TCR activation kinetics and feedback regulation in primary human T cells.

1.3 MB·English

by  Poltorak, Mateusz

Poltoraketal.CellCommunicationandSignaling2013,11:4
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RESEARCH Open Access
TCR activation kinetics and feedback regulation in
primary human T cells
Mateusz Poltorak, Boerge Arndt, Bhavani S Kowtharapu, Amarendra V Reddycherla, Vanessa Witte,
Jonathan A Lindquist, Burkhart Schraven and Luca Simeoni*
Abstract
Background: Signaling through theTCRis crucial for the generation ofdifferent cellular responses including
proliferation, differentiation,and apoptosis. A growing body of evidence indicates that differences in themagnitude
and the duration ofthe signal are critical determinantsin eliciting cellular responses.
Results: Here, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation
induced upon TCR/CD28 ligation inprimary humanT cells. We used two widely employed methods to stimulate T
cellsin vitro, antibodies either cross-linked insolution (sAbs) or immobilized onmicrobeads (iAbs). A comparative
analysis of thesignaling properties ofiAbs and sAbs revealed that,under proliferation-inducing conditions,
feedback regulation is markedly different from that leading to an unresponsivestate. In fact, upon iAbs stimulation
TCR-mediated signaling is prolongedby a positive feedback loop involving Erk, whereas sAbs strongly activate
inhibitory molecules thatlikely terminate signaling. We additionally found that, byenhancing thephosphorylation
ofSrc family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated.
Conclusions: In summary, our analysis documents TCRsignaling kinetics and feedback regulation under conditions
ofstimulation inducing either unresponsivenessor proliferation.
Keywords: TCR-mediated signaling, Feedback regulation, Cell-fate specification, T-cell activation, Lck, Erk, Signaling
dynamics, CD4crosslinking
Background that NGF and EGF elicit different feedback regulation of
Ligation of theTcell receptor (TCR) triggers intracellular the Ras-Erk cascade, which in turn results in distinct
signals which may result in the initiation of markedly dif- temporalprofilesofErkactivity[4].WhereasEGFtriggers
ferent cellular programs leading to differentiation, activa- anegativefeedbackshuttingoffRaf-1activity,NGFstimu-
tion, survival, or apoptosis of T cells. One of the major lation induces a positive feedback regulation of Raf-1.
questionsin cell biologyis how the activation of the same Differences in the duration of Erk activity are sensed by
canonical signaling cascades dictates distinct biological downstreamtranscriptionfactors,thusalteringtheexpres-
outcomes. How signals areinterpreted and translated into sion of specific genes required to carry out the cellular
specific cellular outcomes has been extensively studied in responses[5].
PC-12 cells [1,2]. In these cells, it appears that differences AsimilardynamicbehaviorofErkactivityseemstoexist
in the magnitude and the duration of the Erk signal are also in thymocytes where strong and transient Erk activa-
critical determinants in eliciting the cellular response. For tion induces apoptosis, whereas moderate but sustained
example, sustained Erk activation upon NGF treatment Erk activity induces differentiation of immatureTcells [6]
causesdifferentiation of the PC-12 cell line, whereas tran- andCD8+TCRtransgenicTcells[7].Howthedynamicsof
sient Erk activation upon EGF stimulation induces prolif- ErkactivationisregulatedinmatureTcellsisnotyetclear.
eration in the same cells [3]. Further studies have shown Here, we have used primary human T cells to analyze
TCR activation kinetics and feedback regulation. T cells
were stimulated with CD3 and CD28 antibodies either
*Correspondence:[email protected]
cross-linkedinsolution(sAbs)orimmobilizedonmicrobe-
InstituteofMolecularandClinicalImmunology,Otto-von-GuerickeUniversity,
LeipzigerStr.44,39120,Magdeburg,Germany ads(iAbs),whicharetwocommonlyusedmethodstostudy
©2013Poltoraketal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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T-cell activation. Stimulation with sAbs induces only a kinetics and cellular responses (Figure 1). Stimulation
transientsignalandanabortiveT-cellresponseresultingin with sAbs resulted in a strong and transient induction of
unresponsiveness, whereas stimulation with iAbs induces global tyrosine phosphorylation, as well as of ZAP70,
sustainedactivationoftheErkcascadeandcellproliferation LAT, and PLCγ-1. In contrast, when primary human
[8]. We provide evidence that feedback regulation under Tcells were treated with iAbs, global tyrosine phosphor-
proliferation-inducing conditions is markedly different ylation and the phosphorylation of ZAP70, LAT and
from that leading to unresponsiveness. We demonstrate PLCγ-1 were weak, but sustained (Figure 1A, B). Add-
that sAbs activate negative feedback loops that terminate itionally, phosphorylation of TCRζ was greatly and rap-
TCR-mediated signaling, whereas stimulation with iAbs idly enhanced upon sAbs. In contrast, iAbs stimulation
resultsintheestablishmentofapositivefeedbackloopin- induces only weak TCRζ phosphorylation (Figure 1C).
