualized by bioanalyzer and depicted the presence of a nucleosomal ladder,whichisconsistentwiththepresenceofhistonemodificationsatnucleosomes.Thenucleosomal ladder yield gradually dissipates with lower cell numbers. Targeted cleavage wasobtainedinaslowas5,000cellsasisevidentbyourabilitytogeneratealibraryandsequencingwithahighsignal-to-noiseratio.TogetherthesefindingspostulateCUT&RUNasanefficientmethodologyforepigeneticprofilingoflymphocytepopulations.

Presenter GangXue 15Title L-4togetherwithIL-1βinducesantitumorTh9celldifferentiationinthe

absenceofTGF-βsignalingEmail gxue@wakehealth.eduCo-Authors GuangxuJin,JingFang,YongLuAffiliation WakeForestSchoolofMedicine

Interleukin-9(IL-9)-producingCD4+Thelper9(Th9)cellsareadistinctsubsetofThcellsinduced from naïve CD4+TcellsbyInterleukin-4(IL-4)togetherwithtransforminggrowthfac-

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tor-b(TGF-b)cytokinesignalingInadditiontorolesinallergicinflammationandautoimmunediseases,themostintriguingfunctionofTh9cellsistheirantitumoractivity.WewereamongthefirsttoreportantitumorfeaturesofTh9cells.Morerecently,wereportedthatTh9cellsrepresentanovelthirdparadigmforTcelltherapy–theyarelessexhausted,fullycytolytic,andhyperproliferative,andonly tumor-specificTh9cellscompletelyeradicated late-stageadvancedtumors,ascenariomore like thatseenclinically.Todate,both IL-4andTGF-b have been considered as the vital cytokines that are indispensable for Th9 cell-priming and differentiation.Herewe,forthefirsttime,showthat“Th9celldevelopment”canoccurintheabsenceofTGF-b signaling.WefindthatwhenTGF-b has been replaced by IL-1b, namely the combination of IL-1b and IL-4,efficientlypromotes IL-9-producingTcells (Th9IL-4+IL-1b).Next, we performed a microarray analysis comparing Th9IL-4+IL-1b cells with other known Th cells. Interestingly, Th9IL-4+IL-1b cells are phenotypically distinct T cells as compared to the classicTh9cells(Th9IL-4+TGF-b)andotherThcells,includingtheenrichmentinIL-1-signalingand NF-kB-signalingsignatures.InhibitionofTGF-b-signalingdoesnotaffectIL-9produc-tion from Th9IL-4+IL-1b cells. Howerer, inhibition of NF-kB pathway, mainly RelA, negates the IL-9 production by Th9IL-4+IL-1b cells. Furthermore, Th9IL-4+IL-1b cells are less-exhausted T cells thathavebeenendowedwithcytotoxicTeffectorgenesignatureandtumorkillingfunction,and exert a superior antitumor response than classic Th9IL-4+TGF-b cells in the in vitro cytolytic killing assay and in mouse melanoma model. Therefore, our study sheds new light on Th9 celldifferentiation,andprovideanavenueforapotentiallymorepowerfulweaponforcancerimmunotherapy.

Presenter: GuanYang1 16Abstract title: Autophagy-relatedproteinVps34controlsthehomeostasisandfunction

of macrophagesEmail: guan.yang@vumc.orgCo-authors: Joshua L. Postoak1, Jian-hua Zhang2,LanWu1,andLucVanKaer1Affiliation: 1VanderbiltUniversitySchoolofMedicine;2UniversityofAlabamaatBir-

mingham

Autophagy plays a central role in regulating immune cell responsiveness to a variety of stimuli, and defects in this process have been linked to a variety of diseases. The phos-phatidylinositol-3 kinase vacuolar protein sorting 34 (Vps34) is a key early player in au-tophagy.OurpreviousstudiesfoundthatVps34isacriticalregulatorthatcontrolsthede-velopment, homeostasis, and function of dendritic cells and T cells. However, the role of Vps34inregulatingmacrophagefunctionsisnotwellunderstood.WeinvestigatedtheroleofVps34inmacrophagebiologyusingmyeloidcell-specificVps34-deficientmice.Althoughtheseanimalsdevelopnormally,theyexhibitsevereongoinginflammation.Vps34ablationin macrophages caused a partial impairment in the homeostatic maintenance of Tim-4+ macrophagesanddefectiveuptakeofapoptoticcells. Inaddition,Vps34-deficientmacro-phages had modestly increased surface levels of MHC class II molecules. Interestingly, this altered macrophage phenotype was associated with an expanded population of regulatory Tcellsinthespleenandperitonealcavity,butnotthymus.Importantly,myeloidcell-specificVps34-deficientanimalsshowedsignificantlyreducedseverityofexperimentalautoimmuneencephalomyelitis(EAE),aprimarilyCD4Tcell-mediatedmousemodelofmultiplesclerosis.Collectively,ourstudiesestablishVps34asanimportantregulatorofmacrophagefunctionsandmacrophage-mediatedregulationofEAE.OurfindingsalsohaveimportantimplicationsforthedevelopmentofimmunotherapiestotreatmultiplesclerosisandotherinflammatorydiseasesbytargetingVps34.

Presenter Esther Zumaquero 17Title IFNg:anunexpectedantibodysecretingcell(ASC)fateinSystemicLu-

pusErythematosus(SLE)Email ezm@uab.eduCo-Authors Christopher D. Scharer, Scott A. Jenks, Anoma Nellore, Betty Mouseau,

Davide Botta, Alexander Rosenberg, Jeremy M. Boss, Ignacio Sanz, Frances E. Lund

Affiliation TheUniversityofAlabamaatBirmingham,EmoryUniversity

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SystemicLupusErythematosus(SLE)isanautoimmunediseasecharacterizedbylossoftolerance,productionofautoantibodies(autoAb)andultimatelysystemicandorgan-specificdisease.ItiswellknownthatBcellsandinflammatorycytokinesplayanimportantroleinthepathologyofSLE.WehaveidentifiedapopulationofT-bethi(IgDnegCD27negCD11c+CXCR5neg)pre-ASCsthatareexpandedinasubsetofSLEwithincreasedsystemicinflammatorycy-tokines, pathogenic autoAb and disease severity. Interestingly, we found that activation of naïvehumanBcells(BN)withallogeneicIFNg-producingTh1effectorsplusIL-2andIL-21orwithacocktailofdefinedstimuliincludinganti-Ig,TLR7/8ligand(R848),IL-2,IL-21andIFNg, promoted the formation of a T-bethi B cell population that was phenotypically, transcription-ally, epigenetically and functionally similar to the SLE-derived T-bethi pre-ASCs. Mechanisti-cally, we found that BN cells activated in the presence of IFNg responded to very low levels of R848anddifferentiate,expressedhigherlevelsofIL-21R,andweremoreresponsivetoIL-21(asmeasuredbySTAT3phosphorylation).Theseresultswereconsistentwiththeidenti-ficationofadifferentiallyaccessibleregion(DAR)intheIL-21R locus following chromatin ac-cessibility examination. Moreover, we observed that the appearance of the IL-21R DAR was dependent on the presence of IFNg and IL-2 in the cultures and contained consensus binding motifsforSTAT5andT-bet(IL-2-andIFNg-inducedtranscriptionfactors).Interestingly,thisIL-21R-associated DAR was also observed in T-bethicellspurifiedfromSLEpatients.Thesedata suggest that IFNg synergizes with IL-2 to poise the cells to proliferate, respond to IL-21 anddifferentiate.Hence,wehaveidentifiedanovelroleforIFNg in ASC development that is specificforasubsetofSLEpatients,andwhichcouldbetherapeuticallytargetedtopreventthedevelopmentordifferentiationofpoisedpathogenicpre-ASCs.

Presenter S. Rameeza Allie1 18 Title Identificationofantigen-specific,lungresidentmemoryBcellsafterin-

fluenzainfectionEmail sithyallie@uabmc.edu Co-Authors JohnE.Bradley,UmaMudunuru,MichaelD.Schultz,FrancesE.Lund,

Troy D. Randall.Affiliation Clinical immunologyand rheumatology,University ofAlabamaatBir-

mingham

B cells display phenotypic and functional heterogeneity in multiple anatomical locations fol-lowingvaccinationorinfection.Influenza-specific(Flu+)memoryBcells(BMEM)arefoundinboth lymphoid tissues and lungs. It is unclear whether these cells represent circulating or residentmemoryBcell(BRM)populations.WehypothesizedthataportionoftheFlu+BMEM cell population in the lung would be non-circulating, BRMs.UsingacombinationofBcellspecificantibodyinfusion(i.v.)andparabiosisofinfluenzain-fected,congenically-mismatchedmiceweidentifiedaprotective,Flu+,non-circulatingBRMpopulation.TheFlu+BRMsinthelungsconsistedof56%IgM+and43.9%isotype-switchedBRMs.Theywereestablishedasearlyas15daysafterinfectionandmaintainedforatleast60days.TheformationofFlu+BRMsrequiredthegerminalcenter(GC),asblockingCD40Lwith MR1 antibody, during the primary infection abrogated BRM. However, MR1-treatment ofmicewithestablishedBRMdidnotaffectBRMsinthelung,eventhoughFlu+GCBcellscouldbedetectedintheLNforupto90days.ThesedatasuggestthatGC-dependentlung-BRMsareestablishedearlyafterinfectionandmaintainedindependentlyofGCs.OngoinganalysesoftheBRMsshowsphenotypicandfunctionaldifferenceduringaviralchallenge,whencomparedtotheir lymphoidcounterparts.Thesefindingsareimportantinthedevel-opment of vaccines that elicit BRMs and they will provide mechanistic information into the functionofantigenspecificBMEM cells residing in the mucosa.

Presenter Jessica E. Thaxton, PhD, MsCR 19Title Metabolic Regulation of Translation Primes T Cell Anti-Tumor ImmunityEmail thaxton@musc.eduCo-Authors KatieE.Hurst,KileyA.Lawrence,ArmanB.Aksoy,MollyG.Sekar,Lee

R. Leddy, Zihai LiAffiliation MedicalUniversityofSouthCarolina

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T cells experience loss of function in tumors, but the cell-intrinsic mechanisms that rapidly disabletumorinfiltratinglymphocytes(TILs)areunknown.HerewerevealthatTcellsthatcommit to high levels of new protein synthesis experience the terminal unfolded protein re-sponse(UPR)andlosetheabilitytocontroltumorgrowth.Incontrast,weshowthatlimitingTcellproteinsynthesisrelievestheterminalUPR,reducescelldeath,andimpartsprofoundcapacityofTcellstocontroltumors.Weevidencethemechanismsthatdeterminetheex-tentofproteinsynthesisinTcellsdirectedbythemasterenergysensorAMPK.Strikingly,usingmousemodelsandsamplesfromcancerpatients,werevealforthefirsttimethattheenvironment of tumors rapidly disables protein synthesis in conventional T cells. In contrast, weshowthatAMPK-primedTcellspossessaremarkablecapacitytoovercomeAMPKcon-strained translation once in the stress of tumors in order to reinvigorate protein synthesis. OurworkrevealsthatthecellresponsetostressdramaticallyshapestheabilityofTcellstosynthesize protein in tumors and we elucidate novel roles for paths to translation to impact CD8 TIL anti-tumor capacity.

Presenter RachelV.Jimenez 20Title C-reactive protein promotes the expansion of myeloid derived cells with

a suppressor phenotypeEmail rjmenez@uab.eduCo-authors Alexander J. SzalaiAffiliation DivisionofClinicalImmunology&Rheumatology,DepartmentofMedi-

cine,UniversityofAlabamaatBirmingham,Birmingham,Alabama

Inpreviousstudiesofacutekidneyinjury(AKI)inC-reactiveproteintransgenicmice(CRPtg)our group showed that human CRP exacerbates renal injury. Importantly, the detrimental action of CRP in CRPtg was associated with their heightened renal accumulation of myeloid derivedcellsthathadasuppressivephenotype(MDSC;abletosuppresstheproliferationofanti-CD3/CD28stimulatedTcellsinco-cultures).HereinweprovideevidencethatCRPmodulates both the development and the suppressive phenotype of MDSCs. In vitro CRP i) dose-dependently increased thegenerationofMDSC fromwild typemousebonemar-rowprogenitorsandii)increasedMDSCproductionofintracellularreactiveoxygenspecies.WhenaddedtoMDSC:Tcellco-cultures,CRPsignificantlyenhancedtheabilityofMDSCstosuppressanti-CD3/CD28stimulatedTcellproliferation.ExperimentsusingMDSCsgener-atedfromFcγRIIBdeficientbonemarrowshowedthatwhileCRP’sabilitytoexpandMDSCsandtriggertheirincreasedproductionofintracellularROSdidnotrequirethisinhibitoryFcreceptor, CRP’s ability to enhance the T cell suppressive actions of MDSCs was entirely FcγRIIB-dependent.OurpreliminaryfindingsindicatethatCRPalsoenablesprimaryhumanneutrophils to suppress the proliferation of autologous human T cells. In their sum these results suggest that CRP might be an endogenous regulator of MDSC development and suppressiveactioninvivo,particularlyinAKI.

Presenter Jessica Peel 21Title GerminalcenterorganizationmediatedbyT-betdependentexpression

ofCXCR3andCCR6Email jnpeel@uab.eduCo-authors Sara L. Stone, Christopher D. Scharer, Jeremy M. Boss, Frances E.

LundAffiliation UniversityofAlabamaBirmingham,EmoryUniversity

Thegerminalcenter(GC)iscomposedofdistinctregionswithproliferationandaffinitymatu-rationofGCBcells(GCB)occurringinthedarkzone(DZ)andantigen-mediatedselectionand egress of the long-lived plasma cell (LL-PCs) precursors occurring in the light zone(LZ).DespitethekeyroleforGCsinestablishingenduringhumoralimmunity,weknowlittleaboutthedynamicsoftheGCBcellresponse.WeshowedthatthetranscriptionfactorT-betisexpressedbyGCBcellsandthatBcellintrinsicT-betisrequiredfortheLL-PCresponsefollowinginfluenza(flu)infection.TotestwhetherthelossofLL-PCsinthesemicewasduetoaroleforT-betinregulatingtheGCBresponse,weinfected50:50Tbx21-/-:pepboy chimeric

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micewithfluandmonitoredGCBcells.WefoundthatTbx21-/- B cells were overrepresented intheGCrelativetotheB6cellsandthatTbx21-/- GCBcellswereenrichedintheLZ.Par-titioningofGCBcellsintheLZ/DZisregulatedbychemokinesandweshowedthatT-betcontrolstranscriptionoftwochemokinereceptors,CXCR3andCCR6,whichareexpressedbyflu-specificGCBcells.Using50:50Tbx21-/-:pepboy chimeras, we tested whether T-bet regulatesGCB cell positioning by inducingCXCR3 and repressingCCR6.We observedthattheCCR6+GCBcellsubset,whichisknowntolocalizeintheLZ,washighlyenrichedin Tbx21-/-GCBcells.Bycontrast,theCXCR3+GCBsubset,whichwasdominatedbyWTcells,wasenrichedintheDZ.TodeterminetheroleforCXCR3inGCBcelllocalization,weinfected50:50Cxcr3-/-:pepboy chimeras and found that the Cxcr3-/- GCBcellsareenrichedintheLZ.Therefore,T-betregulatesGCBcelllocalizationandpotentiallythedynamicsoftheGCresponsebycontrollingCXCR3andCCR6expression.

Presenter: NathanKlopfenstein 22Title: Inhibition ofSOCS1 improves outcomes during subcutaneousMRSA

skin infectionEmail: nathan.klopfenstein@vanderbilt.eduCo-Authors: Stephanie L. Brandt, C.H. SerezaniAffiliation: VanderbiltUniversityMedicalCenter

Methicillin-resistantStaphylococcusaureus (MRSA) is theprimarycauseofskinandsofttissue infections inhealthcaresettingsand thosewithdiabeteswithin theUnitedStates.MRSA skin infections are characterized by the formation of a neutrophillic abscess that is the hallmark of pyogenic infections to prevent bacterial spread to deeper tissues. However, the events and factors that drive ideal host defense during these infections remains to be fully elucidated.HereweexaminedtheroleoftheproteinSOCS1anditsimpactofhostdefenseduringMRSAskininfectioninbothhyperglycemicandeuglycemicmice.Wehavepreviouslyshown that hyperglycemic mice are unable to control MRSA skin infection, and demonstrate poorabscessformation.OurdataindicatethatSOCS1expressionishigherinhyperglyce-mic mice throughout the course of infection, correlating with their poor infection outcome. PharmacologicalorgeneticinhibitionofSOCS1inbothcontrolandhyperglycemicmicede-creased lesion size and improved bacterial clearance in the skin, resulting in better infection outcome.InhibitionofSOCS1throughoutinfectioncorrelatedwithanincreaseintheproduc-tionofpro-inflammatorycytokinesTNF-a and IL-1b. Furthermore, both phagocytosis and intracellular bacterial killing were increased in macrophages isolated from hyperglycemic andcontrolmiceuponinhibitionofSOCS1.Overall,thesedataindicatethatthethresholdofSOCS1expressionmaynegativelyimpactskinhostdefenseinbothhyperglycemicandother susceptible patient populations.

Presenter: MeaganJenkins 23Title: Lung-migratoryDendriticCellstrafficintothespleenafterinfluenzavirus

infectionEmail: meaganj@uab.eduCo-Authors: Holly Bachus1, Beatriz Leon-Ruiz2 and André Ballesteros-Tato1

Affiliation: 1Department of Medicine, Division of Clinical Immunology and Rheuma-tology,UniversityofAlabamaatBirmingham,Birmingham,AL35294.;2DepartmentofMicrobiology,UniversityofAlabamaatBirmingham,Bir-mingham,AL35294

InitiationofCD8+Tcellresponsestoinfluenzavirusrequiresthetraffickingoflung-migra-torydendriticcells(mDCs)fromthelungintothelung-drainingLN(mLN).Assuch,whenmDCs are absent or are unable to migrate into the mLN, T cell responses are severely compromised. Importantly, it is generally considered that mDCs die shortly after reaching themLN.Thus,thecurrentparadigmsuggeststhatprimingofinfluenza-specificTcellre-sponses by lung-mDC takes place entirely in themLN.We showhere, however, that afraction of the lung mDCs egress the mLN, enter the blood circulation, and subsequently

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traffictothespleen,wheretheyprimeinfluenza-specificCD8+Tcellresponses.Mechanisti-cally, the exit of mDCs from the mLN is controlled by an S1PR dependent mechanism. As a consequence, blockade of S1P/S1PR interactions prevents the egress of mDCs from mLN, thereby precluding the priming of T cell responses in the spleen. Collectively, our results demonstrateanovelDC traffickingpathwayandsupportanewparadigm forhowTcellresponsestoinfluenzaareinitiated.

Presenter: Ashlyn Anderson 24Title: UnderstandingtheroleofSTAT4inCD4Tcellmediatedneuroinflam-

mation Email: aea1294@uab.eduCo-authors: IanL.McWilliams,BoyoungShin,JoyShepard&LaurieE.Harrington.Affiliation: UniversityofAlabamaatBirmingham

MultipleSclerosis(MS)isanautoimmunediseasethataffectsovertwomillionpeopleworld-wide. MS is characterized by the demyelination of axons in the central nervous system (CNS), leading tovisionproblems,muscleweaknessandpoorcoordination. Among thevarious immune cells that contribute to the disease, a subset of CD4 T cells, Th17 cells has been associated with MS pathogenesis. Recently, there has been a distinction between ho-meostatic Th17 cells, which do not directly contribute to disease and pathogenic Th17 cells thatdirectlyinfluenceinflammation.Importantly,IL-23signalinginTh17cellsisessentialforthe pathogenicity. The transcription factor STAT4 has been historically correlated with Th1 cellsand isdownstreamof IL-23signaling. AsSTAT4deficiencydoesnotpreventTh17differentiation,butstilllimitsinflammation,wehypothesizethatSTAT4isnecessaryforthepathogenicpropertiesofTh17cellsinMS.TostudytheinfluenceofSTAT4inthecontextofneuroinflammation,ourlaboratoryusestheadoptivetransfermodelofexperimentalautoim-muneencephalomyelitisEAE.Ourlaboratoryalsoutilizesin vitro CD4TcelldifferentiatetoidentifyhowSTAT4influencestheexpressionofgenesinCD4Tcellsculturedin“homeo-static”Th17cultureconditionscomparedto“pathogenic”Th17conditions.WefindthatwhileSTAT4 expression does not impact the development of Th17 cells but is necessary to induce CNSinflammation.Furthermore,globalgeneexpressionanalysisindicatesthatSTAT4reg-ulates the recently described pathogenic and nonpathogenic Th17 gene signatures. In the absence of STAT4, the levels of pathogenic Th17 genes including Tbx21, Il22 andCxcl3 are significantlyreduced,whiletheexpressionofnonpathogenicgenesincluding Il10 and Ahr is increased.Furthermore,wefindasignificant increaseofgenesthatrelyonbothIL-23andSTAT4whencomparedtogenesthatrelysolelyonSTAT4inTh17differentiatedCD4Tcells.ThisleadsustofurtherhypothesizethattheIL-23/STAT4signalingaxisisvitaltoneuroinflammation.Together,thesedatarevealanovelroleofSTAT4incontrollingTh17pathogenicity, which may provide a promising therapeutic target for MS patients.

Presenter: MatthewH.Collins 25Title: Epitope targets of the human antibody response to Zika virus infectionEmail: matthew.collins@emory.eduCo-Authors: Matthew H. Collins1,2, Huy A. Tu3,4,CiaraGimblet-Ochieng5,Guei-Jiun

Alice Liou5, Ramesh S. Jadi5,StefanW.Metz5, Ashlie Thomas5, Ben-jamin D. McElvany4, Edgar Davidson6, Benjamin J. Doranz6, Yaoska Reyes7, Natalie M. Bowman2, Sylvia Becker-Dreps8, Filemón Bucardo7, Helen M. Lazear5, Sean A. Diehl3,4, Aravinda M. de Silva5

Affiliations: 1DepartmentofMedicine,EmoryUniversity;2Department of Medicine, UniversityofNorthCarolina-ChapelHill;3Cellular, Molecular, and Bio-medicalSciencesProgram,UniversityofVermont;4Department of Mi-crobiologyandMolecularGenetics,UniversityofVermont;5Department ofMicrobiologyandImmunology,UniversityofNorthCarolina;6Integral Molecular, Inc.; 7DepartmentofMicrobiology,NationalAutonomousUni-versity of Nicaragu; 8Departments of Family Medicine and Epidemiol-ogy,UniversityofNorthCarolina-ChapelHill

Zikavirus (ZIKV) transmissionbecameaglobalpublichealthemergencyafter the recent

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epidemic in Latin America and beyond revealed rare but dire manifestations of infection suchasseverebirthdefectsandGuillain-Barrésyndrome.ZIKVemerged inareaswhereotherflavivirusessuchasdenguearealreadyendemic,andantibody(Ab)cross-reactivityamongrelatedflavivirusescreateschallengesinaccuratelydiagnosinginfections,conduct-ing reliable surveillance, as well as in understanding the distinguishing aspects of the host immuneresponse toZIKV.Vaccines representakeystrategy forpreventionof infectiousdiseases and typically rely on robust antibody responses. To promote vaccine development andgeneratefundamentalknowledgeregardingtheprotectiveZIKV-specificAbresponse,wesoughttoanalyzethedurableantibodyresponsesinindividualsinfectedbyZIKVasafirst flavivirus infection.Weobservedcomplexpopulationsofantibodies thatbind toepit-opes on intact virions, simpler epitopes on envelope protein monomers as well as envelope subdomains. Moreover, strong neutralizing antibody responses that minimally cross-react with dengue viruses were consistently detected. To better understand the molecular deter-minantsof theneutralizingantibodyresponsetoZIKVandtodeveloptoolsthatcouldaidvaccinedevelopment,weisolatedtwopotentlyneutralizingmonoclonalantibodies(mAbs)fromoneprimaryZIKVcaseandmappedkeyaminoacidresiduesinvolvedinmAbbindingand neutralization by multiple complimentary methods including generation of neutralization escapemutantsandalaninescanningmutagenesis.ThemAbsrecognizedifferentepitopescentered on domain I and domain II of the viral envelope protein. Functionally, both mAbs wereprotectiveinalethalmousemodelofZIKVinfection.OngoingworkisexaminingtheprevalenceofthesespecificAbresponsesatthepopulationlevel.Thisworkprovidesnewknowledge and tools that may be useful as diagnostic reagents or as therapeutics and will advance vaccine development.

Presenter: AmynMurji 26Title: Design and Development of Empirical and Rational Epitope-Focused

HIV-1VaccineCandidatesEmail: amyn.murji@vanderbilt.edu Co-Authors: JulianaQin,IanSetliff,NagarajanRaju,AllieGreenplate,ClintHolt,Iv-

elinS.GeorgievAffiliation: VanderbiltUniversity

HIV-1continuestoimposeaglobalhealthburden.CandidatevaccinesusingHIV-derivedan-tigenshavenotproveneffectivetodate,andeffortstowardprotectionagainstnewinfectionsremainahighpriorityinHIV-1research.Inrecentyears,strategiesthattargettheelicitationof broadly neutralizing antibodies that are capable of neutralizing a large fraction of circulating HIV-1variantshaveemergedasapotentialavenuetoaprophylacticHIV-1vaccine.Thesoletargetoftheseneutralizingantibodiesistheenvelopeprotein(Env)ofHIV-1.However,duetotheextensiveglobaldiversityofHIV-1,Env-basedvaccinecandidatessofarhaveonlyledto the elicitation of antibodies with limited neutralization breadth. To address this challenge, we propose to develop technologies for the presentation of diverse antigens to the immune system. In doing so, we developed candidate multivalent immunogens that recombinantly presentonthesurfaceofself-assemblingproteinnanoparticles:1)multiple,diverseEnvsor2)conserveddomainsoftheenvelopeprotein.Here,wepresentprogresstowardthedesignandvalidationofanumberofthesetechnologies.Wehavesuccessfullydesignedandde-velopedcandidatevaccinesdisplayingEnvsfromtwodifferentclades(BG505andCZA97).Nanoparticle-basedimmunogens,eitheras(a)acocktailofBG505-onlynanoparticlesandCZA97-only nanoparticles or (b)multivalent nanoparticles simultaneously displaying bothBG505 andCZA97Envs, elicitedmore robust antibody responses inmice, compared to(c)solubleBG505and(d)BG505andCZA97trimercocktails.Toincreaseourimmunogenproductionefficiency,wehavealsodevelopedmulticistronicsystemsthatcanproducemul-tivalentnanoparticleimmunogensfromasingletranscript.WhileofparticularsignificanceforHIV-vaccinology,thetechnologiesthatwehavedevelopedwillbegeneralizabletovaccinedesign for other viruses that exhibit high levels of sequence diversity.

Presenter: AubreySchonhoff 27Title: Activatedborder-associatedmacrophagesmediateperipheralcellinfil-

trationinanAAV2α-syn model of Parkinson Disease

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Email: aschon@uab.eduCo-authors: WilliamsGP,StandaertDG,HarmsASAffiliation: UniversityofAlabamaatBirmingham

Parkinsondisease(PD)ischaracterizedbyprogressivelossofdopamine-producingneuronsinthesubstantianigraparscompacta(SNpc)andwidespreadintracellularinclusionsoftheproteinalpha-synuclein(α-syn).Recentevidencehighlightstheimmunesystemasapos-siblemediatorofPDprogression.Inbothhumanpatientsandrodentmodels,α-synpathol-ogyisaccompaniedbymicroglialactivation,Tcellinfiltration,hyper-reactivemonocytes,andincreased cytokine and chemokine release. However, the triggers responsible for initiating this immune response and mediating peripheral cell recruitment to the CNS remain poorly understood. Additionally, many previous studies have not distinguished between the resident macrophagepopulationsofthebrain:microgliaandborder-associatedmacrophages(BAMs,includingperivascular,meningeal,andchoroidplexusmacrophages),complicatingthestudyof innate immune mechanisms. BAMs are uniquely positioned for monitoring the borders be-tween the CNS and periphery, and may act as important antigen presenting cells or cytokine/chemokine producing cells in the disease process. To determine the role of BAMs in models ofPD,weutilizedanadeno-associatedvirus (AAV) thatoverexpresses full-lengthhumanα-syninneuronsoftheSNpc.WeinjectedthisintotransgenicreportermiceinwhichthefirstexonofCX3CR1 is replacedwithGFP.Usingflowcytometryand immunohistochemistry,weexaminedtissueresidentCX3CR1+cells (microgliaandBAMs) foractivationmarkersandproliferation.Wefoundthatα-synledtoanincreaseinthenumberofBAMsandintheirexpressionofMHCII.TodeterminethefunctionalroleofBAMs,wespecificallydepletedtheBAMswithoutaffectingothercellpopulations,suchasmicrogliaorperipheralmonocytes,usingclodronateliposomesinjectedi.c.v.DepletionofBAMshadananti-inflammatoryeffect,asitsignificantlydecreasedinfiltrationofperipheralimmunecellsandpreventedupregula-tionofMHCIIbymicroglia.WealsoinvestigatedthespecificityofadditionalBAMtargetingtechniques using anti-CSF1R antibodies. Furthermore, we used a CCL2 reporter mouse to provide evidence of CCL2 cytokine production by BAMs in response to α-syn. These results demonstratetheimportanceofBAMsintheinitiationofneuroinflammationandtherecruit-mentofperipheralimmunecellssubsetsinanα-synPDmodel.

Presenter: Shanel Tsuda 28Abstract title: Identifying key transcriptional regulators that establish T cell memory

during viral infectionEmail: stsuda@scripps.eduCo-authors: HDiao,JJMilner,AWGoldrath,SCrotty,MEPipkinAffiliation: TheScrippsResearchInstitute–Florida

Memory T cells provide enhanced immunity to previously encountered pathogens. During aprimaryinfection,activationofnaïveCD8+Tcellsleadstodifferentiationofmemorypre-cursoreffector cells,whichultimatelydevelop into long-livedmemorycells.However, thetranscriptionfactors(TFs)thatestablishandmaintainthememoryTcelldevelopmentalpro-gram are incompletely understood. Motifs encoding binding sites for both ETS- and bZIP-family TFs are highly enriched within cis-regulatory regions whose chromatin accessibility is induced in naïve CD8 T cells during primary TCR stimulation and that are also accessible inmatureeffectorandmemoryTcellstates.TheETS-andbZIP-familiesareencodedbyalargenumberofgenes(28and60genes,respectively),andRNA-seqanalysesindicatetheyaredynamicallyexpressedinnaïve,earlyeffector,andmemoryCD8+Tcellsubsetsduringacuteviralinfection.ToclarifytheirrolesduringmemoryCD8Tcellsdifferentiationduring acute viral infection, I am applying an in vivo pooledRNAinterference(RNAi)screentargetingallgenesfrombothfamiliesinnaïveCD8+Tcells.PreliminaryexperimentsindicatethatinsufficiencyofoneofthemostlyhighlyexpressedETSmembers,Ets1impairseffectorcellaccumulationandincreasestherelativefrequenciesofterminaleffectorcells,whichissimilartothephenotypesinducedbyRNAiofTFsCbfbandRunx3.InhibitingexpressionofEts1 also decreases the expression of all the Runx-family members, as well factors that are downstreamofRunx3suchasT-bet.Co-transductionofEts1shRNAmirsandRunx3cDNAlargely rescues the Ets1 RNAi phenotype. These results suggest that Ets1 might function upstreamofRunx3,andthatbothTFscooperatetoestablishamemoryprograminrecently

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activatedCD8+Tcells.Furthermore,thepooledRNAiapproachwill likelyclarifythecon-tribution of additional TFs to memory CTL programming, and lead to information that might ultimately inform strategies to manipulate T cells in therapies to enhance T cell function dur-ing infections and cancer.

Presenter: Zhenda Shi, Ph.D. 29Title: Segmented Filamentous Bacterial Present and Cure Rotavirus InfectionEmail: seasonda@yahoo.comCo-authors: Zhenda Shi1, Jun Zou1, Zhan Zhang1, Xu Zhao1, Juan Noriega1, Benyue

Zhang1,RichardK.Plemper1, Chunyu Zhao2, Harshad Ingle3,KyleBit-tinger2, Lisa M. Mattei2, Andrea J. Pruijssers4, Timothy J. Nice5, Megan Baldridge3, Terence S. Dermody6, Benoit Chassaing1, 7, and Andrew T. Gewirtz1

Affiliation: 1CenterforInflammation,ImmunityandInfection,InstituteforBiomedi-calSciencesGeorgiaStateUniversity,Atlanta,GA;2Gastroenterology,Hepatology,andNutrition,Children’sHosp.ofPhil.Philadelphia,PA;3DepartmentofInternalMedicine,WashingtonUniversitySchoolofMed-icine,St.Louis,MO;4DepartmentofPediatrics,VanderbiltUniversitySchoolofMedicine,Nashville,TN;5DepartmentofMicrobiologyandImmunology,OregonHealthSciencesUniversity,Portland,OR;6De-partmentofPediatrics,UniversityofPittsburghSchoolofMedicineandUPMCChildren’sHospitalofPittsburgh,Pittsburgh,PA;7NeuroscienceInstituteGeorgiaStateUniversity,Atlanta,GA

Entericvirusesencounterepithelialcellsamidstdiversemicrobiota.Wethushypothesizedthatourunintentionalgenerationofrotavirus(RV)-resistantRag1-KOmicereflectedmicrobi-otainfluencingRVinfection.Accordingly,suchRV-resistancewastransferredbyco-housingandfecaltransplant.InterrogationofmicrobiotasconferringRV-resistanceviaantimicrobialagents,heat,andfiltration,followedbylimitingdilutiontransplanttogermfreemiceandsub-sequentfecalDNAsequencingrevealedacentralroleforsegmentedfilamentousbacteria(SFB),whichwassufficient toprotectmiceagainstRV infectionandassociateddiarrhea.Suchprotectionwas independentof lymphocytes (innateandadaptive), interferon, IL-17,andIL-22.IncubationofSFB-containingfeceswithRVreducedRVinfectivity,suggestingdi-rect disabling of this virus. Additionally, colonization of ileum by SFB induce changes in host geneexpressionandacceleratedepithelialcellturnover,whichcanreduceRVburden.Thus,irrespectiveof itseffectson immunecells,SFBconfersprotectionagainstcertainchangeenteric viral infections and associated diarrheal disease.

Presenter: RebeccaL.Crepeau 30Title: Impact of CD28 Signaling on the Homeostatic Repopulation of T Cell

SubsetsinSecondaryLymphoidOrgansandPeripheralTissuesEmail: rcrepea@emory.eduCo-authors: Danya Liu, Mandy L. FordAffiliation: EmoryUniversity

Belatacept,aCTLA4Ig fusionprotein,offerspromising long termgraftsurvivalbenefits totransplant patients. However, it is also associated with increased rates of acute rejection which currently limits its clinical use. This increased rejection is potentially due in part to the unintended blocking of CTLA4-mediated coinhibition. Thus, a non-cross-linking Fc-devoid anti-CD28domainantibody (dAb)wasdeveloped toselectivelyblockCD28costimulationwhile leaving CTLA-4 coinhibition intact. Anti-CD28 domain antibody was shown to exhibit increasedefficacy inmouseandNHPmodels of transplantationwhen compareddirectlywith CTLA4Ig. These results have led to the initiation of an NIH-funded clinical trial at Emory University to test theuseofselectiveCD28blockade inkidney transplant recipients.Pa-tients receiving kidney transplants are routinely treated with lympho-depleting agents prior totransplant.ItisunclearwhateffectselectivelyblockingCD28inthissettingwillhaveontheimmuneprofileofthesepatientsfollowinghomeostaticrepopulationoftheirimmunecellcompartments.Toaddressthisquestion,wegeneratedamousemodelinwhichC57BL/6

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mice were treated with CD4+ and CD8+ T cell depleting agents at the time of Balb/c skin transplantation in the presence or absence of anti-CD28 dAb every other day for 7 days. Mice receiving only skin transplantation and no depletion or immunosuppressive therapy servedascontrols.WefoundthatinthissystemCD4+ and CD8+ T cell compartments be-gantorepopulateat4weekspost-depletion.By6weekspost-depletion,overallnumbersand frequencies of splenic and peripheral tissue CD4+ or CD8+Tcellswerenotdifferentbetween mice receiving CD28 blockade and those receiving depletion alone. However, our preliminaryresultsindicatethatblockadeofCD28signalinginthissettingleadstosignificantalterations in the expression of T cell activation markers and costimulatory/coinhibitory mark-ers, with the most profound impact observed within T cells found within peripheral tissues. Alterations in the composition and phenotypes of T cell populations in peripheral tissues following homeostatic reconstitution could have implications for protective immunity of these patients following transplantation.

Presenter Paulo José Basso1,2 31Title TargetingAMPKtomodulateBcellmetabolismandtreatInflammatory

Bowel DiseasesEmail paulo.basso@vumc.orgCo-Authors Thiago M. Steiner1; Fernanda F. Terra1;ShawnaK.McLetchie2; Meire

I.Hiyane;ViníciusAOliveira1, Rafael. R. Almeida1; Mark R. Boothby2; NielsO.S.Câmara1.

