Immunoregulatory CD4-CD8- T cells as a potential therapeutic tool for transplantation, autoimmunity, and cancer.

0.95 MB·English

by  Hillhouse, Erin E.

REVIEWARTICLE
published:24January2013
doi:10.3389/fimmu.2013.00006
− −
Immunoregulatory CD4 CD8 T cells as a potential
therapeutic tool for transplantation, autoimmunity,
and cancer
ErinE.Hillhouse1,2,Jean-SébastienDelisle2,3,4 andSylvieLesage1,2*
1DepartmentofMicrobiologyandImmunology,UniversityofMontreal,Montreal,QC,Canada
2ResearchCenter,Maisonneuve-RosemontHospital,Montreal,QC,Canada
3DepartmentofMedicine,UniversityofMontreal,Montreal,QC,Canada
4DivisionofHematologyandOncology,Maisonneuve-RosemontHospital,Montreal,QC,Canada
Editedby: Acentralobjectiveinorgantransplantationandthetreatmentorpreventionofautoimmune
LucienneChatenoud,UniversitéParis diseaseistheachievementofantigen-specificimmunetolerance.Anadditionalchallenge
Descartes,France
in bone marrow transplantation for the treatment of hematological malignancies is the
Reviewedby:
prevention of graft-vs-host disease (GVHD) while maintaining graft-vs-tumor activity.
JulianDyson,ImperialCollege Interestingly, CD4−CD8− (double negative, DN)T cells, which exhibit a unique antigen-
London,UK
BinLi,ChineseAcademyofSciences, specific immunoregulatory potential, appear to exhibit all of the properties to respond to
China these challenges. Herein, we review the therapeutic potential of immunoregulatory DN
*Correspondence: T cells in various immunopathological settings, including graft tolerance, GVHD, cancer,
SylvieLesage,ResearchCenter,
andautoimmunity.
Maisonneuve-RosemontHospital,
5415Boulevarddel’Assomption, Keywords:graft-vs-hostdisease,autoimmunity,cancer,grafttolerance,immunoregulation,doublenegativeTcells
Montreal,QCH1T2M4,Canada.
e-mail:[email protected],
[email protected]
INTRODUCTION proliferate vigorously against both MHC-sufficient and MHC-
− −
CD4 CD8 doublenegative(DN)Tcellscomposeapproximately deficientstimulatorcells,butnotintheabsenceofstimulatorcells
1–3% of total T cells in both mice and humans (Strober etal., (VanLaethemetal.,2007;Tikhonovaetal.,2012).Infact,arecent
1989; Abraham etal.,1992; Fischer etal.,2005). Phenotypically, study has revealed the native self-protein CD155 as at least one
this rare T cell subset expresses a polyclonal αβT cell receptor of the ligands recognized by quad-deficient DN T cells via their
(TCR)repertoire,lackstheexpressionofFoxp3aswellasnatural TCRandintheabsenceof antigen-processing(Tikhonovaetal.,
killer (NK) cell markers and mostly presents with a naïve T cell 2012).Therefore,thequad-deficientmousemodelhashelpedto
phenotype(recentlyreviewed;HillhouseandLesage,2012).Inter- demonstratethatDNTcelldifferentiationcanproceedinthethy-
estingly,theirdistinctphenotype,namelythelackofCD4andCD8 mus and that DN T cell activation can occur independently of
co-receptors,isalsobelievedtoinfluencethemethodbywhichDN co-receptors.
Tcellsrecognizeantigensandsubsequentlysignalthroughtheir DoublenegativeTcellshavenotonlybeenobservedinquad-
TCR. In fact, major histocompatibility complex (MHC) restric- deficientmice. Forinstance,anincreasednumberof DNTcells
tionisthecardinalfeatureofantigenrecognitionbyTcells,where has been observed in many TCR transgenic models (Hillhouse
theCD4andCD8co-receptorsrespectivelyfacilitatetheinterac- andLesage,2012).IntheseTCRtransgenicmodels,duetoforced
tionwithMHCclassIIandImolecules.AsDNTcellslackboth expressionof anαβTCRtransgene, theDNTcellsdorecognize
CD4andCD8co-receptors, themeansbywhichtheαβTCRon peptide–MHC complexes in the absence of co-receptor expres-
theDNTcellrecognizesMHCligandswithsufficientaffinityand sion,suggestingthattheyexhibitahighaffinityfortheseantigenic
aviditytoprovideactivationofDNTcellsisunclear.Ithasbeen complexes. Moreover, in the absence of CD28 co-stimulation
recently proposed that DN T cells recognize non-MHC ligands. which is required for clonal deletion of thymocytes (Punt etal.,
Indeed,usingquad-deficientmicethatlacktheexpressionofCD4 1994; Kishimoto and Sprent, 1997; Lesage etal., 1997), some
andCD8co-receptorsaswellasMHCclassIandIIexpression,Van thymocytes that strongly recognize self-ligands survive negative
Laethemetal.(2007)demonstratedthatTcelldevelopmentcould selectionandultimatelydevelopintomaturethymicDNTcells
proceedinanMHC-independentmanner.Infact,intheabsence (Pobezinskyetal.,2012). Together,theseresultssuggestthatDN
ofCD4orCD8co-receptorexpression,theintracellulartyrosine Tcellsmayexhibitarelativelystrongaffinitytowardtheircognate
+ +
kinaseLckisnolongersequesteredbytheco-receptorsandisthus ligands. This property is reminiscent of CD4 Foxp3 regula-
availabletopromoteMHC-independentTCRsignalinginthymo- toryTcells(Tregs)andNKTcells,twoimmunoregulatoryTcell
cytes.Subsequently,thesethymocytesdevelopintomatureDNT subsetswhichundergoagonistselectioninthethymus(Baldwin
cellsthatentertheperiphery(VanLaethemetal.,2007).Theabil- etal.,2004).
