RESEARCHPAPER OncoImmunology1:2,141–151;March/April2012;G2012LandesBioscience T cells and T cell tumors efficiently generate antigen-specific cytotoxic T cell immunity when modified with an NKT ligand Yeonseok Chung,1,5 Young-Hee Lee,1 Yongliang Zhang,1 Natalia Martin-Orozco,1 Tomohide Yamazaki,1 Dapeng Zhou,2 Chang-Yuil Kang,4 Patrick Hwu,2 Larry W. Kwak3 and Chen Dong1,* 1DepartmentofImmunology;CenterforCancerImmunologyResearch;UniversityofTexasMDAndersonCancerCenter;Houston,TXUSA;2DepartmentofMelanomaMedical Oncology;CenterforCancerImmunologyResearch;UniversityofTexasMDAndersonCancerCenter;Houston,TXUSA;3DepartmentofLymphomaandMyeloma;Centerfor CancerImmunologyResearch;UniversityofTexasMDAndersonCancerCenter;Houston,TXUSA;4CollegeofPharmacy;SeoulNationalUniversity;Seoul,RepublicofKorea; 5InstituteofMolecularMedicine;UniversityofTexasMedicalSchool;Houston,TXUSA Keywords: cytotoxic T cell, iNKT cell, a-galactosylceramide, cellular vaccine, tumor immunity, CD1d VariousInvariantNKT(iNKT)cellligandshavebeenshownaspotentadjuvantsinboostingTcellreactivatestoantigens on professional APC. Non-professional APC, such as T cells, also co-expressing MHC class I and CD1d, have been © u2nattrac0tive ce1ll vacc2ine car rieLrs dueato thneir podor immeunogsenicity . HBere, wie roeport tshat Tccellsiasewell ans T cecll e. lymphoma can efficiently generate antigen-specific cytotoxic T lymphocytes (CTL) responses in mice in vivo, when formulatedtopresentiNKTliganda-galactosylceramide(aGC)ontheirsurfaceCD1d.VaccinationwithaGC-pulsedEG-7 T-cell lymphoma induced tumor-specific CTL response and suppressed the growth of EG-7 in a CD8 T cell-dependent manner.InjectionofaGC-loadedCD4Tcellsinmiceefficientlyactivated iNKTcellsinvivo.WhileTcellsloadedwitha classI-restrictedpeptideinducedproliferationbutnoteffectordifferentiationofantigen-specificCD8Tcells,injectionof Tcellsco-pulsedwithaGCstronglyinducedIFNcandGranzymeBexpressioninTcellsandcompletelysisoftargetcells Do not distribute. invivo.PresentationofaGCandpeptideonthesamecellswasrequiredforoptimalCTLresponseandvaccinatingTcells appeared to directly stimulate both iNKT and cytotoxic CD8 T cells. Of note, the generation of this cytotoxic T cell response was independent of IL-4, IFNc, IL-12, IL-21 and costimulation. Our data indicate that iNKT cell can license a non-professionalAPCtodirectlytriggerantigen-specificcytotoxicTcellresponses,whichprovidesanalternativecellular vaccinestrategy against tumors. Introduction the T cell immunity in this case.4 Furthermore, injection of DC- associated aGC induces the activation and expansion of iNKT CD1d-restrictedinvariantNKTcells(iNKT)recognizeglycolipid cells and NK cells in vivo in tumor patients.5,6 antigens presented on non-classical MHC class I-like molecule, Non-professional APC in immune system, such as T cells, CD1d. Recent studies revealed a crucial role of iNKT cells in express both MHC class I and CD1d. The expression of MHC the host defense against pathogens and tumors through direct classIonthesecellsmayallowthemtobeprotectedfromNKcell- interaction with microbial antigens or indirect activation.1 mediated cytotoxic attack through interaction with inhibitory Moreover, iNKT cell acts as a bridge between innate and receptors.7 The role of CD1d expression on peripheral T cells in acquired immune system by regulating T cell activation and iNKTcellbiologyisnotyetclear,althoughitiswellestablishedthat function. For instance, iNKT ligands have been shown with CD1dexpressiononthymicTcellsisessentialforpositiveselection strong potency in enhancing T cell responses to soluble protein ofiNKTcell.8,9DChasbeenshowntobethemostefficientAPC antigens or tumor antigens presented by professional antigen- foriNKTcell primingwhenaGCwasinjectedas a solubleform, presentingcells(APC).Co-administrationofa-galactosylceramide while other types of cell expressing CDld suppress iNKT cell (aGC)andproteinantigensgeneratestheantigen-specificCD4T activationinthiscondition.10InadditiontoDC,vaccinationwith and CD8 T cell immunity in vivo.2,3 The generation of antigen- BcellscopulsedwithaGCandtumor-derivedpeptidegeneratesan specific adaptive immunity depends on the activation and efficientanti-tumorCD8Tcellresponseinseveralmurinetumor maturation of dendritic cells (DC) after aGC injection which models.11Furthermore,ourrecentstudyshowedthatautologousB triggers IFNc and TNFa expression from iNKT cells.2,4 lymphoma vaccine, manipulated to present aGC, can establish Costimulation such as CD40 and CD80/CD86 is required for memory anti-tumor T cell immunity.12 Of note, Shimizu et al. *Correspondenceto:ChenDong;Email:[email protected] Submitted:08/29/11;Revised:10/16/11;Accepted:10/19/11 http://dx.doi.org/10.4161/onci.1.2.18479 www.landesbioscience.com OncoImmunology 141 recently showed that non-professional APCs, such as T and NK together raise an interesting question if iNKT ligand-loaded non- cells, can stimulate iNKT cell activation when they presented professionalAPCcouldactasanefficientAPCingeneratingCD8 