Adaptive Nanoparticle Platforms for High Throughput Expansion and Detection of Antigen-Specific T cells - Hickey et al. - Unknown - Unkn

《Adaptive Nanoparticle Platforms for High Throughput Expansion and Detection of Antigen-Specific T cells - Hickey et al. - Unknown - Unkn》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

SUPPORTINGINFORMATIONAdaptiveNanoparticlePlatformsforHighThroughputExpansionandDetectionofAntigen-SpecificTcellsJohnW.Hickeya,b,c,d,e,#,ArielIssera,b,c,#,SebastianF.Salathef,KaylaM.Geea,Meng-HsuanHsiaog,WasamahShaikhc,NkechiC.Uzoukwub,c,JoanGlickBielerb,c,Hai-QuanMaoa,d,e,i,andJonathanP.Schneckb,c,j,*aDepartmentofBiomedicalEngineering,SchoolofMedicine,bInstituteforCellEngineering,SchoolofMedicine,cDepartmentofPathology,SchoolofMedicine,dTranslationalTissueEngineeringCenter,eInstituteforNanoBioTechnology,fDepartmentofBiology,KriegerSchoolofArtsandSciences,gDepartmentofPathobiology,SchoolofMedicine,iDepartmentofMaterialsScienceandEngineering,WhitingSchoolofEngineering,jDepartmentofMedicine,SchoolofMedicine,JohnsHopkinsUniversity,Baltimore,MD,USA#Denotesco-firstauthorship*Correspondenceshouldbeaddressedto:JonathanP.Schneck:733N.Broadway,BRB639,DepartmentofPathology,SchoolofMedicine,JohnsHopkinsUniversity,Baltimore,MD21205,USA.Email:jschnec1@jhmi.edu,Phone:410-614-4589.S1

1SUPPLEMENTALFIGURES1-13S2

2SupplementalFigure1:Enrichingandexpandingrareantigen-specificTcellpopulationsdirectlyfromsplenocytesandcomparingtostartingfrompurifiedCD8+Tcellpopulations.(a)Schematicofexperimentalsetupforcomparingdifferentstartingpopulations(splenocytevs.purifiedCD8+Tcells).Wedividedharvestedsplenocytesintotwoequalparts:onepopulationthatwentthroughastepforCD8+Tcellisolationandtheotherthatdidnot.Enrichingfromsplenocytesdoesnotalterantigen-specific(b)phenotypeor(c)cytokineproductiononday7(errorbarsshows.e.m.;nsp>0.05,n=3,one-wayANOVAwithTukey’sposttest).(d-e)Enhancementsinenrichmentandexpansionofantigen-specificCD8+Tcellsfromsplenocytestartingpopulationsdonotcomefromincreasesinlevelsoffoldenrichmentorpercentcellrecoveryofantigen-specificTcellsonday0.Dopingfluorescently-labeled(CFSE)antigen-specificCD8+Tcells(2CorPMELCD8+Tcells)at(1:104)inendogenoussplenocytesallowcomparisonof(e)foldenrichmentand(e)percentcellrecoveryof50nmaAPCsthatarenotdifferentfromfoldenrichmentandcellrecoveryinCD8+Tcellpopulations(errorbarsshows.e.m.;n=5).(f)Percentand(g)numberofantigen-specificTcells(TRP2+)resultingfromaAPCenrichmentandexpansiontwodifferentstartingpopulationsofcells(CD8+Tcellpurified,splenocytes)atday7(errorbarsshows.e.m.;*p<0.05,n=4,Student’st-test,two-tailed).S3

3SupplementalFigure2:Importanceofanti-CD28forenrichmentandexpansionofCD8+Tcellsfromsplenocytes.(a)Percentageand(b)numberofKbSIYspecificCD8+TexpandedwithKbSIY+anti-CD28vs.KbSIY-onlyaAPCs(errorbarsrepresents.e.m.;*p<0.05,n=3–4,Student’st-test,two-tailed).S4

4SupplementalFigure3:ContributionofendogenousantigenpresentingcellstoenhancedoutputfromSplenocyteE+E.(a)Percentand(b)Numberofantigen-specificTcellsresultingfromaAPCenrichmentandexpansionofsplenocytesdepletedofBcells(CD19+)macrophages(F4/80+)anddendriticcells(CD11c+)(errorbarsshows.e.m.;*p<0.05,n=3–4,one-wayANOVA).S5

