Professional antigen presenting cells in human herpesvirus 8 infection.

0.89 MB·English

by  Knowlton, Emilee R.

REVIEWARTICLE
published:21January2013
doi:10.3389/fimmu.2012.00427
Professional antigen presenting cells in human herpesvirus
8 infection
EmileeR.Knowlton1,Lauren M.Lepone1,JunLi1,GiovannaRappocciolo1,FrankJ.Jenkins1,2 and
CharlesR.Rinaldo1,2*
1DepartmentofInfectiousDiseasesandMicrobiology,GraduateSchoolofPublicHealth,UniversityofPittsburgh,Pittsburgh,PA,USA
2DepartmentofPathology,SchoolofMedicine,UniversityofPittsburgh,Pittsburgh,PA,USA
Editedby: Professional antigen presenting cells (APC), i.e., dendritic cells (DC),
GuidoPoli,Vita-SaluteSanRaffaele monocytes/macrophages, and B lymphocytes, are critically important in the recognition
University,Italy of an invading pathogen and presentation of antigens to the T cell-mediated arm of
Reviewedby: immunity. Human herpesvirus 8 (HHV-8) is one of the few human viruses that primarily
FranciscoVeas,Institutde
targetstheseAPCforinfection,alteringtheircytokineprofiles,manipulatingtheirsurface
RecherchePourleDéveloppement,
France expressionofMHCmolecules,andalteringtheirabilitytoactivateHHV-8-specificTcells.
AdrianoBoasso,ImperialCollege ThiscouldbewhyTcellresponsestoHHV-8antigensarenotveryrobust.OftheseAPC,
London,UK onlyBcellssupportcomplete,lyticHHV-8infection.However,bothcompleteandabortive
*Correspondence: virus replicationcyclesinAPC coulddirectlyaffect viralpathogenesisand progressionto
CharlesR.Rinaldo,Departmentof
Kaposi’ssarcoma(KS)andHHV-8-associatedBcellcancers.Inthisreview,wediscussthe
InfectiousDiseasesand
Microbiology,GraduateSchoolof effects ofHHV-8infectiononprofessionalAPC andtheirrelationshiptothedevelopment
PublicHealth,Universityof ofKSandBcelllymphomas.
Pittsburgh,A419CrabtreeHall,
130DeSotoStreet,Pittsburgh, Keywords:humanherpesvirus8,Kaposi’ssarcoma,multicentricCastleman’sdisease,primaryeffusionlymphoma,
PA15261,USA. dendriticcells,monocytes/macrophages,Blymphocytes,CD4andCD8Tlymphocytes
e-mail:[email protected]
INTRODUCTION disease(MCD)(Soulieret al.,1995)and primaryeffusion lym-
Human herpesvirus 8 (HHV-8) or Kaposi’s sarcoma (KS)- phoma (PEL) (Cesarman et al., 1995; Nador et al., 1996). This
associatedherpesvirus,istheetiologicagentofKS(Changetal., review covers various aspects of HHV-8 targeting of APC and
1994), aneoplasmofendothelial originthat occursin fourdis- Tcellresponsesinhostcontrolofthisinfectiousandoncogenic
tinct epidemiologic forms(Dedicoat and Newton,2003;Jessop, process.
2006): classic or Mediterranean KS, epidemic or AIDS-related HHV-8hasbeenreportedtobetransmittedtocommonmar-
KS,endemicorAfricanKS,andiatrogenic ororgantransplant- mosets and cause persistent infection with rare, KS-like skin
associated KS. KS is the most common cancer associated with lesions(Changetal.,2009).However,thereisasyetnoconsensus
HIV-1 infection and AIDS (Cohen et al., 2005). Although the that this or other simian models (Jung et al., 1999; Fickenscher
incidence of KS in HIV-1 infected persons declined with the and Fleckenstein, 2001; Rosenwirth et al., 2011) recapitulate
advent of antiretroviral therapy (ART) (Gallafent et al., 2005), humanHHV-8infectionanddevelopmentofKSorothercancers
KS can occur in persons on ART with suppressed HIV-1 infec- associatedwiththisherpesvirus.Thus,althoughinvitromodels
tion (Maurer et al., 2007). The success of ART in treating aresuspecttolackingcertaininvivocharacteristics,wewillfocus
HIV-1-associated KS has also been countered by the occasional in this review on HHV-8infection of humanAPC asbeing the
occurrenceofanimmunereconstitutioninflammatorysyndrome mostrelevanttothishumanspecies-specificherpesvirus.
(Felleretal.,2008).Thisisasevere,temporaryenhancementof
KSlesionsduetoanincreaseininflammationandimmunologic HHV-8INFECTIONOFPROFESSIONALAPC
recoveryafterART. Aswiththeotherhumangammaherpesvirus,EpsteinBarrvirus
The discovery of HHV-8 and its causal role in KS develop- (EBV)(Ning,2011),HHV-8targetsAPCbothinvivoandinvitro.
mentopenedthepotential forprophylaxisandtreatment ofthe Indeed, the primary tropism of B cells by these gamma her-
infectionandcancerwithantiviraldrugs,andpreventionofboth pesviruses is uncommon among human virus infections. This
withavaccine.Strategiestoachievetheseendsrequireanintimate sets the stage for development of their associated cancers both
knowledge of the pathogenesis and immune control of HHV-8 indirectly, through alteration of host immunity dependent on
infection.WepostulatethathostcontrolofHHV-8infectionand APC function, and directly via neoplastic effects of the virus.
development of KS is linked to T cell interactions with HHV-8 HHV-8 infection within KS tumors is primarily found in spin-
infected, professional antigen presenting cells (APC), i.e., den- dlecells,whichareofmixedvascularandlymphaticendothelial
dritic cells (DC), monocytes/macrophages, and B lymphocytes. cell and macrophage origin (Regezi et al., 1993; Bubman and
Similarly,APC-Tinteractionsarelikelytobecentrallyinvolvedin Cesarman,2003).However,HHV-8isalsodetectedinmonocytes
theHHV-8-associatedBcellneoplasmsmulticentricCastleman’s thatarefoundinproximitytoKSlesions,andcirculatingBcells
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Knowltonetal. APCinHHV-8infection
of KS patients (Blasig et al., 1997; Monini et al., 1999). InPEL, transmission with a doxycyline (DOX)-inducible cell line har-
HHV-8 is found in immunoblastic cells expressing plasma cell boringrecombinantHHV-8(rKSHV.219)(MyoungandGanem,
markers, andin plasmablasticcells ofa lessterminally differen- 2011c). This indicates that viral entry can be achieved despite
tiated state in MCD (Du et al., 2007). The intimate association lackofexpressionofamajorHHV-8receptor.Thereisalsoevi-
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of HHV-8 with such professional APC in the KS lesion and dencethatHHV-8caninfectCD34 stemcellprecursorsofDC
in other HHV-8-associated cancers suggests a major role for invitrobyasyetundefinedreceptors(Henryetal.,1999;Larcher
virus-APCinterplay.Moreover,anti-HHV-8Tcellimmunitythat et al., 2005). Itislikelythatthere areless prominentalternative
presumably is critically dependent on such virus-APC interac- receptorsforHHV-8thataccountforasmallpercentageofDC-
tions, is present in HIV-1 infected and uninfected persons who SIGN negative APC and cell lines that can be infected by this
areseropositiveforHHV-8(Robeyetal.,2010).Achievingabet- virus.
ter understanding of the role of HHV-8 in inducing-associated
cancerscouldgreatlybenefitfromayet-to-be-developedinvitro BCELLINFECTIONWITHHHV-8
modelofprimaryHHV-8infection ofanaturaltargetcell.This Suggestive evidence that HHV-8 is B-cell tropic in vivo is that
modelshouldconsistentlyreflectHHV-8lytic,latent,andreacti- HHV-8 DNA is detected in B cells from patients with KS
vationinfections.HHV-8infectionofAPCcouldprovidesucha lesions (Ambroziak et al., 1995) and some HIV-1/HHV-8 coin-
model. fected individuals (Murayama et al., 1994). Further evidence
that HHV-8 targets B cells is the isolation of immortalized B
HHV-8RECEPTORSONAPC cell lines from patients with PEL that are infected with HHV-8
InfectionofAPCinvitrorevealsdifferentcyclesofHHV-8repli- (Cesarman et al., 1995). The first in vitro evidence that HHV-8
cation that are likely to relate to pathogenesis of the virus. caninfectBcellswasthatvirusproducedbythesePELcelllines
HHV-8initiallytargetscellsurfacereceptorsforinfection,which couldbetransmittedtoneonatalcordbloodBcells(Mesrietal.,
represent the first level of APC alteration. Herpesviruses use 1996). We speculate that the lack of further evidence for B cell
more than one receptor to infect the same cell (Heldwein and infectioninthoseearlyyearswasthatsuchinfectionrequiresDC-
Krummenacher,2008).Useofthesereceptorsbyherpesvirusesis SIGNexpressionthatisenhancedbyanactivatedstateinBcells.
