Adoptive γδ T cell therapy controls cytomegalovirus infection in preclinical transplantation models

Domain: chemistry
Published: 2026-02-17
Editorial outcome: minor_revision

Abstract
γδ T cells show promise for anti-tumoral therapies but have yet to be evaluated to treat infectious diseases. In this preclinical study, we assess a Vδ1+ γδ T cell-based adoptive cell therapy, named Delta One T cells, to treat cytomegalovirus (CMV) infection in high-risk transplant recipients. Even when expanded from CMV-naïve healthy donors, Delta One T cells efficiently control CMV dissemination in vitro. CMV recognition is independent of the γδ TCR but requires LFA-1 co-stimulation. In an in vivo model, adoptive transfer of mouse γδ T cells recapitulating Delta One T cell features protects mice against lethal murine CMV infection. Importantly, CMV-reactive Delta One T cells can be successfully generated from kidney transplant recipients undergoing refractory CMV infections and maintain their functionality in the presence of immunosuppressive drugs. These findings broaden the scope of γδ T cell therapies to infectious diseases and uncover a universal adoptive T cell therapy to treat refractory CMV infections.

Introduction
Humancytomegalovirus(CMV)infectionremainsthemostcommonopportunistic infection following transplantation. In solid organ transplantation (SOT), CMV disease is more frequent in CMV-naïve recipients with a CMV-seropositive donor (D+R-, 15–20%), than in CMV-seropositive recipients (R+, 5–10%)1,2. CMV disease is associated with an increased risk of graft loss in all solid organ transplant recipients (SOTR) and a higher mortality in lung transplant recipients3,4. In hematopoietic stem cell transplantation (HSCT), D-R+ patients have the highest risk of CMV disease and a decreased survival5.

Besides,inpatientswithCMVdiseaserecurrence(15–30%)6,7orrefractoryCMVinfection(up to 10%)5,8, second-line therapies are associatedwithsignificantadverseeffects.MaribavirhasrecentlybeenapprovedasacurativetherapyforrefractoryorresistantCMVdiseaseinboth HSCT and SOT9. Nevertheless,its efficacyis limited sinceit isnotabletoclearCMVat8weeksin45%ofpatients,andisassociatedwithmorethan 30% of recurrenceand 26% of emergent mutations associatedwithresistance10.ThereisthusanurgentneedforstrategiesabletoinducearobustandpersistentCMV-specificimmuneresponse,whichisessentialforlifelongviralcontrol11.

AdoptivetransferofCMV-specificCD4+andCD8+αβTcellshas emerged as an alternative therapy in both hematopoietic stem cell transplant recipients (HSCTR) and SOTR, with the goal to restore a potentanti-CMVadaptiveimmunity12,13.Third-partyCMV-specificαβT cell adoptive transfer was recently shownto improve survival upon refractoryCMVinfectioninHSCTR14andSOTR15.InSOTR,autologous CMV-specificαβTcelltherapymayalsobenefitpatientswithrecurrent orresistantCMVinfections16,17.However,αβTcelltherapiesarehuman leukocyteantigen(HLA)-restrictedandarethereforecurrentlylimited, in term of feasibility and efficacy, to only a fraction of transplant recipients,raisinginterestforcomplementary,HLA-independent,cel- lularimmunotherapies. It is notably challenging to obtain sufficient numbersofCMV-specificαβTcellsfromCMV-naïvepatients,whoare thosewiththehighestrisktodeveloprefractoryCMVinfections.

The unique capability ofγδT cells to kill tumorcellsindepen- dentlyofmajorhistocompatibilitycomplex(MHC)presentationand theirnaturaltraffickingtotissuesrecentlyputthemattheforefrontof immunotherapystrategies,withmanyongoingclinicaltrialsincancer. However,clinicaluseofγδTcellsininfectiousdiseaseshasyettobe tested,despitetheirwell-demonstratedprotectivefunctionsinseveral infectioussettings18–20.HumanγδTcellshavetraditionallybeendivi- ded into two subsets according to their T cell receptors (TCR): Vγ9Vδ2Tcells(alsocalledVδ2posTcells)andnon-Vγ9Vδ2Tcells(also calledVδ2negγδTcells).Vδ2negγδTcells,mainlycomposedofVδ1and Vδ3 T cells, were shown to be involved in the anti-CMV immune responseinHSCTR21andSOTR22,23.CMV-inducedVδ2negγδTcellsare mainlycomposedofactivatedTeffectormemoryCD45RA+(TEMRA) cells24,andVδ2negγδTcellclonesorcelllineswereshowntoinhibit CMVreplicationandkillCMV-infectedcellsinvitro25,26.InSOTR,Vδ2neg γδTcellexpansionwasassociatedwithrecoveryfromCMVinfection27 andthisprotectiverolewasconfirmedbyseveralstudiesinmice28,29. Importantly, while αβ T cells only recognize antigens presented by classIorclassIIMHC(HLAinhumans),γδTcellscanrecognizeanti- gens via their T cell receptor (TCR) without MHC/HLA restriction, allowing allogeneic cell therapy strategies. Altogether, these data support the use of Vδ2neg γδ T cells as a potential adoptive immu- notherapy for refractory CMV infections, especially in high-risk D- R+HSCTRandD+R-SOTR.

