Rapidantigen-reactiveTcellenrichment(RapidARTE) -1-

Introduction

Antigen (ag)-specificTcellsplayacentral role inmediatingspecificimmuneresponsesaswellasinthe formation of immunological memory.Information about their frequency, phenotype,andfunctionalcapacityisessentialtoestimatethespecific immune status of an individual, tounderstand the mechanisms of protectiveimmunity or immunopathology and to predictimmune protection or diagnose immune-relateddiseases. Due to the very low frequency of ag-specific T cells in peripheral blood the reliabledetection, enumeration and phenotypicalcharacterizationrequiretheprocessingofhighcellnumbers and highly specific analysis methods.CD154 is transiently up-regulated on activatedCD4+ T cells and plays an important role as acostimulatorymoleculeinTcellag-presentingcellinteractions through ligation of CD40.Due to itstransientexpressionwithinhoursafteractivation,CD154canbeusedasamarkerforactivatedag-specific CD4+ T cells. Adding a CD40-blockingantibody during cell stimulation prevents down-regulationofCD154expression.CD154isinducedby the interaction with CD40 expressed on ag-presentingcells.Combining magnetic cell enrichment andmultiparameter flow cytometry analysis ofCD154+CD4+Tcellsallowdirectexvivodetectionandcharacterizationofrareag-specificTcells.

Workflow

PBMCstimulation

Magneticandimmune-fluorescentsurfacelabeling

Magneticenrichmentandintracellularstaining

Flowcytometryanalysis

6h

60min

40min

Directexvivocharacterizationofhumanantigen-specificCD154+CD4+TcellsRapidantigen-reactiveTcellenrichment(RapidARTE)

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Materials

PBMCcultivation• RPMI1640medium• 100´L-Glutaminestocksolution

(200mM)• HumanABSerum• 24-wellplate(e.g.Gas-permeable

CulturePlate,#150-000-362)▲Note:WiththeGas-permeableCulturePlate(#150-000-362),upto2.5´107PBMCs/well/mLcanbestimulatedasopposedto1´107PBMCs/well/mLinstandard24-wellplates.

PBMCstimulation

• CD40pure–functionalgrade,human(#130-094-133)

• Reagentsforag-specificTcellstimulation(e.g.PepTivator®BKVVP1–researchgrade,human,#130-097-272andPepTivatorBKVLT–researchgrade,human,#130-096-504)

• brefeldinA• FcRBlockingReagent,human

(#130-059-901)Buffer(standardwashanddilutionbuffer)

• autoMACS®RinsingSolution(#130-091-222)

• Bovineserumalbumin(BSAStockSolution,#130-091-376)

Magneticandimmunofluorescentsurfacelabeling

• CD154MicroBeadKit,human(#130-092-658)

• InsideStainKit(#130-090-477)• Orbitalshaker• CD3-VioBlue®,human(#130-094-363)• CD4-APC,human(#130-092-374)• CD8-PerCP,human(#130-094-972)• CD14-PerCP,human(#130-094-969)• CD20-PerCP,human(#130-094-976)

Magneticenrichmentandintracellularstaining

• MSColumns(#130-042-201)• MACS®SeparatorforMSColumns

(e.g.MiniMACS™Separator#130-042-102)

• MACSMultiStand(#130-042-303)• CD154-FITC(#130-096-233)• Anti-cytokineantibodiesfor

intracellularstaining(e.g.Anti-IFN-g-PE,#130-091-653orAnti-IL-17A-PE,#130-094-521)

MaterialpreparationPBMCcultivationmediumPrepareasolutionof500mLRPMI1640and5mL100´L-Glutaminestocksolution(2mMfinalconcentration).Add25mLhumanABSerum(5%finalconcentration).Buffer(standardwashanddilutionbuffer)PrepareasolutionofPBS,pH7.2,2mMEDTAand 0.5% BSA by diluting MACS BSA StockSolution1:20withautoMACSRinsingSolution.ReconstitutionofPepTivator®PeptidePoolFor reconstitution of the lyophilized peptidepooltakethevialfrom–20°Candwarm-uptoroomtemperature.▲Note:Donotopenthevialbyremovingtherubberplug.2. Todissolve the 6 nmol packingunit of thePepTivator® Peptide Pool fill a sterile syringe(0.5 mL) with 200 μL of sterile water. Todissolve the 60 nmol packing unit of thePepTivatorPeptidePoolfillasterilesyringe(5mL)with2mLofsterilewater.3.Slowlyinjectthewaterwithasterileneedlethroughthecenteroftherubberplugintothevialcontainingthelyophilizedpeptidepool.