aGC in a cell-associated form.11,13 They also showed that tumors Tcell responseagainstthepeptidesloadedontheirsurfaceMHC lackingcostimulatorymolecules,such as B16 melanoma andEL4 classI.Ifthisistrue,itmightbepossibletousetumorcellitselfor thymoma, can induce the activation of iNKT cells and NK cells pathogen-infected cells to generate tumor- or pathogen-specific in vivo when formulated to present aGC. These observations cytotoxic T cell immunity, which would be much easier, cost- effectiveandpracticalinclinicalsettings. Thereforeinthepresentstudy,wetestedwhetherconventional T cells- as a surrogate CD1d-expressing non-professional APC- can actively interact with iNKT cells and CD8 T cells in the presence of an iNKT cell ligand and MHC class I-restricted peptide. Our results showed an efficient activation of iNKT cells and antigen-specific CD8 T cells in mice vaccinated with T cells presenting both aGC and peptide on the same cells, which induced an effective immunity against microbial infection and tumor. Moreover, vaccination with aGC-pulsed thymoma generated a tumor-specific cytotoxic T cell response in vivo. Thus, in the presence of iNKT cell help, non-professional APC, suchasTcells,canstimulatecytotoxicTcellfunction,providing © 2012 Landeansaltern atBive meainsoof cellsvaccincationimeethod.nce. Results Vaccination with aGC-loaded EG-7 generates CD8+ T cell- dependentantitumoractivity.Wefirstaskedwhethervaccination with tumor cells of T cell origin can trigger anti-tumor activity, Do not distribute. whenmanipulatedtopresentaGC.Totracktumor-specificTcell responses more efficiently in vivo, we employed ovalbumin- expressingEL4thymoma(EG-7)asourtumormodel.Toaddress protective antitumor activity by vaccination with aGC-loaded EG-7, we vaccinated mice with irradiated EG-7 co-cultured with 1 mg/mL of aGC overnight. EG-7 cells incubated with the same volume of solvent (5 ml/mL of 0.5% polysorbate) were used as control (EG-7/veh). After extensive washing, these cells were i.v. injected into syngenic naïve mice. These mice were subcutaneously implanted with live EG-7 and the tumor volume and survival were monitored daily. Mice were sacrificed when tumor diameters reached 20 mm. As depicted in Figure1A and B, the vaccination with EG-7/aGC significantly reduced the growth of solid tumors and induced prolonged survival in comparison to those of EG-7/veh group. Tumors affect myelopoiesis and induce the expansion of CD11b+Gr-1+ Figure1.VaccinationwithaGC-loadedEG-7generatedpreventive antitumoractivity.(A–D)EG-7cellswereco-culturedovernightwith 1mg/mlofaGC(EG-7/aGC)orvehiclefollowedbyirradiation(50Gy). C57BL/6mice(n=7pergroup)werevaccinatedwithEG-7/veh, EG-7/aGCoruntreated(Nil).Oneweeklater,allmicewere subcutaneouslyinjectedwith1!106liveEG-7cellsandthesurvival (A)andtumorsize(B)werechecked.(C)Atthe3weekslater,splenocytes wereisolatedfromtumorbearingmiceandanalyzedbyflowcytometer afterstainingwithanti-CD11b,anti-Gr1,anti-CD19,anti-CD4and anti-CD8antibodies.(D)SplenocyteswererestimulatedwithSIINFEKLin thepresenceofGolgi-Plugfor5hbeforeintracellularstainingofIFNcon OVAspecificTcells.*p,0.05,**p,0.005,pvalueswerecalculated with2-wayANOVA(A),Kaplan-Meiermethod(B)orStudent’st-tests (CandD)incomparisonwiththeEG-7/vehgroup. 142 OncoImmunology Volume1Issue2 myeloid-derived suppressor cell (MDSC) in the bone marrow, whereas EG-7/veh did not (Fig.3A; -0.37% and 72.8% lysis, blood, spleen.14 Of note, we also observed a great decrease of respectively). To examine if CD8+ T cells in the vaccinated mice CD11b+Gr-1+population inthespleenofEG-7/aGCvaccinated directlykilltargetcellsinanantigen-specificway,weperformedan mice (, 4%) compared with that of EG-7/veh group (. 20%) invitroCTLassaywithpurifiedCD8+Tcellsfromthevaccinated (Fig.1C), another indicative of antitumor activity induced by mice by analyzing caspase-3 activation in the target cells. As the vaccination. Importantly, mice vaccinated with EG-7/aGC depicted in Figure3B, the CD8+ T cells from the EG-7/aGC showed higher numbers of CD8+ T cells producing IFNc upon vaccinated mice induced a significantly higher percentage of SIINFEKL stimulation (Fig.1D). These results overall indicate caspase-3cleavageinthetargetcellsthanthosefromtheEG-7/veh the induction of anti-tumor activity as well as tumor-specific vaccinated mice. In addition, the effective target lysis in EG-7/ CD8+ T cell responses upon the vaccination with EG-7/aGC aGC-vaccinated mice was associated with increased antigen- in vivo. specific IFNc+ granzyme B+ CD8 T cells in the spleen (Fig.3C). Therefore, we next examined if the observed anti-tumor To further characterize the antigen-specific CD8 T cells, we activitybyvaccinationwithEG-7/aGCdependsonCD8+Tcells. adoptively transferred CFSE-labeled OT-I T cells (CD45.2) into We injected anti-CD8 depleting antibody into the vaccinated congenic mice (CD45.1). The recipients were vaccinated with micebeforewetransplantedliveEG-7cellsintothem.Depletion either EG-7/veh or EG-7/aGC and CD45.2+ cells were analyzed of