5SupplementalFigure4:UnderstandingthecontributionofCD4+Tcellsinenhancingantigen-specificCD8+Tcellactivation(a)Comparisonstainingofpopulationsofsplenocytes,PanTcells,CD8+isolation,andCD4+depletionusedforE+Eexperiments.(b)Percentofantigen-specificTcellsresultingfromaAPCenrichmentandexpansionfromtwodifferentstartingpopulationsofcells(CD8+Tcellpurified,PanTcellpurified)onday7(errorbarsshows.e.m.;**p<0.01,n=4,Student’st-test,two-tailed).S6

6SupplementalFigure5:Establishingtheproperdoseof300nmaAPCstousetoenrichantigen-specificTcells.(a-b)Dopingantigen-specificCD8+Tcellsat(1:104)inendogenoussplenocytesallowcomparisonof(a)foldenrichmentand(b)percentcellrecoveryof300-nmaAPCsatdifferentratiosofaAPCstocells(errorbarsshows.e.m.;*p<0.05,**p<0.01,***p<0.001,n=5,one-wayANOVAwithTukey’sposttest).(c)BydopinginThy1.1+,transgenicPMELCD8+TcellsintoThy1.2+miceata1:1000ratio,wedetermineeffectiveaAPC:CellratiosneededforS7

7thenewenrichmentandexpansionprotocol(errorbarsshows.e.m.;*p<0.05,n=5,one-wayANOVAwithTukey’sposttest).(d)Comparingantigen-specificTcellfrequencyonday7froma96-wellplateformatstartingpopulationofsplenocytesorpurifiedCD8+Tcells(errorbarsshows.e.m.;**p<0.01,n=8,Student’st-test,two-tailed).S8

8SupplementalFigure6:Schematicforcomparingexperimentalset-upforcomparingbatchedtoindividualantigen-specificCD8+Tcellenrichmentandexpansions.S9

9SupplementalFigure7:Titrationofdetectionbead:cellratiostoevaluateoptimalstainingconcentrationforstainingantigen-specificTcellsonday7oftheenrichmentandexpansionprotocolwithalowfinalpercentageofantigen-specificTcells.(a)Flowcytometryplotsofpeptide-loadedAdaptiveaAPCs(Adaptive+Peptides)andunloaded(Adaptive-Peptides)detectionbeads(b)Percentageofcontrolstaining(Adaptive-Peptide/non-cognate)weresubtractedtoevaluatefinalpercentageofantigen-specificTcellsonday7andcomparetotraditionalbiotinylateddimerstainingreagents.S10

10SupplementalFigure8:Titrationofdetectionbead:cellratiostoevaluateoptimalstainingconcentrationforstainingantigen-specificTcellsonday7oftheenrichmentandexpansionprotocolwithanintermediatefinalpercentageofantigen-specificTcells.(a)Flowcytometryplotsofbothpeptide-loadedAdaptiveaAPCs(Adaptive+Peptides)andunloadedaAPCs(Adaptive-Peptides)detectionbeads(b)Percentageofcontrolstaining(Adaptive-Peptide/non-cognate)weresubtractedtoevaluatefinalpercentageofantigen-specificTcellsonday7andcomparetotraditionalbiotinylateddimerstainingreagents.S11

11SupplementalFigure9:Titrationofdetectionbead:cellratiostoevaluateoptimalstainingconcentrationforstainingantigen-specificTcellsonday7oftheenrichmentandexpansionprotocolwithahighfinalpercentageofantigen-specificTcells.(a)Flowcytometryplotsofbothpeptide-loadedAdaptiveaAPCs(Adaptive+Peptides)andunloaded(Adaptive-Peptides)detectionbeads(b)Percentageofcontrolstaining(Adaptive-Peptide/non-cognate)weresubtractedtoevaluatefinalpercentageofantigen-specificTcellsonday7andcomparetotraditionalbiotinylateddimerstainingreagents.S12

12SupplementalFigure10:CombinationofmultiplexedAdaptiveaAPC,96-wellplateenrichmentandexpansionstartingfromapopulationofsplenocytes,anddetectionbyAdaptivedetectionbeads.Representativestainingonday7bydetectionbeadofantigen-specificTcellsafterenrichmentandexpansionforeachantigenusingunloadedadaptivedetectionbeads(Adaptive-Peptide)asanegativecontrol.S13