Affiliation 1Department of Immunology, Institute of Biomedical Sci-ence, University of São Paulo, São Paulo, Brazil.; 2Depart-ment of Pathology, Microbiology, and Immunology, Vander-bilt University Medical Center, Nashville, TN, United States.

zAMPKintegratesagroupofmetabolicsensorsthatcontrolseveralintracellularprocessessuchasmetabolism,cellgrowth,autophagyandpost-translationalmodifications.However,its role in B cell metabolism has been little assessed despite the relevance of these cells in bothhumoralandcellularresponses.Thus,ouraimwastoevaluatetheroleofAMPKinBcellmetabolismandhowtheBcell-specificmetabolicchangescouldimpactimmune-mediat-edinflammatorydiseases.SortedsplenicBcellsfromcontrol(CT)andBcell-specificAMPKknockout(AMPKΔB)micewereculturedwithLPS.Boththelactate/glucoseratioandactiva-tionmarkersweremainlyincreasedinAMPKΔBcellsafterLPS,whilefewornodifferenceswereobservedwithotherstimuli (CpG,anti-IgMoranti-CD40+ IL-4).Moreover,AMPKΔB cellspresentedasharplyincreaseinIL-10andIL-6synthesis,CD40/CD86expressionandglucoseuptakecomparedtoCTcellsbutdecreaseddifferentiation intoplasmablasts.Ac-cordingtotheseresults,CTBcellstreatedwithAMPKagonistdecreasedIL-10secretionandCD40/CD86expressionbutincreasedplasmablastdifferentiation.SimilarresultswereobservedinmTORC1-deficientBcells,adownstreamtargetnegativelyregulatedbyAMPK.AMPKΔBcellsalsoincreasedmRNAexpressionofGLUT1,glycolyticenzymesandPDK1aswell as decreased mitochondrial activity, basal respiration and maximal respiratory capac-ity.DSS-inducedcolitisinAMPKΔB mice showed a marked improvement of the clinical signs andincreasedIL-10levelsingutcomparedtoCTmice.Finally,Bcell-deficientmMT mice that receivedadoptively transferredAMPKΔBcells had more benign disease scores when compared to those that received CT B cells. These results provide evidence of a regulatory roleofAMPKinglycolysis,activationthresholdandplasmablastdifferentiationofBcells.WeproposethatBcell-specificAMPKinhibitionisapotentialtargettotreatinflammatoryboweldiseases.Financial support: FAPESP; CAPES; CNPq

Presenter AnaBeatrizEnriquez 32Abstract Title Mycobacterium tuberculosis modulation of Notch ligand expression im-

pedes dendritic cell-T cell crosstalk and limits Th17 polarizationEmail ana.enriquez@emory.eduCo-Authors JonathanKevinSia,MelanieQuezadaandJyothiRengarajanAffiliation EmoryVaccineCenter,EmoryUniversity,Atlanta,GA30329

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Mycobacterium tuberculosis (Mtb), thecausativeagentof tuberculosis(TB), isamongtheleadingcausesofdeathworldwide.While IFN-γandCD4+Th1 T cell responses are nec-essarytomountanimmuneresponsetoMtb,theyarenotsufficienttoprovideprotection.Studies from several groups, including our own, have highlighted an important role for IL-17 and Th17 responses in immunity to Mtb infection. Previous research from the laboratory has shown that Mtb restricts Th17responsesbydampeningdendriticcell(DC)responsesandhasidentifiedCD40-mediatedco-stimulationascriticalforgeneratingTh17 responses in response toMtb.WeshowedthatexogenousCD40engagementonMtb-infectedDCsenhancesTh17 polarization and reduces Mtb burden in the lungs in a vaccination model. However, the DC mechanisms that mediate CD40-dependent Th17 polarization and protection have not been defined.HereweshowthatNotchsignalinginDCsmodulatesDC-Tcellcrosstalkandinflu-ences T cell polarization during infection. Engaging the CD40 pathway on Mtb-infected DCs increases the mRNA and protein expression of Notch ligands DLL4 and Jagged1 over the course of infection. Blockade of Jagged1 during a DC-T cell co-culture lowers Th1 responses whileblockadeofbothDLL4andJagged1significantlylimitsTh17 polarization. These results reveal that during infection, Mtb restricts expression of Notch ligands, which impedes Th17 responses during TB.

Presenter UmaShanmugasundaram 33Title: M. tuberculosis-specificTcellresponsesduringLatentTBinfectionof

Rhesus MacaquesEmail: ushanmu@emory.eduCo-Authors: UmaShanmugasundaram,AllisonBucsan,ChrisIbegbu,Vijayakumar

Velu,DeepakKaushal,JyothiRengarajanAffiliation: YerkesPrimateResearchCentre,EmoryUniversity Introduction: Mycobacterium tuberculosis(Mtb)istransmittedthroughaerosolrouteandin-fectionoccursinthelung.Mtbinfectioncaneitherleadtoactiveorlatenttuberculosis.WhileactiveTBisassociatedwith ineffectivecontrolofbacteria,90%oftheindividualsdeveloplatentTBinfection(LTBI),wherebacteriaaresuccessfullycontainedinsidelunggranulomas.However, the immune responses associated with immune control of LTBI are not known and the CD4 and CD8 T cell responses in blood and lung compartments are poorly understood. WehavedevelopedamodelofLTBIinrhesusmacaquesthatrecapitulatesmerelyallas-pectsofhuman infection.Using thismodelwehavecharacterized theMtb-specificTcellresponses involved in immune control during TB latency. Materials and methods: Rhesus macaques(6animals)were infectedthroughaerosolroutewith lowdoseMtbstrainCDC1551. InfectionwasassessedusingTuberculinSkin test,andInterferonGammaReleaseAssays (IGRA)and latencywasdeterminedbychestx-rayand lackofclinical signsandsymptoms. Animals were longitudinally followed until week 24 we studied the phenotype andfunctionofMtbspecificCD4andCD8Tcellsinperipheralblood,BALandlungbyflowcytometry.Results:Mtbantigenspecific(MtbCellwall-andESAT6/CFP10-specific)Tcellsproducing IFNgand IL-17weresignificantlyhigher inBALcomparedtoPBMCandCD4+TcellresponseswerehighercomparedtoCD8+Tcells.ThepeakMtbspecificresponseswere seen at week 7 and were maintained at higher levels throughout the latent infection inBALandinPBMC.MtbantigenspecificTcellsweremainlyofcentralmemoryandex-pressedbothCCR6andCXCR3.MtbspecificactivatedTcellsexpressingCD38declinedovertime in PBMC whereas in BAL high frequencies were maintained till week 24 of latent infection. Conclusion: Mtb latent infection was characterized by high frequency of IFNg and IL-17producingTcellsandantigenspecificcentralmemoryT-cellsinBAL.AntigenspecificactivatedCD4+TcellshomedtoBALandpersistedinBALthroughoutthelatentinfection.

Presenter: StephenKirchner 34Title: Antiviral Protein Expression in the Skin is Regulated by the Circadian

ClockEmail: sjk33@duke.eduCoauthors: CHandfield,MCoates,XLing,JShannon,PMariottoni,DLCorcoran,

AS MacLeodAffiliation: Department of Dermatology, Duke University, Durham, NC, USA

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Antiviralproteins(AVPs)suchastheoligoadenylate-synthase(OAS),MX,andIFITMfami-lies are expressed in epidermal keratinocytes, dendritic cells, and cutaneous T cells in the skin.WhileinflammatorypathwayscaninduceAVPexpressionsuchasIL-27signaling,theexact temporal pattern of constitutive expression and induction of these proteins in response toviralchallengeisunclear.UponexaminationofexpressionofconstitutiveAVPsinprimaryhuman keratinocytes, we found that their expression levels varied over a 24hr timespan. The circadiangeneticclockofBMAL1,CLOCK,andPER/CRYproteinsstronglyinfluencescellu-larimmunity,butits’roleinantiviralproteinproductionisunknown.Wehypothesizedthatthecircadian clock may play a pivotal role for innate antiviral immunity. Here, we show by qPCR that in primary human keratinocytes the expression of several antiviral peptides exhibit cir-cadianoscillationsover24hours.OAS2,MX1,andIFITM1cyclealongwithcircadiangenesBMAL1,CLOCK,andPER2,withAVPpeakexpressionlevelscorrelatedtopeakexpressionofthepositivecircadianregulatorBMAL1(r=.736,p<.005)andAVPexpressiondecreasedwith low levelsofBMAL1expression.siRNAknockdownofBMAL1alsodecreasedAVPexpression.Wenextcorroboratedthesefindingsin vivo. Mouse studies displayed a marked differenceinAVPproductionuponskinwoundingdependingontimeofday.Furtherexplora-tion of this relationship was undertaken with Bmal1-/- mice. Both in silico and in vivo analysis revealed that skin from Bmal1-/-miceexpressedlowerlevelsofAVPsrelativetocontrolwildtypemice(p<0.05,n=4)inresponsetowounding.Weconcludefromthesestudiesthatcir-cadianregulationinfluencesexpressionofdistinctantiviralproteinsbothin vitro and in vivo. Future directions for this project include determining the mechanism by which the circadian clockinfluencesAVPexpressionandthefunctionalroleofthisphenotypeforviralinfections.Better understanding of this mechanism is relevant in understanding how our skin responds toviralchallenges,andmayhelpusunderstandandmanipulatethedifferentialresponsetocutaneous viral infection in relation to daily timing.

Presenter CourtneyHegner 35Title DeterminingtheroleofchromatinregulatorsinthedifferentiationofCD8

T cellsEmail hegne22c@mtholyoke.eduCo-Authors S.Tsuda,R.Chen,H.Diao,M.A.Frederick,J.Milner,A.W.Goldrath,S.

Crotty and M.E. Pipkin Affiliation ScrippsResearchInstitute,NSF

MemorycytotoxicTlymphocytes(CTLs)developfromnaïveCD8+Tcells,andcanprovidelong-term immunity against intracellular infections and tumors. During a typical acute infec-tion,naiveCD8Tcellsdifferentiateintoalargenumberofterminaleffectorcells(TE)thatdonot become memory CTLs after the infection is cleared. At the same time, a smaller number ofmemoryprecursorcells(MP)develop,whichgiverisetolong-livedmemoryCTLs.Thisdif-ferential process is regulated by transcription, but the chromatin regulators and transcription factorsthatspecifyhowandwhennaiveCD8TcellsdifferentiateintoeitherCTLpopulationare still unknown. To gain insight into this problem, we developed and applied a pooled in vivo loss-of-function screen using RNAi to individually suppress all putative chromatin regulatoryproteinsinCD8Tcellsrespondingtoacutelymphocyticchoriomeningitis(LCMV)infection.Analysisofthescreenindicatedthatsuppressionofmultipledifferenthistoneacetyltransferases(HATs)andbromodomainproteinsimpairedformationofTECTLs,whereasadistinct set of HATs and bromodomains impaired development of MP CTLs. Suppression ofBrwd3,abromodomain-containingproteinpreviouslyimplicatedintheJak/Statsignalingpathway inDrosophila appeared to skew differentiation toward TECTLs and away fromMPCTLs.SuppressionofBrwd3withmultipleindividualshRNAmirsinvivoincreasedthefrequencyofTE-likecellsatearlytimesafterLCMVinfection,andincreasedthenumberofTE,MPandDPcellsat thepeakof infection.Moreover,afterthepeakresponse,Brwd3-suppression decreased the fractions of central memory-like cells, and enhanced those of peripheralandeffectormemory-likeCD8Tcells.Theseresults indicate thatBrwd3mightnormallyrestrainthedifferentiationofeffectorcells,toensurenormalMPdevelopment.Giv-en that histone acetylation in chromatin is read by bromodomain-containing proteins, which canrecruitfactorsthatregulatetheefficiencyofRNAPolymeraseIIelongationfromtargetgenepromoters,ourresultssuggestthatdifferentialutilizationofbromodomaincontaining

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proteinsmightregulatetranscriptionthatgovernsthedifferentiationofactivatedCD8Tcellsduring infection.

Presenter: AdamGetzler 36Title: ChromatinregulatorMll1initiatesmemoryTcelldifferentiationandro-

grams stem like properties Email: agetzler@scripps.edu Co-authors: Megan Frederick, Huitian Diao, Runqiang Chen, Ananda Goldrath,

Shane Crotty, Matthew Pipkin Affiliation: ScrippsResearch

Duringacuteviralinfections,naïveCD8+Tcellsactivateanddifferentiateintobotheffectorandmemorylineages.Thisdifferentiationisdefinedbydistinctchangesinchromatinacces-sibility and epigenetic marks. Failure to properly establish these changes results in impaired immunological memory and is a hallmark of dysfunctional T cell responses. However, little is known about how chromatin regulators, one of the main class of chromatin modifying pro-teins, control these processes. To address this question, we performed an in vitro shRNAmir screenagainstallknownmurinechromatinregulatorsfortheireffectontheexpressionofco-inhibitory receptors (i.e.PD1,TIM3,andLAG3),whoseexpression is tightly regulatedduring typicalTcelldifferentiation.We found thatdeficiencyofhistonemethyltransferaseMll1 (Kmt2a),acatalyticsubunitof theSET/COMPASSfamily,significantly increasedex-pressionoftheco-inhibitorygenes.RNA-SeqshowedMll1tobesignificantlyupregulatedinmemory cells relative to exhausted T cells and most highly expressed during the naïve and memory phases of T cell responses, while ATAC-Seq showed a loss of accessibility for Mll1 ineffectorandexhaustedcellstates.ThisdataleadustohypothesizethatMll1isplayingarole in maintaining naïve and naïve-like memory cell states, consistent with its role in main-taininghemopoieticstemcells,anditsdownregulationisrelatedtoterminaldifferentiation.To test this hypothesis, we diminished either Mll1 or controls via shRNAmirs in antigen spe-cificCD8Tcells(P14),transferredthesecellsintonaïvemiceandinfectedwithlymphocyticchoriomeningitisvirus(LCMV).CotransfersofcellsshowedearlyincreasesinaccumulationandterminaldifferentiationofMll1deficientcellsfollowedbyarapidcontractionandlossofmemory precursor cells at the peak of T cell response. This contraction was consistent into memorytimepointsanduponre-challengeMll1deficientcells failedtore-differentiate intoeffectorcells.TogetherthisdatapointstoaroleforMll1inmaintainingTcellstemnessandensuring the proper formation of functional memory CD8 T cells.

Presenter: Mingyong Liu1 37Title: T cell receptor sequencing reveals recruitment of regulatory T cells from

the periphery into omental tumors Email: miliu@uab.eduCoauthors: Dmytro Starenki, PhD2; Sara J. Cooper, PhD2; Troy D. Randall, PhD1;

Selene Meza-Perez, PhD1

Affiliation: 1Department of Medicine – Clinical Immunology and Rheumatology,UniversityofAlabamaatBirmingham, 2HudsonAlpha Institute for Bio-technology.

The omentum is an apron-like fatty tissue that connects the spleen, stomach and pancreas, and contains lymphoid tissues termed milky spots. In the advanced stages of ovarian can-cers, tumor cells frequently metastasize to the peritoneal cavity and invade the omental adi-pose tissue. This is accompanied by an increase in CD4+CD25+FOXP3+ regulatory T cells (Tregs)intheomentum,whichsuppressantitumorimmunity.Despitetheincreasingunder-standing of the roles Tregs play in the context of cancer, how tumor-associated Tregs accu-mulate in the omentum requires further study. To address this, we intraperitoneally injected EG7thymomasintosyngeneicmiceandsortedTregsforTcellreceptor(TCR)sequencing.Although omental Tregs and their splenic counterparts from naïve animals have low degrees of overlap in their TCR repertoires, the similarity between them becomes increased by tu-mors in the omentum. Due to the phenotypic characteristics shared by Tregs in the omentum and colon, we also examined their repertoire similarity and found it elevated by tumors as

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well, albeit more moderately. This indicates Treg recruitment from the periphery during can-cer progression. Moreover, since we did not observe dominant Treg clones in the omenta of tumor-bearing mice, the augmentation of Tregs may not be primarily due to local clonal expansion. In fact, we found tumor development increases the repertoire diversity of omental Tregs.GiventhatTregclonotypesinthespleenarehighlydiverse,thesedatasuggestTregsfrom secondary lymphoid organs may contribute to the omentum compartment during tumor progression.Inlinewiththesefindings,thefrequencyofKi67+ proliferating Tregs is higher inthetumor-freeomentumthaninthespleen,butitissignificantlyreducedintheomentumwithtumors.Interestingly,FTY720treatmentdoesnotaffectthenumberofomentalTregsintumor-bearingmice,indicatingTregtraffickingintotumorsmaybeindependentofthesphin-gosine-1 phosphate receptors. Collectively, these results suggest that regulatory T cells from theperipheryarerecruitedtotheomentumduringtumordevelopment.SpecificmechanismsbywhichTregtraffictotumorsmaythusbetargetedtoenhanceantitumorimmunityandtreatomental tumors.

Presenter: LaceyR.Lopez 38AbstractTitle: YersiniabactindrivesfibroticinflammatoryboweldiseaseEmail: laceylop@email.unc.edu Co-authors: Lopez, Lacey R.1; Moore, Lyndsey1; Broberg, Christopher A.1; Tibbs,

Taylor N.1; Rothemich, Aaron1; Arthur, Janelle C.1

Affliliation: 1.MicrobiologyandImmunology,UniversityofNorthCarolina-ChapelHill,ChapelHill,NC,UnitedStates.

Background:Over30%ofinflammatoryboweldisease(IBD)patientsdevelopintestinalfibrosis(scarring).Adherent-invasiveEscherichia coli(AIEC)arelinkedtoIBD;however,itisunknownhowAIECpotentiateIBD-fibrosis.AIECsecretemetal-chelatingmolecules(siderophores)inmetal-limitedenvironments, like the inflamedgut.TheYersiniabactin (Ybt) siderophore isof key interest, as the Ybt biosynthetic gene cluster is prevalent in the microbiota of IBD patients.OurlabdevelopedanIBD-fibrosismodel,usinggnotobioticInterleukin-10-deficientmice mono-associated with Ybt-producing AIEC. Ybt-producing AIEC are visualized in the intestinalsubmucosa,wherefibrosisdevelops.Fibrosis requiresYbtbiosynthesis,butnotbacterial uptake of Ybt. This suggests Ybt targets the host. WeproposethatYbtmanipulateshostmetalstatusandinfluenceshostcellactivation/death,whichpotentiatesfibrogenesis. Methods: Inculturedfibrosis-associatedcell types (i.e.epithelialcells,macrophages,andfibroblasts),wemanipulatedmetalavailabilityandevaluatedexpressionofmetalresponsive/extracellular matrix genes and cell death. Metal availability was altered using chelators and purifiedYbt in thepresence/absenceofexogenousmetals (e.g. ironandzinc).Ybt-metalbinding was validated via mass spectrometry. GeneexpressionwasevaluatedbyqPCRandcelldeathbylactatedehydrogenaserelease. Results: Ybt induced upregulation of NDRG1, a gene linked to iron starvation. How-ever, more cell cytotoxicity occurred under zinc-depleted versus iron-depleted con-ditions. Ybt-induced cell death was not rescued by any one metal, suggesting mul-tiple biologically important metals are involved in this process. Lastly, manipulating metal in cultured fibroblasts minimally induced expression of extracellular matrix genes. Conclusions:OurresultsdemonstratethatYbtislinkedwithhumanIBD-fibrosisanddrivesfibrosisinarelevantmousemodel.Althoughthemechanismsremainunclear,alteringmetalavailabilityimpactscelldeathandactivationinfibrosis-associatedcelltypes.Furthermore,Ybt induced upregulation of a host iron responsive gene and cell death. Therefore, host metaldisruptionbyYbtmaypotentiateIBD-fibrosis.

Presenter: TaylorTibbs 39Abstract title: Escherichia coli siderophore production induces intestinal fibrosis in

Il10-/- miceEmail: ttibbs@email.unc.eduAuthors: Taylor N. Tibbs1, Aaron Rothemich1, R. Ian Cummings2, Lacey R. Lo-

pez1, Christopher A. Broberg1, Yen-Rei A. Yu2, Robert M. Tighe2, & Janelle C. Arthur1,3,4

Affiliations: 1DepartmentofMicrobiology&Immunology,UniversityofNorthCaro-

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lina,ChapelHill,NC,USA2Department of Medicine, Division of Pulmo-naryandCriticalCareMedicine,DukeUniversityMedicalCenter,Dur-ham,NC,USA3LinebergerComprehensiveCancerCenter,UniversityofNorthCarolina,ChapelHill,NC,USA 4Center forGastrointestinalBiologyandDisease,UniversityofNorthCarolina,ChapelHill,NC,USA

Inflammatoryboweldisease(IBD)affectsover3millionAmericans.Crohn’sdisease(CD),asubtypeofIBD,isnotoriousfordevelopingfibroticstrictures,whicharelifethreateningandrequire surgical removal. Even with surgery, strictures often reoccur, suggesting that there maybeunknownmechanismsintheintestinalmicroenvironmentdrivingfibrogenicpathol-ogy. Adherent invasive Escherichia coli(AIEC)areassociatedwithCDandmanyharborabiosyntheticgeneclusterfortheproductionofthesiderophoreyersiniabactin(Ybt);however,theroleofYbtinCDprogressionandfibroticdevelopmentisunknown.We have developed a gnotobioticmurinemodel whereby fibrosis develops in germ free129 Il10

-

/-

micemono-colonizedwithYbtcompetentAIECNC101.Colitisandfibrosiswasevaluated by H&E and Sirius red stained colon swiss roll sections using established semi-quantitativescoringmethods.Colonictissueswerefurtherassessedusingimmunofluores-centmicroscopy,flowcytometry,andRNAseq.HumanfullthicknesscolonsectionsfromCD,ulcerativecolitis,diverticulitis,andunaffected/normalpatientswerealsoevaluatedbyH&E,Siriusred,andimmunofluorescentmicroscopy.Metagenomesandmetatranscriptomesoffe-cal samples from CD and non-IBD donors were analyzed from publicly available consortium dataviaMetaHITandtheHumanMetagenomeProject2(HMP2).Evaluation of metagenomes and metatranscriptomes from human fecal samples revealed a higher abundance of Ybt genes and transcripts in CD patients compared to non-IBD pa-tients.Inourmurinemodel,fibrosiswasdependentoncolonizationwithYbt+AIEC.Fibrosisincidencewasincreasedinmicemono-colonizedwithYbt+AIECdeficientintheYbtuptakesystem, FyuA. Histological analysis of colon sections from our mouse model compared to human colon resection tissues revealed collagen-rich fibrotic expansions within the sub-mucosaofthecolon,mimickingfibroticlesionsinhumanCD.RNA-Seqdatarevealedanabundanceofextracellularmatrixandmacrophagegenesincolontissuefromfibroticmice,confirmingourhistologicalfindings.Tofurtherexplorethemacrophagepopulationinvolvedinfibrosis,weperformed immunofluorescentmicroscopycoupledwithflowcytometryandidentifiedasignificantexpansionofF4/80

hi

CD11bhi

MHCIIlo

macrophageswithinfibroticco-lons.WepredictthatthesemacrophagesarerecruitedbyYbt-dependentmechanismsandmayberesponsibleforinitiatingacascadinginflammatoryresponsethatactivatesfibroblaststo deposit collagen.

Presenter Doan C. Nguyen 40Title Human bone marrow plasma cell survival is independent of APRILEmail doan.c.nguyen@emory.edu Co-Authors Celia Saney, Iñaki Sanz, F. Eun-Hyung LeeAffiliation EmoryUniversity

Afractionofthecirculatingantibody(Ab)-secretingcells(ASC)maturesintolong-livedplas-macells(LLPC)inthebonemarrow(BM)microniches.PreviousstudiesshowedthatASCsurvival and longevity require APRIL, which upon binding its receptors, BCMA or TACI, ac-tivatesPI3K/Akt(anditsdownstreammTOR)pathwaysandupregulatestheexpressionofantiapoptotic proteins Mcl-1 and Bcl-2. Later work revealed that APRIL binding to SDC-1 (CD138),acell-surfaceheparinsulfateproteoglycan(HSPG),alsoenablesdeliveryofsur-vival signals. Recently, we showed that APRIL is crucial in the ex vivo survival of early-minted human blood ASC. Here, using an in vitro cell-free BM microniche system, we demonstrate thedifferentialrolesofAPRILinthesurvivalofhumanbloodASCandBMplasmacells(PC)and LLPC. APRIL substantially enhanced the survival of bothCD138+ andCD138- ASC populations in the blood, which highly expressed BCMA. In contrast, it had no survival ad-vantageonBMPCorLLPC,despitetheirhighBCMAexpression.Additionally,anti-CD138Ab inhibited thesurvivalofbothbloodASCandBMPCandLLPC, includingCD138- cell

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subsets,suggestinginhibitingthelaterCD138upregulationalsoaffectsbloodASCsurvival.These data suggest that APRIL is important for the survival of early-minted blood ASC but not BM PC or LLPC. In conclusion, APRIL is an important survival and imprinting factor of blood ASC but is not required for LLPC maintenance.

Presenter: Jaime Sancho 41Abstract title: Proteomic analyses of exosome fractions present in peritoneal exudates

frompristane-inducedlupusmicerevealsignificantdifferencesinCD47andTSP-1abundancebetweenCD38-deficientandWTmice

Email: granada@ipb.csic.esCo-Authors: José-Ángel Robles-Guirado 1; Elena González-Paredes 1; Alex Mas-

Ciurana 1; Miguel-Ángel Palacios-Pedrero 1; Carolina Franco-Herrera 1; VictoriaLongobardo2; Antonio Lario 2; Ana-Belén Jodar 3; Francisco J. Blanco 4;ViviandelosRíos5; Ignacio Casal5; and Mercedes Zubiaur ,1.

Affiliation:1 Department of Cellular Biology and Immunology, Instituto de Parasi-tologíayBiomedicina López-Neyra(IPBLN-CSIC),ParqueTecnológicodeCienciasdelaSalud(PTS),Av.Conocimiento17,18016Granada,Spain. 2ProteomicsUnit, IPBLN-CSIC,18016Granada,Spain.3 Fac-ultad de Físicas. Universidad deGranada (UGR).Granada, Spain. 4 FacultaddeMedicina.UniversidaddeGranada(UGR).Granada,Spain.5 Functional Proteomics Laboratory. CIB-CSIC. Madrid, Spain.

Exosomesareextracellularvesiclesthoughttobepresentinallbodyfluids.Shedbycells,theirmolecularmake-upreflectsthatoftheircelloforiginand/ortissuepathologicalsituation.TheincreasedNAD-dependentdeacetylaseactivityofsirtuinsinCD38-deficient(CD38ko)micewillcauseprofoundchangesintheprofileofacetylatedlysines(acK)atspecificsites(acetylome)inanumberofproteinsinvolvedinsignalingpathwaysrelatedwithinflammationandautoimmunity.Therefore,itwillbedistinctfromthatinWTmice,inparticularoncethelupusdiseasefullydevelops.Thisimportantpost-translationalmodificationcanonlybeiden-tifiedbyhigh-resolutionquantitativeproteomics,whichinvolvestheenrichmentinacKpep-tides.Ourworkinghypothesisisthatanalyzingthecomposition(proteinsandmicroRNAs)andfunctionofexosomesreleasedbyspecificcelltypesinthepristaneexperimentallupusmodel will allow us to identify predictive or diagnostic biomarkers that might discriminate theautoimmunityprocessinlupusfrominflammatoryreactionsand/ornormalphysiologicalprocesses.Onefirstobservationofourproteomicdata in thepristane lupusmodel is thepresence ofCD47 and one of its ligands, thrombospondin-1 (TSP-1), in exosomes fromperitonealexudates,particularly inCD38komicewhichdevelopamilderautoimmunedis-ease.TSP-1wasreportedtopromotetheresolutionofinflammatoryprocessandtofacilitatephagocytosisofdamagedcells.Therefore,TSP-1mayplayananti-inflammatoryandimmu-noregulatory role in SLE autoimmunity. CD47 is also the ligand for signal regulatory protein alpha (SIRPa,alsoknownasCD172a),and theCD47-SIRPα interaction initiates the “donot eat me” signal that inhibits phagocytosis. This may facilitate circulation for a longer time through the bloodstream of CD47-positive exosomes and allow them to reach farther places where they can exert their biological action. This circumstance has been used successfully to direct exosomes to tumor tissues for therapeutic purposes. The functional importance of dif-ferentialCD47andTSP-1expressiondetectedinperitonealexosomesfromCD38koversusWTpristane-lupusmicerequiresfurtherstudies.

Presenter: KaitlinA.Read 42AbstractTitle: Ikaroszincfinger transcription factorsasnovel regulatorsofThelper

celldifferentiationandSTATfactoractivityEmail: kread@vtc.vt.eduCo-authors: BharathK.Sreekumar,MichaelD.Powell,JawadZafar,DevinJones,

ChandraE.Baker,MichaelFox,IrvingC.Allen&KennethJ.OestreichAffiliation: Fralin Biomedical Research Institute, Virginia Tech; Bio-

medical and Veterinary Sciences Graduate Program, Virgin-ia-Maryland College of Veterinary Medicine, Virginia Tech

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Cytokinesignalingisimportantforthedifferentiationandfunctionofanumberofcelltypes,includingTlymphocytepopulations.Onemechanismbywhichcytokinesignalsarepropa-gated is via phosphorylation-mediated activation of Signal Transducer and Activator of Tran-scription (STAT) factors,whichdirectly regulatecell type-specificgeneprograms.We re-centlyidentifiedanovelregulatorymodulecomposedofSTAT3andtheIkarosZincFinger(IkZF)transcriptionfactorAiolosinCD4+ T cells, which positively regulates the T follicular helper (TFH) gene program.Given conservation between individualmembers of the IkZFand STAT families, we hypothesized that additional IkZF/STAT complexes may regulate the developmentofotherTcellsubsets.Indeed,wefindthatexpressionoftheIkZFfactorEosiselevated in TH1 cells compared to TFH and naive CD4+ T cell subsets. Furthermore, expres-sion of TH1markers,includingBlimp-1,IL-2rα,andIL-2rβ,aswellasIFN-γproduction,weresignificantlydiminished inEos-deficientTH1cells.Additionally,Eos-deficientCD4+ T cells activated in response to Toxoplasma gondii infection display decreased expression of these markersatthetranscript levelascomparedtotheirwildtypecounterparts.Finally,wefindthatEosphysicallyinteractswithSTAT5andthatthesefactorsareco-enrichedatthePrdm1 and Il2ra gene loci in TH1cells.Curiously,wealsofindthatIkZFfactorexpressioncorrelateswith STAT activation, suggesting that, in addition to directly regulating gene expression, IkZF proteinsmayalsoinfluenceThelpercelldifferentiationthroughtheregulationofSTATfac-toractivity.WefindthatoverexpressionofEos,butnotIkarosorAiolos,resultsinincreasedactivationofSTAT5. Importantly,whenweperformed thesestudiesusinganEosmutantincapableofinteractingwithSTAT5,theobservedincreaseinSTAT5phosphorylationwaslost.WealsofindthatinCD4+Tcells,EosdeficiencycorrelateswithreducedSTAT5activa-tion in TH1cells,whileAiolos-deficientTFHcellsdisplayreducedSTAT3activation.Finally,weobservedifferentialIkZFfactorexpressionacrossCD4+Tcellsubsets.WhencoupledwithknowndifferencesinSTATfactoractivation,thesefindingssuggestthatIkZF/STATregula-torymodulesmaybroadlyregulateThelpercelldifferentiation.

Presenter KimberlyCooney 43Title Regulation of Adenosine Receptor 2A and its Role in the Development

of Neutrophil Extracellular Trap FormationEmail kimberly.ann.cooney@emory.eduCo-Authors CooneyKA,XuK,WangL,GinnSC,DeppenJN,LevitRDAffiliation Division of Cardiology, Department of Medicine, Emory University

SchoolofMedicine,101WoodruffCircle,Suite319,Atlanta,Georgia,30322

Introduction: Neutrophils are a sub-type of leukocytes that play a key role in the innate im-mune system. During times of tissue injury and infection, neutrophils are recruited to the siteofinflammationwheretheyarecapableofrespondingtodamagedtissue.Neutrophilsreleasetheirchromatin,knownasneutrophilextracellulartraps(NETS),intotheextracellularenvironmentwhichcancauseinflammationandtriggerahostofpro-inflammatorypathwaysbyaprocessknownasNETosis.Wediscoveredthatco-cultureofmesenchymalstemcells(MSCs)withhumanneutrophilsdecreasedNETosisandhypothesizedastowhetherMSC-produced adenosine was involved. Recent literature suggests possible mechanisms that may contribute to NET formation. However, it remains unclear how NETs are regulated and whatfactorscontributetotheirproduction.Methods:QuantificationofcAMPproductionwasused to assess adenosine signaling and determine whether cAMP is a regulator of NETosis inbothrestingandstimulatedneutrophils.Otherbiochemicalassaysandtechniquesinclud-ing liquid chromatography and mass spectrometry will be used to examine potential enzymes implementedinNETformation.Results:Wehavefoundthatadenosinereceptor2a(A

2aR)

mediates the anti-NET effects of adenosine. In addition, treatment with an A2a receptoragonistiseffectiveinincreasingcAMPsignalinginisolatedhumanneutrophils.Conclusion:These preliminary experiments suggest that A2a receptor activation is involved in suppres-sionofNETsandthatregulationofdownstreamsignalingfactors,suchascAMP,mayinflu-ence their production. Furthermore, MSCs can reduce NETs in vitro. Reducing NETs in vivo mayimprovefunctionalrecoveryafterMI/Rbyreducinginflammation,preventingthrombo-sis, and other complications. Better understanding of NET properties and examination of downstream mediators may identify therapeutic targets for disease states.

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Presenter: Leila Abdelhamid1 44Title: Retinoic acid exerts time-dependent effects on renal inflammation in

pristane-induced lupus Email: leila88@vt.eduCo-authors Brianna Swartwout2, Xavier Cabana-Puig1, Xin Mimi Luo1

Affiliation: 1DepartmentofBiomedicalSciencesandPathobiology,CollegeofVet-erinaryMedicine,VirginiaTech,Blacksburg,VA24061,USA2Transla-tionalBiology,Medicine,andHealthGraduateProgram,VirginiaTechCarilionResearchInstitute,VirginiaTech

All-trans-retinoic acid (tRA), anactivemetabolite of vitaminA, canact asanadjuvant toexacerbate autoimmunity, even though it is immunosuppressive under steady state. Previ-ousfindingsfromourgroupindicatedthattRAmightexpandthepre-existingimmunogenicenvironment in individuals genetically prone to develop lupus. Here, we show that tRA dif-ferentiallyaffectstheinitiationvs.thecontinuationphaseof lupus.ToexploretherolesoftRA before and after an existing immunogenic status, we utilized the pristane-induced lupus modelinBalb/cmice.OraldosesoftRA(1mg/kg)vs.vehiclecontrolweregivendaily,from3weeksofagetothetimeoflupusinductionat3months(pre-treatment)orfrom3monthsto theexperimentalendpointat6monthspost lupus induction(post-treatment).Pre-treat-ment with tRA, but not the post-treatment, aggravated glomerulonephritis as indicated by increased proteinuria and higher anti-double-stranded DNA autoantibodies levels. Mecha-nistically,tRAinducedtheactivationandtheinflammatoryphenotypeofdifferentsubsetsofconventionaldendriticcellsandexpandedboththeeffectormemoryandgammadelta(γδ)double-negative T cell populations over pristane alone. Additionally, tRA further upregulated the renalmRNAexpression of inflammatory cytokines and chemokines including TNF-α,IL-1β,MCP-1,MIP-1-alphaCCL2,andCCL3.Similarly,upregulationofIntegrinalphal-do-mainofthelymphocytefunction-associatedantigen-1(LFA-1)integrinwasalsoobserved.Moreover,tRAupregulatedtherenaltranscriptlevelofthematrixproteinLamininβ1,whichis known to be associated with mesangial proliferative glomerulonephritis. These results in-dicateenhanced immunecellmigrationand infiltration to therenalcompartmentwith tRApre-treatment in thepristane-induced lupusmodel.Collectively, our findings suggest thattRA promote the initiation of glomerulonephritis by exacerbating the renal immunogenic en-vironment.Thetranscriptprofileinducedbythepre-treatmentwillshedlightonthemolecularmechanism by which it worsened pristane-induced lupus and is currently under investigation.

Presenter: RebeccaM.Brock 45Abstract title: Irreversible Electroporation: A solution to pancreatic cancer’s immuno-

suppressive tumor microenvironmentEmail: rebecmb@vt.eduCo-authors: Rebecca M. Brock, Natalie White, Veronica M. Ringel-Scaia, Sheryl

Coutermarsh-Ott, Kristin Eden, Navid Manuchehrabadi, Rafael V.Davalos, Irving C. Allen

Affiliation: VirginiaPolytechnicInstituteandStateUniversity

Thedevastatinglylowsurvivalrateofpancreaticcancer,atonly8%,isfurthercompoundedby the limitationsofcurrentcancer treatmentoptions.Only15%ofpancreaticcancerpa-tients are candidates for surgical resection due to the location of the primary tumor near or even wrapped around major arteries and bile ducts. The tumor microenvironment is also highly immunosuppressive due to large populations of tumor-associated immune cells such as tumor-associated macrophages and myeloid-derived suppressor cells that have made im-munotherapyoptionsineffectiveinclinicaltrials.Furthermore,pancreaticcancerhasahighrate of metastasis that can lead to organ failure and death in most patients.Irreversible electroporation (IRE) offers a novel, non-thermal approach to tumor ablation.IRE utilizes short, high frequency electrical pulses to form permanent pores in cancer cell membranes. This leads to a loss of homeostasis and the induction of programmed cell death. Inclinicalapplication,IREhasshownsignificantsuccessandiscurrentlybeingfast-trackedby the FDA as a treatment option for pancreatic cancer. However, the evaluation of IRE’s biologicaleffectsonhealthytissue,thetumormicroenvironment,andontumorprogression

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outside of patient survival has been limited.WehypothesizethatIREcanalterpancreaticcancer’simmunosuppressivetumormicroenvi-ronmenttoinhibittumorprogression.Weutilizedheathyporcinetissues,humanpancreaticcancer patient-derived xenograft models, and immunocompetent in vivo murine models to evaluate theeffects of IREon the tumormicroenvironment, immunosuppressive immunecellpopulations,andondiseaseprogression.OurstudiessuggestthatIREcaninducepro-inflammatorycelldeathinpancreatictumorsandalterimmunosuppressivecellpopulations.IRE also leads to increased progression-free survival and lower disease burden with few treatment side effects, potentially due to increased immunosurveillance. These findingscould greatly impact clinical application of IRE for pancreatic patients and allow us to identify potential co-therapy options or targets to increase pancreatic cancer patient survival.

Presenter: AshleighN.Riegler 46Title: The role of necroptosis in the development of adaptive immunity to air-

way infection is pathogen type-dependent. Email: ariegler@uab.eduCo-Authors: Michael J. Fuller, Meagan M. Jenkins, André Ballesteros-Tato, and Car-

losJ.OrihuelaAffiliation: TheUniversityofAlabamaatBirmingham

Necroptosis, a programmed form of lytic cell death, is initiated by various viral and bac-terial pathogens through irreparable ion dysregulation and energy depletion. This cellular damage results in the activation ofRIPK1,RIPK3, consecutively, and the activation andmembrane targeting ofMLKL, the latter responsible for lysis. Recently we have demon-strated that necroptosis of airway epithelial cells is key in the development of the adaptive im-mune response to asymptomatic colonization by Streptococcus pneumoniae(Spn).Briefly,necroptoticdeficientanimalsorwildtypeanimalscolonizedwithSpn lacking the necroptosis-triggering pneumolysin toxin, failed to recruit CD11c+ leukocytes to the sites were Spn were present, had dampened serum IgG responses,were delayed in bacterial clearance, andweremore susceptible to subsequent lethal re-challenge [Front Immunol. 2019, 10:615].Herein we examined the role of necroptosis in the generation of an adaptive immune re-sponse to influenzaA (Flu),aviralpathogenalsoshown to inducenecroptosisofairwayepithelialcells.Wildtype,RIPK3KO,andMLKLKOmicewerechallengedintranasallywith6,500PFUofFlustrainA/PuertoRico/8/1934(i.e.PR8).Weight losswasmonitoredoverthecourseof15daysasameasureofdiseaseseverity.Atday15,miceweresacrificedandlungsandmediastinallymphnodes(mLN)werecollectedforenumerationofimmunecellspresent. Incomparison towildtypeandMLKLKO,RIPK3KOmiceexperienced themostsevereweight loss, indicatinga role forRIPK3activation independentofMLKL-mediatedcell lysis. Enumeration of immune cells by flow cytometry showed no differences in Flu-specificeffector(CD62L-CD69+CD25+),centralmemory(CD127+CD62L+),effectormemory(CD127+CD62L-),andtissueresidentmemory(CD69+CD103+)lungandmLNCD8+ T cell re-sponses.Similarly,nodifferenceswereobservedinFluspecificmLNBcells(CD19+CD95+ CD38+ IgG1+), ormLNand lungT regulatory cells (CD19-CD25+FOXP3+) andT follicularhelpercells(CXCR5+PD1+BCL6+).Theseresultssuggestfundamentaldifferencesintherolefor necroptosis in the development of adaptive immunity against viral versus bacterial patho-gens.Ongoing studies are focused on determining the contribution of necroptosis to theinnateimmuneresponsetoFluinordertoexplaintheobserveddifferencesinRIPK3andMLKLdeficientmice.

