Differential Tissue-Specific Regulation of Antiviral CD8 T-Cell Immune Responses during Chronic

23 Pages·2004·2.37 MB·English

JOURNALOFVIROLOGY,Apr.2004,p.3578–3600 Vol.78,No.7
0022-538X/04/$08.00(cid:1)0 DOI:10.1128/JVI.78.7.3578–3600.2004
Copyright©2004,AmericanSocietyforMicrobiology.AllRightsReserved.
(cid:1)
Differential Tissue-Specific Regulation of Antiviral CD8 T-Cell
Immune Responses during Chronic Viral Infection
Shenghua Zhou,†‡ Rong Ou,† Lei Huang, Graeme E. Price, and Demetrius Moskophidis*
InstituteofMolecularMedicineandGenetics,MedicalCollegeofGeorgia,Augusta,Georgia30912
Received25March2003/Accepted3December2003
The hallmarks of the immune response to viral infections are the expansion of antigen-specific CD8(cid:1)
cytotoxicTlymphocytes(CTLs)aftertheyencounterantigen-presentingcellsinthelymphoidtissuesandtheir
subsequentredistributiontononlymphoidtissuestodealwiththepathogen.Controlmechanismsexistwithin
CTL activation pathways to prevent inappropriate CTL responses against disseminating infections with a
broaddistributionofpathogeninhosttissues.Thisisdemonstratedduringoverwhelminginfectionwiththe D
noncytolyticmurinelymphocyticchoriomeningitisvirus,inwhichclonalexhaustion(anergyand/ordeletion) o
w
ofCTLspreventsimmune-mediatedpathologybutallowspersistenceofthevirus.Themechanismbywhichthe
n
immune system determines whether or not to mount a full response to such infections is unknown. Here we lo
present data showing that the initial encounter of specific CTLs with infected cells in lymphoid tissues is a
d
critical for this decision. Whether the course of the viral infection is acute or persistent for life primarily
e
depends on the degree and kinetics of CTL exhaustion in infected lymphoid tissues. Virus-driven CTL d

expansioninlymphoidtissuesresultedinthemigrationoflargequantitiesofCTLstononlymphoidtissues, f
r
where they persisted at stable levels. Surprisingly, although virus-specific CTLs were rapidly clonally ex- o
m
haustedinlymphoidtissuesunderconditionsofchronicinfection,asubstantialnumberofthemmigratedto

nonlymphoid tissues, where they retained an effector phenotype for a long time. However, these cells were h
t
unable to control the infection and progressively lost their antiviral capacities (cytotoxicity and cytokine tp
secretion) in a hierarchical manner before their eventual physical elimination. These results illustrate the :/
/
differentialtissue-specificregulationofantiviralT-cellresponsesduringchronicinfectionsandmayhelpusto jv
understandthedynamicrelationshipbetweenantigenandT-cellpopulationsinmanypersistentinfectionsin i.a
humans. s
m
.
o
r
g
A cardinal feature of the adaptive immune response to vi- undergo apoptosis, and a stable, long-lived, but numerically /

o
rusesistheactivationofspecificTcellsinthelymphoidtissues reduced memory T-cell population is established. While the
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after they encounter virally infected antigen-presenting cells massive expansion of antigen-specific T cells at the onset of A
(APCs), such as dendritic cells (DCs) (12, 17, 31). For most infectionprovidesamechanismforimprovedsurvivaloddsfor p
viral infections, CD8(cid:1) T cells form a crucial arm of the im- the host by rapid control of the pathogen, an important limi- ril
1
mune response through the actions of effector cytokines and tation to this strategy is the potentially lethal tissue damage ,
cytolysis (20, 21, 27, 69). In addition, CD4(cid:1) T cells provide thattheimmuneresponsecancause. 2
help for both CD8(cid:1) T-cell and B-cell responses (53). The Thecurrentparadigmmaintainsthattheimmunesystemis 01
activation of T cells proceeds to proliferative expansion and remarkablyflexibleandcapableofrespondinginqualitatively 9
b
differentiationintoeffectorTcellsthatarecapableofpromot- and quantitatively distinct ways to different infections, with y
ingarapidresolutionoftheinfection.Becauseinfectionswith tight regulatory mechanisms to ensure both protection and g
u
most viruses are not initiated in or confined to lymphoid tis-
minimal associated pathological consequences (82, 83). This e
sues, the initial antigen exposure and activation of specific T s
considerationisofparticularimportanceduringpersistentvi- t
cellsinlymphoidtissuesarefollowedbytheirmigrationtosites
ral infections, in which antigen-specific T cells (especially
of virus replication in nonlymphoid tissues. This migration CD8(cid:1) T cells, which play a pivotal role in the control or
facilitates a rapid protective response and is regulated by the
eradication of persistent viruses such as Epstein-Barr virus,
expression of homing and adhesion molecules such as selec-
cytomegalovirus [CMV], hepatitis B virus [HBV], hepatitis C
tins,integrins,andchemokinereceptors(9,66,71).Afterthe
virus [HCV], and human immunodeficiency virus [HIV]) (10,
initialproliferativeburst,whichproduceslargequantitiesofT
29, 45, 62, 75) fail to contain virus replication as a result of
cells with diverse subspecificities for viral peptides and clear-
different mechanisms. Evasion mechanisms, utilized to vari-
ance of the pathogen, the majority of antigen-specific T cells
able degrees by different viruses, can counteract cytotoxic T-
lymphocyte(CTL)immuneresponses,enablingavirustosur-
viveandpersistinthehost.Forexample,thefailureofCD8(cid:1)
*Corresponding author. Mailing address: Institute of Molecular
Medicine and Genetics, Medical College of Georgia, 1120 15th St., Tcellstocontrolinfectionatanearlystagecanleadtoshifts
CB-2803,Augusta,GA30912-3175.Phone:[phone].Fax:(706) inimmune-mediatedselectivepressureandtheemergenceof
721-8732.E-mail:[email protected].