13****100I80FMd60ezilam40roN200UnloadedMCMVSIYVDWSupplementalFigure11:PeptideStabilizationAssaytoDetermineRelativeBindingAffinityofSIYandVDWforKbMHCmolecule.RMA-ScellshavesignificantlymoreKbproteinstabilizedandexpressedontheirsurfacewhenpulsedwith1µgofhighaffinitySIYpeptide,comparedtonopeptide,1µgofDb-restrictedMCMVpeptide,or1µgneoantigenVDWpeptide(errorbarsshows.e.m;****p<0.0001,n=3,one-wayANOVAwithTukey’sposttest).S14

14SupplementalFigure12:InVivoPeptidevaccinationwithVDWandSIYpeptide.(a)Representativeand(b)summaryofnon-cognateOVAandcognateSIYandVDWdimerstainingofspleensandlymphnodesofunvaccinatedandvaccinatedmice.ThefrequencyofSIYandVDWspecificCD8+TcellsissignificantlyincreasedbyD15inthespleensofvaccinatedmice(errorbarsshows.e.m;*p<0.05,**p<0.01,****p<0.0001,n=5,one-wayANOVAwithTukey’sposttest).S15

15SupplementalFigure13:EnrichmentandExpansionofantigen-specificCD8+Tcellsforviral(M-1andCMV)andtumor(MART-1)antigensusingAdaptiveaAPCs.(a)Representativeexpansiondatawith100nmAdaptiveaAPCs.(b)Representativeexpansiondatawith300nmAdaptiveaAPCs.S16

16EXPERIMENTALSECTIONMiceB6,2C,andPMELtransgenicmiceweremaintainedperguidelinesapprovedbytheJohnsHopkinsUniversity’sInstitutionalReviewBoard.C57BL/6JmicewerepurchasedfromJacksonLaboratories(BarHarbor,ME,USA).2CTcellreceptortransgenicmicewerekeptasheterozygotesbybreedingonaC57BL/6Jbackground.MHC-IgandPeptidesSolubleMHC-Igdimersloadedwithpeptides(“Pre-loading”)includingDbIg,KbIg,andA2Igwereproducedin-houseasdescribed1,2.Peptidesforexperimentsusedinclude:GP100:KVPRNQDWL,SIY:SIYRYYGL,SVY:SIYRYYGL,OVA:SIINFEKL,TRP2:SVYDFFVWL,VDW:VDWENVSPEL,MCMV:YPHFMPTNL,CMV:NLVPMVATV,M1:GILGFVFTL,MART-1:ELAGIGILTV.PeptideswerepurchasedfromGenScript(NewJersey,USA).2CTcelltransgenicmicearecognateforSIYpeptideloadedintoKbIgandPMELtransgenicmicearecognateforGP100peptideloadedintoDbIg.Pre-loadedArtificialAntigenPresentingCellsProductionArtificialantigenpresentingcells(aAPC)wereproducedin-houseasdescribed2,3.Briefly,loadedantigen-specificdimericMHC-Igandequimolaranti-CD28,clone37.51(BioXCell.WestBebanon,NH,USA)wereconjugatedtothesurfaceofmagneticparticlesfunctionalizedwithNHSsurfacegroupsatabasedparticlesizeof200nm(OceanNanotech,Springdale,AR,USA)perthemanufacturer’srecommendations.AdaptiveArtificialAntigenPresentingCellsProductionS17