volving Erk-mediated phosphorylation of Lck prolonging Interestingly, the phosphorylation kinetics of PAG/Cbp,
TCR-mediated signaling. Collectively, our analysis pro- a transmembrane adaptor protein running at about
videsnovelinsightsintotheregulationofthedynamicbe- 70 KDa which is dephosphorylated upon TCR stimula-
havior of theTCR signaling module that controls cell-fate tion [9], is comparable under both stimulation condi-
specificationinprimaryhumanTcells. tions (Figure 1A). We next analyzed the signaling
kinetics of the Erk cascade. Surprisingly, we found that
Results Erk was very strongly activated under both conditions of
SustainedTCR-mediatedsignalingcorrelateswith stimulation. However, the activation induced by iAbs was
proliferation,whereastransientsignalingparallelswith sustained and lasted up to 90 minutes whereas, upon
unresponsiveness stimulation with sAbs Erk activation was transient, peaked
Peripheral human Tcells were stimulated using sAbs or at1–5minutes,andrapidlydeclinedthereafter(Figure1D).
iAbs. These stimuli induce markedly different activation Thus, despite the weak activation of proximal signaling
Figure1AnalysisofthesignalingsignatureandfunctionaleffectsofsAbsandiAbsstimulation.A-D)PurifiedhumanTcellsweretreated
witheithersoluble(sAbs)orimmobilized(iAbs)CD3xCD28mAbsfortheindicatedtimeperiods.Totalcelllysates(A,B,andD)orTCRζ
immunoprecipitatesC)werepreparedandanalyzedbyWesternblottingusingtheindicatedAbs.Onerepresentativeimmunoblotofatleast3
independentexperimentsisshown.ThephosphorylationofTCRζwasquantifiedusingthe1DImageQuantsoftwareandthevalueswere
normalizedtothecorrespondingtotalTCRζsignal.Datarepresentthemeanofthephosphorylationlevelsshownasarbitraryunits±SEMof3
independentexperiments.E)TcellswerelabeledwithCFSEandstimulatedasindicated.Proliferationwasassessedafter72hbyanalyzingCFSE
contentonaFACSCalibur.Onerepresentativeexperimentofthreeindependentexperimentsisshown.
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molecules, iAbs are capable of inducing strong and pro- TCRsignalosomeandcanregulatetheamplitude,thedur-
longedactivationofdownstreamsignalingpathways. ation, and the specificity of the signal (reviewed in Acuto
It is generally accepted that transient signals triggered etal.[13]).Weaskedthequestionofwhetherthestimula-
by soluble antibodies cannot induce productive T-cell tion with sAbs induced the activation of negative regula-
responses. Indeed, we and others have demonstrated that torymoleculesthatmayterminatesignaling,thusresulting
human peripheral Tcells, mouse OT-I transgenic Tcells, in the transient signal observed above. Among the many
and cytotoxic T-lymphocyte clones are not activated and inhibitory molecules organizing negative regulatory cir-
do not differentiate upon stimulation with antibodies cuits,wedecidedtofocusonc-Cbl,anE3ubiquitinligase
cross-linked in suspension [7,8,10]. Here, we have stimu- belonging to the CBL family, and the adaptor protein
latedprimaryhumanTcells witheithersAbsor iAbs and Dok2, which regulate TCR-mediated signaling through
analyzed their functional responses. Treatment with sAbs two different mechanisms. Whereas members of the CBL
failed to induce T-cell proliferation (Figure 1E). Con- family are involved in the downregulation of signaling
versely, Figure 1E shows that treatment of T cells with molecules via ubiquitination [14], Dok2 and its homolog
iAbsledtoastrongproliferativeresponse. Dok1inhibitthe activation of signalingpathwaysby com-
peting for binding to SH2 domains or by recruiting other
negative regulators, such as SHIP1 and RasGAP, to the
Transientsignalingisregulatedvianegativeregulatory TCR signalosome [15]. The activity of both c-Cbl and
feedbacks Dok2havebeenreportedtoberegulatedbytyrosinephos-
Thedatapresentedabove,showthatsAbsinducedarapid, phorylation [15-17] and can be easily monitored by using
buttransientTCR-mediatedsignalingkinetics,whichcan- anti-c-Cbl and anti-Dok2 phosphospecific antibodies, re-
notinduceproductiveT-cellresponse,whereasstimulation spectively.Figure2BshowsthatuponsAbsstimulation,T
with iAbs resulted in a sustained activation of a variety of cells very rapidly and strongly phosphorylated both c-Cbl
signalingmoleculesandledtoproliferation.Thesedatain- andDok2,whereas, treatmentof humanTcellswithiAbs
dicate that there may be different regulatory mechanisms resulted only in a very weak phosphorylation of both
induced upon sAbs vs. iAbs stimulation. Thus, we next molecules.
investigated how TCR-mediated signaling is differentially c-Cbl targets many signaling molecules for degradation,
regulatedunderthetwoconditions.Wehypothesizedthat includingZAP70[18].Thus,wenexttestedwhethersAbs,
a fast internalization of the availableTCR molecules upon in addition to inducing strong c-Cbl phosphorylation,
stimulation with sAbs could provide an explanation for wouldalsoinduceZAP70ubiquitinationanddegradation.
the rapid termination of TCR-mediated signaling. There- WehavepreviouslyshowninmouseOT-ITcellsthatubi-
fore, we compared the expression levels of the TCR after quitination of ZAP70 results in the appearance of ZAP70
stimulation with either sAbs or iAbs by flow cytometry. bands displaying retarded migration in SDS-PAGE [7].