CD8+ cells greatly decreased the protective anti-tumor activity five days later. OT-I T cells in mice vaccinated with EG-7/veh by vaccination with EG-7/aGC (Fig.2A) and the survival of underwent an extensive proliferation, but expressed little IFNc vaccinated mice (Fig.2B). Taken together, these results indicate (,10%)orgranzymeB(,3%).Bycontrast,significantlyhigher that antitumor immunity generated by aGC-loaded EG-7 is, at population of OT-I T cells in EG-7/aGC-vaccinated mice ©least i n p2art, me0diated1by CD28 T ce llsL. andeexspresse d IBFNc (.i5o0%) sand gcranzyimeeB (.n8%) c(Fig.e3D). . Vaccination with aGC-loaded EG-7 induces ova-specific Taken together, these data demonstrate that aGC-loaded EG-7 cytotoxic responses. Our EG-7 tumor models (Fig.1) and thymomainducedtumor-specificCD8+Tcellstodifferentiateinto CD8depletionexperiment(Fig.2)raisedahypothesisthattumor cytotoxic effector T cells expressing high levels of IFNc and cells ofT cell origin, when they present iNKT ligand, are able to granzymeBinvivo. induce cytotoxic T cell response specific for endogenous tumor aGC induces cytotoxic T cell response to T cell-associated antigens. To test our hypothesis, we co-cultured EG-7 cells with antigens.Ourstudythusfarshowedthat,withhelpofaGC,tumors Do not distribute. aGC or vehicle before being irradiated (EG-7/aGC and EG-7/ ofTcellorigincantriggertumor-specificcytotoxicTcellresponses. veh, respectively). To increase the expression of peptide/MHC-I Therefore we next asked if conventional T cells can also act as complex, EG-7 cells were first pre-treated with IFNc. One week antigenpresentingcellsandtriggercytotoxicTcellresponsesinvivo, after vaccination, we performed an in vivo CTL assay to deter- whenformulatedtopresentaGC.ApreviousstudybyShimizuetal. mine the antigen-specific cytotoxicity against SIINFEKL-loaded showed that T cells, purified by negative selection using magnetic syngenic splenocytes11. When injected into naïve syngenic mice, beadsandwhenloaded with aGC, stimulateiNKT cellsinvivo.13 EG-7/aGC resulted in an evident peptide-loaded target cell lysis To further assess iNKT cell activation by T cells, we first sorted Figure2.CD8Tcellsplayacriticalroleinmediatingantitumoractivity.(AandB)C57BL/6micewerevaccinatedwithEG-7/vehorEG-7/aGConday-7 (n=5pergroup).EG-7/aGCvaccinatedmicewereintraperitoneallyinjectedwithCD8depletingAb(563.8)onday-3and-1.Onday0,allmicewere subcutaneouslyinjectedwith1!106liveEG-7cellsandthetumorvolume(A)andthesurvival(B)werechecked.*p,0.01,**p,0.005,pvalues werecalculatedwith2-wayANOVA(A)orKaplan-Meiermethod(B)incomparisonwithEG-7/vehandEG-7/aGC+rIgGorEG-7/aGC+rIgGand EG-7/aGC+anti-CD8group. www.landesbioscience.com OncoImmunology 143 © 2012 Landes Bioscience. Do not distribute. Figure3.VaccinationwithaGC-loadedEG-7generatesOVA-specificcytotoxicTcellresponseinvivo.(AandB)EG-7cellswerecoculturedovernightwith 1mg/mlofaGC(EG-7/aGC)orvehicle(0.5%polysorbate,EG-7/veh)followedbyirradiation(50Gy).C57BL/6mice(n=3pergroup)werevaccinatedwith EG-7/veh,EG-7/aGCorleftuntreated(Nil).(A)Oneweeklater,aninvivoCTLassayforSIINFEKLwasperformed.CFSEhigh,peptide-pulsedtarget;CFSElow, peptide-unpulsedcontrol.(B)Oneweekaftervaccination,CD8+Tcellswereisolatedandincubatedwith1mg/mLpeptide-pulsedsplenocytesthathad beenlabeledwithDDAO-SEfor2h.Thecellswerefixed,permeabilizedandstainedwithanti–cleavedcaspase-3mAb.Thelevelsofcleavedcaspase-3 intheDDAO-SE-positivecellswereanalyzedbyflowcytometry.DatashownarerepresentativeFACSplots(upperpanels)andmean±SE(lowerpanel). *p,0.05,incomparisonwithEG-7/vehgroup.(C)Oneweekafterthevaccination,splenocyteswereisolatedandrestimulatedwithSIINFEKLfor5hin theprecenceofGolgi-PlugbeforeintracellularstainingofgranzymeBandIFNc.(D)Ovalbumin-specificCD8Tcellswereisolated,labeledwith10mmol/L CFSEandi.v.transferredintotheirsyngenicmiceasdescribedin Figure1.Onthefollowingday,micewerei.v.injectedwithirradiatedEG-7cells manipulatedinvitrowithindicatedconditions.Fivedayslater,lymphoidcellsfromthespleenoftherecipientmicewererestimulatedwithSIINFEKLfor 5hbeforeintracellularstainingofgranzymeBandIFNc.Dataarerepresentativeofatleasttwoseparateexperiments.*p,0.05,**p,0.01,in comparisonwithnon-treated(Nil)group. NK1.12CD82CD192CD11b2CD11c2Gr-12I-Ab2CD4+ cells We next asked whether T/aGC can induce antigen-specific fromthelymphoidcellsofC57BL/6mice.Thesecellswerevirtually cytotoxicity when they were additionally pulsed with MHC class allCD3+CD1d+,indicatingnocontamination ofprofessionalAPC I-restricted peptide. SIINFEKL peptide (1ng/mL) was added to (Fig.4A).ThesortedcellswereloadedwithaGC(T/aGC)orwith Tcells in thelastone hourwhen they were incubated with aGC vehicle (T/veh) and were i.v. injected into syngenic naïve mice. or vehicle (T/aGC/pep and T/veh/pep, respectively). After When we analyzed splenocytes six hours after the injection, we extensive washing, C57BL/6 mice were immunized with T/veh/ observed that CD1d-tetramer+ cells in mice receiving T/aGC pep or T/aGC/pep. One week later, we performed an in vivo produced IFNc+ whereas the same population in mice receiving CTL assay to determine the antigen-specific cytotoxicity against T/veh did not produce IFNc (Fig.4B). Therefore CD4+ T cells SIINFEKL-loaded syngenic splenocytes. Mice vaccinated with efficiently triggered iNKT cell activation in vivo when they were T/aGC/pep showed complete lysis of target cells while mice manipulatedtopresentaGC. vaccinated with T/veh/pep did not (Fig.4C). 