17ForAdaptiveaAPCs,MHC-Igwasconjugatedtoparticles(withthesamemethodasPre-loaded)exceptwithoutpreviouslyloadinginaspecificpeptide.DetectionBeadProductionFordetectionbeads,BNF-Starch-greenF100-nmmagneticparticleswithaminesurfacegroups(Micromod,Rostock,Germany)werefunctionalizedwithSulfo-SMCC(Proteochem,Hurricane,UT,USA)anddimericMHC-IgwasthiolatedwithTraut’sreagent(2-iminothiolane)(SigmaAldrich,St.Louis,MO,USA)andthenmixedwiththefunctionalizedparticlesperthemanufacturer’srecommendations.Foradaptivedetectionbeads,MHC-Igwasconjugatedtoparticleswithoutpreviouslyloadinginaspecificpeptide.ArtificialAntigenPresentingCellCharacterizationTheamountofproteinconjugatedsuccessfullytothesurfaceoftheparticleswasquantifiedthroughfluorescentstaining.TheamountofMHC-IgwasquantifiedbystainingwithFITC-conjugatedratanti-mouseIg1,2,3lightchain,cloneR26-46(BDBiosciences,SanJose,CA,USA),andtheamountofanti-CD28wasquantifiedbystainingwithFITC-conjugatedmouseanti-ArmenianSyrianhamsterIgG,cloneG192-1(BDBiosciences).Particleswerestainedwith1µLoftheantibodyfor1hat4C,washedthreetimes,andthenfluorescencewasreadonSynergyHTXMulti-modeflorescentplatereader(BioTek,Winooski,VT,USA).Proteinwasquantifiedbycomparisontofluorescentstandardcurveofstainingantibodies,andparticlenumberwasquantifiedbyabsorbanceusingaspectrophotometeratawavelengthof405nm.AdaptiveaAPCsandDetectionBeadPeptideLoadingToloadAdaptiveaAPCsorDetectionBeads,analiquotofaAPCs(1.51010particles)orDetectionBeads(3.81010particles)wasaliquotedintoafinalvolumeof100µLofPBSina96-UbottomedS18

18plate.Then1µgofpeptidewasaddedtotheparticlesovernightat4C.Theparticleswerewashedthreetimesonthe“Ring”magnetwith200µLofPBSandimmediatelyused.SupplementedMediaandTCellGrowthFactorSupplementedmedia(B’Media)wasmadewithPBSbufferand0.5%bovineserumalbumin(BSA)(Gemini,Sacramento,CA)and2mMEDTA.TheTcellgrowthfactor(TCGF)wasmadewithRPMI1640mediawithglutamine,1xnon-essentialaminoacids,1mMsodiumpyruvate,0.4vitaminsolution,92µM2-mercaptoethanol,10µMciprofloxacinand10%fetalbovineserum(FBS)(AtlantaBiologicals,FloweryBranch,GA).SpecificCellIsolationandDepletionMurinecellswereobtainedfromadultfemaleandmalemouselymphnodesandspleens.ObtainedcellsweretreatedwithACKlysingbuffertolyseredbloodcellsandfilteredthroughcellstrainerstoisolatesplenocytes.PBMCsfromhealthyhumandonorswereisolatedbyFicoll-PaquePLUSgradientcentrifugation(GEHealthcare,Chicago,IL,USA).ForisolatingCD8+Tlymphocytes,CD4+Tlymphocytes,PanTcells,thesecellswereisolatedfromsplenocytesorPBMCsbynegativeselectionusingCD8+,CD4+,andPanTcellisolationkitsandmagneticcolumnsfromMiltenyiBiotech(Auburn,CA,USA)accordingtothemanufacturer’sprotocol.MemoryCD8+TcellswerenotdepletedforallCD8+isolations,inordertomaintainconsistencywithCD8+populationswewouldencounterinisolatingantigen-specificTcellsfromsplenocyteorPBMCsources.Todepletespecificcellpopulations,biotinylatedantibodywasaddedtosplenocytesfor5minat4C,followedbyratioofanti-biotinmagneticbeadsconsistentwithpreviousmanufacturerrecommendedamountsfromisolationkits(MiltenyiBiotech);forCD4+Tcells,cloneGk1.5(eBioscience),forCD11c+cells,cloneN418(Biolegend,SanDiego,CA,USA).S19