ityofDNTcellstorecognizenon-MHCligandsisalsosupported Inadditiontotheirdistinctphenotype,DNTcellsalsoexhibit
bythefindingthatmatureDNTcellsfromquad-deficientmice a unique antigen-specific immunoregulatory potential. Indeed,
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the immunoregulatory function of DN T cells was first identi- priortotheskingraftefficientlyinducedantigen-specificallograft
fied almost 25 years ago, when Strober etal. (1989) successfully tolerance(Yangetal.,1998,1999;Figure1B).Theantigen-specific
clonedDNTcellsfrommiceandsubsequentlydemonstratedthat tolerance to skin allografts induced by the transfer of donor T
DN T cells mediate suppressor activity in a mixed-lymphocyte cells was proposed to be mediated by 2C TCR transgenic DN
reaction (MLR). Subsequently, Dr. Zhang’s group was the first T cells as only the 2C TCR transgenic DN T cell subset, but
+ +
torevealtheantigen-specificimmunoregulatorypotentialofDN not the 2C CD4 or 2C CD8 T cell subset, was able to sup-
Tcells(Zhangetal.,2000). Specifically, theyshowedthatDNT pressanMLRresponseinvitro(Zhangetal.,2000).Accordingly,
cellsfrom2CTCRtransgenicmicesuppresstheproliferationand Zhang’sgroupshowedthattheinjectionof2CTCRF1DNTcell
+
cytotoxic activity of 2C TCR CD8 T cells in vitro, but not of clones was sufficient to induce both prolonged survival of both
+
CD8 T cells carrying other antigen specificities (Zhang etal., skinandcardiacallografts(Zhangetal.,2000;Chenetal.,2003b;
2000; Young and Zhang, 2002). Moreover, the antigen-specific Figure 1C). Importantly, the allograft tolerance was antigen-
propertyof2CTCRDNTcellsisconferred,atleastinpart,bytheir specific,asfullMHC-mismatchedthirdpartygraftswererapidly
abilitytoacquirepeptide–MHCcomplexesfromantigenpresent- rejected (Zhang etal.,2000; Figure 1C). Collectively, these data
ingcells(Zhangetal.,2000;Priateletal.,2001;YoungandZhang, demonstrate that 2C DN T cells are sufficient to induce both
2002;FordMcIntyreetal.,2008),aprocessknownastrogocytosis skin and cardiac allograft survival, suggesting that immunoreg-
(JolyandHudrisier,2003). Notably,theinvitroantigen-specific ulatoryDNTcellscontributetothebenefitsof DLIonallograft
+
suppressiveactivityof 2CTCRDNTcellstowardCD8 Tcells survival.
was replicated using non-transgenic mice and humans (Zhang Tofurtherunderstandthemechanismbywhich2CDNTcells
etal.,2000;YoungandZhang,2002;Fischeretal.,2005).Together, promote antigen-specific allograft tolerance, Young etal. (2002)
these observations described a unique antigen-specific mode of undertook the examination of the leukocytes found within the
immunoregulationprovidedbyDNTcells,leadingtotheantigen- toleratedskingrafts. Indoingso,theydiscoveredthat2CDNT
+
specificeliminationofCD8 Tcells(Zhangetal.,2000;Youngand cellsarethepredominantleukocytefoundwithintheacceptedskin
Zhang,2002). allografts.Moreover,the2CDNTcellsisolatedfrommicewhich
The immunoregulatory potential of DN T cells has since hadreceiveddonorspleencellspriortotheskingraftdemonstrate
+
been shown to extend beyond T cells. Indeed, TCR transgenic anenhancedsuppressivefunctiontoward2CCD8 Tcellsinvitro
and non-transgenic DN T cells can also inhibit NK cells (He (Youngetal.,2002).Finally,thecomparisonoftranscriptomepro-
etal., 2007; Su etal., 2012), B cells (Zhang etal., 2006; Hill- filesbetween2CTCRF1DNTcellsclonesthatareableorunable
houseetal.,2010;FordMcIntyreetal.,2011),anddendriticcells toconfercardiacallografttolerancerevealedFcRγasapotential
(Gaoetal.,2011). Thecombinationof theirdistinctphenotypic moleculeinvolvedindefiningthetolerogenicpotentialof2CDN
characteristics and their unique antigen-specific immunoregu- Tcells(Leeetal.,2005).TheimportanceofFcRγexpressionon2C
latory properties toward multiple cellular targets has prompted DNTcellsfortheinductionofallografttolerancewasconfirmedas
investigators to examine the role of DN T cells in various dis- theadoptivetransferof FcRγ-sufficient, butnotFcRγ-deficient,
ease models. Herein, we will review the promising therapeutic 2C DN T cells prior to transplantation increased skin allograft
potential of DN T cells in the context of various disease set- survival(Thomsonetal.,2006).Altogether,theseresultsdemon-
tings.Morespecifically,wewilldescribetheimpactofDNTcell stratethat2CDNTcellsparticipateinallografttolerance,likely
+
transferontheinductionofgrafttoleranceandthepreventionof by inhibiting pathogenic 2C CD8 T cell responses, at least in
autoimmunityaswellaspresenttheirdualroleinpreventinggraft- theMHCclassI-restrictedgrafttolerancemodel(Hillhouseand
vs-hostdisease(GVHD)whilepromotinggraft-vs-tumor(GvT) Lesage,2012).