Figure 2A shows that sAbs induce a slow rate of TCR WecheckedwhetherstimulationwithsolubleCD3xCD28
downregulation,whichbecameevidentafter30minutesof Abs also resulted in the appearance of ZAP70 bands
stimulation.Itisimportanttonotethatthemajorityofthe running at a higher molecular weight in primary human
signaling molecules that we have tested reverted to the T cells and we found that activation/phosphorylation of
dephosphorylated/inactive state already 15 minutes after c-Cbl upon stimulation with sAbs indeed correlates with
sAbs stimulation (Figure 1). Therefore, termination of retarded ZAP70 migration (Figure 2C). Additionally, the
TCR-mediated signaling occurs before TCR internaliza- data presented in Figure 2C suggest that stimulation with
tion. On the other hand, the data presented in Figure 2A sAbsalsoinducedZAP70degradation.Conversely,stimu-
show that stimulation with iAbs does not reduce, but ra- lation with iAbs did not significantly induce either c-Cbl
ther slightly increasesTCR levels. This is likely due to the phosphorylationorretardedmigrationanddegradationof
fact that Abs bound to a solid matrix limit TCR internal- ZAP70(Figure2C).Thus,itappearsthatstimulationwith
ization, but do not interfere with its transport to the sAbs activates inhibitory feedback loops that may be re-
plasma membrane. Moreover, we have previously shown sponsibleforterminatingTCR-mediatedsignaling.
that sustained TCR-mediated signaling and proliferation In addition to inducing a strong tyrosine phosphoryl-
can occur under conditions of stimulation inducing TCR ation of c-Cbl and Dok2, stimulation with sAbs also
downregulation [7]. Thus, on the basis of these observa- results in a strong phosphorylation of TCR proximal sig-
tions, we exclude that TCR internalization induced by naling molecules including TCRζ, ZAP70, and LAT
sAbsisthecauseoftransientsignaling. (Figure 1B, 1C). Therefore, we investigated whether sAbs
Havingruledoutthispossibility,wenextfocusedonthe induce a stronger activation of the tyrosine kinases Lck
analysis of feedback regulation events, which have been andFyncomparedtoiAbs.WeimmunoprecipitatedTCRζ
shown to play a crucial role in T-cell activation [11-13]. and assessed the level of active Lck and Fyn associated
Proximal negative feedback loops can be activated by the with the TCR. As shown in Figure 2D, sAbs stimulation
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Figure2sAbs,butnotiAbs,induceinhibitoryfeedbackloops.PurifiedhumanTcellsweretreatedwitheithersoluble(sAbs)orimmobilized
(iAbs)CD3xCD28mAbsfortheindicatedtimepoints.A)MeasurementofTCRinternalization.TheexpressionoftheTCRwasassessedby
PE-conjugatedanti-TCRαβmAbstaininganalysisbyflowcytometry.Datarepresentthe%ofthemeanfluorescenceintensity(MFI)oftheTCR
expressionrelativetotime0of4independentexperiments.B)Thephosphorylationofc-CblonY731andDok2onY351wasdeterminedby
Westernblotting.Thephosphorylatedc-CblandDok2bandswerequantifiedusingthe1DImageQuantsoftwareandthevalueswerenormalized
tothecorrespondingβ-actinsignal.Datarepresentthemeanofthephosphorylationlevelsshownasarbitraryunits±SEMofatleast4
independentexperiments.C)TheexpressionofZAP70wasdeterminedbyWesternblotting.Equalloadingisshownbyreprobingimmunoblots
withantibodiesspecificforβ-actin.ZAP70bandswerequantifiedasabove.Datarepresentthemeanoftheexpressionlevelsshownasarbitrary
units±SEMof6independentexperiments.D)TyrosinephosphorylationofFynandLckintheactivationloopwasdeterminedbyWestern
blottingusingthepSrc(Y416)antibody.Bandswerequantifiedusingthe1DImageQuantsoftwareandvalueswerenormalizedtothe
correspondingtotalFynandLcksignals.Dataonthegraphrepresentthemeanofthephosphorylationlevelsshownasarbitraryunits±SEMof4
independentexperiments.Asterisks(*)indicatetheIgheavychain.Statisticalanalysis**P<0.01;ns,notstatisticallysignificant.
significantlyenhancesthelevelofLckandFynphosphory- Sustainedactivationisregulatedbypositivefeedback
latedontheactivationloop,whichisbelievedtobeasign loops
of an active enzyme. Conversely, this significant increase Wenextinvestigatedwhetherpositivefeedbackloopsmay
inLckandFynphosphorylationisnotobserveduponiAbs be triggered by iAbs, thus leading to sustained activation
stimulation. Hence, the data suggest that, in marked con- of TCR-mediated signaling. In particular, we explored the
trasttoiAbs,sAbsstimulationenhancesLckandFynacti- regulatory circuit involving Lck phosphorylation by
vation. We postulate that the enhanced activation of Lck activated Erk [11]. This model is based on observations
andFynmayresultinastrongertyrosinephosphorylation showing that the Erk-mediated phosphorylation of Lck
of downstream molecules (including negative regulators, on serine 59 alters Lck mobility and the ability of the
such as c-Cbl and Dok2), which might imbalance TCR- SH2 domain of Lck to bind phosphotyrosines [19-21].
