UNIVERSITÄTSKLINIKUMHAMBURG-EPPENDORF
InstitutfürImmunologie
Prof.Dr.MarcusAltfeld
Expression,modulationandfunctionoftheP2X7
receptoronhumanimmunecells
Dissertation
zurErlangungdesDoktorgradesDr.rer.biol.hum./PhDanderMedizinischenFakultätderUniversitätHamburg.
vorgelegtvon:
ArnauSerracantPrat
ausBarcelona,Spanien
Hamburg2018
TABLEOFCONTENTS
1. INTRODUCTION 11.1 THEIMMUNESYSTEM 1
1.1.1 Theimmuneresponse 11.1.1.1 Theinnateimmuneresponse 11.1.1.2 Theadaptiveimmuneresponse 31.1.1.3 Innate-likelymphocytes 5
1.2 PURINERGICSIGNALLING 91.2.1 P1receptors 111.2.2 P2receptors 11
1.2.2.1 P2Yreceptors 111.2.2.2 P2Xreceptors 12
1.3 THEP2X7RECEPTOR 121.3.1 GeneticvariationsofthehumanP2RX7gene 131.3.2 ExpressionofP2X7intheimmunesystem 141.3.3 P2X7-mediateddownstreameffectsinimmunecells 14
1.3.3.1 Assemblyoftheinflammasomeandreleaseofproinflammatorycytokines 141.3.3.2 Sheddingofsurfaceproteins 151.3.3.3 Poreformationandinductionofcelldeath 15
1.3.4 RoleofP2X7signallinginTcellbiology 171.3.5 P2X7inhost-defenceanddisease 181.3.6 Therapiesandtools 21
2. AIMSOFTHESTUDY 23
3. MATERIALANDMETHODS 243.1 MATERIALS 24
3.1.1 Bloodandtissuesamples 243.1.2 Generalequipment 243.1.3 Materialsforcellculture 253.1.4 Materialsformolecularbiology 263.1.5 Materialsforisolationofimmunecellsfromhumanguttissue 293.1.6 Materialsforflowcytometry 303.1.7 Materialsforenzyme-linkedimmunosorbentassay(ELISA) 333.1.8 Generalconsumables 343.1.9 Software 34
3.2 METHODS 353.2.1 Donors 35
3.2.1.1 Humanblood 353.2.1.2 Humangastrointestinaltissue 353.2.1.3 Tcelllines 35
3.2.2 Isolationofhumancells 363.2.2.1 Isolationofperipheralbloodmononuclearcells 363.2.2.2 Isolationofinfiltratingimmunecellsfromhumanguttissue 36
3.2.3 Magnetic-activatedcellsorting 373.2.3.1 Enrichmentofhumanmonocytes 37
3.2.4 Flowcytometry 373.2.4.1 Stainingofsurfacemolecules 383.2.4.2 Exclusionofdeadcellsandcellcounting 393.2.4.3 Stainingofintracellularmolecules 393.2.4.4 Sortingofimmunecelltypes 393.2.4.5 Dataacquisition 40
3.2.5 GenerationofhumanTcelllines 403.2.6 Invitroassays 41
3.2.6.1 ActivationofhumanTcells 413.2.6.2 ProliferationofhumanTcells 423.2.6.3 StimulationofhumanTcellswithretinoicacid 423.2.6.4 Uptakeof4',6-diamidino-2-phenylindole(DAPI) 423.2.6.5 SheddingofCD62Lextracellulardomain 433.2.6.6 SuboptimalstimulationofTcells 433.2.6.7 Inductionofinflammasomeassembly(ASCspecks) 443.2.6.8 StimulationofNF-κB-inducedgenetranscription 443.2.6.9 Stimulationofhumanmonocytes 44
3.2.7 Enzyme-linkedimmunosorbentassay(ELISA) 453.2.8 Geneexpressionassessment 45
3.2.8.1 IsolationofDNAfromhumanblood 453.2.8.2 IsolationofRNAfromhumancells 453.2.8.3 QuantificationofpurifiedDNAandRNA 463.2.8.4 SynthesisofcDNA 463.2.8.5 Realtimepolymerasechainreaction 47
3.2.9 Genotyping 483.2.9.1 PolymeraseChainReaction 483.2.9.2 AgarosegelelectrophoresisofPCRproduct 493.2.9.3 ExtractionofDNAfragmentsfromanagarosegel 503.2.9.4 SequencingofDNA 50
3.2.10 Statisticalanalysis 50
4. RESULTS 514.1 EXPRESSIONANDFUNCTIONOFP2X7ONHUMANMONOCYTESANDMACROPHAGES 51
4.1.1 MonocytesshowhighexpressionofP2X7onthecellsurface 514.1.2 P2X7blockadebyDano1impairsATP-dependentoligomerisationoftheinflammasome 534.1.3 Dano1abolishesATP-inducedreleaseofIL-1βbymonocytes 564.1.4 Dano1exhibitshigherpotencyforblockingP2X7comparedtocurrentlyusedantagonists 574.1.5 Multimericnanobodiesexhibitenhancedpotencythanthemonomericconstructs 584.1.6 Dano1canalsoalterthetranscriptionofgenesregulatedbytheNF-κBpathway 59
4.2 EXPRESSIONANDFUNCTIONOFP2X7ONHUMANLYMPHOCYTES 604.2.1 NKcellsexhibitthehighestexpressionofsurfaceP2X7amongalllymphocytes 604.2.2 CD4+CD25+CD127-regulatoryTcellsexpresslowerlevelsofP2X7thanconventionalCD4T
cells 624.2.3 P2X7ispreferentiallyexpressedineffectorandmemoryCD4Tcells 634.2.4 Innate-likelymphocytesexhibithigherlevelsofP2X7thanconventionalTcells 674.2.5 γδTcellsalsoexhibithigheramountsofP2X7atthemRNAlevel 704.2.6 Innate-like T cell-derived cell lines exhibit higher levels of P2X7 than CD4 and CD8
conventionalcelllines 714.2.7 RNAtranscriptionofP2RX7increasesuponTcellactivation 724.2.8 TheexpressionofP2X7inhumanintestinalTcellsissimilartoperipheralTcells 734.2.9 RetinoicaciddoesnotinduceP2X7upregulationonhumanTcells 754.2.10 γδTcellsexhibithighersensitivitytoATP-inducedsheddingofCD62L 774.2.11 ATP-inducedsheddingofCD62LismediatedbyP2X7 824.2.12 Innate-likelymphocytesexhibithighersensitivitytoATP-inducedporeformation 824.2.13 Dano1efficientlyblocksATP-inducedporeformationinimmunecells 874.2.14 EffectorTcellsaremoresensitivetoATP-inducedporeformationthannaïveTcells 884.2.15 BlockadeoftheP2X7receptordoesnotaffectactivationorproliferationofTcellsinvitro 894.2.16 P2X7doesnotprovidecostimulationundersuboptimalstimulationoftheTCRinvitro 93
5. DISCUSSION 955.1 P2X7EXPRESSIONANDFUNCTIONINHUMANIMMUNECELLS 955.2 DISCREPANCIESBETWEENHUMANANDMURINEP2X7 995.3 THEP2X7NANOBODYDANO1ASAPOTENTIALTHERAPEUTICDRUG 102
6. ABSTRACT 108
7. ZUSAMMENFASSUNG 109
8. ABBREVIATIONS 111
9. REFERENCES 117
10. ACKNOWLEDGEMENTS 144
11. EIDESSTATTLICHEVERSICHERUNG 145
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1. INTRODUCTION
1.1 THEIMMUNESYSTEM
Theinteractionoforganismswiththeenvironmentandthesubsequentexposuretomultipleinsults
hasdriventheevolutionofamulti-layereddefencesystem,theimmunesystem.Apivotalfeatureof
the immune system is to distinguish between foreign (non-self) antigens and self-components
(DelvesandRoitt,2000).Whilemostof the inferiororganismsonly react to threatsby recognizing
conserved structures displayed by pathogens (Smith, 2016), the immune system of vertebrates is
able to mount a specific response to pathogens and to generate long-lived memory cells.
Immunologicalmemory results in highly efficient responses following re-encounterwith the same
pathogen(FlajnikandKasahara,2010;Boehm,2012).
1.1.1 Theimmuneresponse
The entrance of a pathogen alerts the immune system, activating several effector molecules and
tissue-resident and/or circulating immune cells that generate an immune response. The immune
response relies on the close cooperation between two distinct parts of the immune system, the
innate and the adaptive arm. Eachbranchhas crucial and special properties, but the coordination
between both ensures protection against pathogens throughout life (Janeway, 1989; Dempsey,
VaidyaandCheng,2003).
1.1.1.1 Theinnateimmuneresponse
The first line of defence is provided by physical barriers (skin, mucous membranes and body
secretions), which prevent pathogens from entering the body. Next, cells of the innate immune
system(phagocytes,naturalkillercellsandotherinnatelymphoidcells)andeffectorplasmaproteins
react immediatelyafterpathogenrecognition.Thepotentialtomountarapidresponsereliesupon
recognitionofhighlyconservedmoleculessharedbyseveral familiesofpathogens.Therecognition
of pathogen-associated molecular patterns (PAMPs) is mediated by germline-encoded pattern
recognition receptors (PRRs). PRRs canbe found attached to cellmembranes, such as the toll-like
receptors (TLRs) and C-type lectin receptors (CLRs) or free in the cytoplasm, such as theNOD-like
receptors (NLRs) and RIG-I-like receptors (RLRs). In addition, plasma proteins such as the
complement systemor serumamyloidA, contribute to the inflammatoryprocessandclearanceof
pathogensbyrecruitingeffectorimmunecellstothesiteofinfection.Activationofthecomplement
system cascade potentiates the recruitment of macrophages and neutrophils and enhances their
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phagocytic ability. Additionally, the binding of different complement factors to the surface of
bacteriainducestheformationofatransmembranechannel(membraneattackcomplex),leadingto
celllysisandbacterialdeath(DunkelbergerandSong,2010).
Neutrophils are one of the first populations to be recruited to the site of inflammation and are
essentialinavoidingthedisseminationofinfection.Uponarrival,neutrophilssenseandphagocytose
microbes,andrelease largeamountsofantimicrobialparticlesandenzymes(Mayadas,Cullereand
Lowell, 2014). Basophils, eosinophils andmast cells are also an important source of antimicrobial
peptides,enzymesandothercytotoxicsubstances,whicharepivotalinthefightagainstextracellular
bacteria and parasites. These cells, however, are involved in the generation of allergic reactions
(Stone, Prussin and Metcalfe, 2010). Phagocytes, namely monocytes, macrophages, conventional
dendritic cells (cDCs)andneutrophils, arekeyplayers in innate immunity for theirability to ingest
andeliminatepathogensinaprocessknownasphagocytosis.Amongtheseimmunecellpopulations,
monocytes,macrophagesanddendriticcells(DCs)caninadditionprocessandpresentantigenstoT
cells via themajor histocompatibility complex (MHC). Antigen presentation to T cells promotes a
specific immune response against a particular antigen epitope. Thus, antigen presentation by
professionalantigenpresentingcells(APCs)toTcellsservesasabridgebetweeninnateandadaptive
immunity(CharlesAJanewayetal.,2001a).
Plasmacytoid Dendritic Cells (pDCs) are a distinct subset of DCs that react to viral infections by
producingmassive amounts of type one Interferons (IFN),mainly IFN-α and IFN-β. The release of
thesemediatorspromotescytotoxicresponsesagainstvirus-infectedcellsbyactivatingnaturalkiller
(NK)andCD8Tcells(Colonna,TrinchieriandLiu,2004).NKcellsaccountforupto15%ofcirculating
lymphocytes,butarealsopresent inhighernumbers inperipheralorgans (Yu, FreudandCaligiuri,
2013).Theyareextremelyimportantintumorsurveillanceandtheirpresenceiscrucialfortherapid
and effective elimination of viral-infected cells. Human NK cells respond through a variety of
activating receptors (killer cell immunoglobulin-like receptors (KIR-S), CD16, natural cytotoxicity
receptors(NCRs),NTBA,2B4orNKG2D)orinhibitoryreceptors(KIR-L,CD94/NKG2A,LILRB1)encoded
in the germline.NK cells attack cellswithabsent or aberrantly low expression ofMHC class I, but
arealsoactivatedbyantibodies,viralpeptides, recognitionofMHCclass IviaactivatingKIRs (Vély,
GolubandVivier,2016),andcytokines like IL-2, IL-12, IL-15and IL-18 (Freemanetal.,2015).Their
activitydependsona finebalancebetweenactivatingand inhibitory signals.NKcell cytotoxicity is
mediated by release ofperforin and granzymes, antibody-dependent cell-mediated cytotoxicity
(ADCC) and Fas/FasL-induced apoptosis of infected cells.They also releasetumornecrosis factorα
(TNF-α)andIFN-γ,whichcanenhancemacrophageactivityandTcellresponses(Vivier,Tomaselloet
al.2008).
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Innate lymphoid cells (ILCs) are lymphoid cells lacking rearranged antigen receptors and myeloid
markers(SpitsandCupedo,2012).Theyinitiallydevelopinthefetalliverorbonemarrow,andthen
migrate to mucosal tissues. ILC subpopulations are identified based on their function and the
transcription factors they express. Group 1 ILCs are inflammatory cells that rely on the T-box
transcription factorT-betandsecretemainly IFN-γ in response to intracellularpathogens.Ofnote,
cytotoxicNKcellshavebeenrecentlyincludedwithinthisgroupowingtothelargeamountsofIFN-γ
they produce (Zhang and Huang, 2017). Group 2 ILCs are under the control of the GATA-binding
protein3(GATA3)andretinoicacidreceptorrelatedorphanreceptor-α(ROR-α)transcriptionfactors,
and produce cytokines such as IL-4,IL-5,IL-9 andIL-13 in response to parasite infections. Group 3
ILCsaredefinedbythesecretionofIL-17and/orIL-22anddependonthetranscriptionfactorROR-γt
(Spits et al., 2013). They are important for themaintenance of intestinal homeostasis. This group
include lymphoid tissue inducers (LTi), an immune cell subset involved in the development and
maintenanceoflymphoidorgans(Meieretal.,2007;VermijlenandPrinz,2014).
1.1.1.2 Theadaptiveimmuneresponse
The adaptive immune response provides a specific response against a particular antigen. Upon
antigen recognition, adaptive T cells clonally expand and differentiate into effector cells.
Consequently,theonsetofanadaptiveresponsedoesrequireseveraldays.Incontrasttotheinnate
PRRs, the specificity of the antigen receptors is not encoded and inherited in the germline, but
acquiredduringlife.ThegeneticrearrangementofalimitednumberofgenesencodingfortheTcell
receptor(TCR)andBcellreceptor(BCR)resultsinanextensivediversityofreceptors.
Lymphocytesarethemajorplayersinadaptiveimmunity.Theyarefoundconstantlyrecirculatingin
bloodandthelymphaticsystem,orasresidentcellsinalmostalltissuesinthebody.EverysingleB
andTcellpresentsauniquespecificityagainstasingularantigen.Uponantigenencounter,naïveor
immature T and B cells become effector cells, leave the secondary lymphoid organs (SLO) and
migrate to target tissues. Some effector cells develop into long-livedmemory cells and remain in
blood or in tissues as tissue-resident memory (TRM) cells (Schenkel and Masopust, 2014).
Immunological memory is a crucial feature of the adaptive immunity, and it provides long-term
protection by inducing faster andmore efficient responses after a possible re-encounterwith the
sameantigen.
1.1.1.2.1 Tcells
Tcellsarepivotalincell-mediatedimmunity.ThemajorityofhumanTcellsundergorearrangement
of thealpha and beta chainsto constitute the TCR, and are therefore termed αβ T cells (Tαβ).
Receptordiversityoriginatesfromrandomrearrangementsofdifferentvariable(V),diversity(D)and
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join(J)segmentsencodedintheTRAandTRBgenes;andtheinsertion,deletionandsubstitutionof
additionalnucleotidesbetweenthesesegments.Theseeventsenable thegenerationofabout1016
differentTCRsoutofa limitednumberof genes (CharlesA Janewayetal., 2001b;Nikolich-Zugich,
Slifka and Messaoudi, 2004). At the final stages of maturation in the thymus, T cells commonly
expressCD3andCD4orCD8ontheirsurface,whichactasco-receptorsthatstabilizetheinteraction
oftheTCRcomplexwithMHCmolecules.Survivingmaturethymocytesleavethethymusandegress
to periphery as naïve T cells. The complete activation of these conventional T cells requires two
further signals: first, a direct cell-cell interaction between the costimulatory molecules CD28 and
CD80/CD86,andsecond,signallinginducedbythecytokinespresentduringantigenpresentation.
CD4+Tcells
CD4+Tcells,alsonamedThelper(Th)cells,provideassistancetootherimmunecellpopulationsand
theyarereferredtoastheorchestratorsoftheimmuneresponse.Thcellfunctionsaremediatedby
releaseofawiderangeofcytokines,whichare involvedinmanyimmunologicalprocesses,suchas
theactivationofcytotoxicCD8+Tcells,Bcellisotypeswitchingandpotentiationoftheantimicrobial
activity of phagocytic cells. CD4+ T cells differentiate into different subtypes depending on the
cytokinesignalsreceived,andproducespecificsetsofcytokines.Th1cellsareproinflammatorycells
thatsecretemainlyIFN-yandarecriticalfortheclearanceofintracellularpathogensuponactivation
ofmacrophages.Th2cells secrete IL-4, IL-5and IL-13, important in the responseagainstparasites.
Th17 cells secrete IL-17 and are essential during infectionswith extracellular pathogens and fungi
(Zhu, Yamane and Paul, 2010). Th1 and Th17 cells play a major role in the development of
autoimmunity (Tabarkiewiczetal., 2015),whileTh2 cells are involved inallergic reactions. Several
othersubtypesofThcellshavebeendescribed,e.g.:Th9cells,Th22cellsandfollicularhelperTcells
(Tfh).Th9cellsare involved in fightinghelminth infections (Licona-Limónetal.,2013)aswellas in
allergic andautoimmune responses (Dengetal., 2017). Th22 cells play a role inhostdefenceand
inflammatorydiseasesintheskin(Eyerichetal.,2009),whileTfhcellsarecrucialfortheformationof
germinalcentresandoptimalBcelldifferentiationandfunction(Maetal.,2012).
CD8+Tcells
CD8+TcellsorcytotoxicTcellsare responsible for theeliminationofcells infectedwithvirusesor
intracellularbacteria,tumourcellsandinjuredcells.UnlikeCD4+Tcells,whicharerestrictedbyMHC
classIImolecules;CD8+TcellsarerestrictedbyMHCclassImolecules.MHCclassIisexpressedinall
nucleatedcellsanddisplaysendogenousantigenstotheextracellularsurface.PeripheralCD8+Tcells
commonlyexpressCD8moleculesasaαβheterodimer,butcanalsoexistasCD8ααhomodimersat
mucosalsurfaces.CytotoxicTcells inducecelldeathbyreleasinggranzymesandperforins,butalso
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throughtheFas/Fas-ligandpathway.Furthermore,theysecreteproinflammatorycytokines,suchas
IFN-γandTNF.
RegulatoryTcells
RegulatoryTcells(Tregs)areessentialinmaintainingperipheraltoleranceandcontrollingimmune
responsesafterantigenclearance.AmajortypeofTregsexpressthetranscriptionfactorForkhead
BoxP3 (FoxP3) andexpress the IL2α chain (CD25) constitutively (Sakaguchi, 2004). FoxpP3+ Treg
may develop in the thymus already as Treg cells (natural Tregs) or can be peripherally-induced
(inducibleTregs)(Hori,NomuraandSakaguchi,2003).ThelatterarisefromCD4+Tcellsundergoing
reprogrammingafterTCRstimulationinthepresenceoftransforminggrowthfactorβ(TGF-β)and
IL-2 (Schmitt and Williams, 2013). They dampen immune responses using different suppressive
mechanisms,namelyreleaseoftheimmunosuppressivecytokinesIL-10andTGF-β, inhibitionofT
cell proliferation by deprivation of IL-2, induction of cell death, inhibition of T cell activation by
inhibitorycostimulatorymoleculesandconversionofadenosinetriphosphate(ATP)intoadenosine
(Ado)(CurottodeLafailleandLafaille,2009;A.Rissieketal.,2015).
1.1.1.2.2 Bcells
Bcellsmediatethehumoralresponsebysecretingantibodies(Abs).IncontrasttoTcells,Bcellsare
able torecognizewholepathogensandsolubleantigens in theirnative formthroughtheBCR;and
therefore,theyarenotrestrictedtopresentationbyMHCmolecules.ActivationofBcellscommonly
occurswiththecooperationofTfhcells,whichresultsinthegenerationofhigh-affinityantibodies.B
cell activationmay also occur independently of T cell interaction, by TLR receptor signalling or by
crosslinkingofseveralBCRs.T-cellindependentactivationofBcellsleadstoaquickerresponse,but
antibodieswithloweraffinity(Vosetal.,2000).ThemajorityofBcellsdifferentiateeventually into
plasma cells and produce large amounts of immunoglobulins (Igs), and then relocate to the bone
marrow. Additionally, B cells may also present antigen to T cells upon BCR-mediated antigen
endocytosis(Malhotraetal.,2009).
1.1.1.3 Innate-likelymphocytes
Beyond the fully adaptive conventional T cells, a small subset of T cells termed innate-like
lymphocytes (ILLs) share properties of both adaptive and innate immunity (Figure 1). On the one
hand, ILLs undergo rearrangement of their TCR, recognize antigens presented by APCs, and
differentiate intomemory cells, as seen in adaptive immunity. On the other hand, they recognize
conserved antigens (Vermijlen and Prinz, 2014), andalso respond shortly after antigen encounter.
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ThebestdescribedILLsareγδ+Tcells,mucosalassociatedinvariantTcells(MAIT)andnaturalKillerT
(NKT)cells.
Figure1.Classificationofimmunecellsubsetsaccordingtotheirroleintheimmuneresponse.Theimmunesystem is split in two distinct arms: the innate and the adaptive; each of them characterised by differentimmune cell types and effector molecules that protect the body against external invaders. Innate-likelymphocytes(ILLs),suchasγδ+Tcells(Tγδ),mucosalassociatedinvariantTcells(MAIT)andinvariantnatural
Specificity Limited: Recognition of conserved patterns present indifferent pathogens
High: recognition of antigenic epitopes (fewaminoacids)
Receptordiversity
Limited: germline-encoded receptors (i.e. TLRs, NLRs,KIRs)
High: TCR and BCR, arising from somaticrecombination (T and B cells)hypermutation (only B cells)
Memory Absent: equal response upon repeated exposure Present: faster and amplified responses uponrepeated exposure
Responsekinetics Immediate (minutes to hours) Slow (several days)
Relevance
• First line of defence for control of pathogens(antimicrobial activity, phagocytosis, cytotoxicity,cytokine production)
• Efficient recruitment of other immune cells to site ofinfection (chemoattractants)
• Activation of adaptive immunity (antigenpresentation by APCs
• Highly specific and effective clearance ofpathogens and infected-cells (cytotoxicity,antibody production)
• Enhancement of the function of otherimmune cells activity by cytokineproduction (i.e. macrophages)
iNKT
MAIT
Tγδ
Tc
Th
Treg
BEosinophil
Neutrophil Basophil
Mo
DC
NK
ILC
Mast cell
Innate immunity
Adaptive immunity
APCs (antigen presenting cells); BCR (B cell receptor); DC (dendritic cell); Mo (monocyte); ILC (innate lymphoid cell); iNKT
(invariant natural killer T cell); KIRs (Killer-cell immunoglobulin-like receptors); MAIT (mucosal associated invariant T cell); NLRs
(NOD-like receptors);Tc (cytotoxic T cell); TCR (T cell receptor); Th (T helper cell); TLRs (toll-like receptors);Treg (regulatory T cell).
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killerTcells(iNKT)sharepropertiesfrombotharmsandarethereforepositionedattheinterfacebetweenbothsystems. ILLs display an invariant or highly restricted TCR repertoire and recognise conserved microbialelements. These cells become activated at early stages of infections and can be activated both in a TCR-dependentorindependentfashion.
1.1.1.3.1 γδ+Tcells
γδ+Tcellsrearrangethegamma(γ)andadelta(δ)chaininsteadoftheconventionalαandβchains.
TheTCRgamma(TRG)andTCRdelta(TRD)genescontain lowernumberofvariablesegmentsthan
theTRAandTRBgenes,thereforegeneratinglowerdiversitythantheαβTCR.ThreemainVδgene
elements(Vδ1,Vδ2andVδ3)arecommonlyusedbyhumanTγδcells,beingVδ2andVδ1themost
represented in peripheral blood. Receptor diversity is also reduced by the preferential pairing of
these three Vδ elements with a limited or exclusive number Vγ elements (Prinz, Silva-Santos and
Pennington,2013).
γδ+Tcellsemergefromthethymusandrepresent1-10%ofcirculatingTcells,buttheycanalsobe
foundintissues,especiallyinepitheliallayers.EarlyγδTcelldevelopmentinmiceoccursindifferent
wavesoffunctionallydistinctγδ+TcellsubsetscarryingspecificVγsegments(Prinz,Silva-Santosand
Pennington, 2013). In humans, it is not yet known whether a similar pattern exists. γδ+ T cells
recognize bothmicrobial and self-derived compounds presented byMHC-like molecules, but also
intactantigenssuchaslipidsandstress-inducedproteins(Born,KemalAydintugandO’Brien,2013).
Vδ2+cellsareoftenpairedtotheVγ9chainandconstituteupto95%oftheγδ+Tcellpopulationin
adult blood. Vδ2+Vγ9+ cells recognize the pathogen-associated phosphoantigen (E)-4-Hydroxy-3-
methyl-but-2-enyl pyrophosphate (HMBPP), but also the self-derived phosphoantigens Isopentenyl
pyrophosphate(IPP)andDimethylallylpyrophosphate(DMAPP) (Tanakaetal.,1995),presentedby
butyrophilins and other non-classicalMHCmolecules (Harly et al., 2012). Vδ2+ cells are crucial in
cytotoxicityagainstinfected,stressedandtumourcells(Wrobeletal.,2007;Marlinetal.,2017).γδ+
TcellsalsopromotethematurationandactivationofDCsandmonocytes(Leslieetal.,2002;Contiet
al., 2005; Eberl et al., 2009); as well as promoting B and T cell responses and impairing the
immunosuppressiveactivityofTregs(Brandes,WillimannandMoser,2005;Petermannetal.,2010;
PetrascaandDoherty,2014).
Vδ2-cells includethosecellscarryingaVδ1orVδ3chain.HumanVδ1+cellsarethemostabundant
γδ+ T cells at epithelial sites, such as the intestine and skin. They are cytotoxic and secrete IFN-γ,
essentialfortheeliminationofviruses,especiallycytomegalovirus(CMV)(Déchanetetal.,1999;Sell
et al., 2015), fungi and tumour cells. Antigen presentation is driven by MHC class I polypeptide-
related protein A (MICA) and B (MICB). Vδ1+ cells can also recognize endogenous phospholipids
presentedbynon-classicalMHCmoleculesoftheCD1family(Adams,GuandLuoma,2015).
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Ofnote,murineγδ+TcellscanalsobeactivatedindependentlyoftheirTCR,inresponsetopathogen
productsthroughTLRs(Martinetal.,2009)andcytokinesviacytokinereceptors(Suttonetal.,2009).
Whetherasimilaractivationpatternalsooccursinhumansisnotyetknown.
1.1.1.3.2 MucosalAssociatedInvariantTcells
Mucosal associated invariant T (MAIT) cells represent 1-10% of total blood-circulating T cells in
healthyadults,andareofgreatimportanceinmicrobialinfections(Salou,FranciszkiewiczandLantz,
2017).MAIT cells develop in the thymus but undergo selection through theMHC-related 1 (MR1)
molecule expressed on CD4+CD8+ double positive (DP) thymocytes (Seach et al., 2013). After
selection,MAITcellsexitthethymusandmigratemainlytomucosaltissues,butalsototheliverand
lungs. MAIT cells express the invariant TCR alpha chain Vα7.2 predominantly paired with the J
segmentJ33(Vα7.2-Jα33),butalsowiththeJ12(Vα7.2-Jα12)andJ20(Vα7.2-Jα20)segments.These
VJelementscombinewitharestrictedsetofTCR-βchainstobuilda functionalTCR.MAITcellsare
mainly negative for CD4 and CD8 or express the CD8 homodimer CD8αα on their surface
(Reantragoonetal.,2013).Theyhaveaneffector-memoryphenotypeandexpressthe lectinfamily
NK receptor CD161, the IL-18 receptor (IL-18Rα), and tissue-homing chemokine receptors, such as
CCR6andCCR9 (ChandraandKronenberg,2015;Koay,GodfreyandPellicci,2018).Likeγδ+Tcells,
MAITcellscanbeactivatedbothinaTCR-dependentorindependentmanner(Slichteretal.,2016).
TCR-activationisrestrictedbyMR1moleculespresentingbacterialtransitoryneo-antigensderivedof
themetabolismofvitaminsB2andB9.UponTCR-engagement,MAITcellssecreteIFN-γ,TNF-αand
IL-17(vanWilgenburgetal.,2016).MAITcellsalsodisplaycytotoxicactivityandreleasegranzymeB
andperforin (Kuriokaetal., 2015).TCR-independentactivation involves thepresenceof cytokines,
such asIL-12, IL-15, IL-18 and IL-23; which promote the secretion of IFN-γ, granzyme B and IL-17
(Ussheretal.,2014;Slichteretal.,2016).Somestudieshavealsoshownasynergisticeffectbetween
inflammatorysignalsandTCRengagement(Slichteretal.,2016).
AfurthergroupofILLssharepropertiesofTandNKcells,andarenamednaturalkillerTcells(NKTs),
representingaveryrarepopulation inhumanblood(lessthan0.1%)(Kennaetal.,2003). Invariant
NKTcells(iNKTs),alsotermedtype1NKTcells,displayaninvariantTCRalphachain(Vα24-J18)that
preferentially combines with the Vβ11 element. iNKTs participate in the clearance of bacteria,
parasite,fungiandalsoviruses(Juno,KeynanandFowke,2012;Kinjo,KitanoandKronenberg,2013).
TheydisplayanactivatedphenotypeandtypicalmarkersoftheNKlineage,suchasCD161,CD56and
CD16.Theyrecognizepathogen-associatedorendogenouslipidspresentedbythenon-classicalMHC
molecule CD1d on APCs. iNKTs sense danger-associated molecular patterns (DAMPs) and rapidly
produce proinflammatory cytokines that promote activation of NK cells,T cells,B cells, DCs and
macrophages. These cells can be sub-classified according to the cytokines they release. Themost
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commonsubsetsareiNKT1,iNKT2andiNKT17,whichmirrortheTh1,Th2andTh17secretoryprofiles
(ChandraandKronenberg,2015).
Similartootherinnate-likecells,iNKTcellscanalsobeindirectlyactivatedthroughcytokinesignalling
orNKreceptors(Reilly,WandsandBrossay,2010;Brennan,BriglandBrenner,2013).
1.2 PURINERGICSIGNALLING
Purine nucleotides and nucleosides are importantmessengers in extracellular cell communication.
Purinergicsignallingplaysasignificantroleinnumerousorgansystems,includingtheimmunesystem
(BurnstockandBurnstock,2006);andtherefore,isconsideredapotentialtherapeutictargetforthe
treatmentofimmune-relateddisorders(Burnstock,2017),suchasmultiplesclerosisorasthma.
Under physiological conditions, purine nucleotides are mostly intracellular, and contribute to cell
metabolismasenergyreservoirsand/orenzymecofactors.ATPispresentathighconcentrations(1-
10mM)intheintracellularcompartment(BeisandNewsholme,1975;Traut,1994;Burnstock,2017),
whereastheextracellularconcentrationofATPisconsiderablylower(nanomolarrange)(Ryanetal.,
1996;Gorman,FeiglandBuffington,2006).ATPcanbephysiologicallyreleasedinanactiveorpassive
fashion to the extracellularmilieu, where it promotes autocrine and paracrine effects. The active
release of purine nucleotides occurs predominantly by exocytosis or through membrane-bound
proteins(Lazarowski,2012).Inaddition,avarietyofstimuli,includinghypoxia,apoptosis,cellstress
andproinflammatorymoleculesmaytriggertheactivereleaseofATPthroughpannexinchannelsand
connexinhemichannels(Doschetal.,2018).
Upon cell damage, injured cells release passively large amounts of ATP to the extracellular
compartment, leading to a rapid increase of extracellular ATP (eATP). High levels of eATP are
recognizedasadangersignalbytheimmunesystem,triggeringaninflammatoryresponse(Idzkoet
al., 2007; Boeynaems, Communi and Robaye, 2012a; Rodrigues, Tomé and Cunha, 2015; Amores-
Iniestaetal.,2017;Cauwelsetal.,2017).eATPisexposedtoseveralectonucleotidasesthatmediate
its degradation to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). These
enzymes include ectonucleoside triphosphate diphosphohydrolases (ENTPDases), such as CD39;
alkaline phosphatases; and ectonucleotide pyrophosphatases/phosphodiesterases (ENPPs)
(Zimmermann,2000).Additionally, theectoenzymeCD38mediates the catabolismofnicotinamide
adeninedinucleotide(NAD+)toADP-ribose,whichisconvertedtoAMPbyENPP1(Horensteinetal.,
2013;Quaronaet al., 2013). Extracellular AMP can be further degraded by the ectoenzyme CD73
(ecto-5´-nucleotidase),whichmediatesthehydrolysisofAMPintoadenosine(Ado)(Antoniolietal.,
2013), a molecule with immunosuppressive properties (Haskó et al., 2008; Haskó and Cronstein,
10
2013).Adenosinergicsignallingistightlyregulatedbyadenosinedeaminase(ADA),anenzymethatis
foundboth in the cytosol andextracellularly; either as amembrane-boundcomplex togetherwith
CD26orsolubleintheplasmaandserum.ADAisresponsiblefortheirreversibledegradationofAdo
into inosine. Ado can also be transported to the cytosol by nucleoside transporters, and further
convertedtoAMPbyintracellularadenosinekinases(CekicandLinden,2016).
During acute inflammation, high levels of ATP accumulate at the site of inflammation, promoting
infiltrationof inflammatory cells (Kronlageet al., 2010) and releaseof proinflammatorymolecules
(Ferrari,LaSala,etal.,2000;Monção-Ribeiroetal.,2014).Tissueinjury, inflammationandpossibly
aninsufficientdegradationratecontributetothemaintenanceofhighconcentrationsofeATPatthe
inflammatorysite.Onthecontrary,duringresolutionof inflammation,theratiobetweeneATPand
eAdoshiftsinfavourtoAdo.Ultimately,highlevelsofAdoandlowconcentrationofATPcontribute
to the resolution of inflammation (Eltzschig, Sitkovsky and Robson, 2012; Cekic and Linden, 2016;
Faas,SáezanddeVos,2017).Therefore,theextracellularpurinergicmicroenvironmentdetermines
thebalancebetweenaproinflammatoryandanti-inflammatorystatus(Faas,SáezanddeVos,2017).
©NorbertSträter
Figure2.ATPand itsdownstreammetabolites inducedifferent responsesthrough signallingviadifferentpurinergic receptors. Damaged or injured cells release large amounts of ATP to the extracellularcompartment. Purinergic receptors are subdivided in three different subtypes: P1, P2X and P2Y receptors.ATP is the only purine nucleotide inducing the activation of P2X receptors, whereas both ATP and ADP,among other purine nucleotides, trigger the activation of P2Y receptors. Released ATP is recognized as adangersignalbytheimmunesystem,promotinginflammatoryresponsesthroughsignallingviaP2receptors.Different ectonucleotidases are responsible for the complete degradation of ATP to ADP and AMP, andultimatelytoadenosine,whichbindstoP1receptorsandinducesvariousimmune-regulatoryeffects.