144 OncoImmunology Volume1Issue2 © 2012 Landes Bioscience. Figure4.InjectionofconventionalCD4TcellscoatedwithSIINFEKLandaGCinducesafunctionalcytotoxicTcellresponse.(A)Lymphoidcellsfrom spleenandlymphnodesofC57BL/6micewerestainedwithFITC-conjugatedanti-CD19,anti-NK1.1,anti-Gr-1,anti-CD11b,anti-CD11c,anti-CD8a,anti-I- Do not distribute. AbantibodiestogetherwithAPC-conjugatedCD4Ab.LineagenegativeandCD4+cellsweresortedbyFACSAria.ThesortedcellswerestainedwithPE- conjugatedanti-CD3oranti-CD1dAb.Filledhistogramisisotypecontrol.(B)SortedCD4+Tcellswereco-culturedwithaGC(1mg/ml).Cellswerewashed andintravenouslyinjectedintosyngenicmice.Sixhourslater,lymphoidcellsfromspleenwerestainedwithCD1d-tetramerandCD19beforeintracellular IFNcstaining.CD1d-tetramer+CD19-cellsweregatedandanalyzed.(C)ThesortedCD4+Tcellswereco-culturedwithaGC(1mg/ml)orvehicle(0.5% polysorbate)for16hincluding1hpulsewithSIINFEKL.Cellswerewashedandintravenouslyinjectedintosyngenicmice.Oneweeklater,syngenic lymphocyteswereeitherloadedwith1mmol/LpeptidesorleftuntouchedbeforebeinglabeledwithCFSEatdifferentconcentrations(10and1mmol/L, respectively).Equalnumbersofthetwopopulationsweremixedandinjectedi.v.intomice.Eighteento24hlater,lymphoidcellsfromspleenandlymph nodeswereanalyzedtoassesspeptide-specifickilling.(D)OT-ITcellswereisolatedusinganti-CD8microbeadsandAutoMacs.Thesecellswerelabeled with10mmol/LCFSEandi.v.transferredintotheirsyngenicmice.Onthefollowingday,micewerei.v.injectedwithTcellsmanipulatedinvitrowith indicatedconditions.Forty-eighthourslater,lymphoidcellsfromthespleenoftherecipientmicewerestainedwithphycoerythrin(PE)-conjugatedanti- Va2antibodyandthenanalyzedbyflowcytometry.ForintracellularGranzymeBandIFNcstaining,cellswererestimulatedwithSIINFEKLfor5hbefore intracellularstainingofthesemoleculesaccordingtomanufacturer’sinstruction.Dataarerepresentativeofatleasttwoindependentexperiments. To analyze whether T/veh/pep vaccination results in cytotoxic T/aGC/Pep vaccination confers protection against Listeria functionofantigen-specificCD8Tcells,weisolatedCD8+Tcells monocytogenes and melanoma. We next tested whether the from OT-I mice, labeled them with 10 mM CFSE before cytotoxic T cell response generated by T/aGC/pep vaccinationis transferringintoC57BL/6mice,whichwerethenvaccinatedwith effectiveenoughinsuppressingthegrowthofintracellularbacteria T/veh/pep or T/aGC/pep. When OT-I cells in the spleen of and tumor in an antigen-specific manner. We first employed recipients wereanalyzed48hoursaftervaccination,bothtypesof L.monocytogenesinfectionmodelsinceclearanceofthisbacterium T cell vaccination induced the proliferation of OT-I T cells in is largely dependent on CD8 T cell response. Mice were vaccin- vivo, although the proportion of T cells that had undergone over ated with T/aGC, T/aGC/pep or peptide-pulsed dendritic cells five times of division was higher in mice receiving T/aGC/pep (DC/pep) as a control. Ten days later, the vaccinated mice were vaccination than those with T/veh/pep (Fig.4D, upper panels). i.v.injectedwithL.monocytogenesexpressingOVAandthebacterial Moreimportantly,OT-ITcellsinmicevaccinatedwithT/aGC/ burden in the spleen and liver was measured. As expected, mice pep displayed remarkably higher expression of granzyme B and vaccinatedwithDC/pepshowedsignificantlylowerbacterialburden IFNc compared with those in T/veh/pep-vaccinated mice in both spleen and liver compared with non-vaccinated mice (Fig.4D, middle and lower panels). These data demonstrate that (Fig.5A). Compared with non-vaccinated group, mice vaccinated in response to an antigen on CD4 T cells as a non-professional with T/aGC showed slightly lower bacterial burden, especially in APC, although antigen-specific CD8 T cells could proliferate, theliver.Incontrast,micevaccinatedwithT/aGC/pepalsoshowed their functional differentiation into cytotoxic cells could only significantly lower bacterial burden in both organs, which is occurr in the presence of iNKT cell ‘help’. comparabletothoseofDC/pep-vaccinatedmice(Fig.5A). www.landesbioscience.com OncoImmunology 145 © 2012 Landes Bioscience. Do not distribute. Figure5.VaccinationwithTcell-basedvaccinegeneratesprotectiveimmunityagainstL.monocytogenesinfectionandtumorchallenge.C57BL/6mice (n=3micepergroup)werevaccinatedwiththeindicatedcellularvaccine(day0)beforetheywerechallengedwith5!104liveL.monocytogenes expressingOVA(day10).Threedaysafterthebacterialchallenge,bacterialburdeninspleenandliverwasmeasured(A).Dataarearepresentativeof threeseparateexperiments.