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Module n° : 402
Title : Intracytoplasmic and cytokine multicolour protocols
Lecturer : Frédéric Clement – Ghent University –
CEVAC
Molecular Biology and Cytometry Course, May 7-8 2009, Mol
VaccinologyandImmunology
Vaccine Candidate Efficacy
Clinical Trials Immunogenicity
Humoral Cellular
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Centralmemory(TCM)andEffectormemory(TEM)Tlymphocytes
TEM cells provide immediate protection against reinfection or reactivation of disease at sites of infection, whereas TCM cells reside primarily in the lymphoid tissue where they can rapidly expand and differentiate to resupply the effector T cells at peripheral sites.
ImmunologicalMemory
Kern F, Lipira G, Gratama JW, Manca F, Roederer M. Trends Immunol. 2005 Sep;26(9):477-84
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Cytokine-producing lymphocytes
Cell-mediated Immunity Different cells
and Different functions
Cytotoxic lymphocytes
Antibody-producing lymphocytes
CD4 and CD8 T
-lymphocytes
B-lymphocytes
ImmunologicalMemory
ICSmethodology
• S=mula=onwithAg(ProteinorPep=de),co‐s=mula=onwithan=‐CD49dandan=‐CD28.
• Proteintransportinhibi=on(BrefeldinA).• Extracellularstaining(CD4‐CD8‐Viability)• Fixa=on–permeabilisa=on.
• Intracellularstaining(CD3–CD40L–Cytokines)
• Acquisi=on
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Cri=calparametersinICS
• Pre‐analy=calprocess.– CryopreservedvsfreshPBMC
– IsolatedPBMCvsWholeblood
Cryopreservedvsfresh
Maecker et Al. J Immunol Methods 2001; 255:27 Suni et Al. J Immunol Methods 1998; 212:89
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Cri=calparametersinICS
• Pre‐analy=calprocess.– CryopreservedvsfreshPBMC– IsolatedPBMCvsWholeblood– Bloodsamplingmaterial
• Avoidany“s=mulatory”source• T‐cellac=va=onisCa++dependent
– LPSfreeheparin!
Cri=calparametersinICS
• Pre‐analy=calprocess.– CryopreservedvsfreshPBMC
– IsolatedPBMCvsWholeblood– Bloodsamplingmaterial– Sampletransfer
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Sampletransfer
• Wholebloodstoredat22‐25°Cfornolongerthan8hoursbeforecryopreserva=on.
• ShipmentandstorageofcryopreservedPBMCinliquidnitrogen(Notdryice).
• Shipmentandstorageoffrozen&fixatedsamplesondryice.
• Transferfollowingna=onal&interna=onalguidelinesforbiological(infec=ous)samples
Cri=calparametersinICS
• Pre‐analy=calprocess.• Analy=calprocess
– Equipmentstability
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Equipmentstability
• Secundaryequipmentshouldbecalibrated,controlledandmaintainedfollowingSOP.
• DailyFLow‐cytometernoise/backgroundassesment
• Dailylaser/op=csstabilityassessment
• Properdailycompensa=onrou=ne
RAREEVENTANALYSIS!!!!!
Background/noiseassessment
• Analysefresh&filtratedsamplediluent.• Acquisi=onspeed=analysisspeed.• Duringnormalacquisi=on=me
Criterion=NOPOSITIVEEVENTS!!!!
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Equipmentstability
• Define&consequentlyperformproperac=onstotakewhencriteriaarenotmet– Background–cleaning/rinseprocedures– Laser/op=csstability–Lifecycle,laserpathway,focus?
• Negociatequickinterven=onsbytechsupport.
Cri=calparametersinICS
• Pre‐analy=calprocess.• Analy=calprocess
– Equipmentstability– Re‐s=mula=onprocedure
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Re‐s=mula=onprocedure
• TimeandTypeofs=mula=oncanalterprofileandmagnitudeofcytokineproducingcells.
• Classicalovernightac=va=onwithbrefeldinA.• ArewesurethatwemeasureonlyT‐cells?