11
Purinergicsignallingismediatedbypurinergicreceptors(Figure2).Todate,threedifferentfamilies
ofpurinergic receptorshavebeen identified: theP1or adenosine receptors; and theP2XandP2Y
receptors,whichbindtoATPand/orothernucleotides(CekicandLinden,2016).
1.2.1 P1receptors
P1 receptors (P1Rs) are membrane-bound proteins composed of seven transmembrane domains
coupledtoG-proteinsontheintracellularside.CouplingtoaspecificG-proteinsubtypedetermines
thesignallingcascadeandtheoutcomeoftheresponse(Shethetal.,2014).Therearefourknown
subtypesofP1receptors(A1,A2A,A2BandA3);allofthembindingtoAdo.A1RandA2ARarehigh
affinityreceptors,whereasactivationoftheA2BRandA3Rreceptorsrequireshigherconcentrations
ofAdo(Fredholmetal.,2001).
Ado signalling in immune cells ismainlymediated by A2A and A2B receptors, and both receptors
havebeenreportedtobeupregulatedfollowinginflammation.Themajorityofimmunecellsexpress
A2ARontheirsurface,whereastheexpressionofA2BRismainlyrestrictedtoDCsandmacrophages
(CekicandLinden,2016).EngagementofAdotriggersdistinctimmunosuppressiveeffects,including
thesecretionofanti-inflammatoryIL-10bymacrophagesandtheinhibitionofsecretionofTNF-αand
IL-12 by DCs and macrophages. Ado signalling also leads to weaker neutrophil responses and
impaired T cell proliferation and cytokine production (Haskó et al., 2008; Haskó and Cronstein,
2013).
1.2.2 P2receptors
P2 receptors (P2Rs) are excitatory receptors present on a variety of immune cells, and in general,
promoteinflammatoryresponses.Todate,eighteenfunctionalnucleotide-bindingP2receptorshave
beendescribedinhumans;classifiedintotwodifferentfamilies:theionotropicP2Xreceptors(P2XRs)
andthemetabotropicP2Yreceptors(P2YRs).
1.2.2.1 P2Yreceptors
P2Y receptors are homo-or-heterotrimeric receptors that belong to the superfamily of G-protein
coupledreceptors.Theyaregrouped intotwosubfamiliesbasedonsequencehomologyandsignal
transduction:thefirstsubfamily(P2Y1,P2Y2,P2Y4,P2Y6andP2Y11)iscoupledtoGq-proteins;while
the second subfamily (P2Y12, P2Y13, and P2Y14) is coupled to Gi-proteins (von Kügelgen and
Hoffmann,2016).ThemostcommonligandoftheP2YRfamilyisATP,althoughsomereceptorsare
morepromiscuousandalsobindtoothernucleotides,suchasADP,uridinetriphosphate(UTP)and
uridinediphosphate(UDP)(Jacobsonetal.,2009).Indeed,ATPconcentrationdeterminesbindingto
12
certain subtypesof P2YRs and theoutcomeof the response. In addition, the samenucleotide can
functionasanagonistorasanantagonistfordifferentreceptors(Idzko,FerrariandEltzschig,2014).
P2YRsareexpressedvirtuallyinallhumantissues,andtheyareinvolvedinphysiologicalevents,such
as lipidmetabolism, platelet aggregation, bone remodelling and distinct responses in the nervous
system (Boeynaems, Communi and Robaye, 2012b). P2YRs are present in different immune cell
types,includingneutrophils,macrophages,endothelialcells,microgliaandDCs.ActivationofP2YRsis
involvedinchemotaxisandimmunecelldifferentiationandmaturation,andtherefore,isassociated
toseveralinflammatoryconditions,suchaspsoriasisandCrohn´sdisease(Jacobetal.,2013;LeDuc
etal.,2017).
1.2.2.2 P2Xreceptors
P2X receptors are ATP-gated membrane-bound ion channels that do not resemble any other ion
channels or proteins at the molecular level. Seven distinct members (P2X1-P2X7) encompass the
P2XRfamily.Functionalreceptorsrequirethearrangementofthreesubunits,whichrangefrom270
aa (P2X4) to 595 aa (P2X7) (Nickeet al., 1998). Interestingly,while P2X7 only forms homodimers,
someotherP2X receptors can formheterotrimeric ionchannels (P2X1/2;P2X1/4;P2X1/5;P2X2/3;
P2X2/6 and P2X4/6) (Cekic and Linden, 2016). Each subunit is characterized by an intracellular
amino-terminus tail, a long intracellular carboxyl-terminus tail, two hydrophobic transmembrane
domains(TM1andTM2)andabigextracellularloopthatcontainsthebindingsiteforATP(Alveset
al.,2014).ActivationofP2XRrequiresthebindingofoneATPmoleculetothebindingpocketofeach
pair of monomers. Ligand binding induces a conformational change and the opening of the ion
channel,allowingfluxofcations(K+,Na+andCa2+)anddepolarizationoftheplasmamembrane(Egan
andKhakh,2004;Samways,LiandEgan,2014).
P2X receptors are widely expressed throughout the human systems and tissues, underlining the
important role of these ionotropic receptors in a variety of tissues and physiological processes,
including neurotransmission, smoothmuscle contraction and immune cell activation (North, 2002;
Kaczmarek-Hájeketal.,2012).
1.3 THEP2X7RECEPTOR
TheP2X7receptor(P2X7),previouslyreferredasP2Z(Gordon,1986),isthemostrelevantandmost
investigated P2X subtype in the field of immunology due to its role in promoting inflammatory
responses (Di Virgilio et al., 2017; Giuliani et al., 2017). Each subunit of the homotrimer has a
molecularweight of 72 kDa and a length of 595 aa (Bartlett, Stokes and Sluyter, 2014). A unique
13
characteristicof theP2X7subunit is itsvery longcarboxyl-terminaldomain,which is indispensable
forporeformation(Adriouchetal.,2002;Smartetal.,2003;Beckeretal.,2008;Alvesetal.,2014).
ATP istheonlyphysiologicalnucleotidethatservesasa ligandforP2X7 inhumans,althoughsome
other non-nucleotide substances, such as bactericidal peptides released during inflammation, can
alsoactivateP2X7(DiVirgilioetal.,2018).UnlikeothermembersoftheP2XRfamily,P2X7hasalow
affinity toATP, and therefore, high concentrations of ATP (EC ≥ 100µM) are required in order to
induce its activation (Surprenant et al., 1996; Donnelly-Robertset al., 2009). The lower affinity of
P2X7toATPisprobablyaconsequenceofthesize,accessibilityandtheaminoacidcompositionof
theATP-bindingpocket(DiVirgilioetal.,2017).Furthermore,thehighactivationthresholdofP2X7is
thought to serve as a control mechanism to prevent its activation under physiological conditions
(Jiang,2009).
InadditiontoATP,extracellularNAD+ (eNAD) triggers theactivationofP2X7 inmice.Activationby
eNADismediatedbythemono-ADP-ribosyltransferaseARTC2.2, whichcatalysesthetransferofan
ADP-ribosemoietyfromaNADmoleculetoanarginineresidueatposition125(Arg125), locatedin
the immediate vicinity of the ATP-binding pocket (Adriouch et al., 2008). In humans, ART2 is a
pseudogene(Haagetal.,1994);thereforeunresponsivenessofP2X7toNAD+inhumansismostlikely
tobecausedbythelackofART2andADP-ribosyltransferaseactivity(Rissieketal.,2017).
1.3.1 GeneticvariationsofthehumanP2RX7gene
TheP2RX7geneislocatedatthechromosomalposition12q24.31andconsistsof13exonsspanning
53 kb (G. N. Buell et al., 1998). P2RX7 is highly polymorphic, with thousands of single-nucleotide
polymorphisms (SNPs) and several naturally occurring splice variants reported to date (Bartlett,
Stokes and Sluyter, 2014; Di Virgilio et al., 2017). Genetic variations can lead to a loss or gain of
function inP2X7. For instance, the SNPsH155Y,H270RandA348T result indifferent sensitivity to
ATP,beingtheallelicvariants155Y,270Rand348T,theonesconferringhighersensitivity(Cabriniet
al.,2005;Stokesetal.,2010).SeveralSNPsontheP2RX7genehavebeenlinkedtosusceptibilityto
infections, such as tuberculosis (Fernando et al., 2007); and to different diseases, such as
cardiovascular diseases (Gidlöf et al., 2012), rheumatoid arthritis (Portales-Cervantes et al., 2012)
andosteoporosis(Bartlett,StokesandSluyter,2014;Kasuyaetal.,2017).
Inhumans,ninealternativesplicevariants(P2XB-P2XJ)ofP2RX7occurnaturally;allofthemdiffering
in their functional properties from the original and full-lengthP2X7A variant (Sluyter, 2017). For
instance, P2XB is a truncated isoformwith a shorterC-terminus, and therefore lacks the ability to
form the largepore (SluyterandStokes,2011;Bartlett, StokesandSluyter, 2014;DiVirgilioetal.,
14
2017). P2X7C, P2X7E and P2X7G are also C-terminally truncated isoforms. Alternative splicing also
generatesvariantsthatlackorcontainadditionalexons,orthatresultsinanullallele.
1.3.2 ExpressionofP2X7intheimmunesystem
P2X7 is expressed in the surface of most cells of the hematopoietic lineage (Idzko, Ferrari and
Eltzschig,2014),butalsoinmanyothertissuesandcelltypes(BurnstockandKnight,2004;Bartlett,
StokesandSluyter,2014),includingepithelialcells(Welter-Stahletal.,2009)andcellsinthenervous
system (Sperlagh et al., 2006).Within the immune system, P2X7 expression is particularly high in
both human andmurinemonocytes, macrophages (Gu et al., 2000; Burnstock and Knight, 2004),
microglia(Inoue,2008;Heetal.,2017)andDCs(Surprenantetal.,1996;Mutinietal.,1999).P2X7is
alsoexpressedonneutrophils,eosinophils,mastcells,Tcells,Bcells,NKcellsandNKTcells(Wanget
al., 2004; Beldi et al., 2008; Idzko, Ferrari and Eltzschig, 2014). In mice, Tregs and iNKTs are the
lymphocytesexpressingthehighestlevelsofP2X7(Heissetal.,2008;Hubertetal.,2010;Rissieket
al., 2014). Up to now, very little is known about the expression pattern and function of P2X7 in
humanTcells.
Inaddition,P2X7isalsofoundinthelipidraftsfromcertainmurinecelltypes,suchaslymphomaor
lungepithelialcells.LeucocytesandplateletsalsoexpresslargeamountsofP2X7intracellularly,and
P2X7hasalsobeenidentifiedinthephagosomesofmacrophagesinmice(Guetal.,2000;Kuehnelet
al.,2009;Sluyter,2017).
1.3.3 P2X7-mediateddownstreameffectsinimmunecells
EngagementofATPinducesareversibleconformationalrearrangementthatallowstheinfluxofCa2+
andNa+ andeffluxofK+ (Surprenantetal., 1996;Bartlett, StokesandSluyter, 2014).Activationof
P2X7leadstodistinctdownstreamresponsesdependingonthecelltype(Figure3).
1.3.3.1 Assemblyoftheinflammasomeandreleaseofproinflammatorycytokines
P2X7activationtriggerstheprocessingandreleaseoftheproinflammatorycytokinesIL-1βandIL-18
bymonocytes,macrophages,DCsandmicroglialcellsviainflammasomeactivation(Perregauxetal.,
2000; Ferrari et al., 2006; Pelegrin and Surprenant, 2006; Englezou et al., 2015; He et al., 2017).
Inflammasomes are a groupofmultiprotein intracellular complexes implicated in cell damage and
immune responses against pathogens. Among all inflammasomes, the NLRP3 inflammasome, also
knownasNALP3,hasbeenassociatedwiththepathogenesisofmultipleinflammatorydiseases(Guo,
Callaway and Ting, 2015). The production of IL-1β and IL-18 requires two different signals. First,
sensing ofmicrobialmolecules, such as lipopolysaccharide (LPS) by the toll-like receptor 4 (TLR4),
15
triggerssynthesisofinactivepro-IL1βandpro-IL18anditsaccumulationinthecytoplasmofthecells.
Thus,asecondstimulusisrequiredfortheprocessingandreleaseofmatureIL-1βandIL-18(Dowling
and O’Neill, 2012). The activation of P2X7 is one of the mechanisms capable of inducing the
relocalisationandassemblyoftheNLRP3inflammasome.Althoughtheexactmechanismbehindthis
isyettobediscovered,theeffluxofK+ionsseemstoberesponsibleforitsrecruitment(Pétrillietal.,
2007). The NLRP3 inflammasome complex consists of the NOD-like receptor NALP3 protein, the
apoptosis-associated speck-like protein containing a card (ASC) adaptor protein and the inactive
enzyme procaspase-1. Dimerization of NLRP3 receptors enables recruitment of the ASC protein
through the pyrin domain (PYD). ASC, which also contains a caspase activation and recruitment
domain(CARD),bindstotheCARDdomainofprocaspase-1;triggeringaconformationalchangeand
the subsequent proteolytic cleavage of procaspase-1 and its conversion to active caspase-1. Free
caspase-1mediatesthecleavageofimmaturepro-IL1βandpro-IL18intobiologicallyactiveIL-1βand
IL-18 (He,Hara andNúñez, 2016) (Figure 3A). The exactmechanismshow IL-1β is released to the
extracellularcompartmentisstilldebated(Giulianietal.,2017).
1.3.3.2 Sheddingofsurfaceproteins
P2X7 activation also leads to the activation of metalloproteases of the ADAM (A disintegrin and
metalloprotease)family,whichareresponsiblefortherapidsheddingoftheectodomainofseveral
proteins fromthecellmembrane(Figure3B).P2X7-mediatedsheddinghasbeendemonstratedfor
proteinssuchasCD62L inmany immunecell types(Gu,BendallandWiley,1998;Scheupleinetal.,
2009),CD27inmurineTcells(Moonetal.,2006),IL6Rinmonocytes(Garbersetal.,2011),TNF-αin
microglia(Suzukietal.,2004)andCD23inBcells(Pupovacetal.,2015).
1.3.3.3 Poreformationandinductionofcelldeath
ATP-P2X7 interaction inducestherapidandreversibleexternalizationofphosphatidylserine(PS)on
the outer leaflet of the plasmamembrane of T cells (Elliott et al., 2005; Scheuplein et al., 2009);
which is associatedwith influxofNa+ andCa2+ (B.Rissieketal., 2015). PSexposurehas alsobeen
reportedinothercelltypes,suchaserythrocytesandmonocytes(Sluyter,ShemonandWiley,2007;
Wardetal.,2010).PersistentstimulationbyATP inducestheopeningofasecondarynon-selective
porepermeabletoorganiccationicmoleculesupto900Da(Steinbergetal.,1987)(Figure3B).The
mechanismbehindpore formation is still unclear: itmaybedirectlymediatedbyP2X7 itselforby
P2X7 inassociationwithotherproteinssuchpannexin1 (Gulbransenetal.,2012).ProlongedP2X7
activationleadstomembraneblebbingandultimatelytocelldeathofespeciallysensitivecelltypes,
suchasmurineTregs(Scheupleinetal.,2009).
16
ASCCaspase-1
NLRP-3pro-IL1βpro-IL18
extracellular
intracellular
Ca2+
Na+
K+
Ca2+
Na+K+
ATP
P2X7
TLR4
IL1βIL18
IL1βIL18
LPS
©AR
© Anne Rissiek
extracellular
intracellular
Ca2+
Na+
K+
Ca2+
Na+K+
ATP
CD62LP2X7
PS©AR
IL6R
CD27
Modified from Anne Rissiek
A
B
Figure3.ExtracellularATPinducesdifferentP2X7-mediateddownstreamresponses.BindingtoextracellularATP induces the opening of the P2X7 ion channel, allowing the passage of Ca2+ and Na+ to the intracellularcompartmentandK+totheextracellularcompartment.(A)SensingofLPSthroughTLR4leadstothesynthesisoftheprecursorformsofIL-1βandIL-18(pro-IL1βandpro-IL18,respectively)bymonocytesandmacrophages.Engagement of ATP to P2X7 on these immune cell subsets triggers activation of the NLRP3 inflammasome,which mediates the conversion of pro caspase-1 to active caspase-1. Activated caspase-1 catalyses theproteolytic cleavage of the precursor forms to active IL-1β and IL-18, which can then be secreted to theextracellularmilieu.(B)P2X7activationinTcells,andalsoinotherimmunecelltypes,inducestheexposureofPSontheouterleafletoftheplasmamembraneandactivationofmetalloproteasesthatcatalysethesheddingof surface proteins such as CD62L and CD27. Prolonged activation of P2X7 leads to the opening of a non-selectivesecondarypore,allowingthepassageof largemolecules,membraneblebbingand,ultimatelytocelldeathofespeciallysensitivecelltypes.
17
1.3.4 RoleofP2X7signallinginTcellbiology
P2X7signallingis involvedinthedevelopment,differentiationandactivationofTcells.Thymocytes
undergoingnegativeselectioninthethymusreleasesubstantialamountsofATP,whichuponbinding
toP2X7,contributetoenhancedprogrammedcelldeath(Lépineetal.,2006).Thestrengthofγδ-TCR
signalling indoublenegative (DN)cells,whichexpressboth thepre-TCRαandγδ-TCR, isadecisive
factorinαβversusγδ-lineagefatedecisionduringTcellmaturationinmice.Strongsignallingthrough
theγδ-TCRcomplexsupportsthecommitmenttotheγδ-lineage,whereasaweakersignallingfavours
development of thymocytes towards the αβ-lineage(Haks et al., 2005; Hayes, Li and Love, 2005).
Also,theATP-P2X7axisprovidesanadditionalsignalfavouringγδ-lineagecommitment.Theabsence
orblockadeofP2X7results in increased transitionofmurineγδTcells to thedoublepositive (DP)
stage.ImmatureγδTcellsexhibithigherexpressionofP2X7andreleasehigherquantitiesofATPto
the extracellular milieu than DN3 stage thymocytes. Therefore, both P2X7 and secreted ATP
contributetoastrongersignalfavouringcommitmenttoγδlineage(Frascolietal.,2012).
Activation of P2X7 induces cell death of murine Tregs (Rissiek et al., 2014) and dampens their
immunosuppressive potential (Schenk et al., 2011). High concentrations of eATP are needed to
inducecelldeath,whereasjustthebriefexposuretolowconcentrationsofNAD+isrequired(Seman
etal.,2003). Ofnote,wildtype(WT)micehavemoreTregsthanP2X7-deficientmice(B.Rissieket
al., 2015). T cells showdifferent sensitivity toATPaccording to their activationanddifferentiation
status. Murine naïve T cells are more sensitive to NAD+-induced cell death (NICD) than activated
effectorTcells(Rissieketal.,2014).Controversially,adifferentstudyshowsthatintestinaleffector
andmemory CD4+ T cells display higher levels of P2X7 than naïve cells, and therefore, aremore
susceptibletoNICD(Hashimoto-Hilletal.,2017).Asimilarscenarioalsoseemstooccurinhumans:
while intermediate concentrations of ATP increase proliferation of activated T cells, high
concentrations of ATP induce apoptosis of these cells but promote proliferation of Tregs. These
opposedeffectsmightbeexplainedbysignallingthroughotherP2receptors(Trabanellietal.,2012).
TheroleofATPintheproliferationofhumanTcellswasfirstdescribedbyBaricordietal,1996.While
highconcentrationsofATPinducecelldeathofimmunecells(Yoonetal.,2007),lowconcentrations
ofATP induceproliferationofconventionalTcells (Adinolfietal.,2005).UponTCRengagement,T
cellsreleaseATPtotheextracellularcompartment,whichcanstimulateP2X1,P2X4andP2X7inan
autocrinemanner,contributingtoTcellproliferation(Schenketal.,2008;Woehrleetal.,2010). In
this scenario, activation of P2X7 induces the mobilization of calcium ions and increases cellular
energystoresandproductionofIL-2;promotingtheactivationandproliferationofTcells(Adinolfiet
al.,2005;Yipetal.,2009).Moreover,upregulationofP2X7uponT-cellstimulationinJurkatcells(Yip
etal2009)andoverexpressionofP2X7receptorsinthehumancelllineHEK293(Adinolfietal.,2005)
18
resultsinincreasedTcellproliferation.ATPreleasedfromactivatedTcellsalsoactsonneighbouring
cells, inducingcalciumwavesthatregulatemotilityofmurineandhumanTcellsthroughP2X4and
P2X7receptors(Wangetal.,2014).
ATPandP2X7signallingarealsoinvolvedinthepolarizationofmurineTcells.P2X7promotesTcell
differentiation towardsaTh17phenotypeand inhibits their conversion toTregs.P2X7antagonism
rescuesthedifferentiationofnaïveCD4+Tcells toTregs(Schenketal.,2011).Similarly,eATPfrom
commensalbacteriainducesthesecretionofIL-6,IL-23andTGF-βbyasubsetofmurineDCscellsin
the intestine,promoting thedifferentiationofTh17cells. Th17celldifferentiationwas inhibited in
thepresenceofunspecificP2receptorantagonists(Atarashietal.,2008),mostlikelyviainhibitionof
P2X7; although this was not tested in this study. In the tumour environment, P2X7-dependent
releaseofIL-1βisrequiredfortheoptimaldifferentiationofIFNγ-producingCD8+Tcells(Aymericet
al.,2010).P2X7alsocontributestothedifferentiationofTh1cellsinamousemodelformalaria.In
this work, the expansion of Th1 cells correlated to increased cell death of Tfh cells (Salles et al.,
2017).Asimilareffectwasseeninanotherstudy,inwhichP2X7activationlimitedthenumberofTfh
cellsinthesmallintestineofmice(Proiettietal.,2014).
1.3.5 P2X7inhost-defenceanddisease
Purinergic signalling is involved in the chemotaxis of inflammatory cells, antimicrobial activity and
releaseofproinflammatorycytokines(DiVirgilioetal.,2017).P2X7isessentialforthehostdefence
againstbacterial,viral,fungalandparasiteinfections.However,differentstudiesusingP2X7-deficient
miceandpharmacologicalinhibitorsofP2X7showthatP2X7activationcaninfactimproveorworsen
thecourseofinfectiondependingonthepathogen,severityandcircumstancesofinfection(Savioet
al.,2018).Table1summarizestheconsequencesofP2X7activationinseveralpathogeninfections.
Table1.EffectsofP2X7modulationininfectiousdiseases.
Disease Species RoleofP2X7 Methodandimmuneconsequences Reference
Sepsis Mice Negative
P2X7KOmice(CLPmodel):- ↑survival- ↓immuneresponse:↓production
ofIL-1β,IL-6,IL-12,IL-17,andIL-4and↓lunginfiltration.
(Santanaetal.,2015)
Septicencephalopathy Mice Negative
P2X7 KO mice (CLP model) and P2X7antagonistA438079:
- ↑survival- ↓immuneresponse(↓production
ofIL-1β)- ↓leucocyteadhesion(↓CXCL1
andCX3CL1)
(H.Wangetal.,2015)
19
Sepsis Mice Positive
P2X7 KO mice (CLP model) and P2X7antagonistoATP:
- ↓survival- ↑immuneresponse(↑production
ofIL-1β,IL-6,TNF-α)- ↑bacterialburden
P2X7 agonists MgATP and bzATP: A438079(CLPmodel):
- ↑bacterialclearancebymacrophages
(Csókaetal.,2015)
Sepsis Human Negative
Gain-of-functionmutations:- Association with severe sepsis and
sepsisshock
(Geistlingeretal.,2012)
Mycobacteriumtuberculosis(H37Rv)
Mice Positive
P2X7KOmice:- ↑Tregs- ↑bacterialburdeninlungtissue
(Santosetal.,2013)
Mycobacteriumtuberculosis(Bejiing1471)
Mice Negative
P2X7KOmice:- Delayedmortality- ↓leucocyteinfiltration- ↓necroticdeathofmacrophages- ↓bacterialburdenandbacillus
dissemination
(Amaraletal.,2014)
MycobacteriumTuberculosis
Human Positive
Loss-of-functionSNPatposition1513:- Increasedsusceptibilitytoextra-
pulmonarytuberculosis- ↓bacterialclearanceby
macrophages
(Fernandoetal.,2007)
Chlamydiatrachomatis
Humanandmice Positive
ATPtreatedmacrophagesinfectedwithChlamydia:
- PLDactivation- Phagolysosomeformation- Acidificationofthephagolysosome
and↑clearanceofbacteria
(Coutinho-Silvaetal.,2003)
Denguevirus Human Positive
Virus-infectedmonocytes:- ↑productionofNO- ↓viralload- ↓productionofTNF,CXCL8,CCL2
andCXCL10.
(Corrêaetal.,2016)
HIV-1virus Human Negative
- ReleaseofHIV-1virusfromVCCofvirus-infectedprimaryMDMs.
(Grazianoetal.,2015)
HIV-1virus Human Negative
P2X7antagonistsoATPandBBG:- ↓HIVreplicationinprimaryhuman
macrophages
(Hazleton,BermanandEugenin,2012)
Influenzavirus Mice Negative
P2X7KOmice:- ↑survival- ↓neutrophilinfiltration- ↓productionofIFNγ,TNFα,IL-6
CXCL8,CCL2andCXCL10.
(Leyva-Gradoetal.,2017)
CLP(cecalligationpuncture);PLD(phospholipaseD);oATP(oxidizedATP);VCC(virus-containingcompartments).
AdaptedfromSavioetal.,2018
20
P2X7 is associated with the pathogenesis of many diseases from different systems in the body;
typically due to enhanced inflammatory responses caused by release of IL-1β and other
proinflammatorycytokines(Savioetal.,2018).Table2describesthecontributionofP2X7tomultiple
diseaseswithinflammatorycomponent.
Table2.ProtectiveeffectsofP2X7geneticablationorpharmacologicalinhibitionininflammatorydiseases.
Disease Mechanism Consequences Reference
Chronicinflammatory&neuropathicpain P2rx7KO
- ↓releaseofIL-1β- Absence of inflammatory and
neuropathichypersensitivity
(Chesselletal.,2005)
Acutelunginjury PI:A438079
- ↓NLRP3inflammasomeactivation- ↓releaseofIL-1β,IL-17AandIFN-γ
(Wangetal.,2015)
Lunginflammationandfibrosis P2rx7KO
- ↓lunginflammation- ↓fibrosismarkers
(Riteauetal.,2010)
Allergicairwayinflammation
P2rx7KO
- ↓asthmaticairwayinflammation- ↓airwayeosinophilia- ↓gobletcellhyperplasia- ↓bronchialhyperresponsivenessto
methacholine
(Mülleretal.,2011)PI:KN62
Experimentalglomerulonephritis
P2rx7KO
- Betterrenalfunction- ↓proteinuria- ↓glomerularinjury
(Tayloretal.,2009)
PI:A-438079
- Prevention of antibody-mediatedglomerulonephritis
PI:13A7
- ↓albuminuria- ↓inflammationmarkers- ↓kidneydamage
(Danquahetal.,2016)
Experimentalautoimmuneencephalomyelitis P2rx7KO
- ↓incidenceofthedisease- ↓astroglialactivationandaxonal
damage
(Sharpetal.,2008)
Type1Diabetes
P2rx7KO
- Preventionofthedisease- ↓immunecellinfiltration- Noalterationofpancreaticislet
numberandarea
(Vieiraetal.,2016)PI:BBG
Allergiccontactdermatitis PI:13A7
- ↓gaininearweight- ↓levelsofIL-6andIL-1β
(Danquahetal.,2016)
Anti-collagen-inducedarthritis P2rx7KO
- ↓susceptibilitytodisease- ↓swellingandrednessofaffected
joints- ↓destructionofcartilage
(Labasietal.,2002)
Experimentalcolitis PI:BBG
- ↓severityofcolitis- ↓myeloperoxidaseactivity- ↓collagendeposition- ↓levelsofIL-1β
(Marquesetal.,2014)
21
PI:A438079
- Attenuatedmurinecolitis.- ↓NF-κBactivation- ↓expressionofactivecaspase-1in
laminapropriaimmunecells- ↓levelsofIL-1βandTNFincolon
tissues
(Wanetal.,2016)
P2rx7KO
- ↓weightloss- ↓tissuedamage- ↑migrationandaccumulationof
Tregsinthecolon
(Figliuoloetal.,2017)
Liverfibrosis
PI:A438079
- ↓cellinjury- ↓inflammatoryinfiltrationandcell
injury- ↓collagenaccumulationintheliver- ↓levelsofTNF-α,IL-1βandCCL2in
theserum- ↓NFκBactivation
(Huangetal.,2014)
PI:BBGoATP
- ↓liverfibrosis- ↓IL-6,TNF-α,IL-1βandother
inflammatorymediatorsintheliver- ↑portal-systemiccollateralvascular
responsiveness
(Tungetal.,2015)
P2rx7-defficientmice(P2rx7KO);PI(pharmacologicalinhibition);BBG(brilliantblue-G);CCL2(chemokine(C-Cmotif)ligand2);oATP(oxidizedATP).
Also,P2X7 ishighlyexpressedbymany tumoursandpromotes tumourcell growth (Adinolfietal.,
2012). Tumorigenesis occurs under physiological concentrations of ATP (Di Virgilio, 2012).
Controversially,highconcentrationsofATPleadtotumourcelldeathinmanytypesofcancer(Savio
etal.,2018)andpotentiatestheefficacyofcytotoxicdrugs(Pachecoetal.,2016).
1.3.6 Therapiesandtools
P2X7antagonistshaveshownpromisingresultsinpreclinicaltrialsforthetreatmentofinflammatory
diseases,suchasglomerulonephritis,multiplesclerosis, inflammatorypainandrheumatoidarthritis
(Arulkumaranet al., 2011;Bartlett et al., 2014;Bhattacharya andBiber, 2016; Carroll et al., 2009;
Guile et al., 2009). However, some compounds, AZD9056 (NCT00520572) and CE-224,535
(NCT00628095)providednoextrabenefitonclinicaltrials(Keystoneetal.,2012;Stocketal.,2012);
whileothershowedlimitationsintermsofselectivity,dosageandadverseeffects(Friedle,Curetand
Watters,2010;Bartlett,StokesandSluyter,2014).
An alternative therapeutic approach is the use of nanobodies (Nbs). Nbs are the smallest antigen
variablebindingdomain (VHH)of heavy chainonly antibodies (hcAbs)present in camelids (Arbabi-
Ghahroudi, 2017). Nbs exhibit numerous advantages over conventional Abs. Their smaller size,
biochemicalcharacteristicsandthelengthandflexibilityoftheirCDR3region,enablesthemtoreach
and bind epitopes otherwise inaccessible to conventional Abs (Muyldermans, 2013). Furthermore,
22
Nbs are weakly immunogenic, and present high stability, solubility and tissue penetration
(Wesolowski et al., 2009; Unciti-Broceta et al., 2013). They are also easy to reformat by genetic
manipulation. The genetic linkage ofmultipleNbswith same specificity (multivalentNb) increases
theavidityoftheconstruct.LinkagetootherNbswithdifferentspecificityand/orlinkagetocertain
proteindomainsprovideadditional functionsorcandirect theNbs towardsaspecific target tissue
(Wesolowski et al., 2009; Farrington et al., 2014). For instance, fusion to an albumin-specific
nanobodyorFc-domainresultsinanincreasedhalf-lifeoftheNb(Tijinketal.,2008).
23
2. AIMSOFTHESTUDY
ThelaboratoryofProfessorKoch-NoltehasgeneratedP2X7-specificnanobodiesforthepositiveand
negativemodulationofP2X7functioninmice.Theyhaverecentlyshownthatinvivoadministration
of P2X7-specific nanobodies ameliorates allergic contact dermatitis and experimental
glomerulonephritis in mice (Danquah et al., 2016), underlining the potential of P2X7-specific
nanobodies as a therapeutic drug for the treatment of inflammatory disorders. Professor Koch-
Nolte´s laboratory has also generated a highly specific nanobody targeting P2X7 in humans;
designatedDano1(Danquahetal.,2016).
Inlinewiththesefindings,themaingoalofthisdoctoralthesisistoassessthepotentialoftheanti-
humanP2X7nanobodyDano1asatooltomodulatethefunctionoftheP2X7receptor inhuman
immunecells.Thespecificaimsare:
1. To assess the expression of the P2X7 receptor on distinct human immune cell
subpopulations.