19PBMCswereobtainedfromblooddrawnfromhealthymalesandfemalesperJHUIRBapprovedprotocols.EnrichmentandExpansionforSmall(50–100nm)NanoparticleaAPCsAllE+Econditionsreceivedthesamenumberofinitialsplenocytesgoingintothevariousisolationconditions.Followingcellisolation,thenanoparticleaAPCswereaddedtocellsbasedontheratioof1011aAPC-bound,peptide-loadedMHC-Igforevery1107splenocytesorforevery106CD8+Tcells.Similarly,E+EswithKbSIY-onlyaAPCsorfromPan-Tisolatewereperformedataratioof1011aAPC-boundpeptide-loadedclassIMHC-Igforevery1x106CD8+Tcells.TheaAPCparticleandcellmixtureswereincubatedfor1hat4CwithcontinualmixinginaPBSbufferwith2 mMEDTAand0.5%BovineSerumAlbumin(BSA)(TermedRunningBuffer).ThemagneticparticleaAPC:cellmixtureswerethenseparatelywashedinaMiltenyiMSmagneticcolumnthreetimes.Themagneticcolumnwaswetwith0.5mLofPBS,thentheparticles/cellswereaddedtothecolumnandwashedusingtwoseparatewashesofB’MediaandthirdwashusingB’Mediawith1%TCGF.Thecellswerecountedusingahemocytometerandplatedina96U-bottomedplatein160µLperwellofB’Mediawith1%TCGFataconcentrationof1106splenocytes/mLor2.5105CD8+Tcells/mL.TheaAPC:cellmixtureswereculturedinahumidified5%CO237Cincubatorfor3days.Onday3,thecellswerefedwith80µLperwellofB’Mediawith2%TCGFandplacedbackintotheincubatoruntilday7.Onday7,thestimulatedcellswereharvestedintoa5-mLroundbottomtubeforcountingandanalyzedforantigenspecificitybyflowcytometry.96-wellPlate-basedEnrichmentandExpansionfor300-nmaAPCsS20

20AsimilarprotocoltotheE+EforsmallaAPCs,asdescribedabove,wasfollowed,withanalternatewashingprocess.Followingthe1hincubationat4C,theaAPC:cellmixtureswereaddedtoa96U-bottomedwellplateandplacedonamagnetfor5min.Twodifferentkindsofmagnetswereusedfortheexperimentsthe“Ring”magnet—“MAGNUM™EXAdaptiveMagnetPlate”(Alpaqua,Beverly,MA)–andthe“Bottom”magnet—“EasyPlateEasySepMagnet”(STEM-cell,Vancouver,Canada).Thebufferwascarefullyremovedfromthewellswithanangledmultichannelpipettetonotdisruptthemagneticpelletonthebottomorinthering.Theplatewasremovedfromthemagnet,andthepelletwasresuspendedin200µLofB’Media.Theplatewasplacedonthemagnetfor2min.Thesupplementedmediawasthencarefullyremovedfromthewells.Theplatewasremovedfromthemagnet,andthepelletwasresuspendedin200µLofB’Mediawith1%TCGF.Thentheplatewasplacedonthemagnetfor2min.Theplatewasremovedfromthemagnet,andthepelletwasresuspendedin160µLofB’Mediawith1%TCGF.Theplatewasplacedinahumidified5%CO237Cincubatorfor3days.Onday3,thecellswerefedwith80µLperwellofB’Mediawith2%TCGFandplacedbackintotheincubatoruntilday7.Onday7,thestimulatedcellswereharvestedintoa5-mLroundbottomtubeforcountingandanalyzedwithantigen-specificstainingforflowcytometry.ForbatchedversusindividualE+Ecomparisons,thesplenocytesweredividedinto8equalportionsinsterileFACStubes.Therespectivefourtypesofantigen-specificaAPCswereaddedtotwodifferentFACStubes.Toformthebatchedcondition,fouroftheindividualconditionswerecombinedintoonetubeandthenprocessedtogetherfromthenon.InthecaseofhumanTcellexpansion,10%ABserumwasusedinsteadof10%fetalbovineserum.Onday3ofculture,cellswerefedwithhalfthevolumeoftheinitialTcellculturemediawithtwicetheconcentrationofTcellgrowthfactorcocktail.Onday7cellswereharvestedcountedS21