• Purityofstainedandgated(sub)popula=ons
Re‐s=mula=onprocedure
BrefeldinAinducesER‐stress
Retainsproducedproteinsin
TheGolgicomplex
BrefeldinAistoxicover16
hours!Inthiscasemonensinshouldbeused
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ClassicalGateseing
Do we miss anything ? Are we acquiring what we think we see ?
ClassicalGateseing
What about blastic transformation ?
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L gate M gate
Desombere I, Clement F, Rigole H & Leroux-Roels G. J Immunol Methods. 2005 Jun;301(1-2):124-39.
Desombere I, Clement F, Rigole H & Leroux-Roels G. J Immunol Methods. 2005 Jun;301(1-2):124-39.
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Desombere I, Clement F, Rigole H & Leroux-Roels G. J Immunol Methods. 2005 Jun;301(1-2):124-39.
Desombere I, Clement F, Rigole H & Leroux-Roels G. J Immunol Methods. 2005 Jun;301(1-2):124-39.
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ClassicalGateseing
Activation down regulates CD4 and CD8 (sensitivity ?)
Not only T-cells secrete Cytokines (Specificity ?)
Selec=onofCD3beforeCD4/CD8selec=on(andexclusionofCD14/CD16&CD19)
Selec=ononCD3cellsusingan=‐CD3‐AlexaFluor700isperformedbeforeselec=ngtheCD4andCD8cells
PresentCD3‐cellsleadstofalseposi=veresults(comparablewithELISPOTproblem).
Ommi=ngDIMCD4andCD8cellsleadstolossofsensi=vity(donwregula=onofCD4/CD8akers=mula=on)
CD3selec=onleadstosensi=veandspecificassay
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Introduc=onofaviabilitymarker
CellsarestainedwithVid.Vidcovalentlybinds,beforefixa=onandpermeabilisa=onaminegroupsonthecellmembrane(livingcells)oronthecellmembraneandintracellularcomponents(deadorapoto=ccells)
Vidwas=tratedandvalidatedagainstPropidiumIodide
Single events Lymphocytes Viable cells
CD3 + Pure & Viable CD3CD4+ or CD3CD8+
CD14/16/19 -
The 9-color assay on LSR II
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Re‐s=mula=onprocedure
Protein / Lysate APC CD4
CD8
Exogenous pathway
Endogenous pathway
MHC II present.
MHC I Present
Re‐s=mula=onprocedure
APC CD4
CD8
Overlapping peptides
MHC II present
MHC I present
Direct binding
15 mer
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Workshop:
Howisitpossibletos=mulateCD8+with15merpep=des?
Why15‐merpep=des?
• Op=malcompromisefors=mula=ngbothCD4+andCD8+(20‐merslooseabilitytos=mulateCD8+)
• CD8+s=mula=onunclear,possibleexplana=ons:
– Bindingof15‐mertosomedegree(protrusion)– Extracellularproteaseclipping– Proteasomeprocessing(lesslikely)
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Cri=calparametersinICS
• Pre‐analy=calprocess.• Analy=calprocess
– Equipmentstability– Re‐s=mula=onprocedure– Reagentvalida=on
Reagentvalida=on
• Serum(background/poscontrol)• Stainingreagents(an=‐mouseIgGbeads)
– 1trial–1lot(totalquant=trial+bridging)– Respectexp.Dateorvalidateexten=on
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Selec=ngan=bodiesPrimary Antigens
Secundary antigens
Tertiary antigens
CD3/CD4/CD8/CD19 etc…
CD27/CD28/CD4RRA-RO/IFNg/IL2
CD25/Chem.Recept./IL4/IL5
Well characterized and identified broad
subsets
Well characterized and identified broad
subsets but expression pattern is a continuum
low level expression
Buy/Evaluate Many Some one or two
You Need High Specificity High Sensitivity
Fluorochromes? Less Bright Brightest
Be careful with tandem-dyes, spectral overlap and variation !
DotheFull‐Minus‐One(FMO)experiment
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SpectralSpill‐over
Workshop:
Whatfluororchromeswouldyouuseforwhatan=gen?