responses. Importantly,theseobservationswerenotlimitedtothe2CTCR
transgenicsetting.Indeed,theinjectionofallogeneicdonorspleen
GRAFTTOLERANCE cellsbearingasingleMHCclassImismatchpriortoskintrans-
Althoughbetterknownfortheiruseinhematopoieticcelltrans- plantationinthenon-transgenicsettingalsoresultedinprolonged
plantationtoestablishdonorchimerismortreatneoplasticrelapse, allograftsurvival(Yangetal.,1999;Youngetal.,2002;Zhangetal.,
donorleukocyteinfusions(DLI)havealsobeenshowntoimprove 2002). Analogous to the 2C TCR model, DN T cells from non-
allograft survival after solid organ transplantation (Fehr and transgenicmicethatareactivatedinvivofollowingtheinjection
Sykes,2004). Amongpossiblemechanismslinkingdonorleuko- of donor spleen cells preferentially accumulate within the skin
+
cytetransferandallografttolerance,DNTcellshavebeenshown allograftandeliminateCD8 Tcellsinanantigen-specificman-
toincreaseallograftacceptanceinvariousexperimentalsettings. ner in vitro (Young etal.,2002). In a full MHC-mismatch heart
In an attempt to understand why DLI has a positive outcome allograft, injection of non-transgenic DN T cells further pro-
on allograft survival, Dr. Zhang’s group took advantage of the motedrapamycin-inducedgrafttolerance(Zhangetal.,2011b).In
antigen-specific2CTCRtransgenicmodel(Yangetal.,1998,1999), addition,invitro-generatedDNTcellscanalsoprovideantigen-
wherethe2CTCRisalloreactivetotheLd MHCclassImolecule specificskinandpancreaticisletallografttolerance(Zhangetal.,
(Shaetal.,1988). Predictably,skingraftsbearingasingleMHC- 2007,2011a).Moreover,withregardstoxenografts,theinjection
mismatchatLdarethusrapidlyrejectedbythe2CTCRrecipient ofnon-transgenicratdonorspleencellsinmicepreventedCD4+
mice due to the expression of Ld MHC class I molecule on the T cell-mediated cardiac xenograft rejection (Chen etal., 2003a,
donor skin cells (Yang etal., 1998, 1999; Figure 1A). However, 2005). Similartothetransgenicsetting, xenografttolerancewas
theinjectionof donorspleencellstothe2CTCRrecipientmice induced by DN T cells, as the transfer of DN T cells that had
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FIGURE1|Inductionofallografttolerancebythepre-transplantation Rubockietal.,1986),togenerate2CF1mice(H2b,d,Ld−)bearingtheanti-Ld
infusionofdonor-specificspleencellsorDNTcells.Thismodeltakes TCR(whitemouse).DuetoasingleMHC-mismatch,skingraftsfromLd+F1
advantageoftheantigen-specific2CTCRtransgenicsystemandskingraftsto micearerapidlyrejectedbythe2CF1mice.(B)However,thetransferof
facilitatethestudyofallografttolerance,wherethe2CTCRtransgeneis donorspleencellsfromLd+F1miceto2CF1mice(Ld−)priortotheLd+skin
alloreactivetotheLdMHCclassImolecule.(A)C57BL/6(H2b)micearebred grafttransplantationresultsingraftsurvival.(C)Immunoregulatory2CDNT
toBALB/c(H2d)micetogenerateF1mice(H2b,d)thusbearingtheLdMHC cellclonesobtainedfrom2CF1TCRtransgenicmice(Ld−)preventthe
molecule(blackmouse).C57BL/62Ctransgenicmice(H-2b)arebredto rejectionofsingleMHC-mismatch,allogeneic(Ld+,inblack)skingrafts.