2. TodeterminewhethertheexpressionofP2X7ismodulateduponactivationofhumanTcells.
3. ToinvestigatetherelevanceofP2X7activationorblockadeinhumanTcells.
24
3. MATERIALANDMETHODS
3.1 MATERIALS
3.1.1 Bloodandtissuesamples
Sample Source Ethicsprotocol
Peripheralbloodfromhealthyadults Volunteers PV5139
Peripheralbloodfromchildren CenterforObstetricsandPediatrics PV5482
Buffycoatsfromhealthyadults BloodBank -
Guttissuefromobesepatients CenterforInternalMedicine PV4889
3.1.2 Generalequipment
Equipment Company,Location
AnalyticalbalanceLA124i VWRInternational,Radnor(PA),USA
Centrifuge5424R Eppendorf,Hamburg,Germany
Centrifuge5810R Eppendorf,Hamburg,Germany
CentrifugeAllegraX-30R BeckmanCoulter,Brea(CA),USA
CentrifugeBiofugefresco Heraeus,Hanau,Germany
Freezer-20°C Liebherr,Bulle,Switzerland
Freezer-80°C ThermoFisherScientific,Waltham(MA),USA
InvertedMicroscope Olympus,Shinjuku,Tokyo,Japan
MicrocentrifugeGalaxyMiniStar VWR,Radnor(PA),USA
Multi-channelpipettes(100μl/300μl) Eppendorf,Hamburg,Germany
Neubauercellchamber Merienfeld,Lauda-Königshofen,Germany
pH-MeterWTWpH523 Xylem,RyeBrook(NY),USA
PIPETBOYacu2 IntegraBiosciences,Biebertal,Germany
Pipettes2.5/10/20/200/1000μl Eppendorf,Hamburg,Germany
Pipettes20/200μl Gilson,VilliersleBel,France
PrecisionbalancePrecisa400M OehmenLabortechnik,Essen,Germany
Refrigerator/freezer Liebherr,Bulle,Switzerland
ScoutProDigitalscale Ohaus,Parsippany(NJ),USA
Thermomixer5436 Eppendorf,Hamburg,Germany
Thermoshakerwithheatedlid CLF,Emersacker,Germany
25
VacuumpumpVacusafe IntegraBiosciences,Biebertal,Germany
Vortexmixer Heidolph,Schwabach,Germany
Vortexmixer Scientificindustries,Bohemia(NY),USA
Waterbath GFL,Burgwedel,Germany
3.1.3 Materialsforcellculture
Chemicalreagentsandtheirmanufacturers
Adenosinetriphosphate(ATP) Sigma-Aldrich,St.Louis(MO),USA
Apyrase Sigma-Aldrich,St.Louis(MO),USA
AZ10606120(P2X7antagonist) Tocris,Ellisville(MO),USA
BenzoylbenzoylATP(bzATP) Tocris,Ellisville(MO),USA
Biocoll Merck,Darmstadt,Germany
Bovineserumalbumin(BSA) ThermoFisherScientific,Waltham(MA),USA
BrefeldinAsolution,1000X eBioscience,SanDiego(CA),USA
Dimethylsulfoxide(DMSO) AppliChem,Darmstadt,Germany
Ethanol(70%)fordisinfection Th.Geyer,Renningen,GermanyEthylenediaminetetraaceticacid(EDTA),0.5M
AppliChem,Darmstadt,Germany
Fetalbovineserum(FBS) Biochrom,Berlin,Germany
Fetalcalfserum(FCS) Merck,Darmstadt,Germany
Humanserum(TypeAB) Merck,Darmstadt,Germany
JNJ47965567(P2X7antagonist) R&DSystems,Minneapolis(MN),USA
L-glutamine,200mM ThermoFisherScientific,Waltham(MA),USA
Lipopolysaccharide(LPS) Sigma-Aldrich,St.Louis(MO),USA
Nigericin Sigma-Aldrich,St.Louis(MO),USAPenicillin(10000U/ml)/Streptomycin(10000µg/ml) ThermoFisherScientific,Waltham(MA),USA
Phosphate-bufferedsaline(PBS)(1x,10x) ThermoFisherScientific,Waltham(MA),USA
Poly-L-lysinesolution0.01% Sigma-Aldrich,St.Louis(MO),USA
RPMI1640withorw/ophenolred ThermoFisherScientific,Waltham(MA),USA
Sodiumdichloroisocyanurate(ChlorClean) CarlRoth,Karlsruhe,Germany
Trypanblue,0.4% Sigma-Aldrich,St.Louis(MO),USA
Tuerksolution Sigma-Aldrich,St.Louis(MO),USA
X-VIVO15medium Lonza,Basel,Switzerland
26
Cellculturemediaandtheircomposition
FullRPMIMedium
10%FCS(heatinactivated)1%penicillin/streptomycin2mML-glutamineinRPMI
Tcellmedium
10%humanserum(heatinactivated)1%penicillin/streptomycin2mML-glutamineinRPMI
FreezingmediumI 90%RPMI,10%FCS
FreezingmediumII 40%RPMI,40%FCS,20%DMSO
Buffersandsolutionsandtheircomposition
10XMACSbuffer 20mMEDTA,5%BSA(w/v)in10XPBS
Permeabilisation/blockingbuffer 1%BSA+0.2%Saponinin1XPBS
Trypanbluesolution 0.4%trypanbluesolution,1:10inPBS
Reagentsforthestimulationofimmunecells
HDMAPPammoniumsalt EchelonBiosciences,SaltLakeCity(UT),USA
HumanrecombinantIL-2(Tecine) Roche,Basel,Switzerland
Ionomycincalciumsalt Sigma-Aldrich,St.Louis(MO),USA
Phorbolmyristateacetate(PMA) Sigma-Aldrich,St.Louis(MO),USA
PhytohemagglutininPHA Sigma-Aldrich,St.Louis(MO),USA
Purifiedanti-humanCD28antibody(CD28.2) BioLegend,SanDiego(CA),USA
Purifiedanti-humanCD3antibody(OKT3) BioLegend,SanDiego(CA),USA
Retinoicacid Sigma-Aldrich,St.Louis(MO),USA
Kitsfortheseparationofimmunecellsandtheirmanufacturers
MonocytesisolationKitII MiltenyBiotec,BergischGladbach,Germany
3.1.4 Materialsformolecularbiology
Isolationkits
NucleospinBloodKit(DNA,RNAandproteinpurification) Macherey-Nagel,Düren,Germany
27
NucleoSpinGelandPCRClean-up Macherey-Nagel,Düren,Germany
QIAshredder Qiagen,Hilden,Germany
RNeasyPlusMiniKit Qiagen,Hilden,Germany
Chemicalreagentsandtheirmanufacturers
DNaseI Qiagen,Hilden,Germany
Ethanol≥99,8% Roth,Karlsruhe,Germany
ProteinaseK Macherey-Nagel,Düren,Germany
β-mercaptoethanol,50mM Invitrogen,Carlsbad(CA),USA
Chemicalreagents,materialsandequipmentforconventionalPCR
6XDNAloadingdye ThermoFisherScientific,Waltham(MA),USA
Biovision3026MXUV-Illuminator BiovisionTechnologies,Exton(PE),USA
Deoxyribonucleotidetriphosphates(dNTPS) Merck,Burlington(MA),USA
DNAgelelectrophoresis40-0708 PeqlabBiotechnologie,Erlangen,Germany
GeneRuler1KbDNAladder ThermoFisherScientific,Waltham(MA),USA
KODBuffer(10X) Merck,Burlington(MA),USA
KODPolymerase Merck,Burlington(MA),USA
LaminarAirFlowClassIII Gelaire,SevenHills(NSW),Australia
LEAgarose BiozymScientific,HessischOldendorf,Germany
Magnesiumsulphate(MgSO4) Merck,Burlington(MA),USA
Nulcease-freewater ThermoFisherScientific,Waltham(MA),USA
Quickload100bpDNAladder Biolabs,Ipswich(MA),USA
Roti-SafeGelStain Roth,Karlsruhe,Germany
ScoutProDigitalscale Ohaus,Parsippany(NJ),USA
T3PCRThermocycler Biometra,Göttingen,Germany
TAEgelrunningbuffer50X ApplicChemGmbH,Darmstadt,Germany
TI1Transilluminator(UV) Biometra,Göttingen,Germany
28
Listofprimers
P2RX7genotyping
P2RX7(Exon5)ForwardPrimer 5’TAGGATGGGCTTGATGGAAG3’
P2RX7(Exon5)ReversePrimer 5’TAGGACCCAGGACTTTGCAG3’
P2RX7(Exon8)ForwardPrimer 5’TGGCTATGCAGGGAGATGT3’
P2RX7(Exon8)ReversePrimer 5’GCCTTGGAAACCAAAATTAGTC3’
P2RX7(Exon11)ForwardPrimer 5’CCAGCTGGTTGTGTACATCG3’
P2RX7(Exon11)ReversePrimer 5’TGATTTGGGCCTAATTTTCG3’
ENTPD1genotyping
ENTPD1ForwardPrimer 5’GTAGAGGGAGGAAATAG3’
ENTPD1ReversePrimer 5’TGGCTACTCATGCTAT3’
EquipmentforDNAandRNAquantification
Nanodrop2000c ThermoFisherScientific,Waltham(MA),USA
MaterialsforcDNAtranscription
0.1MDithiothreitol(DTT) Invitrogen,Carlsbad(CA),USA
DEPC-H2O Invitrogen,Carlsbad(CA),USA
dNTPS(dATP,dCTP,dGTP,dTT;100mM) Invitrogen,Carlsbad(CA),USA
FirstStrandBuffer5X Invitrogen,Carlsbad(CA),USA
M-MLVReverseTranscriptase Invitrogen,Carlsbad(CA),USA
Randomprimers Invitrogen,Carlsbad(CA),USA
Materialsforreal-timePCR(Taqmanassays)
GAPDHtaqmanprobe;Hs02578991_g1 ThermoFisherScientific,Waltham(MA),USA
IL1Btaqmanprobe;Hs00174097_m1 ThermoFisherScientific,Waltham(MA),USA
IL6taqmanprobe;Hs00985639_m1 ThermoFisherScientific,Waltham(MA),USA
MaximaProbe/ROXqPCRMasterMix ThermoFisherScientific,Waltham(MA),USA
TNFtaqmanprobe;Hs01113624_g1 ThermoFisherScientific,Waltham(MA),USA
29
Materialsforreal-timePCR(SYBRGreenassays)
MaximaSYBRGreen/ROXqPCRMasterMix ThermoFisherScientific,Waltham(MA),USA
Equipmentforrealtime-PCR
StepOnePlusRealTimePCRsystem AppliedBiosystems,FosterCity(CA),USA
Listofprimers
ReferenceGenes
RPL13AForwardPrimer 5’ATCTTGTGAGTGGGGCATCT3’
RPL13AReversePrimer 5’GCTTGCTGTTGTACACAGGG3’
18SForwardPrimer 5’CGGCTACCACATCCAAGGAA3’
18SReversePrimer 5’GCTGGAATTACCGCGGCT3’
Genesofinterest
P2RX7ForwardPrimer 5'TCTTCGTGATGACAAACTTTCTCAA3'
P2RX7ReversePrimer 5'GTCCTGCGGGTGGGATACT3'
3.1.5 Materialsforisolationofimmunecellsfromhumanguttissue
Chemicalreagentsandtheirmanufacturers
CollagenaseIVfromClostridiumhistolyticum Sigma-Aldrich,St.Louis(MO),USA
Dithiothreitol(DTT) AppliChem,Darmstadt,Germany
DNaseI AppliChem,Darmstadt,Germany
Hanks´balancedsaltsolution10X(HBSS) ThermoFisherScientific,Waltham(MA),USA
HEPES Roth,Karlsruhe,Germany
Percoll GEHealthcare,LittleChalfont,UK
Buffersandsolutionsandtheircomposition
DTTsolution(for500ml)
50ml10XHBSS50mlHEPES-bicarbonatebuffer50mlFBS350mldH2O15.4mgDTT/100ml(Fc:1mM)
30
HEPES-bicarbonatebuffer10X
23.8gHEPES(Fc:100mM)21gNaHCO3(Fc:250mM)in 1 litre of ddH2O (pH adjusted to 7.2 withHCl)
Collagenasesolution(for500ml)
500mlRPMI55mlFBS5.5ml100XHGPG1ml0.5MofCaCl21ml0.5MMgCl2100U/mlcollagenase
HGPG100X
59.6gHEPES14.6gL-glutamine1x106Upenicillin1gstreptomycinIn 500 ml RPMI (pH adjusted pH to 7.2 withHCl)
3.1.6 Materialsforflowcytometry
Fluorochrome-conjugatedreagentsanttheirmanufacturers Live/deaddyes L/DsuccinimidylesterPacificOrange Invitrogen,Carlsbad,CA,USAL/DsuccinimidylesterAF750 ThermoFisherScientific,Waltham(MA),USA Proliferationdyes ProliferationdyeeFluor670 ThermoFisherScientific,Waltham(MA),USAFluorochrome-conjugatedantibodiesandtheirmanufacturers
Specificity Fluorochrome Clone Company
Lineagemarkers
CD2 FITC RPA-2.10 BectonDickinson,FranklinLakes(NJ),USA
CD3 APC-Cy7 UCHT1 BioLegend,SanDiego(CA),USA
CD3 PerCP-Cy5.5 OKT3 ThermoFisherScientific,Waltham(MA),USA
CD3 V510 OKT3 BioLegend,SanDiego(CA),USA
CD3 APC-Cy7 OKT3 BioLegend,SanDiego(CA),USA
CD3 BV650 UCHT1 BioLegend,SanDiego(CA),USA
CD4 APC SK3 BioLegend,SanDiego(CA),USA
CD4 AF488 RPA-T4 BioLegend,SanDiego(CA),USA
CD4 V500 RPA-T4 BectonDickinson,FranklinLakes(NJ),USA
CD4 APC-Cy7 RPA-T4 BioLegend,SanDiego(CA),USA
31
CD8α V510 RPA-T8 BioLegend,SanDiego(CA),USA
CD8 BV421 RPA-T8 BioLegend,SanDiego(CA),USA
CD8α PacificBlue RPA-T8 BioLegend,SanDiego(CA),USA
CD8β PE 2ST8.5H7 BectonDickinson,FranklinLakes(NJ),USA
CD8 PE-Cy7 SK1 BioLegend,SanDiego(CA),USA
CD8α FITC RPA-T8 BioLegend,SanDiego(CA),USA
CD14 V450 MφP9 BectonDickinson,FranklinLakes(NJ),USA
CD14 FITC M5E2 BioLegend,SanDiego(CA),USA
CD16 APC-Cy7 3G8 BioLegend,SanDiego(CA),USA
CD19 PE-Cy7 HIB19 BioLegend,SanDiego(CA),USA
CD20 V450 L27 BectonDickinson,FranklinLakes(NJ),USA
CD20 FITC 2H7 BioLegend,SanDiego(CA),USA
CD24 PerCP-Cy5.5 ML5 BioLegend,SanDiego(CA),USA
CD33 BV421 WM53 BioLegend,SanDiego(CA),USA
CD45 V510 HI30 BioLegend,SanDiego(CA),USA
CD56 PE-Cy7 B159 BectonDickinson,FranklinLakes(NJ),USA
CD56 BV421 HCD56 BioLegend,SanDiego(CA),USA
CD56 APC HCD56 BioLegend,SanDiego(CA),USA
CD127 PerCP-Cy5.5 A019D5 BioLegend,SanDiego(CA),USA
CD161 BV421 HP-3G10 BioLegend,SanDiego(CA),USA
CD161 PE HP-3G10 BioLegend,SanDiego(CA),USA
CD163 PerCP-Cy5.5 GHI/61 BioLegend,SanDiego(CA),USA
Activationmarkers
CD25 PE M-A251 BectonDickinson,FranklinLakes(NJ),USA
CD25 BV421 BC96 BioLegend,SanDiego(CA),USA
CD80 PE L307.4 BectonDickinson,FranklinLakes(NJ),USA
HLA-DR FITC G46-6 BectonDickinson,FranklinLakes(NJ),USA
Maturationmarkers
CD27 APC-Cy7 O323 BioLegend,SanDiego(CA),USA
CD28 PE-Cy7 CD28.2 BioLegend,SanDiego(CA),USA
CD45RA PE-Cy7 HI100 BioLegend,SanDiego(CA),USA
CD45RA BV421 HI100 BioLegend,SanDiego(CA),USA
CD62L FITC DREG-56 BioLegend,SanDiego(CA),USA
CD62L PE DREG-56 BectonDickinson,FranklinLakes(NJ),USA
CD69 APC-Cy7 FN50 BioLegend,SanDiego(CA),USA
32
CD197(CCR7) AF647 G043H7 BioLegend,SanDiego(CA),USA
Ectoenzymes
CD38 PerCP-Cy5.5 HIT2 BioLegend,SanDiego(CA),USA
CD38 AF488 HIT2 BioLegend,SanDiego(CA),USA
CD39 PE-Cy7 A1 BioLegend,SanDiego(CA),USA
CD73 PE AD2 BioLegend,SanDiego(CA),USA
Chemokinereceptors
CD183(CXCR3) AF488 1C6/CXCR3 BectonDickinson,FranklinLakes(NJ),USA
CD194(CCR4) PE-Cy7 L291H4 BioLegend,SanDiego(CA),USA
CD196(CCR6) PerCP-Cy5.5 G034E3 BioLegend,SanDiego(CA),USA
CX3CR1 PE-Cy7 2A9-1 BioLegend,SanDiego(CA),USA
Tcellreceptorchainmarkers
Tγδ FITC 11F2 BectonDickinson,FranklinLakes(NJ),USA
Tγδ PE 11F2 BectonDickinson,FranklinLakes(NJ),USA
Tγδ PE-Cy7 11F2 BectonDickinson,FranklinLakes(NJ),USA
Vδ1 FITC TS-1 ThermoFisherScientific,Waltham(MA),USA
Vδ2 APC 123R3 MiltenyBiotec,BergischGladbach,Germany
Vδ2 PE B6 BectonDickinson,FranklinLakes(NJ),USA
Vδ2 FITC B6 BioLegend,SanDiego(CA),USA
Vα7.2 APC 3C10 BioLegend,SanDiego(CA),USA
Vα7.2 PE 3C10 BioLegend,SanDiego(CA),USA
Vγ9 FITC IMMU360 BeckmanCoulter,Brea(CA),USA
Vα24 FITC 6B11 BioLegend,SanDiego(CA),USA
Otherantibodies
rhIgG1Fc - 110-HG R&DSystems,Minneapolis(MN),USA
ASC - N-15 SantaCruzBiotechnology,Dallas(TX),USA
RabbitFc(rbFc) PE - Dianova,Hamburg,Germany
IgD PE IA6-2 BioLegend,SanDiego(CA),USAFluorochrome-conjugatednanobodiesandtheirproviders
Specificity Fluorochrome Clone Provider
hP2X7(Dano1) AF647/- 3c23 ProfessorDr.FriedrichKoch-Nolte,UKE
mARTC2.2 AF647/- s+14 ProfessorDr.FriedrichKoch-Nolte,UKEToxinA,C.difficile
- L+8 ProfessorDr.FriedrichKoch-Nolte,UKE
33
Chemicalreagentsandtheirmanufacturers
Bovineserumalbumin(BSA) ThermoFisherScientific,Waltham(MA),USA
Cleansolution BectonDickinson,FranklinLakes(NJ),USA
Flowsheathfluid BectonDickinson,FranklinLakes(NJ),USA
Rinsesolution BectonDickinson,FranklinLakes(NJ),USA
Sodiumazide(NaN3) Sigma-Aldrich,St.Louis(MO),USA
Fixationbuffer eBioscience,SanDiego(CA),USA
PermeabilisationBuffer10X eBioscience,SanDiego(CA),USA
Paraformaldehyde(PFA) MorphistoGmbH,Frankfurt,Germany
DAPI Merck,Darmstadt,GermanyFlowcytometrybuffersandtheircomposition
FACSbuffer 0.1%BSA,0.02%NaN3in1XPBSFlowcytometryequipmentandmaterialsandtheirmanufacturers
FACSAriaFusion(FACSCoreFacility,UKE) BectonDickinson,FranklinLakes(NJ),USA
FACSAriaIIIu(FACSCoreFacility,UKE) BectonDickinson,FranklinLakes(NJ),USA
FACSCantoII BectonDickinson,FranklinLakes(NJ),USA
FACSCelesta BectonDickinson,FranklinLakes(NJ),USA
3.1.7 Materialsforenzyme-linkedimmunosorbentassay(ELISA)
Buffers,solutionsandtheircomposition
ELISAwashbuffer 0.05%Tween-20in1XPBS
Stopsolution 2NH2SO4
Commerciallyavailablekits
HumanIL-1betaELISAReady-SET-Go eBioscience,SanDiego(CA),USA
Equipment
PlatereaderVictor31240 PerkinElmer,Waltham(MA),USA
34
3.1.8 Generalconsumables
Material Company
Cellstarpolypropylenetubes(15ml,50ml) Greinerbio-one,Kremsmünster,Austria
5mLpolystyreneroundbottomtubes Sarstedt,Nümbrecht,Germany
Cellscraper TPP,Trasadingen,Switzerland
Cellstrainer Sigma-Aldrich,St.Louis(MO),USA
epT.I.P.S(1000µl) Eppendorf,Hamburg,Germany
Examinationgloves UKE,Hamburg,Germany
MilliporeExpressPLUS0.22μm Merck,Darmstadt,Germany
Petridish Nunc,Roskilde,Denmark
Pipettetips(10µl,200µl,1000µl) Sarstedt,Nümbrecht,Germany
Reagentreservoir Starlab,Hamburg,Germany
SafeSealtubes(1.5ml,2ml) Sarstedt,Nümbrecht,Germany
Scalpel CardinalHealth,Dublin(OH),USA
Serologicalpipettes Sarstedt,Nümbrecht,Germany
S-MonovetteZ-Gel/K3E Sarstedt,Nümbrecht,Germany
3.1.9 Software
Material Company
4peaks NucleobytesB.V.,Aalsmeer,TheNetherlands
AdobeIllustratorCS2 AdobeSystems,SanJose(CA),USA
FACSDiva BectonDickinson,FranklinLakes(NJ),USA
FinchTV Gospiza,Inc.,Seattle(WA),USA
FlowJo10.4 FlowJoLLC,Ashland(OR),USA
GraphPadPrism6.07 GraphPadSoftware,LaJolla(CA),USA
Mendeley(Desktop1.18andweb) Elsevier,NewYorkCity(NY),USA
MicrosoftOfficeProfessional2010 Microsoft,Redmond(WA),USA
Nanodrop2000/2000csoftware ThermoFisherScientific,Waltham(MA),USA
StepOneSoftware2.3 AppliedBiosystems,FosterCity(CA),USA
35
3.2 METHODS
Allbuffersandsolutionswerepreparedusingpurifiedwater (ddH2O)orphosphate-bufferedsaline
withoutcalcium(Ca2+)andmagnesium(Mg2+)(PBS-/-),ifnotstatedotherwise.
3.2.1 Donors
3.2.1.1 Humanblood
Human peripheral blood from adult healthy individuals at the UKE was freshly drawn in EDTA or
heparincollectiontubes.BuffycoatsfromhealthyindividualswerekindlyprovidedbytheBloodBank
attheUKE.Alldonorswereoflegalage.Informedconsentwasobtainedfromallvolunteers.Ofnote,
blood samples were obtained and handled according to corresponding ethics protocol
(EthikkommissionderÄrztekammerHamburg,RegulationsmechanismenvonImmunzellen;PV5139).
Humanperipheralbloodfromachildwithcryopyrinassociatedperiodicsyndrome(CAPS)waskindly
provided by Dr.med. Kai Lemberg from the Center for Obstetrics and Pediatrics at the UKE, and
handled according to corresponding ethics protocol (Frühkindliche, medizinische Eingriffe,
AuswirkungenaufdieEntwicklungdesImmunsystems;PV5482).
3.2.1.2 Humangastrointestinaltissue
Humangastrointestinal tissuewasobtainedasdiscardedmaterial fromthe ileumpartof thesmall
intestinefrompatientsundergoinglaparoscopicgastricbypasssurgeryduetomorbidobesityatthe
UKE.GutsampleswerekindlyprovidedbyProf.NicolaGaglianifromtheCenterforInternalMedicine
in the UKE. Of note, human tissue was obtained and handled according to corresponding ethics
protocol(PV4889).
3.2.1.3 Tcelllines
Tcelllineswerederivedfromhumanperipheralbloodmononuclearcells(PBMCs)fromadulthealthy
donors.
36
3.2.2 Isolationofhumancells
3.2.2.1 Isolationofperipheralbloodmononuclearcells
PBMCs fromperipheralbloodorbuffycoatswere isolatedbydensitygradientcentrifugationusing
Biocoldensitygradientmedium.Biocol is ahydrophilicpolymer thatallows the separationof cells
accordingtosize,densityandshape.BloodwasdilutedwithtwopartsofPBS-/-atroomtemperature
(RT).Afterwards,30mlofdilutedbloodwerecarefullylayeredover20mlofBiocolina50mlFalcon
tube, and centrifuged for 25 min at 800 xg with slow acceleration and break at RT. After
centrifugation,mononuclearcellsconstituteamonolayerattheinterphasebetweenplasmaandthe
separationmedium,whereaserythrocytesdepositasasubstantialpelletatthebottomofthetube.
ThemonolayerwasthenrecoveredbyaspirationandwashedtwicewithcoldPBS;firstfor10minat
650xgandafterwardsfor5minat450xgat4°C.Thetotalcellnumberwasmanuallyassessedusing
a Neubauer counting chamber. Dead cells were excluded by dye exclusion using Trypan blue.
Subsequently,theformulaN°ofcellsxDilutionfactorx10000=n°ofcells/mlwasusedtocalculate
thetotalamountofcells.IsolatedPBMCswereimmediatelyusedforfurtherexperiments.
3.2.2.2 Isolationofinfiltratingimmunecellsfromhumanguttissue
Piecesofsmallintestinetissuewereobtainedasdiscardmaterialfrompatientsundergoingbariatric
surgery.TheintestinaltissuewasplacedintoaPetridishcontainingRoswellParkMemorialInstitute
medium(RPMI).Circularfoldsofgutmucosawereseparatedfromthemuscularlayerandfattissue,
cutintosmallpiecesofapproximately0.5cmandincubatedin1mMdithiothreitol(DTT)solutionfor
20minat37°C,whileshaking.Afterwards,theremainingtissueandmediumwerefilteredthrougha
metal cell strainer and washed twice with PBS supplemented with 1% fetal bovine serum (FBS)
(450xg, 5 min 4°C). The supernatant, which contains the intraepithelial lymphocytes (IELs), was
collected and kept at 4°C. To further isolate the laminapropria lymphocytes (LPLs), the remaining
pieces of small intestine were collected, minced with a scalpel and treated with collagenase IV
solution(1mg/ml)containingfreshlyaddedDNase I (10U/ml) for45min, ina5%CO2 incubatorat
37°C,whileshaking.Thedigestedintestinaltissuewasfurtherhomogenizedthroughametalstrainer,
washed once with PBS supplemented with 1% FBS, and the flow-through was collected. The
supernatantscontainingIELsorLPLswerepooledtogetherandpelletedbycentrifugationfor10min,
at515xg, at4°C. From thepooled cells, leukocyteswereadditionallyenrichedbyPercoll gradient
centrifugation.First,Percollosmolaritywasadjustedbyadding1part10XPBSto9partsofPercoll.
Percollsolutionwasfurtherdilutedwith6partsofRPMIsupplementedwith1%FBStocreatea40%
Percoll solution, and used to resuspend the cell pellet. Next, 4 ml of the cell suspension were
37
carefully layeredover4mlof isotonicPercoll solution (67%)and centrifugedat450xg for20min
with low acceleration and break. Infiltrating lymphocytes were collected from the interphase by
aspiration, washed once with 1X PBS supplemented with 1% FBS, at 450 xg for 5 min, and
resuspendedinRPMImediumsupplementedwith10%FBS.
3.2.3 Magnetic-activatedcellsorting
Magnetic-activated cell sorting (MACS) is a method that allows the separation of distinct cell
populationsbasedonmagneticnanoparticlesthatarecoatedwithantibodiesagainstspecificsurface
molecules.Magnetically-labelledcellsarethenretainedtoseparationcolumns.Usingthistechnique,
cellscanbepositivelyornegativelyselected.Positiveselectionisachievedbylabellingofthecellsof
interest,whereasnegativeselectionisachievedbytargetingalltheotherpopulationsbuttheoneof
interest(Miltenyietal.,1990).
3.2.3.1 Enrichmentofhumanmonocytes
Human monocytes were enriched from PBMCs by negative selection using the human monocyte
Isolation Kit II (Milteny Biotec) according to themanufacturer´s protocol. Briefly, a total of 4· 107
PBMCswerepassedthrougha30µMcellfiltertoavoidthecloggingofthecolumn,andcentrifuged
for10minat290xg.Next,cellswereincubatedinasolutioncontainingMACSbuffer,andantibodies
againstFc-receptors(FcR)andbiotin-conjugatedantibodiesagainstthefollowingantigens:CD3,CD7,
CD16, CD19, CD56, CD123 and CD235a, for 10 min at 4°C. Afterwards, anti-biotin magnetic
MicroBeads,whichbindtothebiotinylatedantibodies,wereaddedtothecellsandfollowedby15
min incubation at 4°C. Cells were washed twice at 290 xg for 10 min and resuspended in MACs
buffer.ThecellsuspensionwasrunthroughaMACSLScolumnandwashedthricewith3mlofMACS
buffer.TheeffluentcontainingthemonocyteenrichedfractionwasthencollectedinaFalcontube.
Cellsretainedinthecolumnswereelutedoutsideofthemagneticfieldandusedascontroltoassess
thepurityoftheprocess.Bothfractionswerestainedusingantibodiesagainstlineagemarkers(Table
3)for15minatRT,andthepuritywasfinallyassessedbyflowcytometry.
Table3.ListofantibodiesusedfortheassessmentofpurityafterMACSisolation.
Fluorochrome
V450 V510 AF488 PE PerCP-Cy5.5 PE-Cy7 APC APC-Cy7Antigen
CD14 CD45 CD4 TCRγδ CD3 CD19 CD56 CD16
3.2.4 Flowcytometry
Flow cytometry is one of the most widely used methods in cell-based research. It allows the
simultaneous analysis of both physical and chemical properties on single cells. The fluidics system
38
forces cells tomove single file in droplets through a beam of light. The system is equipped with
mirrors,filtersandopticaldetectorsthatcollectthelightscatteredbythecells,andphotomultipliers
(PMTs) that convert the optical signal into electrical pulses. Electrical pulses are then further
processedandconverted intodigitaldata. Eachcell emits the light from the laser in twodifferent
ways:theForwardScatter(FSC),providing informationaboutthesizeorarea;andtheSideScatter
(SSC),whichrelatestotheinternalcomplexityofthecell(Adanetal.,2017;Cossarizzaetal.,2017).
Usingthistechnique,distinct immunecellpopulationscanbeidentifiedbyusingantibodiesagainst
differentcellmarkers.Tothisend,antibodiesareconjugatedtofluorochromes,chemicalcompounds
thatabsorblightatcertainwavelengthsandre-emititatadistinctwavelength.Theemittedlightis
detectedbydifferentfluorescencedetectors.Ofnote,somefluorochromesareexcitedoremitlight
atasimilarwavelength,whichmaycauseinterferenceamongthem.Thisphenomenonisknownas
spillover, and the method applied to correct the overlapping emission spectra is known as
compensation. Flow cytometry also allows the sorting of live cells based on fluorescence labelling
[section3.2.4.4].
Unlessotherwisestated,washingstepswereperformedwith1XPBS-/-at450xgandatRT.
3.2.4.1 Stainingofsurfacemolecules
Atotalof0.5·106to1·106cellswereusedforstainingofmoleculesexpressedonthecellplasma
membrane.CellsweretransferredtoFACStubeswashedoncewithPBSorRPMIandresuspendedin
PBS or RPMI. To block unspecific binding of the antibodies, cellswere incubatedwith human IgG
(hIgG) for 5 min at RT. Cells were further incubated with antibodies against markers of interest
dilutedinFACSBufferfor30min.Afterwashing,cellswereresuspendedin150–200µlFACSbuffer
andanalysedbyflowcytometry.
Incaseof stainingwholeblood,50µlofbloodwerestainedasdescribedabove instead.After the
incubationwith the staining antibodies andwashingwithPBS, erythrocyte lysiswasperformedby
additionof1ml1Xlysingsolutionfor10minatRTinthedark.
StainingofhumanP2X7wascarriedoutusingthefollowingstrategy.Duetoalowexpressionofthe
receptor,astainingcontrolforP2X7wasperformed.Tothisend,twotubeswererequiredforeach
staining. In the first tube,cellswerestainedwithanantibodycocktail containing theP2X7-specific
nanobody Dano1, whereas the cells from the second tube were stained with the same antibody
cocktail containing ananobody againstmouseARTC2.2 instead.Asmentioned in the introduction,
ARTC2.2 isnotexpressed inhumans.Thus, thesecondtubeservesas isotypecontrol (IC),which is
neededtoensurethespecificityoftheP2X7signal.
39
3.2.4.2 Exclusionofdeadcellsandcellcounting
Dead cellswere excludedusing the amine-reacting life/dead (L/D) fluorescent dyes PacificOrange
(PacO,1µg/ml)orAlexaFluor750(AF750,1µg/ml). Incellswith intactcellmembranes, thesedyes
cannot penetrate the plasmamembrane and bind only to amines present on the cellmembrane.
However,disruptionof theplasmamembraneoccurringduringcelldeath facilitatesbinding to the
aminespresentinsidethecell,thereforeresultinginamoreintensesignal.
L/Dwasadded10minaftertheadditionofthesurfacestainingantibodies,andthecellsuspension
waskeptat4°Cforthefollowing20min.CellswerethenwashedoncewithFACSbufferorRPMIto
removetheexcessofreactivedye.
3.2.4.3 Stainingofintracellularmolecules
For the intracellular staining (ICC) of cytokines and/or transcription factors (TFs), cells were
resuspended inserumfreeX-VIVO-15mediumandstimulatedwith50ng/mlofphorbol-myristate-
acetate(PMA)and1µg/mlofionomycin(Iono)for5hoursintheincubatorat37°Cand5%CO2.To
preventthereleaseofcytokines intothemedium,10µg/mlofbrefeldinA(BfA)wasadded30min
afterstimulationwithPMA/Iono.BfAisasubstancethatinhibitsthetransportofproteinsfromthe
endoplasmic reticulumto theGolgiapparatus, resulting inanaccumulationof cytokines inside the
cell.
After stimulation cells were washed and stained for cell surface molecules and live/dead dye as
describedinsections3.2.4.1and3.2.4.2.Afterwashingtwice,firstwithcoldPBSandthenwithPBS
with 0.2% bovine serum albumin (BSA), pelleted cells were resuspended in 1 ml of 1X
fixation/permeabilisationbufferandfurtherincubatedfor1hat4°Cinthedark.Afterwashingtwice
with1Xpermeabilisationbuffer,antibodiesagainstthecytokinesand/orTFsofinterestwereadded,
andthecellsuspensionwasincubatedfor30minat4°Cinthedark.Finally,cellswerewashedonce
with FACS Buffer, resuspended in 200 µl FACS buffer and analysed by flow cytometry [section
3.2.4.5].
3.2.4.4 Sortingofimmunecelltypes
Flow cytometry also enables the separation of different cell populations according to their
fluorescentproperties.Asmentionedbefore,theliquidstreamconsistsofacascadeofdropletsthat
are screenedoneafter theother.Whenacellmatches thedesiredcriteria for sorting, inourcase
based on the expression of certain surface markers; an electrical pulse is applied to the droplet.
40
Consequently,thecellwillbedeflectedfromthestreamandcollectedseparatelyfromtheothercells
(IbrahimandvandenEngh,2007).
Priortosorting,cellswerestainedforsurfacemarkers[section3.2.4.1]andthenfilteredthrougha
30 µM cell strainer. Sorted cells were collected in FBS-coated sterile FACS tubes. Details on the
immunecellpopulationsofinterestandthemarkersusedforthesortingarelistedinTable4.
Table4.Listoftheimmunecellpopulationsofinterestandthemarkersusedfortheircharacterisation.
3.2.4.5 Dataacquisition
FlowcytometrywasperformedonaBDFACSCanto IIorFACSCelesta flowcytometer.Cell sorting
wasperformedon aBD FACSAria IIIu flow cytometer.Whenneeded for further cell culture, cells
weresortedonaBDFACSAriaFusionundersterileconditions.AllflowcytometersusetheFACSDiva
software.FACSdatawerelateranalysedwithFlowJosoftware.
3.2.5 GenerationofhumanTcelllines
HumanTcell linesareuseful tools tostudy thephenotypeand functionofTcells invitro. For this
study,TCD4+,Tγδ+ (Vδ1+andVδ2+)andMAITcell linesweregenerated.PBMCswere isolatedfrom
bloodofhealthydonors[section3.2.2.1]andFACS-sorted[section3.2.4.4]usingthemarkerslistedin
Table4.
Sorted cells werewashed, resuspended in T cellmedium and seeded into individual wells onto a
round-bottom96-wellplateatadensityof2 ·104 -1 ·105cells/well.TosimulateAgpresentation,
sortedcellswereculturedwith2 ·105heterologoushumanPBMCs irradiatedwith39grays for13
min(3grays/min)inordertoavoidtheirproliferationduringinvitroculture.Eachcellpopulationwas
culturedunderspecificconditions(Table5)andincubatedina5%CO2incubatorat37°C.TCD4cells
were stimulated with phytohaemagglutinin (PHA, 2.5 µg/ml,) and self-produced IL-2 supernatant
(Dilution:1:2000).Tγδ+Vδ1+cellswerestimulatedwithPHA(2.5µg/ml)andIL-2supernatant(1:400).
Tγδ+Vδ2+cellswereculturedwith(E)-4-hydroxy-dimethylallylpyrophosphate(HDMAPP,10nM)and
IL-2 supernatant (1:400).MAIT cells (CD8+, CD161+, Vα7.2+)were stimulatedwith soluble anti-CD3
(0.4 µg/ml,), recombinant IL-2 (rec IL-2, 100 U/ml) and recombinant IL-7 (rec IL-7, 10 ng/ml). Cell
Immunecelltypes Markers Immunecelltypes Markers
TCD3+cells CD3+ Tγδ+Vδ2- CD3+TCRγδ+Vδ2-
TCD4+cells CD3+CD4+ Tγδ+Vδ1+ CD3+TCRγδ+Vδ1+
TCD8+cells CD3+CD8+ MAITcells CD3+CD8+CD161+Vα7.2+
Tγδ+ CD3+TCRγδ+ NKcells CD3-CD56+
Tγδ+Vδ2+ CD3+TCRγδ+Vδ2+ Monocytes GatedaccordingtoFSC-AandSSC-A
41
growthandcellculturecontaminationwerecheckeddailywithaninvertedmicroscope.Every3to4
days,approximatelyhalfof themediumwasremovedandexchangedfor freshmediumcontaining
freshIL-2.Cellpassagewasperformedwhenrequiredinordertomaintaintheexponentialgrowthof
the cell lines. To this end, half of the cell suspension was transferred into a new well and fresh
mediumwithfreshIL-2wasadded.After12to14daysinculture,cellswererestimulated.Cellswere
harvested,washedoncewith 1XPBS andmanually counted. 1 · 104 cells fromeach cell linewere
thenseededintoanew96-wellplatewith2·105feedercellsandstimulatedasspecifiedabove.The
remaining cellswere frozenandputback into culturewhenneeded.Cellswere restimulated fora
maximumof5rounds,andrestimulationroundswereannotatedtokeeptrackoftheculture.