Presenter: KaliCrofts 47Title: A novel mechanism for IL-4 mediated self-tuning of functional avidity in

CD8+ T cellsEmail: kali.crofts@uqconnect.edu.auCo-authors: BethC.Holbrook,DavidR.Soto-Pantoja,DavidA.OrnellesandMartha

A. Alexander-MillerAffiliation: WakeForestSchoolofMedicine

Functionalavidity isacriticaldeterminantof theabilityof cytotoxic lymphocytes (CTL) to

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function in vivo. High avidity CTL are sensitive to low peptide/MHC, while low avidity CTL require high peptide/MHC. The increased sensitivity of high avidity cells results in improved virusandtumorclearance.Whereastheimportanceoffunctionalavidityiswellestablished,themechanismsthatdeterminethesensitivityofaTcelltopeptide/MHCarelessso.WehaveidentifiedanewprocessthroughwhichCD8+ T cells can actively modulate their func-tionalavidity.WefoundCD8+ T cells sense and respond to the level of TCR engagement throughtheregulatedproductionofIL-4.StimulationofOT-ICD8+ T cells with high, but not low amounts of peptide induced autocrine IL-4 that resulted in establishment of a lower avid-ity setpoint. Ca2+ signaling was critical for IL-4 production as demonstrated by promotion of IL-4 production by ionomycin and reduced production following addition of cyclosporine A. High TCR engagement resulted in increased nuclear NFATC1, which has been reported to regulate IL-4 production in CD4+Tcells.Ourdatasupportamodelwherein thepertinentdownstreameffectofencounterwithstrongTCRengagementisanincreaseinNFATC1inthe nucleus resulting in IL-4 production. These data reveal a novel self-tuning mechanism to control the activation set point of CD8+ T cells through regulated autocrine production and response to IL-4.

Presenter: Robin Rolader 48Title: Comparing Clinical and Biological Aspects of Pruritus in the Immune-

Mediated Diseases Atopic Dermatitis and Bullous Pemphigoid Email: rrolade@emory.eduCo-Authors: TarynDeGrazia,YuanLiu,YanyanXing,LiangHan,BridgetBradley,

CeciliaThompson,SandyFrancois,SafiyyahRasheed,SarahChisolm,Suephy Chen, Ron Feldman

Affiliation: EmoryUniversitySchoolofMedicine,DepartmentofDermatology

Skinconditionsthatresultinintenseitchinghaveasignificantimpactonpatientqualityoflife(QOL).Atopicdermatitis(AD)andbullouspemphigoid(BP)aretwomechanisticallydistinctimmune-mediated diseases both characterized clinically by intense chronic itching. Although ADandBPhavedifferencesinclassicpresentation(eczematouseruptionversusurticariaandblisters)andpopulationcharacteristics(youngtomiddleagedpersonsversuselderly),early stages of BP can appear strikingly similar to AD clinically and histologically. Preliminary data from our group have demonstrated that both AD and BP share similar itch symptoms andQOLimpactdespitedifferencesinoverallclinicalpresentation.However,themecha-nisms for this enhanced pruritus in both diseases are unclear. Previous data have suggested that patients with AD demonstrate an increase in cutaneous nerve endings which may ex-plaintheenhanceditchsensation.Weexploredtheroleofthesensoryneuronandpotentialinfluenceson immune-mediatedpathways thatmaybeperpetuatingand/or responding topruritus in these twoseeminglydifferentcohortsofpatients.Biopsieswereobtained fromperilesional skin in patients with AD and BP, examined histologically to detect alterations in intraepidermalnervefibers,and thencompared toskin fromhealthycontrols.Sampleswere obtained from patients with broad demographic diversity, from various body sites, and withitchdurationrangingfrom7months–15years.NervedensitywascalculatedbasedonthenumberofPGP9.5stainedfibersintheepidermis.Interestingly,theaverageepidermalnervedensityintheskinofpatientswassignificantlyreducedcomparedtohealthycontrols(6.89fibers/mm±2.44vs11.49fibers/mm±3.69).Thesedatasuggestthatseeminglydiffer-ent immunologic mediated skin diseases share common clinical endpoints of severe itching, quality of life impact, and reductions in epidermal sensory nerve innervation. It is possible that the decreased epidermal nerve density over time is due to immune-mediated damage; alternatively, active downregulation of sensory nerve function may occur to dampen the itch response. Further researchevaluatingalterations incorresponding itchspecificsignalingpathwaysinthesechronicinflammatorydiseasesmayelucidatecommonpathwaysandar-eas for therapeutic intervention.

Presenter Matthew Cottam 49Title Adipose tissue CD4+ and CD8+ memory T cells are retained with

weight loss and CD8+ memory T cells expand during weight regainEmail Matthew.A.Cottam@vanderbilt.edu

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Co-Authors ArionJ.Kennedy,AlyssaH.HastyAffiliation VanderbiltUniversity

Bouts of weight loss and weight regain, referred to as “weight cycling”, have been associated with increased risk of cardiovascular disease progression and increased risk of development of type 2 diabetes mellitus in humans. To identify causes and correlation of the progressive metabolic dysregulation observed in humans who weight cycle, we have developed a mouse modelofweightcyclinginwhichalternating60%highand10%low-fatdietsinducecyclesofweightgainandweightloss.Glucosetoleranceinweight-cycledanimalsisimpairedcom-pared to age, weight, and adiposity-matched high-fat fed control mice. In conjunction with thisfinding,wehaveobservedanenrichmentforCD8+CD62LlowCD44+ memory T cells, but not CD4+CD62LlowCD44+ memory T cells, following weight cycling in mice. Therefore, we hypothesize that the observed impairment in glucose tolerance is due to re-activation of primed memory T cells during weight regain. To test this, we are characterizing T cells and investigating their role in AT insulin resistance during periods of weight gain, weight loss, and weight cycling. In mice that have lost weight, despite a reduction in body mass and adiposity and a concordant resolution of impaired glucose tolerance, CD4+ and CD8+ T cells increased dramatically.Furthermore,CD62LlowCD44+ memory T cells in both CD4+and CD8+ popula-tionswereelevatedinweight lossmicecomparedtoobesemice.ThesefindingssuggestT cells are retained during weight loss even in the absence of metabolic dysregulation or alteredadiposityandmaybeprimedtorespondtosubsequentweight regain.With thesefindingsinmind,ongoingworktoidentifyATTcellreceptordiversityandcommonclonotypesis being performed.

Presenter: TarynMcLaughlin 50Abstracttitle: CD4+TcellsinMycobacterium tuberculosis and Schistosoma manso-

ni co-infectedindividualsfromKisumu,Kenyaremainfunctionaldespitealtered lineage phenotypes

Email: taryn.mclaughlin@emory.edu Co-authors: JeremiahKhayumbi2,JoshuaOngalo2, Joan Tonui2,FelixHayaraOdhi-

ambo2, Cheryl L. Day1,3

Affiliation: 1Emory Vaccine Center, Emory University, Atlanta, GA; 2Center for Global Health Research, KenyaMedical Research Institute, Kisumu,Kenya; 3DepartmentofMicrobiology& Immunology,EmoryUniversitySchoolofMedicine,Atlanta,GA

Itiswellestablishedthataneffectivetype1Tcellimmuneresponseisnecessarytocontrolinfection with Mycobacterium tuberculosis(Mtb).Determiningfactorsthatmodulatetype1immuneresponsesisthereforecriticalinfurtherdefiningcorrelatesofprotectionduringMtbinfection. Helminths stimulate a strong type 2 immune response, which has been shown to antagonizetype1CD4+T(TH1)cells.Assuch,wesoughttoevaluatewhetherco-infectionwith the parasitic helminth Schistosoma mansoniimpairsCD4+TcellresponsestoMtbinacohortofHIV-uninfectedadults inKisumu,Kenya. Individualswerecategorized intosixgroups by Mtb and S. mansoni infectionstatus:healthycontrols(HC), latentMtbinfection(LTBI)andactivetuberculosis(TB),withorwithoutconcomitantS. mansoni infection.WeevaluatedthefrequencyandlineagephenotypeofCD4+TcellresponsestoMtb in PBMCs usingflowcytometry.Compared toHC, individuals in theLTBIandTBgroupshadmorediverseMtb-specificcytokineresponses,includingproductionofIL-4andIL-13.AcrossallgroupsMtb-specificCD4+TcellswerecharacterizedbyexpressionofbothclassicalTH1markers,CXCR3andT-bet,andTH2markers,CCR4andGATA3.Additionally,theywereenrichedforCCR4+CXCR3+cellsascomparedtobulkCD4Tcellsfromthesameindividual.Tofurthercharacterizethesecells,westimulatedMtb-specificCD4+Tcellsforfivedaystoevaluateproliferation,cytokineproduction,andlineagestate.CD4+Tcellproliferationwashigher in LTBI individuals as compared to HC and TB; however, proliferation capacity did not vary by S. mansonistatus.Similartotheshort-termstimulation,Mtb-specificCD4+TcellsexpressedbothTH1andTH2lineagemarkers.TogetherthesedatashowthatMtb-specificCD4+TcellsarenottraditionalTH1cellsandthatthisphenotypepersistsascellsproliferateinvitro.ThissuggeststhatMtb-specificimmuneresponsesmaybeflexibleinresponsetoantigen. In high pathogen settings where co-infection is common and reoccurring, this may

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beimportantinpreservingMtb-specificTH1responses.

Presenter LoganMelot 51Title Examination of measles virus replication in primary human B cellsEmail lmelot@emory.eduCo-Authors Melissa Coughlin, Bettina BankampAffiliation CentersforDiseaseControlandPrevention,EmoryUniversity

Wild-typemeasles virus (MeV) infection generates a state of immunosuppression,whichdoes not occur after vaccination. In vivo data in the rhesus macaque model suggest that B lymphocytes are a target of wild-type measles virus. However, the contribution of B cells to the pathogenesis of measles and the role B cells may play in measles virus-induced immu-nosuppressionisnotknown.PreviousdatausingarecombinantGFP-expressingvirussug-gests that B cells are infected in vivo and in vitro; however, the replication of measles virus in B cells has not been investigated. This study seeks to examine the replication of wild-type and vaccine strains of measles virus in vitro in isolated primary human B cells. Replication has been investigated by several methods. The expression of viral and recombinant proteins wasevaluatedbyflowcytometryusingananti-MeVhemagglutininantibody,RT-qPCRusingaTaqmanassayspecificformeaslesvirusNgeneandaccumulationofreporterproteinus-ingarecombinantmeaslesvirusencodingGFP.TheproductionofinfectiousvirusbyBcellswasmeasuredbyendpointdilution(TCID50).ViralreplicationwasnotdetectedinprimaryB cells that have been infected in vitro by any of the methods used. Preliminary data show accumulationofGFPinBcellswhenwholeperipheralbloodmononuclearcells (PBMCs)areinfectedwithrecombinantmeaslesvirusencodingGFP.However,replicationwithinBcells could not be demonstrated by the accumulation of viral N gene RNA after isolation from infectedPBMCs.ThesefindingssuggestthatBcellsmaynotbeproductivelyinfectedwithmeasles virus, but are acquiring antigen from infected cells.

Presenter AlexisSponaugle 52Title Generationandcharacterizationofbothclassicallyandnon-classically

restricted CD8+TcellclonesthatdirectlytargetHIV-infectedcells.Email sponaugl@email.unc.eduCo-Authors Alexis Sponaugle1, Maria Abad1, Carolina Herrera1, Maxwell Departee1,

Yinyan Xu1,JoannaWarren1, Ed Browne1,NiluGoonetilleke1

Affiliation 1UniversityofNorthCarolina,NC.USA.

IncontrastwiththehighlypolymorphicMHC-Ia(i.e.MHC-A,-Band-C)locus,non-classicalMHC-Econtainsonlytwoalleles,designatedMHC-E*01:01andMHC-E*01:03,throughoutthe entire human population. The nonsynonymous mutations existing between these two al-leles are outside of the peptide-binding groove, making MHC-E essentially non-polymorphic. In natural infection, MHC-E restricted CD8+ T cells are rarely reported. Recent studies have shown that vaccine-induced MHC-E restricted CD8 T cell responses mediated potent protec-tionagainstchallengewithpathogenicSIV.OurgoalistocloneMHC-ErestrictedCD8+ T cellsinducedbyHIVinfectionwiththeeventualgoalofdevelopingMHC-ErestrictedCD8TcellimmunotherapeuticsagainstHIV.Here, we present an adapted high throughput protocol to generate both classically and non-classically restrictedCD8T cell clones.CD8T cellswere isolated, plated at 1500 cells/well thenexpanded300-500-foldwithCD3/CD28activatingbeads.Tcells fromeachwellweretestedinELISpotforHIV-andHCMV-specificTcellresponses.WecollectedCD8Tcells fromreactivewells,stimulatedthecellsandsinglecellsortedIFNγ+TNFα+cells.Weexpanded the CD8+ T cells 1-8 million-fold by allogenic and cytokine stimulation over the next8weeks,successfullyyieldingclonalHIV-andHCMV-specificclassicallyrestrictedTcellclones.ThismethodisbeingadaptedtoisolateandcloneHIV-specificMHC-ErestrictedCD8+TcellsfromHIVinfected,ARTsuppressedparticipants.

Presenter SkyeOpsteen 53Title Evaluating biomarkers of microbial translocation in an HIV cohort to

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identifypersistentinflammationEmail sao678@uab.eduCo-Authors KeithChampion,LynnPrichard,SonyaL.HeathAffiliation UniversityofAlabamaatBirmingham

Approximately37millionpeopleareinfectedwithHIVglobally,andwhileeffectivetreatmenttosuppressviralreplicationismorereadilyavailable,peoplelivingwithHIV(PLWH)havesignificant comorbidities driven by inflammation including cardiovascular and neurocogni-tivediseases.Microbialtranslocation(MT)isasignificantdriverofinflammationcontributingtoHIV pathogenesis even in virally suppressed patients on anti-retroviral therapy (ART).Translocatedgram-negativebacteria release lipopolysaccharide (LPS) from theircellwallinto the blood, which stimulates and activates monocytes resulting in the release of soluble CD14(sCD14).Generalizedimmuneactivationalsoresultsinthereleaseofcytokinessuchas TNF-αfrommacrophages.GiventhatMTisakeydriverofchronicimmuneactivationinHIV,wesoughttoevaluatebiomarkersofMTintheplasmaofPLWHwith(n=17)andwithout(n=26)viralsuppressioncomparedtohealthycontrols(n=8)bymeasuringLPSvialimulusamoebocytelysate(LAL)assaysandTNF-αandsCD14levelsviaELISAs.InPLWH,LPS(p<0.01)andTNF-α(p<0.001)weresignificantlyhigherthanhealthycontrols.sCD14waselevatedinPLWHcomparedtohealthycontrolswhowereoffARTandhadcompromisedCD4Tcellcountsof<500cells/mL3(p<0.05).However,therewasnocorrelationbetweenthe increase in LPS levels and sCD14 or TNF-α in this small study. GiventhatbiomarkersofMTremainelevateddespiteviralsuppression,thesestudiessug-gestthepotentialfortheiruseinmonitoringinflammationcontributingtocomorbiddiseases.Future studies to evaluate this pathway could aid in biomonitoring, identifying patients that wouldbenefitmost fromtargetedtherapiestomodulate inflammationandimprovepatientoutcomes.

Presenter: DennisNeeld 54Title: PD-1 is negatively regulated by LSD1 through interactions with Blimp-1

during acute viral infectionEmail: dneeld@emory.eduCo-authors: AlexanderP.R.Bally,PeiyuanLu,ParimalMajumder,BenjaminG.Bar-

wick, and Jeremy M. BossAffiliation: EmoryUniversity

Prolonged exposure to antigen, such as during chronic viral infections and cancer, leads to sustained TCR signaling and ultimately T cell exhaustion, which is in part mediated by up regulationofProgrammedcelldeath1(PD-1).AntibodyblockadeofPD-1doesnotworkinall instances and can lead to autoimmunity, thus identifying novel tools to manipulate PD-1 are needed. Therefore, we sought to understand the genetic and epigenetic mechanisms that regulate PD-1 during acute and chronic antigen conditions in order to identify additional therapeutictargetstomodulatePD-1.WedeterminedthechromatinstatusatthePD-1locusandfoundthatactivatingH3K4me1andH3K4me2epigeneticmodificationswerestillpresentatday8duringachronicbutnotacuteLCMVinfection.Thissuggestedafailuretoremovetheactivehistonemarksinchronic infections. Indeed,CD8Tcells lackinglysine-specifichistonedemethylase1A(LSD1),aH3K4demethylase,displaysignificantlyhigherlevelsofPD-1 mRNA and protein during acute but not chronic infection. Molecular analysis revealed that Blimp-1 recruits LSD1 to the PD-1 locus to induce silencing during acute infection. De-spiteBlimp-1binding to thePD-1 locusduringbothacuteandchronicLCMV infection, itfailstorecruitLSD1duringthechronicsetting.Additionally,failuretoremoveH3K4meth-ylation resulted in less DNA methylation at known PD-1 control regions compared to wild-typecells,consistentwiththedifferentialexpressionofPD-1.Takentogether,theseresultsdemonstrate that LSD1 downregulates PD-1 by interacting with Blimp-1 during acute, but not chronicLCMVinfection,facilitatingfullepigeneticrepressionofthelocus.

Presenter: AllisonDyevoich 55Title: TLR and CLR agonists induce antitumor B-1a cell and monocyte re-

sponses in peritoneal carcinomatosis

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Email: adyevoic@wakehealth.eduCo-Authors: MarcelaHaro,KarenM.HaasAffiliation: Dept.ofMicrobiology&Immunology,WakeForestSchoolofMedicine

Peritoneal carcinomatosis (PC) is a conditionwhereprimary tumorsmetastasize into theperitoneal cavity and induce malignant ascites. Due to the high mortality rate associated with this disease and the lack of curative treatments available, it is imperative that more effective treatmentsbedeveloped forPC.The immunesystemplaysan important role intumor elimination, and one strategy currently being explored to treat malignancies involves leukocyteactivation throughpattern recognition receptors (PRRs), includingToll-LikeRe-ceptors(TLRs)andC-typeLectinReceptors(CLRs).OurdatashowsthatmicetreatedwithacombinationofaTLRandCLRagonistareaffordedsignificantprotectionwhenchallengedwithanaggressiveascites-formingmammarytumorcellline(TA3-Ha).However,whenmicelackB-1acells,theprotectionislost.Consistentwiththis,thetreatmentinducesasignificantincreaseinIgMandcomplement(C’)boundtotumorcells,bothofwhicharecriticalcom-ponentsintheantitumoreffect.Furthermore,treatmentinducesarapidincreaseinLy6C+monocytes into the peritoneal cavity that is proceeded by an increase in Ly6Clow macro-phages.Ly6C+monocyteswererequiredfortheantitumorresponse,andtheirrecruitmentdependentontypeIinterferon(IFN-I)signaling.Interestingly,IFN-Idoesnotmediatetheac-tivation of B cells or secretion of IgM, nor do B-1a cells mediate the recruitment of monocytes into the peritoneal cavity, suggesting an uncoupled mechanism between B-1a cell activation and monocyte function. In summary, our data demonstrates pathogen associated molecu-lar patterns can induce rapid natural IgM production that elicits anti-tumor protection via C’ activation. Furthermore, treatment causes essential IFN-I-mediated recruitment of Ly6C+monocytes into the peritoneal cavity that are recruited independently of B-1a cell activation. Ourfindingsprovideapossiblemechanisminwhichtumormetastasisintheperitonealcav-ity can be targeted for clearance in an inherently immunosuppresive microenvironment. This workwassupportedbyaDepartmentofDefenseGrant,W81XWH-15-1-0585,awardedtoK.M.H,andaT32traininggrant,2T32AI007401-26A1awardedtoA.M.D. Presenter PauloHenriquedeMelo 56Title miR-21drivesproinflammatoryandglycolyticprogramofmyeloidcells

during sepsis Email paulo.d.melo@vumc.orgCo-Authors Annie Roccio Pineiros, Carlos Henrique SerezaniAffiliation VanderbiltUniversityVanderbiltUniversityMedicalCenter

Background:Sepsisisassociatedwithahyperinflammatorystate,switchofmetabolicprofileof immune cells and impaired innate immune functions of phagocytes, collectively leading to organ damage and lethality. Identification and regulation ofmoleculeswith pleiotropiccan both prevent exaggerated inflammatory response and decreasesmorbidities associ-atedwith sepsisMicroRNA-21 (miR-21) is oneof themost abundantmicroRNAs (smallnoncodingRNA)inphagocytesandplaysimportantrolesinbothincreasingordampeninginflammation.HowevertherolesofmiR-21intheinnateimmunesystemduringsepsisareinconclusive. Aim: Here, we aim to identify the targets and mechanisms by which miR21 influencesglycolyticmetabolismand inflammatory profile ofmacrophageandneutrophilsduringsepsis.WehypothesizedthatmyeloidmiR21inhibitstheproductionofanti-inflamma-torymediators, leadingtoaberrantglycolysis, inflammatoryresponseandanimal lethality.Results and conclusion:WT (C57BL/6),miR21fl/fl and miR21Δmyel mice were subjected to cecal ligation and puncture (CLP) and peritoneal lavage, serum, bronchi alveolar lavagefluid(BALF)werecollectedwithin18handtheanimalsurvivaldeterminedovertime.Sepsisenhanced miR21 expression in neutrophils and macrophages from peritoneal cavity and lung afterCLP.MiR21deficientmyeloidcellsshowincreasedanimalsurvival,whichcorrelatedwithincreasedbacterialclearanceinboththesiteofinfection(peritonealcavity)andsystemi-cally(blood)anddecreasedproductionofinflammatorycytokines,suchasIL6andIL1b,andreducedofheartandliverdamageasshowbyreducedCK-MBandTGO,respectively.Next,wesoughttotestwhetherdecreasedinflammationinmiR21deficientcellsisdependentonthe glycolytic metabolic program in macrophages/neutrophils during sepsis. Both peritoneal andBALFcellsfromsepticmiR-21deficientmyeloidcellsshoweddecreasedexpressionof

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glycolyticgenes(Hif1a, Scl2a1, Hk1/2, Pkm, Ldha, Mct4).Also,decreasedHK1proteinwasassociatedwithlowerlactatereleasethansepticWTmice.Corroboratingthe in vivo data, LPS-challenged BMDM and peritoneal macrophage frommiR21Δmyel also exhibited lower ex-pressionofglycolyticenzymesandinflammatorycytokines(TNF,IL-1andIL-6)thanmiR21fl/fl cells. Both,macrophages (BMDM and Peritoneal) and neutrophils were submitted toglycolyticstressassay (Seahorse)afterLPSstimulation inorder toconfirm the functionalimpact of miR-21 over glycolysis. Similarity in both cells the lack of miR-21 leads to decrease of ECAR. In conclusion, higher expression of miR-21 after sepsis and LPS stimulation leads toassemblyofinflammatoryandglycolyticmetabolicprograminmyeloidcells.

Presenter AndreaShiakolas 57Title High-throughput mapping of B-cell receptor sequences to antigen speci-

ficityEmail andrea.r.shiakolas@vanderbilt.eduCo-authors IanSetliff*,KelseyA.Pilewski,AmynA.Murji,NagarajanRaju,Larance

Ronsard,CharissaMynhardt,RutendoZiki,KatarzynaJanowska,Ma-saru Kanekiyo, Juliana Qin, Kevin J. Kramer, Allison R. Greenplate,BarneyS.Graham,PriyamvadaAcharya,MarkConnors,LynnMorris,DanielLingwood,IvelinS.Georgiev

Affiliation VanderbiltUniversity

The human B cell repertoire is a rich source of broad and potent neutralizing antibodies againstinvadingpathogens.Recentadvancesinnext-generationsequencing(NGS)allowfor high-throughput interrogation of the antibody repertoire, including paired heavy and light chainsequencing.However, little isknownabout theantigenspecificityof theantibodiesidentifiedinNGSdatasets.Toovercomethis limitation,wedevelopedatechnologytore-coverantigenspecificityfromhigh-throughputsequencingofnatively-pairedheavyandlightchainsofhumanB-cell receptors (BCR).BcellsaremixedwithDNA-barcodedantigens,suchthatboththeantigenbarcode(s)andBCRsequencearerecoveredduringsequenc-ing,enablingdirectcouplingofantigenspecificityandpairedheavy/lightchainmonoclonalantibodysequences.Usingapanelofrecombinantglycoproteinantigens,includingdiverseHIV-1SOSIPtrimersandinfluenzahemagglutinin,wevalidatedthismethodusinghumanB-cell lineswith knownBCRsequencesand specificities. In this experiment,wedemon-strated the ability to simultaneously map thousands of B cells to their cognate antigens with highaccuracy,includingforBcellscross-reactivetomultipleSOSIPvariants.Additionally,wehaveappliedthismethodtostudyantibodyresponsesinmultipleHIV-infectedindividu-als,andwereabletoidentifybothknownandnovelcross-reactiveantibodylineages.Withminimalmodifications,thetechnologyshouldbeeasilyscalabletoenablescreeningofcloseto10^5singleBcellsagainsteachofdozensofantigens,overcomingthelimitsoftraditionalantigen-specificB-cellsorting.Theabilitytointerrogateantibody-antigeninteractionsusingasequencing-basedreadoutwillbehighlybeneficial to thefieldsof immuneprofilingandantibody discovery.

Presenter AaronSilva-Sanchez 58Title NeonatallungCD103intefferocyticdendriticcellspreventgenerationof

effectorCD8TcellsEmail asilva@uab.eduCo-authors Selene Meza-Perez1, Frances Lund2, Sara L. Stone2, Alex Rosenberg2,

Troy Randall1Affiliation TheUniversityofAlabamaatBirmingham,DepartmentofMedicine1

and Department of Microbiology2.

Vaccinationisoneofthemostimportantadaptationsofhumansocietytoreducesusceptibil-itytoinfectiousdiseases.However,vaccinationisoftenmuchlesseffectiveinnewbornsandinfants and the mechanisms that limit the induction of protective immune responses, par-ticularly in the lung, are not completely understood. Here, we established a murine model of pulmonary vaccination to examine the role of lung dendritic cells in the generation of antigen-specificCD8+Tcellsinadultsandneonates.Wefoundthatthelungsandmediastinallymph

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node(MedLN)ofneonatalmicecontainedamajorityofBatf3-dependentXCR1+ DCs, while adult mice contained a majority of SIRPa+ DCs. However, adult XCR1+ DC express high lev-elsofCD103,whiletheneonateXCR1+DCscontaintwodistinctsubsetswithdifferentlevelsofCD103expression-theCD103intDCsandtheCD103hiDCs.TheCD103int DCs have a maturephenotype(CCR7+, PDL-1+, CD40+)thatoccursindependentlyofmicrobiotaorTLRsignaling.AlthoughCD103int DCs spontaneously migrate to the draining lymph node, they failtocaptureandcross-presentexogenousantigens,asaconsequenceOVA-specificCD8Tcellsprimedinneonatesfailtodifferentiateintoeffectorcytotoxiclymphocytes.Transcrip-tomeanalysisofCD103int DC showed increased expression of genes associated with the phagocytosisofapoptoticcells.ThenumberandactivationstatusofCD103int DCs was re-ducedinmicewithdeficientrecognitionofapoptoticcells(MerTK-/-).PrimingofOVA-specificCD8TcellsintheMedLNofMerTK-/-neonatemiceproducedmoreeffectorCD8+ T cells than intheMedLNofcontrolmice.Onthecontrary,usingapoptoticcellstodeliverAgandprimeCD8+TcellinadultmiceproducedpooreffectorCD8Tcells.Overall,ourresultsshowthatthepresenceofactivatedefferocyticCD103int DCs in newborn lungs prevents CD8+ T cell activation by limiting the cross-presentation of exogenous antigens.

Presenter ArielSpurrier 59Title ContributionofB cell subsets toTI-2Ag-specificantibodyproduction

and memoryEmail mspurrie@wakehealth.eduCo-Authors ChristinaA.Daly,JamieJennings-Gee,KarenM.HaasAffiliation WakeForestSchoolofMedicine

Antibody(Ab)responsestoTcellindependenttype2antigens(TI-2Ags)dependoninnate-likeBcellsubsets,includingB-1bcellsandmarginalzone(MZ)Bcells.However,therolesBcellsubsetsplayinclonalexpansion,isotypeswitching,andmemoryBcelldifferentiationinresponsetotheseAgshavebeenunderstudied.UsingBcellsfrommiceexpressingtheVHB1-8geneknockingene,weevaluatedthecontributionofB-1b,MZ,andfollicular(FO)B cell subsets to the TI-2 Ag, NP-Ficoll. All B cell subsets divide in response to NP-Ficoll; nonetheless,Ag-specificB-1bcellsmorerapidlyclassswitchtoIgGandhaveincreaseddif-ferentiationintoAbsecretingcells.AllsubsetsformedmemoryBcells(CD138negCFSEneg)and expressedmemorymarkers previously identified for TDmemory B cells. B-1b cellscontributed to the highest number of memory cells in the spleen and peritoneal cavity, which includedhigher frequenciesof IgGandCD80-expressingcells,whileMZBandFOBcellswerefoundmorefrequentlyinlymphnodesandbonemarrow.Despitesignificantmemoryformation, secondary immunization of recipient mice 4 weeks after primary immunization did notincreaseNP-specificIgGlevels.However,boostinginB-1bcell-recipientmiceoccurredwhenIgGlevelsdeclined.Finally,useofaTI-2Ab-boostingadjuvantenabledsignificantlyhigherprimaryandsecondaryIgGresponsesinB-1bcell-recipients.Collectively,thesefind-ingsdemonstratehighaffinityB-1bcellsgenerateafunctionalmemorypopulationwhichcanbestimulatedtoproducesignificantlevelsofTI-2Ag-specificAbeitherunderconditionsoflowAg-specificIgGorinthepresenceofselectadjuvants.Nonetheless,MZBandFOBsub-setsalsosignificantlycontributetotheTI-2Ag-specificmemoryBcellpool,whichmayhaveimplications for regulation of recall responses.

Presenter: ShakyraRichardson 60Title: ImmunomodulatorycapabilityofVCGinChlamydia immunityEmail: srichardson@msm.eduCo-Authors: RoshanPais,YusufOmosun,StephanieLundy,JosephU. Igietseme

andFrancisO.EkoAffiliation: MorehouseSchoolofMedicineandCenterforDiseaseControl

ImmunizationwithUV-inactivatedChlamydia trachomatiselementarybodies (UV-EB), theinfectious form of Chlamydia, does not protect against infection with live Chlamydia.Vibriocholerae ghosts(VCG),whichareemptyV.choleraecellenvelopesderivedbygeneticinac-tivation,constituteaneffectivedeliveryvehicleforclonedvaccineantigensandpromotestheinduction of protective immunity in the absence of added adjuvants. In this study, we sought

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to investigate theability ofVCG toenhance the innateandadaptive immune responsesinducedbyUV-EB.Thus,bonemarrow-deriveddendriticcells(BMDC)werepulsedwithUV-EBinthepresenceorabsenceofVCGandtheinductionofinnateimmuneresponses,TcellproliferationandabilitytoaffordprotectionagainstChlamydia following adoptive transfer into susceptiblemicewasevaluated.TheresultsshowthatVCGefficientlymodulatedtheimmu-nostimulatorypropertiesofDCsasindicatedbyincreasedsecretionofproinflammatorycyto-kines and expression of toll-like receptors and co-stimulatory molecules associated with DC maturation.Also,UV-EB-pulsedDCsthatwereexposedtoVCGandadoptivelytransferredintomiceresultedineffectivechlamydialantigenpresentationandprotectiveimmunity.TheresultsdemonstratethatVCGactivatethematurationofDCsleadingtoenhancementofin-nate and adaptive immunity to an otherwise non-protective antigen, suggesting their utility as adjuvants for enhancement of protective immunity to inactivated vaccine antigens.

ThisworkwassupportedbygrantsfromNIH(R01AI126897,1C06RR18386)andresourcesupport from CDC.

Presenter SarahMosure 61Title InvestigatinghemeregulationofREV-ERBα activity in Th17 cellsEmail smosure@scripps.eduCo-Authors SeanCampbell,DouglasKojetin,LauraSoltAffiliation ScrippsFlorida

T helper 17 (Th17) cells are important for protective immunity at mucosal barriers, buttheyarealso implicated inautoimmuneandchronic inflammatorydiseasepathogenesis.WerecentlydemonstratedthatthenuclearreceptorREV-ERBα is a cell-intrinsic negative regulator of Th17 cell development in vitro and Th17 cell pathogenicity in mouse models of autoimmunity in vivo.Mechanistically, REV-ERBα represses core Th17 cell genes by competingforbindingatDNAresponseelementssharedwiththeTh17celllineage-definingtranscriptionfactorRORγt. As ligand-regulated transcription factors, nuclear receptors like REV-ERBα are susceptible to regulation by natural, or endogenous, ligands, but it remains unclearwhether theendogenousREV-ERBα ligand,heme, regulatesREV-ERBα activity in Th17 cells. Identifying the roles of endogenous ligands contributes to our understanding of signaling pathways that govern Th17 cell homeostasis and pathogenicity. To investigate aroleforheme,wetreatedprimaryTh17cellsderivedfromREV-ERBα/βfl/fl(WT)orREV-ERBα/βfl/flxCD4-Cre(DKO)mice.WhilehemetreatmentrepressedthedifferentiationofbothWTandDKOTh17cells,therepressionwasnotasgreatintheDKO(orREV-ERBα single KO)cells.ThesedataindicatethathememayhavearoleinregulatingREV-ERBα activity. Totestthispossibility,weretrovirallytransducedaREV-ERBαmutant(H603F)incapableofbindinghemeinprimaryTh17cells.Relativetowild-type(WT)REV-ERBα,theH603Fmu-tant exhibited reduced repression of target genes, despite being present at the protein level ingreateramountscomparedtoWTREV-ERBα.TheincreaseinH603FproteinrelativetoWT indicated thathemebinding targetsREV-ERBα for proteasomal degradation in Th17 cells, consistent with previous studies using other cell types. Together, these results indicate thathemeregulatesREV-ERBa activity by multiple mechanisms in Th17 cells, enhancing bothREV-ERBα-mediatedrepressionofcoregenesandREV-ERBα protein turnover. Thus, heme may function as a key regulatory molecule coupling Th17 cell signaling pathways to geneexpressionthroughmodulationofREV-ERBα activity.

Presenter: KarlaNavarrete 62Title: Interchromosomal templated mutagenesis accounts for mutation clus-

ters in B cell non-Ig genesEmail: karla.navarrete@emory.eduCo-authors: GordonA.Dale,DanielJ.Wilkins,JoshyJ.JacobAffiliation: EmoryUniversity

The immunoglobulin(Ig)genesofgerminalcenterBcells(GCB)undergosomatichyper-mutation in order to generate antibody diversity. During this process, non-immunoglobulin (non-Ig)genescanalsoaccumulatemutations.Therearetwogenerallyacceptedmethods

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bywhichmutationsareintroducedintotheIgandnon-Igloci.Thefirstisthroughtheaccumu-lation of pseudorandom point mutations that occur as a result of activation-induced cytidine deaminase (AID)-dependent double strandedbreaks (DSB),whichare repairedbyerror-prone polymerase. The second is through the process of gene conversion, which involves the use of a template sequence to repair a DSB resulting in the template being copied at the site of repair. It is generally accepted that un-templated mutation occurs in humans and mice, whiletemplatedmutationoccursinchickensandlargefarmanimals.However,findingsfromourlabsuggestthatsomemutationsinthenon-IggenesofmurineGCBarisethroughageneconversion-likemechanism,henceforthreferredtoastemplatedmutagenesis.UsingGCBfromthePeyer’spatchesofagedmice(>6mo.),weamplified~500bpsectionsfromnon-Iggenes including CD79B,RHOH,BTG1,EBF1 and sequenced them using Illumina MiSeq 2x250bp.Wefoundthatthesenon-Iggenesaccumulatemutationsthattendtobeclusteredratherthandiffuse.OurlabhasdevelopedacomputationalpipelinecalledTemplateRec-ognitionviaAnalysisofMonteCarloExperiments(TRACE)thatisabletomatchmutationsclusters within an input sequence to partially homologous donor templates elsewhere in the genome.TRACE-identifieddonor templatesarestatistically likely tohavebeenused inatemplated mutagenesis event which led to the mutation of the input sequence. Through this analysis, we found that unlike gene conversion observed in chickens, murine templated mutagenesis occurs between sequences on different chromosomes. Furthermore, donortemplate sequences are enriched in certain locations in the genome and tend to originate in intronsandintergenicregions,ratherthanexons.Thesefindingshaveopenedanavenueinto the controversial study of non-Ig templated mutagenesis in murine B cells.

Presenter JustinJacobse 63Abstracttitle Interleukin-23receptorsignalingmodulatesthestabilityandfunctionof

FOXP3+ regulatory T cellsEmail justin.jacobse@vanderbilt.eduCo-authors JingLi,RajeevTyagi,JeremyA.GoettelAffiliation VanderbiltUniversity,LeidenUniversityMedicalCenter

Background: The cytokine interleukin 23 (IL-23) has been implicated in themultifactorialpathogenesisofinflammatoryboweldisease(IBD).IL-23haspreviouslybeenassociatedinthe maintenance of Th17 cell responses. However, its pathogenic role in IBD is likely inde-pendent of Th17 cell biology as IL-17 blockade with Secukinumab exacerbated disease. The IL-23receptor(IL-23R)ishighlyexpressedonasubsetofcolonichighlysuppressiveregula-toryTcells(Tregs)expressingforkheadboxP3(FOXP3).WehypothesizethatIL-23Rsig-nalinginTregsmodifiesthetranscriptionallandscapeoftheFoxp3 locus leading to reduced function of Tregs, and thereby driving immune dysregulation in the intestine.

Methods:ToassesswhetherexogenousIL-23activatesTregs in vitro, murine Tregs were stimulatedwith IL-23or IL-6andactivationofSTAT3wasassessedbyFACS.Thesup-pressive capacity of Tregs was examined by labeling naïve CD4+withCellTraceVioletandaCD3aCD28stimulation inpresenceorabsenceofexogenousIL-23,andwithorwithoutWTCD25+ Tregs. The in vivoroleofIL-23RspecificallyinTregswasdeterminedusingIl23r-floxFoxP3YFP-Cre mice.ToexplorewhetherexpressionofIL-23RalteredTregstability/survival,YFP+cellsfrommiceheterozygousorhomozygousforthefloxedIl23rallelewereinjectedinto lymphocytedeficientRag1-/- mice. Four weeks later, T cells were recovered from pe-ripheralcompartmentsandproliferationofdonorTregswasassessedbyEdUincorporation.