BALB/c(H2dm2)mice,whichareaBALB/cLdlossmutant(H2d,Ld−; However,fullMHC-mismatch,thirdpartygrafts(ingray)wererejected.
been isolated from mice which had received xenogeneic donor variant of this cardiac xenograft model, it was also shown that
spleen cells, was sufficient to ensure cardiac xenograft tolerance DN T cells can eliminate B cells, leading to a reduction in anti-
(Chen etal., 2003a). In this xenogeneic model, DN T cells effi- donor-specific antibody levels and delayed graft rejection (Ma
+ +
cientlysuppressedtheproliferationof bothCD4 andCD8 T etal.,2008).Takentogether,thesefindingssuggestthat,asopposed
cell anti-donor reactive cells (Chen etal.,2003a,2005). Using a totheMHCclassI-restricted2CTCRDNTcells,DNTcellsfrom
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+ +
non-transgenic mice can suppress CD4 and CD8 T cells as and, as mentioned above, can efficiently induce graft tolerance
well as B cells in an antigen-specific manner and potently sup- (Ford etal., 2002). Hence, as DN T cell-mediated suppression
pressbothskinandcardiacallograft,aswellascardiacxenograft, depends on interactions between FasL on DN T cells and Fas
rejection. on target T cells (Ford etal., 2002), it has been suggested that
Fas–FasL interactions have been proposed as the molecular DN T cells accumulate in an attempt to compensate for their
mechanismbywhichDNTcellseliminateanti-donorTcells,while inability to suppress autoimmune T cells in lpr/lpr mice (Ford
the elimination of B cells is considered to be perforin-mediated etal.,2002).
(Zhangetal.,2000,2006;Priateletal.,2001;Maetal.,2008;Ford
McIntyre etal., 2011). Interestingly, an accumulation of DN T SYSTEMICLUPUSERYTHEMATOSUS
cellsisobservedinbothFas-andFasL-deficientmice(Watanabe- Similar to ALPS patients, DN T cells are also found in greater
Fukunagaetal.,1992;Takahashietal.,1994),suggestingthatthe numbers in the peripheral blood of patients with SLE (Crispin
Fas–FasLpathwaymayregulatethenumberofDNTcellsinvivo. etal., 2008). However, in the case of SLE, these DN T cells are
By taking advantage of lpr mice, which exhibit a Fas-deficiency, major producers of IL-17 (Crispin and Tsokos, 2009), which is
Ford etal. (2002) demonstrated that DN T cells from lpr mice pathogenicinthisdisease(Wongetal.,2000;Doreauetal.,2009).
can mediate their immunoregulatory function through the Fas Moreover,IL-17-producingDNTcellscanbefoundinthekid-
pathwayaslongasthetargetTcellsexpressafunctionalFaspro- neysectionsofpatientswithlupusnephritis(Crispinetal.,2008).
tein.Indeed,DNTcellsfromlprmicecouldeffectivelydelayboth TheseresultsleadCrispinetal.(2008)tosuggestthatDNTcells
single MHC class I- and single MHC class II-mismatched skin themselves are pathogenic in SLE.Yet, it must be noted that, in
allograftrejection(Fordetal.,2002).Insummary,DNTcellscan these studies, DN T cells were defined as CD4−CD8−TCRαβ+
− −
clearlyinduceanantigen-specifictolerancetobothallograftsand cells, which may contain contaminating CD4 CD8 NKT cells
xenografts by eliminating or inhibiting the function of various expressing a TCR restricted to CD1d molecules (Hillhouse and
targetimmunecells. Lesage,2012). Of relevance,NKTcellsdoindeedincludeasub-
− −
setof CD4 NK1.1 IL-17-producingcells(Coquetetal.,2008).
AUTOIMMUNITY Moreover, NKT cells have been implicated in SLE pathogenesis
The antigen-specific immunoregulatory potential of DN T cells wheretheactivationofNKTcellsthroughtheadministrationof
in graft tolerance suggests that they may also participate in the α-GalCerexacerbateddiseasewhereastheinhibitionofNKTcell
inductionofimmunetoleranceinvariousautoimmunesettings. activationusingananti-CD1dblockingantibodyresultedindis-
As a result, the role of DN T cells has been mostly explored easeamelioration(Zengetal.,2003). Thus,NKTcellscannotbe
inautoimmunelymphoproliferativesyndrome(ALPS),systemic ruledoutasthetrueIL-17-producingcellsubsetinthisparticular
lupuserythematosus(SLE)andmousemodelsoftype1diabetes study. Nevertheless, IL-17-producing DN T cells have also been
(T1D),asdescribedbelow. showntoplayaprotectiveroleagainstbacterialinfections(Cow-
leyetal.,2005;Riol-Blancoetal.,2010).Altogether,anuncertain,
AUTOIMMUNELYMPHOPROLIFERATIVESYNDROME yetpossible,roleforDNTcellsinpatientswithSLEremainstobe
Autoimmune lymphoproliferative syndrome is a rare disorder defined.