hierarchical,basedlargelyondifferentialexpressionoftherecep- Thus, we showed that once blood-derived B cells are activated
tors in specific cell types and states of cell activation. Extensive to express DC-SIGN, HHV-8 can effectively establish infection
in vitro evidence indicates that the ubiquitous cell surface pro- andelicitfull-cycleproductionofinfectiousvirionsinthesecells
teoglycan, heparan sulfate, serves as an initial binding receptor (Rappoccioloetal.,2008).
for HHV-8 on endothelial cells and fibroblasts, as well as APC The fact that HHV-8 cannot infect Raji LCL or K562 cells
(Akula et al., 2001b, 2002; Chandran, 2010; Keruret al., 2010). expressingDC-SIGNthatlacksitstransmembranedomainsup-
Multiple integrins are subsequentlyinvolved in HHV-8 binding ports that viral entry requires DC-SIGN-mediated endocytosis.
andentry (Keruretal.,2010).Athird levelofdifferential selec- Moreover, infection can be blocked by pretreatment of B cells
tionhasbeenidentified frominvitrostudiesofthe threemajor with anti-DC-SIGN monoclonal antibody (mAb) or mannan,
typesofprofessionalAPC.ThetypeIIC-typelectin,DC-specific but not Ab specific for the amino acid transporter protein xCT
ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) serves as a (Rappocciolo et al., 2008). HHV-8 has been reported to use
receptorforHHV-8onbothDCandBcells(Rappoccioloetal., xCT forinfection ofsurfaceadherenthumancells(Kaleebaand
2006,2008).Recently anewentry receptorforHHV-8hasbeen Berger,2006)andinapost-entrystageofhumanendothelialcell
discoveredonendothelialandepithelialcells(Hahnetal.,2012), infection as part of a complex of heterodimeric membranegly-
i.e.,ephrinreceptortyrosinekinaseA2.Thistyrosinekinasefunc- coproteinCD98andtheα3β1andαVβ3integrins(Veettil etal.,
tionsinneovascularizationandoncogenesis,andhasnotyetbeen 2008).
assessedinHHV-8infectionofAPC. Notably,HHV-8infectionisnotrestrictedtoblood-derivedB
The role of HHV-8 binding to APC receptors for entry and cells,astonsillarBcellsconstitutivelyexpressDC-SIGNandcan
infection isbeingclarifiedwithaccumulatingevidencethatcer- be lytically infected with the virus in vitro (Rappocciolo et al.,
tain C-type lectins and integrins are essential to this process. 2008; MyoungandGanem,2011a).ItisprobablethatBcellsin
For example, the Raji B lymphoblastoid cell line (LCL) and such inflamed tonsillar tissue are in an endogenously activated
the myeloblastoid K562 erythroleukemia cell line constitutively state,resultinginenhancedexpressionofDC-SIGN.
expresslittleornoDC-SIGNorα β integrin(Rappoccioloetal., The in vitro activated, B cell model for measuring HHV-8
3 1
2006). Thus, these cell lines do not support detectable produc- infectivityandreplicationsupportstheconceptthatDC-SIGNis
tion of HHV-8 virions (Blackbournet al., 2000b; Bechtel et al., a major receptor for this virus (Rappocciolo et al., 2006, 2008;
2003;Rappoccioloetal.,2006).However,transfectionofthecell Chandran,2010).Thisaddstothewealthofevidencethatshows
lineswithDC-SIGNrendersthemhighlypermissiveforHHV-8 that, in contrast to previous reports (Ganem, 2007), DC-SIGN
infection asmeasuredbyproductionofviralproteinsandDNA is required for highly efficient infection of natural APC tar-
(Rappoccioloetal.,2006).Moreover,infectionofthesecelllines gets with HHV-8. This is in addition to certain integrins that
can be blocked by anti-DC-SIGN mAb, soluble DC-SIGN, and are also involved in HHV-8 entry (Akula et al., 2001a,b, 2002;
mannan,anaturalligandofDC-SIGN.Interestingly, fourBcell Birkmann et al., 2001; Veettil et al., 2008). However, there is
lines (BJAB, Ramos, BCBL1, JSC1) and two T cell lines (Jurkat stillneedforimprovedreliable,quantitativemeasuresofHHV-8
and SupT1) are susceptible to infection through cell-mediated replicationtobetterdefineBcellinfection.Theseshouldinclude
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Knowltonetal. APCinHHV-8infection
combinations of real time polymerase chain reaction (PCR) the λ light chainat2.5–3.5 dayspost-HHV-8 infection in vitro.
assays for cell-associated and non-cell-associated copy numbers Thesecellsareplasmablast-likewithincreasedinterleukin(IL)6
of HHV-8 encapsidated DNA, flow cytometry assays for enu- receptorexpressionandincreasedproliferativeresponsetoIL-6,
merating the number of monoclonal Ab (mAb)-stained cells with 7–36% expressing CD27. This molecule is a member of
expressingvirallyticandlatencycycleproteins,andmostimpor- the tumor necrosis factor (TNF)-receptor superfamily, and is
tant, cell culture-based assays for quantitating the number of involvedinregulationofBcellactivation.Itisnotknownwhether
+
infectious virus particles, e.g., a 50% tissue culture infectious HHV-8directly infectsthese IgM memoryBcellsoraprecur-
dose assay. It is remarkable that almost 20 years after discovery sor of these cells. Also, there are no data on which subset of B
ofHHV-8wedonothavesuchbasicassaystostudythevirus. cells supports a complete lytic cycle of replication with virion
It is postulated that HHV-8 infection drives B cells to an formation and death of the cell. Likewise, there is the possibil-
early plasmablast-like state in MCD and a preterminal plasma ity that HHV-8 infection in these B cell subsets results in an
cell stage of differentiation in PEL (Frizzera et al., 1983; Miller abortivereplicationcycle,leavingmemoryBcellsthatsurviveand
etal.,1984;Cesarmanetal.,1995;Nadoretal.,1995;Agematsu maintainlatentvirusinfection. WespeculatethatHHV-8infec-
et al., 1997; Matolcsy et al., 1998; Dupin et al., 2000; Du et al., tion of naïve and IgM memory B cells leads to establishment
2001; Klein et al., 2003; Chadburn et al., 2004, 2008; Hassman of latency in a portion of cells, resulting in virus-driven plas-
etal.,2011).Hassmanetal.(2011)recentlyshowedthatlatency- mablast differentiation, while some cells support the viral lytic
+
associated nuclear antigen (LANA) B cells express IgM and cycle(Figure1).
FIGURE1|HHV-8 targets B cell subpopulations for infection. The into the lytic cycle to begin transcription of lytic-associated proteins in
B cell target for HHV-8 infection is unknown. However, evidence activated B cells (red outline). The lytic cycle may stop prior to virion
suggests that naïve and/or IgM memory B cell subsets are susceptible production, resulting in an abortive replicative cycle as seen in DC,
to HHV-8 infection. HHV-8 is endocytosed after binding to cell surface endothelial cells, and fibroblasts. The virus in these cells likely enters
entry receptors. The virus then enters latency (left) or initiates lytic latency or may result in B cell apoptosis. Some cells, however, will
replication (Rappocciolo et al., 2008). Latently infected cells drive support full lytic cycle replication, resulting in lytic protein synthesis, and
differentiation toward a plasmablast phenotype that is responsive to the increases in viral DNA that correspond to infectious HHV-8 progeny and
proliferative cytokine, IL-6. The alternative pathway is the entry of HHV-8 subsequent release through cell lysis.
