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Received: 12 November 2020 | Revised: 18 February 2021 | Accepted: 27 February 2021
DOI:
ORIGINALARTICLE
Senescence-associatedβ-galactosidaserevealstheabundance
ofsenescentCD8+Tcellsinaginghumans
ínez-Zamudio1,2 |,4,5 |ThemistoklisVasilopoulos1,2,3 |
LisaGittens-Williams6 |PatriciaFitzgerald-Bocarsly4,5 |UtzHerbig1,2
1Center for Cell Signaling, Rutgers- New Jersey Medical School, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, New Jersey, USA
2DepartmentofMicrobiology,Biochemistry,andMolecularGenetics,Rutgers-NewJerseyMedicalSchool,RutgersBiomedicalandHealthSciences,Rutgers
University, Newark, New Jersey, USA
3RutgersSchoolofGraduateStudies,Rutgers-NewJerseyMedicalSchool,RutgersBiomedicalandHealthSciences,RutgersUniversity,Newark,NewJersey,
USA
4CenterforImmunityandInflammation,Rutgers-NewJerseyMedicalSchool,RutgersBiomedicalandHealthSciences,RutgersUniversity,Newark,New
Jersey, USA
5DepartmentofPathology,Immunology,andLaboratoryMedicine,Rutgers-NewJerseyMedicalSchool,RutgersBiomedicalandHealthSciences,Rutgers
University, Newark, New Jersey, USA
6DepartmentofObstetrics,GynecologyandWomen'sHealth,Rutgers-NewJerseyMedicalSchool,RutgersBiomedicalandHealthSciences,Rutgers
University, Newark, New Jersey, USA
Correspondence
UtzHerbig,CenterforCellSignaling,Abstract
Rutgers-NewJerseyMedicalSchool,Aging leads to a progressive functional decline of the immune system, rendering the
RutgersBiomedicalandHealthSciences,
RutgersUniversity,Newark,NewJersey,
,however,sincemarkers
Email:******@
thatlabelimmunecellswithclassicalfeaturesofcellularsenescenceaccuratelyand
-generationfluorogenic
National Institutes of Health, Grant/Award
Number: 5T32AI125185, R01AI026806, substrateforβ-galactosidaseandmulti-parameterflowcytometry,wedemonstrate
R01AI106125, R01CA136533 and herethatperipheralbloodmononuclearcells(PBMCs)isolatedfromhealthyhumans
R21AG067368
increasinglydisplaycellswithhighsenescence-associatedβ- galactosidase (SA- βGal)
-associatedincreaseswereob-
served in CD8+ T- cell populations, in which the fraction of cells with high SA- βGal
activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high
SA- βGal activity, but not thosewith low SA- βGalactivity,werefoundtoexhibitfea-
turesoftelomeredysfunction-inducedsenescenceandp16-mediatedsenescence,
wereimpairedintheirabilitytoproliferate,developedinvariousT-celldifferentia-
tionstates,andhadageneexpressionsignatureconsistentwiththesenescencestate
,weproposethat
senescent CD8+ T cells with classical features of cellular senescence accumulate to
levelsthataresignificantlyhigherthanpreviouslyreportedandadditionallyprovidea
ínez-.
Patricia Fitzgerald- Bocarsly and Utz Herbig shared senior authorship.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
providedtheoriginalworkisproperlycited.
© 2021 The Authors. AgingCell published by the Anatomical Society and John Wiley & Sons Ltd.
;20:e13344. | 1of15
/:.
2of15 | MARTÍNEZ-ZAMUDIOETAl.
simple yet robust method for the isolation and characterization of senescent CD8+ T
cellswithpredictivepotentialforbiologicalage.