characterized by mutations in either Fas or FasL (Rieux-Laucat
etal., 1995; Del-Rey etal., 2006), which results in defective TYPE1DIABETES
Fas-mediated apoptosis and, consequently, the abnormal accu- Type1diabetesoccursasaresultoftheantigen-specificelimina-
mulation of activated lymphocytes. ALPS patients on average tionofpancreaticinsulin-producingβcells.Itthuspresentsasa
presentwitha10-foldincreaseinDNTcellproportion(Bleesing relevantmodeltoinvestigatetheantigen-specificimmunoregula-
etal., 2001a; Bristeau-Leprince etal., 2008; Magerus-Chatinet torypotentialofDNTcells.
etal.,2009)andasizeableproportionof DNTcellscanalsobe The tolerogenic role of DN T cells in the prevention of
found in the lymphoid organs of the murine models for ALPS, T1D was first revealed using the P14/RIP-gp transgenic mouse
namely lpr/lpr (Fas-deficient) and gld/gld (FasL-deficient) mice model(Ohashietal.,1991),wheretheMHCclassI-restrictedP14
(Cohen and Eisenberg, 1992; Watanabe-Fukunaga etal., 1992; TCRtransgenerecognizesthelymphocyticchoriomeningitisvirus
Takahashi etal., 1994). Moreover, a greater proportion of DN (LCMV)protein, gp33–41(gp33), whichisexpressedunderthe
T cells from ALPS patients, in comparison to healthy controls, ratinsulinpromoter(RIP)forcingitsexpressioninthepancreatic
expresstheBcellantigen,B220(Bleesingetal.,2001a),whichis tissue.RIP-gpmicethatarecrossedtoP14micecarryaveryhigh
consistent with findings in lpr/lpr mice (Davidson etal., 1986). percentageofgp33-reactiveTcells,yettheresultingP14TCR:RIP-
Accordingly, B220 expression is a general feature of proliferat- gpmicedonotspontaneouslydevelopdiabetesontheC57BL/6
+
ing T cells (Bleesing etal., 2001b), which explains why B220 background(Ohashietal.,1991).Indeed,P14/RIP-gptransgenic
DN T cells can also be found in healthy individuals. Although micerapidlydevelopdiabetesupontreatmentwithgp33peptide
it cannot be denied that a drastic increase in the proportion andanti-CD40agonisticantibody,whichresultsintheinfiltration
+
of DN T cells can be observed in lpr/lpr and gld/gld mice, as ofpancreaticisletsbyactivatedCD8 Tcellsandthesubsequent
well as ALPS patients, there is arguably no evidence to sug- destruction of the insulin-producing β cells (Garza etal.,2000).
gest that the accumulation of DN T cells is pathogenic. Indeed, Usingthismodel,itwasshownthatthetransferofgp33activated
Fas-deficient DN T cells found in lpr/lpr mice, but not FasL- P14DNTcells1daypriortodiabetesinductioncaninhibitdia-
deficient DN T cells found in gld/gld mice, remain functional betesdevelopment(Fordetal.,2007).Theseresultswerethefirst
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indicationthatDNTcellsmayparticipateinthepreventionofa biological function of these cells in the prevention of autoim-
+
CD8 Tcell-drivenT1Dpathology,furtherlendingsupportfora munediabetesindiabetes-susceptibleNODmice.Notably,further
+
potentialroleforDNTcellstowardtheeliminationof CD8 T investigation is still needed to verify the role of DN T cells in
cellsinvivo. T1Ddevelopmentusingnon-lymphopenicNODmice,thusunder
The protective role of DN T cells in diabetes development more physiological and spontaneous conditions. Nevertheless,
wasfurtherinvestigatedbyourgroupusingthe3A9TCR:insHEL theseresultsdoprovideusefulinformationregardingthecritical
transgenicsystem,inwhichtheMHCclassII-restricted3A9TCR timeoftreatmentinitiationaswellasevidencethatnon-transgenic
transgenerecognizesapeptidefromhenegglysozyme(HEL)pre- DNTcellsmayexhibitthepotentialtoinhibitT1Ddevelopment
sentedbyI-Ak,whiletheinsHELtransgeneforcestheexpression innon-transgenicmice.
of HELinthepancreatictissue. AlthoughTCR:insHELBALB.K A more recent study further evaluated the role of non-
micearerelativelyresistanttoT1D,CD47-deficientTCR:insHEL transgenic DN T cells in T1D. Here, it was shown that 50%
+
BALB.Kmicehaveahighandspontaneousincidenceofdiabetes. of CD4 T cells isolated from an MLR had been converted to
− − +
Using this model of spontaneous T1D, a single transfer of 3A9 CD4 CD8 T cells. These CD4 T cell-converted DN T cells
DN T cells in the TCR:insHEL CD47-deficient BALB.K trans- sorted by flow cytometry were shown to delay T1D onset when
genic model was able to significantly inhibit the development adoptively transferred to NOD.SCID mice in combination with
of T1D (Dugas etal., 2010). Notably, a 3A9 DN T cell trans- diabetogenicTcells(Zhangetal.,2011a).Thisdelaywasfurther
+
ferledtoasignificantreductioninantigen-specificautoantibody enhanced when using GAD65 antigen to stimulate the CD4 T
serumlevels(Dugasetal.,2010),suggestingtheinvivoelimina- cells, thereby likely generating a higher proportion of GAD65-
tion of autoreactive B cells. We subsequently demonstrated that specificDNTcells.Moreover,asingletransferofGAD65-specific
3A9 DN T cells efficiently eliminate HEL-loaded B cells in vitro DNTcellsspecificitywasabletopreventdiabetesdevelopmentin
(Hillhouseetal.,2010).Althoughthismodelisimperfectinthatit 5-week-oldNODmiceanddecreasebloodglucoselevelsinnew-
isaCD47-deficientmodel,whereCD47isimplicatedinapopto- onset diabetic NOD mice (Zhang etal., 2011a). Therefore, this
sis,phagocytosis,cellmigration,andTcellresponses(Oldenborg, studyoffersapotentiallytranslatabletherapeuticapproachforthe
2004; Chao etal.,2012), it is nevertheless a spontaneous model generation of antigen-specific DNT cells in the prevention and
ofT1DwhereintheresultscomplementthefindingsofFordetal. treatmentofT1D.
