Characterization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell lymphoma with large granular cell morphology

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Characterization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell

lymphoma with large granular cell morphology

C. Bonnefont-Rebeix, C. Fournel-Fleury, Frédérique Ponce, S. Belluco, D.

Watrelot, S. E. Bouteille, S. Rapiteau, D. Razanajaona-Doll, J. J. Pin, Caroline Leroux, et al.

To cite this version:

C. Bonnefont-Rebeix, C. Fournel-Fleury, Frédérique Ponce, S. Belluco, D. Watrelot, et al.. Charac- terization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell lymphoma with large granular cell morphology. Immunobiology, Elsevier, 2016, 221 (1), pp.12-22.

�10.1016/j.imbio.2015.08.007�. �hal-02192918�

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ContentslistsavailableatScienceDirect

Immunobiology

j ou rn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / i m b i o

Characterization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell lymphoma with large granular cell morphology

CatherineBonnefont-Rebeixâ,∗,CorinneFournel-Fleuryâ,FrédériquePonceâ, SaraBellucoâ,DorothéeWatrelotâ,SylvieEBouteilleâ,SylvieRapiteauâ,

DianeRazanajaona-Doll^b,Jean-JacquesPin^b,CarolineLeroux^c,ThierryMarchal^a

aUPSPICE2011-03-101,Oncologie,VetAgroSup,CampusVétérinaire,1AvenueBourgelat69280Marcyl’Etoile,France

bDendritics,60AvenueRockefeller,69008Lyon,France

cINRA,UMR754“RétrovirusetPathologieComparée”,UniversitéLyon1,50AvenueTonyGarnier,69007Lyon,France

a r t i c l e i n f o

Articlehistory:

Received20March2015

Receivedinrevisedform15June2015 Accepted11August2015

Availableonline24August2015

Keywords:

T-celllymphoma Cellline Dog IL-17 CD56mRNA

a b s t r a c t

Dogswithlymphomaareestablishedasgoodmodelforhumannon-Hodgkinlymphomastudies.Canine celllinesderivedfromlymphomasmaybevaluabletoolsfortestingnewtherapeuticdrugs.Inthiscontext, weestablishedacanineT-cellline,PER-VAS,fromaprimaryaggressiveT-celllymphomawithlarge granularmorphology.Flowcytometricanalysisrevealedastableimmunophenotype:PER-VAScellswere positivelylabelledforCD5,CD45,MHCIIandTLR3,andwerenegativeforCD3,CD4andCD8expression.

Althoughunstablealongthecultureprocess,IL-17andMMP12proteinsweredetectableaslateasat passages280and325i.e.respectively24and29monthspostisolation.Atpassage325,PER-VAScells maintainedtheexpressionofIL-17,CD3,CD56,IFN␥andTNF␣mRNAsasshownbyRT-PCRanalysis.

StablerearrangementoftheTCR␥genehasbeenevidencedbyPCR.PER-VAScellshaveahighproliferation indexwithadoublingtimeof16.5handweretumorigenicinNudemice.Comparedtothecaninecell linesalreadyreported,PER-VAScellsdisplayanoriginalexpressionpattern,closetoNKTcells,which makesthemvaluabletoolsforinvitrocomparativeresearchonlymphomas.

1. Introduction

NonHodgkinlymphomasrepresentthemostfrequentcanine hematopoietictumourwithanincidenceof15–30/100000sim- ilartohuman(Marconatoetal.,2013;VailandMacEwen,2000).

Moreover,dogsdevelopspontaneouslymphomasresemblingthe human tumour regarding the type, grade, site of involvement andclinicalfeatures.ThecanineNHLclassificationfollowedthe humanclassificationbygroupinglymphomasaccordingtotheir cytological characteristics (updated Kiel classification (Lennert, 1991))andmorerecentlybasedontherevisedEuropean–American (Harris et al., 1994) and World Health Organization (WHO) classification (Jaffe, 2001; Pileri et al., 1998; Swerdlow, 2008) takinginto accountphenotypic,genetic andmolecular features (Fournel-Fleury et al., 1997,2002; Ponce et al.,2010; Turinelli et al., 2005; Valli et al., 2011). Importantly,dogs and humans

∗Correspondingauthor.