Results:ExogenousIL-23activatedSTAT3inTregstothesameextentasIL-6invitro.More-over,theabilityofWTTregstosuppressproliferationofnaïveCD4+ T cells was attenuated in presenceofIL-23in vitro. In vivo, Il23rflox/+Foxp3YFP-Cre mice had less YFP+ Tregs compared to Il23flox/floxFoxp3YFP-Cremiceinthecoloniclaminapropria.FollowingadoptivetransferofIL-23R-sufficientor-deficientYFP+ Tregs into Rag1-/- recipient mice, a decrease in YFP+ cells was observedinthemesentericlymphnodeandcoloninmicereceivingIL-23RsufficientTregs,whichwasnotattributedtoincreasedproliferationofIL-23R-deficientcells. Conclusions:Thesedatasuggest that IL-23RsignallingdestabilizesFOXP3directlyorbymodulatingsurvivalorfunctionofFOXP3+ cells in vitro and in vivo. However, the underlying

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mechanism(s)remainstobedefined.

Presenter LyPham 64Title Carnitine-palmitoyltransferase1a(CPT1a)activityisnecessaryforneu-

trophilactivationandtraffickingtothesiteofinfectionEmail Ly.Pham@Vanderbilt.eduCo-Authors Ly Pham, Timothy Thayer, Joshua Fessel, Evan Brittain, and Michael-

NotoAffiliation VanderbiltUniversity

Increasingly it is recognized that host genetic variability contributes to infection susceptibility andoutcomes.Thus, therehasbeengreateffortsmade towards leveraginggenomeandphenome-wide association studies to identify genetic factors that impact infection outcome. Ourunbiasedphenome-wideassociationstudyidentifiedanassociationbetweenpolymor-phisms in thegeneencodingcarnitine-palmitoyl transferase1a(Cpt1a)andan increasedrisk of infection, including bacterial pneumonia. CPT1a is a mitochondrial outer membrane proteinthatisrequiredforlong-chainfattyacidentryintothemitochondriafor(FA)oxidation.To examine whether CPT1a is a host determinant of infection outcome, a murine pneumonia model was used in which animals were treated with the inhibitor of CPT1a, etomoxir, or ve-hicle control prior to intranasal infection. Results using multiple bacterial pathogens indicate that animals treated with etomoxir exhibited increased mortality and bacterial burdens when compared with vehicle treated animals. Flow cytometric and histological analyses indicate thatanimalstreatedwithetomoxirhadasignificantreductioninneutrophilsinthebloodandrecruited to the lungs. To investigate the role of CPT1a in neutrophil recruitment in the setting of bacterial pneumonia, animals treated with etomoxir prior to intranasal infection were as-sessed for neutrophil production, activation, and egress from the bone-marrow. FACS analy-sis of the bone-marrow compartment determined that CPT1a function does not play a major roleinneutrophildevelopment.However,completebloodcount(CBC)revealedthatanimalstreated with etomoxir had fewer circulating neutrophils than PBS treated animals. Serum lev-elsofchemoattractantfactorsindicatethatetomoxirtreatmentdidnotaffectsignalsrequiredfor neutrophil mobilization during infection, however, FACS analysis shows reduced expres-sion of mobilizing chemokine receptors on neutrophil. These results suggest that CPT1a function may be critical for neutrophil activation and egress from the bone-marrow. Consis-tent with this, preliminary in-vitro chemotaxis data demonstrate that inhibiting CPT1a function reduced neutrophil migration towards the chemotactic peptide, fMLP, further suggesting that CPT1afunctionisinvolvedinneutrophiltrafficking.TogetherthesedataidentifyCPT1aasanovel host determinant for infection outcome.

Presenter Zheng-RongTigerLi 65Title QuantifyingtheselectiononinfluenzaAvirusimposedbyCD8TcellsEmail tiger.li@emory.eduCo-authors Veronika I.Zarnitsyna,AniceC.Lowen,JacobE.Kohlmeier,Rustom

AntiaAffiliation EmoryUniversity

An essential question regarding the T cell-inducing influenza vaccine is whetherthe cellular immunity may drive the virus to rapidly evolve and escape. In our previ-ous modeling study, we have shown an CD8 T cell escape-variant of influenza A vi-rus (IAV) invades extremely slowly given empirical HLA allele frequencies and mod-est selection pressure. Guided by the model, we aim to quantify the selection fromCD8 T cells using a novel digital droplet PCR system under different in vivo settings. MicewereintranasallyprimedwithHKx31(H3N2)andchallengedwitha1:1mixtureofwild-typeandmutantPR8,whichharborsanN370QescapingmutationinitsNP366-374 immunodom-inantepitope,onday30post-priming.Theareaunderthein vivoviralgrowthcurve(AUC)isusedtomeasurethefitnessofwild-typeandmutantviruses.BasedontheAUCsofC57BL/6(H-2b/b)andCB6F1(H-2b/d),theestimatedselectiveadvantageofthismutationis0.28[0.20,0.36]and0.21[0.10,0.34],respectively.Interestingly,althoughtheintramuscularlyprimedC57BL/6gaveasimilarestimatefor theadvantage(0.22[0.16,0.30]), theadvantagebe-

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camemuchmoreapparentonday6and8afterPR8challenge.ThisimpliesthelungresidentmemoryCD8TcellsmaybethesourceofselectionduringtheinitialstageofIAVinfection. Incombinationwiththemodelprediction,anescape-variantofIAVthatcarriesamutantim-munodominantepitopemayneed100to500generations(correspondingto1to5yearsiftheinfectionperiodisassumedtobe4days)toreach50%ofprevalencegiventhefrequencyofcognateHLAallelesis20%.WeconcludedthattheselectionfromCD8TcellsmaynotbesufficienttodrivetheCD8TcellepitopesofIAVtoevolverapidlyliketheantibodyepitopes.

Presenter: Michael A. Raddatz 66Title: Macrophages Promote RUNX2 Activation and Calcification in Aortic

ValveCellsEmail: michael.a.raddatz@vanderbilt.eduCo-Authors: TessaM.Huffstater,BradleyI.Reinfeld,JeffreyC.Rathmell,W.David

MerrymanAffiliation: VanderbiltUniversityImmunecellinfiltrationcharacterizescalcificaorticvalvedisease(CAVD),yetthereislittleunderstanding of the role of these cells in pathophysiology. The most common immune cells inboththehealthyandthediseasedaorticvalve(AV)aremacrophages,whichareknowntopromoteheterotopiccalcificationinotherdiseases.Tostudytheroleofmacrophagesinthecalcificationofaorticvalveinterstitialcells(AVICs),AVICswereisolatedfromwild-type(WT)mice,andbonemarrow-derivedmacrophages(BMMs)weregeneratedfromWTandNotch1+/-(N1+/-)mice.NOTCH1haploinsufficiencyleadstoCAVDinmiceandhumans.Cellswereseededinmonoculture,intrans-wellculture,orinco-cultureatphysiologicratio(1BMM:7AVICs)andassayedviaimmunofluorescence(IF),quantitativepolymerasechainreac-tion(qPCR),Westernblot(WB),andcalcificnodule(CN)assays.Eachcelltypewasthenenrichedfromco-cultureviamagnetic-activatedcellsorting(MACS)tobeassayedviaqPCRand/orWB.Separately, toassess the roleofhematopoieticcellsat large inCAVD,bonemarrowwasisolatedfromWTandN1+/-C57BL/6miceandinjectedintoirradiatedWTandN1+/- mice. Mice were evaluated by echocardiography and aged on high-cholesterol diet for 6months.MicewerethenevaluatedagainbyechocardiographyandeuthanizedforisolationoftheAV.AVswerestainedforcalcificationviavonKossa,fibrosisviaMasson’sTrichrome,immunecellinfiltrationviaIFforCD3andCD68,andcell-signalingviaIFforRUNX2.Co-cul-tureofBMMsandAVICsincreasesCNformationandexpressionofRunx2, a key transcrip-tionfactorinosteoblastdifferentiation,andosteopontin(Spp1),amarkerofosteogeniccalci-fication.IFstainingrevealsthatRUNX2signalingisincreasedintheAVICs,notbyadditionofRUNX2-expressingBMMs.MicereceivingN1+/- bone marrow transplant had an increased rateofdiseaseprogressiononechocardiographyandincreasedvalvularcalcificationbyvonKossastaining.Interestingly,calcificationco-localizedwithCD68-positivity.Translatingthisimpact of N1+/- macrophages to the in vitromodel,co-cultureofWTAVICswithN1+/- BMMs increasesexpressionofosteopontin.Thesefindingssuggest thathematopoieticcellsandspecificallymacrophagesplayasignificantroleinpromotingcalcificationinCAVD.

Presenter: ZahraaMohammed 67Title: MiR-155PositivelyandNegativelyRegulatesMastCellMediatorRe-

leaseEmail: zahraa.mohammed@uscmed.sc.eduCo-Authors: GregorioGomezAffiliation: UniversityofSouthCarolina

Allergicdiseaseisthe6th leadingcauseofchronicillnessintheU.S.(cdc.gov).Mastcellsplay a central role in allergic disease through the release of various biological mediators such as pre-stored mediators like histamine, serine protease, lipid- derived mediators like prostaglandins and leukotrienes, and cytokines and chemokines that contribute to allergic inflammation.MicroRNAs (miRNAs)areshortnon-encodingRNAs that regulategeneex-pression of various inflammatorymediators by suppressing translation or throughmRNAdegradation. Several miRNAs have been implicated in the regulation of allergic disease in-cludingmiR-155,whichhasbeenimplicatedinallergicasthma.However,theroleofmiR-155inregulatingmastcellsfunctionremainsunclear.Therefore,theaimofthisstudywas

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tocharacterize thespecificroleofmiR-155 inmastcellactivationandreleaseofallergic/inflammatorymediators.Humanskin-derivedmastcellsthatwereisolatedfromnormaltis-sue,andbonemarrow–derivedmastcells(BMMCs)thatweregenerated in vitro from wild type(WT)andmiR155knockout(KO)micewereusedasourexperimentalmodels.Micro-arrayanalysisandqRT-PCRwereused todetect thechanges in themiR-155andgeneexpression.Degranulationwasdeterminedbyβ-hexosaminidasereleaseassay.PGD2 and leukotrieneC4(LTC4)weremeasuredbyenzymeimmunoassay.TheeffectofmiR-155onp38MAPkinase(MAPK),p42/44(ERKs),proteinkinaseB(Akt)andphosphorylatedpro-teinsexpressionwasexaminedbywesternblot.MiR-155expressionwasinducedfollowingFcεRIcrosslinkingwithmultivalentantigeninhumanskinmastcellsandBMMCs.WefoundthatmiR-155hadnoeffectonFcεRI-inducedmastcelldegranulation.Ontheotherhand,FcεRI-inducedCOX-2wassignificantlyinhibitedintheabsenceofmiR-155suggestingthatmiR-155playsacriticalroleinPGD2biosynthesis.Importantly,wefoundthatATF3mRNAexpressionincreasedinBMMCsKOcomparedwithWT,suggestingthatATF3isatargetgeneofmiR-155inPGD2-COX-2pathway.Interestingly,miR-155hadnoeffectonFcεRI-induced LTC4. Accordingly, arachidonate 5-lipoxygenas (ALOX5) was expressed at thesamelevelsinWTandKOBMMCs.FcεRI-inducedcytokineproductionwasdiminishedinmiR-155KOBMMCscomparedtoWT.Interestingly,cytokineproductionfrommiR-155KOBMMCswasincreasedcomparedtoWTfollowingLPStreatment.ThesefindingsindicatethatmiR-155actsasapositiveregulatorintheFcεRIpathway,andanegativeregulatoroftheTLR4pathwayinmastcells.ThephosphorylationofAKTwassignificantlydecreasedinmiR-155KOcomparedwithBMMCsWT,whereasp38,andp42/p44phosphorylationwerethesameinbothtypeofmastcells.ThesedatademonstratethatmiR155isbothapositiveand negative regulator of mast cell mediator release.

Presenter MatthewMadden 68Title GlutaminaseinhibitionaltersCD8Tcelldifferentiationforcancerimmu-

notherapy.Email matthew.z.madden@vanderbilt.eduCo-Authors MarcJohnson,GongboLi,MarcoDavila,JeffreyRathmellAffiliation VanderbiltUniversityMedicalCenter

Cancer immunotherapyefficacydependsontumor-specificTcellactivity.Tcellsdramati-cally increase their metabolic activity upon activation and T cell subsets utilize distinct meta-bolicprograms.Glutaminolysis,thebreakdownofglutamineimportantforanaplerosisandother anabolic processes, is upregulated on T cell activation. Previous studies demonstrate thatinhibitionofanabolicpathways,suchasglycolysisandmTORC1signaling,decreaseseffectorfunctioninCD8Tcells.Incontrast,hereweshowthatblockingglutaminolysisbytreatmentwithGlutaminaseinhibitorCB839duringactivationofmurineCD8Tcellsincreas-eseffectorfunction.GranzymeBandPerforinlevelsincreasewithCB839treatment,asdoeffector-associatedtranscriptionfactorsTbetandEomes.Intriguingly,GlutaminaseinhibitionalsoincreasesKi67,BCL2,TCF1,andCD62Llevels,suggestiveofincreasedproliferation,survival,andTcellmemory.MitochondriainCB839-treatedTcellshavereducedinnermem-brane potential and demonstrate decreased turnover, potentially indicative of improved mi-tochondrialfitness.Baselineoxygenconsumptionrelativetoglycolysisalsoincreases.Cor-respondingly,murinechimericantigenreceptor(CAR)TcellstransientlytreatedwithCB839sustain higher peripheral blood levels in vivo than non-treatedCART cells.Overall, ourresultssuggestthatGlutaminaseinhibitionbyCB839increasesCD8Tcelleffectorfunctionaswellasinvivopersistence,makingTcellGlutaminaseanattractivetargetinanti-tumorim-munity.FuturedirectionsincludecomparingtransientversuschronicGlutaminaseinhibitioninTcellsandfurtherrefiningthemitochondrialphenotypeofCB839-treatedTcells.Invivo,wewillassesstheroleofGlutaminaseinTcellmemoryformation,conductleukemiasurvivalstudiestoconfirmincreasedefficacyofCARTcellstreatedwithCB839,andmeasuretheeffectofCB839treatmentonintratumoralmetabolitecompetitionbetweenTcellsandtumorcells in vivo using 18F-labelled glucose and glutamine.

Presenter: PaulDunbar 69AbstractTitle: CD8+TCellsRequireIL-27Rforoptimalgenerationofaneffectorre-

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sponseduringinfluenzainfectionEmail: pdunbar@emory.eduCo-Authors: Zheng-Rong Tiger Li, Sarah L Hayward, Jenna Lobby, Laurel Lawrence,

KirstenKost,JacobE.KohlmeierAffiliation: EmoryUniversityGDDBS-IMP

PreviousreportshavedemonstratedtheeffectsofIL-27onCD4Tcellactivationanddiffer-entiation, in addition to a role for IL-27 in cancer therapies mediated by cytolytic CD8 T cells. However, as IL-27 is produced by many cell types during infection, and the IL-27 receptor (IL-27R)iswidelyexpressedonactivatedCD8TcellsaswellasonactivatedCD4andNKcells,thedirecteffectsofIL-27signalingonCD8Tcellfunctionarelesswellunderstood.Todirectly test the role of IL-27R on CD8 T cells following viral respiratory infection in vivo we usedamurinemodelofinfluenzainfectionincombinationwithco-transferofcongenicwildtype(WT)andIL-27R-/-OT-ICD8Tcells.Weobservedadramaticreductioninthefrequencyand number of IL-27R-/-OT-IcomparedtotheirWTOT-Icounterpartswithinthesamehostfollowinginfluenzainfection,indicatingaCD8Tcell-intrinsicroleforIL-27Rinthegenera-tion of an acute anti-viral response. Further, these IL-27R-/-OT-Isexpresshigherlevelsofexhaustion markers, fail to proliferate in situ and enter apoptosis at a greater frequency. This effectisnotlimitedtonaiveIL-27R-/-OT-Is,asmemoryIL-27R-/-OT-IsalsofailtoaccumulatetothedegreeoftheirWTcounterpartsonheterologousre-challenge.ThisstudyrevealsanewroleforIL-27inmediatingtheproductionofaneffectiveanti-viralCD8Tcellresponsefollowing respiratory viral infection.

Presenter: Maria Abad 70Title: GenerationofUC-stromalcellstoinvestigateHCMVprimingofCD8T

cellsEmail: maria_abad@med.unc.eduCo-Authors: Erik Lenarcic, Yinyan Xu, Jason Wong, Genevieve Clutton, Joanna

Warren,BarbaraSavoldo,NathanielMoorman,NiluGoonetillekeAffiliation: UniversityofNorthCarolinaatChapelHill

Humancytomegalovirus(HCMV)-specificCD8+ T cells are characterized by a unique, non-exhausting,effectormemoryphenotype.HowHCMVinducesthisphenotypeispoorlychar-acterized.We hypothesized that HCMV-infected fibroblastsmaymodulate T cell primingproducing functionally distinct CD8+ T cells.

Toinvestigatethisquestion,wehavegeneratedmesenchymalcells(MSCs)isolatedfromumbilicalcord(UC).MSCandfibroblastsexpressmanyofthesamephenotypicmarkers.WestandardizedculturemethodsofMSC fromUCcollagenase-treatedWharton´sJelly (UC-WJ)resultinginreproducibledetectionofUC-stromalcellswithin10-15daysofinitialculture.Wecorroboratedthatderivedcells(passage#3-6)displayedfibroblastoidmorphologyandsurfacemarkers,suchasCD73,CD90,CD105andCD44,with levelscomparable to thefibroblastprimarycellline,MRC-5.

PreviousstudieshavereportedIFN-γtreatmentoffibroblasts inducesMHC-IIexpression,leadingtosuggestionsthatfibroblastscanacquireAPC-likeactivity.Weusedflowcytom-etrytoexaminetheeffectsofHCMVinfection+/-IFN-γonMHC-II,CD40,CD80andCD86surfaceexpression.InfectionwitheithertheHCMVisolateTB40\EortheHCMVlaboratory-adaptedstrainAD169failedtoinduceMHC-IIorcostimulatorymolecules.HCMVinfection+/-IFN-γinducedMHC-IIbutcostimulatorymoleculesremainedundetectable.ThissuggestsHCMV-infectedfibroblastsdonotacquireAPC-likephenotypeandthatfibroblastsareun-likely to directly prime CD8 T cells.

HCMV-infectedUC-fibroblastsmayindirectlymodulateTcellprimingthroughthereleaseofcytokinesorextracellularvesicles.Fibroblastsreleaseextracellularvesicles(EVs),whichareimportantmediatorsofcellsignaling,includingantigencross-presentation.WeusedelectronmicroscopytoconfirmthatstromalcellsreleaseEVs.Imagingflowcytometryshowedthatfollowing5.5-8%ofEVcollectedfromtheculturesupernatantofAD169-GFPinfectedMRC-5 fibroblasts wereGFP-positive.Ongoing studies are investigatingwhether UC-Fmodu-

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late mDC priming of CD8+ T cells through direct cell-cell contact and/or indirectly, including throughthereleaseofcytokinesorEVs.

Presenter: Allison E. Norlander, PhD 71Title: Prostaglandin I2 signaling is important for Treg function during allergic

inflammationEmail: allison.e.norlander.1@vumc.orgCo-Authors: Melissa H. Bloodworth, PhD, R. Stokes Peebles Jr., MDAffiliation: VanderbiltUniversityMedicalCenter

Our laboratory reported that prostaglandin I2 (PGI2) promotes immune tolerance inmice.Thus,wehypothesizedthatPGI2 promotes the functionality of Tregs in vivo.WeutilizedmicedeficientinthereceptorforPGI2,IP(IPKO),aswellastwoseparateadoptive-transferbasedmodelsofallergicinflammation.Inthefirstmodel,splenicovalbumin(OVA)-specificTregswereisolatedfromwild-type(WT)andIPKOmice.OVA-specificCD4+effectorTcells(Teff)wereisolatedfromthespleensofWTmice.RAG1deficientmicereceivedequalamountsofTeffandeitherWTorIPKOnaturalTreg(nTreg).AnothergroupofRAG1deficientmiceweretransferredonlyTeff.RAG1deficientrecipientswerechallengedintranasallywithOVA1daypriortoandfor3consecutivedaysposttransfer.Onday4posttransferthebronchoalveolarlavagefluid(BAL)washarvestedandexaminedforthepresenceofIL-13byELISA,whilethelungswereexaminedbyflowcytometry.ComparedtomicetransferredonlyTeff,totalIL-13intheBALwasreducedby46%inthemicetransferredWTnTregs;however,therewasnodecreaseintotalIL-13inthemicetransferredIPKOnTregs.Furthermore,comparedtomicetransferredonlyTeff,totalOVA-specificCD4+IL-13+cellsinthelungswerereducedby32%inthemicetransferredWTnTregs,however,therewasnodecreaseinthesecellsinmicetransferredIPKOnTregs.Inthesecondmodel,WTmiceweresensitizedwithanOVA-alum solution to prime an allergic immune response. Two weeks after sensitization, mice receivedeitherWTorIPKOOVA-specificdifferentiatedinducibleTreg(iTreg),ornoiTregatall.ThemicewerethenchallengedwithnebulizedOVAfor3consecutivedays.Onday4posttransfertheBALwasharvestedandexaminedforthepresenceofIL-13byELISA.Com-paredtomicethatreceivednoiTreg,totalIL-13intheBALwasreducedby60%inthemicetransferredWTiTregs;however,therewasnodecreaseintotalIL-13inthemicetransferredIPKOiTregs.OurdatademonstratethatPGI2 signaling promotes Treg function in vivo and suggeststhatPGI2 may serve as a novel treatment for allergic diseases.

Presenter: Jing Zhu 72Title: Manipulation during B cell maturation ameliorates lupus nephritis in

BXSB miceEmail: jing817@vt.eduCo-authors: Alayna Hay, Ashley Potter, Janie Adkins, Caroline LeethAffiliation: VirginiaTech,DeptofAnimalandPoultrySciences,Blacksburg,VA

Systemic lupus erythematosus (SLE) is a complex autoimmune disease associatedwiththe production of autoantibodies. Previous studies demonstrated the majority of pathogenic autoantibodiesaremutatedIgG,suggestingcrucialrolesofaffinitymaturationinSLEpatho-genesis.OurhypothesisisthatabrogationofthismechanismwillretardSLEdevelopmentbypreventingtheproductionofhighaffinityauto-antibodies.Activation-inducedcytidinede-aminase(AID,genenameAicda)isanenzymerequiredforaffinitymaturation.Inourstudy,we genetically targeted AicdainBXSBmicetoexaminetheeffectsofBcellmaturationonSLE.IntheabsenceofAID,miceshowedsignificantlyreducedautoantibodiesandextendedlifespan.While similar levels of immune complex deposition were seen in the glomeruli,AID-deficientmiceexhibitedimpairedcomplementactivationandamelioratednephritis.Con-sistentwithothermodels,AIDdeficiencyresulted inenlargedgerminalcenters.However,the increase in germinal center B cells did not lead to a corresponding increase in follicular helperTcells.Inaddition,lowaffinityBcellsdidnotfailtosurviveordifferentiateintoplasmacellssincenosignificantdifferencewasfoundinplasmacellgenerationeitherin vivo or in vitro.Inconclusion,thesuccessoftargetingaffinitymaturationandclassswitchtoalleviateSLE, especially nephritis, provides potential innovations in SLE treatment.

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Presenter: NathanC.Winn 73Title: Myeloid targeted depletion of ferroportin disrupts glucose homeostasis

in miceEmail: Nathan.winn@vanderbilt.eduCo-authors: Matthew A. Cottam, Julia Pinette, Alyssa H. HastyAffiliation: DepartmentofMolecularPhysiologyandBiophysics,VanderbiltUniver-

sity

Iron is essential for many fundamental biological processes including, cellular respiration, DNA synthesis, proliferation, host defense, and cell signaling, and therefore iron homeosta-sis is an important determinant of immunometabolic health. Tissue iron overload is associ-ated with insulin resistance and glucose dysregulation in rodents and humans; however, the mechanisms or cell types that mediate this phenotype are not completely understood. GiventhatMɸsaretheprimaryiron-handlingcells,itisreasonabletopositthatperturbedMɸiron metabolism is a precipitating factor in metabolic dysfunction. Accordingly, we tested the postulatethatdisturbedirontraffickingbyMɸsdrivessystemicglucoseintoleranceinmice.Micewithtargeteddeletionofferroportin(Fpn)–theonlyknownmammalianironexporter– inmyeloid cells (FpnΔMɸ)weregeneratedusingCre-Lox recombinationunder control oftheLysMpromoterandcomparedtoFpnflox/flox(Fpnfl/fl) littermatesascontrols.Allmicewere fed rodent chow and had access to water ad libitum. Body weight, fat mass, and lean masswere not different between Fpnfl/fland FpnΔMɸmice.Compared to floxedmice, Fpnexpressionwas≈5-fold lower inbonemarrow-derivedMɸs inFpnΔMɸanimals.ToconfirmthatFpndepletioncausesironretention,asubsetofmicewereperfusedandfixedwithaniron-stainingsolution(Pearl’sPrussianBlue).Grossobservationrevealedthat thespleen,liver, and thymus exhibited positive iron stain that was markedly exacerbated in FpnΔMɸ mice,

confirminganironloadedphenotype.Histologicalevidenceinisolatedliversectionsrevealedthat iron accumulation in FpnΔMɸ mice was localized to extrahepatic cells, presumably resi-dentKupffercells.Inadditional,livertriglycerideswereelevatedinFpnΔMɸ compared to Fpnfl/fl animals. Relative to Fpnfl/fl controls, blood glucose concentrations in response to an intraperi-toneal glucose load were increased in FpnΔMɸ animals. Collectively, these data suggest that disturbedirontraffickinginMɸscontributestoglucoseintoleranceinleanmice.Additionalresearchisrequiredtotease-outthemechanism(s)bywhichbluntedironhandlingbyMɸsdisrupts glucose homeostasis.

Presenter: Robin S Lindsay 74Title: NKcellsalterTcellpresenceandfunctionwithinthetumormicroenvi-

ronmentEmail: rl8fb@virginia.eduCo-Authors: MaritMMelssen,AmberNWoods,andVictorHEngelhardAffiliation: DepartmentofMICandTheCarter ImmunologyCenter,Universityof

Virginia

Whilecheckpointblockadetherapieshavedemonstratedamazingsuccesstreatingavarietyofcancerpatients,significantnumbersofpatientsdonotrespondtotherapy.Whileitiscur-rently unknown what determines a patient’s response to therapy, increased numbers of T cells within tumors is correlated with longer patient survival and positive response to check-pointblockadetherapy.Tcellpresencewithinthetumorcanbeaffectedbyentrythroughtheendothelium,aswellasproliferationorsurvivalwithinthetumor.WhatregulatesTcellpresencewithinthetumormicroenvironment(TME)remainsanopenareaofstudy.Innateimmune cells have been studied for their role in determining T cell activation or suppres-sion within the TME, but their role in driving T cell presence within the tumor has not been thoroughly examined.Among the innate immune cells,NK cells are of particular interestas theycanproduce inflammatory cytokines (IFNγandTNFα) thatalterhoming receptorligand expression on the vasculature. Increased expression of homing receptor ligands may increaseT cell entry into tumors.Using an implantable subcutaneousmurinemelanomamodel,NKcellsonlyproducedIFNγatearlytimepointsduringtumordevelopment.Tode-terminewhateffectNKcellshaveonimmunecellpresencewithintheTME,wedepletedNK

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cells prior to the tumor being established. Homing receptor ligand expression was lowered onthevasculaturefollowingNKdepletion.Inaddition,depletionofNKcellsresultedinlowernumberofTcellswithin thetumoronlyat latetimepoints.SurprisinglyNKcelldepletion,while still lowering homing receptor ligand expression, resulted in increased numbers of T cellsatearlystagesoftumorgrowth.However,TcellswerelessfunctionalwhenNKcellsweredepleted,particularlywithinearlystagetumors.OveralltheseresultssuggestthatNKcells drive increased levels of T cell numbers and function within late stage tumors, possibly by deleting dysfunctional T cells at early stages of tumor growth. Developing therapies that increaseNKcellfunctionwithintheTMEmayresultinincreasedTcellfunctionandimprovetheeffectivenessofcheckpointblockadetherapies.

Presenter MaritMelssen 75Title Formation and function of retention integrin expressing CD8 T cells in a

murine breast cancer model Email mm2xz@virginia.eduCo-authors Robin Lindsay, Amanda Briegel, Anthony Rodriguez, Salwador

Cyranowski, CornelisMelief, Sjoerd van der Burg, Craig Slingluff Jr,VictorEngelhard

Affiliation UniversityofVirginia

Integrins CD49a and CD49b can mediate retention of lymphocytes in peripheral tissues by binding to collagens type IV and type I, respectively. Their expression is upregulated onCD8+tumorinfiltratinglymphocytes(TIL)comparedtocirculatinglymphocytesandpresenceofCD49a+TIL improvespatientoutcome.Little isknownabouthowexpressionof theseintegrinsisregulatedandthefunctionalcapacityofthecellsexpressingthem.Wehypoth-esizedthatCD49aexpressionidentifiesmorefunctionalTcellsinthetumormicroenviron-ment(TME)andexpressionisinducedoneffectorsand/orearlymemorycells.Toaddressthese hypotheses, CD8 TIL from an implantable breast carcinoma model were evaluated atday(d)14andd23forexpressionofCD49a,CD49bandfunctionalmarkers,andtumorswereassessedforcollagenexpression.Inearlystagetumors(d14),Tcellswerepredomi-nantlyCD49bsinglepositive(SP)orCD49aCD49bdoublepositive(DP).Later(d23),CD49bSP cells largely disappeared and CD49a SP cells appeared, while DP cells remained un-changed.After treatmentwith FTY720, to block further T cell infiltration, the switch fromCD49b SP to CD49a SP cells was even more pronounced, suggesting the change is not duetonewly infiltratingCD49aSPcells. Interestingly,regardlessof timepoint,CD49bSPcells were antigen-responsive, whereas DP and CD49a SP cells had features of exhaustion. These data suggest that, contrary to our hypothesis, during tumor progression, CD8 TIL gain CD49a and lose CD49b as they become dysfunctional. To test whether this switch in integrin expression depends on antigen engagement or environmental factors, we transferred acti-vated,non-specificCD49bSPCD8Tcellsintotumor-bearingmice.Inthesetumors,CD49bSP cells upregulated CD49a, followed by downregulation of CD49b. This suggests that the switch in integrin expression is dependent on themicroenvironment, not antigen-specificdifferentiation.Importantly,preliminarydatashowedthatexpressionofcollagenIdecreasedover time, suggesting changes in ligand availability may play a role in the decrease of an-tigen-responsiveCD49b+TIL.Futureexperimentswillassesswhichenvironmentalfactorsdirectly play a role in CD49a upregulation, and how processes of retention integrin expres-sion relate to the antigen-dependent generation of exhaustion.

Presenter AmandaM.Lulu 76Title Pre-existing immune memory to cancer-associated phosphopeptides in

healthy donors suggests ongoing immune surveillanceEmail Aml2cu@virginia.eduCo-Authors KaraL.CummingsandVictorH.EngelhardAffiliation University of Virginia, Department of Microbiology, Immunology and

CancerBiology,Charlottesville,VA

Identifyingappropriateantigensisessentialtodevelopingeffectivecancervaccines.Proteinphosphorylation plays a major role in signal transduction pathways that are dysregulated

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andcontributetothemalignantphenotypeofcancercells.Ourlabhasshownthatphospho-peptides derived from these proteins can be presented by Major Histocompatibility Complex Class-Imoleculesandrepresentanewclassofcancer-associatedneoantigens.Weprevi-ously reported that healthy donors without evident prior cancer had unusually robust CD8 T cell responses to a cohort of HLA-B7+ leukemia-associated phosphopeptides, suggest-ingthattheymightbeduetopre-existingphosphopeptidespecificmemoryTcells.Totestthis, we evaluated CD8+CD45RO+memoryTcellspurifiedfromthebloodofhealthydonors.Thesecellsshowedrobustresponsesto2-25%ofHLA-A2+and0-23%ofHLA-B7+ cancer-associated phosphopeptides. This demonstrates that healthy individuals with no evident can-cer history have pre-existing immune memory to some cancer-associated phosphopeptides. We identified 3 “immunodominant”HLA-A2 associated phosphopeptides, in thatmemoryresponses were evident to them in the majority of analyzed donors. Such immunodominance suggests that these phosphopeptides result from signaling processes that occur commonly in transformation- or viral infection-related processes. In contrast, immunodominant phos-phopeptideswerenotevidentinthecontextofHLA-B7.While2donorsshowedpre-existingimmune memory to a large number of shared targets, other donors showed memory to only small numbers of non-overlapping phosphopeptides. This suggests that the mechanisms driving expression of these phosphopeptide antigens among healthy donors are much less common.Wealsofoundthatresponsestosomephosphopeptidescouldbedetecteddirectlyex vivo. These results suggest that some phosphopeptides in some donors may be the tar-getsofrecentorongoingimmuneresponsescomposedofeffectororeffectormemorycells.These results provide evidence of regular, ongoing immune surveillance in healthy individu-als to cancer-associated phosphopeptides.

Presenter: Anthony B. Rodriguez1,2 77Title: Adaptive and innate immunity orchestrate tertiary lymphoid structure

formation in tumorsEmail: abrodrigu@virginia.eduCo-authors: J. David Peske1,2†, Katie M. Leick1,3, Ileana S. Mauldin1,3, Samuel J.

Young1,3, Marit M. Melssen1,3, Salwador Cyranowski1,GeoffreyR.Parri-ott1,AmberN.Woods1,2, Yang-Xin Fu4,CraigL.Slingluff,Jr1,3.andVictorH. Engelhard1,2

Affiliations: 1BeirneB.CarterCenter for ImmunologyResearch,UniversityofVir-giniaSchoolofMedicine,Charlottesville,VA22908,USA;2Department ofMicrobiology,ImmunologyandCancerBiology,UniversityofVirginiaSchool of Medicine, Charlottesville, VA 22908, USA; 3Department of Surgery,UniversityofVirginiaSchoolofMedicine,Charlottesville,VA22908,USA;4DepartmentofPathology,UniversityofTexasSouthwest-ernMedicalCenter,Dallas,TX75235,USA;†Current Address: Depart-mentofPathology,JohnHopkinsUniversity,Baltimore,MD21218,USA

Tumor-associatedtertiarylymphoidstructures(TA-TLS)areectopiclymphoidaggregatesre-semblingconventionalsecondarylymphoidorgans(SLO).Inpatients,thesestructureshavebeenassociatedwithhighdensitiesof tumor-infiltrating lymphocytes (TIL) andenhancedsurvival.Wepreviously foundthatnaïveT-cellsenter tumorsviahighendothelialvenules(HEV),whicharecrucialcomponentsofTA-TLS;thus,TA-TLSmaybesitesforgenerationof intratumoral immune responses. However, the mechanisms that underlie the formation of TLS in tumors are largely unknown1. Here we show that formation of TA-TLS in murine B16melanomasisorchestratedbypodoplanin+(PDPN+)fibroblaststhatresemblelymphoidtissueorganizercells(LTo)andappeardistinctfromconventionalcancer-associatedfibro-blasts(CAF).ThesePDPN+ cells establish a reticular network within TA-TLS and express elevatedlevelsoftherecruitmentchemokinesCCL21andCXCL13andB-cellsurvivalfac-torsBAFFandAPRIL.Whileexpressionoftheselymphoidtissueorganizermoleculesde-pendsontumornecrosisfactorreceptor(TNFR)signaling,fibroblastproliferationandreticu-lar network formation depends on intratumoral T- and B-cells and lymphotoxin-b receptor (LTbR)signaling.Identificationofthesepathwaysandtheirimpactonintratumoralfibroblastsprovide a platform for manipulating the formation of these structures as a new strategy for cancer immunotherapy.

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Presenter Scott Jenks 78Title Chronic cutaneous lupus erythematosus patients have a breakdown in

autoreactiveVH4.34antibodytolerancewhilemaintainingtolerancetodsDNA and chromatin.

Co-Authors ReginaBugrovsky,XiaoqianWang,AishaHill,ChungwenWei,S.SamLim, Ignacio Sanz, Cristina Drenkard

Affiliation EmoryUniversity

Whilethecontributionofhumoral immunitytosystemiclupuserythematosus(SLE)iswellestablished,theroleitplaysinlupusconfinedtotheskin,chroniccutaneouslupuserythema-tosus(CCLE),islessclear.OnecharacteristicofSLEisabreakdownoftoleranceinauto-reactiveVH4.34antibodiesthatarerecognizedbytheratanti-humanidiotypicantibody9G4(9G4+).Theseantibodieshaveagermlineencodedautoreactivitytoglycolipidsfoundonredblood cells and naïve B and are tightly regulated in healthy individuals. In SLE patients this toleranceisbrokenresultinginhighlevelsofserum9G4+IgGwhichisassociatedwithhighdiseaseactivityandcorrelateswithanti-dsDNA.Thisstudycompared9G4+IgGandassoci-atedSLEauto-antibodiesbetweenCCLEpatientsandSLEpatients.Wefound57%ofSLEpatientswerepositivefor9G4+IgGandSLEpatientshadasignificantlyhigherserumcon-centrationthanHC.Surprisingly,CCLEpatientsalsohadhighlevelsof9G4+IgG,48%werepositiveandserumconcentrationsdidnotstatisticallydifferfromSLEpatients.Incontrast,while many SLE patients had anti-dsDNA and anti-chromatin, few CCLE patients were posi-tiveforthesespecificities.Consequently,9G4+IgGconcentrationwashighlycorrelatedwithboth anti-dsDNA and anti-chromatin concentration in SLE patients but not in CCLE patients. CCLEpatients,however,didhaveauto-reactive9G4+,asBcellandapoptoticcellbinding9G4+antibodiesweresimilarbetweenSLEandCCLEpatients.Thissuggestsatwo-stepmodelof9G4tolerance inSLE, thebreakdownofgeneral toleranceofgermlineencodedautoreactiveVH4.34antibodiesandasubsequentdevelopmentof9G4+IgGspecificfords-DNA.CCLEpatientsclearlyhaveadefectinthefirststepandthemechanismofthisdefectis potentially shared between CCLE and SLE. However, the second step is not shared, as CCLEpatientswith9G4+IgGdonothavehighlevelsofanti-dsDNA.Regulationofthissec-ond step may help determine if otherwise immunologically similar patients may develop SLE orCCLE.Becausethespecificityof9G4+inCCLEisunknown,itremainstobedeterminedwhether these antibodies contribute directly to CCLE disease.