(2007)suggestingthatDNTcellsmaybeof therapeuticinterest Altogether, these findings point toward an antigen-specific
forT1D. immunoregulatory role for DN T cells in autoimmune diseases.
Interestingly, the proportion of DN T cells is significantly The therapeutic potential of these cells certainly merits further
reduced in diabetes-prone mice in comparison to diabetes- investigationinadditionalpre-clinicalmodels.
resistantmiceinboththetransgenicandnon-transgenicsystems
(Dugasetal.,2010).Itisofrelevancethatboththenon-transgenic
GRAFT-vs-HOSTDISEASEANDCANCER
and TCR:insHEL transgenic diabetes-prone non-obese diabetic
(NOD) mouse models exhibit a low number of DN T cells rel- In the treatment of hematological malignancies, allogeneic
ative to other diabetes-resistant strains as it suggests that low hematopoieticcelltransplantation(AHCT)caneradicateseveral
DN T cell numbers are associated with diabetes susceptibility. bloodcancersthatareincurablebychemotherapyalone.Despite
Despiteareductionincellnumber,3A9DNTcellsfrom3A9TCR indisputable successes, the efficacy of AHCT is still limited by
transgenicNOD.H2kmicecarryingadiabetes-pronegeneticback- cancer recurrence and the development of GVHD (Raiola etal.,
ground (Lesage etal., 2002) exhibit an equally potent cytotoxic 2003).WhiletheacuteformofGVHDistriggeredbydirectTcell
functionincomparisonto3A9DNTcellsfromdiabetes-resistant recognitionofhistocompatibilityantigens,thepathophysiologyof
3A9TCRtransgenicB10.Brmice(Hillhouseetal.,2010).There- chronicGVHDremainsmoreelusiveandreliesonseveralimmune
fore,theassociationof DNTcellswithdiabetessusceptibilityis cell types (Shlomchik, 2007). Currently, 40–80% of patients
due to a deficiency in DN T cell number rather than function. developchronicGVHDafterAHCT,whichbringssubstantialmor-
Altogether, these results further suggest that the restoration of bidityandmortality(Lee,2005).Severallinesofevidencesuggest
DN T cell number can prevent T1D development in otherwise that immunoregulatory immune cells are paramount to GVHD
+
diabetes-susceptiblemice. prevention. Indeed, both CD4 Tregs and NKT cells have been
The role of DN T cells in the prevention of T1D was also suggested as major contributors of allotolerance in hematopoi-
recentlyevaluatedusingnon-transgenicDNTcells.Indeed,Dun- eticcelltransplantation(Hoffmannetal.,2002;Kohrtetal.,2010;
can etal. (2010) have demonstrated that non-transgenic DN T Di Ianni etal., 2011; Chaidos etal., 2012). However, the ability
cells can protect from autoimmune diabetes. Specifically, dia- of Tregs to maintain their anti-tumor T cell responses remains
betes induction was inhibited if DN T cells were transferred controversial(Onizukaetal.,1999;Shimizuetal.,1999;Liyanage
into recipient NOD.SCID (severe combined immunodeficiency) etal.,2002;Somasundarametal.,2002;Kohrtetal.,2010).Here,
mice1monthpriortoco-infusionwithdiabetogenicspleencells, wereviewthepotentialof DNTcellsatinhibitingGVHDwhile
whereasthesimultaneousco-infusionofdiabetogenicspleencells specificallypromotinganti-tumorresponses.