CD3 ? FITC CD4 ? PE CD8 ? PerCP IFNg ? APC IL2 ? APC-Cy7
CD25 ? PE-Cy7 AlexaFluor-700
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Workshop:
Whatfluororchromeswouldyouuseforwhatan=gen?
CD3 AlexaFluor-700 CD4 Per-CP CD8 APC-Cy7 IFNg FITC IL2 APC
CD25 PE
Cri=calparametersinICS
• Pre‐analy=calprocess.• Analy=calprocess
– Equipmentstability– Re‐s=mula=onprocedure– Reagentvalida=on– Consistencyofcompensa=onprocedure
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Compensa=onUnstained, SEB stimulated PBMC
Compensa=on
Single color stained, SEB stimulated
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Workshop:Whichplotrepresentsthecorrectcompensatedmeasurement?
Bright fluorescent populations tend to have a greater standard deviation than dimly fluorescent populations !
Cri=calparametersinICS
• Pre‐analy=calprocess.• Analy=calprocess
– Equipmentstability– Re‐s=mula=onprocedure– Reagentvalida=on– Consistencyofcompensa=onprocedure– Purityofselectedpopula=on– Ga=ngconsistency
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Ga=ng
• Centrallaboratory ‐Equipmentmonitoring
‐Reagentvalidated
‐Gatesarelekuntouched
• Interlaboratory ‐Auto‐snapga=ng
Cri=calparametersinICS
• Pre‐analy=calprocess.• Analy=calprocess
– Equipmentstability– Re‐s=mula=onprocedure– Reagentvalida=on– Consistencyofcompensa=onprocedure– Purityofselectedpopula=on– Ga=ngconsistency– Countedevents
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Workshop:
Howmanyeventsisenough?Areyouposi=ve?
IfasamplehasONEeventinapar=culargate,isthateventreal?
3majorrules:Valida=on,valida=onandvalida=on!
• Daily(orless)set‐upcontrols(CytometerSet‐upandTrackingBeads,(CS&T))
Setsvoltageoftheinstrumenttoboosteventsabovenoisewithminimalvaria=on
• Compensa=onsetupusingcells(orbeads)
Useunstainedandsinglestainedcells
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3majorrules:Valida=on,valida=onandvalida=on!
• Specificitycontrols
Includeanuns=mulatedcontrolforeachsample
• Isotypecontrols
• FMOcontrols(lessfrequentasextracheck)
3majorrules:Valida=on,valida=onandvalida=on!
• Biologicalcomparisoncontrols
• Mitogen(SEB)s=mulatedcontrol,posi=vecontrol• Providesproofofcorrectstainingprocedures
• Longitudinalcontrols
• Providesproofthattheassayisundercontrol• Repeatsofwellcharacterizeddonor• Repeat1or2samplesfromthepreviousrun
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3majorrules:Valida=on,valida=onandvalida=on!
Thereisnotheore=calreasontoemployanar=ficialthresholdnumberofevents,belowwhichafrequency
isdeemednega=ve.
Theassessmentofposi=vitycanonlybemadebycomparisonofthemeasurementagainstasetofcontrolsamples,usingstandardsta=s=caltoolsto
comparethefrequencies
Countedevents
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Cri=calparametersinICS
• Pre‐analy=calprocess.• Analy=calprocess
– Equipmentstability– Re‐s=mula=onprocedure– Reagentvalida=on– Consistencyofcompensa=onprocedure– Purityofselectedpopula=on– Ga=ngconsistency– Countedevents– Whatwillwemeasure?
Whatwillwemeasure
Objec=veinclinicaltrials:
‐ Measurethemagnitudeoftheimmuneresponse.‐ Provideinforma=ononphenotypeprofile.
‐ Func=onalprofiling‐ CytvsHelp‐ Th1vsTh2‐ TcmvsTem
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Whatwillwemeasure
Vaccina&oninhumansgeneratesbroadTcellcytokineresponses.DeRosaSC,LuFX,YuJ,PerfeDoSP,FalloonJ,MoserS,EvansTG,KoupR,MillerCJ,RoedererM.JImmunol.2004Nov1;173(9):5372‐80.