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4. Vortex the solution to completely dissolvethelyophilizedpeptidepool.TheconcentrationofthestocksolutionofPepTivator®Peptidesis30nmol(approximately50μg)ofeachpeptidepermL.5. Remove the rubber plug and aspirate thestocksolutionwithapipette.6. To avoid repeated freeze-thaw cyclesprepare working aliquots from the stocksolution.7.Storetheworkingaliquotsat–80°C.StainingMixFreshlyprepare200μLofstainingmixforeachwell:

• 12.5μLCD3-VioBlue®• 25μLCD4-APC• 25μLCD8-PerCP• 25μLCD14-PerCP• 50μLCD20-PerCP• 25μLCD154-Biotin

(fromCD154MicroBeadKit)• 37.5μLFcRBlockingReagent

Methods1.InvitrostimulationforinductionofCD154expression1. Sterile preparation of PBMCs from freshbuffycoatsorwholebloodusingFicoll-Paque™.▲ Note: To remove platelets after density gradientseparation,resuspendcellpelletinbufferandcentrifugeat200´g for10−15minutesat20 °C.Carefullyaspiratesupernatant.Repeatwashingstep.2.Resuspendcellsatadensityof107cells/mLinPBMCcultivationmedium.3. For each non-stimulated control andantigen-stimulatedsampletransfer107cellstoonewellofa24-wellplate.4.Add10μLofCD40pure–functionalgrade(1μg/mL;1:100)toeachwell.5. Add antigen according to manufacturer’srecommendation to the antigen-stimulatedwells, but not to control wells. When usingPepTivators:Add20µLofreconstitutedstocksolutionperwell.6.Incubatecellsfor4hoursat37°C,5%CO2.7.AddbrefeldinAtoafinalconcentrationof2µg/mL.8.Incubatecellsforanother2hoursat37°C,5%CO2.

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2.Magneticandimmunofluorescentsurfacelabeling1.Centrifugeplate for 5minutes at 300´g atroomtemperature.2.Verycarefullyaspirate800μLofcellculturesupernatant.Avoidresuspensionofthecells.3.Add200μLofantibodystainingmixtoeachwell:

• 12.5μLCD3-VioBlue®• 25μLCD4-APC,• 25μLCD8-PerCP• 25μLCD14-PerCP• 50μLCD20-PerCP• 25μLCD154-Biotin

(fromCD154MicroBeadKit)• 37.5μLFcRBlockingReagent

4.Mixwellfor2minutesusinganorbitalshaker.5. Incubatefor3minutes inthedarkatroomtemperature.6. Add 20 μL of Anti-Biotin MicroBeadsUltraPure(fromCD154MicroBeadKit)toeachwell. Mix well for 2minutes using an orbitalshaker.7.Incubatefor13minutesinthedarkatroomtemperature.8.Add300μLofInsideFix(fromInsideStainKit)toeachwell.Mixwell for2minutesusinganorbitalshaker.9.Incubatefor13minutesinthedarkatroomtemperature.10.Add1mLofbuffertoeachwellandmixwell.11. (Optional) Take an aliquot of 200 μL cellsuspensionforstainingoftheoriginalfraction.

3.Magneticenrichmentandintracellularstaining(oncolumn)1.PlaceMSColumninthemagneticfieldofasuitableMACS®Separator.2.PrepareMSColumnbyrinsingwith500μLofbuffer.3.Applycellsuspensionontothecolumn.4.Washcellsbyrinsingthecolumnwith500μLofbuffer,followedby2´500μLofInsidePerm(fromInsideStainKit).5. Prepare a solution of 10 μL of CD154-FITCand90μLofInsidePerm(fromInsideStainKit).6.(Optional)Addadditionalstainingantibodiesto the solution, e.g., for the staining of cellsurface antigens internalized upon cellactivationorantigenswhichaccumulateinthecell.▲Note:Donotexceedthetotalvolumeof150μL.7. Apply the solution onto the column andincubatefor10minutesatroomtemperature.▲Note:TheMACSColumnhasaflow-stopmechanismthatwillretainthesolutioninthecolumn.8.Washcellsbyrinsingthecolumnwith2´500μLofInsidePerm(fromInsideStainKit)followedby500μLofbuffer.9. Remove column from the separator andplaceitonasuitablecollectiontube.10.Pipette500μLofbufferontothecolumn.Immediatelyflushoutthemagneticallylabeledcells by firmly pushing the plunger into thecolumn.11.Cellsarenowreadyforanalysis.Storecellsat 2–8 °C in the dark until analysis.Mix wellbeforeflowcytometricacquisition.▲Note:Samplesmaybestoredat2–8°Cinthedarkforupto24hours.▲Note:Donotusepropidiumiodide(PI)or7-AADstainingingeneral.