E-mailaddress:catherine.bonnefont@vetagro-sup.fr(C.Bonnefont-Rebeix).

sharingthe same environment, the cartography of human and canine lymphomas related to outdoor carcinogens contributes to the evaluation of environmental risk factors (Pastor et al., 2009).

In human, peripheral T-cell lymphomas (PTCL) represent a groupofheterogeneouslymphomascharacterizedbyclonalexpan- sionofTorNKcellswithgenerallypoorerprognosisthanB-cell lymphomas.Standardtherapeuticregimensresultedininsufficient patient outcomes witha rapid relapse and poor 5-years over- all survival rates (Coiffier et al., 2014). Since PTCL account for approximatively10–15%ofallnon-HodgkinlymphomasinWest- erncountries,therapeuticstrategyoflargeclinicaltrialscouldnot beconductedasforB-celllymphomascounterparts(Gooptuetal., 2015;Jainetal.,2015).Therefore,newtherapeuticapproachesare required,especiallyforhighlyaggressiveclinicalformsshowing multidrugresistancelikeextranodal NK-Tcell lymphomas(Gill etal.,2010).Unfortunately,thelackofvaluablepreclinicalmodel aswellasthedifficultiestoderivecelllinesphenotypicallycloseto theprimarytumorsrestrictedtheopportunitiestotestpotentially newreagentstowardsPTCL.

http://dx.doi.org/10.1016/j.imbio.2015.08.007

by-nc-nd/4.0/).

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Indogs,PTCLalsorepresentaheterogeneousgroupwithvari- ousprognoses(Fournel-Fleuryetal.,2002;McDonoughandMoore, 2000).Amongthem,LargeGranularLymphomas(closetohuman extranodalNK/T-celllymphomas) displayedaveryhighaggres- siveclinical course,often associated with hemophagocytic and macrophagesactivationsyndromesandverypooroverallsurvival rates(Turinellietal.,2005).Therefore,theopportunitytodevelopa preclinicalspontaneouscaninemodelortoobtaincanineNK/T-cell linetoassessnoveltherapeuticapproachesforsuchlymphomas constitutesamajorchallenge.

To date, twelve still available canine cell lines have been described and characterized according to their morphology, immunophenotype, pattern of TCR genes status and ability to inducetumoursinmice(Kisseberthetal.,2007;Momoietal.,1997;

Nakaichietal.,1996;Rutgenetal.,2010;Steplewskietal.,1987;

Suteretal.,2005;Umekietal.,2013;Yamazakietal.,2008).Impor- tantly,cellphenotypemaychangeeitherfromtheprimarymaterial totheestablishmentofthecellline(Rutgenetal.,2010)orduring invitromaintenance(Umekietal.,2013),leadingtodifferencesin thepatternofexpressionofspeciﬁcmarkers.Inthiscontext,long- termstudyofex-vivoderivedcellsiscrucialtoadequatelyestablish theirphenotype.

Inthepresentstudy,wefollowedthemorphologyaswellasthe expressionoftheCD3,CD56,IL-17,TNF␣andINF␥markersinthe PER-VAST-cellline,derivedfromanaggressivelargegranularT- celllymphoma,along325invitropassages(overfouryears).We describedanewlymphoma-derivedcelllineexpressingmarkers similartothetumouritoriginatedfrom.