“Thepasernofcytokineprofilesdifferbasedontheimmunogen,adjuvantandinfec=ousagent”
Therecanbesubstan=aldifferencesfrompersontopersonTherefore,op=malmeasurementsofimmunogenicityofavaccineorofimmune
responsestoapathogenwillrequirethemeasurementofmul=pledis=nctfunc=onaloutcomes.
Findcompromisebetweensensi=vity,amountofinforma=onrequiredandavailableequipment
Whatwillwemeasure
• IFNg:
– IFN‐gammahasan=viralandan=parasi=cac=vi=es– influencescellmediatedmechanismsofcytotoxicity– modulatorofT‐cellgrowthandfunc=onaldifferen=a=on– …
• IL2:– DuetoitseffectsonT‐cellsandB‐cellsIL2isacentralregulatorofimmuneresponses
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Ac=on!
• CryopreservedPBMC• Pep=dics=mula=on(20hours,BrefAlast18hours)
• Co‐s=mula=onan=CD49d&an=CD28• CD4/CD8• IFNg/IL2• FacsCalibur‐CellQuest• Meanviability(96%,minimum88%)
IFNγ
IL2
Only IL-2
Only IFNγ
IL-2 and IFNγ double positives
=> 4 measures per cell
phenotype
IL-2 and/or IFNγ
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ICS4colors
Ag1 Ag2 Ag3 Ag1 Ag2 Ag3 Ag1 Ag2 Ag3
Ag1 Ag2 Ag3 Ag1 Ag2 Ag3 Ag1 Ag2 Ag3
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ICS4colors
Ag1 Ag2 Ag3 Ag1 Ag2 Ag3 Ag1 Ag2 Ag3
ICS4colors
Ag1 Ag2 Ag3 Ag1 Ag2 Ag3 Ag1 Ag2 Ag3
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ICS4colors
Ag1 Ag2 Ag3 Ag1 Ag2 Ag3 Ag1 Ag2 Ag3
ConclusionsonTRIALXwithICS4colors
• GoodCD‐4responseAker1dose(Ag1>Ag2>Ag3).• Responsedeclinedin=mebutremaineddetectable.• IL2produc=onsugges=ngmemoryresponse.• ModerateIFNgproduc=on(10%oftotalresponse)sugges=ngeffectorresponse.
• NoCD8response
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ConclusionsonTRIALXLymphoprolifera=onassay
• GoodCD‐4responseAker1dose(Ag1>Ag2>Ag3).• Responsedeclinedin=mebutremaineddetectable.• IL2produc=onsugges=ngcentralmemoryresponse.• ModerateIFNgproduc=on(10%oftotalresponse)sugges=ngeffectorresponse.
• NoCD8response
Higher specificity leads to restored sensitivity
Evolu=on(?)….Morecolors….
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Newplayers
• TNFa:
‐pro‐inflammatorycytokine‐cytotoxicity,an=‐viral
• CD40L(CD154):– S=mulatesB‐cellprolifandIgGproduc=on.– inducescytokineproduc=on– cos=mulatesprolifera=onofac=vatedT‐cells
ICS6colors
Ag High Dose + Adjuvant
Ag Low Dose + Adjuvant Ag High Dose Ag Low Dose Adjuvant
dIL2 = IL2 + (TNFa or CD40L or INFg)
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ICS6colors
Ag High Dose + Adjuvant
Ag Low Dose + Adjuvant Ag High Dose Ag Low Dose Adjuvant
dIFNg = IFNg + (TNFa or CD40L or IL2)
ICS6colors
Ag High Dose + Adjuvant
Ag Low Dose + Adjuvant Ag High Dose Ag Low Dose Adjuvant
dTNFa = TNFa + (IL2 or CD40L or INFg)
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ICS6colors
Ag High Dose + Adjuvant
Ag Low Dose + Adjuvant Ag High Dose Ag Low Dose Adjuvant
dCD40L = CD40L + (TNFa or IL2 or INFg)
ICS6colors
Ag High Dose + Adjuvant
Ag Low Dose + Adjuvant Ag High Dose Ag Low Dose Adjuvant
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ICS6colors
Ag High Dose + Adjuvant
Ag Low Dose + Adjuvant Ag High Dose Ag Low Dose Adjuvant
ConclusionsonTRIALYWithICS6colorinfo
• HighlevelofCellularImmuneresponse• HighlevelofIL2‐CD40Lproduc=on• Sugges=veformemoryTcells*• LowlevelsofINFgproduc=on• ModeratelevelsofTNFaproduc=on• Sugges=veforTh1• NoCD8Tcellresponse
*F.Sallusto,D.Lenig,R.Forster,M.LippandA.Lanzavecchia,TwosubsetsofmemoryTlymphocyteswithdis=ncthomingpoten=alsandeffectorfunc=ons,Nature401(1999),pp.