(B)C57BL/6mice(n=3micepergroup)werevaccinatedwiththeindicatedcellularvaccine(day0).Tendayslater,recipients wereintravenouslychallengedwithlive2!105B16-OVA.Twoweeksafterthetumorchallenge,tumorfociinthelungweremeasured.(C)PBMCwas isolatedinmicevaccinatedwiththeindicatedcellularvaccineandrestimulatedwithSIINFEKLinthepresenceofGolgi-Plugfor5hbeforeintracellular stainingofIFNc.Dataaremean±SE*p,0.05,**p,0.01,***p,0.001incomparisonwithnon-treated(Nil)group.Dataarerepresentativeoftwo separateexperiments. To assess if the vaccinated mice were also resistant to tumor sought to elucidate the mode of action in the efficient induction growth,wei.v.injectedOVA-expressingB16melanomacellsinto of peptide-specific cytotoxicity during T/aGC/pep vaccination. the vaccinated mice. Fourteen days later, we counted tumor foci When we vaccinated CD1d-deficient mice with T/aGC/pep, we in the lung of recipients. Compared with non-vaccinated mice, did not observe peptide-specific cytotoxicity in our in vivo mice vaccinated with T/aGC had less tumor foci (Fig.5B). On CTL assay (Fig.6A). Therefore, the antigen-specific cytotoxicity the other hand, fewer tumor foci were found in mice vaccinated elicited by T/aGC/pep requires iNKT cells in vivo. with T/aGC/pep or DC/pep (Fig.5B). Intracellular staining of Next we asked if vaccinated T cells directly stimulate CD8 peripheral blood mononuclear cells after peptide restimulation TcellsorrequirehostAPC.Weutilizedbm-1mousewhosecells revealed that both DC/pep and T/aGC/pep vaccinations are able to load SIINFEKL onto their MHC I, but the resulting efficiently induced peptide-specific IFNc-producing CD8 T cells complex cannot be recognized by OT-I TCR due to a mutation (Fig.5C), which correlated well with anti-Listeria and anti- in the H-2K region.15 In this experiment, vaccination with metastaticactivity inthevaccinatedmice.Collectively, thesedata T/aGC/pep using T cells from bm-1 mice failed in induction demonstrate that vaccination with T/aGC/pep established of antigen-specific target cell lysis (Fig.6B), indicating that protective immunity against intracellular bacteria and tumor in appropriate MHC/peptide complex on the vaccinating T cells is a peptide-specific manner. essential and that they probably directly primed OT-I cells. Tcells simultaneouslypresentingiNKT andclass I-restricted Since aGC and class I peptide are both presented by the same ligands directly induce antigen-specific cytotoxicity. We next Tcells,thissuggeststhatiNKTligandmayprovidea“dangerous” 146 OncoImmunology Volume1Issue2 © 2012 Landes Bioscience. Do not distribute. Figure6.PeptideandaGConthesameTcellsarerequiredfortheoptimalprimingofCTLbyiNKT-mediatedTcellvaccine.(A)C57BL/6(WT)orCD1d2/2 micewerevaccinatedwithTcellsco-pulsedwithaGCandSIINFEKL(1!106permouse).(B)TcellsfromWTorbm-1micewereco-pulsedwithaGCand SIINFEKLbeforebeingi.v.injectedintoWTmice.(C)C57BL/6micewerevaccinatedwithTcellsco-pulsedwithaGCandSIINFEKL(1!106permouse)or ‘acombinationofTcellspulsedwithSIINFEKLandofTcellspulsedwithaGC’(1!106each)orTcellspulsedwithSIINFEKLplusfreeformofaGC(i.p.). CFSEhigh,peptide-pulsedtarget;CFSElow,peptide-unpulsedcontrol.Dataarearepresentativeofatleasttwoseparateexperiments. signal to the immune system. To test this hypothesis, C57BL/6 vivoCTLassay.Asdepictedin Figure7A,weobservedacomplete mice were vaccinated with T/pep in combination with either peptide-specific CTL activity in all of cytokine-deficient mice we soluble aGC (2mg, i.p.) or T/aGC. In mice vaccinated with the tested.Thus,noneofthesecytokinesisnecessaryinthegeneration former combination, little peptide-specific CTL was generated ofCTLactivitybytheTcellvaccine. (Fig.6C, 13.4% vs. 97.2% lysis in T/aGC/pep-vaccinated mice). Costimulatory signals are essential in regulating the activation Moreover, mice vaccinated with T/aGC plus T/pep only showed andexpansionofiNKTcells.Moreover,CD28andICOSsignals a moderate peptide-specific CTL, significantly less efficient than are required for the anti-tumor activity of activated iNKT thatofT/aGC/pep(Fig.6C,47.2%lysisvs97.2%inT/aGC/pep- cells such as cytotoxicity and anti-metastatic activity.2,21-23 vaccinatedmice).Thus,thesedatademonstratethatthevaccinating Activated T cells express CD80 and CD86 and may regulate TcellsactasdirectstimulatorsforbothiNKTandCD8Tcellsin immune response through T:T interaction.24,25 Therefore we vivoandthatpresentationofaGCandpeptidebythesameTcellis asked whether these costimulatory factors were necessary for the themostefficientregimeningeneratingantigen-specificCTL. induction of CTL in our model. T cells were isolated from Cytokine and costimulation requirements in the CTL C57BL/6(WT), B72/2 (B7.12/2B7.22/2), B7h2/2 or B7B7h2/2 response elicited by T/aGC/Pep. Upon activation, iNKT cells and loaded with aGC and SIINFEKL ex vivo. The T/aGC/pep promptly produce a wide range of cytokines including IL-4 and were injected into WT mice and in vivo CTL assay was IFNc. IL-4 produced by activated iNKT cells was shown to performed.Asdepictedin Figure7B,weobservedanormaltarget promote CD8 T cell proliferation.16 IFNc and IL-12 produced cell lysis