T-cellescapevariantswithmutationsinthepresentedpeptides
†S.Z.andR.O.contributedequallytothiswork.
‡Present address: Department of Medicine, University of Massa- (8,57).Inaddition,theexpressionofcertainviralproteinscan
chusettsMedicalSchool,Worcester,MA01605-2324. perturbantigenprocessingandpeptidepresentation,resulting
3578
VOL.78,2004 CD8(cid:1) T-CELL DYNAMICS DURING PERSISTENT VIRAL INFECTION 3579
inimpairedT-cellrecognitionbyinfectedcells(72).Acombi- withLCMVispresentedinthisstudy,revealingacentralrole
nationofthesefactorsleadstoastateofrelativedeficiencyin for the viral load in lymphoid tissue in the induction and
theantiviralactivityofspecificCD8(cid:1)Tcells,whichisachar- maintenance of clonal exhaustion. The data strongly suggest
acteristic immunological feature in individuals suffering thatCD8(cid:1)Tcellsmaybedifferentiallyregulatedintheenvi-
chronicviralinfections.Increasingevidencesuggeststhatsuch ronments of lymphoid and nonlymphoid tissues, and the pat-
deficiencies may be a consequence of the functional dysregu- tern of T-cell exhaustion observed with mice is likely a com-
lation and/or physical deletion (clonal exhaustion) of virus- monfeatureoftheimmuneresponseduringchronicinfections
specific CD8(cid:1) T cells exposed to a high viral load or may be inhumans.
due to the selection of antigen-specific T cells with altered
functions(3,14,16,19,36,38,47,52,60,76,79,80).Thefact
that antigen-specific CD8(cid:1) T cells with a functional antiviral MATERIALSANDMETHODS
phenotypeareoftendetectableinchronicallyinfectedindivid- Animalsandvirus.C57BL/6andC57BL/6-CD4(cid:2)/(cid:2)micewerepurchasedfrom
JacksonLaboratory(BarHarbor,Maine).Micewereinfectedintravenouslywith
uals suggests that functional inactivation or physical deletion
arelativelylowdose(102PFU)orahighdose(2(cid:3)106PFU)ofLCMV-Docile
may occur at variable degrees to reduce or compromise the
toinitiateanacuteorpersistentinfection,respectively.LCMV-Docile(avariant
levelsoffunctionalvirus-specificeffectorCD8(cid:1)Tcellsindif- isolated from an LCMV-UBC carrier mouse) was obtained from C. J. Pfau
D
ferentpersistentinfections.However,theimpactofviralanti- (Troy,N.Y.)asaplaque-purifiedsecond-passagevirus(56).Virustiterswere
o
genpersistenceonthepopulationdynamicsandfateofvirus- determinedbyuseofanimmunologicalfocusassay(5). w
Isolation of lymphocyte populations from nonlymphoid tissues. Mice were n
specificTcellswithrespecttotheirpatternsofmigrationand
perfused with phosphate-buffered saline (PBS) containing heparin (75 U/ml) lo
abilitiestocontroltheinfectionwithindifferenttissuesofthe priortotissueremoval.Lungtissuesweremincedandthentreatedat37°Cfor a
infected host remains poorly characterized. In particular, it is 1hwithcollagenase(150U/ml;Gibco)inRPMIwith5%fetalcalfserum(FCS). d
e
unknown whether the kinetics and characteristics of T-cell The resulting suspension was pelleted by centrifugation, resuspended in 44% d
exhaustion (anergy and apoptotic deletion) determined for Panedrccoellnt(rPifhuagremdaactia6)0i0n(cid:3)PBgSfowri2t0hmheinpaarti2n0(°2C0.0LUym/mphl)olcayyteersewdeorenh6a7r.5ve%stePderfcroomll, fro
lymphoid tissues reflect those of the overall population of thegradientinterfaceandwashedextensivelybeforeuse.Liverorkidneytissues m
antigen-specificTcellsindifferenthosttissues.Moreover,the weremashedthrougha70-(cid:4)m-pore-sizestrainerinRPMIwith5%FCS.The h
impactofpersistingviralantigenonthefunctionalcharacter- resultingcellsuspensionswerecentrifuged,andthepelletwasresuspendedina t
t
visetricssusonfovnilryums-pspheocidifitcisCsuDes8(cid:1)anTd-icteslrleplaotpiounlashtiiopntsoifnunlycmtiopnhaolildy 36080.8%03%(cid:3)Peagrmcfoomlrlo2sno0iluummtiioncnhalstou2rpi0dp°elCe.mtoCereneltlmesdohwvaeritvhreeshdteebdplaofrroiondm(c2et0hl0lesUapn/emdllel)wtaewnreedrweceatnsrhetraeitdfeudgeexwtdeintah-t p://jv
activememoryCD8(cid:1)Tcellsgeneratedafteranacuteinfection sivelyinRPMIwith5%FCSbeforeuse. i.a
remain important but unresolved issues. An evaluation of Antibodiesandviralpeptides.AntibodieswerepurchasedfromBDPharm- s
these parameters with animal models is of particular interest ingen.PeptidesweresynthesizedattheMCGGenomicsCoreFacility(Augusta, m
becausethestudyofchronicinfectionsinhumansislimitedby Ga.) in a Perkin-Elmer Applied Biosystems (Berkeley, Calif.) 433A peptide .o
synthesizer.ThepeptidesusedforthisstudyweretheimmunodominantH-2Db- r
the inability to recover virus-specific T cells from different restricteddeterminantsforLCMV-specificCD8(cid:1)T-cellresponsesderivedfrom g/

tissues,forcingarelianceupontheanalysisofantigen-specific theviralglycoprotein(GP1)GP133-41(KAVYNFATC)ortheviralnucleopro- o
n
T-cellpopulationsrecoveredfromperipheralblood(PBL). tein NP396-404 (FQPQNGQFI). Experiments measuring virus-specific T-cell