Table5.Specificconditionsforthetreatmentofthedistinctcelllines.
TCD4+ Vδ1+ Vδ2+ MAIT
ReagentsPHA PHA HDMAPP solubleanti-CD3IL-2supernatant IL-2supernatant IL-2supernatant recombinantIL-2(rIL-2)
3.2.6 Invitroassays
3.2.6.1 ActivationofhumanTcells
ActivationofCD4+andCD8+TcellswasdeterminedbytheexpressionoftheactivationmarkersCD25
andCD69onthecellsurface.PBMCsfromhealthydonorswereresuspended inTcellmediumand
stimulated with soluble anti-CD3 (0.5 µg/ml). To assess the influence of P2X7 blockade on T cell
activation,cellswerethenseededontoaround-bottom96-wellplateat0.5-1·105cellsperwell in
theabsenceorpresenceofDano1(100nM)oracontrol-NbagainstmouseARTC2.2 (100nM) ina
finalvolumeof200µl.Nbswereusedindifferentformats,suchasmonomers(mon),dimers(dim),
fusedtotherabbitFc-portionofIgG(rbFc)orlinkedtothealbumin-bindingnanobodyAlb8(dimAlb).
Nbswereaddedasecondtimeafter2daysofculture.After4daysincultureina5%CO2incubatorat
37°C,cellswerestainedwithantibodiesagainst themarkers listed inTable6.Subsequently, the in
vitroactivationoflivingTcellswasmonitoredbyflowcytometry.
Table6.ListofantibodiesusedfortheassessmentofTcellactivationinvitro.
Fluorochrome V450 V500 FITC PerCP-Cy5.5 PE PE-Cy7 APC-Cy7Marker CD25 live/dead CD4 CD3 TCRγδ CD8 CD69
FACS-sortedCD4,CD8andγδTcellsfromhealthydonorswereresuspendedinfullRPMImediumand
seededontoaround-bottom96-wellplatepre-coatedwithanti-CD3atacelldensityof1·105cells
perwell.Coatingwasperformedwith200µlofPBScontaining1µg/mlsolubleanti-CD3,at37°Cfor3
hours. Before seeding of the cells, the 96-well plate was washed twice with PBS under sterile
conditions.Cellswerefurtherstimulatedwith5µg/mlofsolubleanti-CD28,and incubated ina5%
42
CO2 incubator for9daysat37°C.CellswerecollectedeveryodddayforRNA isolation[seesection
3.2.8.2].Asacontrol,FACS-sortedcellsatday0andPBMCswerealsocollected.
3.2.6.2 ProliferationofhumanTcells
Proliferation of CD4+ and CD8+ T cells was determined by eFluor dye dilution by flow cytometry.
PBMCs from healthy donors were resuspended in eFluor670 (2 µM) and incubated in a 5% CO2
incubatorat37°Cfor10min.eFluorlabellingwasstoppedbyadditionoffullRPMIandanincubation
onicefor5min.Afterwards,cellswerewashedtwiceandresuspendedinfullRPMIintheabsenceor
presenceof solubleanti-CD3 (0.4µg/ml).Cellswere seededontoa round-bottom96-wellplateat
0.8-1·105cellsperwellinafinalvolumeof200µl,intheabsenceorpresenceofDano1oracontrol
NbagainstmouseARTC2.2(100nM).After4to5daysinculture,cellswerestainedwithantibodies
againstCD4andCD8andproliferationoflivingTcellswasassessedbyflowcytometry(seeTable6
forFluorochrome-conjugatedantibodies).
3.2.6.3 StimulationofhumanTcellswithretinoicacid
PBMCswereresuspendedinfullRPMImediumandseededontoaround-bottom96-wellplateata
densityof2·105cellsperwellinafinalvolumeof200µl.Cellswerestimulatedwithsolubleanti-CD3
(0.5µg/ml),andculturedintheabsenceorpresenceof1,10or100nMofretinoicacid(RA)ina5%
CO2incubatorat37°Cfor5days.
Cellswereharvesteddaily,andtransferredtoaneppendorftube.Aftercentrifugation(450xg,5min),
supernatantswere collected and frozen at -20°C. Cellswere then resuspended in PBS, transferred
into FACS tubes and stained for the following markers (Table 7). The expression of P2X7 was
monitoredovertimebyflowcytometry[section3.2.4.1].
Table7.ListofantibodiesusedfortheassessmentofRAeffectinTcellactivationinvitro.
Fluorochrome BV421 V500 FITC PerCP-Cy5.5 PE PE-Cy7 APC APC-Cy7Marker CD25 Live/dead CD2 CD38 CD8β CD8α P2X7 CD4
3.2.6.4 Uptakeof4',6-diamidino-2-phenylindole(DAPI)
4',6-diamidino-2-phenylindole (DAPI) is a fluorescentdye that binds to double stranded DNA. The
uptakeofDAPIwasusedasreadouttoassessP2X7-dependentporeformationondifferentimmune
cell types. PBMCs or gut-infiltrating leukocytes were isolated [sections 3.2.2.1 and 3.2.2.2] and
resuspendedinRPMImedium.Atotalof1·106PBMCswereincubatedintheabsenceorpresenceof
Dano1 (100 nM) or Control Nb (100 nM) at 4°C for 30 min. As a control condition, cells were
incubatedwith10U/mlofapyraseina5%CO2incubatorat37°Cfor20min.Afterwards,cellswere
43
loadedwithDAPIandincubatedforfurther30minintheabsenceorpresenceof0.5,1.5or4.5mM
ATPinawaterbathpre-heatedat37°C.Immediatelyafter,cellswereplacedat4°Cinordertostop
thereaction,washedwithcoldRPMImedium,andstainedfor30minat4°Cwithoneoftheantibody
cocktails listed inTable8.Afterwashing twicewithcoldRPMImedium, cellswere resuspended in
100µlFACSbuffer,keptoniceandmeasuredbyflowcytometry.
Table8.ListofantibodiesusedfortheidentificationofimmunecellsandassessmentofuptakeofDAPI.
Fluorochrome BV421 V500 FITC PerCP-Cy5.5 PE PE-Cy7 APC APC-Cy7
(1)Marker DAPI CD8 Vδ1;Vγ9;Vα24 CD3 CD161 TCRγδ Vα7.2
Vδ2 CD4
(2)Marker DAPI CD3 CD8;CD20 CD127 CD25 CD39 CD56 CD4
3.2.6.5 SheddingofCD62Lextracellulardomain
ThesheddingoftheextracellulardomainofCD62Lwasalsousedasreadouttoassessthefunctionof
theP2X7receptorinvitro.Cellsweretreatedasdescribedinsection3.2.6.4.Theantibodiesusedfor
flowcytometricanalysisarelistedinTable9.
Table9.ListofantibodiesusedfortheidentificationofimmunecellsandassessmentofCD62Lshedding.
Fluorochrome BV421 V500 FITC PerCP-Cy5.5 PE PE-Cy7 APC APC-Cy7
(1)Marker CD56 CD8 Vδ1;Vγ9;Vα24 CD3 CD62L TCRγδ;
CD19 Vδ1 CD4
(2)Marker CD161 CD3 CD62L CD127 CD25 CD8 Vα7.2 CD4
3.2.6.6 SuboptimalstimulationofTcells
The role of P2X7 as co-stimulatory molecule under suboptimal stimulation of the TCR was
determinedbyproliferationofCD4+andCD8+TcellsandexpressionoftheactivationmarkersCD25
andCD69overtime.First,PBMCSwerelabelledwitheFluordyeasdetailedinsection3.2.6.2.Next,a
total of 1 · 105 PBMCswere resuspended in fullRPMImedium and seeded onto a 96-well round-
bottomplateatafinalvolumeof200µl/well.Cellswerestimulatedwithdifferentconcentrationsof
solubleanti-CD3(0.1;0.5;1;10and100ng/ml)undertheabsenceorpresenceofATP(1.5;4.5mM),
Dano1(100nM)orapyrase(10U/ml),andincubatedfor4daysina5%CO2incubatorat37°C.Tcell
proliferationandactivationwereassessedafter36hand96hbyflowcytometryusingtheantibodies
listedinTable10.
Table10.ListofantibodiesusedfortheassessmentofTcellactivationandproliferationinvitro.
Fluorochrome BV421 BV500 FITC PerCP-Cy5.5 PE PE-Cy7 APC APC-Cy7Marker CD25 L/D CD3 CD4 Tγδ CD8 eFluor CD69
44
3.2.6.7 Inductionofinflammasomeassembly(ASCspecks)
TheactivationoftheinflammasomewasassessedbyformationofASCspecks.Thismethodrelieson
therecruitmentofASCintolargeproteincomplexesinthecytosol(Sesteretal.,2015).Tothisend,a
totalof1 ·106PBMCs fromhealthydonorswerestimulatedwith100ng/mlLPS in theabsenceor
presenceof200nMofDano1oracontrolnanobodyagainstToxinAfromClostridiumdifficile(clone
L+8)for2hat37°C.Immediatelyafter,cellswerefurtherstimulatedeitherwith2.5or5mMATP,or
with 10 µM or 20 µM nigericin for 30 min at 37°C. Afterwards, cells were fixed with 2%
paraformaldehyde(PFA)for10minon ice(4°C),washedwithFACSbufferat450xgfor5min,and
subsequently permebealised with 0.2% saponin. Cells were stained with rabbit anti-ASC primary
antibodyfor90minat4°C,washedwithcoldFACSbufferfor5minat450xgandstainedwithPE-
conjugated anti-rabbit secondary antibody and a BV421-conjugated antibody against CD33 for
further45minat4°C.Afterstaining,cellswerewashedoncewithFACSbufferandmeasuredbyflow
cytometry.ASCspecksweredefinedbyalowerpulsewidthtopulseareaaftergatingonCD33+cells.
3.2.6.8 StimulationofNF-κB-inducedgenetranscription
Atotalof2.5·104purifiedmonocytes[section3.2.3.1]wereseededontoa24flat-bottomwellplate
coatedwith300µlof0.01%poly-L-lysinesolutionfor25minatRT.CellswerestimulatedwithLPS(1
µg/ml) inRPMImediumintheabsenceorpresenceofdifferentconstructsofDano1(100nM)ora
controlNb(100nM)at37°C,5%CO2.Afterincubationforaperiodof3h,cellswerelysedwithlysing
solutionRLTbufferanddetachedfromthewellplateusingascrapper.Cellswerethencollected,and
resuspended in RLT + 1% β-mercaptoethanol, and stored at -80°C until day of use. mRNA was
isolated from frozen samples and transcribed into complementary deoxyribonucleic acid (cDNA)
accordingtothemanufacturer'sinstructions;asdetailedinsections3.2.8.2and3.2.8.4,respectively.
cDNAwas further used for real-timepolymerase chain reaction (Real-timePCR) [section3.2.8.5.1]
andgeneexpressionoftheIL6,IL1BandTNFgenesweredetermined.
3.2.6.9 Stimulationofhumanmonocytes
Aliquots of heparinized blood from healthy donors were stimulated with LPS (1 µg/ml) in the
presenceorabsenceof serialdilutionsofDano1,acontrolNbor thesmallmolecule inhibitors JNJ
47965567 and AZ10606120, in a 5% CO2 incubator for 1.5 h at 37°C. Blood samples were further
incubated for 30minuteswith the indicated concentrations of ATP in awater bath pre-heated at
37°C.Occasionally, theP2X7receptor agonist benzoyl-ATP (bzATP),whichexhibits greaterpotency
than ATP, was used instead. To bring the reaction to an end, blood aliquots were placed on ice
immediatelyafterincubation.Plasmawasaspiratedaftercentrifugationofthebloodat736xgfor5
45
minat4°C.Finally,plasmawasdiluted1:4inRPMImediumandstoredovernightat4°C.Thelevelsof
IL-1βweredeterminedthenextdaybyenzyme-linkedimmunosorbentassay(ELISA)[section3.2.7].
3.2.7 Enzyme-linkedimmunosorbentassay(ELISA)
Levels of IL-1β in plasma were determined by sandwich ELISA according to the manufacturer´s
instructions(Ready-SET-Go,eBioscience).Treatmentofthesamplesandobtentionoftheplasma is
described in section 3.2.6.9. Briefly, a Maxisorp ELISA plate was coated overnight with capture
antibody against IL-1β at 4°C. The procedure consisted of a capture antibody, the sample and a
detection antibody conjugated with biotin. Afterwards avidin conjugated with horseradish
peroxidase(HRP)wasadded.Theadditionoftetramethylbenzidine(TMB)asasubstratestartedthe
oxidativereaction,whichwaslefttoproceedabout10min,oruntiltheliquidturnedintodarkblue.
The reactionwas stoppedusing2NH2SO4.Washingwasperformed3-5 timeswith300µlofwash
buffer between all steps. Standards were diluted in RPMI, and RPMI was also used as blank.
Absorbancewasreadat450nminaplatereaderVICTOR3spectrophotometer.Thestandardcurve
coveredtherangeof3.9pg/mlto2000pg/ml.DatawereanalysedusingMicrosoftExcel2010.
3.2.8 Geneexpressionassessment
3.2.8.1 IsolationofDNAfromhumanblood
GenomicDNAwasextractedfromwholebloodusingtheNucleospinBlood(DNA,RNAandprotein
purification) Kit following the steps detailed in themanufacturer‘s instructions (Macherey-Nagel).
Briefly,200µlofblood,25µlofproteinaseKand200µlofaspecificbuffer(Buffer3)wereincubated
at 70°C for 15 min. After incubation and addition ethanol (100%), the mix was pipetted into a
NucleoSpin blood column and centrifuged at 11363 xg for 1 min. After two washing steps, the
residualethanolwasremovedbyanadditionalcentrifugationat11363xgfor1min.Finally,DNAwas
eluted in 100 µl elution buffer pre-heated at 70°C, after a 1 min centrifugation 11363 xg. DNA
concentrationwasdetermined[section3.2.8.3]andsampleswerestoredat-20°Cuntiluse.
3.2.8.2 IsolationofRNAfromhumancells
PBMCsandsortedimmunecellswerecentrifugedat1800rpmfor10minat4°Candresuspendedin
RLT lysing solution + 1% β-mercaptoethanol. The cell suspension was then snap-frozen in liquid
nitrogenandimmediatelystoredat-80°Cuntildayofuse.RNAextractionwasperformedusingthe
RNeasy Plus Mini Kit, following the steps detailed in the protocol provided by the manufacturer
(Qiagen). Frozen samples containing cell lysatewere removed from the -80°C and left at RT until
46
completelythawed.ThelysatewasthentransferredintoaQIAshredderspincolumn,centrifugedat
fullspeed(15870xg)for2minandtheflow-throughwascollected.Afterwards,1volumeofethanol
(70%) was added to the lysate until complete homogenization was achieved. Next, the mix was
transferredtoanRNeasyspincolumn,centrifugedat13520xgfor15secandtheflow-throughwas
discarded.Following,bufferRW1wasaddedintothecolumn,andthemixwascentrifugedasstated
before.InordertoremoveanytraceofDNA,themixwasincubatedwithDNaseI(375U/ml)for15
minatRT.Subsequently,bufferRW1wasaddedontothespincolumn,followedbyacentrifugation
at13520xgfor15sec,andtheflow-throughwasdiscarded.AftertwowashingstepswithbufferRPE,
thecolumnwascentrifugedat full speed for1min.Finally,RNAwaseluted in30µlofRNase-free
wateraftercentrifugationat9391xgfor1min.RNAconcentrationwasdetermined[section3.2.8.3]
andimmediatelyusedforcDNAsynthesisorfrozeninliquidnitrogenandstoredat-80°C.
3.2.8.3 QuantificationofpurifiedDNAandRNA
TheconcentrationofDNAandRNAfromhumanblood,PBMCsandpurified/sortedimmunecellswas
determined by spectrophotometry at 260 nm. To this end, the following conversion factors were
used:A260=1=50µg/mlorA260=1=40µg/mlforDNAandRNAsamples,respectively.Thepurity
was determined with the ratio of the absorbance at A260 divided by the absorbance at A280;
consideringaspurearangebetween1.8-2.0and2.0-2.2forDNAandRNA,respectively.
3.2.8.4 SynthesisofcDNA
IsolatedmRNAfromPBMCs,sortedimmunecellsorpurifiedmonocyteswasusedastemplateforthe
synthesisof cDNA ina reverse transcriptionpolymerase chain reaction. To thatend, twodifferent
reactionswereperformed;thefirstonebyincubatingtheRNAwithrandomprimersanddNTPsfor5
min at 65°C, and afterwards by incubating the mix with First Strand Buffer, DTT, diethyl
pyrocarbonate(DEPC)treatedwaterandM-MLVreversetranscriptase,for50minat37°Cand15min
at70°C.ThefinalconcentrationandvolumeforeachreagentaredetailedinTable11.
Table11.ListofreagentsforthesynthesisofcDNA.
Reaction1
Reaction2
Reagents
Volume
Reagents
VolumeRNA 10µl Reaction1Mix 12µlRandomhexamers(3µg/µl) 1µl FirstStrandBuffer(1x) 4µldNTPs(30mM) 1µl DTT(0.1M) 2µl Ʃ=12µl H2O 1µl MLV-RT(200U/µl)
1µl
Σ=20
47
3.2.8.5 Realtimepolymerasechainreaction
Real-Timepolymerasechainreaction(Real-TimePCR)wasperformedusingTaqManorSYBRGreen
assays.
3.2.8.5.1 TaqManassays
TaqMan assayswere run tomeasure gene expression of the interleukin 6 (IL6, Hs00985639_m1),
interleukin-1β (IL1B, Hs00174097_m1), Tumor Necrosis Factor (TNF, Hs01113624_g1) and
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs02758991_g1) genes on purified
monocytes.EachTaqManassay includesamixwithasetofprimersanda reporterprobe labelled
with fluorescence. Real-time PCR reactions were carried out on a 96 Fast PCR-well plate using
MaximaProbe/ROXqPCRmastermix,agene-specificTaqManprobeandcDNAinafinalvolumeof
20µl.TheplatewasthenmeasuredonaStepOnePlusReal-TimePCRSysteminaccordancewiththe
manufacturer’sinstructions(AppliedBiosystems);bydenaturationat95°Cfor10min,followedby40
cyclesat95°Cfor15secand60°Cfor40sec.Meltingcurveanalyseswereperformedtoverifythe
amplificationspecificity.Informationontheconcentrationandvolumeforeachreagentisdetailedin
Table12.TherelativegeneexpressionwascalculatedusingtheΔΔCtmethod.Fortheanalysis,data
werenormalisedtotheexpressionofthereferencegeneGAPDH.NonLPS-stimulatedsamplesinthe
absenceofnanobodieswereusedascalibrator.
Table12.ListofreagentsforqPCRreactionusingaTaqManassay.
3.2.8.5.2 SYBRGreenassays
SYBR Green assays were run tomeasure gene expression of 18S ribosomal RNA (18S), ribosomal
proteinL13a(RPLA13)andthesevenmembersoftheP2Xreceptorfamily(P2RX1-P2RX7)onPBMCs
and sorted immune cells. SYBR Green is an intercalating dye that binds preferentially to double-
strandedDNA.Real-timePCRreactionswerecarriedoutona96FastPCR-wellplateusingMaxima
SYBRGreen/ROXqPCRmastermix,apairofspecificprimersandcDNAinafinalvolumeof20µl.The
optimalpre-dilutionof thecDNAwaspreviouslycheckedaccordingto thereferencegenesRPLA13
and18S for thedistinctsorted immunecell types.Reactionproductsweremeasuredfollowingthe
protocol mentioned in section 3.2.8.5.1. Information on the concentration and volume for each
Reagents
Volume
MaximaProbe/RoxqPCRmastermix
10µl Gene-specifictaqManprobe(3µg/µl) 1µl Nuclease-freewater 4µl cDNA 5µl
Ʃ=20µl
48
reagent is detailed in Table 13. Data were normalised to the expression of the reference genes
RPLA13and18SandtotalPBMCswereusedascalibrator.
3.2.9 Genotyping
Three prominent SNPs that confer different sensitivity to ATP have been identified in the human
P2RX7 gene. These SNPs, which are encoded in exons 5, 8 and 11, result in the variation of one
aminoacidat the followingpositions:155 (HàY),270 (HàR)and348 (AàT) (Stokesetal., 2010).
Similarly,aSNPlocatedinthefirstintronoftheENTPD1genecorrelateswiththeexpressionofCD39
on Tregs and conventional CD4+ T cells (A. Rissieket al., 2015).DNAwas isolated fromperipheral
bloodfromhealthyvolunteers[section3.2.8.1]andgenotypedusingPCR.
3.2.9.1 PolymeraseChainReaction
PCRisatechniqueusedtoamplifyaparticularregionofDNA.Thismethodreliesontheusageofan
enzymetermedDNApolymerase,whichisabletosynthesizemultiplecopiesofDNAbyadditionof
deoxyribonucleotide triphosphates (dNTPs) to aDNA region that hasbeenpreviously flankedby a
specific pair of primers (Mullis et al., 1986). PCR reactions typically consist of 25-30 cycles of an
initialdenaturationstepofthedoublestrandedDNA(dsDNA)at90-95°C,followedbytheannealing
oftheprimersat50-65°CandtheelongationofthecomplementarystrandofDNAata69-72°Cfor
45 sec. The PCR reaction was carried in a suitable buffer with required salts and magnesium
sulfate(MgSO4).Detailson thePCRreactionsandtheprogramfor theoptimalamplificationof the
ENTPD1geneandexons5,8and11oftheP2RX7genearetobefoundin Table14and Table15,
respectively.ThePCRreactionwastransferredintoa0.2mlPCRtube,andDNAtemplatewasadded
andmixedcarefully.Afterwards,PCRtubeswereplacedinaPCRThermocycler.
Table13.ListofreagentsforqPCRreactionusingaSYBRGreenassay.
Reagents
Volume
MaximaSYBRGreen/ROXqPCRmastermix 10µl Forwardprimer 1µl Reverseprimer 1µl Nulcease-freewater 3µl cDNA 5µl
Ʃ=20µl
49
3.2.9.2 AgarosegelelectrophoresisofPCRproduct
ProductsfromPCRwerefractionedduetotheirsize inanagarosegelelectrophoresis.Gelswith1-
1.5% agarose were made with 1X TAE buffer containing 0.5 µg/ml of Rotisafe. Samples were
preparedwith6Xloadingbuffer,loadedintoindividualpocketsofthegelandranatanintensityof
90V.Asareferenceformolecular-weightsizemarker,8µlofDNAladder100bpor1kbwereused.
DNAbandswerevisualizedusingaUV-Transilluminatorandphotographedfordocumentation.DNA
bandswerethenexcisedfromthegelforfurtherprocessing[section3.2.9.3].
Table14.ListofreagentsfortheamplificationofSNPspresentintheP2RX7andENTPD1genes.
P2RX7 ENTPD1
Reagents Volume[µl] Volume[µl]
H2O 31 13.2
KODBuffer(10X) 5 4
dNTPS(2mM) 5 4
MgSO4(25mM) 2 2.4
ForwardPrimer(10µM) 2 1.2
ReversePrimer(10µM) 2 1.2
KODPolymerase(1U/µl) 1 0.8
DNATemplate 2 13.2
Finalvolume 50 40
Table15.ProtocolfortheamplificationoftheSNPtargetingregion.
Step Temperature[°C] Time(min)
1 94°C 5min
2 94°C 30sec
3 60°C 30sec
4 72°C 1min
5 Step2to4x10timeswithadecreaseof1°Catstep3ateachcycle
6 94°C 30sec
7 50°C 30sec
8 72°C 1min
9 Step6to8x25times
10 70°C 10min
50
3.2.9.3 ExtractionofDNAfragmentsfromanagarosegel
TheelutionofDNA fragments fromagarosegelswascarriedoutwith theNucleoSpinGelandPCR
Clean-up according tomanufacturer’s instructions (Macherey-Nagel). DNA fragmentswere excised
fromagarosegelunderUVlightusingacleanscalpel,andincubatedwithNT1bufferat58°Cfor15
min, until dissolved. Afterwards, themix was transferred onto a Nucleospin column, andwashed
twice with Buffer NT3 at 15870 xg for 1 min in a microcentrifuge. To ensure that no residual
carryoversofbuffersarepresent, thecolumnwascentrifugedat15870xg for1min.PurifiedDNA
was finally eluted in 15 µl of de-ionized water by a 1 min centrifugation at 15870 xg. Before
sequencing,DNAconcentrationwasdeterminedasdescribedinsection3.2.8.3.
3.2.9.4 SequencingofDNA
SequencingwasperformedbythecompanyEurofinsGenomicsusingtheSangerSequencingmethod
(Sanger, Nicklen and Coulson, 1977). The sequencing reaction consists of 150 ng of purified DNA
dilutedinde-ionizedwaterand2µlofthespecificprimer(10µM)inafinalreactionvolumeof17µl.
The mix was transferred to labelled-specific safe-lock tubes (Mix2Seq Kit) provided by the
manufacturer.Resultswereanalyzedusingthesoftware4Peaks.
3.2.10 Statisticalanalysis
DatarepresentationandstatisticalanalysiswereperformedusingtheGraphPadPrism6.0software.
Thetwo-tailedStudent'sTtestwasusedforcomparisonsbetweentwogroups;bothforpairedand
unpaireddata.Forcomparisonsamongmultiplegroups,theanalysisofvariance(One-wayANOVA)
withBonferronipost-testwasusedforunpaireddata,andrepeatedmeasures(RM)onewayANOVA
wasusedforpaireddata.Ap-value≤0.05wasconsideredsignificant.
51
4. RESULTS
The results of this thesis will be presented in two sections. In the first part, I will address the
expressionandfunctionoftheP2X7receptoronhumanmonocytes.Here,Iwillalsoshowevidence
ofthepotentialoftheP2X7-specificnanobodyDano1asatherapeuticdrugcandidateinhumans.In
thesecondpart,IwillshowtheexpressionandfunctionofP2X7onhumanlymphocytes,focusingon
theTcellcompartment.
4.1 EXPRESSIONANDFUNCTIONOFP2X7ONHUMANMONOCYTESANDMACROPHAGES
4.1.1 MonocytesshowhighexpressionofP2X7onthecellsurface
P2X7isexpressedbymost immunecells,particularly inmonocytes(Wangetal.,2004). Inhumans,
circulating monocytes are classified according to the expression of CD14 and CD16 in classical
(CD14+CD16-), intermediate (CD14+CD16+) and non-classical monocytes (CD14dimCD16+) (Ziegler-
Heitbrock, 2000).Weused flowcytometry to investigatewhether thereareanydifferences in the
expressionofP2X7onthedifferentmonocytesubpopulations.Thegatingstrategyfortheanalysisis
shown inFigure4A. Leucocyteswere identifiedamong live single cells by theexpressionofCD45,
andmonocytesweregatedaccordingtotheexpressionofCD14andCD16.TheexpressionofP2X7
wasassessedusing theP2X7-specificnanobodyDano1,as itwillbedone inallexperiments in this
thesis.Inordertoensurethatthesignalobtainedisspecific,wecomparedtheMedianFluorescence
Intensity(MFI)ofDano1stainingwiththeMFIofanirrelevantnanobodycontrol,namelyananobody
recognizingmurineARTC2.2,amoleculewhich isnotexisting inhumans.Thecomparisonbetween
thecontrolandspecificstainingisshownforallexperiments,andthestatisticalanalysiscomparing
differentcelltypesisperformedontheresultingsignalaftersubtractionofthecontrol.
In linewith published data,we observed high expression of P2X7 on the surface of all peripheral
monocyte subtypes (Figure 4). Of note, allmonocyte subpopulations show a similar level of P2X7
expression(Figure4B,C).However,whenthespecific(subtracted)signalofthethreesubpopulations
iscompared,itbecomesevidentthatCD14dimCD16+(non-classical)monocytesexpresslessP2X7than
the other subpopulations, and this difference is significant when compared to CD14+ CD16+
monocytesFigure4E).
52
Figure4.AllmonocytessubtypesexpresshighlevelsofP2X7onthecellsurface.(A)DotplotsshowthegatingstrategyfortheidentificationofdifferentmonocytessubtypesinPBMCsamples.(B)ExpressionofsurfaceP2X7(MedianFluorescenceIntensity(MFI)inwholemonocytesandinCD14dimCD16+,CD14+CD16+,andCD14+CD16-monocytesubtypes.(C)ComparisonofP2X7expressionandbackgroundforthedifferentmonocytesubtypes.PvaluesweredeterminedbypairedStudent’sttest***=<0.001.(D-E)BoxandwhiskersplotsforMFI±SDoftheIC(D)andP2X7expressionaftersubtractionoftheMFIoftheIC(MFIP2X7staining–MFIIC)(E).PvaluesweredeterminedbypairedRMone-wayANOVA,followedbyBonferronipost-test:*=<0.05.Datacorrespondtoonerepresentativedonor(B)orsixhealthyindividuals(C-E).
Livingcells Leucocytes
FSC-A
SSC-A
FSC-AFSC-H
CD14
CD16
CD45
SSC-A
P2X7
Counts
Monocytes CD14+ CD16-CD14+ CD16+CD14dim CD16+B
C
A
Monocytes
CD14+CD16+
CD14dimCD16+
CD14+CD16-
D E
P2X7expression[M
FI]
ARTC
2.2expressio
n[M
FI]
ΔP2X7expressio
n[M
FI]
Monocytes
CD14
dimCD16
+
CD14
+ CD16
+
CD14
+ CD16
-0
2000
4000
6000
8000 ****** *** ***
Monocytes
CD14
dimCD16
+
CD14
+ CD16
+
CD14
+ CD16
-0
2000
4000
6000
8000
**
Monocytes
CD14
dimCD16
+
CD14
+ CD16
+
CD14
+ CD16
-0
2000
4000
6000
8000
*
Isotype control hP2X7 staining
53
4.1.2 P2X7 blockade by Dano1 impairs ATP-dependent oligomerisation of the
inflammasome
LPSstimulationofmonocytesinducesthesynthesisoftheIL-1βandIL-18precursors,resultinginthe
accumulation of pro-IL-1β in the cytoplasm. A second stimulus is required for the processing of
mature IL-1β.ATP-gatingofP2X7canprovide sucha second signal, triggering the recruitmentand
polymerisation of ASC proteins into large complexes termed ASC specks. The binding of ASC to
procaspase-1inducestheproteolyticcleavageandconversionofprocaspase-1intoactivecaspase-1,
which mediates the maturation of immature pro-IL-1β and pro-IL-18 into active IL-1β and IL-18,
respectively(Ferrarietal.,2006;Chengetal.,2009).ToassessthepotentialofP2X7nanobodiesfor
human therapy,we first investigated theabilityof theP2X7-specific nanobodyDano1 forblocking
theformationofASCspecks.Forthis,weusedarecentlydescribedflowcytometry-basedprotocol
(Sesteretal.,2015),whichrelieson thedetectionofchanges in thepulseofemitted fluorescence
uponinflammasomeactivation.TheauthorsshowedthattherelocalisationofASCintodensespecks
inducesan increasedheightof the fluorescentpulseassociatedwithadecrease in the fluorescent
pulse width, which is easy to detect by flow cytometry and, in contrast to microscope-based
strategies,allowsforexactquantificationofASCformation.
LPS-stimulatedPBMCswere stainedwith themyeloidmarkerCD33andwith an antibodydirected
againsttheintracellularASCadaptorprotein.Flowcytometryanalysiswasperformedinaccordance
withthegatingstrategydescribedintheoriginalpaper(Sesteretal.,2015).Briefly,afterexclusionof
celldebrisanddoublets,cellsweregatedaccordingtotheexpressionofCD33andASC(CD33+ASC+
cells).ASC specksweredefinedbyASC-WversusASC-A, consideringas speck+ cells thepopulation
displayingareductioninthefluorescentpulsewidth(Figure5).
FSC-A
SSC-A
FSC-A
FSC-W
FSC-A
FSC-H
ASC-A
CD33-A
ASC-A
ASC-WSinglecells
ASC+
monocytes Speck+
SinglecellsCells
Figure 5. Gating strategy for the detection of ASC specks in human blood monocytes for monitoring ofinflammasomeoligomerisation.PBMCswerefixedand stainedwithanantibodyagainst theadaptorproteinASC,followingtreatmentwithLPS(100ng/ml)andATP(2.5or5mM).CellsweregatedbasedontheirsizeandgranularityintheplotFSC-A/SSC-Ainordertoexcludedebrisfromtheanalysis.DoubletswereexcludedintheFSC-A/FSC-W (W=width) and FSC-H/FSC-A plots.Monocyteswere identified using themyeloidmarker CD33,and the formation of ASC specks (black circle) was calculated as the percentage of ASC-positivemonocytes(bluegate).
54
As expected, primingwith LPS alonedid not result inASC-speck formation in healthy donors, and
neither did stimulationwith ATP alone (Figure 6A). In contrast, stimulation of LPS-primed human
PBMCs with low (2.5 mM) or high (4.5 mM) concentrations of ATP induced the recruitment and
assembly of the inflammasome into specks (Figure 6). Furthermore, addition of Dano1, but not a
control nanobody, prior to stimulation with ATP, resulted in a significant blockade of ASC-speck
formation(Figure6B).
Figure6.Dano1blocksATP-dependentactivationof the inflammasome.HumanPBMCswere treated for 2hourswithLPS(100ng/ml)andwithATP(2.5mMand5mM)for0.5hoursintheabsenceorpresenceoftheP2X7-specificNbDano1-mFc(fusedtoFcdomainofmouseIgG1)oracontrolNb-mFcagainstToxinAfromC.difficile(200nM).(A)RepresentativeFACSplotsshowingtheformationofASC-specks:upperrowfromlefttoright(unstimulated,+LPS,+ATP);lowerrowfromlefttoright(LPS+ATP(5mM),LPS+ATP(5mM)+Dano1-mFc,LPS+ATP(5mM)+controlNb-mFc).(B)PercentageofASC-speck+monocytes(Mean±SD,n=3donors)uponstimulationwith5mM(left)or2.5mMATP(right).Dataisrepresentativeofoneexperimentofasetoftwo(n=6).Pvaluesweredeterminedbyone-wayANOVA,followedbyBonferronipost-test:***=<0.001.
A
B
Speck+
cells[%
]
Speck+
cells[%
]
ASC-A
ASC-W
Speck+0.84%
Speck+79.2%
Unstimulated
LPS+ATP
Speck+2.77%
Speck+82.4%
ATP
LPS+ATP+Ctrl-Nb
Speck+1.66%
Speck+5.61%
LPS
LPS+ATP+Dano1
5mMATP 2.5mMATP
0
20
40
60
80
100
LPSATP
--
+-
++
++
++
****
-+
0
20
40
60
80
100 ***
LPSATP
--
++
+-
++
-
Dano1
Control
55
Inflammasomes are also activated by a large variety of microbial molecules, danger signals and
crystalline substances, in a P2X7-independent fashion (Cullen et al., 2015). To further confirm the
specificity ofDano1 toblockATP-dependent inflammasomeactivation,we stimulatedPBMCswith
differentconcentrationsofnigericininsteadofATP.SimilartoATP,nigericininducedrecruitmentand
oligomerisationofASCinLPS-primedPBMCsbutnotinunprimedPBMCs(Figure7).Nigericininduces
the opening of Pannexin-1 channels, leading to a decrease in intracellular levels of K+ and the
subsequent activation of the inflammasome (Pelegrin and Surprenant, 2007). Interestingly,
stimulationwithnigericindidnotresultinsuchaclearpopulationofspeck+cells,asitwasthecase
uponstimulationwithATP (Figure6A,7A).Asexpected,Dano1wasonlyeffectiveafter treatment
with ATP Figure 6B), but had little if any effect on P2X7-independent inflammasome activation
inducedbynigericin(Figure7).Additionally,preincubationwithacontrolnanobodyalsodidnotalter
inflammasome activation. These results indicate that Dano-1 blocks inflammasome activation in
responsetoATPinhumanmonocytes.