2. Materialsandmethods 2.1. Casereport

AsevenyearoldmaleLabradorwasinitiallydiagnosed with Leishmaniasis(assessedwithserologicalandPCRtests),andwas presented to the veterinary hospital for severe dyspnea and asthenia.Thedogshowedmoderateregenerativenormochromic normocytic anemia, with the following hemogram values: red cells:3.53tera/L;hemoglobin:7.8g/dL;hematocrit:22.3%;VGM:

63fL; CCMH: 34.8g/dL; TCMH: 22pg; reticulocytes: 0.5%. No abnormallymphoid cells werepresent onthe peripheralblood smear. The ultrasonic scan conﬁrmed a pleural and pericardial extravasationwithcardialtamponaderelatedtothemainclinical signs(severedyspnea andasthenia) and theabsenceof abnor- malneoplasticmass.Theabdominalultrasonicexaminationgave noadditionalinformation. Cytologicalexaminationshowedthat pericardialeffusionwasmainlycomposedoftumoralcellswithlab- oratoryvaluesindicatingcellcount:29.5×10³/mm³andprotein:

6.4g/dL.Pleuraleffusion displayeda mixof mesothelial,histio- cytic,lymphocytic,neutrophilicandraretumoralcells,withcell count:2.67×10³/mm³andprotein:5.8g/dL.Axillaryandpopliteal lymphnodeswereslightlyenlargedatthefirstclinicalexamina- tion,butfineneedleaspirationofthesesuperficiallymphnodes showednosignsoftumoralcells.AnaggressiveT-celllymphoma withlargegranularcellmorphologywasdiagnosedoncytologi- calexaminationofpericardialandpleuraleffusionaspirations.The diseaseprogressedrapidlytowardsdeathbeforeanychemother- apy.Nohistopathologicalinformationwasavailablebecausethe autopsywasunauthorizedbythedog’sowner.

2.2. EstablishmentofPER-VASlymphomacellline

Cellsfromthepericardialeffusionwereculturedat10⁶cells/ml and 5×10⁵cells/ml in complete RPMI medium (RPMI 1640 medium supplemented with 20% fetal calf serum, 2mM l-

glutamine,1%penicillin/streptomycin)in75cm²ﬂasksat37^◦Cin humidiﬁedatmospherewith5%CO₂.Passageswereconducted3 timesaweekbyreplacinghalfoftheculturewithfreshcomplete RPMI medium.Primary effusionconsisted ofa mixed cellpop- ulationincludinglymphocytes,histiocytes,mesothelialcellsand atypicallymphocyteswithlargeacidophiliccytoplasmicgranules.

After3weeksofculturewith3passagesaweek,cellsstartedto growinsuspensionformingfloatingcellclusters.Thecellswere maintainedincontinuouscultureduring9monthswithhighpro- liferative growth, and werethen stored in liquid nitrogen at a concentrationof10×10⁶cells/mlin90%FCS,10%DMSOatpassage 142.Inasecondperiod,PER-VAScelllinewasthawed4months laterforculturingfrompassages143to199during7months,and thenwasfrozenatpassage199.Inathirdperiod,thecelllinewas thawed2monthslaterfrompassages200to280during8months beforefreezing.Thefourthperiodbeganoneyearandahalflater frompassages281to325during5monthsandimmunophenotype wasassessed.Splittingwasconducted15timespermonthduring thefirstcyclebyreplacinghalfoftheculturewithfreshcomplete RPMImedium.SVFwasmaintainedat20%inthemediumuptopas- sage150andthendecreasedto10%forthefollowingcycles.From thesecondcycleandfurther,5×10⁵cellswereseededin25cm² flasksandweresubcultured8–10timespermonthbyreplacing atleastthree quartersoftheculturewithfreshcompleteRPMI medium.Finally,PER-VAScelllinewasculturedalong325passages overfouryearsthrough4freezing/thawingcycles.

PER-VAScellswereclonedatpassage200byseedingataden- sityof≤1cellperwellin96-wellﬂatsbottomcultureplates(Nunc).

Twenty fouroutof the32 clonesobtainedwereexpanded and analysedforantigenreceptorrearrangementsandphenotype.

Fordoublingtimecalculation,cellswereseededintriplicateat 2×10⁴cells/wellin96-wellplate;viablecellswerecountedevery 24hfor3daysusingtrypanbluedyeexclusion.Thelogarithmic leastsquaresﬁttingtechniquewasusedforthecalculation(Roth, 2006).