mediatedsignaling,thusdampeningT-cellactivation. Stefanova et al. further demonstrated that Erk-mediated
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phosphorylation of Lck prevents SHP-1 binding, thus phosphorylation or prevent phosphorylation, respectively.
interfering with SHP-1-mediated Lck inactivation [11]. Weused the followingmutantsS59D,S59A, S42D,S42A,
According to this model, active Erk would feedback to and S42A/S59A, which were expressed in the Lck-
Lck to sustain signaling. To assess whether stimulation deficient Jurkat T-cell line J.CaM1.6. As shown in
withiAbstriggersthisErk-mediatedpositivefeedbackloop, Figure3D,mutationsofS42donotaffectthemobilityshift
Tcells were stimulated with iAbs and sAbs and the phos- of Lck either in unstimulated or iAbs stimulated cells.
phorylation of Lck on S59 was detected by the appearance Conversely, the S59D mutation results in a constitutive
of a new Lck band running at 59 kDa by Western blot shift to p59 Lck, thus indicating that phosphorylation on
[11]. As shown in Figure 3A and B, stimulation of Tcells this siteplays a major rolein theregulation of Lckmobil-
with iAbs clearly resulted in the formation of p59 Lck (up ity. Accordingly, the S59A substitution, which results in a
to 50% of total Lck), whereas this shift in the molecular non-phosphorylatable mutant, prevents the generation of
weightofLckwasbarelydetectableuponsAbstreatment. the59kDaformofLckuponiAbsstimulation(Figure3D).
To demonstrate that the appearance of p59 Lck in- In summary, these data demonstrate that Erk-mediated
deed depends on Erk-mediated phosphorylation,Tcells phosphorylationofLckat S59 resultsinitsretarded mobil-
werestimulatedwithiAbsfor 30mininthepresenceor ityonSDS-PAGE.
absence of U0126 or MEK Inhibitor I, inhibitors of the To check whether the inhibition of Erk-mediated Lck
Erk activator MEK. This treatment has previously been phosphorylationalsoresultedinareductionofitsactiv-
shown to abolish the conversion of Lck to the p59 form ity, we investigated phosphorylation levels of down-
[11]. In agreement with these observations, we also stream signaling molecules that are substrates of Lck,
found that treatment of iAbs-stimulated T cells with such as thetyrosinekinase ZAP70 and the adaptorpro-
U0126 or MEK Inhibitor I completely abolished both tein LAT whose phosphorylation depends on ZAP70. T
Erk activation and the shift of Lck to the p59 form cells were stimulated for 30 min with iAbs. Subse-
(Figure3C).Wenexttestedwhetherthemolecularshift quently, Erk activity was blocked by the addition of the
of Lck upon iAbs stimulation is indeed induced by MEK inhibitor U0126. The data presented in
phosphorylation of S59. To assess this issue, we took ad- (Figure 4A, B) show that the phosphorylation of both
vantage of Lck constructs carrying S to D and S to A ZAP70 and LAT is reduced upon MEK inhibition, thus
mutations at this position, which mimic constitutive indicating that Erk-mediated Lck phosphorylation may
Figure3iAbsinduceanErk-mediatedpositivefeedbackloop.A)PurifiedhumanTcellsweretreatedwitheithersoluble(sAbs)or
immobilized(iAbs)CD3xCD28mAbsfortheindicatedtimepoints.Lckexpressionwasdetectedincelllysatesbyanti-Lckimmunoblotting.B)
Bandscorrespondingtop56orp59LckwerequantifiedasdescribedinFigure2.Datarepresenttheratioofthelevelsofp56andp59,andtotal
(p56+p59)Lckshownasarbitraryunits±SEMof5independentexperiments.C)PurifiedhumanTcellsweretreatedwithimmobilized(iAbs)
CD3xCD28mAbsfortheindicatedtimeperiodsinthepresenceorabsenceofMEKInhibitorIorU0126.SampleswereanalyzedbyWestern
blottingusingtheAbsindicated.Onerepresentativeimmunoblotof4independentexperimentsisshown.D)J.CaM1.6cellsweretransfected
withvariousconstructscarryingdifferentmutations(S42A,S42D,S59A,S59D,S42A/S59A).Aftertransfection,cellswereeitherleftunstimulatedor
stimulatedwithiAbsfor45min.SampleswereanalyzedbyWesternblottingusingtheindicatedAbs.Onerepresentativeexperimentoffive
independentexperimentsisshown.