and DN T cells does not protect from disease (Duncan etal.,
2010). However, as NKT cells were not fully excluded from the GRAFT-vs-HOSTDISEASE
cellular preparation, additional studies using NKT cell-depleted Mousemodelsof GVHD,althoughtheyadmittedlydonotfully
non-transgenic DN T cells are warranted to firmly establish the replicatehumandisease,facilitatetheexaminationofthecellular
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Hillhouseetal. ImmunoregulatoryDNTcellsfortherapy
processamidtheallogeneicresponseswhichgiverisetoaGVHD- THEANTI-TUMORRESPONSE
likepathologyinvivo(Shlomchik,2007).ApotentialroleforDN In addition to preventing GVHD, DN T cells demonstrate an
T cells in the prevention of GVHD can be traced back to over anti-tumor activity without causing GVHD in recipient mice
two decades ago when Bruley-Rosset etal. (1990) demonstrated (Young etal., 2001, 2003b). Indeed, >90% of sublethally irra-
that transfer of spleen cells from pre-immunized mice was able diated immunodeficient SCID mice that were co-infused with a
to inhibit GVHD only when DN T cells were included in the lethal dose of A20 lymphoma cells together with allogeneic 2C
cellularpreparations. AlthoughtheDNTcellswerenotvoidof TCRtransgenicspleencellssurvivedindefinitelyintheabsenceof
contaminatingNKTcells,theseresultsstillsuggestedthatDNT GVHD.Interestingly,recipientmiceexhibiteda15-foldincrease
cellsplayaroleinGVHDprevention.Becauseoftheirprominent inthenumberofDNTcellsintheirspleen(Youngetal.,2003b),
antigen-specificimmunoregulatorypotentialaswellastheirabil- suggestinganassociationbetweenDNTcellsandtheinhibitionof
ity to promote allotolerance, the contribution of DN T cells at GVHDaswellastumorgrowth.Accordingly,Youngetal.(2003b)
preventing GVHD was later investigated using the 2C TCR sin- demonstrated that the injection of either 2C DN T cell clones
gle MHC-mismatch model (Young etal.,2003a), for which DN ornon-transgenicDNTcellswassufficienttopreventA20lym-
Tcellshavebeenshowntoeffectivelypreventallograftrejection phoma tumor growth, without inducing clinical or histological
(Zhangetal.,2000;Youngetal.,2002;Chenetal.,2003b;Figure1). signsofGVHD.Altogether,theseresultsfurtherdemonstratethe
Specifically, micebearingasingleMHC-mismatch(Ld)injected anti-tumoralpotentialofDNTcells.
withLd-specific2CTCRtransgenicspleencellssurvivedformore TofurtherdefinethepotentialuseofDNTcellsinthetreatment
than 150 days without any clinical or histological signs of acute of hematological malignancies, Merims etal. (2011) performed
orchronicGVHD(Youngetal.,2003a;Figure2A).Thetolerance a pre-clinical study in acute myeloid leukemia (AML) patients
of donor spleen cells may be explained, at least in part, by the undergoing complete remission following standard chemother-
accumulationof2CTCRDNTcells,whichcaneffectivelyinhibit apy. In this study, they demonstrate that DN T cells can be
+
pathogenicCD8 Tcellresponses,inrecipientmice(Youngetal., readily isolated from the blood of AML patients. Moreover,
2003a). Subsequently, the role of non-transgenic DN T cells in the isolated DN T cells maintain their phenotype following
bothparenttoF1andfullyMHC-mismatchedbonemarrow(BM) a proficient cellular expansion reaching nearly 500-fold in 2
transplantationfollowingeitheramyeloablative(Heetal.,2007) weeks (Merims etal.,2011). Of interest, the DN T cells isolated
or non-myeloablative regimen (Su etal., 2012) was investigated and expanded in vitro demonstrated efficient cytolytic activity
+ +
(Figure 2B). In stark contrast to CD4 and CD8 T cells, the towardtheautologousleukemiccellsinsixoutofsevenpatients
co-injection of non-transgenic DN T cells with T cell-depleted (Merims etal., 2011). Importantly, the anti-tumoral activity of
BM into a sublethally irradiated host ensured prolonged recipi- DN T cells may extend beyond hematological malignancies, as
ent survival in the absence of clinical signs of GVHD (He etal., melanoma-reactiveDNTcellshavealsobeensuccessfullyisolated
2007; Su etal.,2012; Figure2B), indicating that DN T cells are from a previously immunized melanoma patient (Voelkl etal.,
not alloreactive nor pathogenic in this context. Moreover, this 2009). The mechanism by which DN T cells are able to confer
ledtostablemixedchimerismand,asopposedtoBMtransplan- antigen-specificimmunetoleranceallthewhileprovidingananti-
tation alone, promoted donor allotolerance (He etal., 2007; Su tumor response is counterintuitive and certainly merits further
etal.,2012;Figure2B).Together,thesefindingsdemonstratethat, investigation.