proTpoeratidedsreosfsvtihruess-espqeuciefisctioCnDs,8(cid:1)weTecvealllusatinedditfhfeerefnutnctitsiosuneasl FfrueAnceTcptMitoo,nrsw(wThCeicrRhe)pewexhritfihobriHtms-e2adDbhb/yiGguhPs1ian3fgfi3n-t4hit1eyisofopnrtoimtDaibzff.eedcTtDheedb-bbinyinttedhrieancgrteipopenlapctoeifdmetehKnetAoTVf-YctheNlel April
duringacuteversuspersistentinfectionswithlymphocyticcho- cysteineatanchorposition9inthenaturalpeptidewithmethionine.Keyfunc- 1
,
riomeningitisvirus(LCMV).ThemurineLCMVsystemisan tional analyses were confirmed by using the natural viral peptide (KAVYN 2
excellent model for the study of the dynamics of virus-host FATC). Note that LCMV-Docile contains an amino acid change in peptide 0
GP2276-286(380N3S)derivedfromviralGP2,anotherimmunodominantde- 1
interactions during an acute or chronic infection (1, 50, 83). terminant for LCMV-specific CD8(cid:1) T-cell responses in C57BL/6 mice. This 9
Infection of mice with a relatively low dose of LCMV-Docile b
leads to a strong and broadly directed virus-specific CD8(cid:1) atombininodaHci-d2Daltbe,raantdionnosuGbPst2a-nsptieaclilfiycreCdDuc8e(cid:1)sTthceeallbsiwliteyreofdtehteecGtaPb2le27in6-t2h8e6ipnefepcttidede y g
T-cellresponsethatisreadilydetectableinthespleen,result- mice. u
ing in the efficient clearance of virus within 2 weeks after Quantitativeanalysisofvirus-specificCD8(cid:1)Tcells.Majorhistocompatibility es
infection. After resolution of the infection, the majority of complex(MHC)-peptidetetramerswerepreparedaspreviouslydescribed(2,49, t
52). Experiments utilized H-2Db tetramers complexed with the GP133-41
expandedantigen-specificCD8(cid:1)Tcellsundergoanapoptotic (KAVYNFATM) or NP396-404 (FQPQNGQFI) peptide. Single-cell suspen-
death phase, and a stable population of memory T cells is sions prepared from the spleen or from lymphocytes isolated from different
established. However, the dynamics of this process change tissues were stained with tetramers along with an antibody specific for CD8
(clone53-6.72)influorescence-activatedcellsorting(FACS)buffer(PBSwith
dramatically under conditions of infection with a relatively
1%bovineserumalbuminand0.2%sodiumazide).Afterbeingstainedfor1h
high dose of LCMV-Docile, which leads to a disseminating
at 4°C, cells were fixed in PBS containing 0.1% paraformaldehyde and were
infection. High levels of antigen stimulation due to the in- measured in a FACSCalibur flow cytometer (Becton-Dickinson, San Jose,
creasedviralloadattheonsetofinfectionresultinatransient Calif.),anddatawereanalyzedwithCellQuestsoftware.
CD8(cid:1)T-cellresponsebywhichantigen-specificCD8(cid:1)Tcells Intracellularstainingforcytokines.Splenocytesorlymphocytesisolatedfrom
tissueswereculturedin96-wellflat-bottomedplatesat106cells/wellin200(cid:4)lof
areinduced,proliferate,andinitiallyexhibitantiviralfunctions
RPMI1640(Gibco)supplementedwith10%FCSinthepresenceorabsenceof
butprogressivelylosethisability.Thelossofprotectiveimmu- theindicatedpeptideataconcentrationof1(cid:4)g/ml(49,52).Forthequantitation
nityresultsinviralpersistence(47).Suchfunctionallydeficient oftotalvirus-specificCD8(cid:1)T-cellresponses,virus-infectedDC(DC2.4,kindly
Tcellssurviveinthehostforlongperiodsbutmayeventually providedbyK.Rock,Boston,Mass.)(68)stimulatorswereusedincombination
withintracellularcytokinestaining.Effectorcells(106)wereincubatedwith4(cid:3)
be physically eliminated (52, 80, 81). A detailed analysis of
virus-specificCD8(cid:1)T-cellresponsesinlymphoidandnonlym- 105DC2.4cellsthatwereuninfectedorhadbeeninfected48hpreviouslywith
LCMVatamultiplicityofinfectionof0.5.Allstimulationreactionswereper-
phoidtissuesunderconditionsofacuteorpersistentinfection formedfor6hat37°Cinthepresenceof10Uofmurineinterleukin-2(IL-2)/well
3580 ZHOU ET AL. J.VIROL.
and1(cid:4)gofbrefeldinA(BD-Pharmingen)/well.After6hofculture,cellswere from the spleen, peripheral (axillary, para-aortic, and mesen-
harvested,washedonceinFACSbuffer,andsurfacestainedwithanantibody teric) lymph nodes (PLN), bone marrow (BM), liver, kidney,
specificformouseCD8(cid:5)(clone53-6-72).Thecellswerethenfixed,permeabil-
lung, and PBL were stained with the appropriate tetramer-
ized(Cytofix/Cytopermkit;BD-Pharmingen),andstainedforintracellularcyto-
peptidecomplex(Db/GP133-41orDb/NP396-404)andanan-
kinesbyuseofafluoresceinisothiocyanate-conjugatedantibodyagainstmurine
gammainterferon(IFN-(cid:6))(cloneXMG1.2)oraphycoerythrin-conjugatedan- tibody specific to CD8(cid:5) to identify antigen-specific CD8(cid:1) T
tibody against murine tumor necrosis factor alpha (TNF-(cid:5)) or IL-2 (clones cellswithintheentirelymphocytepopulation.Thefunctioning
MP6-XT22andJES6-5H4,respectively).Cellswerethenanalyzedbyflowcy- of virus-specific T cells was measured by staining for IFN-(cid:6)
tometryasdescribedabove.
secretionafterpeptidestimulation.