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Knowltonetal. APCinHHV-8infection
Currently, the main in vitro models that recapitulate HHV-8 Transcription of HHV-8 lytic genes occurs during either a
infectionofAPCareBlineagecelllinespersistentlyinfectedwith primary infection of susceptible cells or during reactivation of
thevirus.Amongthem,thebodycavity-basedlymphomacellline latentlyinfected cells. Thequestionremainswhether thekinetic
(BCBL-1), a B cell line derived from a patient with PEL which gene activation in a chemically induced cell line (BCBL-1) or
islatentlyinfected withHHV-8andEBVnegative(Changetal., the naturally targeted RTA (TREx-BCBL1-RTA) will reflect the
1994;Cesarmanetal.,1995),isthemostcommonlyutilized.In cascade events of natural infection of B cells or other APC. To
latently infected cells of KS tumors and in such cell line mod- identify the true gene transcription and reactivation events in
els, the virus exists as circular or episomal DNA with limited HHV-8infection, primarycellssusceptibletoHHV-8shouldbe
expressionoflatentgenes(Schulz,2006).Therefore,HHV-8lytic used.OnlythencantheobservationsfromTPA-inducedBCBL-1
and latent infections cannot be defined conventionally in these andDOX-inducedTREx-BCBL1-RTAcelllinesbevalidated.
systems starting with the total absence of infectious viral parti- In 2005, a cluster of microRNA (miRNA) coded by HHV-8
cles,asthereisalwaysalowlevelofpersistentvirusproduction. wasdiscovered(Caietal.,2005;Samolsetal.,2005).Thisshort,
However,latencycanbedisrupted,triggeringthelyticcascadeof 22 nucleotide, non-coding miRNA silences mRNA expression
viralreplication,andlyticgenesexpressedsequentiallyasimme- through a silencing complex (miRISC). HHV-8 miRNAs are
diateearly(IE)genes,early(E)genes,andlate(L)genes,resulting expressedduringlyticandlatencycyclesofvirusreplication,and
in production of encapsidated virions. Such lytic viral replica- act on both cellular and viral transcriptomes (Gottwein, 2012).
tion islargely irreversible(Chandran,2010; Wenand Damania, StudiesofHHV-8miRNAhaveutilizedPELcelllines,aswellas
2010). foreskinfibroblastsandendothelialcells.Theseindicateamulti-
HHV-8 lyticgeneprofilinginthese persistently infected PEL factorial role in maintaining viral latency, regulating lytic virus
celllineshasbeenextensivelyaccomplishedusingtilingmicroar- replication, and enhancing cell survival. As miRNA activity is
ray (Chen et al., 2009), DNA microarray (Agematsu et al., dependentonitslevelandtargetswithinspecificcell types, itis
1997; Chandriani et al., 2010; Lock et al., 2010), and high- imperativethatmiRNAbeassessedinprimaryBcells.
throughputreal-timePCR(FakhariandDittmer,2002;Dittmer, Finally,asHHV-8isoneofthefewhumanvirusesthatprimar-
2003). However, most studies on latency-lytic reactivation of ilytargetsBcells,anindepthunderstandingoftheeffectsHHV-8
HHV-8usevariouschemicalstoinduceviralreplication(Myoung infection has on these cells should be established. However, lit-
and Ganem, 2011b). The question is whether such reactivation tledataexistconcerningBcellactivationstatesorsurfacemarker
reflectsnaturalHHV-8virallyticreactivationfromlatency,since expressionuponHHV-8infection.Likewise,interactionsbetween
+
chemical agents such as 12-O-tetradecanoylphorbol-13-acetate HHV-8 infected B cells and CD4 T helper cells are yet to be
(TPA) have pleiotropic effects on host cell signaling and chro- defined.ConsideringthatthemajorfunctionofBcellsisproduc-
matin structure. How they affect cell signaling pathways is also tionofAbthatpreventandameliorateinfection,thereisneedto
unknown. Thus, the natural reactivated cascade of lytic tran- assessthequalityandquantityofAbproductionoverthecourse
scriptsofHHV-8stillwaitstoberevealed. ofHHV-8infectionanddevelopmentofKS.Yet,thereisnocon-
Instead of using chemical inducers, Nakamura et al. (2003) sensusassayfordetecting ortitering anti-HHV-8 Ab. Detection
developedanengineeredBCBL-1celllinethatinduciblyexpresses of anti-LANA Ab by immunofluorescence assays has low sen-
the replication transactivator protein, RTA, encoded by ORF50, sitivity (as low as 64%) among individuals with KS (Rabkin
i.e., TREx-BCBL1-RTA. RTA is necessary and sufficient for the et al., 1998; Corchero et al., 2001; Nascimento et al., 2007).
switchbetweenHHV-8latencyandlyticreplication(Dengetal., ELISAandWestern blotassaysforanti-latent (LANA)andlytic
2000).Infact,mutationoftheRBP-JksiteswithintheRTApro- (ORF65orK8.1)Abhavehighersensitivities andareoftenused
moter is enough to enhance latency in transformed-293 cells for serologic testing, yet can have low specificities (Nascimento
and peripheral blood mononuclear cells (PBMC) (Lu et al., et al., 2007). These conventional methods for serologic testing
2012). In the TREx-BCBL1-RTA cell line, RTA expression is therefore lack standardization and can be unreliable, under-
under the control of a DOX-inducible promoter. Treatment of lining the necessity for more accurate methods of quantifying
TREx-BCBL1-RTAcellswithDOXresultsinexpressionofRTA, anti-HHV-8Ab.
whichinturninducesviralreplication(Nakamuraetal.,2003). Regardless of the vagaries of anti-HHV-8 Ab assessments,
While the role of RTA in causing a switch from latency to humoral immunity to HHV-8 infection has been described for
viral replication has been demonstrated, the mechanisms regu- several cohorts. A luciferase immunoprecipitation system that
latingcoordinateinductionofexpressionofmostoftheHHV-8 quantifiesAbresponsetomultipleantigenswasusedtocompare
lyticgenesduringthisreactivation havenotbeenevaluatedina profilesofKS,MCD,andPELpatients(Burbeloetal.,2010).The
systematicfashion. study showed significantdifferences inAb responsesamongthe
Virus reactivation events have been studied in primary and groups,includinghigheranti-K8.1AbdetectedinPELandMCD
immortalized microvascular endothelial cells (MVEC) with the comparedtoKSandhigherAbtiterstoORF65inPELcompared
recombinantvirus,rKSHV.219(VieiraandO’Hearn,2004).This to KS. Likewise, higher Ab titers against v-cyclin were observed
virusexpressesagreenfluorescentprotein(GFP)underanEF-1α in KS and PEL compared to MCD, and higher anti-LANA Ab
promotertoindicateinfectionandaredfluorescentproteinunder titersweredetectedinKScomparedtoMCD.Anexplanationfor
aPANpromotertoindicatelytictranscription.Thismodelshould thedifferenceinAbresponsesinindividualswiththeseHHV-8-
beconsideredforamoredetailedevaluationofAPCinfectionand associatedcancersiscurrentlyunknown,butislikelyareflection
reactivation. of the differential expression oflatent and lytic viral genes. The
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Knowltonetal. APCinHHV-8infection
qualityandquantityofanti-HHV-8responsemaychangeoverthe Ab-secreting plasma cells. M2 macrophages express more DC-
courseofdiseaseprogressionorafteranti-viraltherapy.Following SIGN than M1 macrophages (Cassol et al., 2012), supporting
ART, increases inAb againstboth latent and lytic proteins have the concept that M2 macrophages are likely to serve as a more
beenobservedforindividualswithorwithoutKS(Gilletal.,2002; efficient target cell for HHV-8 infection. Importantly, multiple
Wilkinson et al., 2002; Bourboulia et al., 2004; Sullivan et al., receptorsareneededforefficientinfectionofthesemacrophages
2010). More in depth studies with larger cohorts and advanced byHHV-8.Indeed,whenDC-SIGNisblockedinIL-13-activated
testing methods shouldbeperformed, whileinvitro modelsfor MDM or the monocytic cell line THP-1, HHV-8 can still bind
HHV-8infection inBcellsanddetection ofantiviral Abshould using heparan sulfate, although virus entry is reduced (Kerur
beestablished. etal.,2010).