in mice vaccinated with costimulation-deficient T cells. upon iNKT and DC interaction mediate anti-tumor activity.17,18 Thus, the induction of peptide-specific cytotoxicity by the T cell RecentstudiesshowedthatIL-21isproducedbyiNKTcellsupon vaccine does not require B7 and B7h costimulation. TcR stimulation and regulates the activation and expansion of iNKT cells.19,20 Therefore, we asked whether these cytokines Discussion producedafteriNKTcellactivationhadanyroleinthegenerationof CTLinourTcellvaccinemodel.WevaccinatedIL-42/2,IFNc2/2, In the present study, we showed that vaccination with con- IL-12p352/2orIL-212/2micewithT/aGC/pepandperformedin ventional CD4 T cells presenting both CD1d-aGC and MHC www.landesbioscience.com OncoImmunology 147 © 2012 Landes Bioscience. Figure7.MolecularrequirementsforefficientindicationofCTLbyiNKT-mediatedTcellvaccine.(A)C57BL/6mice(WT)orvariouscyokine-deficientmice withC57BL/6backgroundwerevaccinatedwithTcellscopulsedwithaGCandSIINFEKL.(B)PureCD4+TcellswereisolatedfromC57BL/6(WT),B72/2, B7h2/2orB7B7h2/2asdescribedin Figure1.IsolatedTcellswereco-pulsedwithaGCandSIINFEKLexvivobeforeinjectedintoWTrecipient.Aweek later,aninvivoCTLassDaywaspoerforme d.nCFSEhigho,peptidte-pu lseddtargeti;CFsSElow,pteptidre-unipulbsedconutrol.Dattaareerepre.sentativeofthreeseparate experiments. class I-restricted peptide triggered iNKT cell activation and is possible that T cells, like other non-professional APC, do not generated an antigen-specific cytotoxic Tlymphocyte response in express high levels of costimulatory molecules, which is required vivo. Mice receiving this novel T cell vaccine were resistant to for CD8 T cell effector function.26 We have also previously L. monocytogenes infection as well as to tumor challenge in an observed this type of non-productive activation in CD8 T cells antigen-dependent manner. Moreover, vaccination with aGC- reactive to a tissue antigen, which is mediated by PD-1-PDL1 pulsed T cell lymphoma also induced CTL response specific for interaction.27 In the presence of aGC and hence iNKT “help,” an endogenous tumor antigen. Mechanistic analyses indicate antigen-specific CD8 T cells were able to undergo effector that the vaccinated T cells directly stimulated peptide-specific differentiationbytheCD4Tcellvaccinethatresultsinprotection cytotoxic T cells and that the CTL responses induced by these against infection and tumor. Since iNKT ligands are either ‘T cell vaccines’ were independent of cytokines such as IFNc, derived from infectious agents or induced during infection, their IL-12 and IL-21. Therefore T cells as well as tumors of T cell presence on non-professional APC may provide a “dangerous” origin can act as antigen-presenting cells that efficiently trigger signaltotheimmunesystemand,throughiNKTcells,“license”a cytotoxic Tcell responses, when they are formulated tostimulate productive T cell activation and their effector differentiation. iNKT cells in vivo. How iNKT cell help CD8 cell effector differentiation remains Dendritic cells loaded with various iNKT ligands are potent unclear at this stage. After activation, iNKT cells promptly inducer of T cell immunity. We recently showed that B cells co- produce a wide range of cytokines which can further activate pulsed with aGC and class I peptide also induce antigen-specific themselves and other immune cells including NK, DC, T and B CTLresponseinvivoasefficientlyaswell.11Moreover,ourrecent cells. IFNc and IL-12 are critical mediators for T cell immunity. study showed that B lymphoma cells loaded with aGC elicited a IFNc produced by iNKT cells and NK cells mediates anti- strong protective immunity that is dependent on CD4 T cells.12 angiogenic activity of aGC.18 IL-12 produced by DC after However, it has been unclear whether non-professional APC in interactionwithiNKTcellsiscriticalforinducingIFNciniNKT the presence of iNKT cell help could induce T cell effector cells.17 However, in our T cell vaccine model, neither IFNc nor function. In the present study, we found that CD8 T cells were IL-12 was required for CTL generation. Recent studies suggest activated by antigens presented by CD4 T cells and proliferated that IL-21 is a crucial cytokine for iNKT cell activation and extensively. However, they did not produce significant amounts function.19,20 However, an efficient induction of CTL response ofIFNcorGranzymeBthatareassociatedwithcytotoxiceffector by ourT cell vaccine inIL-21-deficientmice excludes theroleof function,indicatingthatCD4TcellsarenotanefficientAPC.It this cytokine in the generation of CTL response in our model. 148 OncoImmunology Volume1Issue2 Moreover, although vaccinated T cells were found to directly Preparation of T and dendritic cells. Conventional CD4 stimulate iNKT and CD8 T cells, this action does not appear to T cells were purified from lymphoid cells of C57BL/6 mice by require costimulation. We recently found that dendritic cells sorting CD82 NK1.12 CD192 CD11b2 CD11c2 Gr-12 I-Ab2 loaded withaGCinduced iNKT cell activationintheabsenceof but CD4+ cells with a FACS Aria1. These cells were . 