ASC-A
ASC-W
Speck+41.8%
LPS+Nigericin
Speck+49.2%
LPS+Nigericin+Ctrl-Nb
Speck+54.4%
LPS+Nigericin+Dano1
A
B
Speck+
cells[%
]
Speck+
cells[%
]
20µMNigericin 10µMNigericin
0
20
40
60
80
100
LPSNigericin
--
+-
++
++
++
0
20
40
60
80
100
LPSNigericin
--
++
+-
++
-
Dano1
Control
Figure 7. Dano1 does not have an effect on P2X7-independent inflammasome activation. Human PBMCswere treated for 2 hours with LPS (100ng/ml) and with nigericin (10µM and 20 µM) for 0.5 hours in theabsence or presence of the P2X7-specificNbDano1-mFc or a control Nb-mFc (200 nM). (A) RepresentativeFACSplotsshowingtheformationofASC-specksunderspecificconditions. From left toright:LPS+Nigericin(20µM); LPS+Nigericin (20µM)+Dano1-mFc; LPS+Nigericin (20µM)+ controlNb-mFc. (B)PercentageofASC-speck+cells(Mean±SD,n=3donors)uponstimulationwith20µM(left)or10µMnigericin(right).Dataare representative of oneexperiment of a setof two (n=6). P valuesweredetermined by one-way ANOVA,followedbyBonferronipost-test.
56
4.1.3 Dano1abolishesATP-inducedreleaseofIL-1βbymonocytes
IL-1βisanimportantmediatoroftheinflammatoryresponse,andisinvolvedinavarietyofcellular
activities, including cell proliferation, differentiation, and apoptosis. In contrast to other
proinflammatorycytokines,IL-1βissynthesizedasaprecursorandcleavedbycaspase-1toproduce
thematureform,whichissubsequentlyreleased(Dinarello,2009).TheroleofP2X7forinducingthe
cleavage of the precursor pro-IL1β has been extensively investigated (Giuliani et al., 2017). To
evaluate thepotentialofDano1 inblocking IL-1βproduction,weusedasurrogate inflammation in
vitro model, which is the stimulation of freshly obtained human blood with LPS and ATP, and
subsequentmeasurementofIL-1βproductionintheserumafterincubation.
First,weaimedtodetermineanoptimalconcentrationofATPtoinduceanadequatereleaseofIL-1β
tobemeasuredbyELISA.AsshowninFigure8A,bothconcentrationsofATP(2.5and5mM)induced
thereleaseofhighamountsofIL-1β.Ofnote,thelackofoneofthetwosignals,eitherprimingwith
LPSortreatmentwithATP,resultedinnoreleaseofIL-1β.Furthermore,preincubationwithDano1
significantlypreventedthereleaseofIL-1β(Figure8A).
Second, to further confirm the capacity of Dano1 to antagonise P2X7 in vitro, we treated human
bloodwiththeP2X7receptoragonistbenzoyl-ATP(bzATP),whichexhibitsgreaterpotencythanATP.
UnlikestimulationwithATP,whereaconcentrationhigherthan0.5mMisneededtoinducerelease
of IL-1β (Figure 8C), the same concentration of bzATP induced a sizeable release of the cytokine
(Figure 8B). Aswe expected, Dano1, but not a controlNb, efficiently blocked the release of IL-1β
afterstimulationwithbothATPandbzATP(Figure8A,B).
Figure8.Dano1blockstheATP-dependentreleaseof IL-1βbyLPS-primedhumanmonocytes.FreshhumanbloodwastreatedwithLPS(1µg/ml)for1.5hoursat37°C,5%CO2,andfurtherincubatedwithATPorbzATPfor0.5hourat37°C.LevelsofIL-1β[ng/ml]inplasmaweremonitoredbyELISA.(A)LPS-treatedhumanbloodwasincubatedwitheither2.5mMor5mMofATPundertheabsenceorpresenceof100nMDano1-rbFc(n=6).(B-C)LPS-treatedhumanbloodwasincubatedwith0.5mMbzATP(B)orATP(C)undertheabsenceorpresenceof100nMofDano1-mFcoracontrolNb-mFc(α-ToxinAC.difficile) (n=3).Pvaluesweredeterminedbyone-wayANOVA,followedbyBonferronipost-test:****=<0.0001.
A
IL-1β[ng/ml]
IL-1β[ng/ml]
B
IL-1β[ng/ml]
C
57
Finally, to assess the specificity of the signal blocked, we used blood of a patientwith cryopyrin-
associatedperiodic syndrome (CAPS). CAPS are rare autoinflammatorydiseases characterizedby a
gain-of-function mutation in the NLRP3 inflammasome that leads to its constitutive activation,
bypassing the signalprovidedbyexternal stimuli suchasATP (Cordero,Alcocer-GómezandRyffel,
2018).ThepatientwithCAPSpresentedaveryhighproductionof IL-1βafterstimulationwithonly
LPS, while stimulation with LPS alone resulted in no production of IL-1β in the healthy control.
Interestingly,Dano1didnotabolishthemassiveproductionofIL-1βinthepatientwithCAPS(Figure
9), demonstrating the specificity of Dano1 for blocking ATP-P2X7-dependent activation of the
inflammasomebutnotconstitutiveactivationoftheinflammasome.
Figure 9.Dano1 specifically blocks ATP-induced release of IL-1β by LPS-primed human monocytes. Freshhumanbloodfromahealthydonor(black)orapatientwithCAPS(grey)wastreatedwithLPS(1µg/ml)for1.5hoursat37°C,5%CO2,and further incubatedwithATP (2.5mM) for0.5hourat37°Cunder theabsenceorpresence of Dano1-mon. Data are of a single experiment. Legend: HD = healthy donor; CAPS: patient withcryopyrin-associatedperiodicsyndrome.
4.1.4 Dano1 exhibits higher potency for blocking P2X7 compared to currently used
antagonists
ToevaluatethepotentialofDano1asatherapeuticdrug,wecomparedtheblockingcapacityofthe
nanobody with two small molecule antagonists that are currently in preclinical development,
JNJ47965567 (Jansen) and AZ10606120 (AstraZeneca). All healthy donors responded to the
treatmentwithgreatsecretionofIL-1β,whichwasabolishedwhenthebloodwaspreincubatedwith
Dano1 (Figure10).Dano1blockedATP-inducedreleaseof IL-1βatsubnanomolar IC50 (0.13±0.05
nM),whilebothsmall-moleculeantagonistswereusedatsubmicromolarIC50(JNJ47965567=253.1
±197.2; andAZ10606120=352.6±31.8). Therefore,Dano1demonstratedup to2000-foldhigher
potencythanthesmall-moleculeantagonistscurrentlyinpreclinicaldevelopment.
0
1
2
3
4
IL-1β
[ng/
ml]
LPSATP
Dano1
+--
++-
+++
---
n.d.
HDCAPS
HD
CAPS
58
Figure10.Dano1blocksATP-inducedreleaseofIL-1βatsubnanomolarIC50values.Freshhumanbloodwastreated with LPS (1µg/ml) for 1.5 hours at 37°C, 5% CO2, and further incubated with 2.5 mM ATP in thepresenceofserialdilutionsofDano1-mFcorthesmall-moleculeantagonistsJNJ47965567orAZ10606120.Dataareoffourhealthydonors(n=4).
Altogether,ourdatademonstratethespecificityofDano1forblockingP2X7-mediated inductionof
IL-1β,underliningitspotentialasaninnovativepre-clinicaldrugcandidate(Danquahetal.,2016).
4.1.5 Multimericnanobodiesexhibitenhancedpotencythanthemonomericconstructs
SincethelabofProf.NoltehadpreviouslydemonstratedthatdimerisationofmousespecificP2X7-
nanobodiesimprovedbindingaffinityandenhancedtheirfunctionalpotency(Danquahetal.,2016),
we assessedwhethermultimerisation of Dano1would also enhance its blocking capacity. Dimeric
nanobodies were obtained by genetic linkage of two monomers with a 35 glycine-serine linker
(Gly/Ser) (Danquah et al., 2016). To investigate their blocking potential, LPS-primed human blood
wasincubatedwithdifferentformatsofthenanobodyDano1:initsmonomericform,asadimerand
asadimerfusedwiththeFcdomainofrabbitIgG.Allnanobodyconstructsshowedagoodantagonist
effectandblockedthereleaseofIL-1β(Figure11).Furthermore,dimerisationofDano1andfusionto
anFcdomainresulted in lower IC50values (Dano1-mon=3.74nM;Dano1-dim=0.57nM;Dano1-
rbFc=0.07nM).Therefore,bivalentNbsexhibithigherpotencythanmonomericconstructs.
Concentration[nM]
IL-1β[ng/ml]
10-² 10-¹ 10⁰ 10¹ 10² 10³ 10⁴0
1
2
3
4
10-² 10-¹ 10⁰ 10¹ 10² 10³ 10⁴0
1
2
3
4
10-² 10-¹ 10⁰ 10¹ 10² 10³ 10⁴0
1
2
3
4
10-² 10-¹ 10⁰ 10¹ 10² 10³ 10⁴0
1
2
3
4
Dano1-mFc AZ10606120 JNJ 47965567
59
Figure 11. Dimerisation of Dano1 results in enhanced blocking of IL-1β release by LPS-primed humanmonocytes. LPS-treatedhumanbloodwas incubatedwith serialdilutionsofDano1-mon (monomer),Dano1-dim(dimer)orDano1-rbFc(fusedtorabbitFc)priortostimulationwithATP(2.5mM).Dataarerepresentativeofasingleexperiment(n=1).
4.1.6 Dano1canalsoalterthetranscriptionofgenesregulatedbytheNF-κBpathway
BeyondIL-1production,whichisdrivenbyinflammasomeactivation,wewantedtoaddresswhether
Dano1wouldexertaneffectonothermajorinflammatorypathways,forinstancethetranscriptionof
genescontrolledbyNF-κBsignalling.Tothisend,westimulatedmonocyteswithLPSandmeasured
the transcription of IL1B, IL6 and TNF by qRT-PCR. For this experiment, we used two different
constructs of the nanobodies, one fused with the Fc-domain of rabbit IgG (rbFc), and the other
constructfusedtothealbumin-bindingnanobodyAlb8(dimAlb);bothresultinginanincreasedhalf-
life of the Nb in vivo (Tijink et al., 2008). Our results show that Dano1 does not impair P2X7-
independenttranscriptionof IL1B (Figure12A)and IL6 (Figure12B).Similarly, treatmentunderthe
presenceofacontrolNbdidalsonothaveaneffectonthetranscriptionofthesegenes(Figure12A,
B).Unexpectedly,wedetectedareducedexpressionoftheTNFgeneaftertreatmentinthepresence
of both Dano1 and a control Nb (Figure 12C). Of note, this effect was reproduced in three
independentexperiments,regardlessofthespecificityandformatoftheNbs.
IL-1β[ng/ml]
Dano1[mM]
10-² 10-¹ 10⁰ 10¹ 10² 10³0
1
2
3
4
+ LPS
Dano1-mon
Dano1-dim
Dano1-rbFc
60
Figure 12.Dano1decreases the expressionofTNF but not IL1B and IL6 in LPS-treatedhumanmonocytes.P2X7-independent activation ofNF-κB–mediated transcription of IL6, IL1B andTNF in purified human bloodmonocyteswasmonitored by quantitative reverse transcription PCR (qRT-PCR) after primingwith LPS for 3hours in the absence or presence of either 200 nM Dano1-mFc or control Nb-mFc (upper row), or Dano1-dimAlborcontrolNb-dimAlb(lowerrow).ControlnanobodywasspecificformouseARTC2.2.(A-C)ThegraphsshowthefoldincreaseinthetranscriptionofIL1B(A),IL6(B)andTNF(C)genes(Mean±SD,n=4).DataarerelativetotheexpressionofthereferencegeneGAPDHandwerecalculatedusingthedelta-deltaCtformula(2–∆∆Ct).Dataare representativeof three independentexperiments.Pvaluesweredeterminedbyone-wayANOVA,followedbyBonferronipost-test:**=<0.01.
4.2 EXPRESSIONANDFUNCTIONOFP2X7ONHUMANLYMPHOCYTES
4.2.1 NKcellsexhibitthehighestexpressionofsurfaceP2X7amongalllymphocytes
InordertodeterminetheexpressionofP2X7 in lymphocytes,wedesignedantibodypanelsforthe
identification of the lymphocyte subsets. We first analysed the expression of P2X7 on the three
majortypesoflymphocytes:Bcells(CD19+),Tcells(CD3+)andNKcells(CD16+CD3-CD19-).Classical
monocytesweregatedbasedontheexpressionofCD14(Figure13A),andusedaspositivecontrol
fortheexpressionofP2X7.
IL1B
[foldincrease]
IL6[fo
ldincrease]
TNF[fo
ldincrease]
IL1B
[foldincrease]
IL6[fo
ldincrease]
TNF[fo
ldincrease]
IL1B IL6 TNFNb
-Fc
Nb-dim
Alb
A B C
0
40
80
120
160
LPS - + ++0
4
8
12
16
LPS - + ++
0
40
80
120
160
LPS - + ++0
4
8
12
16
LPS - + ++
0
6
12
18
24**
LPS - + ++
0
6
12
18
24****
LPS - + ++
- + Dano1 + Control Nb
61
Figure13.P2X7isdifferentiallyexpressedamongdistinctlymphocytepopulations.(A)Gatingstrategyfortheidentificationofthedifferentlymphocytesubtypesandclassicalmonocytes.(B)P2X7expression(MFI)onthesurface of B cells (CD19+), T cells (CD3+), NK cells (defined as CD3-CD19-CD16+ in the lymphocyte gate) andclassical monocytes (CD3-CD19-CD14+). The number of cells in the histogram plot was normalised for eachpopulation. (C)ExpressionofP2X7(MFI)andbackgroundsignaldetectedonthe IC for thedifferent immunecellsubtypes.(D)ExpressionofP2X7(MFI±SD)aftersubtractingtheMFIoftheICfromtheMFIoftheP2X7staining(MFIP2X7-MFIIC).Histogramsin(B)arerepresentativeofonedonorwhilethirteenhealthydonorsare shown in (C-D). P valuesweredeterminedbypairedStudent’s t test,orbypairedRMone-wayANOVA,followedbyBonferronipost-test:*=<0.05;**=<0.01;***=<0.001;****=<0.0001.Legend: red= innateimmunecells//blue:adaptiveimmunecells.
DuetothecomplexityoftheP2X7staining,wefirstdeterminedforeachcellsubsetwhetherthere
aredifferencesbetween theMFIof theP2X7 staining and theMFIof the isotype control (IC).We
found significant differences between the MFI of both stainings in all immune cell populations
(Figure 13C). Of note, we did not find any differences in the MFI among isotype controls. All
lymphocyte subsets displayed relatively low amounts of P2X7 compared to classical monocytes
(Figure13B,C).Interestingly,NKcellsexpressedsignificantlyhigherlevelsofP2X7thanTandBcells,
thelattershowingnegligiblespecificstaining.Tofurtherconfirmtheseresults,wesubtractedtheIC
fromtheP2X7staininginordertoobtainasingleMFIvalueforthecomparisonofP2X7expression.
A
B
Bcells
Tcells
CD3
CD
19Classicalmonocytes
NKcells
CD16C
D14
P2X7expression[M
FI]
ΔP2X7expressio
n[M
FI]
C
D
P2X7
Counts
B cells T cells
NK cells Classical monocytes
Isotype control
hP2X7 staining
hP2X7 staining
B cells T cells NK cellls0
400
800
1200
1600
*******
****
Cl. Monocytes0
2000
4000
6000****
B cells T cells NK cells0
300
600
900
1200****
****
**
Isotype control hP2X7 staining
62
Aftersubtraction,weconfirmedthehigherexpressionofP2X7onNKcellscomparedtoTandBcells
(Figure13D).
Our data suggest a distinct pattern of expressionwithin the lymphocyte compartment,where NK
cellsexpressthemostP2X7ontheirsurface,followedbyTcellsandultimatelybyBcells.
4.2.2 CD4+ CD25+ CD127- regulatory T cells express lower levels of P2X7 than
conventionalCD4Tcells
WenextaimedtoassessthepatternofexpressionwithintheTcellcompartment.Tregsweregated
accordingtoexpressionofCD25andCD127(CD4+CD25highCD127-)withintheCD4Tpopulation.The
remainingCD4Tcellswereconsidered"conventionalCD4Tcells"(Figure14A).
Figure14.HumanTregsexpresslowerlevelsofP2X7thanconventionalCD4Tcells(A)GatingstrategyfortheidentificationofdistinctT cell subtypes. (B)P2X7expression (MFI)on the surfaceofCD8T cells (CD3+CD8+),conventional CD4 T cells (TCD4conv., CD4+ CD25-/low CD127+) and Tregs (CD4+CD25+CD127-). Number of cellswasnormalised.(C)ExpressionofP2X7(MFI±SD)andbackgroundsignaldetectedontheICofdistinctTcellsubsets. (D) Expressionof P2X7 (MFI ± SD) after subtractionof theMFI of the IC from theMFI of theP2X7staining(MFIP2X7-MFIIC)onthesurfaceofdistinctTcellsubsets.Dataarerepresentativeofone(B)orseven
CD4
CD
8
TCD8
TCD4
Single cells
CD25
´CD1
27
TCD4conv.
Tregs
TCD4
HLA-DR
CD
45R
A
Naive
Activated
Tregs
CD25
CD
45R
A Memory
Tregs
CD25
CD
39
CD39+
CD39-
TregsA
B
P2X7
Counts
TCD4 conv. Tregs TCD8
P2X7expression[M
FI]
Δ P2X7expression[M
FI]
C D
Isotype control
hP2X7 staining
TCD4conv.
Tregs TCD80
300
600
900
1200
******
****
TCD4conv.
Tregs TCD80
300
600
900
1200
*
Isotype control
hP2X7 staining
63
(C-D)healthydonors.PvaluesweredeterminedbypairedStudent’sttest,orbypairedRMone-wayANOVA,followedbyBonferronipost-test:*=<0.05;**=<0.01;***=<0.001;****=<0.0001.Legend:blue=adaptiveimmunecells(C-D).
AllthreeTcellsubsets(conventionalCD4,TregsandCD8)showedlowlevelsofP2X7ontheirsurface
(Figure 14B, C), but still significantly different from the negative control (Figure 14C). At first, it
seemsthatCD8cellshadlowerlevelsofP2X7,however,whenthenegativestainingwassubtracted,
CD8 T cells showed similar expression of P2X7 than CD4 T cells, while Tregs had the lowest
expression(Figure14D).Therefore,theapparentdifferencesseeninFigure14Cwererathercaused
by the lower fluorescent signal of the stainings in CD8 T cells using this constellation of surface
markers.
4.2.3 P2X7ispreferentiallyexpressedineffectorandmemoryCD4Tcells
Wenextinvestigatedatwhichstageofmaturation/differentiationdoTcellsexpresshigherlevelsof
P2X7.Tothisend,weusedCD27andCD45RAtodefinenaive(CD27+CD45RA+),centralmemory(CM,
CD27+ CD45RA-), effectormemory CD45RA positive (TEMRA, CD27-CD45RA+) and effectormemory
(EM,CD27-CD45RA-)phenotypes inCD4andCD8Tcells. Inaddition,we identifiedeffectorTcells
based on the lack of expression of CD27 and CD28 (CD27-CD28- cells). The gating strategy for the
identificationofthedistincteffectorsubsetsisrepresentedonFigure15A.TEMRACD4Tcellswere
notanalysedduetoinsufficientcellnumber.
All cell subsetswere significantlypositive (althoughwith lowexpression) forP2X7 (Figure15B,C).
Interestingly,CMCD4TcellsshowedhigherexpressionofsurfaceP2X7thanthewholepopulationof
CD4 T cells (Figure 15C). These results were confirmed by comparison of P2X7 staining after
subtractionof IC (Figure 15D).Wealsoobserved that CMand EM cells expressedhigher levels of
P2X7thannaïveCD4Tcells(Figure15D).
64
Figure 15. CD4 T cells with effector phenotype express higher levels of P2X7 than naïve cells. (A) GatingstrategyfortheidentificationofdifferentTcelleffectorsubtypes.(B)P2X7expression(MFI)onthesurfaceofnaive (CD27+CD45RA+), central memory (CM, CD27+CD45RA-), effector memory (EM, CD27-CD45RA-) andeffectors(CD27-CD28-)CD4Tcells.(C)Expression(MFI±SD)ofP2X7andbackgroundsignaldetectedontheIConthesurfaceofdifferentCD4effectorsubsets.(D)ExpressionofP2X7(MFI±SD)aftersubtractionoftheMFIoftheICfromtheMFIoftheP2X7staining(MFIP2X7-MFIIC)onthesurfaceofdifferenteffectorCD4Tcelleffectorsubsets.Dataarerepresentativeofone(B)orseven(C-D)healthydonors.PvaluesweredeterminedbypairedStudent’sttest,orbyRMone-wayANOVA,followedbyBonferronipost-test:*=<0.05;**=<0.01;***=<0.001;****=<0.0001.Legend:blue=adaptiveimmunecells(C-D).
Similarly,we also determined the pattern of P2X7 expressionwithin distinct Tregs subpopulations
according to maturation status (Figure 14A); and observed that activated (HLA-DR+), memory
(CD45RA) and CD39+ Tregs expressed higher levels of P2X7 than naïve Tregs (CD45RA+) (data not
shown).
SinceP2X7isinvolvedinthepolarizationofTcellstowardstheTh1andTh17phenotypes(Schenket
al., 2011; Salleset al., 2017),we assessed the expressionof P2X7ondistinct Th subtypes defined
CD28CD
27
Effectors
TCD4A
P2X7
Counts
B
CD45RA
CD27
Naive
TEMRA
TCD4CM
EM
C
P2X7expression[MFI]
EM
Naive CM
Effectors
Isotype control hP2X7 staining T CD4 Naïve CM EM Effectors0
300
600
900
1200
*** *** *** ******
DΔ P2X7expression[M
FI]
Isotype control hP2X7 staining
T CD4 Naïve CM EM Effectors0
300
600
900
1200
***
***
****
***
65
according to expression of different chemokine receptors (Becattini et al., 2015). Th17 cells were
identifiedasCCR6+CCR4+CXCR3-,Th1/Th17cellsasCCR6+CCR4-CXCR3+,Th2asCCR6-CCR4+CXCR3-
andTh1asCCR6-CCR4-CXCR3+(Figure16A).AllThelpercellsubtypes(Figure16C)tendtoexpress
higherlevelsofP2X7thanwholeCD4cells,whichcontainthenaïvesubset,andP2X7expressionwas
significantlyhigheronTh1andTh17cells(Figure16D).
Figure 16. Proinflammatory Th1 and Th17 cells express higher levels of P2X7. (A) Gating strategy for theidentification of distinct T helper cells. (B) P2X7 expression (MFI) on the surface of Th17 cells (CCR6-
CCR4+CXCR3-),Th1/Th17(CCR6-CCR4-CXCR3+),Th2(CCR6+CCR4+CXCR3-)andTh1cells(CCR6+CCR4-CXCR3+). (C)ExpressionofP2X7(MFI±SD)andbackgroundsignaldetectedontheIConthesurfaceofdistinctThelpercells.(D)ExpressionofP2X7(MFI±SD)aftersubtractionoftheMFIoftheICtotheMFIoftheP2X7staining(MFIP2X7-MFIIC)onthesurfaceofdistinctThelpercells.Dataarerepresentativeofone(B)orsix(C-D)healthydonors. P values were determined by paired Student’s t test, or by RM one-way ANOVA, followed byBonferronipost-test:*=<0.05;**=<0.01;***=<0.001.Legend:blue=adaptiveimmunecells.
CD3
CD
4
TCD4
FSC-H
CXCR3C
CR
4
Th17
Th1/Th17
TCD4 (CCR6+)A
Th1
P2X7
Counts
Th2 Th17B
C D
CCR6
CCR6-
TCD4
CCR6+
CXCR3
CC
R4
TCD4 (CCR6-)Th2
Th1
Th1/Th17
Δ P2X7expression[M
FI]
P2X7expression[MFI]
Isotype control hP2X7 staining
TCD4 Th1 Th2 Th17 Th1/Th170
300
600
900
1200******
*****
TCD4 Th1 Th2 Th17 Th1/Th170
200
400
600
*
**
Isotype control hP2X7 staining
66
IncontrasttoCD4Tcells,wedidnotfindanydifferencesontheexpressionofP2X7onthedifferent
CD8effectorsubsets,neitherbeforenoraftersubtractionoftheIC(Figure17).
Figure17.AllCD8TcellsubsetsexpresssimilarlevelsofP2X7onthecellsurface.(A)Gatingstrategyfortheidentification of different T cell effector subtypes. (B) P2X7 expression (MFI) on the surface of naive(CD27+CD45RA+), central memory (CM, CD27+CD45RA-), effector memory CD45RA positive (TEMRA, CD27-
CD45RA+),effectormemory(EM,CD27-CD45RA-)andeffectors(CD27-CD28-)CD8Tcells.(C)Expression(MFI±SD) of P2X7 and background signal detected on the IC on the surface of different CD8 effector subsets. (D)ExpressionofP2X7 (MFI±SD)aftersubtractionof theMFIof the IC fromtheMFIof theP2X7staining (MFIP2X7-MFIIC)onthesurfaceofdifferenteffectorCD8Tcelleffectorsubsets.Dataarerepresentativeofone(B) or seven (C-D) healthy donors. P valueswere determined by paired Student’s t test, or by RMone-wayANOVA,followedbyBonferronipost-test:****=<0.0001.Legend:blue=adaptiveimmunecells.
In summary,CD4Tcellswithanactivated,effectorormemoryphenotypeexpresshigher levelsof
P2X7withinCD4Tcells.Thispatternwasalsoobserved inTregs,even though theyshowvery low
amountsofP2X7.Incontrast,theeffectorphenotypedoesnotcorrelatewiththeexpressionofP2X7
onCD8Tcells,whichexpressedsimilaramountsofP2X7independentlyoftheireffectorphenotype.
A
B
CD28
CD27
Effectors
TCD8
CD45RA
CD27
Naive
TEMRA
TCD8
CM
EM
TEMRANaive CM EM Effectors
P2X7
Counts
Δ P2X7expression[M
FI]
P2X7expression[MFI]
C DIsotype control hP2X7 staining
T CD8 Naïve CM TEMRA EM Effectors0
300
600
900
1200
************************
T CD8 Naïve CM TEMRA EM Effectors0
300
600
900
1200
Isotype control hP2X7 staining
67
4.2.4 Innate-likelymphocytesexhibithigherlevelsofP2X7thanconventionalTcells
Since NK cells express the highest levels of P2X7 within the lymphocyte compartment, we asked
whether other innate lymphocytes or innate-like lymphocyteswould also express higher levels of
P2X7.Inhumans,1-6%ofcirculatingTcellsbelongtotheγδlineage,beingTcellsharbouringtheVδ2
chainthemostabundant(50-90%)γδTcellsubtype.ThegatingstrategyfortheidentificationofγδT
cellsisshowninFigure18A.
T cells from the γδ lineage expressed higher levels of P2X7 on their surface when compared to
conventionalCD4andCD8Tcells(Figure18).WithinγδTcells,Vδ2+cellsweretheonesexpressing
thehighestlevelsamongallTcellsubsets(Figure18B).Aftersubtractionoftheunspecificsignal,we
confirmed the higher expression of P2X7 in both γδ T cells (all) and Vδ2+ cells compared to
conventionalTcellsandVδ2-cells(Figure18D).
AnotherTcellpopulationwithasemi-invariantTCRandinnatepropertiesareMAITcells, identified
asCD3+Vα7.2+CD161+ inFigure19A. Interestingly,MAITcellsexhibitedsimilarexpressionofP2X7
thanγδTcells(Figure19B-D),andthereforehigherthanconventionalTcells.
We next assessed P2X7 on the whole population of innate lymphoid cells (ILCs), defined as CD3-
CD127+CD161+,andthetwomajorNKcellsubsets:NKdim(CD3-CD56dim)andNKbrightcells (CD3-
CD56high).Thegatingstrategyforthe identificationofall immunecellpopulations is represented in
Figure 19A.We observed higher expression of P2X7 on ILCs and bothNK subsets than αβ T cells
(Figure19E,F).
68
Figure 18. γδ T cells express higher levels of P2X7 than conventional T cells. (A) Gating strategy for theidentification of different immune cell populations. (B) P2X7 expression (MFI) on the surface of CD4(CD3+CD4+),CD8 (CD3+CD8+), γδ (CD3+TCRγδ+),Vδ2+ (CD3+TCRγVδ2+),Vδ2- (CD3+TCRγVδ2-)T cells.Numberofcells was normalised. (C) Expression of P2X7 (MFI ± SD) and background signal detected on the IC on thesurfaceofdistinctTcellsubsets.(D)ExpressionofP2X7(MFI±SD)aftersubtractionoftheMFIoftheICfromtheMFIoftheP2X7staining(MFIP2X7-MFIIC)onthesurfaceofTcellsubsets.Dataarerepresentativeofone(B) or seven (C-D) healthy donors. P valueswere determined by paired Student’s t test, or by RMone-wayANOVA,followedbyBonferronipost-test:**=<0.01;***=<0.001;****=<0.0001.Legend:orange=innate-likelymphocytes//blue=adaptiveimmunecells.
TCRγδ
Vδ2
Vδ2+
Tγδ
Vδ2-
TCD8
Singlecells
CD3
TCRγδ
Tαβ
Tγδ
TCD8
CD4
CD8 TCD4
TαβA
B TCD4
P2X7
Counts
Tγδ Vδ2- Vδ2+
Isotype control hP2X7 staining
P2X7expression[MFI]
ΔP2X7expression[M
FI]
C D
TCD4 TCD8 Tγδ Vδ2- Vδ2⁺0
500
1000
1500****
**** ****
****
***
TCD4 TCD8 Tγδ Vδ2- Vδ2⁺0
500
1000
1500
** *******
****
hP2X7 staining
hP2X7 staining
Isotype control
69
CD3
TCRγδ ´CD3-
Tαβ
CD45+cells
CD127CD
45
ContainsILCs
CD3-
CD161
CD56
ILCs
ContainsILCs
CD161
Vα7.2
MAIT
TαβA
CD56
FSC-H NKdim
CD3-
NKbright
B
ILCs
Tαβ Tγδ
NKbright
MAIT
NKdim
P2X7
Counts
ΔP2X7expression[M
FI]
P2X7expression[MFI]
P2X7expression[MFI]
ΔP2X7expression[M
FI]
C D
E F
Isotype control hP2X7 staining
hP2X7 staining
hP2X7 staininghP2X7 staining
Isotype control
Tαβ Tγδ MAIT0
400
800
1200
1600
****
********
Tαβ Tγδ MAIT0
400
800
1200
1600
´
***
**
Tαβ Tγδ ILCs NK dim
NK bright
0
400
800
1200
1600
****
**** ****
****
****
Tαβ Tγδ ILCs NKdim
NKbright
0
400
800
1200
1600
****
***
***
70
Figure 19. Innate-like lymphocytes and innate like cells express higher levels of P2X7 than conventional Tcells.(A)Gatingstrategyfortheidentificationofdifferentimmunecellpopulations.(B)P2X7expression(MFI)onthesurfaceofαβ(CD3+TCRγδ-),γδ(CD3+TCRγδ+),MAIT(CD3+CD161+Vα7.2+),ILCs(CD3-CD127+CD161+),NKdim(CD3-CD56dim)andNKbrightcells(CD3-CD56high).Numberofcellswasnormalised.(CandE)ExpressionofP2X7(MFI±SD)andbackgroundsignaldetectedonthe IConthesurfaceofdistinct immunecells. (DandF)ExpressionofP2X7 (MFI±SD)aftersubtractionof theMFIof the IC fromtheMFIof theP2X7staining (MFIP2X7-MFIIC)onthesurfaceofdistinctimmunecells.Dataarerepresentativeofone(B)orseven(C-F)healthydonors. P values were determined by paired Student’s t test, or by RM one-way ANOVA, followed byBonferronipost-test:**=<0.01;***=<0.001;****=<0.0001.Legend:red=innateimmunecells//orange=innate-likelymphocytes//blue=adaptiveimmunecells.
Ourdatademonstratesthatinnatecells(NKcells,ILCs)andinnate-likelymphocytes(MAITcells,γδT
cells) express higher levels of P2X7 on the cell surface than conventional T cells. Therefore, P2X7
expressionrevealsadistinctpatternthatcorrelatestothe“innateness”ofthelymphocytes.
4.2.5 γδTcellsalsoexhibithigheramountsofP2X7atthemRNAlevel
Tofurtherconfirmthispattern,wedeterminedtheexvivoexpressionofP2X7atthemRNAlevelin
sorted T cell subsets by qRT-PCR. The gating strategy for the identification and sorting of the
differentT cell subpopulations is shown inFigure20A. The sample“CD3T cells” includedall T cell
subpopulations; and therefore was used as experimental calibrator. P2X7 mRNA expression was
more than threefoldhigher in γδ T cells than in conventional T cells (Figure20B), thus confirming
that innate-like γδ T cells exhibit higher levels of P2X7 both at the surface and mRNA level. As
representedinthegraph,theexpressionofP2RX7variesamongthedifferentdonors,whichmaybe
aconsequenceofgeneticvariations(SNPs)intheP2RX7gene.
Figure20.γδTcellsexhibithigherexpressionoftheP2RX7genethanconventionalTcells.TheexpressionofP2RX7insortedCD3+Tcells,CD3+CD4+Tcells,CD3+CD8+TcellsandCD3+TCRγδ+TcellswasmonitoredbyqRT-PCR.(A)GatingstrategyfortheidentificationandsortingofTcellsubsets.(B)Foldincreaseinthetranscriptionof P2RX7. Data are relative to the expression of the reference gene RPL13A; using CD3+ T cells (TCD3) ascalibrator.Relativegeneexpression(Mean±SD)wascalculatedusingthedelta-deltaCtformula(2–∆∆Ct).Dataare representativeof five healthydonors (n=5). P valuesweredeterminedbyone-wayANOVA, followedbyBonferronipost-test:***=<0.001.
Tαβ
LiveTCD3
CD3
Live/dead
CD3
TCRγδ
Tαβ
Tγδ
CD4
CD8
TCD8
TCD4
P2RX
7[fo
ldincrease]
A B
TCD3 TCD4 TCD8 Tγδ0
2
4
6
8 ***
***
***
71
4.2.6 Innate-likeTcell-derivedcell linesexhibithigherlevelsofP2X7thanCD4andCD8
conventionalcelllines
Since innate-like lymphocytesare rareevents inperipheralblood inmanydonors,wegeneratedT
celllinesderivedfromsortedγδTcells,MAITandalsofromCD4Tcellsfromhumanperipheralblood
inordertoobtainsufficientcellsformechanisticstudies.
Thepurityofthecelllineswasalwayshigherthan70%fortheMAIT-derivedcelllineandhigherthan
90%fortheotherTcell-derivedcelllines(Figure21A).Similartowhatwesawinperipheralblood,γδ
andMAIT-derivedTcell linestendtoexpresshigher levelsofP2X7thantheCD4-derivedTcell line
(Figure21B).Furthermore,withintheγδ-derivedTcellline,cellsexpressingtheVδ2chainexhibited
higherlevelsofP2X7thanTγVδ2-cells(Figure21B).
Inlinewithourpreviousfindings,thesedataconfirmthedistinctpatternofP2X7expressionwithin
theTcellcompartment.However,theγδTcelllinescreatedlosttheexpressionofP2X7afterseveral
roundsofstimulation,andtherefore,couldnotbefurtherusedfortestingP2X7function.