Interestingly, thereareonlyminimaldataonneutralizingAb Although studies are lacking for coordinated expression of
inHHV-8infection.Thefirstsuchevidencewasthatrabbitpoly- HHV-8 ORFs in monocytes/macrophages, HHV-8 establishes
clonalneutralizingAbtogBpreventHHV-8infectionofprimary productive infection in THP-1 cells with an ordered expression
humanforeskinfibroblasts(Akulaetal.,2001a)andoralepithe- oflatencygeneORF73andlyticgeneORF50.Infact,theHHV-8
lial cells (Duuset al., 2004). Concurrently, it was demonstrated genomewasreported to persistfor30daysinthese cells(Kerur
thatserafrompersonswhowereseropositiveforHHV-8asshown etal.,2010).Suchlimitedexpressionoflyticgenestogetherwith
by anti-LANA immunofluoresence assay also had neutralizing the persistence of latency genes is believed to be unique for
Ab that inhibited virusinfection of transformed dermal MVEC HHV-8(Krishnanetal.,2004).
(Dialynaetal.,2004).UsingarecombinantHHV-8(rKSHV.152) Of interest is that ORF K14 of HHV-8 encodes a surface
thatexpressesGFP,Kimballetal.(2004)foundsignificantlylower glycoprotein vOX2 that is homologous to cellular OX2 (Chung
neutralizingAbtiters toHHV-8intheserumofHIV-1infected et al., 2002), and which inhibits macrophage function (Foster-
personswithKScomparedtothosewithoutKS.Thisisincontrast Cuevas et al., 2004). The vOX2 glycoprotein could be central
toInoueetal.(2004)whoreportedthattherewerenodifferences toHHV-8immunopathogenesisinthatitstimulatesproduction
inneutralizingAbtitersbetweenHIV-1infectedpatientswithor of inflammatory cytokines IL-1β, IL-6, monocyte chemoattrac-
without KS. However, the latter study used an HHV-8 reporter tant protein 1 (MCP-1), and TNF-α in primary monocytes,
celllineT1H6treatedwithpolybreneintheirvirusneutralization MDM,andmonocyte-derivedDC(MDDC)(Chungetal.,2002).
assay.Polybreneresultsinreceptor-independentinfection(Davis Furthermore,expressionofvOX2onBcellsstimulatesmonocytes
et al., 2002), thus potentially obscuring interpretation of virus toproduceinflammatorycytokines.MDMtransfectedwithvOX2
neutralizationassays.Finally,itisevidentthatthereisaneedfor produceinflammatorycytokines andhaveenhanced phagocytic
indepth,longitudinalstudiesofneutralizingAbandotherantivi- activity,whileinhibitingtheimmunomodulatoryeffectsofIFN-
ralAbsuchasthosethatmediateAb-dependentcellcytotoxicity, γ and down-regulating MHC class I and class II expression
inrelationto progressionofHHV-8infection anddevelopment on macrophages (Salata et al., 2009). It was recently reported
HHV-8relatedcancers. that vOX2-transfected APC co-cultured with T cells results in
suppressed IFN-γ production and mobilization of the cytolytic
MONOCYTE/MACROPHAGEINFECTIONWITHHHV-8 granulemarkerCD107athroughinhibitionofERK1/2phospho-
Macrophages in several body compartments naturally express rylation(Misstearetal.,2012).
DC-SIGN (Granelli-Piperno et al., 2005; Kamada et al., 2009),
aswell asintegrinsincludingα β (Ammonetal.,2000),which HHV-8INFECTIONOFMYELOIDDC
3 1
presumably renders them susceptible to HHV-8 infection. An Evidence of infection of human DC in vivo with HHV-8 has
earlyreportshowedthatmonocyte-derivedmacrophages(MDM) been limited (Rettig et al., 1997; Olsen et al., 1998). However,
fromnormaldonorsthatarestimulated invitro withallogeneic there is no a priori reason why human DC should not take up
PBMCcanbeinfectedbyHHV-8,butthisrarelyresultedincom- HHV-8andsupportatleastabortiveinfectioninvivo.Tissueresi-
plete, lytic replication (Blackbourn et al., 2000b). In addition, dent,myeloidDCconstitutivelyexpressDC-SIGN(Soilleuxetal.,
treatmentofbloodmonocytesfromKSpatientswithproinflam- 2002).MDDCexpressDC-SIGNinvitro,againindicatingacen-
matory cytokines invitro resultsinHHV-8 persistence (Monini tralroleforthisreceptorinHHV-8infectionofAPC.Studiesare
etal.,1999). needed to determine whether this C-type lectin is required for
MDM become susceptible to HHV-8 infection in vitro after HHV-8infectionoftissueDCinvivo.
activation with IL-13, which enhances DC-SIGN expression WhenMDDCareinfectedinvitrowithHHV-8,virallyticpro-
(Rappocciolo et al., 2006). IL-13 is an anti-inflammatory, Th2 teinsareproducedwithlittleviralDNAproduction(Rappocciolo
cytokine that induces alternatively activated (M2) macrophages et al., 2006), similar to abortive HHV-8 infection of vascular
(Haoetal.,2012).Thesecontrastwithclassicallyactivated(M1) endothelial cells (Renne et al., 1998; Vieira et al., 2001; Akula
macrophages which have preferential expression of proinflam- et al.,2002; Naranattetal., 2003; Raghuet al.,2009). Although
matory cytokines, chemokines, and effector molecules, such as HHV-8 infection does not significantly alter MDDC viability,
IL-12, IL-23, tumor necrosis factor α (TNF-α), inducible nitric it decreases MDDC function, i.e., lowers their capacity to acti-
+
oxide synthase (iNOS), and MHC class I and II. In contrast, vate antigen-specific CD8 T cell responses. Moreover, HHV-8
M2 macrophages express a wide array of anti-inflammatory infected MDDC have impaired antigen uptake, with a signif-
molecules, including IL-10, and transforming growth factor β icant decrease in endocytic capacity and DC-SIGN expression
(TGF-β). IL-13 also promotes differentiation of B cells into within 24h after infection. DC-SIGNinternalization in MDDC
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Knowltonetal. APCinHHV-8infection
is associated with lytic HHV-8 gene expression (Rappocciolo reactivation of HHV-8 from latency in B cells. That is, ago-
et al., 2006). In addition to MDDC, HHV-8 in vitro infec- nists specific for TLR7/8 reactivate latent HHV-8 and induce
tion of IL-13-treated MDM results in a loss of DC-SIGN sur- viral lytic gene transcription and replicationin latently infected
face expression, suggesting that HHV-8 binding to DC-SIGN PEL cell lines of B cell origin (Gregory et al., 2009). This has
triggers internalization. Hence, alteration of DC-SIGN expres- important implications for host control of HHV-8 infection,
sion could be a strategy used by HHV-8 to escape immune as signaling through the TLR1/2/6 complex, TLR7, TLR9, and
defensesandleadtoanon-robustimmuneresponse(Wangetal., TLR10 affects multiple stages of B cell activation, proliferation,
2002). cytokine secretion, terminal differentiation, and Ab secretion in
response to T cell-dependent antigens (Bekeredjian-Ding and
HHV-8INFECTIONOFLANGERHANSCELLS(LC)AND Jego,2009).
INTERSTITIAL-DERMALDC(iDDC)
TheskinandmucosacontainstwomajortypesofDC:(1)langer- SUMMARY
hanscells(LC),whichresideintheepidermisinclosecontactwith Utilizingheparansulfate,cellsurfaceintegrins,andDC-SIGNfor
keratinocytesand(2)interstitial-dermalDC(iDDC),residentin binding and entry, HHV-8 establishes infection in professional
thedermisandmucosallayers.LCandiDDCprocesscutaneous APC that are essential to processing and presenting antigen. As
antigens and migrate to draining lymph nodes to present anti- theseHHV-8-targetedAPCinitiateTandBcelladaptiveimmune
genstoTandBcells.BecauseofthestrategicpositionofLCand responses,thiscoulddemonstrateanevolutionarymechanismto
iDDCandtheirabilitytocapturepathogens,thesecellscouldrep- establish viral latency and persistence in the host. Accordingly,
resent potential targets for HHV-8 infection. Furthermore, due regulationofviralproteinexpressionlimitsdetectionofHHV-8
to the expression of the C-type lectins, i.e., langerin (CD207) inAPCbyTcells,resultinginsustainedlatency.Althoughvalu-
and DC-SIGN, on LC and iDDC, respectively, it is tempting able conclusions have been drawn from immortalized cell lines
to speculate that HHV-8 could utilize the same entry mecha- as surrogates for these APC, primary cell models such as blood
nisms as seen in MDDC (Rappocciolo et al., 2006). Therefore, and tissue APC provide a more natural account of the qual-
it is important to determine if these APC are also targeted by ity of HHV-8 infection, better displaying the mechanisms of
HHV-8 and whether they support full lytic replication or an latency and abortive and non-abortive virus replicative cycles.
abortive cycle. LC and iDDC can be generated from pluripo- ThisisreflectedbythefactthatamongprofessionalAPC,HHV-8
+
tent cord blood CD34 cells (Caux et al., 1997), which could undergoesfull lyticreplicationonlyinactivated Blymphocytes,
provetobevaluabletoolstostudy HHV-8infection andsubse- yet can bind to, enter and alter various functions of DC and
quentantigenprocessandpresentationtoTcells(Colletonetal., monocytes/macrophages.