708–712
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ConclusionsonTRIALYWithICS4colorinfo
• HighlevelofCellularImmuneresponse• HighlevelofIL2‐CD40Lproduc=on• Sugges=veformemoryTcells
• LowlevelsofINFgproduc=on• ModeratelevelsofTNFaproduc=on
• Sugges=veforTh1• NoCD8Tcellresponse
ConclusionsonTRIALYWithlymphoprolifinfo
• HighlevelofCellularImmuneresponse• HighlevelofIL2‐CD40Lproduc=on• Sugges=veformemoryTcells
• LowlevelsofINFgproduc=on• ModeratelevelsofTNFaproduc=on
• Sugges=veforTh1• NoCD8Tcellresponse
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Today’sstatusoftheassay
• 9colorexperiment
Antigen Fluorochromes Laser CD3 AlexaFluor-700 633 CD4 Per-CP 480 CD8 APC-Cy7 633 IFNg FITC 480 IL2 APC-Cy7 633
TNFa PE-Cy7 480 CD40L PE 480 Viability AMCyan 405
Dump Channel Pacific Blue 405
Today’sstatusoftheassay
• 15resultcombina=onspersample!(AND)
IFNG IL2 TNFa CD40L IFNG IL2 TNFa IFNG TNFa CD40L IFNG IL2 CD40L IFNG IL2 IFNG TNFa IFNG CD40L IFNG IL2 TNFa CD40L IL2 TNFa IL2 CD40L TNFa CD40L CD40L IL2 TNFa
Andallit’sbooleancombina=ons….(OR)
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Today’sstatusoftheassay
• 15resultcombina=onspersample!(AND)
• Andallit’sBooleancombina=ons….(OR)
• Leadstothegenera=onof3–4gigabyteofdataperday(1experimentwith150samples)
• Automatedrou=nesareessen=altoanalyzedata
Morefunc=onalinforma=onavailable
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Acknowledgments
CEVAC CORE LAB TEAM (Past and Present)
Andrea Verwulgen Jan De Schilder Agnes Van De Putte Annelies Goussaert Freya Van Houtte Caroline Buysschaert Manuela Cousin Lore Dewulf Marijke Van Nimmen Filip Clinckspoor Petra Premereur Ellen Demeyere Sabrina Verlee Anja Coen Sofie Librecht Miryam Hautera Sibyl Couvent Yvonne Gijbels David Thonnard Thora Vanderstock Peter Vanderlinden
Prof. Dr. G. Leroux-Roels
Frederic Clement
Center For Vaccinology Ghent University
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Back‐upslides
Analysisofreproduc=bility&repeatability(R&R)
Requirements:
– mimicactualworkingcondi=ons– mimicnormalworkload– blind.
Condi=ons:
– 3operators– 10samples(2repeats/day)– 4days– 4s=mula=oncondi=ons(3Ag+Bkg)– Totalof80tubes/operator/day
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R&RresultsPo
sitiv
e CD
4 /
10e 6
CD
4
R&Rresults
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R&Rresults
R&Rresults
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R&Rconclusion
anova3
N obs N miss obs mean est mean cv repeat cv repro
operator
cv RR opera
tor cv repro
day cv RR day cv RR
measure
211 29 1107 1175 43.35 20.00 47.74 18.75 47.23 47.74
95% Prediction Interval
value lower upper 1000 -100 2100
1 -1098.8386 1100.8386