21andre-platedatadensityof50,000cellsperwellwithanadditionaldoseofaAPCs,whileasubsetwastakenforantigen-specificstaining.Onday10ofculture,cellswerefedwithhalfthevolumeoftheinitialTcellculturemediawithtwicetheconcentrationofTcellgrowthfactorcocktail.Cellswereharvestedonday14,counted,andstainedforantigen-specificity.AntigenSpecificStainingOnday7ofculture,thenumberofcellswerecountedusingahemocytometer.Aftercounting,lessthan500,000cellswerecollectedandplacedintotwo5-mLroundbottomtubesforantigen-specificstaining.Onetubewasusedforthecognatepeptide-MHCstain,andtheothertubewasusedforthenon-cognatestaintodeterminebackgroundstaining.Tothetwoconditions,1µgofcognateornon-cognatebiotinylatedMHC-Igin100µLofPBSwith0.05%sodiumazideand2%FBS(FWB)for1hat4C.TheexcessbiotinylatedMHC-IgwithPBSwaswashedthroughcentrifugation.Thesampleswerethenstainedwitha1:350ratioofPE-labeledstreptavidin,with1:100APC-conjugatedratanti-mouseCD8a,clone53-6.7(Biolegend,SanDiego,CA,USA),andwith1:1000ratioofLIVE/DEAD®FixableGreenDeadCellStain(ThermoFisher)for15minat4°C.Excesssecondaryandlive/deadstainwerewashedbycentrifugationandresuspendedwith150µLofPBSbufferwithFWBtoreadonaBDFACSCaliburflowcytometer.Todeterminethepercentofantigen-specificcells,thefollowinggateswereusedintherespectiveorder:live+,lymphocyte+(forwardscatterbysidescatter),CD8+,andDimer+.TheDimer+gatewasdeterminedbycomparingnon-cognatetothecognatestain.Todeterminethepercentageofantigen-specificcells,thepercentageofDimer+ofthecognateMHC-Igstainwassubtractedfromthenon-cognateMHC-Igstain.Toobtainthenumberofantigen-specificcells,thisnumberwasmultipliedbythepercentageofCD8+Tcellsandthenumberofcellscounted.S22

22Detectionofantigen-specifichumancellswasdonesimilarly,exceptinsteadofstainingwithbiotinylateddimer,theantigen-specificcellswerestainedwithpurchasedPE-labeledtetramer(MBLInternational,Woburn,MA)for30minatroomtemperature,thenwashedandstainedwithAPC-conjugatedanti-humanCD8a,cloneSK-1(Biolegend),and1:1000ofLIVE/DEAD®FixableGreenDeadCellStainfor15minat4°C.FluorescentMagneticBeadAntigen-specificStainingOnday7ofculture,thenumberofcellswerecountedusingahemocytometer.Aftercounting,lessthan500,000cellswerecollectedandplacedintotwo5-mLroundbottomtubesforantigen-specificstaining.Toeithertube,pre-loadedMHC-Igfluorescentbeadsoradaptivedetectionbeads+/-peptideswereaddedattheindicatedamountsandallowedtobindfor45minat4°C.Thenasolutionof1:100APC-conjugatedratanti-mouseCD8a,clone53-6.7(Biolegend,SanDiego,CA,USA)(formousestains)andwith1:1000ratioofLIVE/DEAD®FixableRedDeadCellStain(ThermoFisher)wasaddedtosamplestostainforanadditional15minat4°C.CellswerewashedandreadonaBDFACSCaliburandantigen-specificitywasdeterminedsimilartobiotinylateddimer-MHCstaining,whileunloadedadaptivedetectionbeadswereusedasthebackgroundstaining.ParticlestainingofhumancellswasdoneusingasimilarprotocolexceptanAPC-conjugatedmouseanti-humanCD8a,cloneSK-1(Biolegend),wassubstitutedfortheratanti-mouseCD8astain.InVivoPeptideVaccinationNaïve8-week-oldfemalemicewereinjectedsubcutaneouslywithamixtureof100µgSIYpeptideandpolyI:Cdilutedinto200µLPBSontheirleftrearflanks,and100µgVDWpeptideandpolyS23