2.3. Primarycellsandcelllinesusedascontrols

A20murineBcelllymphomaandJurkathumanT-cellleukemia cell lines were obtained from the CelluloNet facility (SFR Bio- Sciences Gerland-Lyon Sud, UMS3444/US8). They were usedas positive controls for western-blot analysis of Per-Vas cells for IL17andMMP12expression,respectivelyandincomparisonwith canineandhumanPBMC.CaninePBMCwereisolatedformhealthy adultbeagledogs(ClaudeBourgelatInstitutoftheVetAgroSupvet- erinarycampus,Lyon,France)inaccordancewiththeinstitutional guidelines.HumanPBMCwereobtainedfromhealthydonorsunder informedconsent(EtablissementsFranc¸aisduSang,Lyon,France).

RT-PCRanalysisofPER-VAScellsweredoneincomparisonwith canineleukemiaT-cells(CD3+/CD5+/CD45+/CD4−/CD8−/CD21−).

LymphoidhyperplasiaT-andB-cellsfromﬁneneedleaspiration oflymphnodeswereobtainedfromdogssampledatVetagroSup Veterinarycampusandwereusedaspositivecontrolforclonality assessment(PARRassay).

2.4. Phenotypeanalysis

PER-VAS cells were analyzed by ﬂow cytometry from pas- sages25to325.Immunocytochemistrywasperformedonprimary pericardialeffusion and onPER-VAScells at passage240using cytospin smears.Cells werelabelled withmonoclonalantibod- ieslistedinTable1.Forﬂowcytometryanalysis,1–5×10⁵cells wereincubatedwithmAborisotypematchedirrelevantIgsfor 30min at 4^◦C. For indirect staining,after a PBS washing, FITC- labelledanti-mousesecondaryantibodywasincubatedfor30min

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Table1

Antibodiesusedinthisstudy.

Speciﬁcity Clone Isotype Conjugation Applicationused Source Referencesforcrossreactivity

HumanCD1a NA1/34-HLK mIgG2a –^a FC Serotec,Oxford,UK Galkowskaetal.(1996)

CanineCD3 CA17.2A12 mIgG1 FITC FC Serotec,Oxford,UK –

HumanCD3⑀ CD3-12 rIgG1 –^a FC,ICC Serotec,Oxford,UK Jonesetal.(1993)

HumanCD3⑀ polyclonal rabbit – ICC Dako,Glostrup,Denmark Jonesetal.(1993)

CanineCD4 YKIX302.9 rIgG2a FITC FC,ICC Serotec,Oxford,UK –

CanineCD5 YKIX322.3 rIgG2a FITC FC,ICC Serotec,Oxford,UK –

CanineCD8 YCATE55.9 rIgG1 FITC FC,ICC Serotec,Oxford,UK –

CanineCD11c CA11.6A1 mIgG1 –^a FC Serotec,Oxford,UK –

HumanCD14 TÜK4 mIgG2a –^a FC Dako,Glostrup,Denmark Drexler(2001)

CanineCD21 CA2.1D6 mIgG1 PE FC Serotec,Oxford,UK –

CanineCD45 YKIX716.13 rIgG2b FITC FC Serotec,Oxford,UK –

CanineMHCII YKIX334.2 rIgG2a FITC FC Serotec,Oxford,UK –

CanineTCR␣␤ CA15.8G7 mIgG1 – ICC P.F.Moore,UCDavis –

CanineTCR␥␦ CA20.8H1 mIgG2a – ICC P.F.Moore,UCDavis –

HumanTLR3 722E2 mIgM –^a FC,WB GiftfromDendritics,Lyon,France Bonnefont-Rebeixetal.(2007)

HumanIL-17 412H6 mIgG1 –^a FC,WB GiftfromDendritics,Lyon,France Thisstudy

HumanMMP12 703D10 mIgG1 –^a FC,WB GiftfromDendritics,Lyon,France Thisstudy

Anti-mouseIg – GoatIg FITC FC Dako,Glostrup,Denmark –

Abbreviations:m=mouse;rat;FC=ﬂowcytometry;ICC=immunocytochemistry;WB=Westernblotanalysis.

aRequiredanti-mouseIg-FITC-labelledsecondaryantibodyforﬂowcytometry.

at4^◦C.IntracellularstainingforCD3(dilution1:100),TLR3,IL-17 andMMP12(16␮g/ml)wasperformedafterpermeabilizationwith 0.3%saponin(SIGMA)inPBS.CellswereanalyzedonaBDAccuri ﬂowcytometer(BDBiosciences).