Analysis of the cytotoxic T-cell response. Direct ex vivo CTL activity was
determinedbya5-h51Crreleaseassayasdescribedpreviously(48).Lymphocytes Inagreementwithearlierfindings,theinfectionofmicewith
were incubated with peptide (GP133-41 or NP396-404)-loaded EL-4 cells or 102PFUofLCMV-Docilecausesanacuteinfectionwithinitial
virus-infectedMC57Gcellsateffector-to-target(E:T)ratiosrangingfrom50:1to viralreplicationinmultipletissues.Viraltiterspeakedbetween
200:1.E:TvaluesshownwerecorrectedforthenumberofGP133-41orNP396-
days 3 and 6, followed by a rapid decline below detectable
404tetramer-positivecellsineachepitope-specificCTLpopulationorforthe
levelsbyday20afterinfectionforalltissuesexamined(Fig.1A
numberofGP133-41andNP396-404tetramer-positivecellsinthetotalvirus-
specific CTL population. CTL precursor activity was determined with a bulk and D). The burst of virus-specific CD8(cid:1) T-cell responses in
culturesystemasdescribedpreviously(48).Briefly,lymphocyteswereprepared lymphoidtissues(spleenandPLN)focusedtowardthedomi-
fromLCMV-infectedmiceattheindicatedtimepoints.Cellswereculturedin nant GP133-41 or NP396-404 epitope accounted for almost D
i2n4f-ewcetelldpplaetreitsonfoeral5mdaacyrsopathaagedsen(1sit(cid:3)yo10f54)i(cid:3)n210m6lpoefrIswceolvlet’osgmetohdeirfiewditDhuvlibruecs-- ((cid:9)80%) the entire expanded CD8(cid:1) T-cell population. The ow
n
cuola’stemdecdeilulsmwseurepprleesmusepnetneddewditinh110m%loFfCmSinainmda1l0esUseonftimalumriendeiuILm-2w/mithl.5R%esFtiCmS- eanntsiugienng-scpoenctirfiacctCioDn8p(cid:1)haTse,ceshllos,wwnabsyfaolnlouwmeedricbayltrheedumcteiomnooryf loa
perculturewell,andserialthreefolddilutionsofeffectorcellsweretestedina phase,duringwhichepitope-specificCD8(cid:1)Tcellspersistedat d
51Crreleaseassay,usingMC57Gcellsinfectedwithvirusorpulsedwith10(cid:4)gof e
stablelevelswhileretainingafunctionalphenotype(Fig.1B,C, d
theindicatedpeptide/mlastargetcells.TheE:Tcellratiosshownwerecorrected
forthetotalnumberoftetramer-positivecellsperculturewell(priortoculture) E, and F). Consistent with several reports demonstrating the fro
by the equation E:T (cid:7) (D (cid:3) P (cid:3) L) (cid:8) N, where P is the percentage of presence of antigen-specific T cells in many tissues that may m
tetramer-positivelymphocytes,Listhetotalnumberoflymphocytesaddedto harbortheinfection(43,44,61),themigrationofvirus-specific
h
eachwellinthebulkculture,Disthedilutionfactor(10,as1/10ofthebulk T cells from lymphoid tissues through the blood to nonlym- t
culturewasusedinthefinal51Crreleaseassay),andNisthenumberoftarget tp
cellsperwell(inthiscase,104)inthe51Crreleaseassay. phoidtissuesproceededrapidly,resultinginefficientcontrolof :/
TCRaffinitymeasurementbydissociationassay.TheTCRaffinitiesofGP133- the infection. As shown in Fig. 1, the kinetics of proliferative /jv
41-andNP396-404-specificCD8(cid:1)Tcellsweremeasuredbyatetramerdissoci- expansion and memory T-cell development were similar for i.
a
ationassayasdescribedpreviously(67).Briefly,lymphocytesisolatedfromthe virus-specificCD8(cid:1)T-cellpopulationsisolatedfromlymphoid s
spleen or liver of infected mice were stained at room temperature with the m
andnonlymphoidtissues.Consistentwiththatobservedforthe
Db/GP133-41orDb/NP396-404tetramerandCD8antibody,asdescribedabove. .