23I:Cdilutedinto200µLPBSontheirrightrearflanksonbothDay0andDay7.OnDay15,mousespleensandlymphnodeswereharvestedfordimerandparticlestaining,followingsimilarprotocolsdescribedabove.SplenocyteImmuneCellFlowCytometryPanelLessthan500,000cellswerecollectedandstainedwitha1:100PBSsolutionofPe/Cy7-conjugatedratanti-mouseCD19,clone6D5(Biolegend),APC-conjugatedratanti-mouseNK-1.1,clonePK136(BDPharmingen),APC/Cy7-conjugatedratanti-mouseCD8a,clonenumber53-6.7(Biolegend),PE-conjugatedratanti-mouseCD4,cloneH129.19(Biolegend),PerCp-conjugatedratanti-mouseCD11c,cloneN418(Biolegend),AmCyan-conjugatedF4/80,clone605(Biolegend),and1:1000ofLIVE/DEAD®FixableGreenDeadCellStain(ThermoFisher)for15minat4°C.CellswerethenwashedwithFACSwashbuffertobereadonBDLSRIIflowcytometerandanalyzedusingFlowJotomeasurethepopulationofBcells(CD19+),NKcells(NK1.1+),CD4+Tcells,dendriticcells(CD11c+),andmacrophages(F4/80+).TCellProliferationAssayCD8+Tcellswereisolatedaspreviousdescribedandresuspendedin1mLTcellculturemedia.Cellsweremixedwith1µLCellTracecarboxyfluoresceinsuccinimidylester(CFSE)dye(ThermoFisher)in1mLofTcellculturemediaper3millioncellsandincubatedat37°Cfor20min.CFSEstainedcellswerewashedwith50mLofTcellculturemediatoremoveunstaineddyeandplated.Onday3ofculture,cellswereharvestedandstainedwitha1:100PBSsolutionofAPC-conjugatedratanti-mouseCD8a,clone53-6.7(Biolegend)for15minat4°C.TheCFSEfluorescenceintensitywasmeasuredusingBDFACSCaliburflowcytometer.CellproliferationwasanalyzedusingFlowJowithdilutedCFSEfluorescencepeakssignifyingpopulationaftereachS24

24roundofcelldivision.Asubsetofthecellswasallowedtoexpandfor7daysandviablecellswerecountedwithahemocytometertodeterminefoldexpansion.TCellPhenotypeAssayOnday7ofculture,thenumbersofcellswerecountedusinghemocytometer.Aftercounting,lessthan500,000cellswerecollectedandstainedwitha1:100PBSsolutionofAPC-conjugatedratanti-mouseCD8a,clone53-6.7(Biolegend),PE-conjugatedratanti-mouseCD62L,cloneMEL-14(BDBiosciences),PerCP-conjugatedratanti-mouseCD44,cloneIM7(Biolegend),and1:1000ofLIVE/DEAD®FixableGreenDeadCellStain(ThermoFisher)for15minat4°C.CellswerethenwashedwithFACSwashbuffertobereadonBDFACSCaliburflowcytometerandanalyzedusingFlowJotomeasurethepopulationofnaïveTcells(CD62L+CD44-),effectorTcells(CD62L-CD44+),andmemoryTcells(CD62L+CD44+).TCellCytokineFunctionalityAssayOnday7ofculture,approximately500,000CD8+Tcellswereisolatedfromeachconditionandseparatedintocognateornoncognategroups.Cellswerestainedwith1µgofeithercognateornon-cognatebiotinylatedpMHC-Igdimerfor1hat4°C.Afterwashing,sampleswerestainedwitha1:350ratioofPE-labeledstreptavidin(BDPharmingen,SanDiego,CA,USA).Then10µLsolutionof1:50FITCanti-CD107a,1:350BDGolgiStopProteinTransportInhibitor(BDBiosciences),and1:350BDGolgiPlugProteinTransportInhibitor(BDBiosciences)inPBSwasaddedtothesamplesandincubatedwith100µLofcompletemediafor37°Cfor6h.Cellswerethenwashedandstainedwith1:100PBSsolutionofPerCP-conjugatedanti-mouseCD8a,clone53-6.7(Biolegend)and1:1000ofLIVE/DEAD®AmCyanFixableAquaDeadCellStain(ThermoFisher)at4°Cfor30min.Cellswerethenfixedandpermeabilizedwith100µLBDS25