Figure 21. Cell lines derived from innate-like lymphocytes express higher levels of P2X7 than cell linesderivedfromCD4Tcells.CD4Tcells,γδTcellsandMAITcellsfromhumanperipheralbloodweresortedbyflowcytometryandculturedunder specific conditions. (A)Purity (percentage)ofeachcell lineuponspecificgating. (B) Overlaid histograms of the expression of P2X7 for the different T cell-derived cell lines. Datacorrespond today3 (MAIT-derivedT cell line)orday11 (CD4-andγδ-derivedT cell lines)after stimulation.Data (Median Fluorescence Intensity (MFI)) are of one representative donor. Legend: dotted line= isotypecontrol;filledhistogram=P2X7expression;blue=adaptiveCD4Tcellsandorange=innate-likelymphocytes.
Vα7.2
CD161
CD4- CD8+cells
MAIT74.3%
TCD4
P2X7
Counts
Vδ2+ Vδ2- MAIT
TCRγδ
Vδ2
Tγδ
Vδ2+
Vδ2-
CD3
TCRγδ
Singlecells
Tγδ98.8%
CD3+cells
CD4
CD8
TCD495.2%
A
B
72
4.2.7 RNAtranscriptionofP2RX7increasesuponTcellactivation
ThehigherexpressionofP2X7onactivated/effectorTcellspromptedustoinvestigatewhetherP2X7
is upregulated upon activation. Since both signals from the IC and P2X7 staining increase upon
activation,weassessedchanges inthetranscriptionofP2RX7uponstimulationofsortedCD4,CD8
andγδTcellswithsolubleαCD3for9days.AllTcellsubsetsfromalldonorsupregulatedP2RX7over
thecourseoftimecomparedtodayzero(Figure22). In linewithcellsurfaceexpression,thebasal
expression of P2RX7was higher on γδ T cells than in CD4 and CD8 T cells, and this differential
expressionwasmaintainedovertime(Figure22).UpregulationofP2X7wascharacterizedbyapeak
onedayafteractivation,followedbyadecreaseonthemRNAlevelsondaythree.Thispatternwas
veryprominentonγδTcells,whichafteradownregulationofP2X7expressiononday2,showeda
steadyincreaseingeneexpression.CD4Tcellsshowedasimilarprofileofupregulation,butreached
themaximumofexpressionalreadyondayone.Nevertheless,theupregulationupondaythreewas
less marked than in γδ T cells (Figure 22). In contrast, CD8 T cells exhibited a progressive
upregulationofP2X7intwoofthedonors(Figure22A,B),reachingthehighestlevelsofP2X7onday
9foralldonors(Figure22).AlldonorsweregenotypedforthreeSNPs(H155Y,H270RandA348T)in
theP2RX7gene,whichresultindifferentsensitivitytoATP;beingtheallelicvariants155Y,270Rand
348T,theonesconferringhighersensitivity(Cabrinietal.,2005;Stokesetal.,2010).Thegenotypes
ofthedifferentdonorsareasfollows:D1(HY/RH/AT),D2(YY/RR/TT)andD3(HH/RR/TT).Although
the pattern of expression between the different cell types was similar among all donors, genetic
variationscouldexplainthedifferencesinP2RX7expression.
Altogether,ourdataindicatethatTcellsupregulateP2RX7expressionuponactivation,andthatthis
effectismorepronouncedinγδTcells.
Figure22.P2RX7geneexpressioninTcellsisupregulateduponTcellstimulationinvitro.SortedCD3+CD4+Tcells,CD3+CD8+TcellsandCD3+TCRγδ+TcellswerestimulatedwithsolubleαCD3antibodies for9days.TheexpressionofP2RX7wasdeterminedbyqRT-PCR.Data are relative to theexpressionof the reference geneRPL13A,andbasalexpressionofP2RX7ofCD4Tcellswasusedasthecalibratorsample. (A-C)RelativegeneexpressionofP2RX7 inCD4Tcells (green),CD8Tcells (blue)andγδTcells (orange) foreachdonor (D1-D3;n=3).
P2RX7
[fold
incr
ease
]
Time (days)
P2RX7
[fold
incr
ease
]
Time (days)
P2RX7
[fold
incr
ease
]
Time (days)
BA CD1 D2 D3
0 1 3 5 7 90
20
40
60
80
0 1 3 5 7 90
20
40
60
80
0 1 3 5 7 90
20
40
60
80
TCD4 TCD8 Tγδ
73
4.2.8 TheexpressionofP2X7inhumanintestinalTcellsissimilartoperipheralTcells
Inthemouseintestine,bothconventionalCD8αβ+andunconventionalintraepithelialCD8αα+Tcells
expressmoreP2X7thantheirperipheralcounterparts (Heissetal.,2008). InpatientswithCrohn´s
disease P2X7 is expressed in higher levels in the mucosa layer of the colon (Neves et al., 2014).
Therefore, we investigated P2X7 expression on human T cells from the small intestine from two
donorsundergoingbariatricsurgery.ThegatingstrategyusedfortheidentificationofdifferentTcell
subsetsisshowninFigure23A.Incontrasttomice,humanintestinalcellsdidnotexhibithighlevels
ofP2X7onthesurface,butrathersimilartothe levelsobservedonTcells intheperiphery(Figure
23B).Again, γδTcells fromthe intestinealso seemtoexpresshigher levelsofP2X7 thanCD4and
CD8Tcells(Figure23B).
BotheATPandpurinergicsignallingareimplicatedinexacerbatedintestinalinflammatoryresponses
(Diezmos, Bertrand and Liu, 2016; Wan et al., 2016; Figliuolo et al., 2017). Indeed, Wang and
colleaguesreportedhigherlevelsofeATPincolontissuesofmicesufferingfromcolitis(Wanetal.,
2016);underliningtheneedforcontrollingthelevelsofATPtopreventexcessiveinflammationinthe
intestine(Friedmanetal.,2009;Kusuetal.,2013).Thus,weinvestigatedtheexpressionoftheATP-
metabolisingectoenzymesCD39andCD73onhumangutTcells.Inperipheralblood,theexpression
ofCD39ishighlyvariable(2-70%)onTregs,whereasCD4andCD8Tcellsexpresslowerlevelsofthis
ectoenzyme(A.Rissieketal.,2015).Ourdatashowthat80%ofintestinalCD8Tcellsfromonedonor
expressedCD39(Figure24B),althoughthiswasnotthecasefortheotherdonor(>5%ofCD39+CD8
T cells). In contrast, expression of CD39was almost negligible in conventional CD4 T cells (Figure
24B).Tregsrepresentalowpercentageofintraepitheliallymphocytes(IELTs),butaremoreabundant
amonglaminapropria lymphocytes(LPLs)(Lutteretal.,2018).Weobservedlowfrequency(>2.5%)
of Tregs in human gut tissue, which included both IELTs and LPLs, and CD39 was only partially
detectedinoneofthedonors(Figure24B).
WefoundthatmostCD8Tcellsfromtheintestineandalsoinperipheryexpressveryhighlevelsof
CD73, while expression of CD73 seems to be higher in intestinal CD4 T cells compared to the
periphery (Figure24). Even thoughCD39andCD73are rarely coexpressed inhumanperipheral T
cells (Figure24A) (Mandapathiletal.,2010),weobservedcoexpressionofthetwoectoenzymes in
nearly75%ofintestinalCD8Tcellsofoneofthedonors(Figure24B).
74
Figure23.HumanintestinalTcellsexpresssimilaramountsofP2X7thanTcellsfromtheperiphery.Exvivoexpression of P2X7 on the surface of human intestinal T cells. (A) Gating strategy for the identification ofdifferentTcellsubsets.(B)P2X7expression(MFI)onthesurfaceofαβ(CD3+TCRγδ-),CD4(CD4+),CD8(CD8+),γδ(CD3+TCRγδ+),Vδ2(Vδ2+)andnotVδ2Tcells (Vδ2-).Numberofcellswasnormalised.Dataareofasingledonor(n=2).
A Allevents
Lymphocytes
FSC-A
SSC-A
FSC-A
FSC-H
Lymphocytes
Singlecells
CD3
FSC-H
Singlecells
CD3- CD3+
CD3
TCRγδ
CD3+
Tγδ
Tαβ
CD4
CD8
αβ Tcells
TCD8
TCD4
TCRγδ
γδ Tcells
Vδ2+
NotVδ2+
P2X7
Counts
NotVδ2+γδ Tcells Vδ2+
CD3+ CD4Tcells CD8TcellsBVδ2
Isotype control hP2X7 staining
75
Figure24.TheectoenzymeCD73ishighlyexpressedinhumanintestinalCD8Tcells.PercentageofCD39andCD73 positive peripheral (A) and intestinal (B) CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD8+) and Tregs(CD4+CD25+CD127-).Dataareofone(A)ortwo(B)individualdonors.
In summary, we report similar expression of P2X7 between intestinal and peripheral T cells in
humans. We also report high and similar expression of the ectoenzyme CD73 between human
intestinalandperipheralCD8Tcells,whereastheexpressionofCD73seemstobehigherinintestinal
CD4Tcells.
4.2.9 RetinoicaciddoesnotinduceP2X7upregulationonhumanTcells
Thehumanintestine iscolonizedbyacomplexpopulationofmicroorganismsand, intestinalTcells
are in constant activation tomaintainhomeostasis. In the intestineofmice,CD8andeffector and
CD73
CD
39
Blood
CD8 T cells TCD4 conv. TregsD1
D2
A
IntestineB
CD73
CD
39CD8 T cells
2.5 0.34
31.6 65.6
TCD4 conv
3.4 0.04
77.7 18.9
Tregs
65.4 1.5
31.2 1.84
2.04 1.79 1.73 0.17 3.68 1.55
25.3 70.8 69.9 28.2 65.9 28.7
23.5 60.1 3.72 1.83 21.1 0
1.94 14.4 50.1 44.3 64.1 1.84
76
memoryCD4Tcellsexpresshigh levelsofP2X7on thesurface,which increases their sensitivity to
extracellular nucleotides and P2X7-mediated cell death (Heiss et al., 2008; Hashimoto-Hill et al.,
2017).Retinoicacid (RA),which isproducedbyepithelialandstromalcells,andDCs, (Iwata,2009;
Halletal.,2011)isabundantinthesmallintestine(Halletal.,2011)andanimportantdeterminantof
gutimmunity(Czarnewskietal.,2017).Inmice,RAmediatesupregulationofP2X7onTcells(Heisset
al., 2008; Hashimoto-Hill et al., 2017). To investigate whether this also occurs in humans, we
polyclonalystimulatedPBMCsanddeterminedtheeffectofincreasingconcentrationsofRAonP2X7
expressioningutTcells,namelyCD4,CD8ααandCD8αβ(Figure25A).
Figure25.StimulationofhumanTcellswithretinoicacid(RA)doesnotaltertheexpressionofP2X7.PBMCswere leftuntreatedor stimulatedwithsolubleαCD3antibodies for4days in thepresenceorabsenceofRA(100nM). Theexpressionof P2X7wasdeterminedby flow cytometryover time. (A)Gating strategy for theidentificationofdistinctTcellsubsets.(B-C)ExvivoexpressionofP2X7(day0)onCD4+,CD8αα+andCD8αβ+Tcells.DatacorrespondtoMFIoftheIC(blackdottedline)andP2X7staining(greyfilledhistogram)(B)oraftersubtractionoftheIC(ΔP2X7)(C).(D)ExpressionofP2X7(MFI)aftersubtractionoftheIC(MFIP2X7-MFIIC)cellsoverthecourseoftime.Dataarerepresentativeoftwoindependentexperiments(n=2).
CD4
CD2
CD8
CD4
CD8βCD
8α
Tcells TCD4
TCD8TCD8αα
TCD8αβ
TCD4 TCD8αα TCD8αβ
P2X7
Counts
ΔP2X7expressio
n[M
FI]
Time(days)
ΔP2X7expressio
n[M
FI]
B
A
D
C
Isotype control hP2X7 staining
0 1 2 3 40
100
200
300
400
0 1 2 3 40
100
200
300
400
0 1 2 3 40
100
200
300
400
TCD4 TCD8αα
TCD8αβ
0
100
200
300
400
Uns$mulated
αCD3
αCD3 + RA
RA
77
AsshownbytheupregulationofCD38andCD25(datanotshown),Tcellsweresimilarlyactivated
under all conditions. All three T cell subsets exhibited low basal expression of P2X7 (Figure 25B),
beingCD8ααTcellstheonesexpressingmore(Figure25C).PolyclonalTcellstimulationresultedina
moderate increase in P2X7 gene expression on CD4 and CD8αα T cells, but not in CD8αβ+ T cells
(Figure25D).Incontrasttomice,additionofRAdidnotresultinupregulationofP2X7onthesurface
ofTcells,evenathighconcentrations(Figure25D).UnstimulatedcellslostexpressionofP2X7over
time,andRAdidnotrevertthistendency(Figure25D).
Incontrasttowhathasbeenreported inmice,humanintestinalTcellsexpress lowlevelsofP2X7,
similar to those observed in T cells from the periphery. Additionally, RA does not modulate the
expressionofP2X7onhumanTcells.
4.2.10 γδTcellsexhibithighersensitivitytoATP-inducedsheddingofCD62L
The distinct pattern of expression found in innate-like lymphocytes prompted us to investigate
whetherahigherexpressionofP2X7wouldimplymoresensitivitytoATP.P2X7activationresultsin
activationofADAMmetalloproteinasesandsubsequentsheddingofprotease-susceptiblemembrane
glycoproteins,suchasCD62L(Scheupleinetal.,2009)andCD27(Moonetal.,2006).
Figure26.Gatingstrategyfortheidentificationofdistinctimmunecellsubsets.γδTcellswereidentifiedasCD3+TCRγδ+cells;andwithinthissubsetVδ2+Vγ9+andVδ1+cellswerealsoidentified.αβTcellswereidentifiedaccording to expression of CD3, CD4 (CD4 T cells) and CD8 (CD8 T cells). CD4 T cells were subdivided inconventional CD4 T cells (CD25-CD127+) and Tregs (CD25+CD127-). NK dim (CD56dim) and NK bright cells(CD56high)weregatedwithinCD3-CD19-cells.
Singlecells
CD3
TCRγδ+CD
19 Bcells Tcells
CD3- CD19-
CD4
TCRγδ
Tcells
Tγδ
Tαβ
Vδ2
Vδ1+Vγ9
Tγδ
Vδ1 Vδ2γ9
CD4
CD8
Tαβ
TCD8
TCD4
CD25
CD127
TCD4
TCD4conv.
Tregs
CD56
CD3
CD3- CD19-
NKdim NKbright
78
Thus,weassessedthesheddingofCD62Lupon incubationofPBMCswithATP.Thegatingstrategy
fortheidentificationofthedifferentimmunecelltypesisshowninFigure26.Althoughthesurface
expression of P2X7was relatively low in Tregs, the dramatic effects of P2X7 activation onmurine
Tregcells(Hubertetal.,2010)promptedustoassesssensitivitytoATPintheirhumancounterparts.
Our readout is thepercentageof cells expressing surfaceCD62L.Due to thehigh variability in the
expressionofCD62Lamongdonors,datawere standardisedand thebaseline frequencyofCD62L+
cells (withoutadditionofexogenousATP)was setat100% foreachdonor.Thus, the frequencyof
CD62L+ cells after addition of different concentrations of ATP is relative to the frequencywithout
additionofATP.
Figure27.ConventionalCD4TcellsandTregsexhibitsimilarsensitivitytoATP-inducedsheddingofCD62L.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandthesheddingofCD62Lonconventional CD4 T cells (TCD4 conv., blue) and Tregs (green) was determined by flow cytometry. (A)ConcatenatedFACSplotsofCD62L+conventionalCD4Tcells (left)andCD62L+Tregs (right). (B)FrequencyofCD62L+conventionalCD4+TcellsandTregsuponstimulationwithATP.(C)FrequencyofCD62L+conventionalCD4 T cells and CD62L+ Tregs upon stimulationwith ATP relative to the basal frequency of CD62L+ cells (noexogenous ATP). Basal frequency of CD62L+ cells is represented by a dotted black line at 100%. Data arerepresentativeofone(AandB)orseven(C)healthydonors.PvaluesweredeterminedbypairedStudent’sttestforthecomparisonoftwogroups,andpairedRMone-wayANOVAfollowedbyBonferronipost-testforthecomparisonofthreeormoregroups.
RelativefrequencyofC
D62L
+cells
ATP[mM]
FrequencyofCD6
2L+cells
ATP[mM]
B C
A
0
ATP[mM]
CD62L
0.5 1.5 4.5
TCD4conv.
0 0.5 1.5 4.5
Tregs
0.5 1.5 4.50
20
40
60
80
100
0
20
40
60
80
100
0.1 1 10
TCD4 conv. Tregs
79
SheddingofCD62Lwasobserveduponadditionof1.5mMofATP(≈40%loss)andstimulationwith
4.5mMofATP,ledtoa70%decreaseofCD62L+cellsinbothTcellsubsets(Figure27A-C).However,
we did not find any significant differences in ATP-induced shedding of CD62L between Tregs and
conventionalCD4TcellsinresponsetoATP(Figure27C).
Next,wecomparedthesensitivitytoATPofinnate-likeγδTcellswithconventionalCD4andCD8T
cells.AllthreepopulationsshowedsheddingofCD62Luponadditionof1.5mMATP,althoughtoa
differentextent. Insomedonors,γδTcells started to reactalreadyuponstimulationwith0.5mM
ATP. Interestingly,weobserved thatuponadditionof1.5mMofATP, sheddingofCD62L isnearly
complete in γδT cells (Figure28),whileonlyhalfofCD4andCD8T cells lostCD62L from the cell
membrane.NoteventhehighestconcentrationofATP(4.5mM)resultedincompletelossofCD62L+
conventionalTcells(Figure28).Ofnote,CD8TcellsprovedtobemoresensitivetoATPstimulation,
in linewith its slightly higher expression levels of P2X7or itmaybebecauseMAIT cellswere not
excludedfromtheCD8gate.
Figure 28. γδ T cells exhibit higher sensitivity to ATP-induced shedding of CD62L. Human PBMCs werestimulatedwith different concentrations of ATP for 30min and the shedding of CD62L on CD4 (blue), CD8(green)andγδTcells(orange)wasdeterminedbyflowcytometry.(A)ConcatenatedFACSplotsofCD62L+CD4(left)andCD62L+CD8(middle)andCD62L+γδTcells(right)stimulatedunderdifferentconditions(B)FrequencyofCD62L+CD4andCD62L+CD8andCD62L+γδTcellsuponstimulationwithATP.(C)FrequencyofCD62L+CD4,
ATP[mM]
CD62L
0 0.5 1.5 4.5
TCD4
0 0.5 1.5 4.5
TCD8
0 0.5 1.5 4.5
Tγδ
FrequencyofCD6
2L+cells
ATP[mM]
Relativefrequencyo
fCD6
2L+cells
ATP[mM]
A
B C
0
20
40
60
80
100
0.1 1 10 0.5 1.5 4.50
20
40
60
80
100****
*****
**
***
TCD4 TCD8 Tγδ
80
CD62L+CD8andCD62L+γδTcellsuponstimulationwithATPrelativetothebasalfrequencyofCD62L+cells(noexogenous ATP). Basal frequency of CD62L+ cells is represented by a dotted black line at 100%. Data arerepresentativeofone(AandB)ornine(C)healthydonors.PvaluesweredeterminedbypairedStudent’sttestfor the comparisonof twogroups, andpairedRMone-wayANOVA followedbyBonferronipost-test for thecomparisonofthreeormoregroups:*=<0.05;***=<0.001;****=<0.0001.
WithinγδTcells,eventhoughVδ2+TcellsexpressmoreP2X7ontheirsurfacethanVδ2-Tcells,Vδ1
cellsprovedmoresensitivetoATPthanVδ2γ9Tcells(Figure29);sinceatthelowestconcentration
ofATP(0.5mM)30%ofVδ1cellshadalreadylostCD62Lfromtheirsurface,whileonly10%didsoin
theVδ2γ9population.AthigherconcentrationsofATP,bothγδTcellsubsetshadcompletelyshed
CD62Lfromthecellsurface(Figure29).
Figure 29. Vδ1 T cells are more sensitivity to ATP-induced shedding of CD62L than Vδ2γ9 T cells at lowconcentrationofATP.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandthesheddingofCD62LonVδ1(red)andVδ2Vγ9(orange)Tcellswasdeterminedbyflowcytometry.(A)FrequencyofCD62L+Vδ1andCD62L+Vδ2γ9TcellsuponstimulationwithATP.(B)FrequencyofCD62L+Vδ1andCD62L+Vδ2γ9TcellsuponstimulationwithATPrelative to thebasal frequencyofCD62L+cells (noexogenousATP).BasalfrequencyofCD62L+cellsisrepresentedbyadottedblacklineat100%.Dataarerepresentativeofone(A)orseven(C)healthydonors.PvaluesweredeterminedbypairedStudent’sttestforthecomparisonoftwogroups,andpairedRMone-wayANOVAfollowedbyBonferronipost-testforthecomparisonofthreeormoregroups.
NK cells are the paradigm of innate lymphocytes. The expression of CD56 differentiates two NK
subsets, themore immatureCD56high, and thehighly cytolytic CD56dim.Wehavepreviously shown
that NK cells express the highest levels of P2X7 at the cell surface among all lymphocytes.
Accordingly,sheddingofCD62LwassignificantlyhigheronbothNKsubsetscomparedtoCD4Tcells
inresponsetoATP(1.5and4.5mM)(Figure30B).WealsoobservedlowerfrequencyofCD62L+cells
ontheNKdimsubsetthaninNKbrightcellsuponstimulationwith1.5mMATP(Figure30B),inline
withitseffectorphenotype.
FrequencyofCD6
2L+cells
ATP[mM]
A
Relativefrequencyo
fCD6
2L+cells
ATP[mM]
B
0
20
40
60
80
100
0.1 1 10 0.5 1.5 4.50
20
40
60
80
100*
Vδ2γ9 Vδ1
81
Insummary,NKcellsarehighlysensitivetohighconcentrationsofATP.Furthermore,thepatternof
CD62LsheddinginNKcellsissimilartothepatternfoundinγδT(Figure30D).
Figure30.NKcellsalsoexhibithighersensitivitytoATPthanconventionalCD4Tcells.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandthesheddingofCD62LonconventionalCD4Tcells(blue),γδTcells(orange),NKdim(yellow)andNKbright(red)wasdeterminedbyflowcytometry.(AandC)FrequencyofCD62L+NKdim,NKbright,CD4Tcells(A)andγδTcells(C)uponstimulationwithATP.(BandD)FrequencyofCD62L+NKdim,NKbright,CD4Tcells(B)andγδTcells(D)uponstimulationwithATPrelativetothebasalfrequencyofCD62L+cells(noexogenousATP).BasalfrequencyofCD62L+cellsisrepresentedbyadottedblacklineat100%.Dataarerepresentativeofone(AandC)oreight-nine(BandD)healthydonors.PvaluesweredeterminedbypairedStudent’sttestforthecomparisonoftwogroups,andpairedRMone-wayANOVAfollowedbyBonferronipost-test for thecomparisonof threeormoregroups:*=<0.05;**=<0.01;***=<0.001;****=<0.0001.
Overall,themagnitudeofresponsetoATPamonglymphocytesisasfollows:NKdimandγδTcells>
NKbright >CD8T cells >CD4T cells, resembling themagnitudeof surfaceexpressionof P2X7on
theseimmunecells.
FrequencyofCD6
2L+cells
FrequencyofCD6
2L+cells
Relativefrequencyo
fCD6
2L+cells
Relativefrequencyo
fCD6
2L+cells
ATP[mM] ATP[mM]
ATP[mM] ATP[mM]
A B
C D
0
20
40
60
80
100
0.1 1 10
0
20
40
60
80
100
0.1 1 10
NK dim NK brightTCD4 Tγδ
0.5 1.5 4.50
20
40
60
80
100
* **
0.5 1.5 4.50
20
40
60
80
100****
***
***
82
4.2.11 ATP-inducedsheddingofCD62LismediatedbyP2X7
Inorder to confirm the roleofP2X7 inCD62L shedding,wepreincubatedPBMCswithDano1or a
controlNbpriortostimulationwithATP.Ascontrol,PBMCswerealsopreincubatedwithapyrase,an
enzymethatcatalyses thehydrolysisofATPtoAMPand inorganicphosphate, thuseliminating the
ligandofP2X7andpreventing its activation.PreincubationwithDano1orapyraseabolishedATP-
induced shedding of CD62L. In contrast, preincubationwith a control nanobody did not have any
effectonATP-inducedsheddingofCD62L(Figure31),confirmingtheroleofP2X7inthisresponse.
Figure 31. Dano1 inhibits P2X7-mediated shedding of CD62L in response to ATP. Human PBMCs werepreincubatedwith100nMofDano1(blue)oracontrolNb(black),10U/mlapyrase(grey)orRPMIascontrol(white)for20minatRTor37°C(apyrase)priortostimulationwithATP(4.5mM)forfurther30min.FrequencyofCD62L+cells(Mean±SD,n=9)wasassessedbyflowcytometry.PvaluesweredeterminedbypairedRMone-wayANOVA followedbyBonferronipost-test for the comparisonof threeormoregroups:*=<0.05; ***=<0.001;****=<0.0001.
4.2.12 Innate-likelymphocytesexhibithighersensitivitytoATP-inducedporeformation
Activation of P2X7 triggers the opening of a secondary non-selective pore permeable to cationic
moleculeswithamolecularmassupto900Da(Steinbergetal.,1987).Therefore,wemeasuredthe
uptake of DAPI as a surrogate approach for P2X7 activation. To study this aspect, we stimulated
PBMCswithATPandmeasured theuptakeofDAPIby flowcytometry. Thegating strategy for the
identificationofimmunecellsisshowninFigure32.Inthiscase,wealsoidentifiedTregsaccordingto
the expression of CD39, andMAIT cells as CD8+CD161+Vα7.2+ cells. SinceMAIT cells are relatively
rareinhumanblood(1-10%)(Salou,FranciszkiewiczandLantz,2017),andthattheymostlyharboura
memory phenotype (characterized by the absence of CD62L) (Chandra and Kronenberg, 2015),
determinationofATP-inducedsheddingofCD62Lwasnotreliableinthissubset.
- Apyrase Dano1 Control
FrequencyofCD6
2L+cells
FrequencyofCD6
2L+cells
FrequencyofCD6
2L+cells
TCD4 TCD8 Tγδ
0
20
40
60
80
100
0 4.5ATP
[mM]
*
0
20
40
60
80
100
0 4.5ATP
[mM]
****
0
20
40
60
80
100
0 4.5ATP
[mM]
***
83
Figure32.Gatingstrategyfortheidentificationofdistinctimmunecellsubsets.γδTcellswereidentifiedasCD3+TCRγδ+ cells; andwithin this subsetVδ2+Vγ9+ andVδ1+ cellswerealso identified.αβT cellsweregatedaccordingtoexpressionofCD3,andCD8TcellsaccordingtoexpressionofCD8.ConventionalCD4Tcellswereidentified as CD25-CD127+ and Tregs (CD25+CD127-)were subdivided according to expressionof CD39.MAITcellswere identified as CD8+CD161+Vα7.2+ cells. NK dim (CD56dim) andNK bright cells (CD56high)were gatedwithinCD3-CD19-cells.
The uptake of DAPIwas induced upon stimulationwith 1.5mMof ATP in both human Tregs and
conventionalCD4Tcells(Figure33).SimilartowhatweobservedforthesheddingofCD62L,bothT
cell subsets followed a similar pattern (Figure 33B), confirming that human Tregs do not exhibit
highersensitivitytoATP(Figure33C).
Singlecells
CD3
TCRγδ
Tcells
CD3-
CD4TC
Rγδ
Tcells
Tγδ
Tαβ
Vδ2
Vδ1+Vγ9
Tγδ
Vδ1 Vδ2γ9
CD56
CD3
CD3-
TCD8
TCD4
CD4
CD8
RestofTcells
TCD4conv.
CD25CD127
TCD4
NKdim NKbright
Vα7.2
CD161
Tαβ
RestofTcells
MAIT
CD4
CD39
Tregs
Tregs
CD39-
CD39+
<DAPI
Counts
TCD4conv. TregsA
0 mM 0.5 mM 1.5 mM 4.5 mM[ATP]:
84
Figure 33. Conventional CD4 T cells and Tregs exhibit similar sensitivity to ATP-induced pore formation.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandtheuptakeofDAPIwasmonitoredbyflowcytometry.(A)UptakeofDAPI(Medianfluorescenceintensity,MFI)byconventionalCD4Tcells(TCD4conv.,left)andTregs(right).(B-C)ComparisonofuptakeofDAPI,MFI(B)andMFI±SD(C),byTCD4conv.(blue)andTregs(green).Dataarerepresentativeofone(A-B)orseven(C)healthydonors.Pvaluesweredetermined by paired Student’s t test for the comparison of two groups and paired RM one-way ANOVAfollowedbyBonferronipost-testforthecomparisonofthreeormoregroups.
Wenext investigatedATP-inducedpore formationof γδ T cells in comparisonwith conventional T
cells (Figure 34A, B). Stimulation with 1.5 mM ATP and 4.5 mM ATP substantially increased the
uptakeofDAPI inallTcellsubsets,althoughtoadifferentextent(Figure34). In linewithprevious
findings, γδTcellsproved tobemoresensitive toATP thanconventionalT cells, regardlessof the
concentrationofATP(Figure34C).
Within γδ T cells, the Vδ1 and Vδ2γ9 subsets exhibited similar profiles upon stimulation with
differentconcentrationsofATP(Figure35A),althoughinthiscase,Vδ2γ9Tcellsweremoresensitive
toATP(1.5mM),astheuptakeofDAPIwasslightlyhigherinthissubset(Figure35B).
DAPIuptake[M
FI]
ATP[mM]
B C
DAPIuptake[M
FI]
ATP[mM]
0.0
5.0×10³
1.0×10⁴
1.5×10⁴
0.1 1 10 0 0.5 1.5 4.50
1×10⁴
2×10⁴
3×10⁴
TCD4 conv. Tregs
85
Figure34.γδTcellsexhibithighersensitivitytoATP-inducedporeformation.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandtheuptakeofDAPIwasmonitoredbyflowcytometry.(A)Uptake of DAPI (MFI) by conventional CD4 T cells (left), CD8 T cells (middle) and γδ T cells (right). (B-C)ComparisonofuptakeofDAPI,MFI(B)andMFI±SD(C),byCD4Tcells(TCD4,blue),CD8Tcells(TCD8,green)andγδTcells(Tγδ,orange).Dataarerepresentativeofone(A-B)oreight(C)healthydonors.Pvaluesweredeterminedby pairedRMone-wayANOVA followedbyBonferroni post-test for the comparisonof threeormoregroups:*=<0.05;**=<0.01;***=<0.001;****=<0.0001.
DAPI
Counts
ATCD4 TCD8 Tγδ
ATP[mM] ATP[mM]
B C
DAPIuptake[M
FI]
DAPIuptake[M
FI]
0
1×10⁴
2×10⁴
3×10⁴
0.1 1 10 0 0.5 1.5 4.51×10²
1×10³
1×10⁴
1×10⁵
**
**
********
****
**
**
0 mM 0.5 mM 1.5 mM 4.5 mM[ATP]:
TCD4 TCD8 Tγδ
ATP[mM] ATP[mM]
A B
DAPIuptake[M
FI]
DAPIuptake[M
FI]
Vδ2γ9 Vδ1
0 0.5 1.5 4.51×10²
1×10³
1×10⁴
1×10⁵*
0
1×10⁴
2×10⁴
3×10⁴
0.1 1 10
86
Figure35.Vδ2γ9TcellsexhibitslightlyhighersensitivitytoATP-inducedporeformation.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandtheuptakeofDAPIonVδ2γ9(orange)andVδ1cells (red) was monitored by flow cytometry. Data are represented by MFI (A) or MFI ± SD (B). Data arerepresentativeofone(A)orsix(B)healthydonors.PvaluesweredeterminedbypairedStudent’sTTestforthecomparisonoftwogroupsandpairedRMone-wayANOVAfollowedbyBonferronipost-testforthecomparisonofthreeormoregroups:*=<0.05.
DAPI uptake could also be measured in the innate-like MAIT cells (Figure 36). In line with other
innate-like cells,MAIT cellsweremore sensitive toATP-inducedpore formation than conventional
CD8Tcells(Figure36C).AlsoNKcellstendtoreactmoreprominentlytohighconcentrationsofATP
thanCD4Tcells(Figure37),althoughwecouldonlyconfirmthistendencyonNKdimcellsinresponse
tothehighestconcentrationofATP(datanotshown).
Figure36.Innate-likeMAITcellsexhibithighersensitivitytoATP-inducedporeformationthanconventionalCD8Tcells.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandtheuptakeofDAPIwasmonitoredbyflowcytometry.(A)UptakeofDAPI(MFI)byCD8Tcells(left)andMAITcells(right).(B-C)ComparisonofuptakeofDAPI,MFI(B)andMFI±SD(C),byMAIT(orange)andCD8Tcells(green).Dataarerepresentativeofone(A-B)ornine(C)healthydonors.PvaluesweredeterminedbypairedStudent’sttestforthe comparison of two groups and paired RM one-way ANOVA followed by Bonferroni post-test for thecomparisonofthreeormoregroups:*=<0.05;**=<0.01.
DAPI
Counts
TCD8 MAITA
DAPIuptake[M
FI]
ATP[mM]
B C
DAPIuptake[M
FI]
ATP[mM]
TCD8 MAIT
0
5×10³
1×10⁴
2×10⁴
2×10⁴
0.1 1 10 0 0.5 1.5 4.51×10²
1×10³
1×10⁴
1×10⁵**
**
*
0 mM 0.5 mM 1.5 mM 4.5 mM[ATP]:
87
Figure37.Innateandinnate-likelymphocytesexhibithighersensitivitytoATP-inducedporeformationthanconventionalTcells.HumanPBMCswerestimulatedwithdifferentconcentrationsofATPfor30minandtheuptakeofDAPIbyCD4Tcells,CD8Tcells,γδTcells,MAITcells,NKdimandNKbrightcellswasdeterminedbyflowcytometry.Data(MFI)arethemeanofatotalof6-8healthydonors.
In line with previous findings, innate and innate-like immune cells are more sensitive to ATP-
mediatedporeformation.Inthiscase,thesequenceofresponsetoATPisthefollowing:γδTcells>
MAITcells>NKdim>Nkbright>CD8Tcells>CD4Tcells.ThesedataresemblethepatternofP2X7
expressionseenbyflowcytometry,eventhoughNKcellsexpressthehighestlevelsofP2X7.
4.2.13 Dano1efficientlyblocksATP-inducedporeformationinimmunecells
ToconfirmtheroleofP2X7inporeformationuponATPengagement,wepreincubatedPBMCswith
Dano1,acontrolNborapyrasepriortostimulationwithATP.ATPinducedtheprominentuptakeof
DAPIbyallimmunecells.Asexpected,preincubationwithDano1orapyrase,butnotacontrolNb,
decreasedtheuptakeofDAPItothelevelsmeasuredinbasalconditions(Figure38).
OurdataconfirmthatP2X7-blockadebyDano1orremovalofATPbyapyrasepreventsATP-induced
poreformationinimmunecells;confirmingtheroleofP2X7insuchcellularresponse.
DAPIuptake[M
FI]
ATP[mM]
0
1×10⁴
2×10⁴
3×10⁴
0.1 1 10
NK dim
NK bright
TCD4
TCD8
MAIT
Tγδ
DAPIuptake[M
FI]
DAPIuptake[M
FI]
DAPIuptake[M
FI]
TCD4 TCD8 Tγδ
1×10⁰
1×10²
1×10⁴
1×10⁶
***
0 4.5ATP
[mM]
1×10⁰
1×10²
1×10⁴
1×10⁶
0 4.5ATP
[mM]
**
1×10⁰
1×10²
1×10⁴
1×10⁶
0 4.5ATP
[mM]
**
- Apyrase Dano1 Control
88
Figure 38. Dano1 inhibits P2X7-dependent pore formation induced by ATP in a variety of T cell subsets.HumanPBMCswerepreincubatedwith100nMofDano1(blue)oracontrolNb(black),10U/mlapyrase(grey)orRPMIascontrol(white)for20minatRTor37°C(apyrase)priortostimulationwithATP(4.5mM)forfurther30min.TheuptakeofDAPI(MFI±SD,n=8)wasdeterminedbyflowcytometry.PvaluesweredeterminedbypairedRMone-wayANOVAfollowedbyBonferronipost-testforthecomparisonofthreeormoregroups:*=<0.05;**=<0.01;***=<0.001;****=<0.0001.