Presenter Denitra Breuer 79Title ActivationofNeurokininReceptor3byAgonistSenktideInducesanIn-

flammatoryPhenotypeinImmuneCellsEmail daevans@ncsu.eduCo-Authors JennaViveiros,MarecaLodge,CaseyNestor,ArionKennedyAffiliation NorthCarolinaStateUniversity

Inflammationisapartoftheimmunesystemthatisactivatedinresponsetoinjuryandinfec-tion,leadingtodiseaseclearanceandwoundhealing.However,chronicinflammationcanhaveanegativeaffectand leadtometabolicdisorders,arthritis,andevensomecancers.Neurokininreceptorshavebeen linkedtomanydiseaseprocessessuchaspain,fibrosis,addictivedisorders,aswellasacuteandchronicinflammation.Publisheddatademonstratethatinflammatorycytokinesandbacterialproductscanelevateneurokininreceptor1(NK1R)expression in murine macrophages, though less is known about neurokinin receptor 2 and 3 (NK2RandNK3R, respectively). Previous results have shown that activation ofNK3Rcanleadtoinflammationinthebrain,butitisunclearhowNK3Ractivationimpactsperiph-eralimmunecellfunction.WetreatedmalesheepwithNK3Ragonistsenktidefor2hoursandexaminedimmunecellpopulationsinthebloodbyflowcytometry.SheeptreatedwithsenktidehadareducednumberofCD44+activatedimmunecellsinthebloodcomparedtosheep treated with vehicle. Based on the reduction in cell number, we examined if senktide regulates immune cell polarization. By microarray analysis, we found that macrophages ex-pressthegenetachykinin-3receptor(Tacr3),whichencodestheproteinNK3R,atlowlevels.WetreatedJ774mousemacrophageswithsenktidefor16hoursandexaminedchangesinproinflammatorygeneresponse. Inflammatorycytokine IL1-βwas increased14-foldwhencells were treated with senktide compared to untreated cells. This data shows that in vivo,

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reductions in immune cell populations by senktide may be due to polarization of activated im-munecellstoaninflammatoryphenotype.ThesedatasuggestthatNK3Ractivationinducesaninflammatoryphenotypeinmacrophagesandisapossibletargetfortreatmentofchronicinflammation.

Presenter Elene Clemens1 80 Title EvaluationofepitopespecificityintheimmuneresponsetoPR8H1N1

influenzavirusinfectioninneonatalandadultAfricanGreenmonkeysEmail eclemens@wakehealth.eduCoauthors Davide Angeletti2, Beth C. Holbrook1, S. Tyler Aycock3, Jonathan Yewd-

ell4, and Martha A. Alexander-Miller1

Affiliations 1WakeForestSchoolofMedicine;2UniversityofGothenburg,Sweden;3UniversityofTennesseeHealthScienceCenter;4NIAID, NIH

Generationofeffectiveantibody responses followingvirus infection ischallenging inneo-nates as a result of impairments in the innate and adaptive immune systems that character-ize early life. This leaves them particularly susceptible to severe disease following virus in-fection,e.g.influenzaAvirus.Apotentialcontributortotheefficacyoftheimmuneresponsegenerated following infection is theepitope specificity of theelicitedantibodies.Previousstudiesinadultmicehavereportedadefinedandreproduciblepatternofimmunodominanceamongantibodiesdirectedtotheneutralizingepitopesontheheadoftheinfluenzahemag-glutinin(HA)molecule.TheimpactofageontheimmunodominancepatternofHA-specificantibodieshasnot beenexplored.Toaddress this, epitope recognitionwasquantified inplasma collected fromnewborn and adult AfricanGreenmonkeys on d14 followingPR8influenzavirus infection.For theseanalysesweusedHAmoleculesengineered tosinglyexpresseachoftheneutralizingepitopesidentifiedontheHAhead.Inaddition,weusedaconstructthatrestrictsrecognitiontothestemregionofPR8HA.OuranalysesrevealedasimilarpatternofrecognitionbyHA-specificIgGbetweeninfantsandadults inthe14dayperiod following infection. Incontrast, theHA-specific IgMpoolsexhibiteddistinctbindingpatternsinthesetwogroups.Themoststrikingofthesedifferenceswasintheproductionofantibodies capable of recognizing the stem portion of HA. These data suggest antibody im-munodominance patterns may be modulated with age and sheds new light on the regulation ofpotentiallybroadlyprotectivestemresponsestoinfluenzavirus.

Presenter: MarissaAGonzales1 81Title: Modulating T cell function through elongation of the mitochondrial net-

worktoimprovecytotoxicCD8+tumorinfiltratinglymphocytefunctionE-mail: mag4bw@virginia.eduCo-authors: LelisaFGemta1, Frank M Mason2, Timothy NJ Bullock1

Affiliation: 1Department of Pathology,University of Virginia,Charlottesville, VA22908;2DivisionofHematologyandOncology,VanderbiltUniversity,Nashville,TN37232

For a number of tumor types, including melanoma, positive clinical outcomes correlate with infiltrationofT-cellsintothetumor.However,failuretocompletelycontroldiseaseprogres-sion often occurs, even with immune responses enhanced by checkpoint blockade or other immunotherapy.WhenT-cellsfailtocontroltumorgrowth,thetumorinfiltratinglymphocytes(TIL)exhibitcharacteristicsofexhaustionsuchaspoorproliferation,pooreffectorfunction,andan increase in inhibitoryreceptorexpression.WenowknowthatmetabolicactivityofT-cellscontributestodifferentiationandeffectorfunction,leadingustoinvestigatethemeta-bolicstateofTIL.WepreviouslyreportedthatmelanomaTILhavelimitedOXPHOScapabil-ityandlowATPlevels.Tounderstandthebasisofthisusingflowcytometry,wefindthatTILhave lower mitochondrial mass, as well as lower mitochondrial membrane potential when compared to functionaleffectorT-cells.Therefore,wehypothesize thatTILareunable toeffectivelyregulatetheirmitochondrialnetwork,leadingtothedegradationofmitochondrialfunction,and it is likely thatoneormore fusionorfissionmachineryproteinshasalteredexpressionorcanbemanipulatedtoimproveTILfunction.WefindthatTILhavepunctatemi-

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tochondriaascomparedtofunctionaleffectorcellsandhypothesizethatelongationofthemi-tochondrialnetworkwillimproveTILfunctionsuchasincreasingthecapacityforOXPHOS,whichisseeninmemoryTcellswithelongatedmitochondrialnetworks.Usingmicelackingthefissionprotein,Drp1,inTcellsasamodelofelongatedmitochondria,weseeincreasedmitochondrialmembranepotential,sparerespiratorycapacityandOXPHOSascomparedtoWTcellswithfissioncapability.Wehaveinvestigatedtheconsequencesoflimitingmi-tochondrialfissiononCD8+Tcellresponsestobothvaccinationandtumors.Additionally,we have investigated pharmacological methods of elongating mitochondrial morphology or enhancing mitochondrial biogenesis to reinvigorate oxidative metabolism in exhausted TIL.

Presenter Aaron D Stevens 82Abstracttitle VaccinationslowstumorgrowththroughalreadyinfiltratingCD8TcellsEmail ads9zf@virignia.eduCo-authors Colleen T Curley, Natasha D Sheybani, Richard J Price, and Timothy NJ

BullockAffiliation UniversityofVirginia

CD8 tumor infiltrating lymphocytes (TIL) are critical for tumor control, and their presenceis a positive prognostic factor in many cancer types including melanoma. However, many patienttumorshavepoorexistingTcellinfiltration.Additionally,evenintumorsthatarewellinfiltrated,theTILaretypicallyexhausted,characterizedbylimitedcytokinerelease,cytotox-icity,andproliferation.InordertoimprovebothTcellinfiltrationandfunction,wehaveusedavaccinationstrategycombiningaCD40agonisticantibody,theTLR3agonistpolyI:C,andtheantigenovalbumininourB16OVAmelanomamodel. Vaccinationsignificantlyslowedtumor growth when administered both early and once tumors are palpable as a single treat-ment and corresponded with more than tenfold increases in both CD8 and CD4 T cells in thetumors.ByblockingtraffickingtothetumorusingtheS1PreceptoragonistFTY720,wehave shown that the newly activated T cells generated in the periphery by vaccination are notrequiredtoslowtumorgrowth.Instead,CD8Tcellsthathavepre-infiltratedtumorbythetimeofvaccinationaresufficienttoslowtumorgrowth.Preliminarystudiesindicatethatthisvaccination regimen works with melanocyte self-antigens and neoantigens expressed by this melanoma model, indicating its potential utility for vaccination regimens targeting mutations in human tumors. Current studies are underway to understand the mechanism by which the vaccination is working within the tumor, with initial data suggesting increased cytotoxicity and proliferation by the CD8 T cells and more mature dendritic cells that improve or maintain the CD8 TIL.Melanoma commonly metastasizes to the brain, resulting in poor prognosis for patients. In contrasttosubcutaneousB16OVAtumors,intracranialB16OVAtumorsarepoorlyinfiltratedbyTcells.WemodifiedthevaccinationtotargetCD8TcellsmoredirectlybyreplacingtheCD40 antibody with a CD27 agonistic antibody, which may be more tolerable in patients. Strikingly, CD27 vaccination was able to both reduce tumor size and vastly increase CD8 infiltrationintointracranialB16OVAtumors.

Presenter: Charu Aggarwal1 83Title: Kinetic differences in the evolution of plasmablast response between

primary versus secondary dengue infections.Email: aggarwal.charu19@gmail.comCo-authors: KaustuvNayak1, Mohit Singla2,SivaramGunisetty1, 6, Elluri Seetharami

Reddy1, Harekrushna Panda1,GuruprasadMedigeshi3, Rakesh Lodha2, SushilKabra2,RafiAhmed4,Murali-KrishnaKaja1,4,5,6, Anmol Chandele1,

Affiliation: 1 ICGEB-Emory VaccineCentre, International Centre forGenetic En-gineering and Biotechnology, New Delhi, India. 2All India Institute of Medical Sciences, New Delhi, India. 3Translational Health Science and Technology Institute, Faridabad, India. 4Department of Microbiology and Immunology,EmoryUniversitySchoolofMedicine,Atlanta,GA.5Emory VaccineCenter,EmoryUniversitySchoolofMedicine,Atlanta,GA.6De-partmentofPediatrics,EmoryUniversitySchoolofMedicine,Atlanta,GA.

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Antibodies are implicated in both protection and pathology against human dengue virus. However, the major cellular factories of the antibody production, plasmablasts, remain poorly characterizedindengue.Here,weprovideafirstdetailedcharacterizationofthemagnitude,specificity, and rapidity of the plasmablasts response in acute febrile dengue patients inIndia. Consistent with previous reports, we found that plasmablasts expand massively. The expansion was highly heterogeneous among individual dengue patients with frequencies ranging fromas lowas1%toashighas85%of theBcells.Stratificationof thepatientsbased on the day of illness suggested that the plasmablast responses peak around 4-6days post onset of symptoms. These plasmablasts were highly proliferating as assessed by Ki67expression.AvastmajorityofthemwereproducingdenguespecificIgG.Atthepeakof the response, the frequency of the plasmablasts was slightly higher in secondary dengue patients compared to the primary dengue patients. By identifying patients who have not yetevolveddenguespecifichumoralresponseatthetimeoftheclinicalpresentation,andbyperformingalongitudinalanalysis,wefoundkineticdifferencesintheevolutionofplas-mablastsinprimaryversussecondarydenguepatients.WefoundthatdenguespecificIgGantibodysecretingcells(ASC)canexpandasmuchas1000-foldinashortwindowofjust24-48hrs. This rapid response was preferentially seen only in patients experiencing secondary dengueinfections.ArapidIgGsecretingplasmablastresponseearlyafteronsetofdenguefever is accompanied by a massive increase of serum neutralizing antibodies against both infecting and heterologous serotypes. By contrast, patients with primary dengue infections showed very little ASC response at the early times after onset of febrile illness and rarely produced dengue neutralizing antibodies. These results suggest that B cell responses might be largely protective during secondary dengue infections.

Presenter Xavier Cabana-Puig 84Abstract title Characterizing the role of Lactobacillus spp. in systemic lupus erythe-

matosusEmail xavier91@vt.eduCo-authors Qinghui Mu, Brianna K. Swartwout, Leila Abdelhamid, Meeta B.

Prakash, Jacob M. Bond, Christopher M. Reilly and Xin M. LuoAffiliation VirginiaPolytechnicInstituteandStateUniversity

Systemiclupuserythematosus(SLE)isamulti-systemautoimmunediseasewithnoknowncure. The crosstalk between the gut microbiota and the immune system plays an important role in the tolerance induction to self-antigens both in the intestinal mucosa and at the sys-temic level. Wepreviouslydemonstrateddifferencesbetweenthegutmicrobiota in lupusmice compared to healthy controls, with a decreased Lactobacillaceae concentration. A mix-ture of L. reuteri, L. oris, L. johnsonii, L. gasseri, and L. rhamnosussignificantlyattenuatedlupus-liked disease in lupus-prone MRL/Mp-Faslpr(lpr)mice,byrestoringtheimbalancebe-tween regulatory T cells and T helper 17 cells. To further understand the role of Lactobacillus spp.,wetreatedMRL/lprmicewiththesupernatantofthesame5culturedstrainscontainingthe secreted metabolites, given that bacterial metabolites may induce an immunosuppres-siveresponse.Theresultsshowedasignificantattenuationintheinflammationofthespleenand renal lymph nodes similar to the bacterial mixture, but not that of the mesenteric lymph nodes. The results also demonstrated that there was a tendency to decrease double-strand-ed DNA autoantibodies. Additionally in our previous studies, L. reuteri and an uncultured Lactobacillus specieswerepredominantly identifiedusing16S rRNAsequencing inMRL/lpr mice. To evaluate the role of L. reuteri in attenuating lupus disease, we treated mice withthesinglestrain.WefoundL. reuteri didnotexertabeneficialeffectinMRL/lprmicephenotypically. This suggests the other remaining strains in the Lactobacillus mixture could have a leading role in ameliorating the disease. Based on these results, we have a better understanding of the pathogenesis of SLE and the role of probiotics Lactobacillus spp. as a potential new therapeutic agent against the disease.

Presenter SarahJ.Dulson 85Title STAT4DirectsProtectiveInnateLymphoidCellResponsestoGastroin-

testinal Infection

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Email sjdulson@uab.eduCo-authors EmilyE.Watkins,LaurieE.Harrington,Ph.D.Affiliation UniversityofAlabamaatBirmingham

Foodborne infection with Listeria monocytogenes(Lm)carriesamortalityrateof20-30%inhigh-risk individuals and leads to serious complications such as meningitis and miscarriage. Therefore, studies evaluating gastrointestinal responses to Lm are needed to better enhance therapeuticinterventions.Ourdatashowthatadaptiveimmuneresponsesaredispensablein the first fivedaysof infection sincebacterial burden is similar betweenRag1-deficientandwildtypemice. InnateLymphoidCells(ILCs)aretissue-resident lymphocytesthatareenriched at barrier surfaces, and we hypothesize that intestinal ILCs are central mediators of theearlyresponsetoLminfection.Usingcytokinereportermice,wedemonstratearobustandearlyIFNɣresponsetoLmbyGroup1ILCs(ILC1s)inthelargeintestinelaminapro-pria.Inaddition,systemicIFNɣresponseasearlyasdaytwopost-infectionissignificantlyreduced in the absence of ILCs. Consistent with this,mice devoid of ILCs suffer highermortality and increased bacterial dissemination compared with ILC-sufficient littermates.Mechanistically,ILC1slackingthetranscriptionfactorSTAT4areunabletoproduceIFNɣ,andSTAT4-deficientmicereadilysuccumbtobacterialdisseminationandmortalityfollow-ing Lminfection.Interestingly,STAT4-deficientanimalswithintactILCpopulationswerenolonger protected against infection compared with ILC-depleted littermates, indicating that STAT4signalingiscriticalforILC-mediatedprotection.UnlikeknownrolesforSTAT4inTcells,regulationofIFNɣbySTAT4inILCsoccursindependentlyofIL-18RαandTbetexpres-sion. Further, ATAC-sequencing demonstrated similar accessibility of the Ifng gene locus in wildtypeandSTAT4-deficientILC1s.However,inhibitionofSTAT4activityimmediatelypriortocytokinestimulationdemonstratesthatSTAT4directlypromotesIFNɣproductioninILC1sin response to cytokine activation. Together, these data illustrate a critical role for ILCs in the early responses to gastrointestinal infection with Lm and identify STAT4 as a central modula-tor of ILC-mediated protection.

Presenter: SamuelStampfer,MD/PhD 86AbstractTitle: Structure-BasedDesignofaNovelEbolaVaccineCandidateEmail: sstampf@emory.eduCo-authors: Stampfer SD, Yang C

EbolavirusesarefiloviruseswhichhavecausedmultipleepidemicsofviralhemorrhagicfeverinAfrica.Therearenohighlyeffectivetreatmentsandcontroloftheseoutbreaksreliesonquarantine, supportive care, and vaccination of individuals at increased exposure risk. Cur-rently-available ebola vaccines are expensive and require cold-chain storage and shipment, limiting the ability to vaccinate the at-risk population. All of these vaccines use the ebola fusionglycoproteinGPastheimmunogen,andtheyareeffective.In vivo, however, ebola transcribesthemajorityoftheGPgeneasanalternatetranscriptthatyieldsasecreted,trun-cated,non-fusogenicproteinsGP.sGPisefficientlysecretedaftersynthesisandthereforemucheasiertoproduceinlargequantitiesthanGP.Moreover,sGPformshomodimersviaintermoleculardisulfidebondsandisstableatelevatedtemperatures,andcouldrepresentamorecost-effectiveimmunogen.MiceimmunizedwithsGPareabletotoleratelethalchal-lengesfromebolavirus,andwesuspectthisisduetosharedepitopespresentonbothsGPandGPthatareknowntoelicitneutralizingantibodies.

WeareexploringmodificationstosGPtoenhanceitsimmunogenicity.Specifically,weareattemptingtodeleteportionsofitsC-terminaldomain,whoseresiduesdifferfromGPandare likely to induce antibodies incapable of binding ebola virions. This domain has a cysteine residuerequiredforsGPtoassumeitsnativeconformation:asadisulfide-linkeddimer.De-letingthedomainislikelytodisruptsGPfoldingandassembly.

ThroughanalyzingtheelectronmicroscopystructureofsGP,weidentifiedputativeresiduesthat, if changed to cysteine, couldpossibly createa replacement intermolecular disulfidebond. To test this idea, we generated an obligate monomer by mutating native cysteine residues toserine,and then introduced twopossiblepairsofcysteines.Onewasable toeffectivelygenerateanintermoleculardisulfidebond.Wethuscreatedarationally-designed

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vaccineantigenwhichwilltoleratedeletionsofepitopesinsGPthatdonotinduceneutral-izingantibodiesagainstGP. It isaprecursor fora futurecost-effective,heat-stableebolavaccine that may one day be used to control and prevent future outbreaks in resource-limited settings.

Presenters AyakaSugiura,GabrielaAndrejeva,JeffreyC.Rathmell 87Title Targeted CRISPR-Cas9 based screening approach to identifying critical

nodes in metabolic pathways

Chronicinflammationinthesettingofautoinflammatorydiseasesandimmunosuppressionin the setting of tumor microenvironments are both characterized by an imbalance between pro-inflammatoryeffectorT(Teff)cellsandanti-inflammatoryregulatoryT(Treg)cellsthatleadstodysregulatedimmuneresponses.Whilemanycurrentlyavailabletherapiesbroadlytargettheimmunecompartment,selectivelytargetingthespecificTcellsubsetsthatcontrib-ute to disease may provide a new avenue for the development of improved immunothera-pies.IntheRathmelllab,wehaveshownthatTeffandTregcellscanbedistinguishedbytheir reliance on distinct metabolic programs, and that this exposes a way to preferentially targetspecificTcellsubsets.Throughuntargetedmetabolomicandproteomicapproaches,we have also recently identified the one-carbonmetabolismpathways to be differentiallycriticaltoTeffandTregcells.Tofurtherdissectthesepathways,Ihavedeveloped in vitro and in vivo approaches to CRISPR-Cas9 based screening in primary T cells. In this method, I design small-scale custom targeted guide RNA libraries, which are transduced into primary T cells. For the in vitro assays, these cells can be cultured under selective pressures, as well as sorted for populations of interest. For the in vivo assays, the transduced cells can beadoptivelytransferredintohostmiceofdifferentinflammatoryandcancermodels,suchasairwayinflammationandmelanoma.Attheendoftheassay,thetransferredTcellsarerecovered from organs of interest and/or tumors, and processed to yield genomic DNA. The guideRNAsequences are thenamplified out and sequenced to identify those that haveeither been enriched or depleted in the assay, corresponding to favorable and deleterious gene knockouts. This approach provides a powerful tool for identifying critical nodes within metabolic pathways of interest in primary T cells that can potentially serve as therapeutic targets.

Presenter: KrystleL.Ong 88Title: Platelet-Neutrophil Interactions Induce a Low-Density Neutrophil Phe-

notypeEmail: krystleo@uab.eduCo-Authors: Harish C. Pal, Ashley N. Connelly, Christian X. Fay, Marcus D. Davis,

Valeriya Kuznetsova, Ashish Dhyani, Richard Huijbregts, E. TurnerOverton,ZdenekHel

Affiliation: TheUniversityofAlabamaatBirmingham

Asubsetofneutrophilsidentifiedco-localizingwithmononuclearcells,knownaslow-densityneutrophils(LDNs),haveincreasedfrequenciesinpatientswithinflammatoryconditionsin-cluding cancer and chronic infections. In cancer, this population is commonly referred to asgranulocyticmyeloid-derivedsuppressorcells(g-MDSCs).Whiletheoriginandfunctionof LDNs are unclear, immunosuppressive functions have been ascribed to this population. T-cell mediated responses can be suppressed by g-MDSCs by the production of reactive oxygenspecies,thereleaseofarginase-1resultinginadownregulationoftheTCRζchain,and induction of regulatory T cells in the tumor microenvironment. The tumor microenviron-ment contains g-MDSCs and activated platelets which have been implicated in promoting metastasisandtumorprogression.Platelet-neutrophilaggregatesarehallmarksofdifferentpathological conditions including inflammatory andmyeloproliferative disorders. Plateletsproduce content which biochemically interacts with neutrophils and the releasate include un-processed arachidonic acid which can be transformed into leukotrienes promoting neutrophil activation. The primary interaction between platelets and neutrophils is P-selectin- depen-dent thatwhenamplifiedcanshiftneutrophils towardsanactivatedphenotype.Currently,themechanismof inductionof the low-densityneutrophilphenotype isunclear.Wehave

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observed a time-dependent increase in the frequency of LDNs following Thrombin Receptor ActivatorPeptide6(TRAP-6)stimulation.LDNinductionwasabrogatedwhenplatelet-neu-trophilinteractionswereblockedviatheP-selectin/PSGL-1axis.AsubsetofneutrophilsfromTRAP-6-stimulatedblooddemonstratedtheabilitytoengulfplateletparticles.Pre-treatmentwith a phagocytosis inhibitor Cytochalasin D reduced the increase in the frequency of LDNs byTRAP-6whileexhibitingnoeffectonplatelet-neutrophilassociations.TRAP-6-stimulatedplateletsactivatedisolatedneutrophilsevidencedbyanincreaseinNETosis.TRAP-6-acti-vated platelets shift neutrophils toward an activated low-density phenotype which is partially dependent on a phagocytic mechanism.

Presenter: Holly A. Morrison 89Title: Elucidating the Role of the Noncanonical NF-kB Signaling Pathway in

Inflammation-Induced Carcinogenesis: Pathway Controls Stem CellNiche in Colonic Mucosa

Email: hamorrison18@vt.eduCo-authors: KristinEden,DanielRothschild,MorganStephens,StephanBrown,Eda

Holl, Irving C. AllenAffiliation: Virginia-MarylandCollegeofVeterinaryMedicine

Colorectalcancerismediatedbyinflammation-inducedtumorigenesis,inwhichunregulatedinflammationcausescellsinthecolontoaccumulatemutationsandepigeneticchangesthatpromotecarcinogenesis.Thusly, thedysregulationof inflammatorypathways in thecolonis attributed to this disease pathogenesis and such signaling may be attributed to NF-kB signaling.Thissignalingisdividedintotwodistinctpathways–thecanonicalandnoncanoni-calpathways.Thenoncanonicalpathwayisunderstudiedincomparisontothewell-definedcanonicalpathway.Currently,inthisfieldofresearch,therehasbeenaresurgenceinstudy-ing the noncanonical NF-kB pathway, particularly the NF-kBinducingkinase(NIK).NIKisapertinentkinaseinthesignalingcascade.Whenactivated,thenoncanonicalNF-kB signaling pathwayisattributedtochronicinflammation,productionofcytokinesassociatedwithinflam-mation and cancer, development of secondary lymphoid structures in the gut, recruitment of immune cells and cell proliferation. In our research, we observe that complete knockout of the noncanonical pathway via whole-body knockout of Nik inmiceresults insignificantchanges in large intestine phenotypes, including reduced stem cell marker expression, di-minishedregeneration/differentiationcapacityupontheseinflammatoryconditions,alteredinmicrobiomecomposition,and increasedpredisposition towards inflammation-inducedcar-cinogenesis.Followingthispreliminaryresearch,noveltissue-specificknockoutmiceweregenerated to elucidate the mechanisms relating to the observed phenotypes. These mice either had deletions of RelA/p65(relatingtothecanonicalpathway)orNik(relatingtothenoncanonicalpathway)intissue-specificregions.Here,weobservedthatthetwopathwayshavedistinctrolesinregulatinginflammationandtumorprogression.Specifically,RelA/p65attenuatesdiseaseprogressioninmyeloidcellsofthegastrointestinal(GI)tract,whileNikpredominately regulates disease progression inGI epithelial cells. Translating thisworktowards human medicine, colonic biopsy samples from human colorectal patients show sig-nificantdownregulationof thenoncanonicalNF-kB pathway. Together, this data suggests that the noncanonical NF-kB pathway has a protective role against colorectal cancer by regulatingimmunesystemhomeostasisintheGItract.

Presenter Mildred Perez 90Title CD151asaTcellactivationmarkerEmail mperez14@uab.eduCo-authors Kelsey Lowman, LillianSeu,ChristopherTidwell,DavidMoylan,Olaf

KutschandSteffanieSabbajAffiliation UniversityofAlabamaatBirmingham

Werecentlydescribedahyper-responsiveTcellsub-populationthatexhibitedanincreased,antigen-independentpropensitytoproliferateinthepresenceofIL-2andwasidentifiedbyexpressionofthetetraspaninCD151.ThefrequencyofCD151expressionwasafunctionof thememory differentiation state (Tnaive < TCM < TEM < TEMRA). CD151was consistently

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expressedonsenescentCD28-CD57+Tcells,butalsoidentifiedinTcellpopulationsthatwereCD28+.WefurtherdemonstratedthatCD151inTcellsisnotapassivemarker,butonce expressed provided an active outside-in signal that changed cell cycle control and apoptoticprocessmotifsofTcells(Seuetal.,J. Immunol.,2017). Wenowdemonstratethat,CD151expressiononunstimulatedTcells isnotcorrelatedwithotherTcellactiva-tionmarkers.CD151expression isupregulated followingTCR/CD3activationandCD151expressioncorrelateswithproliferation capacity. This findingwouldbeconsistentwithapreviousreportthatCD151congregatesattheT-cellsideoftheimmunologicalsynapseandenhancessignaling.ThefindingfurtherestablishesCD151asapotentialmarkerofcurrentlyand previously activated T cells. To test this hypothesis, we compared T cell frequencies in healthycontrolindividualswiththethoseofHIV/ARTpatients,whoareknowntoexhibitsignsofpersistentimmunehyperactivation.WefoundtheCD4+CD151+TcellpopulationinHIV/ARTpatientsisexpanded,suggestinganassociationofCD151+Tcellfrequencieswiththeimmuneactivationstatusoftheindividual.Furthermore,weprovideevidencethatCD151+Tcellscanhaveaninflammatoryphenotype,aswefoundbaselinegranzymeBexpressioninCD4+andCD8+TcellstobeassociatedwithCD151expression.InregardstoHIV/ARTpatients,ourfindingssuggestthatCD151+TcellsareacandidatepopulationthathasthepotentialtoserveasaTcellcorrelateofimmunehyperactivation.Overall,ourdataprovideadditionalevidencethatCD151,inacontextdependentmanner,actsasaTcellactivationmarker.

Presenter WeirongChen 91Title Abnormal maturation of antibody secreting cells in patients with active

systemic lupus erythematosus Email Weirong.chen@emory.eduCo-authors Sohee Hong, Eun F. Lee, Ignacio SanzAffiliation EmoryUniversity

Systemiclupuserythematosus(SLE)isaseveresystemicautoimmunediseasecharacter-ized by multiple B cell abnormalities and production of a variety of autoantibodies against nuclear,cytoplasmicandcellsurfaceauto-antigensbyantibodysecretingcells(ASCs).Inthe normal non-disease state, the frequency of circulating ASCs in total B cells is extremely low; while following immunization, ASCs increases in a tightly regulated manner. However, during active SLE, ASCs are dysregulated and exhibits a dramatic increase in the circulation. Indeed, the frequency of ASCs in the peripheral blood is correlated with disease activity, as measuredbytheSLEDAIscoringsystem.Despitetheirclinicalsignificance,theheteroge-neity, morphology, and molecular mechanisms of circulating ASCs in SLE remain largely unknown. In this study, peripheral ASCs were divided into 4 subsets based on the surface expressionoftheCD19andCD138:CD19+CD138-(pop2),CD19+CD138+(pop3),CD19-CD138-(pop4),andCD19-CD138+(pop5).CD19+subsets,andtoalargerextent,CD19-subsets, were markedly increased in active SLE patients compared to healthy controls post flu-vaccination.Next,electronmicroscopyshowedultrastructuralchangesinthecirculatingASCs subsets from active SLE patients in comparison to vaccinated healthy controls, includ-ing tighter nucleus, enhanced endoplasmic reticulum volume, increased numbers of mito-chondria, and presence of autophagosomes, features which are reminiscent of bone marrow plasma cells. Interestingly, phenotypic characterization of circulating ASCs suggested that thosefromactiveSLEpatientshadsignificantlyelevatedexpressionofCXCR4,areceptorfor the homing and survival of plasma cells in the bone marrow, when compared to healthy controls post-vaccination. Furthermore, next generation sequencing was used to analyze the clonality and connectivity between circulating ASC subsets from active SLE patients. Highly polyclonalrepertoireandclonalrelatednessinallASCsubsetswereobservedinmostflareSLE patients. Together, these data show the increase of ASCs accompanied by the changes ofultrastructuralmorphologyandupregulationofCXCR4during theflareSLE, indicatingtheir potential of homing to bone marrow and becoming long-lived plasma cells.

Presenter: Shirin Masjedi 92Title: Olfactory Receptor Expression in Tumor AssociatedMacrophages in

Breast Cancer

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Email: shirin.masjedi@vanderbilt.eduCo-Authors: Evan BGlass, StephanieODudzinski, Laurence J Zwiebel, ToddD

GiorgioAffiliation: VanderbiltUniversity,Nashville,TN,USA

Macrophages are abundant within breast tumors and correlate to tumor progression. They may adopt pro- or anti-tumor (inflammatory) phenotypes due to tumormicroenvironmentinteractions.Tumor-associatedmacrophage(TAM)characteristicsaremodulatedbyspecificactivationofthenuclearfactor-kappaB(NF-kB)pathway.Activatingcanonicalversusalter-native arms of the NF-kB pathway is one controller of immunostimulatory function in mac-rophages.Wehavereportedtranscriptabundanceofolfactoryreceptor(OR)genesamonginvasive breast carcinomapatients.ORsaremembers of theGProteinCoupledProteinReceptor(GPCR)familythatfacilitatethesenseofsmell.ThemechanismbehindtheectopicexpressionofORgenesinbreasttumorsortheirpotentialroleinbreastcancerremainun-known.TheaimofthisstudyistovalidateandcharacterizeORexpressioninTAMscorrelat-edwithNF-kBpathwayactivation.Macrophageswithtumorigenic(M2)orinflammatory(M1)characteristics were generated by ex-vivo stimulation of bone marrow-derived macrophages (BMDMs).Macrophageswerealsoisolatedfromadouble-transgenicmousemodelwithex-pressionofCSF1promoterandP52bydoxycyclineinduction,correspondingtoactivationof the alternative NF-kB pathway. In addition, TAMs were harvested from primary mammary tumorsof12-weekoldPyMT-MMTVmice.ORgeneexpressionwasmeasuredbyDNAmi-croarrayandqPCR.MacrophageinflammatorycharacteristicswereevaluatedbyqPCRandflowcytometry.Amongthetop78significantlyupregulatedgenesinthedouble-transgenicmacrophages,21wereORs (foldchange≥1.2).Olfr60,Olfr1487andOlfr598showed thegreatestlevelsofupregulation.ComparisonamongtheORexpressionindifferenttypesofmacrophagesshowedthatTAMsdemonstrated47-foldincreaseinOlfr60comparedtoregu-larmacrophagesand21-foldcomparedtoM2BMDMs.TAMsshowedincreaseinCD206and Arg-1 compared to regular macrophages, exhibiting more M2-like tumorigenic behavior. However,theirlevelsofCD206werecomparabletoM2s.Double-transgenicmacrophagesshowed1.4-fold increase inOlfr60expressioncompared to regularmacrophagesbutnotsignificantlydifferentthanM2sandsignificantlylowerthanTAMs.TheyalsohadcomparablelevelsofCD206andArg-1genestoM2macrophages.OurfindingssuggestthatORexpres-sion is altered in tumorigenic macrophages, and the tumor microenvironment is critically essentialforexpressionofORsinbreasttumors.

Presenter: JessicaShartouny 93Title: Optimizingfrog-derivedantimicrobialpeptidesforuseasantiviralsEmail: j.r.shartouny@emory.eduCo-Authors: Joshy JacobAffiliation: EmoryUniversity

Influenzainfectionsareresponsibleformuchoftheannualdiseaseandeconomicburdenworldwide, occurring both as seasonal epidemics and sporadic pandemics. Each year, ill-nessesworldwidenumberinthemillionswith250-500,000deaths,soreliableanti-influenzamethodsareimportant.Thefirstdefenseagainstinfluenzaisvaccination,howeverinpan-demics or when the predicted vaccine strains are mismatched, antiviral drugs become the next option. As resistance to conventional antiviral agents can occur, the development of novel anti-viral compounds is necessary to continue to treat influenza infections. Antimi-crobialpeptides(AMPs)areonelargepotentialpooloftherapeutics,astheyareproducedinnatelybymostformsoflifeandoftenhavebroadactivities.Wehavesynthesizedafrog-AMP-derivedpeptide,Yodha,whichisaneffectiveinhibitorofvariousH1N1andH3N2influ-enza viruses in in vitro focus-forming assays. The peptide appears to form large conglomer-atesoffibrilsinsolutionthatcanbeimagedwithelectronmicroscopy,however,whichisahindrancetoinvivodelivery.Wehavedesignedandscreenedseveralpanelsofmodifiedpeptides based on Yodha’s primary structure to examine the structure-function relationship of the antiviral activity. From these screens, we have determined that polymerization is not requiredforanti-viralactivityandseveralmodifiedpeptideshavebeenfoundtoout-performwildtype Yodha and are primary candidates for the development of a deliverable drug for seasonalandpandemicsubtypesofinfluenza.

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Presenter: Anoma Nellore1 94 Title: FcrL5expressioninantigen-specificmemoryBcellsmarksaT-bet+ ef-

fector memory B cell population that correlates with long-lived antibody productionafterfluvaccination.

Emial: anellore@uabmc.eduCo-Authors: Scharer CD2, Tipton CM3, Zumaquero E4, Rosenberg A4,5, Fucile C5,

Mousseau B4, Zhou F4, Bradley JE4,GoepfertPA1,4, Boss JM2, Sanz I3, Randall TD6, Lund FE4.

Affiliation: UAB

T-bet, a cytokine-inducible master transcriptional regulator, is expressed by both T and B lymphocytes.WhileT-betisknowntoregulateTh1lineagecommitmentandeffectorTcellterminaldifferentiation,thesignificanceofT-betexpressionbymemoryBcells(Bmem)re-mainsunclear.WehypothesizedthatT-betexpressingBmemareendowedwith“effector-like” properties that distinguishes them from other Bmem subsets. To test this hypothesis, we immunized immunologicallyhealthyadultswith the inactivated influenzavaccine (IIV).Withinonemonthofvaccination,we identified twodistinctpopulationsofcirculating influ-enzahemagglutinin-specific (HA+)Bmem thatdifferentiallyexpressedT-bet. Interestingly,the presence of the T-bethi HA+ Bmem correlated with the magnitude of the long-lived vac-cineserologicresponse.Wealsore-vaccinatedIIVsubjectsthefollowingyearwiththenewlyformulatedIIVthatcontainedthesameHA(H1)antigenfromthepriorseason.Weobservedthat the Bmem response to the H1 vaccine antigen shifted from a predominant T-bethi Bmem response in year 1 to T-betlo Bmemresponseinyear2.UsingFcrL5asasurrogatemarkerto divide the HA+ Bmem cells into T-bethi and T-betlo subsets, we performed RNA-seq and B cell repertoire analyses.We found that the FcrL5pos (T-bethi)HA+ Bmem transcriptome wasenrichedineffectormemorygenesetsrelativetotheFcrL5neg(T-betlo)HA+ Bmem tran-scriptome.Moreover,weobservedthatFcrL5pos HA+ Bmem were clonotypically distinct from FcrL5neg HA+Bmem,indicatingthatthesecellswerelikelyderivedfromdifferentprecursors.Finally,inapatientrevaccinatedwithIIV,wefoundthatclonotypesfromtheyear1FcrL5pos HA+ Bmem subset were preferentially recalled, relative to the year 1 FcrL5lo HA+ Bmem clonotypes, into the year 2 early plasmablast response. Collectively, the data suggest that FcrL5+(T-bethi)HA+BmemmayrepresentapoisedeffectormemoryBcellsubsetthatcanrapidlydifferentiateintoASCsafterantigenre-challenge.1DivisionofInfectiousDiseases,UniversityofAlabamaatBirmingham,2Department of Mi-crobiology,EmoryUniversity, 3Division of Rheumatology, Lowance Center for Human Im-munology,EmoryUniversity,4DepartmentofMicrobiology,UniversityofAlabamaatBirming-ham, 5InformaticsInstitute,UniversityofAlabamaatBirmingham,6Division of Rheumatology/Immunology,UniversityofAlabamaatBirmingham

Presenter: SarahLHayward 95Title: Unrelatedrespiratoryinfectionscompromiseestablishedcellularimmu-

nity by promoting apoptosis of pre-existing lung-resident memory CD8 T cellsEmail: sarah.louise.hayward@emory.edu

Co-authors: ZRTigerLi,JennaLLobby,JoelO.P.Eggert,andJacobEKohlmeierAffiliation: EmoryUniversitySchoolofMedicine

Lung-residentmemoryTcells (lungTRM)arecritical forprotectiveheterosubtypic immuni-tyagainst influenzaviruses.However, theefficacyofcellular immunityagainstrespiratorypathogenssuchas influenzawanesover timedue to thegradual lossofflu-specific lungTRM.Onepossiblemechanism for thisdecline is frequentexposure toenvironmentalandbiologicalinsultsresultinginlocalizedinflammationthatpromotesthedeathofestablishedlung TRM.We investigatedwhetherunrelated infectionscouldexacerbate the lossofpre-existing,flu-specificlungTRM andreducetheefficacyofcellularimmunitytosubsequentin-fluenzachallenge.Infectionofinfluenza-immunemicewithSendaivirus,anunrelatedmurineparainfluenzavirus,resultedinsignificantlyhigherviraltitersandgreatermorbidityfollowinginfluenzachallengecompared toPBS-treatedcontrols.This lossofprotectivecellular im-

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munitycorrespondedtoasignificantdecreaseinthenumberofpre-existingflu-specificlungCD8 TRM compared to PBS controls due to increased apoptosis. This loss of pre-existing lung TRM following Sendai infection was not due to competition for limited resources or tis-suenichesbetweenflu-andSendai-specificlungTRM. Sendai infection had no impact on thenumberofsystemicflu-specificmemoryCD8Tcellsinthespleen.ThelossoflungTRM requiredanactiveinfection,asbothSendaiandLCMVinfectionbutnotintranasaldeliveryofTLRagonistsdidsignificantlyreducethenumberofpre-existingflu-specificlungCD8TRM. Together, these data suggest that tissue damage induced by unrelated respiratory infections can promote the loss of pre-existing lung TRM and compromise cellular immunity against respiratory pathogens.