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Figure4AnErk-LckfeedbackloopregulatesTCR-mediatedsignaling.A)PurifiedhumanTcellsweretreatedwithiAbsalonefor30min
andtheneitherDMSOortheMEKinhibitorU0126wasaddedandincubatedforanadditional30to60min.SampleswereanalyzedbyWestern
blottingusingtheindicatedAbs.B)BandsinA)werequantifiedandthevalueswerenormalizedasdescribed.Graphsshowthemeanofthe
phosphorylationlevelsofErk1/2,ZAP70,andLATorthelevelofp59Lckasarbitraryunits±SEMof4independentexperiments.C)Purified
humanTcellswerepreincubatedeitherinthepresenceofDMSOortheMEKinhibitorU0126andsubsequentlystimulatedwithsAbsforthe
indicatedtimepoints.SampleswereanalyzedbyWesternblottingusingtheindicatedAbs.D)BandsinC)werequantifiedasdescribedabove
andthedatafromatleasttwoindependentexperimentsareshown.
enhance its response. Conversely, treatment of sAbs- EnhancementofSrckinasesphosphorylationconverts
stimulated Tcells with the MEK inhibitor reduced Erk sustainedintotransientsignal
phosphorylation, as expected, but not ZAP70 or LAT Thedata presented above suggestthat sAbs andiAbs in-
phosphorylation(Figure4C,D). duce qualitatively different signals and feedback regula-
Collectively, these data suggest that stimulation with tion which are translated into distinct cellular responses.
iAbs activates an Erk-mediated positive feedback loop How the cell senses the quality of the signal is not yet
whichisrequiredforproperT-cellresponseandprolifera- fully understood. Our data suggest that sAbs induce
tion. Importantly, the regulatory circuit induced by iAbs stronger Src kinases activation and a stronger tyrosine
seems to mimic a previously described mechanism that is phosphorylation pattern compared to iAbs stimulation
inducedinTcellsuponphysiologicalstimulation[11]. (Figure 1). These observations may suggest that Src
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kinases are involved in deciphering the nature of the sig- In summary, we have shown that stimulation with
nal.TotestthecontributionofLck,themajorSrckinase iAbs induces different feedback regulation than sAbs
in T cells, in the regulation of signaling dynamics, we treatment (Figure 6). sAbs lead to strong and rapid acti-
suppressed its expression by RNAi in Jurkat Tcells and vation of Src kinases and subsequently to the phosphor-
evaluated the effects on Erk activation. Figure 5A shows ylation of inhibitory molecules (e.g. c-Cbl, Dok2), which
that cells expressing low amount of Lck displayed pro- terminatesignaling.Ontheotherhand,iAbsinduceonly
longed Erk1/2 activation. These observations are in line slight increase in kinase activity and an Erk-Lck positive
with previous studies showing that knockdown of Lck in feedback loop, which may be required to prevent rapid
Jurkat and primary human T cells prolonged Erk phos- Lck dephosphorylation by SHP-1 or other phosphatases,
phorylationandtranscriptionalactivation [22,23]. andtherefore leadtosustained activation.
We next decided to investigate whether strong phos-
phorylationofLckandFynmayconvertasustainedintoa Discussion
transientsignal.Tothisaim,CD4+primaryhumanTcells Signaling through a variety of plasma membrane-
werestimulatedwithiAbsforashorttimeperiodandsub- associated receptorsleads to cell decision processes such
sequently CD4 was cross-linked using soluble anti-CD4 as cell proliferation, differentiation, survival, and motil-
mAbs. It is known that CD4 crosslinking results in trans- ity. Considerable evidence suggests that the magnitude
phosphorylation of Lck, thus strongly enhancing its activ- and the duration of a signal determine the functional
ity. As presented in Figure 5B, CD4 crosslinking indeed outcome. As little is known on the mechanisms regulat-
resulted in a strong induction of Lck phosphorylation ing signaling kinetics correlating with cellular responses
measured using an anti-pY416Src antibody. Most import- in T cells, we have analyzed TCR-mediated signaling
antly,enhancedLckphosphorylationparalleledwithasig- under conditions leading to either T-cell unresponsive-
nificant reduction in Erk phosphorylation (Figure 5B). ness or to proliferation. We employed sAbs and iAbs
Accordingly,wefoundthatalsoCD69expressionandpro- stimulation which induce qualitatively different signals
liferation were strongly reduced upon CD4 crosslinking and T-cell responses [8]. We found striking differences
(Figure 5C, D). These data suggest that strong Src-family in TCR signaling kinetics and feedback regulation. In-
kinase activity may result in the activation of inhibitory deed, under proliferation-inducing conditions, TCR-
signalssuppressingT-cellactivation. mediated signaling is prolonged by a positive feedback
Figure5LckphosphorylationcorrelateswithdecreasedT-cellactivation.A)JurkatTcellsweretransfectedwithLcksiRNAduplexorsiRNA
control(Ctrl)andculturedfor48h.Subsequently,cellswerestimulatedwithsolubleCD3mAbs(cloneOKT3)fortheindicatedtimes.Celllysates
wereanalyzedbyimmunoblottingusingtheindicatedAbs.ImmunoblotverifyingLckdownregulationisshown.Onerepresentativeexperiment
ofthreeindependentexperimentsisshown.B)-D)PurifiedhumanCD4+Tcellsweretreatedwithimmobilized(iAbs)CD3xCD28mAbsinthe
presenceorabsenceofcross-linkedCD4mAbasindicated.B)ThephosphorylationlevelsofErk1/2andSrckinasesweredeterminedbyWestern
blotting.Thephospho-specificbandswerequantifiedusingthe1DImageQuantsoftwareandthevalueswerenormalizedtothecorresponding
β-actinsignal.Dataonthegraphrepresentthemeanofthephosphorylationlevelsshownasarbitraryunits±SEMof3independent
experiments.C)24hafterstimulation,theactivationofCD4+TcellswasanalyzedbystainingwithCD69andflowcytometry.Dataonthegraph
representthemeanoftheexpressionlevelsshownasarbitraryunits±SEMof3independentexperiments.D)CD4+Tcellswerelabeledwith
CFSEandstimulatedasindicated.Proliferationwasassedafter72hbyanalyzingCFSEcontentonaLSRFortessa.Onerepresentativeexperimentof
3independentexperimentsisshown.