4.2.14 EffectorTcellsaremoresensitivetoATP-inducedporeformationthannaïveTcells
BasedonthehigherexpressionofP2X7inthesurfaceofeffectorCD4Tcells,wehypothesizedthat
effectorcellsmightalsoexhibithighersensitivitytoATP.Tothisend,wedeterminedtheuptakeof
DAPI on different T cell subsets, identified according tomaturationmarkers (Figure 15A, 17A), in
response to ATP. Of note, we only analysed those populations with a frequency higher than 5%.
EffectormemoryCD4TcellsexhibitedhigheruptakeofDAPIthannaïveandcentralmemoryCD4T
cells(Figure39A).Comparably,CD8Tcellswithaneffectorprofile(EM,TEMRAandeffectors)were
alsomoresensitivetoATPthannaïveCD8Tcells(Figure39B).Althoughthistendencywasobserved
indifferentdonors,wedidnotfindsignificantdifferencesamongthem.Additionally,wereconfirmed
that ATP-induced pore formation occurs through P2X7 signalling, as preincubation with Dano1
preventedtheuptakeofDAPIinallpopulations(datanotshown).
DAPIuptake[M
FI]
ATP[mM]
DAPIuptake[M
FI]
ATP[mM]
DAPIuptake[M
FI]
ATP[mM]
DAPIuptake[M
FI]
ATP[mM]
A
B
D1 D2
0
3×10³
5×10³
8×10³
1×10⁴
0.1 1 10
0
4×10³
8×10³
1×10⁴
2×10⁴
0.1 1 10
TCD4EMCMNaive
TCD8EMCMNaiveTEMRAEffectors
0
5×10³
1×10⁴
2×10⁴
2×10⁴
0.1 1 10
0
5×10³
1×10⁴
2×10⁴
2×10⁴
0.1 1 10
89
Figure39.EffectorTcellsexhibithighersensitivitytoATP-inducedporeformationthannaivecells.HumanPBMCs were stimulated with different concentrations of ATP for 30min and the uptake of DAPI on naïve,centralmemory(CM),effectormemory(EM),effectorsandTEMRACD4(A)andCD8(B)Tcellswasmonitoredby flow cytometry.White circles represent thewhole population of CD4 and CD8 T cells. Data (MFI; n = 2donors;D1,D2)arerepresentativeofsixhealthydonors.
Theexpressionof theATP-metabolisingenzymeCD39 inTregs isgeneticallydetermined, therefore
somedonorshaveahighproportionofCD39+Tregs (>40%)while insomeothers less than20%of
Tregs express CD39 (A. Rissiek et al., 2015). Moreover, CD39 is an activation marker for T cells
(Maliszewski et al., 1994). Therefore, we compared the sensitivity of Tregs expressing CD39 with
thosenotexpressing theectoenzyme in response toATP (Figure40), andobserved, thatactivated
Tregs(CD39+Tregs)reactstronglytoATPthannon-activatedTregs(CD39-Tregs)(Figure40).
Figure 40. CD39+ Tregs exhibit higher sensitivity to ATP-induced pore formation than CD39- Tregs cells.HumanPBMCswere stimulatedwith different concentrations ofATP for 30min and the uptakeofDAPI onCD39+ (blue) and CD39- Tregs (green) was monitored by flow cytometry. Data are represented by Medianfluorescenceintensity(MFI)(A)orMFI±SD(B).Dataarerepresentativeofone(A)orsix(B)healthydonors.PvaluesweredeterminedbypairedStudent’sttestforthecomparisonoftwogroupsandpairedRMone-wayANOVAfollowedbyBonferronipost-testforthecomparisonofthreeormoregroups:*=<0.05;**=<0.01.
Theseresults indicatethatactivatedandeffectorTcellsexhibithighersensitivitytoATPthantheir
naïveandnon-effectorcounterparts.
4.2.15 BlockadeoftheP2X7receptordoesnotaffectactivationorproliferationofTcells
invitro
Uptonow,ourexperimentsdescribedtheimmediateconsequencesofP2X7-activationinresponse
toATP.Thenextobviousaimwastodeterminethe influenceofprolongedactivationofP2X7onT
cell responses. It is known that high concentrations of ATP induce P2X7-mediated cell death of
murine T cells (Yoonet al., 2007). In contrast,when low concentrations of ATP are present, P2X7
promotes the activation and proliferation of T cells through P2X7 signalling (Adinolfi et al., 2005;
Schenket al., 2008; Yipet al., 2009). Thus,we first investigatedwhether blockadeof P2X7would
ATP[mM] ATP[mM]
DAPIuptake[M
FI]
DAPIuptake[M
FI]
A B
0 0.5 1.5 4.51×10²
1×10³
1×10⁴
1×10⁵
*
*
**
Tregs CD39+
Tregs CD39-
0
3×10³
6×10³
9×10³
0.1 1 10
90
havean impactonTcellactivation.Forthis,westimulatedhumanPBMCswithαCD3antibodies in
thepresenceofDano1oracontrolNb,andthenassessedtheexpressionoftheactivationmarkers
CD25andCD69.ThegatingstrategyfortheidentificationofCD4andCD8TcellsisshowninFigure
41A.
WedetectedhighlevelsofCD25andlowerlevelsofCD69onthesurfaceofCD4andCD8Tcellsfour
days after stimulation (Figure41B,D).CD25expression increasedprogressively over the courseof
time, reaching thehighest levels 96hours after stimulation (Figure41C,E). In contrast, CD69was
upregulated one day after stimulation, and started to decrease after 72 hours (Figure 41C, E).
BlockadeofP2X7byDano1didnotimpairorincreaseactivationofTcells(Figure41C,E).
Figure41.Dano1doesnotaffect activationofhumanT cells in vitro.HumanPBMCswere stimulatedwithαCD3antibodies(0.5µg/ml)intheabsenceorpresenceof100nMofDano1oracontrolNb(anti-mARTC2.2).TcellactivationwasdeterminedbytheexpressionofCD25andCD69onthecellsurface.Nanobodieswereusedasdimers,andaddedondayzeroanddaytwo.(A)GatingstrategyfortheidentificationofCD4(CD2+CD4+)andCD8Tcells(CD2+CD8+).(BandD)ExpressionofCD25(left)andCD69(right)inCD4(B)andCD8(D)Tcells.Data(MFI)correspondtoday0(blackdottedline)anddayfour(grayfilledhistogram).(CandE)Percentage(Mean±SD,n=3)ofCD25+(left)CD69+(right)CD4(C)orCD8Tcells(E).Dataarerepresentativeofonedonorfromtwoindependentexperiments.
Asasecondapproach,weinvestigatedTcellproliferationonPBMCsbyeFluordyedilutionbyflow
cytometry(Figure42).ThegatingstrategyfortheidentificationofproliferatingCD4andCD8Tcellsis
Lymphocytes
FSC-A
SSC-A
FSC-A
FSC-H
Singlecells
CD2
Live/d
ead
LiveTcells
CD4
CD8
TCD8
TCD4
CD25
Counts
CD69
Counts
TCD4
%CD2
5+cells
%CD6
9+cells
Time(h) Time(h)
B
A
C
- Dano1 Control
- Dano1 Control
Uns-mulated
+ α-CD3
0 24 48 72 960
20
40
60
0 24 48 72 960
20
40
60
80
CD25
Counts
CD69
Counts
TCD8D E
%CD2
5+cells
%CD6
9+cells
Time(h) Time(h)0 24 48 72 96
0
20
40
60
0 24 48 72 96020406080100
Day 0 Day 4
91
showninFigure42A.StimulationwithαCD3antibodiesinducedtheproliferationofCD4andCD8T
cells(Figure42)afterthreedaysinculture.Consequently,Tcellproliferationwasalsonotaffectedby
thepresenceofDano1oracontrolNb(Figure42B,C).
Figure42.Dano1doesnotaffectproliferationofhumanTcellsinvitro.HumanPBMCswerestimulatedwithαCD3antibodies(0.5µg/ml)andATP(1.5mMor4.5mM)intheabsenceorpresenceof100nMofDano1oracontrol Nb (anti-mARTC2.2). T cell proliferation was monitored by flow cytometric analysis of eFluor dyedilution.Nanobodieswereusedasmonomers(M),dimers(D)orfusedtothealbumin-bindingnanobodyAlb8(Alb);andaddedondayzeroanddaytwo.(A)GatingstrategyfortheidentificationofCD4andCD8Tcells.(B)Overlaid histograms (MFI) represent the amount of CD4 (left) and CD8 (right) T cells in proliferation. (C)Percentageofproliferatingcells(Mean±SD,n=3).Pvaluesweredeterminedbyone-wayANOVAfollowedbyBonferronipost-test.Dataareofonerepresentativedonorofatotaloffourdonors.
Next,we testedwhetherATP-mediated activationof P2X7would influence T cell function. To this
end, we stimulated human PBMCs with αCD3 antibodies or with HDMAPP (for γδ T cells), and
assessed the upregulation of activation markers and T cell proliferation in the presence of
exogenously added ATP.While addition of 1.5mM of ATP did not alter upregulation of CD25 on
conventional T cells, it seemed to reduce activation of γδ T cells (Figure 43A). At higher
concentrationsofATP,allthreeTcellsubsetsshowedlittleactivation,likelyduetoinductionofcell
death.ThiseffectcouldbepartiallyblockedwithDano1,butonlyonconventionalTcells,andnoton
γδTcells(Figure43A),underliningthehighersensitivityofthesecellstoATP.Asimilarpicturewas
obtainedfortheupregulationofCD69(Figure43B).
A
Livecells
CD8
Live/dead
CD4
CD8
TCD8
TCD4
eFluor670
CD8
TCD8
Cellsindivision
CD4
eFluor670
Cellsindivision
TCD4
Proliferatio
n[%
]
Proliferatio
n[%
]
B CTCD4
eFluor670
Counts
eFluor670
Counts
TCD4 TCD8 TCD8
0
20
40
60
80
αCD3 --
+-
+M
+D
+Alb
+AlbNb
0
20
40
60
αCD3 --
+-
+M
+D
+Alb
+AlbNb
- + Dano1 + Control NbUns$mulated
αCD3
αCD3 + Dano1
αCD3 + Ctrl-Nb
92
Figure 43. ATP stimulation impairs T cell activation in vitro. Human PBMCs were stimulated with αCD3antibodies (0.5 µg/ml) and ATP (1.5mMor 4.5mM) in the absence or presence of 100 nM of Dano1. TheactivationofCD4(left),CD8(middle)andγδTcells(right)wasdeterminedbytheexpressionoftheactivationmarkersCD25 (A)andCD69 (B)onthecell surface.Nanobodieswereusedasmonomers,andaddedondayzeroanddaytwo.Data(Mean±SD,n=2)arerepresentativeofasingledonor.
Similarly,ATPalsoimpairedTcellproliferationinadose-dependentmanner, leadingtoacomplete
lackofproliferationuponadditionof thehighest concentrationofATP (4.5mM) (Figure44). Even
though only live cellswere included in the analysis, it is likely that the high concentration of ATP
triggers the initiation of apoptosis, so that even the cells that survive are not able to proliferate
normally.Additionally,blockadeofP2X7didnot inhibit theeffectscausedbyATP,regardlessof its
concentration(Figure44).
These data suggest that blockade of P2X7 does not have an impact on T cell activation or
proliferation in vitro. However, we cannot exclude the contribution of other P2 receptors in this
scenario.
%CD2
5+cells
Time(h)
%CD6
9+cells
Time(h)
%CD2
5+cells
Time(h)
%CD6
9+cells
Time(h)
%CD2
5+cells
Time(h)
%CD6
9+cells
Time(h)
TCD4 TCD8 TγδA
B
0 24 48 72 96 1200
20
40
60
80
100
0 24 48 72 96 1200
20
40
60
80
100
0 24 48 72 96 1200
20
40
60
80
100
0 24 48 72 96 1200
20
40
60
80
100
0 24 48 72 96 1200
20
40
60
80
100
0 24 48 72 96 1200
20
40
60
80
100
- 4.5 mM 4.5mM + Dano11.5 mM 1.5 mM + Dano1
93
Figure 44. ATP stimulation impairs T cell proliferation in vitro.Human PBMCswere stimulatedwith αCD3antibodies (0.5 µg/ml) and ATP (1.5mMor 4.5mM) in the absence or presence of 100 nM of Dano1. TheproliferationofCD4 (A), CD8 (B) andγδT cells (C)wasmonitoredby flowcytometric analysisof eFluordyedilution.Nanobodieswereusedasmonomers,andaddedondayzeroanddaytwo.Percentageofproliferatingcells(Mean±SD,n=2)arefromasingledonor.
4.2.16 P2X7doesnotprovide costimulationunder suboptimal stimulationof the TCR in
vitro
Finally, we hypothesized that P2X7 might act as a costimulatory signal under suboptimal TCR-
stimulation, especially on innate-like lymphocytes, since they respond independently of TCR
stimulation. To this end, we determined T cell activation and proliferation on human PBMCs in
response to increasing concentrations of αCD3 and ATP (Figure 45), mimicking a condition of
suboptimal stimulation.At1nMofαCD3, the stimulationof conventionalCD4T cellswasoptimal,
whileγδTcellscouldonlybefullystimulatedupon10nMofαCD3.AdditionofexogenousATP(1.5
mM) had little if any effect on T cell activation, since surface levels of CD69 and CD25 remained
similar(Figure45A,B);butresultedindiminishedTcellproliferation(Figure45C).
PresenceoftheATP-degradingenzymeapyrasealsoresultedinimpairedTcellproliferation(Figure
45C), whereas blockade of P2X7 using Dano1 did not alter ATP-induced effects (Figure 45);
suggestingthatotherP2receptorsmaycomeintoplay.
Althoughweonlyanalysedonedonor,ourresultssuggestthatprolongedinteractionofTcellswith
ATPaffectsTcellproliferationandactivationinadose-dependentmanner,regardlessofthestrength
of TCR stimulation. Furthermore, blockade of P2X7 does not seem to be sufficient to prevent the
effectsinducedbyprolongedstimulationwithATPinlong-termassays.
Proliferatio
n[%
]
Time(h)
TCD4
Time(h)
Proliferatio
n[%
]
TCD8
Proliferatio
n[%
]
Time(h)
TγδA B C
0 24 48 72 96 1200
20
40
60
80
100
0 24 48 72 96 1200
20
40
60
80
100
0 24 48 72 96 1200
20
40
60
80
100
- 4.5 mM 4.5mM + Dano11.5 mM 1.5 mM + Dano1
94
Figure45.ATPdoesnotactasacostimulatorysignalundersuboptimalstimulationoftheTCR.eFluorlabelledPBMCswerestimulatedwithdifferentconcentrationsofαCD3antibodiesandATPintheabsenceorpresenceofDano1(monomer).ThepercentageofCD69+(A),CD25+(B)orproliferating(C)CD4(upperrow)andγδTcells(bottomrow)wasdeterminedbyflowcytometry.Dataarefromasingledonor.
TCD4
A B
%CD6
9+cells
αCD3[ng/ml]
%CD6
9+cells
αCD3[ng/ml]
%CD2
5+cells
αCD3[ng/ml]
%CD2
5+cells
αCD3[ng/ml]
Proliferatio
n[%
]
αCD3[ng/ml]
Proliferatio
n[%
]
αCD3[ng/ml]
CTγδ
0.1 1 10 100 10000
20
40
60
80
100
0.1 1 10 100 10000
20
40
60
80
100
0.1 1 10 100 10000
20
40
60
80
100
0.1 1 10 100 10000
20
40
60
80
100
0.1 1 10 100 10000
20
40
60
80
100
0.1 1 10 100 10000
20
40
60
80
100
Apyrase1.5 mM + Dano11.5 mM-
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5. DISCUSSION
ATPispredominantly locatedintracellularly inphysiologicalconditions.Damagedcellsreleaselarge
quantitiesofATPtotheextracellularcompartment,whichcanbindtopurinergicP2receptorsonthe
surfaceofimmunecells,triggering,inmostofthecases,inflammatoryresponses(Faas,Sáezandde
Vos,2017).P2signallinginducesavarietyofresponses,suchasactivationandproliferationofTcells,
degranulation of neutrophils, and production of reactive oxygen species (ROS) and inflammatory
cytokines bymonocytes andmacrophages (Idzko et al., 2007; Boeynaems, Communi and Robaye,
2012a;Pettengilletal.,2013;Rodrigues,ToméandCunha,2015;CekicandLinden,2016;Danquahet
al.,2016;Amores-Iniestaetal.,2017;Cauwelsetal.,2017).AmongallP2receptors,P2X7isthemost
studied in the fieldof immunologydue to its striking contribution to inflammation.Thebindingof
ATP to P2X7 triggers the activation of the inflammasome andmaturation of the proinflammatory
cytokine IL-1β (Lister et al., 2007), which is essential for the host-response and resistance to
pathogens, but it also exacerbates damageduring chronic disease and acute tissue injury. Indeed,
P2X7isassociatedwiththepathogenesisofavarietyofinflammatorydisorders,thusattractingthe
attentionofmanyresearchersasapotentialtargetfortherapy(Mehtaetal.,2014;Burnstock,2017;
BurnstockandKnight,2018).
Inthisstudy,weidentifiedinnate-likelymphocytes(ILLs)asthecelltypeexpressingthehighestlevels
of P2X7 and reactingmore promptly to ATP among all T cell subsets. Furthermore, we show the
potentialof theP2X7-specificnanobodyDano1asapromisingdrugcandidate for thetreatmentof
humandisease.
5.1 P2X7EXPRESSIONANDFUNCTIONINHUMANIMMUNECELLS
Wefirst setout toassess theexpressionofP2X7on immunecell typesusingananobody (Dano1)
thatrecognizeshumanP2X7withhighspecificity(Danquahetal.,2016).ThelowexpressionofP2X7
in several immune cell subtypes still required the use of a stringent negative control in order to
ensurethespecificityoftheP2X7signal.
P2X7 isvirtuallyexpressedbyall immunecellpopulationsandmost tissues in thebody (Burnstock
andKnight,2004;Sperlaghetal.,2006;Welter-Stahletal.,2009;Bartlett,StokesandSluyter,2014;
Idzko, Ferrari and Eltzschig, 2014). Within granulocytes, human eosinophils (Ferrari, Idzko, et al.,
2000)andmastcells(Warehametal.,2009)expressP2X7.Theexpressionofafunctionalreceptorin
neutrophils is still controversial (Martel-Gallegos et al., 2010). Although there are no studies
reportingitsexpressioninhumanbasophils,P2X7isexpressedbybonemarrow-derivedbasophilsin
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mice(Tsaietal.,2015).DataarisingfromourlabshowslittleifanyexpressionofP2X7inthesurface
ofall granulocyte subtypes (datanot shown),and thediscrepancycouldbedue toa falsepositive
signalarisingfromthehighautofluorescencesignalcommontoallgranulocytes.Inagreementwith
publisheddata,weobservedthehighestexpressionofP2X7inhumanmonocytes,bothonthecell
surface and at mRNA level. We detected lower expression of P2X7 in non-classical monocytes
(CD14dim CD16+) compared to the classical (CD14+ CD16-) and intermediate (CD14+ CD16+) subsets.
Non-classicalmonocytes release loweramountsof IL-1βafterexposure toLPSorLPS + bzATP than
classical and intermediatemonocytes due to a higher degradation rate of the pro-IL-1β transcript
(Hadadietal.,2016).WepostulatethatthelowerexpressionofP2X7onthissubsetmightcontribute
tolessefficientprocessingofpro-IL-1βtoactiveIL-1β,suggestingacorrelationbetweenexpression
andfunctionofP2X7.
WhilemurinelymphocyteshavebeencharacterizedintermsofP2X7expression,verylittleisknown
abouttheexpressionofP2X7inhumanlymphocytes.AccordingtoGuandcolleagues,humanNK,T
and B cells show similar expression of P2X7; low on the cell membrane, but much higher
intracellularly(Guetal.,2000).Inourhands,NKcellsexpressthehighestlevelsofP2X7,bothonthe
surface and at themRNA level. In contrast, P2X7 is functional on B cells even though it is barely
detectableat thecell surface (Guetal.,2000;Pupovacetal.,2015).Thecontroversialdata in this
field might be explained by the usage of different reagents targeting the human P2X7R. The
monoclonalantibody(mAb)´L4´isthemostwidelyusedantibodyforthedetectionofhumanP2X7
by flow cytometry. MAb L4 partially blocks P2X7 function in human monocytes, while Dano1
completelyblocksitsfunctioninvitro(G.Buelletal.,1998;Danquahetal.,2016),whichmightbea
consequenceofbindingtodifferentepitopes(Danquah,unpublished).
ThereisgeneralconsensusthathumanTcellsexpressP2X7(Budagianetal.,2003;SluyterandWiley,
2014),however,whetherdistinctTcellsubsetsexhibitdifferentlevelsofP2X7hasnotbeenreported
yet.OurdatashowadistinctpatternofP2X7expressionamonghumanTcells,being ILLsandcells
withaneffectorphenotypetheonesexpressingthehighestlevelsofthereceptor.ILLs,includingγδT
cells,mucosalassociatedinvariantTcells(MAIT)andinvariantNKTcells(iNKTs),areaclassofTcells
that exhibit properties from both innate and adaptive immunity (Vermijlen and Prinz, 2014). In
contrasttoconventionalαβTcells,ILLsdisplayahighlyrestrictedTCRrepertoire:humanMAITcells
expresstheVα7.2chainpredominantly incombinationwiththeVβ2andVβ13chains(Tilloyetal.,
1999;Leporeetal.,2014),whilehumaniNKTscombinetheVα24-Jα18preferentiallywiththeVβ11
chain(Dellabonaetal.,1994).Duetotheirlowfrequencyinhumanperipheralblood,iNKTcellswere
notincludedinthiswork(Montoyaetal.,2007).γδTcellsareuniqueinthattheyrearrangetheTCR
γandTCRδchainsinsteadoftheclassicalTCRαandTCRβchains,beingmostofγδTcellscirculating
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in blood Vγ9δ2+ cells. ILLs are not restricted by classicalMHC class I or IImolecules, but by non-
classical MHC molecules instead. MR1 molecules present vitamin B metabolites to MAIT cells.
Antigen presentation to iNKT cells ismostlymediated by CD1dmolecules;whileMICA,MICB and
butyrophilins present antigen to γδ T cells (Harly et al., 2012; Prinz, Silva-Santos and Pennington,
2013;Reantragoonetal.,2013;Adams,GuandLuoma,2015).Remarkably,ILLsrespondrapidlyupon
antigenencounterandthereforebecomeactivatedattheearlystagesofinfections.
Inmice,TcellsexpressinghigherlevelsofP2X7exhibithighersensitivitytoP2X7-inducedcelldeath
andP2X7-dependentsheddingofCD62Lfromthecellsurface(AswadandDennert,2006).However,
Safya and colleagues have recently reported that T cell sensitivity toATP does not strictly rely on
P2X7expressionbut rather dependson the activation anddifferentiation statusof conventional T
cells.Bothactivated(CD69+)naïveandmemoryTcellsexhibithigherexposureofphosphatidylserine
(PS) and pore formation in response to ATP than their non-activated (CD69-) counterparts. In
contrast, non-activated T cells exhibit higher sensitivity to ATP-induced shedding of CD62L than
activatedTcells;underliningdissimilitudebetweenthedifferentP2X7-mediatedcellularresponsesin
distinctTcellsubsets(Safyaetal.,2018).WeobservedacorrelationbetweenP2X7expressionand
sensitivitytoATP inhumanTcells.Thus, ILLsdonotonlyshowthehighestexpressionofP2X7but
also exhibit the highest sensitivity to ATP within the T cell compartment. We also observed a
correlationbetweenTcellactivationstatusandsensitivitytoATP,beingeffectorconventionalTcells
more sensitive to ATP than naïve T cells.We also report upregulation of P2RX7 expression upon
activation, especially in γδ T cells. The higher expression and sensitivity of P2X7 in T cellswith an
effector profile might serve as a “checkpoint” to limit excessive inflammatory responses. Higher
expression of P2X7 in activated T cells would also support the role of P2X7 as a regulatory
mechanismto inducecelldeathofTcells,whenastrongeffectorresponse isno longernecessary.
Indeed,P2X7-inducedcelldeathofeffector intestinalCD4T cells isdeterminant for the controlof
experimentalcolitis(Hashimoto-Hilletal.,2017),andgeneticablationofP2X7worsensexperimental
autoimmune encephalomyelitis (EAE) by impairing apoptosis of activated lymphocytes (Chen and
Brosnan,2006);highlightingthecontributionofP2X7inlimitingeffectorTcellresponses.
P2X7-mediatedcelldeathofimmunecellsoccursathigh(millimolar)concentrationsofATP(Yoonet
al.,2007),compatiblewithaninflammatorymilieu.ExtracellularNAD+(eNAD+)canalsoactivateP2X7
inmice; and just thebrief exposure to low concentrationsofNAD+ induces cell deathof sensitive
murineTcellsubtypes,especiallyTregsandiNKTcells(Semanetal.,2003;Hubertetal.,2010;Rissiek
etal.,2014). Instead, lowconcentrationsofATPpromotetheactivationandproliferationofTcells
throughP2X7 signalling (Baricordietal., 1999;Adinolfietal., 2005; Schenketal., 2008; Yipetal.,
2009).AhigherexpressionofP2X7couldimplystrongercontributionofP2X7duringTCRactivation.
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ILLscanbeactivatedinaTCR-independentmannerviastimulationofinnatereceptors(NKreceptors,
TLRs) or through cytokine signalling (Martin et al., 2009; Sutton et al., 2009; Reilly, Wands and
Brossay, 2010; Brennan, Brigl and Brenner, 2013; Ussher et al., 2014; Slichter et al., 2016). We
speculatethatP2X7signallingcouldactasacofactorundersuboptimalstimulationof theTCRand
modulatethefunctionofTcellsduringinflammation,especiallyofILLs.
ExtracellularATP(eATP)doesnotonlyinducetheactivationofP2X7,butalsoofallP2Xandseveral
P2Y receptors,namelyP2Y1,P2Y24,P2Y11andP2Y13 (CekicandLinden,2016). SinceP2X7 shows
thelowestaffinityforATPamongallP2receptors,wehaveusedhighconcentrationsofATPinthis
study,whichmighthaveactivatedotherP2receptors.Additionally,eATPisexposedtotheactivityof
ectonucleotidases, such as CD39 and CD73, which mediate its hydrolysis into adenosine (Ado); a
moleculethat inducesimmunosuppressivefunctionsonimmunecellsuponbindingtoP1receptors
(Haskó et al., 2008). Thus, different concentrations of ATP and its downstream metabolites may
triggercontrastingresponsesthroughsignallingviadifferentreceptors (Idzko,FerrariandEltzschig,
2014; Spaans et al., 2014), revealing the complex network of purinergic signalling.We report that
specificblockingofP2X7doesnotimpairproliferationoractivationofhumanCD4andCD8Tcellsin
vitro.SincephysiologicalconcentrationsofATP(1-50nM)donotinducetheproliferationofactivated
CD4 T cells (Trabanelli et al., 2012), we attributed the lack of effect in our assays to insufficient
releaseofATPbyTcells,andthereforetheinabilitytoactivateP2X7;althoughwedidnotmeasure
the concentration of ATP present in the media. Upon TCR engagement, both P2X1 and P2X4
receptorstranslocatetotheimmunesynapseandbindpericellulareATP.EngagementofATPinduces
Ca2+entry,activationofnuclearfactorsofactivatedTcells(NFATs)andsynthesisof IL-2;whichare
crucialdownstreamresponses for theactivationandproliferationofTcells. InhibitionofP2X1and
P2X4 receptorsprevents theuptakeofCa2+ anddiminishes the synthesisof IL-2. Theseeffectsare
moreprominentwhenusingnon-selectiveP2antagonistsorP2X4-specificantagoniststhaninhibitors
of P2X1 or P2X7 (Schenk et al., 2008; Woehrle et al., 2010). Ledderose and colleagues recently
showedthatnaïveTcellsreleaseATPinresponsetothechemokineSDF-1α,andthatATPbindingto
P2X4contributestothemigrationofhumanandmurineTcells(Ledderoseetal.,2018),underlining
theroleofP2X4inTcellbiology.ItisthenplausiblethatP2X7blockadedidnotaltertheproliferation
oractivationofT cells inourexperimentsdue to thecompensatoryeffectofotherP2X receptors,
mostlikelyP2X4.StimulationofTcellswithhighdosesofATPprominentlydecreasedtheactivation
andproliferationofTcells,probablyduetoATP-inducedcelldeathofTcells.Inlinewithourfindings,
stimulationofactivatedCD4TcellswithahighconcentrationofATP(1mM),butstilllowerthanthe
concentrationsusedinourstudy,impairsTcellproliferation(Trabanellietal.,2012).Thesamestudy
showedthatstimulationofactivatedCD4Tcellswith250nMATP inducesTcellproliferation,and
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thatthiseffectwascounteractedupondegradationofATPbyapyrase.Weobservedthatblockadeof
P2X7wasnotsufficienttoinhibittheeffectsofhighconcentrationsofATPonTcellproliferationand
activation, againunderlining thepromiscuoususeofP2 receptors.Whether the inhibitionofP2X7
upon stimulationwith lowor intermediate concentrations of ATPwould prevent the activation or
proliferationofTcellsneedstobefurtherinvestigated.
Activationofmetalloproteinasesandsubsequentsheddingofsusceptiblemembraneglycoproteins,
andtoalesserextentporeformationoccurrelativelyshortlyafterP2X7activation.Incontrasttothe
lackofeffect seenon long-termassays likeT cellproliferation,weobserved thatblockingofP2X7
efficiently inhibitedsheddingofCD62Landpore formationon immunecellsuponstimulationwith
ATP.Inlong-termassays,ATPisproduced,metabolisedanditengages,inasustainedorintermittent
manner,withdifferentP2receptors.BlockingP2X7withDano1doesnotexcludethecontributionof
ATP and its metabolites to other purinergic receptors (Cekic and Linden, 2016). For instance,
activationoftheP2Y11receptorimpairsP2X7-mediatedporeformationinducedbyeATP;butdoes
not affect the influx of Ca2+ (Dreisig et al., 2018). Thus, studying theproximal effects immediately
after P2X7 activation, such as influx of Ca2+ ions,wouldmarkedly reduce prolonged interaction of
ATPwith other P2 receptors. Usingmulticolour flow cytometry,we could still distinguish the Ca2+
signallingondifferentimmunecellsubsets,andclarifywhetherhighersensitivityofP2X7toATPon
ILLsoccursimmediatelyorrequiresalongertimeofinteraction.Alternatively,toelucidatetheroleof
asinglereceptorsuchasP2X7wouldrequiretheuseofspecificinhibitorsforallotherP2receptors.
TheuseofaP2X7-specificagonistorthegenerationofP2X7-specificNbsthatpotentiatethefunction
ofhumanP2X7,asit isthecaseoftheNb14D5inmice(Danquahetal.,2016),wouldalsoprovide
valuableinformationaboutwhatactuallyoccursafterexclusivelyactivatingP2X7.Finally,itshouldbe
takenintoaccountthatthesedataarisefrom invitrostudies,andmightnotcompletelymirrorthe
concentrationsorinteractionsthatoccurinvivo.
5.2 DISCREPANCIESBETWEENHUMANANDMURINEP2X7
The murine P2X7 receptor shares 80% sequence identity to the human P2X7 receptor (Bartlett,
Stokes and Sluyter, 2014). However, several discrepancies exist between the two species. For
instance, ATP is the only purine nucleotide able to induce P2X7 activation in humans,whereas in
mice, theecto–ADP-ribosyltransferaseARTC2.2canalso trigger itsactivation in response toeNAD+
(Seman et al., 2003; Adriouch et al., 2008). The relevance of this pathway in humans is not yet
completely understood, since a premature stop codon in the ART2 pseudogene prevents its
expressioninhumancells(Haagetal.,1994)and,consequently,NAD+doesnotinduceapoptosisof
human Tregs or PBMCs (Cortés-Garcia et al., 2016). Four additional variants of ART (ART1, ART3,
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ART4 and ART5) exist in humans (Glowacki et al., 2009). Within the immune system, ART1 is
expressed in lymphocytes, monocytes and granulocytes (Grahnert et al., 2002; Koch-Nolte et al.,
2006;Cortés-Garciaetal., 2016).ART3 is expressed indifferent tissues, suchasmuscleandbrain;
whileART4 ishighlyexpressedbyerythrocytesandslightlyexpressedbymonocytes(Koch-Nolteet
al.,2006).ART5isfoundinsimilartissuesasART3(Koch-Nolteetal.,2006),but isalsodetected in
lymphocytes(Cortés-Garciaetal.,2016).Fromthesevariants,however,onlyART1andART5exhibit
ADP-ribosyltransferaseactivity(Koch-Nolteetal.,2006).SinceART5lackstheC-terminalGPIanchor
andonlyexists insolubleform(Glowackietal.,2001); it isgenerallybelievedthatART1istheonly
one able to parallel the ribosyltransferase functions of murine ARTC2.2 (Koch-Nolte et al., 2006;
Cortés-Garciaetal.,2016).
GeneticvariationsaffectboththehumanandmurineP2X7,leadingtodifferencesintheexpression,
sensitivity and function of P2X7 (Wiley et al., 2003; Bartlett, Stokes and Sluyter, 2014). A distinct
allelicvariant(P451L)resultinginthesubstitutionofaprolinebyaleucineintheC-terminalcytosolic
tailofP2X7leadstolowersensitivitytoATP.BothC57BL/6andDBAmice,amongotherstrains,carry
the 451Lmutation and exhibit lower sensitivity to ATP thanmany other strains ofmice, such as
BALB/candnon-obesediabetic (NOD)mice (Adriouchetal., 2002).Additionally, different immune
cell types express different P2X7 splice variants. Murine T cells preferentially express the highly
sensitive P2X7 variant (P2X7k), whereas macrophages predominantly express the classical P2X7
variant(P2X7a)(Schwarzetal.,2012).Ofnote,eNAD+onlyinducesARTC2.2-mediatedactivationof
theP2X7kvariant(Schwarzetal.,2012).ThelowersensitivityofTcellsfromC57BL/6micemightalso
be attributed to a lower expression of P2X7 on the cell surface (Hubert et al., 2010). The
identificationofdifferentsplicevariants revealed that twodifferentstrainsofP2X7-deficientmice,
namely theGSK (GlaxoSmithKline) andPfizer lines, arenot complete knockouts for P2X7 (Bartlett,
StokesandSluyter,2014).SincetheP2X7kvariantoriginatesfromanovelexon1,TcellsfromGSK
miceexpressafullyfunctionalP2X7receptor(Nickeetal.,2009). InPfizermice,bothP2X713band
P2X71c splice variants escaped genetic deletion, although in this particular case, P2X7 is not
functionalon immunecells (SimonR J Tayloretal., 2009). It isnot knownwhetherananalogous
splice variant to the highly sensitive P2X7K also exists in humans. Polymorphisms in the human
P2RX7 gene can also lead to a loss-or-gain of function of P2X7. To date, three different gain-of-
function (GOF) SNPs (H155Y, H270R, and A348T) and several loss-of-function (LOF) SNPs (V76A,
G150R,R276H,R307Q,T357S,E496A,I568N)havebeenidentified(Cabrinietal.,2005;Fulleretal.,
2009; Stokesetal., 2010;Bartlett, Stokes andSluyter, 2014;Caseleyetal., 2014;DiVirgilioetal.,
2017). Someother SNPs, such as theQ460R, do not lead to significant changes in the functionof
P2X7 per se (Roger et al., 2010); but when co inherited with other GOF SNPs, generate GOF
101
haplotypes (Cabrini et al., 2005; Barden et al., 2006; Stokes et al., 2010). Many of these
polymorphismsareassociatedwithsusceptibilitytoinfection,suchastoxoplasmosis(Jamiesonetal.,
2010)andtuberculosis(Fernandoetal.,2007);andalsowiththepathogenesisofnumerousdiseases,
such as rheumatoid arthritis (Al-Shukaili et al., 2011), ischemic stroke and ischemic heart disease
(Gidlöf et al., 2012), chronic pain (Sorge et al., 2012), multiple sclerosis (Gu et al., 2015) and
osteoporosis(Gartlandetal.,2012).