+ +
asopposedtoCD4 orCD8 Tcells, thetransferof allogeneic
DN T cells does not cause GVHD. Rather, DN T cells prevent CONCLUSION
GVHD, induce mixed chimerism and promote donor-specific Immunoregulatory DN T cells, which compose approximately
allotolerance. 1–3%of TcellsinhumanPBMCs(Fischeretal.,2005),demon-
Notably,thereisaccruedevidencethatDNTcellsalsoprevent strate a unique antigen-specific therapeutic potential, such that
GVHD in humans uponAHCT. Indeed, the stem cells used for theyprovideafavorableoutcomeingrafttolerance,autoimmu-
AHCTarecurrentlymostcommonlyobtainedfromtheperiph- nity and GVHD. In addition, DN T cells exhibit an interesting
eral blood mononuclear cells of granulocyte colony-stimulating anti-tumoralpotential.ThisseeminglybroadapplicabilityofDN
factor (G-CSF)-treated donors, where G-CSF is used to mobi- Tcellsatconferringantigen-specificimmunetolerancemustbe
+
lizeCD34 hematopoieticstemcellsintotheblood(Shlomchik, interpretedwithcaution. Indeed, DNTcellscomposeahetero-
− − +
2007).Interestingly,inadditiontoskewingtowardaTh2pheno- geneous subset of cells, which include all CD4 CD8 CD3 T
type,G-CSFtreatmentalsoresultsinanincreaseintheproportion cellsubsets,namelyTCRγδTcells,NKTcellsandimmunoregu-
of immunoregulatory DN T cells in the blood (Kusnierz-Glaz latoryTCRαβDNTcells(HillhouseandLesage,2012). Despite
etal., 1997). The combination of these two phenotypes may theheterogeneityinthedescriptionofDNTcellsinvariousstud-
explain why the transfer of CD34+ stem cells isolated follow- ies,whichdonotalwaysexcludeTCRγδand/orNKTcells,most
ing G-CSF treatment does not increase the incidence of acute studiesinTCRtransgenicandnon-transgenicmiceaswellasin
GVHD relative to classical BM transplants. More recently, two humanspointtowardanimmunoregulatoryroleforDNTcells.
studiesalsonotedaclearcorrelationbetweenanelevatedpropor- Afewnotableexceptionstothisarethedescriptionofpathogenic
tion of DN T cells and a low incidence of GVHD development IL-17-producing DN T cells in SLE (Crispin and Tsokos, 2009)
following AHCT (McIver etal., 2008; Ye etal., 2011). Together, andthecharacterizationofIL-23responsiveDNTcellscontrol-
theseobservationssupporttheviewthathumanDNTcellsmay ling bacterial infections (Cowley etal., 2005, 2010; Riol-Blanco
preventGVHD. etal., 2010). Clearly, additional studies aimed at identifying
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Hillhouseetal. ImmunoregulatoryDNTcellsfortherapy
FIGURE2|DoublenegativeTcellspromotetolerancewhileinhibiting cellsovertime.(B)BALB/c(H2d)miceunderwentmyeloablative(toppanel,
GVHD.(A)ImmunodeficientSCIDF1mice(H2b,d,Ld+)weresublethally sublethalirradiation)ornon-myeloablative(bottompanel,busulfantreatment)
irradiatedandreconstitutedwith2CF1(H2b,dm2,Ld−)Ld-specificspleen conditioningpriortobeingreconstitutedwithacombinationofC57BL/6(H2b)
cells.Threeweekslater,recipientmiceweregivenskingraftsfromF1(Ld+, DNTcellsandTcell-depleted(TCD)C57BL/6(H2b)bonemarrow(BM)cells.
inwhite)andfullMHC-mismatch,thirdparty(ingray)mice.Thethirdparty Subsequently,recipientmicereceivedskingraftsfromdonorC57BL/6(H2b,
skingraftwasrejected,whereastheskingraftfromtheF1(Ld+)donor inblack)andthirdparty(H2k,ingray)mice.Regardlessofthetypeof
mousewasaccepted.Importantly,therecipientmiceshowednosignsof conditioningregimenutilized,therecipientmiceacceptedallC57BL/6(H2b,
GVHDandexhibitedaprominentincreaseinthelevelsofdonor-derivedDNT inblack)donorgraftswhilethirdparty(H2k,ingray)graftswererejected.
uniquemarkersfortheseuniquecelltypeswillhelpclarifywhich exciting advances in the pre-clinical setting. As the function of
specific cellular subset contributes to each specific biological DN T cells is mainly antigen-specific, the use of DN T cells in
setting. immunotherapyshouldleadtofewerside-effectsandadecreased
In conclusion, immunoregulatory DN T cells exhibit great riskofinfections,whichisamajorconcernintheapplicationof
potentialasacellulartherapyforvariousmodelsofdisease,with broadimmunosuppressiveregimens.Moreover,DNTcellsexhibit
www.frontiersin.org January2013|Volume4|Article6|7
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Hillhouseetal. ImmunoregulatoryDNTcellsfortherapy
apotentimmunosuppressivepotentialinmultiplemodelsystems, from the Fonds de recherche du Québec - Santé (FRQS) Junior-
fromtransplantationtoautoimmunity,suggestingavastarrayof 1clinician-scientistandissupportedbytheLeukemia/Lymphoma
applicabilityofDNTcellsincellulartherapy. Society of Canada. Sylvie Lesage holds a Canadian Insti-
tutes of Health Research New Investigator scholarship. Sylvie
ACKNOWLEDGMENTS LesageandJean-SébastienDelislearebothindependentlyfunded
Erin E. Hillhouse is recipient of a Diabète Québec scholarship by the Foundation of the Maisonneuve-Rosemont Hospital
andwasrecipientofaPhDscholarshipfromtheCanadianInsti- and National Sciences and Engineering Research Council of
tutes of Health Research. Jean-Sébastien Delisle is an awardee Canada.
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