Presenter LevelleD.Harris 96Title PhenotypicandfunctionalprofilesofNKcellsinindividualswithlatent

Mycobacterium tuberculosis infectionEmail ldharr3@emory.eduCo-authors JeremiahKhayumbi2,JoshuaOngalo2, Joan Tonui2, Loren E. Sasser1,

FelixHayaraOdhiambo2, Cheryl L. Day1,3

Affiliations 1EmoryVaccineCenter,EmoryUniversity,Atlanta,GA,USA; 2Center forGlobalHealthResearch,KenyaMedicalResearchInstitute,Kisumu,Kenya; 3DepartmentofMicrobiology& Immunology,EmoryUniversitySchoolofMedicine,Atlanta,GA,USA

Mycobacterium tuberculosis(Mtb)isthecausativeagentoftuberculosis(TB).In2017ap-proximately 9 million individuals developed active TB disease and it is estimated that one quarteroftheworld’spopulationislatentlyinfected.LatentMtbinfection(LTBI)isdefinedasindividuals who have been infected with Mtb but do not have clinical signs or symptoms of ac-tiveTBdisease.AlthoughthecorrelatesofimmuneprotectiontoMtbarenotwelldefined,itis known that numerous innate and adaptive immune subsets are important in mediating con-trolofMtbinfection.NKcellsareinnateimmunecellsthatcankillMtb-infectedmacrophagesin vitro by releasing granulysin, activate macrophages through production of IFNy, and regu-late CD4 T cells responses through direct and indirect mechanisms. A greater understanding ofNKcellbiomarkersinLTBIwillprovidebetterinsightsintofuturetherapeuticinterventions.Inthisstudy,weevaluatedNKcellphenotypicandfunctionalprofilesassociatedwithLTBIinKenyanadults.PBMCswerecollectedfromcohortsofMtb-uninfectedhealthycontrols(HC,N=30)andindividualswithLTBI(N=31)inKisumu,Kenya.IndividualsclassifiedasLTBIhadapositiveQuantiFERON-TB(QFT)test,whereasHCwereQFT-negative.FlowcytometrywasperformedonPBMCstophenotypeNKcellsubsetsandcharacterizeNKcellresponsestogenericstimuli(MHCclassI-devoidcellsandantibody-coatedtargetcells)andMtbanti-gens(Mtbwholecelllysate,Mtbcellmembrane,andMtbcellwall).WefoundthatLTBIisassociatedwithdecreasedNKcellexpressionofthenaturalcytotoxicityreceptorNKp46anddecreasedexpressionof the inhibitory receptorTIGIT, comparedwithHC.WhileNKcellfunctionalcapacitytogenericstimuliissimilarbetweenLTBIandHC,NKcellsfromindividu-alswithLTBIexhibitdiminisheddegranulation(CD107a)andactivation(CD69)inresponsetoMtbantigenstimulation,comparedwithHC.Takentogether,ourresultsindicatethatNKcells are phenotypically altered in LTBI and that these cells have decreased response to Mtb antigens when compared with Mtb-uninfected individuals.

Presenter Casey Butrico 97Title Neutrophil dysfunction in comorbid Staphylococcus aureus osteomyeli-

tis alters bacterial metabolism and abscess architecture Email casey.e.butrico@vanderbilt.edu Co-Author Aimee Potter, Jacob Curry, and Jim CassatAffiliation VanderbiltUniversity

Staphylococcus aureus is a leading cause of antibiotic-resistant bacterial infections and can infect nearly every organ of the human body. One commonmanifestation ofS. aureus diseaseisinvasiveboneinfection,knownasosteomyelitis.Osteomyelitisisconsideredone

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of themostdifficult to treat infectionsandnecessitates long-termantibiotic treatmentandsurgicalintervention.Uniquetoskeletalinfections,S.aureusclustersaroundnecroticbonefragments, known as sequestra. These distinct communities are encompassed by innate immune cells and strongly associated with progression to chronic disease. To better under-stand how S. aureus survives during osteomyelitis, we conducted transposon sequencing (TnSeq) to identify genes essential for staphylococcal growth in vivo. TnSeq identified anumber of central metabolic pathways as crucial for bacterial survival. Mono-infections with S. aureus mutants inactivated for various central metabolic pathways revealed that S. aureus reliesheavilyonglycolysisduringosteomyelitis. Interestingly, the tricarboxylicacid (TCA)cycleandgluconeogenesisweredispensableforbacterialgrowth.Inlinewiththisfinding,anapleurotic(replenishing)reactionswerealsocrucialforbacterialsurvivalinbone.Tofur-therdelineatetheroleofcentralmetabolismpathwaysduringdifferentstagesof infection,we created a pool of select S. aureus strainswithdifferentialmetaboliccapabilities.Usingthis pool, we discovered that mutants which were nonviable during monoinfection were able to persist during a competitive infection. This suggests the ability of S. aureus to resource shareor residewithin different abscessniches in vivo. In addition to intra-bacterial influ-ences during osteomyelitis progression, infiltrating neutrophils regulateS. aureus growth and physiology in a spatially and temporally resolved manner. Previous work suggests that neutrophildepletionmodifiesabscessstructureduringS. aureus infection. To elucidate the role of neutrophils in S. aureus metabolic regulation, we modeled neutrophil depletion during osteomyelitis. Abscess architecture, infection kinetics, and dissemination to distant organs were altered in the absence of functional neutrophils. Moving forward, novel imaging ap-proachesandfluorescenttranscriptionalreportersforcentralmetabolismgeneswillbeusedto spatially resolve bacteria physiology in vivo. Together, our data suggest that abscess architectureandneutrophilfunctioninfluenceS. aureus central metabolism.

Presenter: Brenna D. Appleton 98Title: TheroleofmicroRNA-22-3pinthedysregulationoflupusTregs and the

pathogenesis of systemic lupus erythematosus.Email: brenna.d.appleton@vanderbilt.eduCo-Authors: AshleyW. Faust,Danielle L.Michell,Michelle J.Ormseth, KaseyC.

Vickers&AmyS.MajorAffiliation: VanderbiltUniversity

Systemic lupuserythematosus(SLE) isanautoimmunediseaseaffectingover1.5millionAmericans and at least 5million individualsworldwide. Evidence demonstrates autoanti-body producing B cells and dysfunctional CD4+ T cells contribute to SLE pathology, however lack of understanding surrounding mechanisms of disease pathogenesis have prevented therapeutic advancement. Studies indicate one mechanism for dysregulated immune ho-meostasisinautoimmunityisthroughmicroRNAs(miRNAs).MiRNAsareshort,endogenouspost-transcriptional regulators of gene expression which act by degradation or translation repressionoftargetmRNAs.OurgrouppreviouslyidentifiedmiR-22-3pasbeingincreasedthree-fold in SLE patient plasma compared to age- and gender-matched heathy controls. MiR-22-3plevelswereincreasedinwholeCD4+Tcells(four-fold)andTregs(three-fold)fromB6.SLE1.2.3micecomparedtoB6controls,andinhibitionofmiR-22-3p,usinglocked-nu-cleicacid(LNA)-22amelioratedkeydiseasepathologiesincludingglomerulonephritis.Therewasalsoa10%reductioninIFN-g+ CD4+ T cells in mice treated with LNA-22. Based on these data,wehypothesizedincreasedmiR-22-3plevelsinSLETcellspropagatedinflammationdirectly by skewing naïve CD4+ T cells towards the Th1 phenotype during polarization, or indirectly by reducing the regulatory capacity of Tregs.ResultsshowneithermiR-22-3pdefi-ciency nor overexpression impact the polarization of Th1 or iTreg cells. However, inhibition ofmiR-22-3p inB6.SLE1.2.3mice increasesTreg IL-10 production, and overexpression of miR-22-3pin vitro decreases Il-10 transcript in iTregs two-fold, suggesting heightened levels ofmiR-22-3pmayaltertheir suppressivefunction.WeconcludethatmiR-22-3poverexpres-sion in Tregs alters their suppressive capacity which indirectly contributes to autoimmune T cell dysregulation in SLE.

Presenter: Heather Koehler 99

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Title: Die to live another day: the role of ZBP1 in virus-induced necroptosis and innate host defenses.

Email: Heather.s.koehler@emory.eduCo-Authors: Edward S. MocarskiAffiliation: Department of Microbiology and Immunology, Emory Vaccine

Center, Emory University School of Medicine, Atlanta, GA 30322, USA.

The primary function of the immune system is to protect the host from invading pathogens. In response, microbial pathogens have developed various strategies to evade detection and elimination by the immune system. Z-Nucleic Acid Binding Protein 1(Zbp1 aka DAI and DLM-1) is an important innate immune protein that was first reported as a cytosolic DNA sensor that leads to increases in type 1 IFN in a redundant fashion to other cytosolic nucleic acid sensors. Later Zbp1 was identi-fied as an adaptor protein for RIPK3 implicated in sensing viral dsRNA leading to virus-in-duced necroptosis ( MCMV, VACV, IAV,HSV). The association of ZBP1 with RIPK3 initiates necroptosis which is a cell death pathway that not only restricts viral replication, but also promotes anti-viral inflammation through the release of damage-associated molecular pat-terns (DAMPs). This plays a significant role in limiting the pathogenesis of viral infections. In response, many viruses have developed countermeasures for the pathway in the form of encoded suppressors of cell death. These evolved countermeasures suggest that ZBP1’s role in anti-viral inflammation is of extreme importance and warrants further investigation. We have recently identified a novel role for Zbp1 in regulating the amplitude of inflamma-tion resulting from viral infections or cytokine stimulation. Zbp1 acts to restrict the magni-tude and duration of NFkB and MAPK signaling. Furthermore Zbp1 dampen the release of inflammatory cytokines such as IL1-b and TNFa following stimulation. Taken together, Zbp1 is an important determinate of favorable of come of viral infections and innate immune responses.

Presenter Jessica D. Hathaway-Schrader 100Title Specific Commensal Gut Bacterium Critically Regulates Commensal

GutMicrobiotaImmunoregulatoryActionsDuringPost-PubertalSkeletalDevelopment

Email hathawa@musc.eduCo-Authors NicoleA.Poulides,JoyE.Kirkpatrick,AmyJ.Warner,BrooksA.Swan-

son,MichaelE.Chew,ElizaV.Taylor,SakamuriV.Reddy,CarolineWestwater,ChadM.Novince

Affiliation MedicalUniversityofSouthCarolina

Thecommensalgutmicrobiotacriticallyregulatespostnatalskeletaldevelopment.Wehaveshown the commensal gut microbiota enhances osteoclastogenesis, which appears to be mediated by TH17/IL17responseeffectsinbonemarrowandliver.Ofinterest,segmentedfilamentousbacteria(SFB)isadistinctcommensalgutbacteriumthatpotentlydirectsTH17/IL17-mediatedimmunity.StudypurposewastodelineatetheinfluenceofSFBoncommen-sal gut microbiota immunomodulatory actions regulating post-pubertal skeletal development. FemaleC57BL/6T germ-free(GF) littermatesweremaintained asGF ormonoassociatedwithSFBat5-weeks-old;euthanizedat9-weeks-old.SFBcolonizationwasconfirmedinSFBmiceby16SrDNAanalysis.IL17wassubstantiallyincreasedintheileumandserumwhile%TH17cellswereelevatedinspleensandliver-lymph-nodes(LLNs)ofSFBmice,validatingSFB induction of TH17/IL17 immunity. Micro-CT analysis revealed SFB mice had decreased trabecular bone volume fraction in the proximal tibia, which was attributed to reduced tra-becularnumber.TRAP+osteoclastsizeandnumberofnucleiwereenhanced inSFBvs.GFmice.TodeterminetheinfluenceofSFBinacomplexgutmicrobiota,female9-week-oldC57BL/6T #excluded-flora(EF)mice (#specific-pathogen-freemicedevoidofSFB)and*murine-pathogen-free(MPF)mice (*specific-pathogen-freemice colonized by SFB) wereinvestigated.Paralleling thefindings fromSFB-monoassociatedmice,16S rDNAanalysisvalidated SFB colonization in MPF mice. MPF vs. EF mice also had increased serum IL17 and ileum Il17a. Trabecular bone volume fraction was blunted in MPF mice. MPF mice had enhancedTRAP+osteoclastnumbersandsize in vivo and in vitro. Flow cytometry dem-onstrated%MDSCsand%TH17 cells were elevated in marrow of MPF vs. EF mice. MPF

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micehad increased%TH17 cells anddecreased%TREGS and%naïveCD4+ T-cells in the spleen.Notably,%M1-macrophages and%MDSCswere enhanced inmesenteric-lymph-nodes(MLNs),while%TH17and%TH1 cells were increased in LLNs of MPF vs. EF mice. MPF mice had upregulated Lcn2 in liver and marrow and elevated LCN2 in serum, which provides novel mechanistic insight about commensal gut microbiota osteoimmune response effects.SFBcolonizationenhances innateandadaptive immunity,upregulatesosteoclas-togenesis,anddrivesboneloss.Thisresearchnotablyrevealsthatspecificmicrobescriti-cally impact commensal gut microbiota immunomodulatory actions regulating post-pubertal skeletal development.

Presenter Beth Holbrook 101Title ANovelR848-ConjugatedInactivatedInfluenzaVirusVaccineIsEffica-

cious and Safe in a Neonate Nonhuman Primate ModelEmail bcholbro@wakehealth.eduCo-Authors JongR.Kim,LanceK.Blevins,MatthewJ.Jorgensen,NancyD.Kock,

Ralph B. D’Agostino, Jr., S. Tyler Aycock, Mallinath B. Hadimani1, S. BruceKing1,GriffithD.Parks,MarthaA.Alexander-Miller

Affiliation WakeForestSchoolofMedicine,WakeForestUniversity1

Influenzaviruscancauselife-threateninginfectionsinneonatesandyounginfants.Althoughvaccinationisamajorcountermeasureagainstinfluenza,currentvaccinesarenotapprovedforuseininfantslessthan6monthsofage,inpartduetotheweakimmuneresponsefol-lowingvaccination.Thus,thereisastrongneedtodevelopnewvaccineswithimprovedeffi-cacyforthisvulnerablepopulation.WedevelopedaninnovativenonhumanprimateneonatemodeltotesttheefficacyofanovelTLR7/8agonistR848-conjugatedinfluenzavirusvac-cine. Newborn animals were housed in a nursery to allow challenge studies to be performed. The results from our study showed that this vaccine induces a robust antibody response afterprimaryvaccination,boost,andchallenge,aswellasapotentIFNγ–producingTcellrecall response. These responses were associated with lessened pulmonary pathology and improved clearance after virus challenge compared with a non-adjuvanted virus vaccine. Wealsodeterminedtheeffectivenessofthisvaccineinmother-rearedinfantsaswellasitsabilitytopromoteimprovedresponsesat6monthscomparedtovaccinationintheabsenceofR848.Inagreementwithournurserystudy,R848conjugatedtoinfluenzavirusinducedahigherantibodyresponsethatwasassociatedwithincreasedantibodyat6monthsfollowingvaccination.ThesedatastronglysupporttheutilityofR848-conjugatedinactivatedinfluenzavirusasaneffectivevaccineinthisvulnerablepopulation.

Presenter: Jenna Petronglo 102Abstracttitle: Theinfluenceofinnateimmuneresponsesonosteoclastdifferentiation

and function during Staphylococcus aureus osteomyelitis Email: jenna.r.petronglo@vanderbilt.eduCo-authors: Nicole Putnam, Jacob Curry, Laura Fulbright, Jim CassatAffiliations: VanderbiltUniversity,MolecularPathologyandImmunologyGraduate

Program Inflammationinbone,alsoknownasosteomyelitis(OM),ismostfrequentlycausedbythebacterial pathogen Staphylococcus aureus.Whiletheinductionofastrongantibacterialre-sponsebyhostcellsisessentialtotheresolutionofbacterialOM,theresultinginflamma-tion drives pathologic skeletal remodeling, which can lead to weak and fracture-prone bone. Boneresorbingosteoclasts (OCs)areessential formaintenanceofhealthybone,butarealsoimplicatedasdriversofinflammatoryboneloss,partiallythroughenhanceddifferentia-tionofmacrophageprecursors intoOCs(osteoclastogenesis). In thisstudy,wesought tounderstandhowinflammatorystimuligeneratedduringinnateimmuneresponsesinfluenceOCdifferentiation.Wefocusedontheinterleukinreceptor1pathwaybecauseofitsestab-lishedrolesinantibacterialdefenseandhomeostaticosteoclastogenesis.Wehypothesizedthat this signaling pathway is a major driver of infection-induced osteoclastogenesis. To test this,westimulatedWTbonemarrowderivedmacrophageswiththeOCdifferentiationfactorreceptoractivatorofNFĸBligand(RANKL)andS.aureussupernatants,withorwithoutIL-

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1R1blockade.ByquantifyingthenumberofOCsusinghistochemicalstaining,weshowthatIL-1R1 blockade significantly diminishes bacterially-induced osteoclastogenesis.We nextassessedtheroleofIL-1R1signalingindrivingOC-mediatedbonelossinanin vivo model. CorticalboneofWTandIL-1receptortype1knockout(Il1r1-/-)femurswereinoculatedwithS. aureus and harvested at day 14 post-infection. Changes to the trabecular bone were as-sessed using histomorphometry to quantify osteoclastogenesis and μCT to measure archi-tecturalchangesinbone.WediscoveredthatlossofIL-1R1abrogatesinfection-inducedOCdifferentiationandinflammatoryboneloss.SinceIL-1R1drivesbonelossbyalteringOCdif-ferentiation,wenextstimulatedOClineagecellswithbacterialsupernatantsfor6hoursandmeasured transcriptional changes in immunologically relevant genes using the Nanostring nCounter platform.We found that cellular responses to bacterial stimulation are stronglydependentontheOCdevelopmentalstage.Overall,thesedatademonstratetheimportanceof IL-1R1 signaling in driving bone loss in S. aureusOMandsupportfurtherstudiesintotheprecisemechanismsbywhichinflammationimpactscellsalongtheOCdifferentiationpath-waytoelicitinflammatoryboneloss.

Presenter: ElizabethL.Frost 103Abstract title: Regulation of myeloid cells by spleen tyrosine kinase contributes to the

pathogenesis of experimental autoimmune encephalomyelitisEmail: ef2r@virginia.eduCo-authors: Catherine R. Lammert, Ashely C. Bolte, Makenzie A. Scanlon, Calli E.

Bellinger, Mariah E. Shaw, and John R. LukensAffiliation: UniversityofVirginia

Multiplesclerosis(MS)isadebilitatingautoimmunediseasethatischaracterizedbyimmune-mediated damage tomyelin in the central nervous system (CNS). Although autoreactiveCD4+TcellsarerequiredforinitiationofMS,myeloidcells—dendriticcells,macrophages,neutrophils,CNS-residentmicroglia—alsoparticipateinpathogenesisatmultiplestagesofdisease.Spleentyrosinekinase(SYK)isanintracellularsignalingproteinthathasrecentlybeenidentifiedtoplaycriticalrolesintheregulationofinflammatorycytokineproductionandtheclearanceofcellulardebris.AlthoughSYKhasbeendescribedtobecentrallyinvolvedinthepathogenesisofnumerousautoimmuneandneuroinflammatorydisorders,itsinvolve-ment in MS currently remains unknown. Interestingly, various molecular factors that have been linked toMSdisease ingenome-wideassociationstudies, includingCD37,TREM2,andmultipleC-typelectinreceptors,areknowntoprimarilysignalthroughSYK.Toinves-tigatethecontributionsofSYKsignalinginMS,wehavegeneratedmultiplenovelgeneticmousestrainsinwhichSYKhasbeendeletedindefinedimmunecellpopulationsthatareknowntoplaykeyrolesinMSprogression.Here,wefindthatdeletionofSYKinmyeloidcellsunderthecontroloftheLysMpromoterreducesEAEseverityandinfiltrationofinflammatoryimmunecellsfromtheperiphery.WeobservemodestdefectsinprimingoftheautoreactiveT cell response along with decreased chemokine levels in the CNS at early stages of EAE. Current work further investigates the mechanisms that contribute to this protective pheno-typeinmicewithSYK-deficientmyeloidcells.

Presenter: Ali Nazmi1 104 Title: Innate CD8αα+ cells and osteopontin promote ILC1-like intraepithelial

lymphocytehomeostasisandintestinalinflammationE-mail: anazmi.abdelnabi@vumc.orgCo-authors: KristenL.Hoek1,MichaelJ.Greer2, M. Blanca Piazuelo3, Nagahiro Mi-

nato4andDanyvidOlivares-Villagómez1,5,6Affiliation: 1Department of Pathology, Microbiology and Immunology, Vanderbilt

University Medical Center, Nashville, TN, USA; 2Department of Bio-medicalInformatics,VanderbiltUniversity,Nashville,TN,USA;3Depart-mentofMedicine,VanderbiltUniversityMedicalCenter,Nashville,TN,USA; 4MedicalInnovationCenter,GraduateSchoolofMedicine,KyotoUniversity, Kyoto 606-8507, Japan; 5Vanderbilt Institute for Infection,Immunology and Inflammation, Vanderbilt University Medical Center,Nashville,TN,USA;6ORCID,0000-0002-1158-8976.

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Innate CD8αα+cells, also referred to as iCD8α cells, are TCR-negative intraepithelial lym-phocytes(IEL)possessingcytokineandchemokineprofilesandfunctionsrelatedtoinnateimmune cells. iCD8α cells constitute an important source of osteopontin in the intestinal epithelium.Osteopontin isapleiotropiccytokinewithdiverseroles inboneand tissuere-modeling, but also has relevant functions in the homeostasis of immune cells. In this report, we present an evidence for the role of iCD8α cells and osteopontin in the homeostasis of TCR-negativeNKp46+NK1.1+IEL(ILC1-like).WeshowthatintheabsenceofiCD8α cells, thenumberof ILC1-like IEL issignificantlyreduced.TheseILC1-likecellsare involved inintestinalpathogenesisintheanti-CD40mousemodelofintestinalinflammation.Thereduc-tion of iCD8α cell numbers and/or osteopontin expression results in a milder form of intestinal inflammation in thisdiseasemodel.Collectively,our resultssuggest that iCD8α cells and osteopontinpromotesurvivalofILC1-likeIEL,whichsignificantlyimpactsthedevelopmentofintestinalinflammation.

Presenter Mei Lan Chen1 105Title Constitutive androstane receptor directs T cell adaptation to bile acids

in the small intestineEmail meichen@scripps.eduCo-Authors HongtaoWang2, Amber Eliason1, Xiangsheng Huang2,GuohuiWang2,

Rui Xiao3, Matthew E. Pipkin1, David D. Moore4, and Mark S. Sundrud1

Affiliation 1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458,USA; 2Department of Pediatrics, Section ofGastroenterology,BaylorCollegeofMedicineandTexasChildren’sHospital, Houston, TX, 77030, USA; 3Department of Molecular and HumanGenetics,BaylorCollegeofMedicine,Houston,Texas77030;BaylorGenetics,Houston,Texas77021;4Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas77030,USA

Immune competency demands that circulating CD4+Thelper(TH)cellscoordinateprotectiveimmuneresponsesindiversetissuemicroenvironments(i.e.,skin, lung,gut).Yetstill, themechanisms enabling TH cell homeostasis and function in discrete non-lymphoid tissues re-mainilldefined.Wehaverecentlydiscoveredthatpro-inflammatorysubsetsofTHcells—in-cluding IFNg-secretingTh1cellsandIL-17A-expressingTh17cells—upregulateexpressionof the xenobiotic transporter Mdr1 upon migration into the small intestine lamina propria to maintainhomeostasisandsuppressCrohn’sdisease-likesmallbowel inflammation in thepresence of detergent-like intestinal metabolites, called bile acids. Further, a subset of small bowelCrohn’sdiseasepatients ischaracterizedbyovertMdr1-deficiency.Thus, immunehomeostasis in the small intestine involves local interaction between mucosal TH cells and mucosa-associated bile acids, which may be exploited for the development of safer and more selective Crohn’s disease therapies. In exploring these THcell—bileacidinteractionsfurther, we have performed an in vivoRNAiscreenandidentifiedtheconstitutiveandrostanereceptor(CAR;genesymbolNr1i3)asanuclearreceptorwhichTH cells use to sense and re-spond to mucosa-associated bile acids in the small intestine lamina propria. TH cells lacking CARtransferseveresmallbowelinflammatorydiseaseinRag1-/- recipients, and that CAR activates the expression of dozens of cytoprotective genes in THcells—includingMdr1—thatare recognized for detoxifying and removing bile acids in hepatocytes. As a whole, these findingssuggestthatTHcellsinfiltratingthesmallintestineacquireahepatocyte-likegeneexpression program to adapt to locally recycled bile acids. In addition, our results shed light on an important new pathway that may be relevant to the understanding and treatment of human Crohn’s disease.

Presenter GordonDale 106Title Distantgenomicsitescontributetosomaticdiversificationofsequences

at the IgH locus in mice and humans via templated mutagenesisEmail Gordon.Dale@emory.eduCo-Authors DanielJ.Wilkins,MichaelJ.Rowley,ChristopherScharer,JeremyM.

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Boss,VictorCorces,IgnacioSanz,andJoshyJacobAffiliation EmoryUniversity

During the maturation of a humoral immune response, B cells engage in a program of or-chestrated mutagenesis known as somatic hypermutation. B cells that undergo this program are localized to a lymphoid structure known as the germinal center, in which a Darwinian evolutionaryeventoccurs.Mutatinggerminal centerBcells, specific foragivenantigen,competeforhigheraffinitybindingovermultiplealternatingroundsofmutationandselection.Thosethatdisplayreducedaffinitycomparedtotheirpeersaredeleted.Ithasbeenshownthat mutagenesis during the germinal center reaction can occur in two separate ways. In chickensandrabbits,diversityoccurs throughgeneconversion(templatedmutagenesis),inwhichadonorsequenceiscopiedintoahomologous,butdifferent,recipientsequence.Conversely, in mice and humans, mutagenesis occurs through the accumulation of pseudo-random point mutations driven primarily by the combination of AICDA and the error-prone polymerasepolη.Inanimalsthatundergogeneconversion,itisknownthatpseudorandompoint mutations can occur. However, in mice and humans, the process of gene conversion is largely thought to not occur. Here we show that such a process is active in murine and humanBcellsbyMonteCarloanalysisofmutationclusterswithinhumanIgHVgenese-quences,aswellasunselectedtransgenesinmiceplacedattheIgHlocus.Weperformeddeep sequencing on the IgHV repertoire of circulatingmemoryB cells from four humandonorsandanalyzedthemurinetransgenedatageneratedfromtheFredAltlaboratory.Wefindthattemplatedeventsaccountforapproximately5-10%ofthemutationloadofcirculat-inghumanmemoryBcells,thatdonorsitesaresignificantlyclosertotheIgHlocusviaHi-Canalysis in germinal center B cells vs. naïve B cells, and that donor sequences are predicted tobeprimarilywithinopenchromatinregionsdetectedviaATAC-seq.Mostsignificantly,wealsofindthatallhumandonorsacquiremutationsinterchromosomallyfromdiscretesitesinthe human genome. These results suggest that templated mutagenesis occurs in human and murine B cells and contributes a non-negligible number of mutations during the matura-tion of humoral immune response.

Presenter Jakob Habib 107Title Investigating the role of type I interferon signaling during costimulation

blockade resistant rejectionEmail jghabib@emory.eduCo-Authors DaveMathews,YingDong,AllisonStephenson,AbheekGhosh,Cindy

Breeden, Abe Matar, Brendan Lovasik, Andrew AdamsAffiliation EmoryUniversity

Purpose:Costimulationblockade(CoB)isapromisingnewtransplantimmunosuppressionstrategyofferingimprovedlong-termpatientandallograftsurvivalwithoutthenephrotoxicityof calcineurin inhibitors. However, increased risks of acute rejection have impeded wide-spreadadoptionofCoB.TypeIinterferons(IFN),producedmainlybyplasmacytoiddendriticcells(pDCs),inducesystemicinflammationthatmayprimetheadaptiveimmunesystemforacute rejection. In this study, we examine the contribution of signaling through the type I IFN receptor(IFNAR)toCoB-resistantallograftrejection.

Methods:WeperformedfullyMHC-mismatchedskingraftsfromBALB/cJdonorstoC57BL/6recipients.MiceweretreatedwithCoB(CTLA4-Ig+anti-CD40L)aloneorinconjunctionwithanti-IFNAR and graft survival was assessed over time. In a subsequent experiment, mice wereeuthanized1,2and3weekspost-transplantandthelymphocyteswereanalyzedbyflowcytometry.Results: Transplanted untreated mice rejected rapidly (mean survival time=11 days),CoB treatment improved survival but resulted inCoB-resistant rejection (MST=23d), andCoB+aIFNARsignificantlyimprovedsurvival(MST>60d).IFNARwashighlyexpressedonmurinepDCs(PDCA1+SiglecH+B220+) relative toconventionalDCsandTcells.Combi-nation treatment withCoB+anti-IFNAR significantly reducedMHC I expression on pDCscompared to CoB alone, and reduced CD80 expression on pDCs at the peak of rejec-tion,suggestinga roleof type I interferonsignaling for theactivationofpDCs.CoB+anti-IFNARtreatmentalsoledtoareductioninthefrequencyofactivated(CD44+)andeffector

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(IFNy+TNFa+)CD8TcellscomparedtoCoBalone.Conclusions: These data suggest that signaling through IFNAR augments pDC activation andisassociatedwithCD8+TcellactivationandeffectorfunctionsinordertopromoteCoB-resistant rejection.

Presenter: MichaelBrandonWare 108Title: Interleukin-6 in biliary tract cancers promotes expansion of immune

suppressive myeloid cells and is associated with increased myeloid cells in patient tumors

Email: mbware@emory.eduCo-Authors: Jennifer Yang, Mohammad Y. Zaidi, Alyssa Krasinskas, Thomas A.

Mace, Matthew R. Farren, Yiman Li, Wanqi Chen, Zhengjia Chen,Gregory S. Young, Omar Elnaggar, Zheng Che, Shishir K. Maithel,TaniosBekaii-Saab,BasselEl-Rayes,GregoryB.Lesinski

Affiliation: EmoryUniversityBiliarytractcancers(BTC)areanaggressivemalignancyfrequentlyassociatedwithelevat-ed immunosuppressive cell types in patients. These cancers can be grouped into distinct subtypeswithuniquemolecularandgeneticsignatures.WehypothesizedthatBTC-derivedcytokines act through distinct pathways to promote immune suppressive features of the dis-ease.Cytokine and chemokine secretion, and activation of associated signaling pathways, were studiedinapanelofBTCcelllines.WeusedflowcytometryandimmunoblottostudytheeffectsofBTCsupernatantsonhumanperipheralbloodmononuclearcells(PBMCs).Basedonresults,ahumanBTCtissuemicroarray(TMA,n=33)wasstainedforIL-6,GM-CSFandCD33+S100a9+myeloidcells.BTCcelllinesdemonstratedactiveSTAT3andSTAT5signaling,andsecretedseveralim-munomodulatory factors including IL-6, GM-CSF, andMCP-1. Antibody neutralization ofIL-6andGM-CSFinthesesupernatantslimitedSTAT3andSTAT5phosphorylationinBTCcell lines and in PBMCs exposed to these supernatants. The expansion of myeloid derived suppressorcells(MDSC)fromPBMCsculturedwithBTCsupernatantswasinhibitedwithantibody neutralization of IL-6 andGM-CSF. TMA analysis revealed significant associa-tionbetweenIL-6expressionandCD33+S100a9+myeloidcellinfiltrationoftumorsamples(p<0.001).Interestingly,analysisofTMAdatastratifiedbydiseasesubtyperevealedasig-nificantcorrelationbetweenhigherlevelsofIL-6andGM-CSFandearlierrecurrence-freesurvival in extrahepatic cholangiocarcinoma, but not in intrahepatic cholangiocarcinoma. ThisstudyhighlightsIL-6andGM-CSFaspotentialtargetsinBTCforfutureclinicaltrials,asthesecytokinespromotetheexpansionofimmunosuppressivecelltypes.Specifically,ourdataidentifiesasubtypespecificcytokineprofileinextrahepaticcholangiocarcinoma.Thisstudysuggestssubtypespecific immunesignatures inBTCthatmaybe leveraged in thetreatment of these deadly diseases.

Presenter Anna B. Morris 109Title Tcell-expressedFcgRIIBfunctionallyregulatesCD8+Tcell immunity

via ligation of Fgl2 Email anna.burcham@emory.eduCo-authors David F. Pinelli, Danya Liu, Jeremy M. Boss, Christopher D. Scharer,

Mandy L. FordAffiliation EmoryUniversity

FcγRIIBisthesoleinhibitoryFcγreceptorandisknownforitsinhibitoryfunctiononBcells,DCs,andmacrophages.Interestingly,wediscoveredthatFcγRIIBisupregulatedonasub-set of CD44hiCD62LloeffectorCD8+ T cells following transplantation and aimed to elucidate its function. TodetermineifFcγRIIBplaysacell-intrinsicrole in inhibitingCD8+ T cells, we generated

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a CD8+T cell conditionalKOsystemandobservedenhancedaccumulationofFcγRIIB-/-

CD8+Tcellsat14(p<0.05)and21(p<0.01)dpost-transplantationrelativetoWTcontrols.RNAseq analysis of FACS-sorted, donor-reactive CD8+ T cells revealed an enrichment of apoptosis-related genes in FcγRIIB+ vs. FcγRIIB- cells. Further, FcγRIIB-/- CD8+ cells ex-hibitedlowerexpressionofactivecaspase3/7onday16followingtransplantcomparedtoWTcells(p=0.0315).TodetermineifFcγRIIBondonor-reactiveCD8+ T cells impacted graft rejection,WTrecipientsofWTorFcγRIIB-/- CD8+ T cells were treated with anti-CD28 domain antibody.Animals that receivedFcγRIIB-/- CD8+ cells exhibited accelerated graft rejection relativetothosereceivingWTCD8+Tcells.ThesedatademonstratethatFcγRIIBfunctionsin a cell intrinsic manner to functionally regulate CD8+ T cell immunity.Todeterminewhetherantibodiesare the functional ligandofFcγRIIBonTcells,weem-ployed AID-/- animals that are unable to generate class-switched antibodies. WT vs.FcγRIIB-/- CD8+TcellswereadoptivelytransferredintoWTvs.AID-/- recipients of skin grafts, and donor-reactive CD8+ T cell responses were measured at day 14. Results indicated that AID-/-recipientspossessedsimilarlyincreasedfrequenciesofFcγRIIB-/- CD8+ T cells relative toWTCD8+ T cells, demonstrating that antibodies are not the sole ligand via which T cell-expressedFcγRIIBregulatesCD8+ T cell immunity. Instead, we found that an alternative ligandforFcγRIIB,Fgl2,inducedcaspase3/7activityinFcγRIIB+butnotinFcγRIIB-/- T cells, suggestingthatFgl2isafunctionalligandforFcγRIIBonCD8+ T cells. Basedontheseexperiments,weconcludethatFcγRIIBisanovel,cellintrinsicCD8+ T cell regulatory pathway that regulates CD8+ T cell apoptosis and functions through ligation of the immunoregulatory cytokine Fgl2.

Presenter: Christopher Peek 110Title: MechanismsofbonelossduringinflammatoryboweldiseaseEmail: christopher.t.peek@vanderbilt.eduCo-authors: Caleb Ford, Nicole Putnam, Jacob Curry, Blanca Piazuelo, Margaret

Allaman,KeithWilson,&JimCassatAffiliation: VanderbiltUniversity:Pathology,Microbiology,andImmunology

Twohallmarksof inflammatoryboweldisease(IBD)aregastrointestinal inflammationandchanges in the intestinal microbiota. However, patients with IBD frequently experience extra-intestinal disease, of which skeletal pathology is among the most common manifestations. TheetiologyofIBD-associatedbonelossremainsunclearbutlikelyreflectsacombinationofmalabsorptivemalnutrition, glucocorticoid therapy, and chronic inflammation.Yet, per-sistent bone loss in nutritionally replete and glucocorticoid naïve patients implicates chronic inflammationandpotentiallyundiscoveredmechanismsasdriversofIBD-associatedboneloss. In this study, we used complementary mouse models of colitis-driven bone loss to test thehypothesisthatIBD-associatedbonelossistriggeredinpartbyinflammatorycytokinesand(PAMPs). Inflammatorycytokines,suchastumornecrosis factoralpha(TNF-a),aresystemicallyelevatedinpatientswithIBDandprofoundlyaffectskeletalbiology.Inadditiontoinflammation,changesinthemicrobiomeinfluenceboneremodeling.Importantly,skeletalcellssenseand respond topathogen-associatedmolecularpatterns (PAMPs)viapatternrecognitionreceptors(PRRs)utilizingtheadaptorprotein,MyD88.Weobservedsignificantreductionsintrabecularbonevolumeovertotalvolume(BV/TV)inmicesubjectedtoachem-icallyinducedcolitiswithdextransodiumsulfate(DSS).Usingmultiplexedcytokineanalysis,we discovered increased abundance of several cytokines which have been implicated in multiplemodelsof inflammatoryboneloss, includingTNF-a,CXCL5,andCXCL10,intheskeletal microenvironment of DSS-treated mice. Adoptive T-cell transfer colitis recapitulated thesefindings.Additionally,IL-12/23p40,thecommonsubunitofIL-12andIL-23,wasfoundtobesignificantlyincreased.Intriguingly,IL-12andIL-23cytokinesshareacommonsub-unit,IL-12/23p40(p40),yetexertopposingeffectsonboneremodeling.IL-12largelyinhibitsosteoclastdifferentiation,whileIL-23leadstoboneresorptionthroughenhancedosteoclas-togenesis. Moving forward, we are using fecal microbiome transplants, gnotobiotic mice, and skeletalspecificpatternrecognitionreceptorgeneticknockoutmicetotestthehypothesisthat the microbiome impacts skeletal homeostasis during colitis. This work will elucidate fun-damental mechanisms by which systemic cytokines and circulating microbial components impact bone biology during colitis and provide a platform to test the role of colitis-associated cytokines in pathologic bone loss.