KEYWORDS
aging,cellularsenescence,immunosenescence,lymphocytes,p16,PBMC,senescence-
associatedβ-galactosidase,Tcells,telomere
1 | INTRODUCTIONDespitedifferencesinpathwayactivation,SCsshareanum-
Cellularsenescenceisastableproliferativearrestthatisencoun-that they secrete numerous NF- kB and p38- MAPK regulated pro-
teredbymammaliancellsinresponsetoavarietyofsignalsandinflammatorycytokinesandothermolecules,collectivelycalledthe
stresses (Hernandez- Segura et al., 2018). In mammals, this response “senescence- associated secretory phenotype” or SASP (Coppe et al.,
functionstosuppresscancerdevelopmentandplaysalsoimport-2008). Although the SASP differs in composition depending on cell
antrolesduringtissuerepair,woundhealing,andembryonicdevel-type,senescence-inducingsignals,andtimeelapsedfollowingse-
opment (Rhinn et al., 2019). Although senescent cells (SCs) can be nescence induction, a primary function of the SASP is to generate a
clearedfromtissuebyadaptiveandinnateimmuneresponsesunderpro-inflammatoryenvironmentthatstimulatesanimmuneresponse
thesecircumstances,notallSCsareclearedand,consequently,(Prata et al., 2019). Another feature common to SCs is that they
accumulateinvarioustissuesduringaging(Prataetal.,2019).Inup- regulate certain heterochromatin proteins, such as macroH2A,
mousemodels,thisage-associatedaccumulationofSCshasasub-leadingtoastablerepressionofcellproliferationgenes(Zhang
stantialnegativeimpactonfitnessandhealth,asitcontributestoet al., 2005, 2007). In addition, SCs develop a greater abundance
agingandthedevelopmentofage-associateddiseases(Bakeretal.,oflysosomalcontentandareductioninlysosomalpH(Kurzetal.,
2016; Munoz- Espin & Serrano, 2014). While it is currently unclear 2000),resultinginincreasedexpressionandactivityoflysosomal
whysomeSCsevadeimmunecellclearanceandaccumulateintis-β-galactosidase;thishallmarkofSCsinparticularallowstheirdetec-
sues,itispossiblethatimmunecellsthemselvesprogressivelybe-tioninculturesandintissue,regardlessofthesenescence-inducing
comesenescentwithage,therebyincreasinglyweakeningimmunesignalorsenescencepathwayactivated(Gorgoulisetal.,2019).
responsesthatwouldotherwiseclearSCsfromagedtissue(PrataOurcurrentunderstandingofcellularsenescencestemsprimar-
etal.,2019).Thishypothesis,however,hasbeenprovenchallengingilyfromstudiesconductedusingmammalianfibroblastcultures.
totestprimarilyduetothelackofauniversalmarkerthatcaniden-Senescencepathwaysinothercelltypes,includingthoseofcircu-
tifyallsenescentmammalianimmunecellsaccuratelyandefficientlylating peripheral blood mononuclear cells (PBMCs), such as CD4+ T
(Goronzy & Weyand, 2019; Pangrazzi & Weinberger, 2020).cells, CD8+ T cells, monocytes, B cells, natural killer (NK) cells, and
Inmammaliancells,attheleasttwopathwaysactivatethese-plasmacytoiddendriticcells(pDC's),arestillincompletelyunder-
,mediatedbythetumorsuppressorstood (Vicente et al., 2016). Although subsets of PBMCs, such as
p53andcyclin-dependentkinase(CDK)inhibitorp21,primarilybe-cytotoxic CD8+ T cells, undergo replicative senescence in culture, to
comes activated in response to a persistent DNA damage response what degree they do so in vivo remains unclear (Chou & Effros, 2013;
(DDR),-Pangrazzi & Weinberger, 2020). A primary reason for this uncertainty
malsomatichumancells,orcellsthatlackdetectabletelomeraseis that specific markers used to identify senescent CD8+ T cells (Song
activity,dysfunctionaltelomeresaregeneratednotonlyduetore-et al., 2018; Xu & Larbi, 2017), such as a loss of the cell surface re-
peatedcelldivisioncyclesthatcauseprogressivetelomereerosion,ceptors CD28 and CD27 and a gain of expression of CD45RA, CD57,
butalsoasaconsequenceofgenotoxicstressesthatcausedouble-TIGIT,and/orKLRG1maynotaccuratelycharacterizeallTcellsthat
stranded DNA breaks (DSBs) in telomeric repeats (Fumagalli et al., havepermanentlylosttheabilitytoproliferateduetoacquisition
2012; Hewitt et al., 2012). A second senescence pathway is acti-ofmacromoleculardamage,upregulationofcyclin-dependentki-
vatedduetoupregulationoftheCDK4/6inhibitorp16INK4a,whichnase(CDK)inhibitors,anddevelopmentofsenescence-associated
resultsinastablepRb-- Galactosidase (SA- βGal)activity,criteriathatdefinethestateof
stressescanactivatethissenescencepathwayincertaincelltypes,cellular senescence (Gorgoulis et al., 2019). In fact, bulk CD8+ T cells
p16INK4aisalsoupregulatedintheabsenceoftelomeredysfunctionthat have lost expression of CD28 and/or that display varying levels
or a persistent DDR (Ben- Porath & Weinberg, 2005; Herbig et al., of CD45RA, CD57, TIGIT, or KLRG1 maintain the ability to proliferate
2004). Although the molecular triggers of this DDR- independent followingappropriatestimulation,whichisincompatiblewithaclas-
senescenceresponsearestilllargelyunclear,p16INK4a-mediatedse-sical senescence response (Chou & Effros, 2013; Song et al., 2018).