spleen and PLN, an equal distribution of epitope hierarchies o
CellswerewashedtwicewithFACSbufferandincubatedoniceinthepresence r
ofsaturatingamountsofaDb-specificmonoclonalantibody(28-14-8s)toallow among virus-specific CD8(cid:1) T-cell populations was observed g/
for tetramer dissociation and binding to the Db antibody. Dissociation was fornonlymphoidtissues. o
monitoredfor0to120min.Half-lives(t1/2)weredeterminedbytheequation(ln TheinfectionofB6micewith2(cid:3)106PFUofLCMV-Docile n
2)/meanslopevalueandwereexpressedinminutes.Themeanslopeofeach A
intervalwasequivalenttothevariableln(F/F)/t,whereF isthenormalized resultedinalong-termpersistenceoftheinfectionindifferent p
afltuothreesecnendcoefatthtehientsetarvrtalo,fatnhdetinistetrhveall,enFgbtihasothfbethneorinmtearlivzaelda(6to7t)a.lfluorescence hNoPs3t9t6is-s4u0e4s-s(pFeicgi.fi2cACaDn8d(cid:1)DT).cTehlles,eixnpitainalsliyonexohfibGitPin1g33a-4f1u-nocr- ril 1
CDD8e(cid:1)pleotrioCnDo4f(cid:1)CTD-8c(cid:1)ellosruCbsDe4ts(cid:1)oTn-cdealyls1u5basefttesrininfveicvtoi.onMbicyeawneirnetrdaeppelreittoedneoafl t(isopnleaelnpahnednoPtLypNe),, bpurotcteheedierdfunracptiiodnly(IiFnN-ly(cid:6)mspehcroeidtionti)sswuaess , 20
injectionofapurifiedspecificantibody(anti-CD8antibody[YTS169]oramix- 1
progressively lost (by day 20) and the cells either persisted at 9
ture of anti-CD4 antibodies [YTS191 plus YTA3.1]), as previously described

(46). The antibody was administrated on days 15 and 17 after infection, and high levels in an anergic stage (GP133-41), as indicated by a b
treatmentwascontinuedatweeklyintervalsthroughouttheexperiment. lack of IFN-(cid:6) secretion upon restimulation, or underwent y
g
Biochemicalandhistologicalanalysisofviralhepatitis.Liverinjurywasmon- physical elimination (NP396-404). In striking contrast, a con- u
itored by immunohistochemical analysis and measurements of serum alanine e
aminotransferase(sALT)andserumaspartateaminotransferase(sAST)activity siderable fraction (10 to 20%) of virus-specific T cells that s
accordingtothemanufacturer’sprotocol(PointeScientificInc.,LincolnPark, migratedtononlymphoidtissueswithintactantiviralfunctions t
Mich.). Liver tissues harvested from infected mice were embedded in OCT retained their functional phenotypes for prolonged times.
compound(SakuraFinetek,Torrance,Calif.),snap-frozeninadryice–2-methyl- Moreover, the fate of NP396-404-specific CD8(cid:1) T cells in
butanebath,sectioned,airdried,andfixedin10%acetone.Sectionsfromeach
lymphoid tissues followed a distinct pattern compared to
tissuespecimenwerestainedwithhematoxylinandeosinandweresubjectedto
that for nonlymphoid tissues. While in the blood and lungs
grossandmicroscopicpathologicalanalyses.
the initial expansion of NP396-404-specific CD8(cid:1) T-cell
populations was followed by their rapid physical elimina-
RESULTS
tion, in other tissues (BM, liver, and kidney) they persisted
Dynamics of virus-specific CD8(cid:1) T-cell response in lym- for prolonged periods, with a fraction of them retaining
phoid versus nonlymphoid tissues during an acute or persis- functional activity. However, antigen-specific CD8(cid:1) T cells
tent LCMV infection. To examine the fate of virus-specific withafunctionalphenotypewereinefficientatviralcontrol,
CD8(cid:1)Tcellsinrelationtoviralloadsinlymphoidcompared and they eventually (by day 250) lost their antiviral proper-
tononlymphoidtissuesduringanacuteorpersistentinfection, ties and either persisted (GP133-41) or underwent deletion
wemeasuredthekineticsofviraltitersandvirus-specificCD8(cid:1) (NP396-404). An extended analysis of the Kb-restricted
T-cell responses in different tissues of B6 mice infected with GP134-43 peptide-specific CD8(cid:1) T-cell response that is
LCMV-Docile (102 or 2 (cid:3) 106 PFU). Lymphocytes isolated prominent during acute infections revealed comparable ki-
VOL.78,2004 CD8(cid:1) T-CELL DYNAMICS DURING PERSISTENT VIRAL INFECTION 3581
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FIG. 1. Persistenceofvirus-specificCD8(cid:1)Tcellsathighstablememorylevelsinnonlymphoidtissuesduringanacuteviralinfection.Analyses
wereperformedtocorrelatethekineticsofvirusreplication(AandD)withthekineticsofvirus-specificCD8(cid:1)T-cellresponses(B,C,E,andF).
C57BL/6micewereinfectedwith102PFUofLCMV-Docile,andvirustitersindifferenttissuesweremeasuredattheindicatedtimes.Datashown
aremeans(cid:10)standarderrorsofthemeans(SEM)oflog PFU/goftissuefor5to10mice.ParalleltotalnumbersofGP133-41orNP396-404
peptide-specificCD8(cid:1)Tcellsweredeterminedbystainin1g0withH-2Dbtetramers(F)ormeasuringintracellularIFN-(cid:6)(E)productionafterthe
stimulationofcellswiththeappropriatepeptide.Valueswerederivedbymultiplyingthepercentagesoftotaltetramer-positivecellsbythetotal
numbersoflymphocytesisolatedfromagiventissue.Datashownaremeans(cid:10)SEMoflog virus-specificTcellsperspleenfor5to10mice.
10
3582 ZHOU ET AL. J.VIROL.
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FIG. 2. Differentialregulationofvirus-specificCD8(cid:1)T-cellresponsesinlymphoidversusnonlymphoidtissuesduringapersistentinfection.
C57BL/6micewereinfectedwith2(cid:3)106PFUofLCMV-Docile,andvirustitersindifferenttissuesweremeasuredattheindicatedtimes(Aand
D).Datashownaremeans(cid:10)SEMoflog PFU/goftissuefor3to5mice.TotalnumbersofGP133-41orNP396-404peptide-specificCD8(cid:1)T
cellsweredeterminedbystainingwithH-120Dbtetramers(F)ormeasuringintracellularIFN-(cid:6)(E)productionafterthestimulationofcellswith
theappropriatepeptide(B,C,E,andF).Datashownaremeans(cid:10)SEMoflog virus-specificTcellsperspleenfor5to10mice.