2009).
CYTOKINESANDCHEMOKINESINHHV-8INFECTIONOFAPC
HHV-8INFECTIONOFPLASMACYTOIDDC(pDC) Cytokines and chemokines produced by inflammatory APC, as
Plasmacytoid DC (pDC) are a lymphoid-lineage subset of APC wellasTcells,playacrucialroleinHHV-8replicationanddevel-
that produce extraordinary amounts of the antiviral protein opmentofKS.InflammatorychangesoccurearlyinKSpriorto
interferon α (IFN-α) in response to virus infection (Liu, 2005). thedetectionofthecancer(Mesrietal.,2010).Proinflammatory
AlthoughpDCdonotexpressDC-SIGN,theyaresusceptibleto processes drive early stage KS to develop into mature, spin-
in vitro infection with GFP-tagged HHV-8 (West et al., 2011). dle cell lesions (Rezaee et al., 2006). Thus, KS tumors are
GFPexpressionwasdetectedin23%ofpDCfromhealthyblood comprised of spindle-shaped cells of endothelial origin (Regezi
donors at 16h post-infection. Also, ORF57 and LANA expres- et al., 1993) in an environment rich in inflammatory cell infil-
sionwasdetected byPCR at48 and72h, respectively. Infection trates, including B cells, monocytes/macrophages, and CD8+ T
ofthepDCresultsinupregulationofactivationmoleculeCD83 cells (Monini et al., 1999). The infiltrating cells produce large
andTcell co-receptorCD86,andinducesproductionofIFN-α. amounts of Th1 polarizing, proinflammatory cytokines (e.g.,
InductionofIFN-αbyHHV-8occursthroughactivationofToll- IFN-γ, IL-1β, TNF-α and IL-6), chemokines (e.g., IL-8), and
likereceptor9(TLR9)signalinginpDC.Atpresent,however,itis growthfactors[e.g.,vascularendothelialgrowthfactor(VEGF)],
unclearwhatreceptorsHHV-8usestoinfectpDC,andwhether which can induce the KS-like phenotype observed in activated
HHV-8infectionofpDCresultsinanabortiveorfullylyticviral endothelialcells(Fiorellietal.,1998;Moninietal.,1999;Ensoli
replicativecycle. etal.,2000).IFN-γistheearliestandmostabundantinflamma-
tory cytokine observed in KS (Fiorelli et al., 1998) and can be
HHV-8INFECTIONANDTLR detected inKSlesionsbeforeevidenceofHHV-8DNA(Monini
Several types of TLR expressed on different APC are emerg- et al., 1999). IL-6 is also found at very high levels in both KS
ing as important factors in the innate and adaptive immune lesions and in circulation of patients with MCD (Ambroziak
response to HHV-8. Notably, virus triggering of C-type lectins, et al., 1995). In MCD, IL-6 induces B cell proliferation and
includingDC-SIGN,incombinationwithTLRtriggeringonDC causesinflammatoryclinicalsymptoms(SchulteandTalat,2010).
induces signaling and cytokine responses. These in turn regu- Observations from a transgenic mouse model demonstrate that
late T cell polarization that is central to host immune control mice expressing viral IL-6 but lacking mammalian IL-6 do
of infections (Van Kooyk, 2008). In addition, TLR have also not experience phenotypic changes (e.g., lymphoadenopathy,
beenimplicatedinreactivationofHHV-8.TRL7/8couldcontrol hypergammaglobulinemia,splenomegaly)associatedwithMCD
FrontiersinImmunology|HIVandAIDS January2013|Volume3|Article427|6
Knowltonetal. APCinHHV-8infection
(Suthauset al.,2012). IL-6,aswell asoncostatinM (OSM)and IFN-α (West et al., 2011). In monocytes, production of IP-10,
IL-10, arealso detected at high levels in PEL cells. Proliferation IFN-β1,MCP-1,andIRF-1occursinconjunctionwithanupreg-
of PEL can be inhibited when receptors for the IL-6 path- ulation of TLR3 expression (West and Damania, 2008). Our
way are blocked (Drexler et al., 1999). Thus, an as yet mini- lab has previously demonstrated that MDDC infected in vitro
mally detailed imbalanceinthe Th1-Th2 milieu duringHHV-8 with HHV-8 secrete IL-6, TNF-α, IP-10, MIP-1α, and MIP-1β
infection appears to be closely linked to APC in driving the (Hensleretal.,2009).WhileIL-12p40expressionincreasespost-
outgrowth of KS endothelial cells, as well as PEL and MCD infection, bioactive IL-12p70 isnot detected inHHV-8 infected
Bcells. MDDC. This suggests a virus-related inhibition of constitutive
Other cytokines and chemokines produced by APC, partic- production of IL-12p35, or a defect in complexing of these
ularly IL-8 and MCP-1, are elevated in serum of KS patients subunits into IL-12p70. Furthermore, the results support an
and have been implicated in many cancers (Sun et al., 2006; intentional skewing of cytokine production in HHV-8-infected
Mehrad et al., 2007). Enhanced expression of MCP-1, but not MDDCtowardinductionofaTh2responsethatcouldenhance
otherNF-kβactivated cytokines(RANTES,IL-8andTNF-α),is developmentofKS.
alsodetectedininvitroinfectedhumanumbilicalveinendothe- WespeculatethatcytokineandchemokineprofilesinHHV-8
lial cells (HUVEC) (Caselli et al., 2007). When bound to its infected Bcellsissimilartothecytokinedysregulationobserved
CCR2 receptor on endothelial cells, MCP-1 results in chemo- inEBV-associateddisease(Gosselinetal.,1992;Kleinetal.,1996;
taxis and mediates angiogenesis in vitro (Galvez et al., 2005; Kurzrock, 2001; Glaser et al., 2006). Elevated levels of IL-1β,
Mehrad et al., 2007). KS tumors are highly vascularized with TNF-α,IL-6,IL-8,andIL-10aredetectedintheserumofpatients
abnormal angiogenesis, leading to enhanced blood flow to the with EBV-associated diseases, while a less favorable outcome
tumor by expanding pre-existing blood vessels (Mesri et al., correlates with increases of IL-6 and IL-10 in Hodgkin’s lym-
2010). IL-1β, TNF-α, IL-8, and IL-6 can also enhance tumor phoma(Fayadetal.,2001).CommonstrategiesbetweenEBVand
cell growth and vascularization (Ensoli et al., 1989; Ensoli and HHV-8, such as NF-κB signaling pathway alterations (Hayden
Sturzl, 1998; Fiorelli et al., 1998) by inducing the expression of and Ghosh, 2008; De Oliveira et al., 2010) and the expression
two angiogenic mediators, i.e., VEGF and fibroblastic growth of virokines (Sin and Dittmer, 2012), imply that an imbalance
factor (Ensoli et al., 1989; Cohen et al., 1996; Cornali et al., ofimmunemediatorsisassociatedwiththeoncogenesisofthese
1996; Monini et al., 1999). In addition to angiogenesis, inflam- gammaherpesviruses.