99.5% B7 and B7h (data not shown). It is unclear at this stage whether CD3+CD4+andCD1d+.Insomeexperiments,CD4Tcellswere there is any signal in addition to TcR that is required for iNKT isolatedfromCD1d2/2orbm-1mice.Bonemarrow-derivedDCs and CD8 T cell activation by our T cell vaccine. weregeneratedbyculturing bone-marrowcellsinthepresenceof Nonetheless, T cells loaded with an iNKT ligand serves as GM-CSF and IL-4 for 6 d. Sorted T cells were co-cultured with a potent cellular vaccination approach. A mild suppression of aGC (1 mg/mL) or the same volume of solvent vehicle (5 ml/mL tumor growth and L. monocytogenes growth was observed in mice of 0.5% polysorbate) overnight. In some experiments, these cells vaccinated with T/aGC, indicating that an effective innate were further incubated with SIINFEKL peptide for 1 h. immunity was induced. However, vaccination with T/aGC/pep EG-7cellswereculturedinthepresenceofIFNc(100ng/mL) was far more effective in eliciting CD8 T cell immunity and for 48 h including overnight culture with aGC or vehicle as specifically suppressing tumor growth and L. monocytogenes described above. These EG-7 cells were washed and irradiated growth, which was comparable to DC vaccination. Based on with Cs source (50 Gy) before i.v. injected into syngenic mice. ourresults,weproposethatTcellsfromindividualsinfectedwith CFSE-labeled OT-I adoptive transfer. Ovalbumin-specific Tcell-tropicpathogens—suchasHIV—couldbeusedasasource CD8 T cells were isolated from the lymphoid cells of OT-I mice of cellular vaccine for triggering pathogen-specific cytotoxic using anti-CD8 magnetic beads and AutoMacs (. 92% are response. Notably, i.v. injection of irradiated EG-7 thymoma Va2+). These cells were labeled with 10 mM of CFSE and i.v. ©did no t i2nduce0an OV1A-spec2ific CT LLrespoanse; hnoweverd, injece- trasnsferr edBinto Ci57oBL/6smicec. In isomee expnerimecnt, Ley5.1 . tion of aGC-loaded, irradiated EG-7 efficiently generated an congenic mice were used as recipients. Recipient mice were i.v. OVA-specific CTL response. As a result, vaccination with aGC- injected with CD4 T cells or irradiated EG-7 cells formulated ex loaded, irradiated EG-7 induced a potent anti-tumor activity vivo with indicated conditions. Two to five days later, lymphoid against challenge with live EG-7, which was CD8+ T cell- cells from the spleen of recipientmice were isolated and cultured dependent.ThereforeweproposethatvaccinationofaGC-loaded with 0.1 mg/mL SIINFEKL in the presence of GolgiPlug Tlymphomamayresultinthegenerationoftumor-specificCTL (1 mL/mL) for 5 h. These cells were stained with phycoerythrin Do not distribute. response. (PE)-conjugated anti-Va2 Ab or PE-conjugated anti-Ly5.2 Ab Our finding is novel in that non-professsional APCs are also before permeabilized and further stained with APC-conjugated able to trigger cytotoxic T cell immunity with the help of iNKT anti-IFNc Ab or Alexa 647-conjugated anti-granzyme B Ab. cell. T cell is a major population in blood and can be easily Total 1 ! 106 events were collected and CFSE+ PE+ cells were expanded and cultured in vitro. Although resting human T cells gated and analyzed by FACS Calibur. express little CD1d, activated T cells as well as thymocytes in iNKTcellstaining.ForintracellularstainingofIFNciniNKT humans express CD1d on their surface.28 Moreover, cutaneous cells, splenocytes and liver mononuclear cells from mice injected T cell lymphomas in humans have been reported to express high withvehicle-pulsedTcells,aGC-loadedTcellswerestainedwith levels of CD1D.29 These observations suggest that our findings PE-conjugatedaGC-loaded/CD1d-tetramertogetherwithFITC- can be directly applicable in clinical settings. Therefore, tumors conjugated anti-CD19 Ab (for splenocytes) or FITC-conjugated that are not professional APC in origin, especially thymoma and anti-TCRβ Ab. These cells were permeabilized and stained T lymphoma, could be used as efficient APC for cytotoxic T cell with APC-conjugated anti-IFNc Ab. CD192CD1d-tetramer+ or anti-tumor immunity when they are formulated to stimulate TCRβ+CD1d-tetramer+ cells were defined as iNKT cells and iNKTcells.OurTcellvaccinestrategywillprovideanalternative analyzed. cellularvaccineregimenthatcaninducetheefficientactivationof InvitroandvivocytotoxicTlymphocytesresponseassay.The both innate and adaptive immunity in vivo. in vitro SIINFEKL- specific cytotoxic T lymphocytes response wasmeasured usingacaspase-3cleavage assay.32In brief,CD8 T Materials and Methods cells were isolated from the vaccinated mice by using anti-CD8 microbeads and magnetic activated cell sorting. Splenocytes from Mice. C57BL/6, OT-I, C57BL/6bm1 (bm-1), IL-42/2, IFNc2/2, syngenicmicewereusedastargetcellsafterlabeledwithDDAO- IL-12p352/2, CD802/2CD862/2 (B72/2) mice were purchased SE and 1 mg/mL peptide. The sorted CD8 T cells were co- from The Jackson Laboratory (Bar Harbor, ME). B6.SJL culturedwiththetargetsplenocytesata1:1ratiofor2hat37°C. mice (Ly5.1) were purchased from Taconic Farm. B7h2/2 and The cells were permeabilized and stained with PE-conjugated B7B7h2/2 mice were generated and backcrossed in our labora- anti-cleaved caspase-3 antibody. The percentage of cleaved tory.26,30CD1d2/2micewerebredinouranimalfacility.IL-212/2 caspase-3-positive cells among DDAO-SE-labeled target cells mice were previously described31 and bred in our animal facility. wereanalyzedbyflowcytometry.TheinvivoSIINFEKL-specific All mice were kept under specific pathogen-free conditions in cytolytic activity of CD8 T-cell responses was measured as our animal facility (MD Anderson Cancer Center). All animal described previously.33 Briefly, splenocytes were either loaded studies were approved by the Institutional Animal Care and Use with1mg/mLpeptideorleftuntouchedbeforebeinglabeledwith Committee ofMD Anderson Cancer Center. CFSEatdifferentconcentrations(10and1mmol/L,respectively). www.landesbioscience.com OncoImmunology 149 Equal numbers of the two populations were mixed and injected challenged with 1 ! 106 live EG-7 cells and the survival and i.v.intomice.18–48hlater,splenocytesfromtherecipientswere tumor volume were monitored. Tumor volume based on caliper analyzed by flow cytometer to assess the peptide-specific killing. measurements were calculated by the modified ellipsoidal The ovalbumin-specific lysis was calculated as follows: r = % formula, 1/2(Length ! Width2). CFSElow / % CFSEhigh and % lysis = [1 – (r / r )] ! Statistics.StatisticalvalueswereassessedbytheStudent’st-test. unprimed primed 100, where r is the ratio. P values were expressed and error bars are SE. The correlation of L. monocytegenes infection and tumor challenge. C57BL/6 tumor volume was analyzed for significance using 2-way analysis mice were vaccinated with the indicated cellular vaccine (day 0) of variance (ANOVA).The Kaplan-Meier method was used to beforetheywerei.v.challengedwith5!104liveL.monocytogenes estimate the survival outcomes and groups were compared with expressingOVA(day10).Threedaysafterthebacterialchallenge, log-rank statistic. bacterial burden in spleen and liver was measured by culturing serially diluted homogenized spleen and liver on brain-heart Acknowledgments infusion agar plate. Peptide-specific IFNc-secreting cells in WethankDr.HaoShenforListeria-Ova,theFACSCoreFacility PBMC were measured after restimulation with SIINFEKL in at the MD Anderson for assistance with cell sorting and the the presence of Golgi-Plug for 5 h followed by intracellular NIH tetramer core facility (Atlanta, GA) for providing PBS57- staining of IFNc. In a tumor challenge model, mice vaccinated loaded CD1d-tetramers. This study was funded in part by grants with indicated cellular vaccines were i.v. challenged with live 2 ! from the National Institutes of Health (C.D.) and an MD 105B16-OVA10daftervaccination.Twoweeksafterthetumor Anderson Interdisciplinary Research Project (C.D.). C.D. is a challenge,tumorfociinthelungwerecountedandOVA-specific Cancer Research Institute Investigator and a Research Trust ©CD8 T 2cells w0ere me1asured2after s taiLning awith PnE-condjugatedeFesllow o f BMD Anideorson CsancercCeniter.eD.Z.nis supcporteed by . OT-I tetramer in combination with FITC-conjugated anti-CD8 MD Anderson Cancer Center and a Developmental Award Ab. To test protective antitumor effect by autologous vaccine, from NCI Joe Moakley Leukemia Spore grant. The Flow aGC loaded EG-7 cells were irradiated before i.v. vaccination Cytometry Core Facility is supported by The University of (1x106 cells). At 7 d later, mice were s.c. challenged 1 ! 106 live Texas MD Anderson Cancer Center Support Grant CA16672 EG-7 cells and then the survival and tumor size of mice were (NIH). monitored. To deplete CD8 T cells, 100 mg of anti-CD8 mAb Do not distribute. (563.8) was i.p. injected twice (on day-3 and -1) into the mice Disclosure ofPotential Conflicts OfInterest Statement vaccinated with EG-7/aGC on day-7. On day 0, mice were i.v. No potential conflicts of interests were disclosed. References 7. Lanier LL. NK cell receptors. Annu Rev Immunol 15. Norbury CC, Basta S, Donohue KB, Tscharke DC, 1998; 16:359-93; PMID:9597134; http://dx.doi.org/ PrinciottaMF,BerglundP,etal.CD8+Tcellcross- 1. TupinE,KinjoY,KronenbergM.Theuniqueroleof 10.1146/annurev.immunol.16.1.359 primingviatransferofproteasomesubstrates.Science naturalkillerTcellsintheresponsetomicroorganisms. NatRevMicrobiol2007;5:405-17;PMID:17487145; 8. 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Fujii S, Shimizu K, Kronenberg M, Steinman RM. 2853-61;PMID:17312129 cytokineproduction.JImmunol2007;178:2827-34; Prolonged IFN-gamma-producing NKT response 14. GabrilovichDI,NagarajS.Myeloid-derivedsuppressor PMID:17312126 induced with alpha-galactosylceramide-loaded DCs. cells as regulators of the immune system. Nat Rev Nat Immunol 2002; 3:867-74; PMID:12154358; Immunol 2009; 9:162-74; PMID:19197294; http:// http://dx.doi.org/10.1038/ni827 dx.doi.org/10.1038/nri2506 150 OncoImmunology Volume1Issue2