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Figure6FeedbackregulationofTCR-mediatedsignaling.sAbsstimulationtriggersstrongphosphorylationofSrckinases,suchasLck,and
leadstostrongactivationofdownstreamsignalingpathways.Inadditiontotheactivationofpositiveregulators,sAbsalsoinduceinhibitory
molecules(c-Cbl,Dok2),whichmightimbalanceTCR-mediatedsignaling,thusrapidlyterminatingT-cellactivation(leftside).Ontheotherhand,
iAbsstimulationresultsintheactivationofanErk-Lckpositivefeedbackloop,whichisrequiredtosustainsignaling(rightside).
loop involving Erk and Lck. Conversely, stimulation with proposed by Nika et al., the pool of constitutively active
sAbs strongly activates inhibitory molecules that likely Lck is sufficient to initiate the signaling cascade [25].
terminatesignaling.Theseobservationsareinagreement This weaker signal will in turn activate positive feedback
with the model proposed by Acuto et al., that the signal loops which enhance the strength and prolong the acti-
amplitudeandkineticsindoublepositivethymocytesde- vation of more distal signaling cascades, thus culminat-
pend on the type of the applied stimulus [13]. Here, we inginproliferation.
show that a similar principle may apply also to matureT How Lck senses the characteristic of the stimulus trig-
cells. gering the TCR, which will in turn result in the gener-
An important question that needs further investiga- ation of the appropriate cellular program, is not yet
tions is how signals with a common origin at the TCR known. However, when we compared sAbs vs. iAbs, we
are split to activate different effector molecules. During found that Lck undergoes different phosphorylation
thymocyte development, it has been proposed that a events. Whereas sAbs enhance phosphorylation of Lck
molecule or complex functioning as “signal splitter” at Y394, which is believed to enhance its kinase activity,
senses the intensity of signals emanating from the TCR iAbs induce phosphorylation of Lck at S59. We propose
and discriminates between negatively and positively that phosphorylation at Y394 induced by sAbs results in
selecting ligands [24]. However, such a molecule has not a hyper-phosphorylation of downstream signaling mole-
yet been identified in immatureTcells. We propose that cules that disturbs the equilibrium between positive and
Lck may function as “signal splitter” in mature T cells negative regulators of TCR-mediated signaling, favoring
directing signals emanating from the TCR toward unre- inhibitory signals (like c-Cbl) that shutdown T-cell acti-
sponsiveness if the signal is at high intensity (i.e. in case vation. This hypothesis is also supported by our observa-
ofstimulation withsAbs,which inducestrongSrckinase tions and previously published data showing that
activation). This safety mechanism could be set in mo- suppression of Lck expression by RNAi strongly impaired
tion in case of an inappropriate stimulation that could the activation of the inhibitory molecules SHP-1 and
lead to T-cell hyperactivation and the development of c-Cbl and alsoprolongeddownstream signaling (i.e. pErk,
autoimmunity. The idea that the molecular switch is NFAT/AP-1) induced by soluble CD3 stimulation [22,23].
located at the apical part of the cascade could represent Thus,strongLckactivationmayhaveinhibitoryeffectson
an advantage of the system. In fact, the termination of T-cell activation. On the other hand, phosphorylation on
the signal at a membrane proximal level will require S59mayberequiredtopreventrapiddeactivationbySHP-
only the activation of a limited number of downstream 1.Moreover, if Lck becomes strongly active, this would in
inhibitory pathways to efficiently stop activation. In case turn shutdown signaling as in the case of sAbs stimula-
of an appropriate stimulus, such as iAbs, Lck activity is tion. Interesting in this regard, we found that iAbs stimu-
not substantially increased over the basal level. As lation not only enhances phosphorylation on S59 but
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concomitantly reduces phosphorylation of Y394 at later by non-Tcell depletion usingTcell isolation kits(Miltenyi
time points after activation (Figure 2D and our unpub- Biotec). The purity of Tcells, determined by flow cytome-
lishedresults). try,wasusuallymorethan96%.