matory cells and cytokines can contribute to viral reactivation
and replication. IFN-γ was shown to induce ORF59 expression HHV-8ENCODEDPROTEINSINVOLVEDINIMMUNEMEDIATOR
inBCBL-1(Blackbournetal.,2000a)andreactivatelatentHHV- RESPONSES
8 in BC-3 PELcells byactivation ofPim-family kinases(Cheng Besidescellularcytokinesandchemokines,HHV-8encodessev-
et al., 2009). Mercader et al. showed OSM, IFN-γ, and hepato- eral proteins that share homology to host genes. These are
cytegrowthfactor/scatterfactor-inducedlyticcycleactivationof involved in inflammation and angiogenesis that contribute to
BCBL-1 resulting in virion production (Mercader et al., 2000). the inflammatory environment observed in KS. Cytokines and
ThisprinciplehasbeendemonstratedinHHV-8infectedPBMC, chemokines encoded by HHV-8 havebeen the focus of numer-
where inflammatory cytokines could maintain or increase viral ous studies and reviews (Nicholas, 2005; Gasperini et al., 2008;
load up to 10-fold when the infected cells were cultured in the Mesrietal.,2010;SakakibaraandTosato,2011;Leeetal.,2012;
presenceofinflammatorycytokines(Moninietal.,1999). SinandDittmer,2012).Thus,vIL-6has24%homologytohuman
In AIDS-related KS, immune dysregulation and induction IL-6 and can induceexpression ofVEGF and MCP-1 (Nicholas
of inflammatory cytokines acts to further enhance KS tumor etal.,1997).Theseinturntriggerangiogenicpathways.Elevated
growth. When BCBL-1 cells that are latently infected with levelsofvIL-6,aswell aslevelsofhumanIL-6andHHV-8viral
+
HHV-8 are cultured with HIV-1 infected CD4 T cells, sol- load, have been associated with a recently described syndrome
uble factors secreted by the T cells cause the virus to enter ofseveresystemicinflammatorysymptoms(Uldricketal.,2010).
lyticreactivation(Mercaderetal.,2000).Inflammatorycytokines However,consistentlyreproducibleassaysforquantitationofvIL6
induced by both HIV-1-infected and HHV-8-infected cells pro- areneededtoextendsuchstudies.
mote expression of receptors for HIV-1 Tat, which acts as a The G-protein coupled receptor (vGPCR) is an early
progressionfactorinKSdevelopment(Ensolietal.,1990;Barillari lytic phase gene homologous to the IL-8 receptor, CXCR-2
et al., 1993) and increasing viral load (Harrington et al., 1997). (Arvanitakis et al., 1997; Nicholas, 2005). vGPCR constitutively
Indeed, serumandcell samplestakenfromKSlesionsofHIV-1 signalsandresultsinenhancedproductionofIL-1β,IL-8,MCP-1,
infected individuals co-infected with HHV-8 show markedly IL-6, and VEGF that can have both autocrine and paracrine
increased levels of inflammatory cytokines, growth factors, and effects (Gershengorn et al., 1998; Schwarz and Murphy, 2001).
angiogenic mediators (Ensoli and Sturzl, 1998; Pugliese et al., K1 and K15 are signal transducing proteins that induce VEGF,
2002). IL-6, and IL-8 (Choi and Nicholas, 2010). LANA and the viral
HHV-8 has a broad cellular tropism in vivo including B flice inhibitory proteins (vFLIP) have been linked to enhanced
cells, endothelial cells, monocytes, keratinocytes, and epithelial cytokineproductionviaactivationoftheMAPKandNF-κBpath-
cells that could result in production of inflammatory medi- ways, respectively (Wang and Boshoff, 2005). vIRF3 expression
ators (Chakraborty et al., 2012). In pDC, HHV-8 induces inhibits MHC class II expression as well as IFN-γ production
enhanced levels of IL-6, IL-8, MIP-1α, MIP-1β, CCL22, and (Schmidt et al., 2011). Finally, viral macrophage inflammatory
www.frontiersin.org January2013|Volume3|Article427|7
Knowltonetal. APCinHHV-8infection
proteins (MIPs) (vCCL1, vCCL2, vCCL3) share homology to (Wheatleyetal.,1998;RedchenkoandRickinson,1999;Subklewe
MIP1-αandRANTESandcaninducemonocytechemotaxisand et al., 1999a,b, 2001, 2005; Lin et al., 2002). Other studies have
+ +
signaltransduction(Arvanitakisetal.,1997;Nakanoetal.,2003; revealed polyfunctional CD8 and CD4 T cell reactivity and
Nicholas, 2005). Given the plethora of such data derived from newMHCclassIepitopesforHIV-1GagandNefusingpeptide-
highlymanipulatedmolecularandcelllinemodels,thechallenge loaded DC(Huanget al.,2010). Importantly, wehaveused this
is to link these unique HHV-8 factors directly to HHV-8 infec- DC model to map epitopes of HHV-8 lytic and latency pro-
tionanddevelopmentofcancersinnaturalcellulartargetsofthe teins with libraries of synthetic, 15mer peptides overlapping by
virus. 11aa (Lepone et al., 2010). Nevertheless, it may be more prac-
tical to generate large numbers of CD40L-activated, autologous
SUMMARY B cells that favorably compare to DC as APC (Schultze et al.,
Adelicatebalanceexistsbetweenprotective immunityinvolving 2004).
+ +
cytokine and chemokine production by host APC and virus- To date, relatively few CD8 and CD4 T cell epitopes
driven induction of cytokines and chemokines that aid in the within only 15 of the over 80 ORFs of HHV-8 have been
∗
dissemination ofinfection andmediate pathogenesis. Severalof identified, and most of these are restricted by HLA A 0201
themediatorsthatareessentialtotheimmuneresponseandacti- (Robey et al., 2010). Information is therefore needed on the
vationoflymphocytescanexacerbateinfectionandcauseclinical broad range of potential antigenic sites in the virus that are
symptoms when over produced in response to HHV-8 infec- restricted by other MHC class I and II haplotypes. Moreover,
tion, including IFN-γ, IL-1β, IL-6, IL-8, TNF-α, and MCP-1. no studies have yet established a hierarchy of naïve and mem-
+ +
The roleofeach cytokine/chemokine inthe developmentofKS ory CD8 or CD4 T cell responses to HHV-8 epitopes in
and the inflammatory environment observed within KS tumors control of HHV-8 infection. There also are minimal data on
likely varies depending on their quantity and origin. Therefore, whether alterations in anti-HHV-8 T cell responses are related
production of these immune mediators by cells the virus nat- to development of KS (Guihot et al., 2006) and whether the
urally targets for infection may better reflect HHV-8-induced, lowerincidenceofKSinHIV-1infectedpersonsreceivingARTis
cytokine/chemokinedrivenpathogenesis. related to increasesinanti-HHV-8Tcell responses(Bourboulia
et al., 2004; Bihl et al., 2007a). Such information is important
APC-TCELLINTERACTIONSINHHV-8INFECTION for development of prophylactic and therapeutic vaccines for
Given our rudimentary understanding of HHV-8-APC interac- HHV-8.
tions, we know even less regarding HHV-8-specific T cell-APC HHV-8 infection alters the capacity of DC to be recognized
interactionsandtheirroleincontrollingviralinfectionanddis- byandactivateCTL.Bothdirectpresentationusingviralproteins
ease.Akeychallengeistoadaptcurrentinvitromodelsusingcell endogenouslyproducedinDC,andcross-presentationpathways
linesandHHV-8constructstosystemsthatallowdecipheringof usingviralproteinsfromexogenoussourcesofvirusarelikelyto
thebasicstepsofnaturalHHV-8infection,andantigenprocess- beoperativeinHHV-8infection.Infact,EBVdoesnotreplicate
+
ingand presentation, invarioustypes ofAPC. Theinteractions inMDDC,which instead activate anti-EBV CD8 Tcells byan
of APC with T cells that underlie the generation of anti-HHV- antigen cross-presentation pathway(Herret al.,2000; Subklewe
8 T cell immunity begin with DC of myeloid origin that take etal.,2001;Popescuetal.,2003).
up viral antigen at local sites of infection, then travel to the ItispossiblethatHHV-8infected,apoptoticendothelialcells,
draininglymphatics,andinduceantiviralTcellresponses(Ueno macrophages, and B cells are recognized as “distressed” cells at
et al., 2007). There are specialized subsets of DC that populate localsitesofinfectionandengulfedbyLCandiDDC(Uenoetal.,
differenttissue sitesandhavedistinct virologicinteractions and 2007).TheseDCcouldthenmigratetolocallymphnodeswhile
immunologicfunctions.Myeloid-derivedLCandiDDCpopulate processing the ingested viral proteins through alternative MHC
+
theepidermisanddermisrespectively,andareassociatedwithKS classIpathwaysforpresentationto CD8 Tcells.Furthermore,
lesions. iDDC are similarin phenotype and function to dermal severalHHV-8proteins,particularlythosecodedbyORFK3and
DC,andarelinkedtosystemicKSlesions.OtherDCsubsetssuch K5, have intriguing properties of altering expression of MHC
asCD141+DCwhicharethehumansurrogatesofmouseCD8α classI,Tcellcoreceptors,andDC-SIGN.Interestingly,cytokines
DC subsets (Bachem et al., 2010; Jongbloed et al., 2010), could released by PELs can interfere with the in vitro differentiation
+
benaturaltargetsforHHV-8.Thesecellsexhibitstrongpriming of immature MDDC from CD14 monocytes (Cirone et al.,
ofTcellstoantigen.Itisimperativethatweassesstranscription 2008).