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4.Intracellularstainingoforiginalsamples1. Wash cells by adding 1 mL of buffer.Centrifugecellsfor5minutesat300´gatroomtemperature.2.Aspiratesupernatant.3. Resuspend cells in 500 μL of Inside Perm(from Inside Stain Kit) and vortex well.Centrifugecellsfor5minutesat300´g.4.Aspiratesupernatant.5. Prepare a solution of 10 μL of CD154-FITCand90μLofInsidePerm(fromInsideStainKit).6.(Optional)Addadditionalstainingantibodiesto the solution, e.g., for the staining of cellsurface antigens internalized upon cellactivationorantigenswhichaccumulateinthecell.▲Note:Donotexceedthetotalvolumeof150μL.7. Add staining solution to the cells andincubatefor10minutesatroomtemperature.8.Wash cells by adding 1mL of Inside Perm(fromInsideStainKit).Centrifugecellsfor5minutesat300´g.9.Aspiratesupernatant.10.Resuspendcellsin250μLofbuffer.11.Samplesarereadyformeasurementnow.▲Note:Samplesmaybestoredat2–8°Cinthedarkforupto24hours.

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ABFigure1:Detectionofvirus-specificTcellswithoutenrichment via Rapid ARTE. (A) and (B) PBMCsfrom six randomly selected healthy donors werestimulated with various peptide pools forimmunodominant antigens of BKV, JCV, CMV,influenza, EBV, and AdV, and for positive controlwithamixtureofCMV/EBV/influenzaMHCclassI–restrictedpeptides.After6hours,IFN-gproductionwithintheCD4+Tcellcompartmentwasanalyzedbyintracellular staining using the Rapid CytokineInspector. CMV-, EBV-, and AdV-specific IFN-g+T cells were clearly detectable in several donorsamples.Incontrast,IFN-g+-,BKV-,andJCV-specificTcellsweredetectableonlyatverylowfrequencies,between 0.01 and 0.03%, in five out of six donorsamples.

Figure 2: Rapid ARTE successfully enriches ag-specificTcells.InbloodsamplesBKV-specificTcellsare present only at very low frequencies, in therange of the detection limit of flow cytometryanalysis. Therefore, we used the Rapid ARTEprotocolforenumerationofBKV-specificTcells.Upto2.5´107PBMCsfrom14healthydonorswereleftuntreated or stimulated with BKV peptide poolscovering the complete sequence of the large Tantigen (LT)andvirionproteinVP1.Bothproteinsare immunodominant targetsforTcell immunity.After six hours, cellswere treated as described inthisprotocol,andtheabsolutenumbersofenrichedCD154+CD4+ T cells were determined. In bloodsamples of each donor, BKV-reactive CD4+ T cellswerefound.Absolutenumbersrangedbetween87and2340per1´107PBMCs.

IFN-g

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ABFigure 3: The Rapid ARTE approach allows toprocesslargercellnumberscomparedtodetectionbyflowcytometryalone.(A)PBMCs(2.5´107)fromsixhealthydonorswereprocessedasdescribed inthis protocol. Our new approach enables thereliablecharacterizationoffunctionalsubsetsoftheentire BKV-reactive CD154+CD4+ T cell pool, i.e.,TNF-α–, IFN-g–, IL-10–,and IL-2–producingT cells.(B) Subsequently, the cytokineprofileof theBKV-reactiveCD4+Tcellswasanalyzed.TNF-αand IL-2were expressed by the majority of BKV-specificTcellsinallsamplesanalyzedforthesecytokines.Incontrast,thefrequencyofIFN-g+cellsamongBKV-reactiveCD4+Tcellsvariedbetween27and74%inthe different donor samples. Furthermore, IL-10–producing T cells could be identified at very lowfrequencies,whichcouldnothavebeendetectedbyflowcytometrywithoutpre-enrichment.

References1.Bacher,P.etal.(2013)J.Immunol.190:3967–3976.2.Bacher,P.etal.(2013)CytometryA83:692–701.

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