10
netics of functional inactivation and physical elimination to tioningoftheantiviralCD8(cid:1)T-cellpopulationdiffersmark-
those observed for NP396-404 peptide-specific T cells in edly in different tissues. Lymphoid tissues provide an envi-
micewithchronicLCMVinfections(datanotshown).Thus, ronment for effective and rapid down-regulation of the
theimpactofachronicinfectiononthedynamicsandfunc- antiviralCD8(cid:1)T-cellresponseduringchronicinfections.In
VOL.78,2004 CD8(cid:1) T-CELL DYNAMICS DURING PERSISTENT VIRAL INFECTION 3583
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FIG. 3. Kineticsoftotalvirus-specificCD8(cid:1)T-cellresponseindifferenttissuesbasedontetramerversusintracellularIFN-(cid:6)secretionafter ,
stimulationoflymphocyteswithDC2.4virus-infectedcellsduringacuteorpersistentinfections.C57BL/6micewereinfectedwith102(A)or2(cid:3) 20
106(B)PFUofLCMV-Docile,andtotalnumbersofvirus-specificCD8(cid:1)Tcells(sumofGP133-41andNP396-404peptide-specificTcells)were 1
determinedbystainingwithDb/GP133-41andDb/NP396-404tetramers(F).Thetotalnumbersofvirus-specificCD8(cid:1)Tcellsfromtissueswere 9
testedfortheabilitytoproduceIFN-(cid:6)aftershort-termculturingwithvirus-infectedDC2.4cellsontheindicateddaysafterinfection(E).Data b
shownaremeans(cid:10)SEMoflog10virus-specificTcellspertissuefor3to6mice. y g
u
e
s
contrast, the mechanisms of T-cell exhaustion operate less tions. Collectively, these data demonstrate that LCMV infec- t
efficiently in nonlymphoid tissues. tioninducesarobustantiviralCD8(cid:1)T-cellresponseinmulti-
To test whether the distinct kinetics of epitope-specific ple host tissues. An illustration of the massive expansion and
CD8(cid:1)T-cellpopulationsobservedinlymphoidversusnonlym- tissue distribution of GP133-41 or NP396-404 tetramer-posi-
phoidtissuesinmicewithacuteorpersistentinfections(Fig.1 tive CD8(cid:1) T cells in different tissues in relation to the total
and 2) reflected the overall response to the virus, we deter- IFN-(cid:6)-positiveCD8(cid:1)Tcellsonday20afteranacuteorper-
minedthekineticsofvirus-specificCD8(cid:1)T-cellpopulationsby sistentinfectionwithLCMV-DocileispresentedinFig.4.
using virus-infected DCs (DC2.4) in combination with intra- Tofurtherexaminewhethervirus-specificCD8(cid:1)T-cellpop-
cellularIFN-(cid:6)staining.Asexpected,inmicewithacuteinfec- ulationsarepreferentiallyaccumulatedand/orretainedintis-
tions, the sum of GP133-41- and NP396-404-specific CD8(cid:1) suesofinfectedmice,weevaluatedtherecruitmentkineticsof
T-cellpopulations,asdetectedbytetramerstaining,essentially specific CD8(cid:1) T cells by calculating the percentages of tet-
comprised the overall LCMV-specific CD8(cid:1) T-cell response ramer-positive T cells within the lymphocyte population in
measured by the IFN-(cid:6) secretion assay (Fig. 3). In addition, eachtissueasafunctionoftime.AsapercentageofCD8(cid:1)T
the data confirmed the distinct pattern of functional loss and cells,thenumberoftetramer-positivecells(thesumofGP133-
deletion during the course of persistent infections that was 41- and NP396-404-specific cells) was higher during an acute
observed for individual epitope-specific CD8(cid:1) T-cell popula- infection than during a persistent infection. Furthermore, tis-
3584 ZHOU ET AL. J.VIROL.
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FIG. 4. Expansionanddistributionofepitope-specificCD8(cid:1)Tcellsinlymphoidandnonlymphoidtissuesinrelationtothetotalnumbersof e
virus-specific IFN-(cid:6)-producing CD8(cid:1) T cells during acute versus persistent infections. C57BL/6 mice were infected with 102 (A) or 2 (cid:3) 106 st
(B)PFUofLCMV-Docile,andlymphocyteswereisolatedfromtheindicatedtissues20daysafterinfection.Thepercentageofantigen-specific
CD8(cid:1)TcellswasassessedbystainingwithDb/GP133-41(toppanels)orDb/NP396-404(middlepanels)tetramersandantibodyagainstCD8(cid:5).
Plotsshownaregatedonlivecells.Totalnumbersofvirus-specificCD8(cid:1)TcellsproducingIFN-(cid:6)afterstimulationwithvirus-infectedDC2.4cells
weredeterminedbyconcurrentanalyses(bottompanels).ThepercentagesofCD8(cid:1)TcellsstainingpositiveforDb/GP133-41orDb/NP396-404
tetramersorproducingIFN-(cid:6)areindicatedinthelowerrightcornersofthecorrespondingpanels.Resultsarerepresentativeofseveralseparate
experiments.