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Presenter Lavinia Franchitti 111Title CD8+lymphocytemediatedsuppressionofHIVexpressioninCD4+T

cellsEmail lavinia.franchitti@emory.eduCo-authors AbigailWillemse,JackYoon,ErickWhite,BryanCox,GuidoSilvestri,

DeannaAKulpaAffiliation EmoryUniversity

ThepersistenceofHIVinfectionunderARTisduetoareservoirof latently infectedcellsthat remain indefinitelydespitesuppressionofvirus replication.Defining themechanismsresponsiblefortheestablishmentandmaintenanceoftheHIVreservoirunderARThasbeenthefocusofeffortsaimedatHIVeradication.SeveralstudieshavedemonstratedthatCD8+TcellsinhibitvirusreplicationduringuntreatedHIV/SIVinfection;however,themechanismsresponsibleforthisantiviraleffectremainpoorlyunderstood.Weusedourprimarycellbasedin vitromodelofHIVlatencytostudytheCD8+Tcellmedi-atedsuppressionofHIVexpression.ToexaminetheimpactofCD8+Tcellsontheestab-lishmentofHIVlatency,memoryCD4+TcellsfromHIVnaïvedonorswereinfectedin vitro andthenco-culturedwithactivatedCD8+Tlymphocytes(1:1or1:5target:effectorratios).ByassessingintracellularGagexpressiononCD4+Tcellsbyflowcytometry,andbyquantify-ingthefrequencyofintegratedHIVDNAbyqPCR,wefoundthatHIVexpressioninCD4+Tcellswasreducedwhenco-culturedwithCD8+Tcellsanaverageof9-fold(p<0.0001)and18-fold (p<0.0001)at1:1or1:5 ratios respectively,withoutsignificantly reducing thefrequencyofHIV-infectedcells(n=21).ToassesstheroleofCD8+Tcellsinlatencyrever-sal, cells generated in our in vitro latency model were stimulated in the presence or absence ofactivatedCD8+T lymphocytes.WeobservedasignificantsuppressionofHIV latencyreversal,a6-folddecreaseat1:1target:effectorratio(p=0.0156)and14-folddecreaseat1:5ratio(p=0.0156).OurstudieshavedemonstratedaCD8+lymphocytemediatedsuppressionofHIVexpres-sioninCD4+Tcellsthatfunctionstoinducetheestablishmentaswellasmaintainlatencyinthepresenceofactivationsignaling.UnderstandingthemechanismsbywhichCD8+lym-phocytessuppressvirustranscriptionandultimatelypromoteHIVlatencyandpersistenceinART-treatedHIV-infectedindividualsmayprovidecriticalinsighttosupportthedesignofnewapproachesforHIVeradication.

Presenter: Allison C. Stephenson 112Title: Addition of Anti-CD127 to Costimulation Blockade Prolongs Allograft

Survival via PD-1Email: acsteph@emory.eduCo-authors: DaveMathews,YingDong,StevenKim,CynthiaBreeden,AndrewAd-

amsAffiliation: EmoryUniversity

Costimulation blockade as a strategy for transplant immunosuppression has given rise to im-provedlong-termoutcomesforthefirsttimeinthirty-years.Patientstreatedwithbelatacept,ahigh-affinityvariantoftheCTLA4-Igfusionproteinenjoysuperiorrenalfunction,andde-creasedriskofdeathorgraftlossat7yearfollowup.Wehavepreviouslyidentifiedasubsetof memory T cells which express high levels of CD127, which are associated with costimula-tionblockaderesistantrejection.Wethereforeinvestigatedwhethersignalingdownstreamof this receptorwasnecessary forcostimulation independent rejection.Weexamined theefficacyofcombinedcostimulationblockadeandanti-CD127directedtherapyinastringentmurineskinallotransplantationmodel.Whilecostimulationblockadeimprovedsurvivalmod-estly,allanimalseventuallyrejected(MST=21days).Theadditionofanti-CD127preventedthedevelopmentofacuteallograftrejectionandledtoindefinitesurvival(p<.0001,MST>80days).SignalingdownstreamofCD127isknowntobecriticalforproliferationandhomeo-stasis.WethereforeinvestigatedtheimpactofCD127blockadeonTcellproliferation.Inamodelofgraft-versushostdisease,CFSElabeledC57BL/6splenocytesweretransferredtoirradiated Balb/C recipients, who were then treated with costimulation blockade, or combined

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costimulationblockade+anti-CD127,orreceivednotherapy.Theadditionofanti-CD127tocostimulationblockadesignificantlyabrogatedallostimulatedproliferativeresponse,nearlyreducingproliferationbyhalf(p=.0002).TofurtherdefinetheimpactofcombinedCTLA4-Ig+anti-CD127ongraftspecificTcells,weutilizedanantigen-specificmodel(Ova-expressingskingraft,withOT-IandOT-IIadoptivetransfer).WefoundmicetreatedwithCTLA4-Ig+anti-CD127demonstratedreducedexpansionandeffectorfunctionofgraft-specificCD8Tcells.Whencharacterized,thesecellsexpresshighlevelsofPD-1andTIGIT,suggestinganexhausted phenotype. By pharmacologically blocking PD-1 in our murine allotransplantation model,theprotectivebenefitofCTLA4-Ig+anti-CD127therapywaslost(p<.0001),suggest-ing that the addition of anti-CD127 to costimulation blockade gives rise to graft tolerance via PD1 mediated T cell exhaustion.

Presenter DonaldMcGuire 113Title DefiningHumanCD8memorysubsetsfollowingacuteviralinfection.Email dcmguir@emory.eduCo-Authors Rama Akondy, Carey Jansen, Shashikala Nagar, Srilatha Edupuganti,

MarkMulligan,HaydnKissick,RafiAhmed.Affiliation EmoryUniversity

Induction of an T cell memory response is an important mechanism in a protective immune response.Thehighlyeffectiveyellowfevervaccinegenerateslastingprotectionandalong-livedCD8memory. Wehavepreviously foundthatYFVspecificCD8Tcelldevelop intoquiescentmemorycellsthatlackmosteffectorproteins.Thesesamecellsalsohaveepi-geneticmarksthatsuggestapasteffectorfunction.Theoriginsandhistoryofthislong-livedmemorypopulationremainsunclear. Totracethe lineageof thesememoryYFVspecificCD8TcellsweutilizedsinglecellRNA-seq.Ourdatafromsamplestaken14daysto14yearsafteryellowfevervaccinationidentifiedtwopopulationsofcells.Thefirstpopulationcloselycorrelatedwithcellsfound6-14yearsaftervaccination.Thesecellsexpressedfewereffectormoleculesandexpressedmarkerssuggestingaffinitytolymphoidstructures.Thesecondpopulationexpressedhighlevelsofeffectormoleculesandlackedmanyofthemark-ersassociatedwithlymphoidhoming.Thesecellsweremoreeffectorlikecellswereabsentin samples taken more than one year after vaccination. Interestingly, both populations re-expressCD45RAaftertheendofviremia.Consequently,thesecellsdonotmeettheformaldefinitionofCMorEMcells.However,thesepopulationsdocloselyresemblethoseofMP/MPECandTE/SLECasdefinedinmice.

Presenter: AnaCarolinaGuertaSalina 114Title: Skin resident macrophage-derived microRNA21 impairs skin host de-

fense. Email: ana.g.salina@vumc.orgCo-authors: Stephanie Brandt, Alexandra Ivo de Medeiros, Carlos Henrique Ser-

ezani Affiliation: VanderbiltUniversityMedicalCenterandUniversityofSaoPaulo

Introduction. Staphylococcus aureus skin infection is orchestrated by the actions of resi-dent and recruited immune cells. Skin-resident macrophages produce a variety of chemoat-tractants to recruit neutrophils and monocytes to the site of infection to form an abscess andcontainthebacteria.However,excessiveproductionofinflammatorymediatorscausesunrestrictedphagocyterecruitmentandtissuedamage.OurlaboratoryaimstoidentifytheroleofmicroRNAsasmallnoncodingRNAthatinhibitmRNAexpressionortranslation.Wehave previously shown that miR21 is a homeostatic regulator of macrophage polarization in vivo.WhethermiR21regulatesmicrobialclearanceisnotwellunderstood.Aim.Here,wehypothesizethatmiR-21depletionenhancestheexpressionofantimicrobialeffectors,whiledecreasesinflammatorycytokineproduction,leadingtooptimalhostdefense.Methodology.C57BL/6ormiR21∆myelwere infectedwith~3x106 S. aureusCFUs.c.. The sizeof thelesion was measured during the time, and skin biopsies were collected and processed for qRT-PCR,chemokineforELISA,colonyformingunit(CFU)and,histology.Results.MiR21expression is enhanced after infection and depletion of skin macrophages, but not neutro-

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philsdecreasedmiR21abundanceduringinfection.SkininfectioninmiR21KOmiceshowedreducedlesionsizeandbacterialburdenwhencomparedtoWTanimals.Wedetectedin-creasedIL-1ß,TNF-α,IL-10andwellasNitricoxide.Importantly,wealsodetectedincreasedexpressionofpro-resolutiongenes,suchasCD36,TIM4andcollagen3and4.Increasedabundanceofinflammatorymediatorsalongwithanti-inflammatorygenesleadtoincreasedneutrophil migration, highly organized abscess with ticker capsule when compared to in-fectedWTmice.Conclusion.OurresultssuggestthatmiR21isanegativeregulatorofmac-rophage actions during skin infection and that treatment of mice with a miR21 antagomir could provide important translational insights to skin infections. Financial Support. FAPESP 2017/04786-0and2018/01622-9,HL-103777andR01HL124159-01.

Presenter: Jessica Shannon1 115Title: AdultEpidermalLgr5+StemCellRegenerativePotentialandImmune

Functions duringCutaneousWoundRepair isControlled by Thymic-Stromal-Lymphopoeitin(TSLP)

Email: jessica.shannon@duke.eduCo-Authors: P Mariottoni2 DL Corcoran3AS MacLeod1,2,3,4

Affiliation 1DepartmentofImmunology,DukeUniversity,Durham,NC,USA;2De-partmentofDermatology,DukeUniversity,Durham,NC,USA;3Genom-ic andComputationalBiology,DukeUniversity,Durham,NC,USA; 4

DepartmentofMolecularGeneticsandMicrobiology,DukeUniversity,Durham,NC,USA

After skin injury, re-epithelialization is an essential hallmark of successful repair of cuta-neous barrier and required for prevention of infections. Among various approaches, stem-cell based therapy has shown great potential in both animal models and human studies to overcome and treat large wounds, such as those observed in burn wound victims and toxic necrolysis patients. However, the underlying mechanisms how stem cell factors contribute to cutaneous wound healing and whether they also contribute to antimicrobial immunity are notknown.Geneticfate-mappingwoundhealingstudiesrevealedthatadultLgr5+ hair follicle stemcells(butnotLgr6+stemcells)upregulatethecytokineTSLP during the mid-phase of the wound healing response. In contrast to constitutive high expression of TSLP receptor (TSLPR)onskin-residentimmunecells,ahairfollicle-associatednon-hematopoieticThy1.2+ population upregulated TSLPR expression upon wounding. Notably, when TSLP was admin-istered into skin wounds, it accelerated wound closure compared to vehicle treated wounds (p<0.01).Furthermore,transcriptionalsignaturesfromTSLP-treatedmouseskinilluminateasetofdifferentiallyexpressedgenesthatoverlapwithtranscriptionalchangesduringwoundhealing in both human and mouse models. This overlap analysis revealed chemotactic factors known to regulate stem cell migration in a time-dependent manner and the RNA helicase DDX6, a protein with known roles in maintaining epidermal stem cell self-renewal. Thisobservationwasinagreementwithourfindingthatstimulationofprimaryhumanepi-dermalkeratinocytesorhumanskinexplantswithrecombinantTSLPsignificantlyincreasedDDX6expression.Overall,ourfindingsimplicateanovelroleTSLPinepidermalregenera-tion which may be mediated by DDX6.

Presenter Shawna McLetchie1 116Abstracttitle Livefast,dieyoung:AMPKα1inBcellsbalancesmodulationofplasma

cell and memory B cell survival against function Email shawna.k.mcletchie@vanderbilt.eduCo-authors Sung Hoon Cho2; Paulo Basso2,3; Ariel Raybuck2; Mark Boothby1,2

Affiliation 1DepartmentofCancerBiology,VanderbiltUniversity;2 Department of Pathology,Microbiology,andImmunology,VanderbiltUniversityMedi-cal Center, Nashville, TN.; 3Department of Immunology, Institute of Bio-medicalScience,UniversityofSãoPaulo,SãoPaulo,Brazil.

UponactivationBcellsundergoproliferation,selection,andeventualdifferentiationintoei-ther memory B cells or antibody-secreting plasma cells. Emerging evidence indicates that

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metabolic programs regulate B cell activation and antibody responses. However, the meta-bolic programs that support the durability of plasma cells and the memory B cell population areunknown.Adenosinemonophosphate-activatedproteinkinase(AMPK)isanevolution-ary conserved serine/threonine kinase that integrates cellular energy status and nutrient availabilitytointracellularmetabolicpathways.HereweshowthatlossofAMPKα1inBcellsleadstonormalmemoryBcellandplasmacelldifferentiationbutincreasedcirculatingan-tigen-specificimmunoglobulininresponsetohapten-carrierimmunizationwhencomparedto wild-type mice. Consistent with the increase in immunoglobulin production observed in vivo intheabsenceofAMPKα1,LPS-activatedAMPKα1-deficientBcellssecretemoreIgG1onapercellbasisthanAMPKα1-sufficientcontrolBcells.Furthermore,despiteenhancedimmunoglobulinproduction,normalplasmacellandmemoryBcellformationinAMPKα1-deficientmice,lossofAMPKα1inBcellsimpairedthelong-termsurvivalofthememoryBcell and plasma cell populations in vitro and in vivo.AMPKα1-deficientBcellsexhibit in-creasedmTORC1signalingandaberrantmitochondrialactivitybothin vitro and in vivo after immunization.Moreover,AMPKα1-deficientB cells sustain increasedmitochondrialROSanddefectsinmitophagy.Collectively,thesefindingsfitamodelwhereAMPKα1inBcellssupports the longevity of the long-lived plasma cell and memory B cell populations by pro-motingmitochondrialturnoveranddampeningmTORC1activity.Surprisingly,however,thefindingsalsorevealevidencethatametabolicsensorregulatestherateofimmunoglobulinproduction by the plasma cell population. NIHR01AI113292,HL106812,R25-GM062459,NCIT32CA009592-29

Presenter Christopher A. Risley1 117Title Interferongammasignalingregulatesthegenerationofinfluenza-spe-

cificmemoryBcellsEmail crisley@uab.edu Co-authors Christopher A. Risley1*, Sara L. Stone1, Christopher Scharer2, Jerry M.

Boss2, Frances E. Lund1

Affiliation 1Dept.ofMicrobiology,Univ.AlabamaatBirmingham,BirminghamAL35243; 2Dept.ofMicrobiologyand Immunology,EmoryUniv.,AtlantaGA30322

Despiteextensiveresearchtopreventorminimizeinfluenzavirus(flu)outbreaks,fluremainsa serious human health concern.While immunization remains ourmost effectivemeansofprotectionfromfluinfection,thevaccinemustbereformulatedannuallyduetoconstantmutationoftheviralhemagglutininproteinthatnegatesantibody(Ab)-mediatedvirusneu-tralization.Althoughneutralizingflu-specificcirculatingAbcanpreventinfection,memoryBcells(Bmem),whichareelicitedwithvaccination,alsoplayakeyroleinreducingmorbidityandmortality by rapidly differentiating into protectiveAb-secreting cells (ASCs) followingreexposuretothevirus.DespitetheimportanceofBmeminimmunitytoflu,weknowlittleaboutthesignalsthatinducetheirformationandprogramtheireffectorproperties.Todate,most mechanistic studies of Bmem have been performed in immunized mice using model haptenated-proteins adjuvanted in alum, which preferentially promotes type 2 immune re-sponses.Thisissignificantlydifferentfromimmuneresponsesgeneratedduringafluinfec-tion that induces a strong host IFN response. Recently, we demonstrated that B cell intrinsic expression of T-bet, an IFNg-inducibletranscriptionfactor,regulatesASCdifferentiationfol-lowingfluinfectionand,whiledispensableformemoryBcelldevelopment,T-betisrequiredforBmemdifferentiationintosecondaryASCs.Here,weshowthatT-betisexpressedacrossall isotypesofBmem, including IgMand IgG1,and that there isaBcell-intrinsic require-ment for IFNgRsignalingtogeneratetheseoptimalflu-specificBmemcells.Consistentwiththisfinding,deletionof theIFNg-inducible transcription factor STAT1 selectively in B cells alsoalters thedevelopmentof theflu-specificBmempopulation.Additionally, IFNgR sig-naling,transducedviaSTAT1,duringBmemgenerationiscriticalforBmemdifferentiationinto ASCs, as their loss dramatically impairs the recall response to heterosubtypic infection. Taken together these data indicate a B cell-intrinsic requirement for IFNg signaling, mediated by STAT1 and T-bet, in generating robust, transcriptionally distinct and functional Bmem populationsfollowinginfluenzainfection.Inthefuture,wehopetousethesedatatodevelopbettervaccineformulationsthatwillmoreeffectivelyelicittheseprotectiveBmemcells.

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Presenter AmberWolabaugh 118Title: Lineage-tracingofBcellsinhumanvaccinationidentifiestranscriptional

signatures governing the fate of individual clonotypes in vivoEmail: amber.nicole.wolabaugh@emory.eduAuthors: AmitA.Upadhyay1,AmberN.Wolabaugh1, Alice Cho2,RobertC.Kauff-

man2, Nirav B. Patel3, Reem A. Dawoud1,GregoryK.Tharp3, F. Eun-Hyung Lee2,4,JensWrammert2 and Steven E. Bosinger1,3,5

Affiliation: EmoryUniversity - 1 Division of Microbiology & Immunology, Yerkes National Primate Research Center; 2 Department of Pediatrics, School ofMedicine,EmoryUniversity.;3YerkesNHPGenomicsCoreLabora-tory, Yerkes National Primate Research Center.; 4 Divisions of Pulmo-nary,Allergy,&CriticalCareMedicine,EmoryUniversity,Atlanta,GA;5 Department of Pathology & Laboratory Medicine, School of Medicine, EmoryUniversity.

BackgroundVaccinationsofferauniqueopportunitytostudythehumanadaptiveimmunesystem.Theadvent of single-cell-RNA-Seq now makes it possible to obtain the transcriptional signatures of individual B cells, and distinguish signatures between clonal families exhibiting desirable functionalqualities.Recently,wedevelopedabioinformaticspipeline(BALDR) thataccu-ratelyreconstructspairedIgH+IgLsequencesfromsc-RNA-Seqdataandallowslinkingoftranscriptional data with clonotypes. Here, we applied this “Clonal lineage-tracing” in the con-text of a human vaccination to investigate the signatures in vaccine-induced plasmablasts that predict entry the memory pool. MethodsTwoindividualswerevaccinatedwith2016/2017Fluarixquadrivalentvaccines.IgHreper-toire sequencing of the naïve and memory populations was performed at Day 0, and at day 28. sc-RNA-Seq of 271 individual plasmablasts at day 7 post-vaccination was performed usingSMART-Seq,theSeuratpackagewasusedforsingle-cellDEGanalysis,andBALDRforIgH+IgLreconstruction.ResultsWeidentifiedseveralclonotypesfromthepre-vaccinenaïveandmemorypopulations,andpost-vaccinememorypoolthatwererepresentedinDay7plasmablasts.Wecomparedgeneexpression between plasmablasts that were expanded at day 7 and with plasmablasts rep-resentedassinglets,andidentifiedauniquesignatureforplasmablastexpansion.Finally,we contrasted gene expression of plasmablasts with clonotypes of naïve origin with those of memoryoriginandidentifiedseveralsignificantdifferentiallyexpressedgenes.ConclusionIn this proof of concept study, we combined repertoire sequencing with sc-RNA-Seq to trace clonotypes in vaccine-induced plasmablasts. Lineage-tracing allowed us to identify tran-scriptional signatures predicting entry into the memory pool. The ability to track B cell gene expression with evolution of antibodies will substantially inform rational vaccine design and allow linking of signatures with B cell and antibody functional properties, enabling unprec-edentedinsightintothemolecularmechanismsgoverningvaccineefficacy.

Presenter: Bradley I Reinfeld, 119Title: Glycolytic Tumor Associated Macrophages significantly contribute to

FDG-PETavidityoftumorsCo-Authors: Mathew Z Madden, Melissa M Wolfe, Allison Cohen, H. Charles

Manning,KatyEBeckermann,JeffCRathmell,andWKimrynRathmellEmail: bradley.reinfeld@vanderbilt.eduAffiliation: VanderbiltUnversityMSTP

F18 fluoro-2-deoxyglucose Positron Emission Tomography (FDG-PET) is a fundamen-talimagingtooltodiagnoseandstagecancer.Forthepast50years,thistechnologyhasbeenused toevaluate tumorburdenbasedon theprinciplesof theWarburgeffect, thattumorcellsconsumemoreglucosethannon-tumorcells.Intriguingly,Warburgmetabolismis implemented by immune cells in order to perform their requisite functions. Because the

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heterogenoustumormicroenvironmentcontainsawidevarietyoftheseinfiltratingimmunecells, I hypothesize that the activation state of tumor resident immunocytes contributes to theFDG-PETavidityoftumors.UsingmagneticbeadcellseparationtechniquesonsinglecellsuspensionsobtainedfromMC38flanktumors,Iamabletosuccessfullypurify3cellfractions:(1)Tcellenriched,(2)myeloidenrichedand(3)tumorcellfractions.BycombingthisseparationtechniquewithFDG-PETimagingandgammacounting,weseethatimmunecellscontributetoapproximately70%ofFDGavidityinthistumormodel.Onapercellbasis,the myeloid fraction is the most avid and is enriched with tumor associated macrophages (TAM,CD45+,CD11b+,F4/80+). Inthismodel,F18-glutmaineaccumulates intheCD45-tumorcells,nottheCD45+immunecells,suggestingthatimmunecellPETavidityisglu-cosespecific.Additionally,thistechniqueillustratesknownaspectsasimmune-metabolism,wheretumorinfiltratingTcellsutilizeglucosetoagreaterextentthannaïvesplenocytes.Insupport of this data, ex vivoseahorseanalysisdemonstratesthatthisCD45+,TAMenrichedfraction is the most glycolytic tumor resident population in this model. I also observe that tumorinfiltratingTcellshaveasignificantlyhigherglycolyticandoxidativemetabolismthentheir naive splenocytes counterparts. Therefore, future work will investigate the role of mac-rophageglycolysisonFDG-PETavidityacrossmouseandhumantumorsandinresponsetonewtreatmentmodalities.Wewillimplementgeneticandpharmacologicaltoolstodepletemacrophagesandevaluatehow tumorFDGaviditychanges in response.ThesefindingschallengethedogmathatFDG-PETavidityissolelydrivenbytumorcellWarburgeffectandhighlightthesignificantroleofimmunecellsglycolysisinclinicalsettings.

Presenter Madhubanti Basu 120Title InductionandrelationshipofHIVEnvelope-specificBcellsintheperiph-

eral blood and bone marrow following vaccination in humansEmail mbasu@uabmc.eduCo-Authors Michael Piepenbrink1, Czestochowa Francois1, Fritzlaine Roche1, Chris-

topher Fucile1, Alexander Rosenberg1, Jane Liesveld2,MichaelKeefer2, andJamesKobie1

Affiliation 1UniversityofAlabamaatBirmingham,Birmingham,AL,2UniversityofRochester, Rochester, NY

Inductionoflong-livedplasmacellsproducingprotectiveHIV-1Envelope(Env)-specifican-tibody(Ab)isaprimarygoalofHIVvaccinestrategies.However,vaccine-inducedEnv-spe-cifichumanplasmacellshavenotyetbeen identified. Inaneffort to identifyEnv-specificbonemarrow plasma cells after vaccination, samples were obtained fromHVTN 105, aphase I trial in which participants were immunized with the same bivalent gp120 protein, AIDSVAXB/E,asusedinRV144,althoughcombinedwithaDNAimmunogen(insteadofALVAC-HIV)invariousprime/booststrategies.PeripheralbloodandbonemarrowsampleswereanalyzedbyELISpot,flowcytometry,inadditiontosinglecellandMiSeqbasedreper-toireanalysis.ParticipantswhoreceivedaboostingregimenthatincludedAIDSVAXB/EhadrobustperipheralbloodplasmablastresponsesasmeasuredbyEnv-specificELISpot(n=21)and flowcytometry (n=20) after the final immunization.TheEnv-specific immunoglobulinrepertoireoftheplasmablasts,assessedbymonoclonalAbgeneration(>60mAbstotalfrom16participants),wasdominatedbyVH1geneusage(~60%,VH1-2>VH1-46>VH1-24),andhadmodestmutationfromgermline(~5%).EpitopemappinghasindicatedV3isadominanttarget;howeverotherregionsincludingV1/V2andC1/C5werealsotargeted.Themajorityof the mAbs are high avidity, and a subset can mediate Ab-dependent cellular phagocyto-sis. To determine if these plasmablast-derivedEnv-specificmAbs persisted in long-livedplasma cells, bone marrow was evaluated ~7 months after immunization. Although ELISpot ofCD138+plasmacells(n=3)couldreadilydetectinfluenzaspecificIgGAbsecretingcells(ASCs),Env-specificIgGASCswerenotconvincinglydetectedbyELISpot.However,VHdeepsequencingofthebonemarrow(n=6)identifiedmultiplemembersofmAbclonallin-eages(n=19),ofwhich~47%werefoundinCD138+plasmacellcompartment.OurresultsindicatethatHIV-1Env-specificBcellsarepresentinlong-livedbonemarrowplasmacellrepertoire after vaccination in human, but may persist at very low frequency.

Presenter Bhrugu Yagnik1,2 121

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Title TherapeuticvaccinationagainstSIVwithorwithoutPD-1blockadegen-eratespolyfunctionalandbroadanti-viralCD8+TcellresponsesinSIVinfected rhesus macaques

Email byagnik@emory.eduCo-Authors Sheikh Abdul Rahman1,2, Alexander P. Bally1,2,KristenMorrow1,2,Gor-

dan J Freeman3,RafiAhmed1,2, Rama Rao Amara1,2

Affiliation 1.Division ofMicrobiology and Immunology, EmoryVaccineCentre,YerkesNationalPrimateResearchCentre,EmoryUniversity,Atlanta,Georgia, USA; 2.Department ofMicrobiology and Immunology, Em-orySchool ofMedicine, EmoryUniversity, Atlanta,Georgia,USA; 3.DepartmentofMedicalOncologyandCancerVaccineCenter,Dana-FarberCancerInstitute,Boston,Massachusetts,USA

AntiretroviralTherapy(ART)againstHIVhasbeensuccessfulinsuppressingviralburdenandimprovingthequalityoflifeforindividualswithHIV/AIDS.However,thehighcostofthistherapy poses challenges of its accessibility, especially in the developing countries. More-over, HAART fails to achieve a cure as virus rebounds immediately upon treatment cessa-tion, demanding a continual dependence on the continued therapy. Persistence of latent viral reservoirs during ART and immune dysfunction mediated by inhibitory immune checkpoint receptors are the two major factors that underlay the failure to clear the virus. Therapeutic interventions aiming to reduce the latent viral reservoirs and enhance antiviral immunity are criticalinachievingdurableremissionofHIV.Toachievethisgoal,wearecombiningthera-peuticCD40L-adjuvantedDNA/MVASIVvaccinevaccination,PD-1blockadewithARTinSIVmac239infectedrhesusmacaque.NineteenIndianrhesusmacaqueswereinfectedwithSIVmac239andtreatedwithART.TheseweredividedintothreegroupswhereinagroupofsixanimalsreceivedCD40L-adjuvantedDNA/MVASIVmac239vaccineandTLR7agonist,another group of seven animals, in addition to vaccine, received primatized anti-PD-1 Ab at the initiation of ART and during DNA vaccinations; and another group of six animals served asacontrolgroup.WeobservedthatPD-1infusionattheinitiationofARTresultedinin-creasedfrequenciesofSIV-specificCD8+Tcells.Therapeuticvaccinationinducedarobustpolyfunctional CD4 and CD8 T cells producing cytokines IFNg, TNFa and IL-2, and targeting anaverageof13CD4epitopesand7CD8epitopes.Theseresponsespersistedforlongerperiod in the PD-1 group. Moreover, vaccine induced CD8 T cells showed improved cytolytic potential depicted by granzyme B and perforin expression. In addition, we also observed increased frequencies ofCXCR5+SIV-specific aswell as totalCD8+T cells suggestinginductionofantiviralCD8+TcellswiththepotentialtohometoB-cellfolliclesandlimitHIVreplication in vivo. Currently, these animals are undergoing ART interruption to evaluate the influenceofvaccinationonSIVrebound.Wehypothesizethatthevaccineinducedpolyfunc-tionalSIVspecificCD8+TcellswillcontributetobluntingofreemergingviremiafollowingART interruption.

Presenter: Erin McLaughlin 122Title: Aproposedrole for Interleukin-6 in thesuppressionofTh2asthmatic

responsesEmail: emarie@uab.eduCo-authors: Beatriz LeónAffiliation: UniversityofAlabamaatBirmingham

TheincidenceofallergicasthmaintheUnitedStatesandotherindustrializedcountrieshasbeenincreasingoverthepastfewdecades.Oneproposedexplanationforthisincreaseisthe hygiene hypothesis, which in the context of asthma, states that a lack of early childhood exposuretohighlevelsofenvironmental lipopolysaccharide(LPS)endotoxinsfromgram-negativebacteria,increasesthesusceptibilitytodevelopingallergicdiseases.Ourlabhasrecently shown that LPS can indeed reduce Th2-driven, allergen-induced airway inflam-mation by inducing the up-regulation of the transcription factor T-bet in CD11b+ migratory dendritic cells, which we found was required to upregulate T-bet in responding CD4+ T cells. Furthermore, we and others have found that T-bet expression in T cells is required to pre-venttheTh2celldifferentiationprogram.SomeevidenceinthefieldsuggeststhatIL-12andIL-18 signaling synergizes to induce robust T-bet expression through the upregulation of

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IFN-γinTcells.Duetothesefindings,ourlabisnowinvestigatingtheroleofIL-12andIL-18 in the LPS-mediated suppression of Th2 cell responses. In addition, studies have shown thatinterleukin-6(IL-6)reducesTh2cellresponsesinmurineasthmamodels,butnoclearmechanism has yet been described. Here, we investigate the interplay of IL-12, IL-18, and IL-6inahousedustmite-inducedasthmamodelinC57BL/6mice.ThroughstudieswithIL-6knockoutandIL-6receptorknockoutmice,wehaveshownthatIL-6signalingupregulatestheexpressionoftheIL-18receptoronTcells,thusaffectingthesynergyofIL-18andIL-12toinduceT-betandpreventTh2responses.WeproposeamodelinwhichIL-6affectsTcellabilitytorespondtoIL-18andIL-12byinfluencingthethresholdsofthesecytokinesneededfor T cell responses.

Presenter: Deepti Maheshwari1 124 Title: Massive depletion of non-classical monocytes in systemic circulation

during dengue febrile illness. Email: deepti.m.3191@gmail.comCo-authors: KaustuvNayak1, Mohit Singla2,SivaramGunisetty1,6,GuruprasadMe-

digeshi3, Rakesh Lodha2,SushilKabra2,RafiAhmed4,5, Anmol Chan-dele1,Murali-KrishnaKaja1,4,5,6

Affiliation: 1 ICGEB-EmoryVaccineCentre,InternationalCentreforGeneticEngi-neering and Biotechnology, New Delhi, India. 2All India Institute of Medi-cal Sciences, New Delhi, India, 3Translational Health Science and Technology Institute, Faridabad, India, 4Department of Microbiology and Immunology,EmoryUniversitySchool ofMedicine,Atlanta,GA.5EmoryVaccineCenter,EmoryUniversitySchoolofMedicine,Atlanta,GA.6DepartmentofPediatrics,EmoryUniversitySchoolofMedicine,Atlanta,GA.

Monocytes in human blood circulation can be divided into three subpopulations according to the expression of themembrane receptor for lipopolysaccharide (CD14), and the lowaffinity FcγRIII-A receptor for the Fc region of IgG (CD16): CD14++CD16− (classical),CD14++CD16+ (intermediate),andCD14+CD16++ (non-classical).Thesemonocytesub-sets display distinct ligand sensing, tissue patrolling and cytokine secretion patterns under normal homeostatic conditions; and are thought to be potential targets for dengue viral in-fection.Whilethenonclassicalmonocytes,whicharealsooftencalledaspatrollingmono-cytes, are implicated in endothelial interactions, the intermediate monocyte subset is impli-cated in shaping the adaptive B cell response. Despite this wealth of information, very few studiesanalyzedtherelativeabundanceofthesedifferentmonocytesubsetsinhumanbloodduring dengue febrile infection in vivo. In this study we analyzed the frequency and number ofeachofthethreemonocytesubsetsindengueconfirmedfebrilepatientsfromIndia.Wefound that non-classical monocytes undergo a massive depletion whereas the intermedi-ate monocyte subset increases substantially. Similar trends were observed in patients with primary versus secondary infections and in patients with severe versus non severe disease. Taken together, these results suggest that massive changes occur in the relative abundance ofthemonocytesubsetsduringdenguefebrileillness;andthatthesechangesarereflectiveof innateresponseto febrile infectionrather thandifferencesmediatedbyprimaryversussecondary dengue infections.

Presenter: LuisEnriqueMuñoz 125Title: Metforminenhancestheefficacyoftumormembranevesicle-based vaccine immunotherapy in a murine model of triple negative breast cancer. Email: uis.munoz@emory.eduCo-Authors: Ramireddy Bommireddy, Haley Lei Huang, and Periasamy Selvaraj.Affiliation: EmoryUniversity

Breast cancer remains the most commonly diagnosed cancers among women, with over 250,000newcasesperyearintheUnitedStates.Immunotherapyhasemergedasapromis-

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ing treatment of breast cancer, however response rates to therapy remain far from ideal. In thisstudy,weinvestigatedthecombinatorialeffectoftumormembranevesicle(TMV)-basedvaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to suppress tumor growth with direct and indirect antitumoreffects.OurresultsshowthatTMVvaccination inhibitsprimarytumorgrowth inpreclinical tumor models and reduced spontaneous metastasis to the lungs. The combina-tion treatment with metformin further inhibited primary tumor growth and metastatic spread. TodissectthemechanismbehindthesynergybetweenTMVvaccinetherapyandmetformin,weprofiledtheexpressionofsurfaceinhibitorymarkersontumorsandobservedamarkeddecreaseinPD-L1expressionaftermetformintreatment.TodetermineitseffectonTcellsinfiltratingthetumorwelookedattumorCD8TcellsandfoundthattheTMVvaccineinducedamassiveincreaseinTcellinfiltration,whilethecombinationwithmetforminreducedthisinfiltrationdespitehavingbettercontroloftumorgrowth.Finally,welookedatthequalityoftheCD8TcellswithinthetumorbysurfacePD-1antTim3expressionaswellascytokineproductionand found thatcombination treatmentproducedmore terminaleffectorCD8Tcellsthatproducedmorepro-inflammatorycytokinescomparedtoTMVvaccinetreatmentalone. This suggests that one way metformin can suppress tumor growth is by reducing the inhibitionexertedonTcellsbytumorPD-L1andthussynergizingwithTMVvaccineimmu-notherapy.

Presenter: RachelMuir 126Title: Enhanced susceptibility to colitis upon exposure to TNF-naive micro-

biotaEmail: rqmuir@uab.edu Co-Authors: BarbaraJ.Klocke,CaseyMorrow,TrentonSchoebandCraigL.May-

nardAffiliation: UniversityofAlabamaatBirmingham

Inflammatoryboweldisease(IBD)encompassesseveralgastrointestinalinflammatorycon-ditions manifesting in susceptible individuals as a result of improper immune regulation of the gutmicrobiota.Anestimated3millionAmericanssufferfromIBD,forwhichthereisnocure.The exact conditions leading to the onset of this multifactorial disease remain poorly under-stood. To date, the most successful therapy is a neutralizing antibody to tumor necrosis fac-toralpha(TNFα),aproinflammatorycytokinethatisupregulatedinIBDpatients.However,30%ofpatientsareprimarynon-responders toanti-TNFα therapyandanadditional30 -40%ofpatientsloseresponsivenesswithinthefirstyearoftreatment.WehypothesizedthatTNFαsignalingisinvolvedinshapingthecompositionofthemicrobiotabothathomeostasisandduringintestinalinflammationtherebyimpactingsusceptibilitytoandchronicityof,mu-rine colitis. Dysregulated colonic IL-10 signaling is known to drive colitis in a microbiota de-pendent manner. Therefore, we employed a model based on transient administration of an IL-10receptorblockingantibody(anti-IL-10R) inotherwiseimmunocompetentmice.Upon16Ssequenceanalysisof fecalpellets,wehave found that themicrobiotaofanti-IL-10Rtreatedmiceareshiftedfromtheirpre-treated,homeostaticfecalmicrobiota,even30daysafterthelastanti-IL-10Rinjection.Anti-IL-10Rinducesmild,acutecolitis inwildtype(WT)miceandsevere,chroniccolitisinTNFdeficient(Tnf-/-)miceasdeterminedbyhistologicalanalysis,immunecellinfiltration,andfecallevelsoftheinflammationbiomarkerlipocalin-2.Uponco-housingTnf-/- andWTmice,anit-IL-10RtreatedWTmiceexhibitmoreseverecolitiscomparedtotheirseparatelyhousedanti-IL-10Rtreatedcounterparts.OurresultssuggestthatexposuretotheTNF-naïvemicrobiotaisunfavorablefortheWTmice,upondisruptionofcolonichomeostasis.Further,thissuggeststhatTNFαsignalingdoeshavearoleincon-ditioning the microbiota.

Presenter: Abraham J. Matar 127Title: Interruption of Notch Signaling via Blockade of Delta-like Ligands 1 and

4 Prevents Co-stimulation Blockade Resistant Allograft Rejection Email: Abraham.Jamil.Matar@emory.eduCo-authors: YingDong,BrendanP.Lovasik,DavidV.Mathews,CynthiaABreeden,

AbheekGhosh, Allison Stephenson,WilliamH. Kitchens, AndrewB.