We also found that crosslinking of CD4 in cells under-
going activation dampen T-cell responses. We propose
that a strong activation of Src kinases induced upon CD4 T-cellstimulation
crosslinking may triggers inhibitory feedback loops in a After isolation, Tcells were cultured overnight in RPMI
similar manner to sAbs. Interestingly, when anti-CD4 is 1640 medium containing 10% FCS (PAN Biotech) and 2
immobilizedtogetherwithanti-CD3onmicrobeads,T-cell μg/mlCiprobay(BayerScheringPharma).Successively,T
activation is enhanced compared to stimulation with anti- cells were stimulated with either soluble or immobilized
CD3 alone. Under this condition, an enhanced Src kinase mAbs as follows. For soluble Ab stimulation, 2x106 cells
phosphorylation was not observed [8]. Previous observa- were loaded with 10 μg/ml biotinylated anti-human CD3
tions had shown that crosslinking of CD4 before stimula- (cloneUCHT1,eBioscience)incombinationwith10μg/ml
tion also impaired T-cell activation [26]. This mechanism biotinylatedanti-humanCD28(cloneCD28.2,eBioscience)
has been implicated in T-cell depletion occurring during mAbs in 100 μl RPMI 1640 for 15 min on ice. After
HIV infection [27]. Our data suggest that crosslinking of washing, receptors were cross-linked by adding 25 μg/ml
™
CD4 by gp120 and anti-gp120 antibodies may shutdown NeutrAvidin (Pierce). For microbead stimulation,
™
T-cell activation also during immune responses in HIV- SuperAvidin -coated polystyrene microspheres (Ø~10
infectedpatients,thuscontributingtoimmunodeficiency. μm, Bangs Laboratories) were coated with biotinylated
In addition to Lck, we have found major differences in CD3incombinationwithCD28mAbs(10μg/mleach)for
theregulationofErkactivationbetweensAbsandiAbs.It 30 min at 37°C in PBS. Antibody-coated microbeads were
has been previously shown that Erk activity in cytotoxic washed twice with PBS, resuspended in RPMI 1640 and
mouseT lymphocytes after stimulation with immobilized incubatedwithTcellsina1:1ratio.Forstimulationofpre-
antibodies depends on nPKCs, whereas, sAbs stimulation activated cells 10 μg/ml of purified IgM anti-human CD4
activates Erk also via cPKCs [28]. Thus, TCR-mediated (cloneMEM16,kindlyprovidedbyV.Horejsi,Academyof
Erk activation under condition of stimulation correlating SciencesoftheCzechRepublic,CzechRepublic)wasused.
with proliferation appears to be not only quantitatively, For Jurkat T cell stimulation soluble CD3 mAbs (clone
butalsoqualitativelydifferentfromthatinducedbysAbs. OKT3)wasused.
Stimulations in the presence of either the MEK inhibi-
Conclusions tor I, U0126 (Cell Signaling Technology) or DMSO
Insummary,weshowthatTCR-mediatedsignalingkinetics (Sigma-Aldrich) were performed by pre-incubating
and feedback regulation under proliferation-inducing con- T-cells for 30 min with 10 μM of the compounds before
ditions (iAbs) are markedly different from those leading to stimulation with mAbs. For indicated microbead stimu-
unresponsiveness (sAbs) and we provide some potential lation, 10 μM of either UO126 or DMSO were added 30
mechanistic insights that may explain this differential be- minafterstimulation.
havior. We hope that the comparative analyses presented
here will inspire further studies aimed at dissecting the Celltransfections
spatio-temporalregulationofT-cellactivation. The Jurkat T-cell line and Lck-deficient variant of the
Jurkats (J.CaM1.6) were maintained in RPMI 1640
Methods medium supplemented with 10% FCS (PAN Biotech) and
HumanEthics antibiotics at 37°C and 5% CO . For cell transfection, we
2
Approval for these studies involving the analysis of used pBos expression plasmid encoding various Lck
TCR-mediated signaling in human Tcells was obtained constructs (S42A, S42D, S59A, S59D, S42A/S59A). For
from the Ethics Committee of the Medical Faculty at the RNAi experiments siRNA Lck duplex containing 21
Otto-von-Guericke University, Magdeburg, Germany nucleodites was purchased from Life Technologies. The
with the permission number [107/09]. Informed consent sequences were as follows, sense: 5’–UAACCAGGUU
was obtained in writing in accordance with the Declar- GUCUUGCAGUG–3 antisense: 5’–CUGCAAGACAAC
ationofHelsinki. CUGGUUAUC–3’. As a negative control we used a
Renilla Luciferase siRNA duplex 5´–CCAAGUAAU
Cellpurification GUAGGAUCAATT–3’. To achieve efficient transfection,
Peripheral blood mononuclear cells were isolated by Ficoll JurkatTcellswereelectroporatedusingtheGenePulserII
gradient (Biochrom) centrifugation of heparinized blood (Bio-Rad) as previously described [29]. 48 h after electro-
collected from healthy volunteers. Total population of porationcellswerecollected,stimulatedwithiAbsorsAbs
humanTcellsorCD4+subpopulationwerefurtherpurified asindicated,andprocessedforWesternblotting.