Immunocytochemistry was performed on cytospin smears using the UltraTek HRP Anti-Polyvalent kit (ScyTek Laborato- ries)withVector^®NovaRED^TM(VectorLaboratories)assubstrate, followingthemanufacturer’sinstructions,withhematoxylincoun- terstaining.Negativecontrolwasperformedwithouttheprimary mAb.TCR␣␤andTCR␥␦stainingwerekindlyperformedbyPrPeter F.Moore,U.C.Davis,California,USA.

2.5. WesternBlot

CanineandhumanPBMC,A20,JurkatandPER-VAS(atpassage 280)cellswerelysedaspreviouslydescribed(Bonnefont-Rebeix et al., 2007).Cell lysates were denatured at 95^◦C for 5min in the presence of 2% ␤-mercaptoethanol and 2% SDS. Recombi- nanthumanMMP12producedinCOP5murinecells(rhuMMP12, giftfromDendritics,Lyon,France) wasusedaspositive control forMMP12detection.Fifteen␮gof proteinswereseparatedon 12or15%SDS-PAGEandblottedontonitrocellulosemembranes.

Membraneswereblockedwith“Protein-FreeT20BlockingBuffer”

(ThermoScientiﬁcPierce)atroomtemperaturefor10min.before incubation for 1h at room temperature and overnight at 4^◦C withanti-IL-17AmAb(9␮g/ml)andanti-MMP12mAb(15␮g/ml) (Dendritics,France).Blotsweredevelopedaspreviouslydescribed (Bonnefont-Rebeixetal.,2007).

2.6. Polymerasechainreactionforantigenreceptor rearrangements(PARR)

For clonality assessment, both immunoglobulin and T-cell receptor␥(TCR␥)genesrearrangementswereinvestigatedusing PARRaspreviouslydescribed(Burnettetal.,2003).Totalgenomic DNAwasextractedfrom5×10⁶frozenprimaryeffusioncellsand PER-VAScellsatpassages145,205and325usingthe“NucleoSpin^® Tissue kit” (Macherey Nagel). Cells from lymphoid T- and B- hyperplasiawithpolyclonalreceptorrearrangementwereincluded inallassaysascontrols.PCRreactionswerecarriedoutat95^◦Cfor 4min,followedby40cyclesat95^◦Cfor45s,55^◦Cfor45sand72^◦C for45s.PCRproductswereseparatedonto2.5%agarosegeland visualizedafterstainingwithGelRed(Thermoscientiﬁc).

2.7. mRNAexpression

TotalRNAwasextractedfromPER-VAScellsatpassages280 and325andfromcanineleukemiaCD3⁺T-cellusingthe“Ambion^® PureLink^® RNAMiniKit”(Life technologies).AfterDNasetreat- ment(“Ambion^®TURBODNA-freeDNaseTreatmentandRemoval Reagents”),100ngoftotalRNAwerereverse-transcribedusingthe

“iScript^TMcDNASynthesisKit”(Bio-Rad).RT-negativecontrolshave beenperformedto conﬁrmtheabsenceof contaminating DNA.

PCRreactionswereperformedin20␮lwith10–200ngofcDNA usingthe“Kapa^TM TaqDNAPolymerase kit”(KapaBiosystems).

Theampliﬁcationwasperformedat95^◦Cfor3min,followedby40 cyclesof95^◦Cfor20s,60^◦C(except63^◦CforIL17)for20sand72^◦C for20susingdog-speciﬁcprimers(0,2␮M)forCD3␦,CD56,INF␥, TNF␣,IL-17,MMP12,andACTBashousekeepinggene(Table2).PCR productsweresize-fractionatedona2%agarosegel.