TheexpressionpatternofP2X7on immunecellsalsodiffersbetween the twospecies.Whileboth
speciesexhibitthehighestlevelsofP2X7onmonocytesandmacrophages,theexpressionpatternis
different within the T cell compartment. Inmice, P2X7 is highly expressed on Tregs and in iNKTs
(Hubertetal.,2010;Rissieketal.,2014);whereasweobservethehighestexpressionofP2X7onILLs
in humans. Remarkably,we found Tregs expressing very low levels of P2X7within human T cells.
MurineTregsarehighlysensitivetoP2X7-mediatedcelldeath(Rissieketal.,2014);whereasneither
ATP(1mM)norNAD(60µM)areabletoinducecelldeathorsheddingofCD62LonhumanCD39+or
CD39- Tregs in vitro (Cortés-Garcia et al., 2016). In contrast to these findings, we did observe
sheddingofCD62LandinductionofporeformationonTregsuponstimulationwithhigherdosesof
ATP (≥ 1.5 mM). Upon stimulation with ATP, murine Tregs are more sensitive to P2X7-mediated
sheddingofCD62Landpore formation than conventional T cells (Safyaetal., 2018).Wehowever
report practically identical sensitivity toATP in humanTregs and conventional CD4 T cells. Also in
contrast to what Cortés-Garcia and colleagues reported, we observed higher levels of P2X7 and
highersensitivitytoATPinCD39+Tregsthanintheirnegativecounterparts.CD39isanectoenzyme
thatconvertseATPintoADPandAMP;whichcanbefurtherdegradedtoAdobyCD73(Antonioliet
al., 2013). Degradation of ATP prevents its binding to P2 receptors and therefore exacerbated
inflammatoryresponses.SinceCD39+Tregsexhibitstronger immunosuppressiveactivity (Borsellino
etal.,2007;Herrathetal.,2014;A.Rissieketal.,2015),weexpectedlowerlevelsofP2X7onthecell
surface of this subset. In addition to its role in the catabolism of ATP, CD39 is considered an
activationmarkerforTcells(Maliszewskietal.,1994).ThehigherexpressionofP2X7onCD39+Tregs
and CD39+ conventional CD4 T cells correlates to our data showing higher expression on
effector/activated T cells.We postulate that higher expression of P2X7 could have a dual role in
humanCD39+Tcells:Duringtheonsetofinflammation,ATPconcentrationishigh,suppressingTreg
function. Towards resolution of inflammation, conventional T cells upregulate CD39 in order to
eliminateATPand facilitateadenosineproduction. LowerconcentrationsofATPcould then induce
proliferation and activation of regulatory T cells, dampening the inflammatory response, and
maintainingTregfunction(Kinseyetal.,2012;Ohtaetal.,2012).CD39+Tregsarealsomoresensitive
to ATP-induced pore formation. Safya and colleagues suggest that ATP-induced pore formation
102
activitybymurineTregs could facilitate releaseofATPand therefore its conversion intoAMPand
ultimatelyintoAdo(Safyaetal.,2018).AlthoughCD39andCD73arerarelycoexpressedinhuman
Tregs(Mandapathiletal.,2010),CD73isalsofunctionalasasolublemolecule.Thus,webelievethat
a similar release of ATP by CD39+ Tregs and subsequent conversion to Ado might also occur in
humans. Tuning theavailabilityofATP is apromising therapeuticoption tomodulate the immune
response,anddeserves further study toelucidate theconsequencesofdifferent concentrationsof
eATP in thedifferenthumanTcell types,according toexpressionofP2X7, threshold foractivation
andactivationstatusofthecells.
In addition to the high levels of ATP released in response to cell damage (Wan et al., 2016), gut
microbiotagenerateandsecrete largeamountsofATP to theextracellular compartment (Iwaseet
al.,2010;Hironakaetal.,2013).Inmice,eATPfromcommensalbacteriainducesthesecretionofIL-
6,IL-23andTGF-βbyDCs,promotingthepolarizationofTcellstowardsaTh17phenotype(Atarashi
et al., 2008). An exacerbated Th17 response is strongly associated with gut inflammation. Thus,
strategiesthatreduceeATPorblockingATP-mediatedTh17differentiationarebeneficialtocontrol
gutinflammation(Friedmanetal.,2009;Nevesetal.,2014;Wanetal.,2016;Figliuoloetal.,2017).
P2X7iswidelyexpressedthroughouttheintestineinmice,bothintheepitheliumandimmunecells
(Diezmos,BertrandandLiu,2016).MurineconventionalCD8αβandunconventionalCD8ααTcellsof
theintestinalepitheliumandlaminapropriaexpresshigheramountsofP2X7thanCD8Tcellsfrom
the periphery (Heiss et al., 2008). In contrast, we report similarly low expression of P2X7 on the
surface of intestinal T cells and peripheral immune cells in humans; althoughwe did not directly
compare theexpressionofP2X7 in intestinalandperipheralT cells fromthe samedonor.Retinoic
acid mediates the upregulation of P2X7 on the surface of murine T cells (Heiss et al., 2008;
Hashimoto-Hill et al., 2017), but we did not observe any upregulation of P2X7 on human T cells.
Nevertheless, individualswithCrohn´sdiseaseshowincreasedexpressionofP2X7inthemucosaof
thecolonandhigherlevelsofproinflammatorycytokines(Nevesetal.,2014),suggestingapotential
role of P2X7 in this disease. Thus, it would beworth investigatingwhether intestinal T cells from
patientssufferingfrominflammatorydisordersof thegastrointestinal tractexpresshigher levelsof
P2X7thanhealthyindividuals.
5.3 THEP2X7NANOBODYDANO1ASAPOTENTIALTHERAPEUTICDRUG
TheroleofP2X7 in thepathogenesisof inflammatorydisorders ismainlydrivenbythematuration
and subsequent release of the proinflammatory cytokine IL-1β (Ferrari et al., 2006; Baroni et al.,
2007;Gicqueletal.,2015;Giulianietal.,2017).Experimentalanimalmodelsareextremelyusefulfor
testingthesafetyandefficacyofnewlygenerateddrugs,aswellasforunderstandingthemolecular
103
mechanismsofthetherapy.SinceP2X7clearlycontributestoexacerbatedinflammatoryresponses,
the blockade or genetic ablation of P2X7 in animal models results in amelioration of the
symptomatology and/or increased survival of the mice (see Table 2). Still, drug effectivity in
experimentalmodelsdoesnot implyasimilaroutcome inhumanclinical trials.TheP2X7 inhibitors
AZD9056(NCT00520572)andCE-224,535(NCT00628095)didnotdemonstrateanyadditionalbenefit
forthetreatmentofrheumatoidarthritisinphaseIIclinicaltrials(Keystoneetal.,2012;Stocketal.,
2012). The AZD9056 inhibitor also failed to translate into benefits for patients suffering from
osteoarthritis (study code: D1522c00001) and chronic obstructive pulmonary disease (study code:
D1521C00002)(A.Stockleyetal.,2008;Arulkumaran,UnwinandTam,2011);butitdidreducepain
intensity inpatients suffering fromchronicpain (EudraCT-2005-002319-26) (Eser et al., 2015). The
reason for this discrepancy couldbedue to specific differences in theexpressionof P2X7and the
protein isoform (determined by different allelic and splice variants), which may lead to different
sensitivitytoATPinmiceandhumans.
Nanobodies(Nbs)arehighlypromisingtoolsnotonlyforresearch,butalsofortherapy(Arezumand
etal.,2017;Bannas,HambachandKoch-Nolte,2017;Kochetal.,2017).Atpresent,eightdifferent
NbsarebeingtestedinclinicaltrialsbythepharmaceuticalcompanyAblynx(Ghent,Belgium).From
these,NbsagainsttheIL-6RandagainstthevonWillebrandfactorshowedpromisingandbeneficial
effectsinphaseIIclinicaltrialsagainstrheumatoidarthritis(NCT01284569)(VanRoyetal.,2015)and
acquiredthromboticthrombocytopenicpurpura(NCT01020383)(Peyvandietal.,2016);underlining
thepotentialofNbsforthetreatmentofinflammatorydiseases.
OwingtotheirsmallsizeandthelengthandflexibilityoftheirCDR3region,Nbsareabletobindto
epitopesthatarenormally inaccessibletoconventionalantibodies(Abs) (Muyldermans,2013).Nbs
induce weak immunogenicity due to their short-half life and a high sequence homology with the
human variable VH domain (Unciti-Broceta et al., 2013), and they can be genetically optimized to
further reduce its immunogenicity (Vincke et al., 2009). Furthermore, the fusion of target-specific
NbstootherNbswithadifferentspecificityand/ortospecificproteindomainsenablestargetingtoa
specific tissueor results in increasedhalf-lifeof theNb (Wesolowskietal.,2009;Farringtonetal.,
2014).Indeed,injectionofananobodyagainstP2X7amelioratedinflammationinglomerulonephritis
andallergiccontactdermatitisinmice(Danquahetal.,2016).
Monocytes, macrophages, DCs and mast cells produce IL-1β, which is a pivotal mediator in
inflammation(RenandTorres,2009).Differentfromothercytokines,whichareproducedasmature
formsandreleased through theclassical secretorypathway, IL-1β isproducedasaprecursor (pro-
IL1β) upon TLR stimulation andbecomes active after cleavageof theprecursor formby caspase-1
(Garlanda, Dinarello and Mantovani, 2013). Both the processing and release of IL-1β are tightly
104
regulated in order to avoid exacerbated inflammation (Charles A. Dinarello, 2011). Signals that
induce the processing of pro-IL1β include nigericin, ATP, flagelin, uric acid and silica crystals, and
aluminium hydroxide, among others (Charles A Dinarello, 2011; Cullen et al., 2015). These
compounds triggervariouscellular signals,although theeffluxofK+ ions seems tobe theoriginof
inflammasomeactivation(Muñoz-Planilloetal.,2013).ProcessingofIL-1βalsooccursindependently
ofcaspase-1.Cellularstress,dectin-1oractivationofFasreceptorscanalso inducethecleavageof
pro-IL1β through activation of caspase-8. Alternatively, proteases released from neutrophils and
mast cells during inflammation can also mediate the processing of pro-IL1β in the extracellular
compartment(CharlesADinarello,2011;Afoninaetal.,2015).
High concentrationsofATPaccelerate the conversionof theprecursor into active IL-1β (CharlesA
Dinarello,2011).ActivationofP2X7andoverproductionofIL-1βmediateexacerbatedinflammatory
responses and are implicated in the pathology of many inflammatory disorders and autoimmune
diseases, such as rheumatoid arthritis, chronic andneuropathic pain, inflammatory bowel disease,
cardiovascular diseases, acute lung injury, lung inflammation and fibrosis, experimental
glomerulonephritis,experimentalautoimmuneencephalomyelitis,multiplesclerosis,type1diabetes
(T1D),allergiccontactdermatitisandcancer (Labasietal.,2002;Chesselletal.,2005;Sharpetal.,
2008;S.R.J.Tayloretal.,2009;Riteauetal.,2010;Cocciaetal.,2012;H.Wangetal.,2015;Vieiraet
al., 2016; Danquahet al., 2016; Di Virgilioet al., 2017; Lin and Edelson, 2017; Chenet al., 2018).
(CharlesADinarello,2011). Inthisthesis,wetestedthepotentialofDano1formodulatinghuman
immunecellsanddemonstratedthespecificityofDano1for theblockingofP2X7 function invitro.
Using a surrogate inflammation model with LPS, we showed that Dano1 efficiently blocks the
assemblyofthe inflammasomeinperipheralmonocytes,aswellasthereleaseofproinflammatory
IL-1β.Dano1exhibitedmuchhigherpotencythanthesmall-moleculeantagonistsJNJ47965567and
AZ10606120, both currently in preclinical development.We confirmed the specificity of Dano1 in
P2X7-induced inflammasome activation, since blockade of P2X7 had no effect in a patient with
constitutiveactivationoftheinflammasome.
IL-1β contributes to the conversion of naïve CD4 T cells towards the proinflammatory Th17
phenotype (Chung et al., 2009; Lasigliè et al., 2011). The engagement of ATP to P2X receptors,
especiallyP2X7,promotespolarizationofmurineTcellstowardstheTh1andTh17helperphenotype
(Atarashietal.,2008;Schenketal.,2011;Purvisetal.,2014;Sallesetal.,2017),whileinhibitingtheir
conversion into CD4+ CD25+ Tregs and type 1 regulatory T cells (Tr1 cells) (Schenk et al., 2011;
Mascanfronietal.,2015).OurdatashowthatproinflammatoryTh1andTh17cellsshowhigherlevels
ofP2X7onthesurface,suggestingaroleofP2X7alsointhepolarizationofhumanTcells;although
such contribution has not been reported so far. P2X7 activation is also involved in cellular
105
metabolism (Di Virgilio et al., 2017). On the one hand, basal activation of P2X7 increases the
concentrationofCa2+ in themitochondriaandcellularenergystores inP2X7-transfectedHEKcells;
thus promoting cell growth (Adinolfi et al., 2005). Moreover, it also enhances the efficiency of
aerobic glycolysis inP2X7-expressingHEKcells (Amorosoetal., 2012), supporting cell proliferation
under glucose-deprived conditions. On the other hand, P2X7 contributes to oxidative
phosphorylationmetabolism,which isnecessary for thegenerationandfunctionofmemoryCD8T
cells(BorgesdaSilvaetal.,2018).IL-1βandIL-23aresufficienttoinduceandmaintainanadequate
rate of aerobic glycolysis needed to support the metabolic needs for Th17 differentiation. It is
plausible that P2X7 may contribute to the shifting towards the glycolytic pathway and promote
polarizationof T cells towards certain Thphenotypes, namely theproinflammatory Th1 andTh17;
althoughthespecificconditionsthatinfluenceitscontributiontoaspecificmetabolicprogramming
havenotyetbeenidentified.
AlthoughwecouldnotelucidatethespecificfunctionofP2X7onTcells,boththecontributionofIL-
1βinTh17polarizationandthehigherexpressionofP2X7inseveraleffectorTcellsubtypesindicate
thatP2X7couldalsobeapotentialtargetforT-celldrivendiseases.Th17cellsplayacriticalrolein
thepathogenesisofautoimmunediseases(Tesmeretal.,2008;Jadidi-NiaraghandMirshafiey,2011;
Hanetal.,2015).Dano1couldbeasuitabledrugforthetreatmentofdiseasesmediatedbyTh1or
Th17cells,suchasrheumatoidarthritis,multiplesclerosis,psoriasisorinflammatoryboweldisease;
notonlyby reducing IL-1βproductionbutalsobydirectlymodulating the functionofTcells.Since
P2X7-mediatedcelldeathofmurineTregsfavoursinflammation(DiVirgilioetal.,2017;Figliuoloet
al.,2017),andthepresenceofATPinhibitstheconversionofmurineTh17cellsintoIL-10producing
cellsinvitro(Fanetal.,2016);itisfeasiblethatblockadeofP2X7couldcounteractsucheffectsonT
cells and suppress inflammation in vivo. Nonetheless, due to differences in the expression and
sensitivity of P2X7onmurine andhumanT cells it is first necessary to fully determine the roleof
P2X7andtheeffectofATPconcentrationondifferenthumanTcellsubsets.
SinceallelicvariantsinfluenceP2X7sensitivitytoATP(Fulleretal.,2009),acomprehensiveanalysis
of the genotype should be performed before Dano1 could go into the clinics. In this study, we
genotypedalldonorsforthreedifferentSNPsassociatedwiththesensitivitytoATP:H155Y,H270R,
and A348T. The originally published reference sequence of human P2X7 (Rassendrenet al., 1997)
contains the variant H-H-A, which completely differs from the ancestral variant (Y-R-T). All three
aminoacidsintheancestralvariantconferhighersensitivitytoATP.Theseallelicvariantsoccurata
frequency of 24.77% for 155Y, 75.23% for R270 and 32.24% for T349 in the Iberian population in
Spain; although thereareprominentdifferencesamongdifferenthumanpopulations (Gibbsetal.,
2015).Weobserved cleardifferences in themagnitudeof the response toATP stimulationamong
106
donors. Nevertheless, Dano1 completely blocked P2X7 function in all donors, regardless of the
genotype.
Our group recently identified a SNP in the CD39 gene (ENTPD1) that strongly correlates with the
expressionlevelsofCD39inthesurfaceofTcells(A.Rissieketal.,2015).Inlinewiththisfinding,we
aimed to investigatewhether theexpressionofP2X7andsensitivity toATP ishigher in individuals
carrying the “high sensitive SNPs”.While levels of CD39 expression on Tregs can be predicted by
analysing a single SNP, the combination of the three GOF SNPs in the P2RX7 gene generates 36
differentviablevariants.Todate,therearenodatareportingwhetherthepresenceofjustoneofthe
aminoacids in the heterozygous form is sufficient to induce higher sensitivity, or whether the
combinationoftwoormoreaminoacidsatthedifferentpositionsisneeded.Althoughthisisavery
interestingaspect,oursamplesizewastoosmalltocontributetothistopic.Still,wedidobservethat
mostofthedonorsdifferinoneormoreaminoacidsatpositions155,270and348(seeTable16).
We believe that once the influence of each of three GOF SNPs in the P2RX7 gene has been
elucidated, it would be worth investigating whether there is a correlation between the different
genotypesoftheENTPD1andP2RX7genesinhumans;forinstancewhetherindividualscarryingthe
SNPassociatedwithhigherexpressionofCD39wouldalsocarryaP2X7variantassociatedwithlower
expressionandsensitivityofP2X7.
107
Table16.Genotypes(SNPsH155Y,H270RandA348T)ofthedifferentdonors.
SNPs
SNPs
Donors H155Y H270R A348T Donors H155Y H270R A348T
1 YY RR AA 24 YY RR AT
2 HY HR AT 25 HY RR AT
3 HY RR AT 26 YY RR TT
4 HY RR AT 27 HY RR AT
5 HH HR AT 28 YY HR AA
6 HH HR AT 29 HY RR AA
7 HY RR TT 30 YY RR AT
8 HY RR AA 31 HY RR AT
9 HY HR AT 32 HY HR AT
10 YY RR TT 33 HY RR AA
11 HH HH AT 34 HY RR AT
12 HY HH AA 35 YY RR TT
13 YY HR AT 36 HY HR AT
14 HH HR AA 37 HH HR AT
15 HY RR TT 38 YY HR AT
16 YY HR AT 39 HY HH AA
17 HY RR TT 40 HY HR AT
18 YY HR AT 41 HH RR TT
19 HY HR AA 42 YY RR TT
20 YY RR AA 43 HH RR TT
21 HY RR AT 44 HY RR AT
22 HH RR AA 45 YY RR AT
23 HY HR AA 46 HY HR AA
108
6. ABSTRACT
Injured anddying cells release large amounts of adenosine triphosphate (ATP) to the extracellular
milieu, which is recognized as a danger signal by the immune system. Released ATP engages
purinergic receptors, such as P2X7, promoting inflammation. Extracellular ATP is exposed to
degradation, therefore ATP and its downstreammetabolitesmay trigger contrasting responses by
signalling through different purinergic receptors. P2X7 is expressed in most human tissues and
immune cells. ATP binding activates P2X7 and induces the influx of Ca2+ andNa+ and efflux of K+,
triggering distinct cellular responses depending on the cell type. Persistent stimulation by ATP
inducestheopeningofanon-selectiveporepermeabletoorganiccationicmolecules,andultimately
leadstocelldeath.Inmonocytes,P2X7activationresultsintheactivationoftheinflammasomeand
subsequent processing of the proinflammatory cytokines IL-1β and IL-18. IL-1β is pivotal for host
immune response against pathogens, but is also associated with exacerbated inflammatory
responses. Experiments performed in P2X7-deficient animal models and using pharmacological
blockers have demonstrated that P2X7 plays a key role in autoinflammatory and autoimmune
diseases,highlightingitspotentialasatargetfortherapy.
WhileinmiceP2X7isprominentlyexpressedonregulatoryTcells(Tregs)andinvariantnaturalkiller
cells (iNKTs);we found that in humanperipheral blood, innate-like lymphocytes (ILLs) express the
highestlevelsofP2X7amonglymphocytes,andarethemostsensitivetoATP.ILLsexhibitproperties
from both innate and adaptive immunity and, in contrast to conventional T cells, become rapidly
activateduponantigenencounter.ActivatedandeffectorTcellsalsoshowhigherexpressionofP2X7
andsensitivitytoATPthannaïveTcells.ThesedatasuggestaroleofP2X7asa“checkpoint”tolimit
excessive effector immune responses. We also demonstrated the high specificity of Dano1 for
blockingP2X7-mediatedcellularresponsesinallimmunecells.Dano1efficientlyinhibitedactivation
of the inflammasome and release of IL-1β in a surrogate model of inflammation with LPS.
Remarkably, Dano1 exhibited up to 2000-fold higher potency in preventing release of IL-1β than
small-moleculeantagonistscurrentlyinpreclinicaldevelopment.
In conclusion, we proved the potential of the P2X7-specific nanobody Dano1 for blocking P2X7
function inhumans,underliningDano1asapromisingdrugcandidate for the treatmentofhuman
inflammatory diseases. Since we observed higher expression of P2X7 on the surface of
proinflammatoryThelper (Th) subsets,wepostulate thatDano1couldalsobeapotential therapy
againstTh1andTh17cell-drivendiseases;notonlybyreducingIL-1βproductionbutalsobydirectly
modulatingthefunctionofTcells.
109
7. ZUSAMMENFASSUNG
Absterbende und gestresste Zellen geben große Mengen an Adenosintriphosphat (ATP) in den
extrazellulären Raum ab, welches dort vom Immunsystem als Gefahrensignal erkannt wird.
Freigesetztes ATP bindet an purinerge Rezeptoren, wie zum Beispiel P2X7, und wirkt auf diesem
Wege entzündungsfördernd. Extrazelluläres ATP kann in unterschiedliche Metabolite abgebaut
werden, die wiederum an andere purinerge Rezeptoren binden und teilweise antagonistische
Immunantworten auslösen. P2X7 wird in den meisten humanen Geweben und Immunzellen
exprimiert.ATPaktiviertP2X7undinduziertdamitdenInfluxvonCa2+undNa+,sowiedenK+efflux.
Damit werden, in Abhängigkeit vom Zelltyp verschiedene Reaktionen ausgelöst. Anhaltende
Stimulation durch ATP induziert das Öffnen von nichtselektiven Zellporen, durchgängig für
organische, kationischeMoleküle,was letztendlich zum Zelltod führt. InMonozyten vermittelt die
AktivierungvonP2X7dieAktivierungvonInflammasomenunddamiteinhergehenddieProzessierung
von entzündungsfördernden Zytokinen, wie IL-1β und IL-18. IL-1β ist entscheidend für die
Immunantwort gegen Pathogene, aber auch assoziiert mit einer Verschärfung der
Entzündungsreaktion. Experimente in P2X7-defizienten Tiermodellen und der Gebrauch von
pharmakologischenAntagonisten konnte zeigen, dass P2X7 eine Schlüsselrolle bei der Vermittlung
von autoentzündlichen Reaktionen und Autoimmunerkrankungen spielt. Diese Ergebnisse
unterstreichendieRollevonP2X7alspotentiellesZielmolekülfürTherapien.
WährendP2X7inderMausprimärvonregulatorischenT-Zellen(Tregs)undinvariantennatürlichen
Killerzellen(iNKTs)exprimiertwird,konntenwirzeigen,dassinhumanemperipherenBlutinnate-like
lymphocytes (ILLs), verglichen mit anderen Lymphozyten, die höchste P2X7-Expression aufweisen
und am empfindlichsten auf ATP reagieren. ILLs weisen sowohl Eigenschaften von Zellen des
angeborenen als auch des erworbenen Immunsystems auf. Anders als konventionelle T-Zellen
werden ILLsunverzüglichnachAntigenkontaktaktiviert.AktivierteundEffektorT-Zellen zeigten im
VergleichzunaivenT-ZellenebenfallseinehöhereP2X7-ExpressionundEmpfindlichkeitgegenüber
ATP auf. Diese Daten legen nahe, dass P2X7 als Kontrollpunkt zur Limitierung von ausufernden
Immunantworten dient.Wir konnten ebenfalls zeigen, dass Dano1, ein therapeutischerNanobody
gegenP2X7,dieP2X7-vermitteltenzellulärenAntworteninallenImmunzellenspezifischblockiert.In
einem LPS-Entzündungsmodell inhibierte Dano1 die Aktivierung von Inflammasomen und die
Ausschüttung von IL-1β sehr effizient. Bemerkenswerterweise zeigte Dano1 dabei eine
zweitausendfachhöhereWirkungbeiderInhibierungderZytokinausschüttungalsniedermolekulare
AntagonistenausderaktuellenpräklinischenEntwicklung.
110
Zusammenfassend haben wir gezeigt, dass der P2X7-spezifische Nanobody Dano1 ein hohes
Potential besitzt, die Funktion von P2X7 in humanen Zellen zu blockieren. Diese Ergebnisse
unterstreichen Dano1 als einen vielversprechenden Kandidaten für die Behandlung von
entzündlichen Erkrankungen im Menschen. Da wir eine höhere Expression von P2X7 auf der
OberflächevonentzündungsförderndenUntertypenderT-Helfer-Zellenzeigenkonnten,postulieren
wir,dassDano1auchpotentiellfürdieBehandlungvonTH1-undTH17-Zell-vermitteltenKrankheiten
therapeutisch eingesetzt werden könnte; nicht nur durch die Reduzierung der IL-1β Produktion,
sondernauchdirektdurchModulationderT-Zellfunktion.
111
8. ABBREVIATIONS
% Percent
18S 18SribosomalRNAgene
Abs Antibodies
ADA Adenosinedeaminase
ADAM Adisintegrinandmetalloprotease
ADCC Antibody-dependentcell-mediatedcytotoxicity
Ado Adenosine
ADP Adenosinediphosphate
Ag Antigen
AMP Adenosinemonophosphate
ANOVA Analysisofvariance
APCs Antigenpresentingcells
ASC Apoptosis-associatedspeck-likeproteincontainingacard
ATP Adenosinetriphosphate
BBG BrilliantBlue-G
BCR Bcellreceptor
BD BectonDickinson
BfA BrefeldinA
BSA Bovineserumalbumin
bzATP Benzoyl-ATP
Ca2+ Calciumions
CAPS Cryopyrinassociatedperiodicsyndrome
CARD Caspaseactivationandrecruitmentdomain
CCL2 Chemokine(C-Cmotif)ligand2
cDCs ConventionalDCs
cDNA Complementarydeoxyribonucleicacid
CLP Cecalligationpuncture
CLRs C-typelectinreceptors
112
CMV Cytomegalovirus
Dsegments Diversitysegments
DAMPS Danger-associatedmolecularpatterns
DAPI 4',6-diamidino-2-phenylindole
DCs Dendriticcells
ddH20 Purifiedwater
DEPC Diethylpyrocarbonate
Dim Dimer
dimAlb Linkedtothealbumin-bindingdomainnanobodyAlb8
DMAPP Dimethylallylpyrophosphate
DMSO Dimethylsulfoxide
DN Doublenegative
DNA Deoxyribonucleicacid
dNTPs Deoxyribonucleotidetriphosphate
DP Doublepositive
dsDNA DoublestrandedDNA
DTT Dithiothreitol
eATP ExtracellularATP
ELISA Enzyme-linkedimmunosorbentassay
eNAD ExtracellularNAD+
ENPPs Ectonucleotidepyrophosphatases/phosphodiesterases
ENTPDases Ectonucleosidetriphosphatediphosphohydrolases
FACS Fluorescence-activatedcellsorting
FBS Fetalbovineserum
FcR Fcreceptors
FoxP3 ForkheadBoxP3
FSC Forwardscatter
GOFSNPS Gain-of-functionSNPs
GAPDH Glyceraldehyde3-phosphatedehydrogenasegene
GATA3 GATA-bindingprotein3
113
GSK GlaxoSmithKline
H2SO4 Sulfuricacid
hcAbs Heavychainonlyantibodies
hIgG HumanIgG
HMBPP (E)-4-Hydroxy-3-methyl-but-2-enylpyrophosphate
HRP Horseradishperoxidase
IC Isotypecontrol
ICC Intracellularstaining
IELs Intraepitheliallymphocytes
IFN Interferons
IFN-γ Interferongamma
IL-18Rα IL-18receptorα
IL-6R Interleukinsixreceptor
ILCs Innatelymphoidcells
ILLs Innate-likelymphocytes
ILTs Innate-likeTcells
iNKT InvariantNKTcell
Iono Ionomycin
IPP Isopentenylpyrophosphate
Jsegments Joinsegments
K+ Potassiumions
kDa Kilodalton
KIRs Killer-cellimmunoglobulin-likereceptor
L/D Life/dead
LOFSNPs Loss-of-functionSNPs
LPLs Laminaproprialymphocytes
LPS Lipopolysaccharides
LTi Lymphoidtissueinducers
MACS Magnetic-activatedcellsorting
MAIT MucosalassociatedinvariantTcell
114
mARTC2.2 MouseARTC2.2
mFc FusedtothemouseFc.portionofIgG
MFI Medianfluorescenceintensity
Mg2+ Magnesiumions
MgSO4 Magnesiumsulfatae
MHC Majorhistocompatibilitycomplex
MICA MHCclassIpolypeptide-relatedproteinA
MICB MHCclassIpolypeptide-relatedproteinB
mM Milimols/liter
Mon Monomer
MR1 MHC-related1
mRNA MessengerRNA
Na+ Sodiumions
NAD Adeninedinucleotide
Nbs Nanobodies
NCRs Naturalcytotoxicityreceptors
NFATs NuclearfactorsofactivatedTcells
NICD NAD+-inducedcelldeath
NKcells Naturalkillercells
NKT NaturalkillerTcell
NLRs NOD-likereceptors
NOD Non-obesediabetic
oATP OxidizedATP
P1Rs PurinergicP1receptors
P2Rs PurinergicP2receptors
P2RX7 P2X7gene(human)
P2rx7KO P2rx7-defficientmice
P2X7 P2X7receptor
P2XRs PurinergicP2Xreceptors
P2YRs PurinergicP2Yreceptors
115
PAMPS Pathogen-associatedmolecularpattern
PBMCs Peripheralbloodmononuclearcells
PBS Phosphate-bufferedsaline
pDCs PlasmacytoidDCs
PFA Paraformaldehyde
PHA Phytohaemagglutinin
PI Pharmacologicalinhibition
PLD PhospholipaseD
PMA Phorbol-myristate-acetate
PMTs Photomultipliers
PRRs Patternrecognitionreceptors
PS Phosphatidylserine
PYD Pyrindomain
RA Retinoicacid
rbFc FusedtotherabbitFc.portionofIgG
RecIL-2 RecombinantIL-2
RecIL-7 RecombinantIL-7
RLRs RIG-I-likereceptors
RNA Ribonucleicacid
ROR Retinoicacidreceptorrelatedorphanreceptor
ROS Reactiveoxygenspecies
RPLA13 RibosomalproteinL13agene
RPMI Roswellparkmemorialinstitutemedium
RT Roomtemperature
SD Standarddeviation
SLO Secondarylymphoidorgans
SNP Singlenucleotidepolymorphism
SSC Sidescatter
TCD4conv. ConventionalCD4Tcells
TCR Tcellreceptor
116
TFG-β Transforminggrowthfactorbeta
Tfh FollicularhelperTcells
TFs Transcriptionfactors
Th Thelpercells
TLRs Toll-likereceptors
TMB Tetramethylbenzidine
TNF Tumornecrosisfactor
TRA Tcellreceptoralphagene(human)
TRB Tcellreceptorbetagene(human)
TRD Tcellreceptordeltagene(human)
Tregs RegulatoryTcells
TRG Tcellreceptorgammagene(human)
TRMs Tissueresidentmemorycells
T1D Type1diabetes
Tαβ αβTcells(conventionalTcells)
UDP Uridinediphosphate
UTP Uridinetriphosphate
Vsegments Variablesegments
VCC Viruscontainingcompartments.
VHH VariablebindingdomainofhcAbs
WT Wildtype
μg Microgram
μL Microliter
μM Micromoles/liter
117
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10. ACKNOWLEDGEMENTS
Well,hereIamwritingthelastsentencesofmyPhDthesis.Itishardtobelievethatthischapteris
comingtoanend.Now,itistimetothankallthepeoplewhohelpedmeduringthisperiodofmylife.
First, Iwant to thankmymentorProf.Dr.EvaTolosa forwelcomingmeaboardandgivingmethe
opportunity to do a PhD in her group. Thanks for the supervision during these years, but most
importantly,thankyouforyourhumanquality.MercipertotEva!
I alsowant to thanksProf.Dr.Med. FriedrichKoch-Nolte for providing all thenanobodies and for
yourinputwheneveritwasneeded.
ThankstoalltheIFIsfortheniceatmosphereandthenicetimeIhadattheInstitute.Specialthanks
gotoallthemembersfromtheofficeandthelabteam(Anna,Anne,Laura,Tina,Enja,Riekje,Romy,
Nora,Jolan,Manu,Kati,Laurenz),Iamgoingtomissyouall.Thanksforyourfriendship,thesupport
andgreattimes.ToAnne,thanksforbeingsohelpfulandalwaysbeingthere.TomylabmateRomy,
alsothanksforallthehelpandthemanyniceconversationsandlaughswehad.ToRiekjeandEnja,
thanks forall the laughsandcountless funnymoments in the lab.And finally, toManu, thanks for
beingsocaringsincetheveryfirstdayIstartedworkinginthelab.Again,Iamreallygratefultoallof
you.
Ofcourse,IwanttothankallmyfriendsinHamburg,speciallyCaroandRachita.Toallofyou,thanks
for making my adventure in Hamburg such a fun and a never-to-be-forgotten experience. It is
needlesstosay,butthankstoallmyfriendsbackhomeforalwaysbeingthere,despitethedistance.
Finally,butmost importantly, I thankallmy family for theirunconditional support. THANKS tomy
parentsandmysisterforalwaysbelievinginmeandencouragingmeineverysinglestepofmylife.I
can’tthankyouenoughforall.Moltesgràciesdetotcor!
Thankstoeveryone!
145
11. EIDESSTATTLICHEVERSICHERUNG
Ichversichereausdrücklich,dassichdieArbeitselbständigundohnefremdeHilfeverfasst,andereals
dievonmirangegebenenQuellenundHilfsmittelnichtbenutztunddieausdenbenutztenWerken
wörtlich oder inhaltlich entnommenen Stellen einzeln nach Ausgabe (Auflage und Jahr des
Erscheinens),BandundSeitedesbenutztenWerkeskenntlichgemachthabe.
Ferner versichere ich, dass ich dieDissertation bisher nicht einem Fachvertreter an einer anderen
Hochschule zur Überprüfung vorgelegt oder mich anderweitig um Zulassung zur Promotion
beworbenhabe.
Icherkläremicheinverstanden,dassmeineDissertationvomDekanatderMedizinischenFakultätmit
einergängigenSoftwarezurErkennungvonPlagiatenüberprüftwerdenkann.
Unterschrift:......................................................................