sue-specific differences were observed in both the magnitude peaked by day 9 (around 30%) but then declined rapidly to
andkineticsoftheLCMV-specificCTLresponseineachcase relativelylowbutstablelevels(4to5%).Inmicewithpersis-
(Fig. 5). Thus, the percentages of tetramer-positive CD8(cid:1) T tent LCMV infections, the percentages of tetramer-positive
cellsincreasedrapidlyinlymphoidtissuesandmoreslowlyin CD8(cid:1)Tcellsvariedsignificantlybetweendifferenttissuecom-
nonlymphoidtissues,includingblood,overthecourseofacute partments. While in the spleen, PLN, kidney, and lung, 5 to
infections before reaching relatively high stable levels (30 to 10%ofCD8(cid:1)Tcellsweretetramerpositive,intheblood,BM,
40%).Surprisingly,inthePLNtherewasatransientincrease and liver, this population was more prominent (peak values
in the number of tetramer-positive CD8(cid:1) T cells, which around30%).Thisdistinctkineticpatternforthepercentages
VOL.78,2004 CD8(cid:1) T-CELL DYNAMICS DURING PERSISTENT VIRAL INFECTION 3585
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FIG. 5. Tissue-specifickineticsofthevirus-specificCD8(cid:1)T-cellresponsebasedonpercentagesofCD8(cid:1)Tcellsthatweretetramerpositive m
duringacuteversuspersistentinfections.ThepercentagesoftotalCD8(cid:1)TcellsspecificforGP133-41orNP396-404intheindicatedtissues,as
h
determinedbytetramerstaining,werecalculatedbasedonthedatapresentedinFig.1foracuteinfections(F)orinFig.2forpersistentinfections t
(E)ofC57BL/6micewithLCMV-Docile.Datashownaremeans(cid:10)SEMoflog virus-specificTcellspertissuefor5to10mice. tp
10 :
/
/
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i.
a
of tetramer-positive CD8(cid:1) T cells in different tissues and the cellsatday90,althoughthisactivitywassubsequentlylost(by s
m
fact that tissues such as the kidney, lung, and liver were per- day200).Similarresultswereobservedforthekidneyandlung
.
fused extensively before the isolation of lymphocytes exclude (datanotshown). o
r
the possibility that such virus-specific CD8(cid:1) T cells were de- For an independent assay of functional activity, we also g
/
rived from blood contamination. When the total numbers of compared the abilities of antigen-specific CD8(cid:1) T-cell popu- o
virus-specificCD8(cid:1)Tcellspertissuewerecomparedwiththe lationsindifferenttissuestodeveloplyticactivityafterstimu- n
A
cumulativesumoftheirnumbersinnonlymphoidtissues(liver, lationinvitro.Thisapproachallowsfordirectfunctionalcom- p
kidney,lung,BM,andblood),thefigureswerecomparableto parisonsbetweenantigen-specificCD8(cid:1)T-cellpopulationsin ril
or even exceeded that of the spleen at both the peak of the different tissues with regard to the capacity for proliferation, 1
,
responseandthememoryphaseforbothacuteandpersistent differentiationtoeffectorcells,andacquisitionofcytolyticac- 2
infections(datanotshown). tivity after stimulation with a viral antigen. For this analysis, 0
1
Distinct patterns of virus-specific CD8(cid:1) CTL activity in cytolytic activity was expressed as a function of the E:T ratio 9

lymphoidversusnonlymphoidtissuesduringacuteorpersis- corrected for the number of tetramer-positive cells initially b
y
tent LCMV infections. To better characterize virus-specific added to each culture well, as described in Materials and
g
CD8(cid:1) T-cell populations during acute or chronic LCMV in- Methods.Cellsisolatedfromthespleensandliversofacutely u
e
fections,wetestedforpossiblefunctionaldifferences,inaddi- infected mice at early times (days 6 and 9) had rapidly up- s
tion to IFN-(cid:6) production, between antigen-specific CD8(cid:1) T regulatedtheirlyticactivity,andtheysustainedthisresponseat t
cells in different tissues. When we performed direct ex vivo relatively high levels after virus elimination over a period of
antigen-specificCTLassays,strikingdifferenceswereobserved 200 days (Fig. 7A). However, at comparable E:T ratios, anti-
among T cells isolated from different tissues. Nine days after gen-specificCD8(cid:1)Tcellsisolatedfromthespleenweremore
acuteinfectionwithLCMV,ahighleveloflyticactivitymea- efficientatlysisofeitherpeptide-loadedorvirus-infectedtar-
suredonvirus-infectedorpeptide-loadedtargetcellsatiden- getcellsthanthoseisolatedfromtheliver,exceptforonday6
tical E:T cell ratios was exhibited by cells from all tissues afterinfection.Asexpected,cellsfromthespleensofmicewith
examined (Fig. 6A and data not shown). However, although chronicinfectionsexhibitedsubstantialcytolyticactivityduring
cells isolated from the spleen and liver exhibited significant theinitialphaseofinfectionbutlosttheirlyticactivitiesrapidly
antigen-specificlyticactivityatlatertimesafterinfection(upto (byday30)(Fig.7B).Experimentsperformedwithcellsfrom
day200),cellsfromPLNandbloodlosttheirlyticactivitiesby the liver confirmed the results of IFN-(cid:6) secretion assays, as
day30afterinfection.Inmicewithchronicinfections,directex thesecellshadsubstantialantigen-specificcytolyticactivityon
vivo lytic activity was detected in lymphoid tissues at early days30and90,butnotday200,afterinfection(Fig.7anddata
times (days 6 and 9) but was lost by day 30. In fact, antigen- notshown).