+
of HHV-8 ORFs in natural targets of the virus, in comparison Anintriguingrecent discoveryisthatactivated CD4 Tcells
to well-documented immunomodulatory properties of HHV-8 suppressHHV-8lyticreplicationintonsillarBcells(Myoungand
expressedincelllinesandartificialconstructs,suchaspersistently Ganem,2011a).Thesuppressiveactivityrequirescell-cellcontact.
infectedBCBL-1(Coscoy,2007). However,itisnotaclassicCTLresponse,asitcanbemediatedby
Interactions of HHV-8 with DC subsets could be critical at TcellsfromHHV-8seronegativepersons,isnotMHCrestricted
the site of virus replication, and be centrally involved in gener- and does not lyse the B cell targets. This is proposed to be a
atingTcellresponsestothevirus.EfficientactivationofHHV-8 pathwaybywhichHHV-8isdrivenintolatencyinBcells.These
+ + +
epitope-specific CD8 T cells requires presentation by peptide- CD4 TcellsarereminiscentofCD8 Tcellsthatexhibitnon-
loaded,autologous,matureDC(Wangetal.,2002).Thisissimilar cytotoxicresponsesthatsuppressHIV-1infection (Killianetal.,
to optimal activation of anti-EBV CTL by peptide-loaded DC 2011).
FrontiersinImmunology|HIVandAIDS January2013|Volume3|Article427|8
Knowltonetal. APCinHHV-8infection
+
ALTEREDHHV-8ANTIGENPROCESSINGANDPRESENTATION CD8 expanded T cells in patients with classic KS that share a
Presentation of HHV-8 proteins to both CD8 (MHC class I TCR-βvariablesubunitbias(Galleuetal.,2012),aphenomenon
restricted)andCD4(MHCclassIIrestricted)Tcellsisimpaired observedinresponsetochronicviralinfections(Trautmannetal.,
+ +
byHHV-8infection.Evidencesuggeststhatanti-HHV-8CD8 T 2005;Wynnetal.,2010).Second,CD8 TcellimmunitytoHHV-
cellresponsescanbeinhibitedbyK3andK5proteinsthatdown- 8proteinsispresentinHHV-8seropositive,healthyindividuals.
+
regulate MHC class I expression (Coscoy and Ganem, 2000; CD8 Tcellsspecificfor5HHV-8lyticcycleproteinsarepresent
Ishido et al., 2000b). Interestingly, K5-encoded MIR2 down- inbloodinthefirstfewmonthsofprimaryHHV-8infectionof
regulates T cell costimulatory molecules ICAM-1 and CD86 normaladults(Wangetal.,2001).ThisprimaryCTLandIFN-γ
(CoscoyandGanem,2001)andIFNγR1(Lietal.,2007b)which responsetoHHV-8peakswithin2yearsofinfection,andwanes
could act to decrease T cell responses to HHV-8. Ishido et al. thereaftertolowbutdetectablelevels.Furthermore,KSdoesnot
+
showed thatK5 also dampensnaturalkiller (NK)cell-mediated commonlyoccurinHIV-1infectedindividualswithhighCD4
cytotoxicity by downregulation of ICAM-1 and CD86 (Ishido Tcellcounts(Strickleretal.,1999).
et al., 2000a). The NK activating receptor, NKG2D, responsi- To date, however, there is little direct evidence for a role of
ble for detecting infected cells, is downregulated by HHV-8 K5 T cell immunity in HHV-8 infection and control ofKS (Hislop
+
(Thomas et al., 2008) via the release of the tumor-associated and Sabbah, 2008). Lower CD8 T cell responses have been
prostaglandinE2(PGE2)fromKScells(Dupuyetal.,2012).This found in persons with KS compared to asymptomatic persons
also results in inhibition of IL-15-mediated NK cell activation (Guihotetal.,2006;Lambertetal.,2006).However,verymodest
and survival, addingto the immuneescapetactics employed by increases in CD8 T cell responses to HHV-8 immunodominant
this virus (Dupuy et al., 2012). Likewise, infection of primary peptides are found in persons on ART (Wilkinson et al., 2002;
fibroblastsresultsinlimitedNKcellactivationandkillingactivity Bourboulia et al., 2004). While progressive increases in HHV-8
(Matthewsetal.,2011).Branderetal.(2000),reportedadecrease load precede development of disease in HIV-1-infected persons
in lysis by HIV-1 peptide-specific CTL clones of cells infected (Campbelletal.,2000;Laneyetal.,2007),evidenceislackingfor
with HHV-8. Thus, it is apparent that K3 and K5 have multi- a direct association between control of HHV-8 load and HHV-
factorialeffectsonimmunecontrolofHHV-8infection.Ofnote 8-specific, T cell immunity (Guihot et al., 2006). Nevertheless,
is that the intracellular load of HHV-8 in infected endothelial anincreased incidence ofKS in organtransplantrecipients and
cells is directly related to their loss of expression of MHC class HIV-1-infected persons (Dedicoat and Newton, 2003) suggest
I and ICAM-1, in association with expression of MIR2 (Adang a role for T cell immunity in prevention of KS, similar to T
etal.,2007).Interestingly, EBVinfectionalsodecreasesrecogni- cell immunity in EBV-related cancers (Gottschalk et al., 2005).
tionoflatentlyinfectedcellsbydownregulationofMHCclassI Reductionofimmunosuppressiveregimenscanresultinsponta-
molecules,particularlyincellsderivedfromBurkitt’slymphoma neousresolution ofKSinorgantransplant recipients (Firoozan
(Hislopetal.,2007). etal.,2005).Similarly,theincidenceofKShasdeclinedaftersup-
MHC class II recognition is dampened by HHV-8 infection. pressionofHIV-1byART(Rabkin,2001),whereTcellnumbers
Sabbahetal.reportedthatLCL,withanintactMHCclassIIpro- and function are partially restored (Rinaldo et al., 2000; Letvin
cessingpathway,couldpresentLANApeptidestoLANA-specific and Walker, 2003; Benito et al., 2004). There are also shorter
+
CD4 T cell clones, whereas PEL cells were not recognized in incubationperiodsfordevelopmentofKSafterHHV-8infection
an IFN-γ ELIspot (Sabbah et al., 2012). PEL express vIRF3, a in HIV-1-infected men compared to men infected with HHV-8
known inhibitor of the MHC class II master regulator CIITA priortoHIV-1infection(Gaoetal.,1996;Jacobsonetal.,2000).
(classIItransactivator)(Schmidtetal.,2011).WhenCIITAfunc- Primary infection with HHV-8 in immunosuppressed persons
+
tionwasrestoredinPEL,CD4 Tcellclonerecognitionwasalso has a more severe outcome than reactivated HHV-8 infection.
restored (Sabbah et al., 2012), supporting a role for HHV-8 in Finally,HHV-8expressesmanyproteinsthathaveimmunomod-
the reduction of MHC class II expression. Interestingly, IFN-γ ulatory functions that could down-regulate T cell immunity
inducible expression of CIITA results in MHC class II expres- (AresteandBlackbourn,2009).
siononendothelialcells,andisimpairedafterHHV-8infection The emerging biology of KS and HHV-8 infection presents
throughinductionofsuppressorofcytokinesignaling3(SOCS3) intriguingfactorsthatinterrelateHHV-8-specificTcellimmunity
(Butleretal.,2012).Thisresultsininhibitionoftheearlyevents to control of the cancer. HHV-8 is found as a latent infec-
intheIFN-γsignalingpathway. tion in most of the spindle cells in the KS lesion (Moore and
In sum, various HHV-8 proteins appearto playa significant Chang, 1995; Foreman et al., 1997; Dupin et al., 1999; Boshoff
role in the disruption of antigen processing and presentation. and Chang, 2001). Since replication of herpesviruses in suscep-
However,furtherdataareneededtounderstandtheextentofviral tiblecellsresultsincelldeath,latencymustbeestablishedeither
protein function in immunopathogenesis of HHV-8 infection verysoonafterinfectionorpossiblyfollowinganabortive(non-
inAPC. productive)infection. Asmallpercentage ofendothelialandKS
spindle cells express a complete replication library of HHV-8
TCELLRESPONSESTOHHV-8:RELATIONTOHHV-8DISEASE proteins early in the disease, whereas the majority of the trans-
PROGRESSION formed cells ultimately express only HHV-8 latency proteins.