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AdamsAffiliation: EmoryUniversity

Co-stimulationblockade(CoB)hasemergedasapromising immunosuppressionstrategywith the advent of belatacept, a novel CTLA4-Ig fusion protein that blocks CD28-mediated T cell co-stimulation. Belatacept confers long-term advantages in graft survival and function in renal transplant recipients compared to traditional calcineurin inhibitor-based immuno-suppressionbutisassociatedwithincreasedratesofearlyacuterejection.Usingamousemodel of MHC-mismatched skin transplantation, we investigated the role of Notch pathway inhibitionviablockadeofDelta-like ligands1and4 (Delta1/4)onCoB-resistantallograftrejection.InourmodelofBalb/CtoC57BL/6skintransplantation,combinedCoB(CTLA-4Ig+anti-CD154)andDelta1/4blockadesignificantlyprolongedskingraftsurvivalcomparedtoCoBalone(MST20daysvs.>83days,p=0.001**).Delta1/4blockadedidnotinhibitTcellproliferationinvivo,butinsteadinducedTcellapoptosis.Anti-donorIgGantibodywasalsosignificantly reduced in thecombined treatmentgroup.Wenextexamineddonor-specificTcellresponsesusingmOVAskingraftsinrecipientswhichreceivedanadoptivetransferofThy1.1+ovalbumin-specificOT-IT cells. CombinedCTLA-4IgandDelta1/4blockadesuppresseddonor-specificCD8+TcellaccumulationindraininglymphnodescomparedtoCTLA-4-Ig alone. Further, the combined blockade regimen suppressed both IFN-gamma andTNFproductionbydonor-specificCD8+Tcells.ThesedatademonstratethatDelta1/4blockadesuppressesdonor-specificTcell responses in thesettingofCoBandpreventsCoB-resistantrejection.WehaveidentifiedtheNotchsignalingpathwayasapromisingtar-get for future large animal and clinical studies of CoB-resistant rejection.

Presenter: Rasha Raheem Alkarkoushi 128Title: Indole-3-carbinolamelioratescolonicinflammationinDSS-treated,He-

licobacter muridarum-infected mice.Email: rashaa@email.sc.eduCo-authors: Ioulia Chatzistamou,UdaiP.Singh,MarpeBam,YvonneHui,Haider

Alrafas, Prakash Nagarkatti, Mitzi Nagarkatti and Traci L. TestermanAffiliation: UniversityofSouthCarolina/Schoolofmedicine/Pathology,Microbiol-

ogy, and Immunology

Enterohepatic Helicobacterspeciesareepidemiologicallylinkedtoincreasedinflammatorybowel disease; however, little research has been done to elucidate potential contributions of individualspecies.WehypothesizedthatHelicobacter muridarum (Hm)wouldalter thecourseofDSS-inducedcolitisandtheresponsetoindole-3-carbinol(I3C),ananti-inflam-matoryphytochemical.We treatedHm-infectedC57BL/6micewith1%DSS+/-40mg/kgI3Candmeasuredinflammatorybiomarkers.WefoundthatH. m exacerbated DSS-induced colitis and increased the percentage of Th17 cells in the mesenteric lymph nodes and spleen and increased IL-17 in colonic tissue compared to the DSS group. Also, we found that Hm bacteria itselfproduced inflammationandpathology. I3C,on theotherhand,amelioratedcolitis and shifted theTreg/Th17balance inDSS+H.m-infectedmice.We found that I3CtreatmentofDSS+H.m-infectedmicedecreasedtheexpressionofpro-inflammatoryIL17andRORCaswellasincreasedanti-inflammatoryFoxp3whencomparedtotheuntreatedgroup.ThedecreasedexpressionofRORCcorrelatedwithincreasedmiR-let7a-2andmiR-29a-3p expression and increased FoxP3 correlated with decreasedmiR-874 expressionfollowing I3C treatment.Moreover, I3C reduced the abundance of certain taxa, such asClostridiales, Actinobacteria, and Erysipelotrichales, and increased the abundance of Ru-minococcus. In summary, H. muridarum causes baseline inflammation and exacerbatescolitisviamicroRNAmediatedincreasesinTh17cells,whileI3Camelioratescolitisvia in-creasedTregpopulations.(SupportedinpartbyNIHgrantsR01AI123947,P01AT003961,P20GM103641,R01AT006888)

Presenter John E. Bradley 129Title GenerationandvalidationofBcelltetramerstoidentifyantigen-specific

B cells.Email johnbradley@uabmc.edu

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Co-authors John E. Bradley and Troy D. RandallAffiliation UniversityofAlabamaatBirmingham,DepartmentofMedicine,Division

of Clinical Immunology and Rheumatology

IndividualTandBcellsexpressuniqueantigenreceptorsthatallowthemtobindtospecificantigens. To identify T cells that recognize particular antigens, immunologists generated recombinant, biotinylated MHC class I and MHC class II molecules, loaded them with an antigenicpeptidesand“tetramerized”thembybindingtofluorescentlylabeledstreptavidin.Thesetetramersalloweddirectcharacterizationofantigen-specificTcellsbyflowcytometry.More recently, we have generated “B-cell tetramers”, which consist of recombinant proteins intheirnativeconfigurationexpressedwithbiotinylationsitestherebyallowingthemtobe“tetramerized”bybindingtofluorescentlylabeledstreptavidin.ThesetetramerslabelBcellsspecific forasingleantigenandallow theBcells tobecharacterizedbyflowcytometry.GeneratingMHC tetramersof different specificities involves swappingout antigenic pep-tides. However,generatingBcell tetramersofdifferentspecificitiesrequiresmakingnewandsometimesverydifferentproteins.Manyoftheantigensofinterestaresurfaceproteinson viruses or bacteria. Moreover, these proteins often bind to mammalian surface proteins or glycans as a way to mediate viral or bacterial entry into cells or evade immune function. Thus, one must be able to distinguish the ability of tetramers to bind BCRs from their ability to bind non-BCR proteins or glycans on the surface of cells. Here, we will show examples ofBcelltetramersandpresentstrategiestoovercomesomeofthedifficultiesencounteredwhen designing and validating B-cell tetramers.

130Presenters: Ariel Ley1, Doan C. Nguyen1, Celia Saney1, Chet Joyner1, Iñaki Sanz2,3, F. Eun-Hyung Lee1,3* Title In vitro maturation of early blood antibody secreting cells (ASCs) to

bone marrow LLPCsEmail f.e.lee@emory.edu Affiliation 1Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine,

2Division of Rheumatology, 3Lowance Center for Human Immunology, DepartmentofMedicine,EmoryUniversity,Atlanta,GA,USA

Long-livedplasmacells (LLPCs)are responsible for thesecretionand long-termmainte-nance of serum antibody levels in the absence of antigen re-stimulation. The generation of LLPCsisprimarilythoughttooccurasaresultofantibodysecretingcell(ASC)differentiationwithin a germinal center response or extrafollicular response; however, additional evidence suggests that further LLPC maturation can occur within the specialized niche of the bone marrow microenvironment. Previous studies showed that ASC survival and maintenance in vitro requires three bone marrow factors: hypoxia, secreted factors from mesenchymal stro-mal cells derived from human bone marrow, and APRIL, a TNF family member that binds to BMCAorTACIpossiblythroughSDC-1(CD138).Usinganin vitro cell-free BM microniche system,weobservedthematurationofbloodASCsincultureoverfourweeks.Wesimul-taneously measured antibody production, transcriptional changes and epigenetic changes. From ex vivo early minted blood ASC and BM LLPC, we demonstrate morphological dif-ferences that include: an increase in the number of mitochondria, condensation of nuclear chromatin, an increase in ER volume and an increase in the number of autophagosomes. Concordantly, we observe similar morphological changes from the population of early minted ASC that mature in the in vitro cell-free BM microniche. Evidence from both in vitro and ex vivo experiments indicate that there are a number of transcriptional and epigenetic programs that regulate the maturation of early blood ASCs within the bone marrow microniche and substantiates a role for ongoing maturation within the bone marrow.

Presenter AnusmitaSahoo 131Title SequenceandstructureguidedHIV-1CladeCimmunogendesignEmail anusmita.sahoo@emory.eduCo-Authors Anusmita Sahoo, Rama Rao AmaraAffiliation EmoryUniversity

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LimitednumberofCladeCHIV-1envelopeproteinshavebeenengineeredintostableim-munogensduetodifficulties in foldingtheenvelopeproteins intothenativetrimeric form.1086Cisonesuchenvelopeswhichislessstudied.Useofrecentstructureguidedstabilizingstrategiesonthisproteinyieldedeitheraggregatesoratrimericproteinwithlowaffinityforapex(V1V2)directedbroadlyneutralizingantibodies.AntibodiesdirectedagainstV1V2re-gionoftheenvelopehasbeenseentobeassociatedwithdecreasedriskofinfection(RV144trial),withmajorcontributionbytheV2hotspotdirectedantibodies.WeanalyzedtheCladeCsequencesandmutatedtheresiduesin1086Cwhichvariedfromtheconsensussequencesin the hotspot region. All possible mutants were generated with the residues changed to the dominant and sub-dominant residues in the consensus sequence at the selected positions. Twovariantswerescreenedwhichshowedsignificant improvement inbinding tomultipleV1V2directedbroadly neutralizing antibodies. Fewmodifications restored theactive sitebindingtooneoftheV1V2specificantibodies,whileotherssynergisticallyhelpedinfoldingtheproteinintobetternativeliketrimers.SequenceguidedmodificationsweretestedontoverywellcharacterizedBG505SOSIPandwerefoundtohaveaninfluenceonthefoldingoftheprotein.Singlesitevariantsat173positionwasseentoinfluencetheabilityoftheim-munogentomodulateandinfluencetheimmuneresponse.Twovariants(immunogens)at173positionyieldedsimilarantibodytitresagainstthesolubleenvelopeprotein.However,theydifferedsignificantlyintheirabilitytorecognizenativeenvelopeonthecellsurface.Thisobservation holds true when binding was monitored against cell surface expressed enve-lopesfromtheHIV-1globalpanel.Wearecurrentlylookingintothedifferencesineffectorfunctionsoftheseantibodiesgeneratedbytheabovediscussedtwovariantsat173position.Structural studies and further analyses will help understand the mechanism of modulation of 173positionontheimmuneresponse.Theseobservationswillbeimportantforengineeringenvelopes as immunogens to drive native envelope directed immune response.

Presenter: AshleyLanduyt 132Title: Cooperation between T-dependent antibodies and IL-10 reduces in-

flammatoryboweldiseasesusceptibilityE-mail: ashland@uab.eduCo-Authors: KlockeBJ1, Schoeb TR2, Maynard CL1

Affiliation: UniversityofAlabamaatBirmingham

Inflammatoryboweldiseases(IBD)arelifelongautoinflammatoryconditionscharacterizedbyloss of immune tolerance to gastrointestinal microbes. Currently, the impact of humoral im-munityonIBDislargelyundefined.Weemployedmousemodelssimulatingloss-of-functionmutations associated with IBD to study the role of T-dependent antibodies in the disease. In-ducibleTcellcostimulatorligand(ICOSL)isencodedbyanIBDsusceptibilitylocus.ItisalsoessentialforformationofT-dependentantibodies.WeobservedfewerIgG-producingcolonicplasma cells, reduced colon-draining lymph node germinal centers, and lower T-dependent IgGin Icosl-/- mice. Surprisingly, however, we detected more CD4 T cells producing IL-10 in the Icosl-/-colon. IL-10 isan immunosuppressivecytokine that, like ICOSL, isencodedbyanIBDsusceptibility locus.Wehypothesizedthat theelevatedIL-10production in theIcosl-/- colon compensates for insufficient T-dependent antibodies in preventing intestinalinflammation.WeinvestigatedtherelationshipbetweenIL-10andT-dependentantibodiesbygeneratingmicedeficientinCD4TcellderivedIL-10(Il10cKO)andICOSL.Icosl-/-Il10cKOpups demonstrated accelerated colitis prior to 4 weeks of age. This early-onset disease could be delayed by fostering Icosl-/-Il10cKO pups to wild type dams. This strongly implies that T-dependent antibodies, even conferred through milk, cooperate with IL-10 to protect fromcolitisinearlylife.Wetheninvestigatedwhetherthiscooperationwasrequiredinadult-hood.Forthis,wetemporarilyeliminatedIL-10producingcellsfromadultmice.Unlikewildtype mice, Icosl-/- mice depleted of IL-10 producing cells rapidly developed colitis. This sug-gests that T-dependent antibodies also maintain intestinal homeostasis during adulthood. As depletion of IL-10 producing cells from Iga-/- mice caused no disease, we determined that theprotectiveeffectwasnotfromT-dependentIgA.However,wediddeterminethatIcosl-/-micehavesignificantlyreducedIgGtowardcolonicmucosa-associatedmicrobes.TreatingIcosl-/-micewith antibiotics againstGram-positive or anaerobic commensals averted thecolitis upon depletion of IL-10 producing cells. Collectively, our data indicate T-dependent IgGtowardmucosa-associatedGram-positiveanaerobessynergizeswithIL-10tolimitsus-

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ceptibilitytointestinalinflammationthroughoutthelifespan.

Presenter: GlennMichaelLaMuragliaII 133Title: Selective CD28 Costimulation Blockade Exhibits Superior Control of

GerminalCenterResponsesThroughPreservation of theCoinhibitorCTLA-4

E-Mail: gmlamuraglia@emory.eduCo-Authors: Mandy L. Ford, I. Raul BadellAffiliation: EmoryTransplantCenter,AtlantaGA,USA

Costimulation blockade of the CD28 pathway with CTLA-4Ig has shown the ability to attenu-ateTcelldependentdonor-specificantibody(DSA)responsesintransplantation.However,CTLA-4Ig’smechanismofbindingligandsCD80/86indiscriminatelyblocksbothCD28co-stimulatory and CTLA-4 coinhibitory pathways, potentially depriving lymphocytes of critical coinhibition.WehavepreviouslydemonstratedthatselectiveCD28blockadeprovidesen-hancedinhibitionofTfollicularhelper(TFH),germinalcenter(GC)BcellandDSAresponsescompared to CTLA-4Ig following transplantation. TFHcellsdifferentiallyexpresssubstantialamounts of CTLA-4 in response to alloantigen and several studies have implicated CTLA-4 asakeyregulatoroftheirdifferentiationandfunction.Therefore,wehypothesizethattheenhanced DSA inhibition observed with selective CD28 blockade is dependent upon pres-ervation of critical CTLA-4 coinhibitory activity. Thus, we utilized a full MHC mismatched BALB/ctoB6skinallograftmodeltodeterminewhetherimprovedinhibitionoftheTFH cell-mediated alloresponse with selective CD28 blockade is CTLA-4 dependent. Recipients re-ceivedanti-CD28domainantibody(dAb)+/-anti-CTLA-4foranalysisofTFH cell-mediated re-sponses and DSA formation. Selective CD28 blockade in the presence of CTLA-4 reversed the inhibition of DSA formation exhibited by anti-CD28 blockade alone with the development of anti-donor antibodies post-transplant. Flow cytometric analysis of graft-draining lymph nodes following transplantation revealedsignificantdevelopmentofTFH(3.35%vs.0.46%),GC-TFH(36%vs.25%),GCB(63%vs.9.7%)andplasmacell(5.3%vs.4%)responsesinCD28dAb+anti-CTLA-4treatedmicecomparedtoanti-CD28monotherapy.TodeterminewhetherthedependenceofCTLA-4isspecifictocognateTFH:B cell interactions and TFH cell intrin-sic, we performed ex vivo TFH:B cell co-cultures in the presence of anti-CD28 dAb with and withoutanti-CTLA-4.CD28dAb-mediatedinhibitionofBcellproliferationanddifferentiationintoIgG+GL7+GC-likeBcells,andIgGantibodyproductionwerereversedwithanti-CTLA-4treatment. Additionally, anti-CTLA-4-mediated reversal was observed when T cells alone (withoutBcells)wereexposedtoanti-CTLA-4.Thus,thesedataindicatethatselectiveCD28blockade-mediatedinhibitionofthealloimmuneGCresponseisCTLA-4dependentandTFH cell intrinsic, supporting the continued development of CD28 blockade-based immunosup-pressivestrategiesthatpreserveCTLA-4coinhibitoryactivitytoimprovecontrolofGCre-sponses and DSA formation following transplantation.

Presenter: SeleneMeza-Perez 134Title: Intestinal microbiota alters omental tumor growthEmail: smeza@uab.eduCo-authors: Aaron Silva-Sanchez, Mingyong Liu, and Troy D. RandallAffiliation: UniversityofAlabamaatBirmingham

Theomentumisanadiposetissuethatcontainsmilkyspots(MS).TheMSaresimilar tosecondary lymphoid organs and can generate B and T cell immune responses to peritoneal antigens. Moreover, the omentum also collects metastasizing tumor cells in the peritoneal cavity and tumors growing in the omentum are associated with poor clinical prognosis. Here

weshowthatintestinalmicrobiotaaffectsTregsactivationprofileandimpairstumorgrowthinomentum.Ourtransplantabletumormodelinwildtype(WT)miceshowsthatperitonealtumorsgrowprogressively in theomentumandperitonealcavity inWTmice,whereas ingermfree(GF)andantibiotictreatedmicetumorsdonotgrow.OmentaltumorgrowthinWTmice is associated with an increase in PD-1+Tregs,whiletumor-specificCD8+ T are reduced. However,inGFmicenumbersofPD1+TregsremainunalteredbuttumorspecificCD8+ T cellsarestillpresent.Afterco-housedGFmicewithWTmice,ex-GFmiceshowedtumor

77

growth and restore the increase in PD-1+Tregswith lownumberofspecificCD8+ T cells. These data suggest that intestinal microbiota has a role in omental Treg activation and in the tolerance to peritoneal tumors.

Presenter Saba Tufail1,2 135Title Ovalbuminself-assemblesintoamyloidnanosheetsthatelicitimmune

responses and facilitate sustained drug releaseEmail sabatufail12@gmail.comCo-authors Mohd. Asif Sherwani3, Shoaib Shoaib1, Sarfuddin Azmi1, Najmul Islam1

Affiliation 1Department of Biochemistry, Faculty of Medicine, Jawaharlal Nehru MedicalCollege,AligarhMuslimUniversity,Aligarh,U.P.-202002, IN-DIA; 2Department of Biochemistry, Faculty of Life Sciences, Aligarh MuslimUniversity,Aligarh,U.P.-202002, INDIA; 3Department of Der-matology,UniversityofAlabamaatBirmingham,AL-35294,USA.

Amyloidfibrilsareassociatedwithmanyneurodegenerativediseases,motivatinginvestiga-tions into theirstructureand function.Althoughnot linked toaspecificdisease,albuminshavebeenreportedtoformmanystructuralaggregates.Wewereinterestedininvestigatinghost immune responses to amyloid fibrils assembled from themodel protein ovalbumin.Surprisingly, upon subjecting ovalbumin to standard denaturing conditions, we encountered giant protein nanosheets harbouring amyloid-like features and hypothesized that these nanosheetsmighthavepotential inclinicalor therapeuticapplications.Wefound that thenanosheets, without the administration of any additional adjuvant, evoked a strong antibody response in mice that was higher than that observed for native ovalbumin. This suggests that amyloid nanosheets have self-adjuvanting property. The nanosheet-induced immune responsewashelperTcell2(Th2)biasedandnegligiblyinflammatory.Whiletestingwheth-er the nanosheets might form depots for the sustained release of precursor proteins, we did observe release of ovalbumin that mimicked the conformation of native protein. Moreover, the nanosheets could load the anticancer drug doxorubicin and release it in a slow and sustained manner. Taken together, our results suggest that amyloid nanosheets should be further investigated as either an antigen delivery vehicle or as a multifunctional antigen and drug co-delivery system, with potential applications in simultaneous immunotherapy and chemotherapy.

Presenter MelissaWolf 136Title FineNeedleAspirationBasedImmuneOrganoidsRecapitulateTMEin

Thyroid CancerEmail Melissa.m.wolf.1@vanderbilt.eduCo-Authors MelissaM.Wolf,BradI.Reinfield,MatthewZ.Madden,W.KimRath-

mell,VivianL.WeissAffiliation VanderbiltUniversity

Replicationofthetumormicroenvironment(TME)isamajorlimitationinorganoiddevelop-ment as a model system for cancer research. Studies aimed at understanding the TME are typicallyperformed inanoversimplified2Dcellculture,orutilizingcomplexmousemodelsystems. Not only are in vivomousestudiestimeconsumingandcostly,significantdiffer-ences in the mouse immune system and TME may hinder the applicability of the results in a translationalparadigm.Here,weshow thathumanspecimensobtainedviafineneedleaspiration(FNA)fromcancerousthyroidandrenalcelltumorscangrowandbemaintainedas immune organoids harboring a diverse lymphoid and myeloid population in a matrigel-based culture model. This system provides several advantages over cocultured cell lines with primary immune cells. In our FNA based method, the immune cell population obtained is obtained directly from the TME and therefore may be more representative of a tumor-as-sociated exhaustive and/or regulatory immune phenotype. In addition, these studies provide aframeworkforfuturedrugdevelopmentandallowfortheevaluationofa3Dorganizationoftumorcellswithimmunecellsthatcouldrecapitulatephysiologicalcancerprocesses.Ulti-mately, there is a translational gap between 2D cell culture and animal model testing; there-fore,evaluationofthe3DorganizationoftumorcellswithinanidealTMEcouldrecapitulate

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physiological cancer processes.

Presenter KevinS.Cashman 137Title Multiple facets of RNA sensing is human systemic lupus erythematosus

B cells.Email kevin.cashman@emory.edu Co-Authors ZoeSimon,RachelH.Lee,DavidShon,KiraE.Smith,VivienWarren,

Scott A. Jenks, F. Eun-Hyung Lee, Ignacio SanzAffiliation EmoryUniversity Anti-nucleotideautoantibodies(e.g.anti-dsDNA)areadefiningserologicalhallmarkforsys-temic lupuserythematosus (SLE),with~70%orhigherofpatientsexhibiting thisspecificautoreactivity. Additionally, loss of self-tolerance is also preeminent towards RNA binding proteins/RNA:protein complexes such as Ro antigens, Smith antigens, or ribonucleopro-teins. Recent evidence from our group suggests that anti-RNA nucleotide responses are significantlyelevated inSLEpatientswith lupusnephritis,andequallyas interesting, thatSLEpatientsexhibitinganenrichmentof effectorB cell populations suchas theCD19hiCD27- IgD-CD21-CD11c+DN2Bcell anda closely relatedantigenexperiencednaïvepopulation, have higher anti-RNA titers. Little is known about the concurrence of anti-DNA and anti-RNA responses in the context of SLE, leading to the overarching question of wheth-er anti-nucleotide responses are mutually exclusive or dependent on one another in the contextofSLE.WithourpreviousfindingthatDN2Bcellsarehyper-responsivetoRNAcontaining TLR agonists such as R848, and lead to a serological enrichment in anti-RNA, anti-Sm, and anti-RNP autoreactivities in SLE patients, it would suggest the potential of exclusive nucleotide activation pathways which could lead to improved targeted therapies in thisheterogenousdisorder.Hereweshow,thatthereisasignificantelevationinanti-ssRNAtiters in SLE versus healthy individuals. Additionally, this did correspond to an enrichment in DN2 and the clinical presentation of lupus nephritis. Both anti-dsDNA and anti-ssRNA responsescorrelatedwellwiththepresentationoftheother,however,therewasastratifica-tion of patient groups which included a patient population with strong anti-ssRNA responses but little-to-no anti-dsDNA titers. Patients with the highest titers of anti-ssRNA antibodies werealsothepatientswiththehighesttiterstowardsdsRNA(e.g.polyI:C),suggestingthepotential of a virally derived disease etiology. Taken together these data initially suggest that anti-RNA autoreactivity can be mutually exclusive in SLE patients, and this may represent a populationofpatientswhoseclinicalfeaturesaredrivenbyaspecificenvironmentaltriggersuch as a viral infection.

Presenter AshleyConnelly 138Title IdentificationandCharacterizationofNovelHumanNeutrophilSubsets

inInflammation-InducedPathogenesis.Email acon@uab.eduCo-Authors KrystleOng,MarcusDavis,HarishPal,ValeriyaKuznetsova,Richard

Huijbregts,ChristianFay,AshishDhyani,E.TurnerOverton,ZdenekHel

Affiliation UniversityofAlabamaatBirmingham

Despitesuccessfulviralcontrol,HIV-1-infectedindividualsmaintainanincreasedriskoflife-threatening conditions including cardiovascular, liver, and other end-organ diseases. This excessrisk isassociatedwithchronic inflammationand immunedysregulation.However,thespecificmediatorsandmechanismsdrivingthesepathologiesarenotyetunderstood.Neutrophils, the most abundant leukocytes, are increasingly recognized as a heterogeneous population with critical roles in both immune regulation and disease pathogenesis. Compre-hensive characterization of neutrophils and neutrophil subsets is hampered by an inherent instability of neutrophils ex vivo, their tendency to form multiplets with other cell types, and highnon-specificstainingduetothereleaseofcationicgranularproteinsfollowingneutrophilactivation. Novel methods of neutrophil characterization developed in our laboratory have facilitatedtheidentificationofseveralnovelneutrophilsubpopulationsincludingtwosubsetsoflow-densityneutrophils(LDNs).LDNsareknowntobeexpandedinmanyinflammatory

79

conditions including autoimmune diseases, trauma, cancer, and chronic infections. The LDN subpopulationsidentifiedinourlaboratory(CD16HighCD64LowandCD16-CD64High)havedis-tinctphenotypicandfunctionalpropertiesthatlikelycontributetoinflammationanddiseaseprogression.WepresentdatademonstratingthatHIV-1/AIDSisassociatedwithsignificantalterations in the phenotype, metabolism, and gene expression of total neutrophils as well as anelevatedfrequencyofCD16-CD64HighLDNsubset.Observedchangesinneutrophilpopu-lationscorrelatewithkeyclinicalparametersincludingCD4+Tcellcount,yearsofexposuretoanti-retroviraltherapy,andlevelofliverfibrosis,akeyriskfactorforliverdiseasedevelop-ment.Overall, thepresenteddata indicateacritical roleof thenewly identifiedneutrophilsubsetsinthepathogenesisofHIV-1/AIDSandpotentiallyotherinflammatorydiseases.

Presenter DeepakTomar 139Title Highthroughputsingle-cellrepertoireprofilingofBcellreceptorinSLE

subjects with lupus nephritisEmail dtomar@emory.eduCo-Authors JenniferHom,ChristopherTipton,MatthewWoodruff,ScottJenks,Ig-

nacio SanzAffiliation EmoryUniversitySystemiclupuserythematosus(SLE)isasystemicautoimmunediseaseinwhichfaultyBcelltolerancepromotesmultipleautoantibodieswithhighdiseasespecificityandclinicalin-volvementinmultipleorgansystems,includingthekidney(lupusnephritis).IndividualswithSLE have elevated levels of Abs encoded by the VH4-34 IgH gene which can be detected bythe9G4idiotype-specificmAb.Inlupusnephritissubjects,generationofpathogenicau-toantibodies results in tissuedamageeither through inflammatory immune complexesorby in situ binding to the tissue antigens. In-depth analysis of the molecular and antigenic propertiesofdifferentBcellsubsets,whichcancontribute to this tissue-basedresponse,is critical for our understanding of SLE and end-organ manifestations. To address this, we used a combination of an ultra high throughput methodology for linking the B cell receptor heavyandlightchainvariableregion(VHandVL)onasingle-celllevel,coupledwithnextgenerationsequencing(NGS)foranalyzingtheBcellrepertoirefromSLEpatients.Plasma-blasts(CD19+IgD-CD27highCD38highCD138neg),switchedmemory(CD19+IgD-CD27+),dou-ble negative (CD19+IgD-CD27-CXCR5-), activated naïve (CD19+IgD+CD27- CD21-CD24-

CD23-)andrestingnaïve: (CD19+IgD+CD27-)Bcells fromSLEpatientswereflowsorted,withsubsequentVHandVLtranscriptlinkageusingemulsionRT-PCR.IgH,IgLandlinkedtranscripts from blood, kidney biopsies and urine B cells were sequenced using the Illumina Miseq platform. Analysis of B cells from lupus nephritis patients showed a highly polyclonal repertoire of all subsets analyzed in the blood, kidney and urine compartments. B cells in the kidney displayed higher average mutation rate as compared to B cells in the blood compart-ment.SomeoftheexpandedBcelllineagespersistedoveraperiodof3yearsinthebloodand were found to be interconnected between the blood, kidney and urine compartments, alongwithsubstantialclonalexpansionsoftheSLE-associatedVH4-34clones.Monoclonalantibodies are currently being made from these expanded lineages to test for their auto-reactivityprofiles,andnephritogenicantigenbindingcapacity.

Presenter Jacob Files 140 Title Impact of predicted CD4 T-cell adaptation on vaccine-induced immune

responsesinHIV-1vaccinesEmail jkfiles@uab.eduCo-Authors Sarah Sterrett, Courtney Mangum, Tim Fram, Nathan Erdmann, Anju

Bansal,PaulGoepfertAffiliation UniversityofAlabamaatBirmingham,DepartmentofMedicine

Background:OurgrouphaspreviouslyusedHLA-IIassociatedHIVpolymorphismstopre-dictCD4T-cellescapeinHIV.InbothacuteandchronicHIVinfection,theadaptedepitopes(AE)ofthesepolymorphismselicitedweakerCD4immuneresponsesthanthecorrespond-ingnon-adaptedepitopes(NAE).TheseresultssuggestthatCD4Tcellsexertimmunepres-

80

sureonHIVduringinfection.SinceCD4AEareencodedbyallcurrentclinicalHIVvaccineinserts, evaluation of their immunogenicity is essential in order to optimize future vaccine design.Methods: 88 human PBMC samples were obtained, including both vaccine recipients and placebocontrols,fromthreedifferentHIV-1vaccinetrials(40samplesfromHVTN106,24fromHVTN502, 24 fromHVTN505). UsingHLA-II binding prediction programs, epitopescontaining the HLA-II associated polymorphic sites were designed that fully matched each respectivevaccineimmunogensequence.Epitopeswerethendividedintosite-specificpoolscontaining2-5peptides,aswellasintolargerAEandNAEpools.Immunogenicitywasthentested using a CD8-depleted IFNgELISpotassay(n=88),aswellasthemoresensitiveflow-basedactivationinducedmarker(AIM)assay(n=33)toidentifyantigenspecificCD4Tcells.Results: In the88vaccinesamples,we identified14 IFNg ELISpot responses to the site-specificpools.Inaddition,wesaw10positiveNAEpoolELISpotresponses,whileweonlyidentified1positiveAEpoolresponse(p=0.013).Outofthe33individualstested,therewasa trend towards more AIM responses to epitopes within the NAE pool, with 12 NAE pool responsesandonly5AEpoolresponses(p=0.09).ThefrequencyofOx40andPDL1co-expressiontoNAEpoolwasincreasedincomparisontotheAEpool(p=0.008).Conclusions:OurinitialdatademonstratethatCD4AEcontainedwithinpasthumanHIV-1vaccine trialsarepoorly immunogenic,suggesting thatvaccine insertscouldbemodifiedto enhance CD4 responses. Such optimization strategies could thereby enhance vaccine-induced antibodies which are felt to be essential for vaccines designed to prevent infection.

Presenter: Sung Hoon 141Title: Hypoxia-InducibleFactors(HIF) inCD4+ T cells promote metabolism,

switch cytokine secretion, and T cell help in humoral immunity Email: sunghoon.cho@vanderbilt.eduCo-authors: ArielL.Raybuck,EdnaKemboi,andMarkR.BoothbyAffiliation: DepartmentofPathology,Microbiology,andImmunology,Vanderbilt

UniversityMedicalCenter

T cell help in humoral immunity includes interactions of B cells with activated extrafollicu-lar CD4+andfollicularThelper(Tfh)cells.EachcanpromoteantibodyresponsesbutTfhcellsplaycritical rolesduringgerminalcenter (GC) reactions.After re-stimulationof theirantigen receptor (TCR)byBcells,helperTcellsactonBcellsviaCD40 ligandandse-creted cytokines that guide immunoglobulin class switching. Hypoxia is a normal feature ofGC,raisingquestionsaboutmolecularmechanismsgoverningtherelationshipbetweenhypoxia response mechanisms and T cell help to antibody responses. Hypoxia-inducible factors(HIF)areprominentamongmechanismsthatmediatecellularresponsestolimitedoxygenbutalsoareinducedbylymphocyteactivation.WenowshowthatlossofHIFinCD4+ T cells compromised essential functions in help during antibody responses. HIF-1a depletion from CD4+Tcellsreducedfrequenciesofantigen-specificGCBcells,Tfhcells,andoverallantigen-specificAb.Further,HIFpromotedCD40LexpressionwhilerestrainingtheFoxP3-positive CD4+cellsintheCXCR5+follicularregulatory(Tfr)population.GlycolysisincreasesT helper cytokine expression, and HIF promoted glycolysis in T helper cells via TCR or cyto-kine stimulation, as well as their production of cytokines that direct antibody class switching. Indeed, interferon-gelaborationbyHIF-deficient invivo-generatedTfhcellswasimpaired.Collectively, the results indicate that HIF transcription factors are vital components of the mechanisms of help during humoral responses.

Presenter Luke Postoak 142Title ThymicepithelialcellsrequiretheclassIIIPI3KVps34forhomeostasis

and CD4 T lymphocyte selection Email joshua.l.postoak@vanderbilt.eduCo-Authors Shiyun Xiao2,GuanYang1,LanWu1, Jinhua Zhang3, Nancy Manley2,

LucVanKaer1Affiliation 1VanderbiltUniversity,2UniversityofGeorgia,3UniversityofAlabamaat

Birmingham

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The generation of a functional, self-tolerant T lymphocyte receptor repertoire critically de-pends upon interactions between developing thymocytes and antigen-presenting cells (APCs)inthethymus.TheselectionofTlymphocytesoccursintwosteps.Inthefirst,thy-mocytes that have successfully rearranged their antigen receptor and weakly interact with self-peptide/MHConcorticalthymicepithelialcells(cTECs)receivepro-survivalsignals.Inthesecondstep,positively-selectedthymocytestraffictothemedullawherethymocytesthatstronglyinteractwithmedullarythymicepithelialcells(mTECs)ordendriticcellsareclon-ally deleted or induced into a regulatory program. Interestingly, in addition to ubiquitously expressed antigen, a subset of mTECs express the transcriptional regulator AIRE, which mediatesectopicexpressionandpresentationoftissue-specificantigens.Thepresentationof self-antigens to developing thymocytes involves a variety of cellular processes, including proteolysis,endocytosis,vesicletrafficking,andautophagy.Inourcurrentstudy,wefocusontheclassIIIPI3K,Vps34,whichhasbeenimplicatedinautophagyandrelatedprocesses.ToaddressthecontributionofVps34toTECfunction,wehavegeneratedmicewithaTEC-specificVps34deficiency.WefounddefectsinthehomeostasisofTECsintheseanimalsespecially in mTECS. Additionally, when TCR transgenic lines were bred to these animals we found profound defects in the positive selection of the CD4 T lymphocyte lineage but not the CD8 T lymphocyte lineage. In future studies, we will further characterize the cellular processesmediatedbyVps34thatcontributethenoteddefectsinTECsurvival.Also,wewillassessthecontributionofVps34tonegativeselectionbyutilizingtheOTII/Rip-mOVAmodelinmicethathaveaninducibledeletionofVps34inAIRE-expressingmTECs.Collectively,thesestudiesmayidentifyaroleforthePI3KVps34inmaintainingTEChomeostasisandcontributing to the repertoire of selecting ligands processed and presented by TECs.

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Abad MariaAbdelhamid LeilaAbreu RodrigoAllie RameezaAnderson AshlynAu-Yeung ByronBasso PauloBrock RebeccaCollins MatthewCooney KimberlyCottam MatthewCrepeau RebeccaCrofts KaliDunbar ZerickDunbar PaulDyevoich AllisonEdupuganti SrilathaEnriquez AnaGeorge-Alexander Lou-EllaGetzler AdamHaines RobertHegner CourtneyJacobse JustinJenkins MeaganJimenez RachelKania AnnaKirchner StephenKlopfenstein NathanLi Zheng-RongLiu MingyongLopez LaceyMadden MatthewMclaughlin TarynMelo PauloMelot Logan Mirlekar BhalchandraMisumi IchiroMohammed Zahraa Mosure SarahMurji AmynNavarrete KarlaNeeld DennisNguyen DoanOpsteen SkyePatterson DillonPeel JessicaPham LyRaddatz MichaelRead KaitlinRen JingjingRichardson ShakyraRiegler AshleighRolader Robin Sancho JaimeSautto GiuseppeAndreaSchonhoff Aubrey

INDEXOFPOSTERNUMBERS-SESSION1

Last Name First Name Poster Number

70446

1824

731452543493047

96955

2321436133563232012342265373868505651111067612662544053

1216466425604648413

27

83

Shanmugasundaram UmaShi ZhendaShiakolas Andrea Silva Sanchez AaronSponaugle AlexisSpurrier ArielSwartwout BriannaThaxton JessicaTibbs TaylorTsuda ShanelVanwinkle PeytonXue GangYang GuanZumaquero-Martinez Esther C.

INDEXOFPOSTERNUMBERS-SESSION2 Last Name First Name Poster Number

332957585259

4193928

8151617

10483

12898

120129

799784137

91105141

8013810685

140111103

8110796

10095

1017899133132130

7476

Abdelnabi AliAggarwal CharuAlkarkoushi RashaAppleton BrennaBasu MadhubantiBradley JohnBreuer DenitraButrico CaseyCabana-Puig XavierCashman KevinChen WeirongChen Mei LanCho Sung HoonClemens EleneConnelly AshleyDale GordonDulson SarahFiles JacobFranchitti LaviniaFrost ElizabethGonzales MarissaHabib JakobHarris LevelleHathaway-Schrader JessicaHayward SarahHolbrook BethJenks ScottKoehler HeatherLaMuragliaIi G.MichaelLanduyt AshleyLey ArielLindsay RobinLulu Amanda

INDEXOFPOSTERNUMBERS-SESSION1(continued)

Last Name First Name Poster Number

84

12492

12711312211675134109

89126125

947188

11090

102142119117

771311141159386

112828713913510873

118136121

72

Maheshwari DeeptiMasjedi ShirinMatar AbrahamMcguire DonaldMclaughlin ErinMcletchie ShawnaMelssen MaritMeza Perez SeleneMorris AnnaMorrison HollyMuir RachelMunoz LuisNellore AnomaNorlander AllisonOng KrystlePeek ChrisPerez MildredPetronglo JennaPostoak JoshuaReinfeld BradRisley ChrisRodriguez AnthonySahoo AnusmitaSalina AnaShannon JessicaShartouny JessicaStampfer SamuelStephenson AllisonStevens AaronSugiura AyakaTomar DeepakTufail SabaWare MichaelWinn NathanWolabaugh AmberWolf MelissaYagnik BhruguZhu Jing

INDEXOFPOSTERNUMBERS-SESSION2(continued)

Last Name First Name Poster Number

85

NOTES:

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Cover Art Description and Credit:

“Wholemountisolationofmurineolfactoryepithelialtissuerevealshematopoieticcells(red,CD45)thatinteractwiththeaxontracksofolfactorysensoryneurons(green,OMP-GFP).These immune cells are essential for barrier defense of the brain and help maintain tissue homeostasis in the nose.”

ImagecourtesyofSebastianWellford,DukeUniversity.

ViewAbstractsusingQRcodeor visit

http://sis.emory.edu/documents/SIS-Abstracts-2019.pdf

For a full version of the SIS book visithttp://sis.emory.edu/documents/SIS-book-2019.pdf