25Cytofix/CytopermFixationandPermeabilizationSolution(BDBiosciences)overnight.Cellswerethenwashedwith1BDPERM/Washbufferwith2%BSAandstainedwith1:100solutionofAPC-conjugatedratanti-mouseIFN-γ,cloneXMG1.2(BDPharmingen)andPE-Cy7-conjugatedratanti-mouseTNFα,cloneMP6-XT22(Biolegend)inPERM/Washbufferwith2%BSAat4°Cfor1h.StainedcellswerereadonBDLSRIIflowcytometer.IFN-γReleaseAssaysOnday7ofculture,25,0002CCD8+Tcellswerere-stimulatedwith300-nmpre-loadedvs.AdaptiveaAPCspre-vs.post-loadedKbSIY/anti-CD28particlesfor18hat37°C,andthenthesupernatantswerecollected.IFN-γwasmeasuredbyELISAusingtheebiosciencemurineIFN-γReady-SET-Go!Kit(SanDiego,CA,USA)PeptideStabilizationAssaysRMA-Scellswereleftat25°overnightandpulsedfor2hwith1μgpeptideandputat37°for2htodegradeunstableMHCmolecules.Cellswerethenstainedwithanti-KbcloneM1/42andanalyzedbyflowcytometryforMHCexpression.DopedEnrichmentExperimentsPMELCD8+TcellswereobtainedbyusingamouseCD8+TcellnegativeisolationkitfromMiltenyiBiotechandfollowingthemanufacturer’sinstructions.PMELtransgenicmicehaveCD8+TcellswiththesameTcellreceptorthatrecognizesthemouseMHCDbloadedwiththegp100peptide.ThePMELCD8+Tcellswerecountedwithahemocytometerandaddedata1:1000ratiotowildtypeB6CD8+Tcellsandmixedthoroughlyinrunningbuffer.ParticleaAPCswereaddedtothismixtureattheindicatedamountsper1×106totalCD8+Tcellsandallowedtobindat4 °Cfor1 h.Theparticlecell-mixturewasthenwashedmagneticallyaspreviouslyS26

26describedwithinthe“EnrichmentandExpansion”experiments.Allparticle-cellmixturescountedviaahemocytometerandstainedwiththeAPC-conjugatedratanti-mouseCD8a,clone53–6.7,(Biolegend)for15 minat4°C,washedandreadonaBDFACSCalibur.FoldenrichmentwasdeterminedbydividingthepercentofPMELpositivecellsintheelutedparticle-cellmixturebythepercentofPMELpositivethenative1:1000dopedmixture.PercentcellrecoverywascalculatedbydividingthenumberofPMELpositivecellsintheelutedparticle-cellmixturebythenumberofPMELpositiveinthenative1:1000dopedmixture.ThePMELcellcountswerecalculatedbymultiplyingthenumberofcellsineachmixturebythemeasuredpercentagesfromflowcytometry.ParticleandBeadBindingParticleaAPCswereallowedtobindwithcognatetransgenicCD8+Tcellsat4°Cfor1hatvariousratiosofparticleaAPCstoTcells.Thismixturewaswashedandstainedwitha1:350ratioofPElabeledrat-anti-mouseIgGfor15minat4°C.PElabeledpolyclonalgoat-anti-mouseIgG1(ThermoFisher)recognizesthemouseIgGofthedimericKb-Igontheparticlestodiscriminatethequantitateparticlesonthesurface.Excessantibodywaswashedawayandthenstainedwitha1:100PBSsolutionofAPC-conjugatedratanti-mouseCD8a,clone53-6.7(Biolegend).CellswerewashedandreadonaBDFACSCaliburtodeterminethepercentofcellsboundwithrespecttothenon-particleboundandnon-cognateCD8+Tcellsofbackgroundstaining.Fluorescentmagneticdetectionbeadbindingandanalysiswasperformedsimilarly,exceptthatthereisnoneedforsecondaryantibodystainingsincetheparticlesthemselvesarefluorescent.S27

27REFERENCES(1)Kosmides,A.K.A.K.;Necochea,K.;Hickey,J.W.J.W.;Schneck,J.P.J.P.SeparatingTCellTargetingComponentsontoMagneticallyClusteredNanoparticlesBoostsActivation.NanoLett.2018,18(3),1916-1924.acs.nanolett.7b05284.https://doi.org/10.1021/acs.nanolett.7b05284.(2)Hickey,J.W.;Schneck,J.P.EnrichandExpandRareAntigen-SpecificTCellswithMagneticNanoparticles.JoVE(JournalVis.Exp.)2018,No.141,e58640.(3)Hickey,J.W.;Isser,A.Y.;Vicente,F.P.;Warner,S.B.;Mao,H.-Q.;Schneck,J.P.EfficientMagneticEnrichmentofAntigen-SpecificTCellsbyEngineeringParticleProperties.Biomaterials,2018,187,105-116.S28