Poltoraketal.CellCommunicationandSignaling2013,11:4 Page10of11
http://www.biosignaling.com/content/11/1/4
Immunoprecipitation T cells were cultured for 72 h at 37°C, 5% CO . Prolif-
2
Primary human Tcells (3×107) were either left untreated eration was assessed by CFSE dilution using a BD
or stimulated with sAbs or iAbs for the indicated periods LSRFortessa, FACSDiva Software 6.1.3 (BD Biosciences),
of time. Cells were lysed in 1% Brj58 or 1% lauryl malto- andFlowJo 7.6.5(TreeStar,Inc.).
side(N-dodecylβ-maltoside),1%IGEPALCA-630,1mM To determine the efficiency of T-cell activation,Tcells
Na VO , 1 mM PMSF, 10 mM NaF, 10 mM EDTA, were stimulated as described above. After 24h, T cells
3 4
50 mM Tris pH 7.5, and 150 mM NaCl, and cleared by were stained with PE-labeled mAbs against CD69 (BD
centrifugation. TCRζ chains were immunoprecipitated Biosciences)andanalyzedbyflow cytometry.
withagarose-conjugatedCD3ζ(SantaCruzBiotechnology)
antibody followed by recombinant protein A-agarose TCRinternalization
beads (Santa Cruz Biotechnology) at 4°C overnight. After To determine TCR internalization, 1×106 cells were sti-
washing, TCRζ immunoprecipitates were resolved by mulated with sAbs or iAbs as mentioned above at 37°C
SDS-PAGE, transferred to a nitrocellulose membrane for 0–60 min. Cells were stained with PE-conjugated
(Amersham), and analyzed by immunoblotting with the TCRαβ mAb (BD Biosciences) for 15 min at 4°C and
indicatedantibodies. analyzedbyflow cytometry.
Abbreviations
Westernblotting
TCR:Tcellreceptor;sAbs:Antibodiescross-linkedinsolution;
Tcells were lysed in buffer containing 1% lauryl malto- iAbs:Immobilizedonmicrobeads.
side (N-dodecyl β-maltoside), 1% IGEPAL CA-630, 1
Competinginterests
mM Na VO , 1 mM PMSF, 10 mM NaF, 10 mM EDTA,
3 4 Theauthorsdeclarethattheyhavenocompetingfinancialandnon-financial
50 mM Tris pH 7.5, and 150 mM NaCl. Post-nuclear interests.
lysates were separated by SDS-PAGE and transferred
Authors’contributions
onto nitrocellulose membranes (Amersham). Mem-
MPcarriedoutthebiochemicalstudies,performedtheproliferationassays
branes were probed with the indicated primary anti- andthestatisticalanalysis;BAparticipatedinthedesignofthestudyand
bodies and the appropriate HRP-conjugated secondary carriedoutthebiochemicalstudies;AVRandBSKcarriedoutthe
biochemicalstudies;VWparticipatedintheanalysisofLckmutants;JALand
antibodies (Dianova) and developed using the ECL detec-
BSparticipatedinthedesignofthestudyandhelpedtodraftthe
tion system (Amersham). The following antibodies were manuscript;LSperformedthestatisticalanalysis,conceivedandcoordinated
used for Western blotting in this study: anti-phospho(p)- thestudy,anddraftedthemanuscript.Allauthorsreadandapprovedthe
T202/Y204 Erk1/2, anti-pY319ZAP70, anti-pY171LAT, anti- finalmanuscript.
pY783PLCγ1, anti-pS338-c-Raf, anti-pS217/221MEK1/2, Authors’information
anti-pS380p90RSK, anti-pY731-c-Cbl, anti-pY351-p56Dok2, B.A.andM.P.contributedequallytothiswork.BApresentaddress,Diabetes
anti-pY416Src (all from Cell Signaling Technology), anti- Centre,DepartmentofMedicineInnenstadt,Ludwig-MaximilianUniversity,
80336Munich,Germany.BSKpresentaddress,VectorologyandExperimental
Lck (from BD Transduction laboratories), anti-Lck (from GeneTherapy,UniversityofRostock,18057Rostock,Germany,VWpresent
Epitomics), anti-Fyn (Fyn01, kindly provided by Vaclav address,MERCK,Munich,Germany.JALpresentaddress,Departmentof
Horejsi), anti-ZAP70, anti-CD3ζ (Santa Cruz Biotechnol- Nephrology,Hypertension,Diabetes,andEndocrinologyOtto-von-Guericke
University,39120MagdeburgGermany,JALandBSaremembersofSYBILLA
ogy), anti-pTyr (clone 4G10)-HRP conjugate (Millipore), [EU7FP]andtheMagdeburgCenterforSystemBiology(MaCS).
and anti-β-actin (clone AC-15) (Sigma-Aldrich). For
Acknowledgements
quantifications of the Western blots, the intensity of the
WearegratefultoTiloBeyerforcriticallyreadingthemanuscriptandhelpful
detected bands was acquired using the Epson Perfection discussionandtoNicoleJüling,InesMeinert,andPatriciaGintschelfor
V700PhotoScannerandanalysiswasperformedusing1D excellenttechnicalassistance.
TheworkwassupportedbygrantsfromtheGermanResearchFoundation
ImageQuant software (Kodak). Unless indicated other-
(DFG),FOR-521[SI861/1],GRK-1167[TP12]andSFB-854[TP19].
wise,β-actinwasusedasaloadingcontrol(typicalloading
errorintheexperiment:±13%). Received:28September2012Accepted:9January2013
Published:14January2013
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