TherecombinantexpressionvectorsaredesignedtoexpresshumanengineeredTcellreceptor(TCR)activatorandhumanPD-L2(GenBankAccession#NM_025239)inmammaliancells.ThetransfectedcellscanbeusedinconjunctionwithPD-1/NFATReporter/JurkatTcells(BPS#60535)tostudytheinteractionsofPD-1withPD-L2ligandinacellularcontextandscreenformodulatorsofthissignalingpathway.
Inthisassay,PD-1/NFATReporter/JurkatTcellsareusedaseffectorcells;HEK293cellsoverexpressingPD-L1(orPD-L2)andanengineeredTcellreceptor(TCR)activatorareusedastargetcells.Whenthesetwocellsareco-cultivated,TCRcomplexesoneffectorcellsareactivatedbyTCRactivatorontargetcells,resultinginexpressionoftheNFATluciferasereporter.However,PD1andPD-L1orPD-L2ligationpreventsTCRactivationandsuppressestheNFAT-responsiveluciferaseactivity.Thisinhibitioncanbespecificallyreversedbyanti-PD1oranti-PD-L1antibodies.PD1,PD-L1,orPD-L2neutralizingantibodiesblockPD1:PD-L1and/orPD1:PD-L2interactionandpromoteTcellactivation,resultinginreactivationoftheNFATresponsiveluciferasereporter.
Components
•HEK293cellsanditsgrowthmediumorothercelllines
•Transfectionreagentformammaliancellline[WeuseLipofectamine™2000(lifetechnologies#11668027).However,othertransfectionreagentsworkequallywell.]
•PD-1/NFATReporter/JurkatTcells(BPSBioscience#60535)
•Opti-MEMIReducedSerumMedium(lifetechnologies#31985-062)
•Assaymedium:RPMI1640+10%FBS+1%Penicillin/Streptomycin
•Anti-PD-1neutralizingantibody(BPSBioscience#71120)
•96-welltissueculture-treatedwhiteclear-bottomassayplate
•One-Stepluciferaseassaysystem:(BPSBioscience#60690)orotherluciferasereagentsformeasuringfireflyluciferaseactivity
•Luminometer