nescenceclearlyhasimportantphysiologicalconsequences,asitsig-Here,wereportanaccurateandefficientmethodtoquantify,
nificantlycontributestoagingandthedevelopmentofage-relatedisolate,andcharacterizelivesenescentimmunecellsfromperipheral
disorders in mammals (Munoz- Espin & Serrano, 2014).:.
MARTÍNEZ-ZAMUDIOETAl. | 3of15
that increasingly develop high levels of SA- βGalactivityinhealthytoas“older”);aroundtheage,thehumanimmunesystemisfoundto
humanswithage,uncoverpotentialcausesforcellularsenescenceexhibitage-associateddeficits(TableS1).Youngerandolderdonors
in circulating CD8+ T cells, and characterize the senescence path-werealwaysrecruitedinpairs,allowingustoprocessandanalyze
ways activated in CD8+ T cells with high SA- ,freshly isolated PBMCs from donor pairs in parallel (Figure 1a and
ourfindingsmayprovideinsightsintotherapeuticopportunitiestoFigure S2a). Cord blood, together with blood from healthy donors in
modulateT-cellsenescenceindisease,infection,,wasalsocollectedandanalyzedforthepresenceofSCs,
reasoningthatifsenescentcellsincreasinglydevelopwithadvanc-
ingageinhumans,infantcordblood,essentiallyrepresentingage0,
2 | RESULTSshould display the fewest numbers of cells with high SA- βGalactivity.
Surprisingly,age-associatedincreasesofmeanfluorescenceintensi-
| AmethodtoisolateliveSCsforsubsequentties (MFIs) of SA- βGalsignalswereobservedinmostPBMCsubsets
analysisanalyzedforsomedonorpairs,includingTlymphocytes,plasmacy-
toiddendriticcells(pDC's),naturalkiller(NK)cells,andmonocytes
OnefeaturethatiscommontoSCsisthattheydevelopincreased(Figure 1b,c and Figure S2b). Most consistent age- associated in-
SA- βGalactivity(Gorgoulisetal.,2019).ThishallmarkofSCsallowscreases in mean fluorescence intensities (MFIs) of fSA- βGal,how-
their detection using chromogenic (X- Gal) (Dimri et al., 1995) or ever, were observed only for CD8+ T cells (Figure S2b,c). In order to
fluorogenic (FDG) (Debacq- Chainiaux et al., 2009) βGalsubstrates,determine which PBMC subsets increasingly develop high SA- βGal
albeitwithlimitationsduetopoorcellpermeabilityorlackofcel-activitywithadvancingage,wequantifiedthepercentagesofcells
lularretention,,wetestedthat display increased fSA- βGalsignalintensitiesbytakingadvan-
asecond-generation,cell-permeable,andself-immobilizingfluoro-tageofthefactthatdonorsintheir20s,andoftenalsoolderdonors,
genic SA- βGal substrate (fSA- βGal) (Nakamura et al., 2017) for its had distinct populations of cells with lower and higher fSA- βGalfluo-
abilitytolabelliveSCsforprolongedperiods,
accurately analyzed, quantified, and isolated by flow cytometry. As populationsmet,designatingthepopulationwithlowersignalinten-
anticipated, incubation with fSA- βGalcausedGM21fibroblastsinsity as “fSA- βGallow”andth
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