Although immunity to HHV-8 isfarless well-defined than that Circulating B cells and monocytes can be positive for HHV-8
to EBV, T cell immunity to HHV-8 likely plays a similar, criti- DNA (Ambroziak et al., 1995; Blasig et al., 1997), and HHV-
+
cal rolein viral control. First, there is anincreasein CD4 and 8-infected macrophages are present in KS tissues (Blasig et al.,
www.frontiersin.org January2013|Volume3|Article427|9
Knowltonetal. APCinHHV-8infection
1997). Th1 cytokines have been implicated in reactivation and CTLlysisofHHV-8infectedcells,includinghowitcomparesto
persistenceofHHV-8inBcellsandmonocytesfromKSpatients otherputative,intransinhibitorsofCTLfunctionsuchasK3and
(Siriannietal.,1998).TcellinfiltratesarecommoninKStissues K5.Moreover,theEBNA1-CTLinhibitionconcepthasundergone
+
(Blasigetal.,1997;Fiorellietal.,1998).CD8 TcellsinKStissues major revision. First, the GAr domains of EBNA1 can inhibit
produceIFN-γandexpressHLADR(Fiorellietal.,1998;Sirianni mRNA translation, which may be more critical to lack of CTL
et al., 1998), suggesting that tumor-infiltrating lymphocytes are recognitionthaninhibitionofproteosomalprocessing(Yinetal.,
respondingtoHHV-8antigens. 2003).Second,EBNA1infectedcellsexpressEBNA1peptidesthat
Comprehensive longitudinal studiesareneeded to accurately canberecognizedbyCTLwhenassessedinmoresensitiveassays
assesstheroleofanti-HHV-8Tcellimmunityindevelopmentof (Lee et al., 2004). This indicates that the effects of LANA1 on
KS.TcellresponsestoHHV-8couldbedirectedatdifferentlytic pathways related to CTL function that use chimeric constructs,
and latency proteins at different stages of infection and disease, indicatorcelllines,etc.,needtobecharacterizedinanaturalcon-
+
similartoEBV(Gottschalketal.,2005;HislopandSabbah,2008). text using CD8 CTL and natural targets that are specific for
By comparison, the immediate early regulatory protein BMLF1 LANA1.
+
and other early and late lytic cycle proteins are targets for CD8 Similar to EBV, CD8 T cell responses to HHV-8 tend to
CTL during primary and latent EBV infection (Bogedain et al., be directed toward lytic antigens (Robey et al., 2009). While
+
1995;Stevenetal.,1997;Hislopetal.,2007). there are much fewer CD8 T cell epitopes known for HHV-8
than EBV, the majority of these epitopes are within the early
TCELLRESPONSESTOHHV-8:RELATIONTOTCELLRESPONSESTO and late lytic proteins (Robey et al., 2010, 2011). With regard
EBV to polyfunctionality, one study found that for both EBV and
We propose that T cell immunity in EBV infection provides HHV-8,Tcellsspecificforlatencyantigensweremorepolyfunc-
lessonsforwhat islikelyoccurringinHHV-8 infection. During tional than those specific for lytic antigens (Bihl et al., 2007b).
mononucleosis caused by a primary infection with EBV, T cells The phenotype of these cells was also found to be different,
specific forbothlyticand latencyEBV proteins arepresent, but withagreaterproportionofeffectormemoryTcellsspecificfor
responsestolyticepitopestendtobestronger(Longetal.,2011). latency antigens than lytic antigens for both EBV and HHV-8.
+
In healthy EBV seropositive individuals, CD8 T cell responses In both EBV and HHV-8-associated malignancies, latency pro-
are also found to be greater for lytic epitopes, with up to 3% teinsarepredominantlyexpressed,soitisthoughtthatresponses
of cells specific for a single lytic epitope and up to 0.5% for to latency proteins could be important in controlling these dis-
a single latency epitope (Hislop and Sabbah, 2008). Anti-EBV eases (Hislop and Sabbah,2008; Taylorand Blackbourn, 2011).
+
CTLresponsesshiftduringlatentinfectiontoEBVnuclearanti- Evidence suggests that there are higher levels of CD8 CTL
gensEBNA3andLMP2,whilestillretainingspecificityforsome specific for EBV and cytomegalovirus (CMV) than HHV-8 in
lytic cycle proteins (Hislop et al., 2002). The hierarchy of CTL the bloodofseropositive individuals(Wanget al.,2002; Guihot
responses to immunodominant epitopes of EBV is related to a etal.,2006).HigherTcellresponsesto EBVandCMVantigens
lower expression of latency proteins in infected cells (Pudney couldberelatedtotheirgreaterviralloadinpersistentlyinfected
etal.,2005).AlthoughHHV-8doesnothavegeneshomologous persons, with more turnover of viral antigen from latent, per-
to EBNA and LMP, HHV-8 latency-associated nuclear antigen sistent reservoirs that maintains a greater level of memory CTL
(LANA or ORF73), kaposin (T0.7 or ORF K12), and K1 are precursors.
+
putative latency and transforming proteins that are targets for Antigen-specific CD8 T cells occupy a lineage of naïve and
CTL (Osman et al., 1999; Brander et al., 2001; Lepone et al., memorycompartmentsthatareinvolvedintheexpansion,effec-
+
2010). tor, and contraction phases of CD8 memory T cells (Halwani
+
Host selection of CD8 T cell epitopes within HHV-8 pro- et al., 2006). Central memory and effector memory T cells are
teins could be based in part on the relative expression of viral contrasted based on expression of surface molecules related to
proteins by the MHC class I endogenous pathway, compara- migration and differentiation. Patients with MCD have more
ble to EBV (Levitsky et al., 1996). However, evidence from the CD8+CD45RA−CCR7−CD27− IFN-γ+ cells (a late memory T
+ − − +
anti-EBV CTLfield indicatesthat CTLreactivity to this gamma cell phenotype) and fewer CD8 CD45RA CCR7 CD27 cells
herpesvirusvariesastotheHLAhaplotype,withdifferentMHC (early and intermediate T cell phenotype) than normal, HHV-
classIhaplotypesexhibitingdifferentCTLreactivitytothesame 8 seropositive controls. This phenotypic shift is not found for
+
EBV proteins (Hislop et al., 2007). Perhaps HHV-8 has mecha- EBV-specific CD8 T cells. Interestingly, HHV-8 viral loads are
nismssimilartotheGly-Ala(Popescuetal.,2003)repeatdomain negatively correlated withearlyandintermediate effector mem-
in EBNA1 that inhibits proteosome processing of viral proteins orycells. Themoredifferentiated Tcell phenotypeisassociated
throughtheMHCclassIpathway(Levitskayaetal.,1995;Hislop with disease, rather than a loss of HHV-8-specific CD8 T cells
et al., 2007), thereby inhibiting generationof EBNA1-specific T orpolyfunctionalactivity,astheHHV-8-specificTcellsaresim-
cells.Infact,LANA1caninhibitproteinprocessingincis(Kwun ilar in function (secretion of IFN-γ, TNF-α, MIP1-β, and/or
et al., 2007; Zaldumbide et al., 2007). Bioinformatic analysis of CD107a)ininfectedpatientsandhealthycontrols(Guihotetal.,
HHV-8sequencessupportsthatlatencyproteinsarelikelyto be 2008).
poorer targets for CTL than immediate early or lytic proteins In healthy, HHV-8 seropositive individuals controlling
(Vider-Shalit et al., 2007). However, it is not yet clear if the in infection, there are both monofunctional and polyfunctional
+
cisfunctionofLANA1isdirectlyinvolvedindown-regulationof CD8 T cells present that are specific for HHV-8 proteins
FrontiersinImmunology|HIVandAIDS January2013|Volume3|Article427|10