2.7. Tumorigenicityassay

Xenografted tumor cell assays were conducted in Cancer Research Center,Toulouse,France, inaccordance withthe Ani- malCareand UseCommittee(approvalnumber 06 563DG-04).

Two adult (7 weeks old) athymic nude mice (RjOrl:NMRI- Foxn1nu/Foxn1nu)wereinoculated subcutaneously intheright sidewith10⁷PER-VAScells(passage60)in0.25mlPBSpermouse.

Micewereobservedweeklyfortumourgrowth.Euthanasiawas conducted11weeksafterinoculation.Tumormasswasexcised fromtheinoculationsite andﬁxedin10%formalinandembed- dedinparafﬁn.Fortissuemorphology,glassslidessectionswere preparedandstainedwithhematoxylinandeosin(HE).

3. Results

3.1. Characteristicsoftheprimaryeffusioncellsand establishmentofPER-VAScellline

The primary malignant pericardial effusion of the 7 year- old male Labrador consistedof a mixtureof median-sized and large lymphoma cells with nucleus twice larger than an ery- throcyte (Fig. 1A) in a hemorrhagic background. These cells displayed a high nuclear/cytoplasmic ratio, irregularly folded nuclei,irregularcoarsechromatin,poorlyvisiblepalenucleoli,and abasophilic cytoplasmcontainingnumerousmicrovacuolesand someazurophilicgranules.ExpressionofCD3^,^CD4,^CD8,^CD79a,

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Table2

OligonucleotideprimersusedforRT-PCRofPER-VAS.

Targetgene Accessionnumber Primer Nucleotidesequence(5-3)

CD3␦ XM536556 FORREV GAACAATCCGACAAAGCACCT

AGTGGCGATTATGTCAGCGA

CD56 AY860627 FORREV TTGTCCCCAGCCAAGGAG

TAGATGGTGAGCGTGGAGGAAGA

INF␥ NM001003174 FORREV GGCTGTAACTGTCAGGCCAT

ACGAAAAGAGACCCACTC

TNF␣ NM001003244 FORREV GCCTGCTGCACTTTGGAGT

GTTGGCCAGGAGGGCATT

IL-17 NM001165878 FORREV CTGAGCCTGGTGGCTATCAT

GGGCCTTCTGGAGTTCGTATT

ACTB NM001009784 FORREV CCAACCGTGAGAAGATGACC

CCAGAGGCGTACAGGGACAG

Fig.1. ComparativefeaturesoftheprimarymalignantpericardiceffusionandtheestablishedPER-VAScellline.

(A).Theprimarymalignantpericardiceffusiondisplayedanimportantcontingentofmedium-sizedandlargelymphomacellswithabasophiliccytoplasmcontaining numerousclearvacuoles(*)andazurophilicgranules(→)inahemorrhagicbackground.(B).PER-VAScellsestablishedfromtheprimarymalignantpericardiceffusion showedlargecellswithahighnucleartocytoplasmicratioverysimilartotheprimaryeffusioncells,andadeepbasophiliccytoplasmcontainingrareazurophilicgranules (→).StainingwithMay-Grünwald-Giemsastain,originalmagniﬁcation×100.

Fig.2. Immunocytochemistryonprimaryeffusioncells(A,B)andPER-VAScellline(C,D)smearsusingmonoclonal(A,C)andpolyclonal(B,D)anti-CD3^.

(A)Primaryeffusioncellsshowingapositivestainingfornormallymphocyte(*)whereasatypicalmedium-sizedandlargelymphocytes(→)arenegative(throughbackground) usingCD3mAb.(B)Positivestainingofprimarymalignanteffusioncellsusingpolyclonalrabbitanti-humanT-cellCD3.(C)NegativestainingofPER-VAScelllineatpassage 325usingCD3mAb.(D)PositivestainingofPER-VAScelllineatpassage325usingpolyclonalanti-CD3.