specific lytic activity was readily detected with both virus-in- HierarchicalfunctionalimpairmentofCD8(cid:1)Tcellsduring
fected and peptide-loaded (GP133-41 or NP396-404) target a chronic viral infection. To further evaluate the functional
3586 ZHOU ET AL. J.VIROL.
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FIG. 6. Virus-specific CD8(cid:1) T cells from nonlymphoid tissues preserve their ex vivo lytic activity for prolonged periods during persistent il
infections.C57BL/6micewereinfectedwith102(A)or2(cid:3)106(B)PFUofLCMV-Docile,andlymphocyteswereisolatedfromtissuesatthe 1,
indicatedtimes.DirectexvivoCTLactivitywasmeasuredonvirus-infectedMC57Gcells(F)oronEL-4cellsloadedwithapeptideGP133-41 2
(Œ)orNP396-404((cid:1))targetatanE:Tratioof200:1foralltissues.TheE:TcellvaluesshownarecorrectedforthenumberofGP133-41and 0
1
NP396-404tetramer-positivecellsinthetotalvirus-specificCTLpopulation(virus-infectedtargets)orforthenumberofGP133-41orNP396-404 9
tetramer-positivecellsineachepitope-specificCTLpopulation(peptide-loadedtargets).Lysisofuntreatedtargetcellswasusually(cid:1)5%atthe
b
highestE:Tratio.However,inafewcases,nonspecificlysisexceedingthe5%levelwassubtractedfromcorrespondinglysisvalues.Resultsare y
representativeofthreeseparateexperiments. g
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properties of virus-specific CD8(cid:1) T-cell populations during tetramer-positive population produced IL-2 upon stimulation
acuteversuschronicinfections,weperformedanalysesofcells with virus or peptide. Costaining for IFN-(cid:6) and TNF-(cid:5) or
isolated from the liver or spleen to establish the kinetics of IFN-(cid:6) and IL-2 during the response to acute infections re-
cytokineresponsesbycomparingnumbersofIFN-(cid:6)-,TNF-(cid:5)-, vealedthatTNF-(cid:5)andIL-2wereproducedbyantigen-specific
and IL-2-producing CD8(cid:1) T cells with the numbers of tet- CD8(cid:1) T cells producing IFN-(cid:6) (data not shown). Similar ki-
ramer-positive CD8(cid:1) T cells with the same specificity by re- neticprofilesforvirus-orpeptide-specificCD8(cid:1)T-cellpopu-
stimulatingcellswitheithervirus(usingDC2.4-infectedcellsto lationswereobservedforcellsisolatedfromtheliver.
stimulatecytokinesecretion)orpeptide(GP133-41orNP396- To examine how chronic infections impacted the ability of
404).AsshowninFig.8Aformicewithacuteinfections,nearly antigen-specificCD8(cid:1)Tcellstoup-regulatecytokineproduc-
theentirepopulationoftetramer-positivecellsfromthespleen tion upon antigen stimulation in vitro, similar analyses were
producedIFN-(cid:6)duringthecourseofinfection.Thenumberof performed with mice that were persistently infected with
tetramer-positiveCD8(cid:1)TcellsproducingTNF-(cid:5)variedfrom LCMV.AsshowninFig.8B,duringtheinitialphaseofinfec-
20 to 80% of the total number of tetramer-positive cells, but tion(day6)theentirepopulationoftetramer-positiveCD8(cid:1)T
the percentages became higher during the memory phase of cells isolated from the spleen possessed the capacity to pro-
infection(days30and140).Incontrast,only5to10%ofthe duceIFN-(cid:6),whileonlyafractionofthemproducedTNF-(cid:5)(10
VOL.78,2004 CD8(cid:1) T-CELL DYNAMICS DURING PERSISTENT VIRAL INFECTION 3587
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FIG.6—Continued. il
1
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2
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1
to20%)orIL-2(1to2%).However,thisabilitywasprogres- hierarchical level of secretion and subsequent loss of IL-2, 9
sivelylostbyday20afterinfection,withsimilarkineticsforthe TNF-(cid:5), and finally, IFN-(cid:6) production. In contrast, in the en- b
y
individual cytokines. Interestingly, the ability of NP396-404- vironmentoflymphoidtissues(e.g.,thespleen),Tcellsexpe-
g
specific CD8(cid:1) T cells to produce IL-2 was completely sup- rience a rapid functional inactivation with similar kinetics for u
e
pressed,evenatearlytimesofinfection.Anessentiallysimilar theindividualcytokines. s
pattern of cytokine production was observed at day 6 after Phenotypicanalysisofantigen-specificCD8(cid:1)Tcellsduring t
infection for antigen-specific T cells isolated from the liver. acute versus chronic infections. The differentiation of naïve
However, marked qualitative differences were observed fur- CD8(cid:1)Tcellsintoeffectorcellsbytheiracquisitionofantiviral
ther in the course of infection. Thus, while the capacity for propertiesandtheirrelocationtodifferenttissuesisacomplex
IFN-(cid:6)productionduringtheinitialphaseofinfectionwaslost process involving a structural reorganization of the cellular
for the majority of virus- or peptide-specific CD8(cid:1) T cells, a membraneandcytoskeletonassociatedwiththeexpressionof
substantial fraction of them (10 to 20%) preserved this func- activationmarkersandanalterationincelladhesionandhom-
tion at stable levels (until at least 140 days after infection). ingreceptorexpression(6,42,66).Toexaminethepatternof
However,theabilitytoproduceIFN-(cid:6)wasalsocompromised expression of such molecules during acute versus persistent
atday250afterinfection(datanotshown).Instrikingcontrast infections,welongitudinallyanalyzedactivationandadhesion
tothis,thefrequencyofantigen-specificCD8(cid:1)Tcellsproduc- molecules on CD8(cid:1) T cells (specific for GP133-41) isolated
ingTNF-(cid:5)progressivelydeclinedandwasessentiallyabsentby from the spleen and liver. As shown in Fig. 9A, in the spleen
day 120 after infection. The capacity for IL-2 production was CD11a (LFA-1) expression was up-regulated on antigen-spe-
abolished more rapidly, during the initial phase of infection. cific CD8(cid:1) T cells during the early phase of an acute or per-
Thus,thesefindingssuggestthatantigen-specificCD8(cid:1)Tcells sistent infection (day 6) and remained at elevated levels
innonlymphoidtissuesexperiencefunctionalexhaustionwitha throughouttheinfection.Ataverylatestageofinfection(day