Recently,arobustclinical-grademethodforselectiveandlarge- scaleexpansionofVδ2negγδTcells,termedDeltaOneT(DOT)cells duetopredominance(>70%)ofVδ1+cellsinthefinalproduct,was developed for adoptive immunotherapy in patients suffering from chronic lymphocytic leukemia30 and acute myeloid leukemia31–33. Leveragingthisexpansionprotocol,weaimedtoevaluateDOTcells against CMV infection in an attempt to extend the applications of adoptive γδ T cell therapies. In this study, we expanded Vδ2neg γδ T cells (hereafter called DOT cells) from both CMV-seropositive (CMVpos)andCMV-seronegative(CMVneg)healthyblooddonors,as well as D+R-SOTR. Ourobjectivesweretocharacterizeandcompare theiranti-CMVreactivityinvitroandinvivo,todissectthemolecular mechanismsunderlyingCMVsensingbyDOTcells,andtoevaluatethe feasibilityofanautologoustherapyinimmunocompromisedkidney transplantrecipients(KTR),towardstheuseofDOTcellsasanadop- tiveimmunotherapyforpost-transplantrefractoryCMVinfection.

Results
DOTcellexpansionfromCMVposandCMVneghealthydonors
Aspreviouslydescribed,Vδ2negγδTcellsfromtheperipheralbloodof healthydonorscanbeselectivelyandrobustlyexpandedinvitroto clinicallyrelevantnumbersusingtheDOTprotocol30,31.Thisprocedure leadstoahighly-polyclonalexpansionstemmingfromnaïveVδ2negγδ Tcells31.However,CMVinfectionstronglydrivesthesecellstowards terminal differentiation, reducing the frequency of naïve Vδ2neg γδ Tcellsfromapproximately70%inCMVnegdonorstoaroundorbelow 30% in CMVpos donors24. We therefore first set out to uncover a potentialnegativeimpactofpreviousCMVexposureontheinvitro expansioncapacityofDOTcells.Forthis,DOTcellswereexpanded from10CMVnegand10CMVposhealthyblooddonorsandanalyzed byflowcytometryasdepictedinFig.1a.After21daysofculture,we obtainedamean±standarddeviation(SD)of4.3±1.8×107Vδ2negγδ T cells for CMVneg donors, compared to 6.2±4.7×107 cells from CMVpos donors (Fig. 1b), with corresponding Vδ2neg γδ T cell fold increases in culture of 1.8±1.5×103 and 1.2±0.7×103 (Fig. 1c). A stronglyenrichedcellularproductwasobtained,asVδ2negγδTcells represented85.8±5.1%and84.8±7.6%ofexpandedcellsforCMVneg and CMVpos donors respectively (Fig. 1d). All theseparametersdid notsignificantlydifferbetweenthetwogroups(two-tailedunpaired t-tests, absolute numbers P=0.25, fold increases P=0.34, and per- centages P=0.71).We alsoconfirmed thatVδ1+ γδT cellswere the mainpopulationobtainedafterinvitroexpansion,followedbyother Vδ2negγδTcellsubtypes,andrareVδ2+γδTcellsandnaturalkiller (NK)cells.ThisrepartitionisillustratedinFig.1efor3CMVnegand3 CMVpos donors and was not significantly impacted by the CMV serotype (determined by two-tailed unpaired t-tests for each cell population: Vδ1+γδ T cells P=0.7463; Vδ2+γδ T cells P=0.3458; Vδ3+γδTcellsP=0.2220;Vδ1/2/3negγδTcellsP=0.2732;CD3negcells P=0.8933;αβTcellsP>0.9999).Altogether,theseresultsshowthat previousCMVinfectiondoesnotaffecttheclinical-gradeexpansionof DOTcells.

DOTcellsdisplayaCMV-independenteffectorprofile
Donor CMV serostatus could potentially influence DOT cell pheno- type, and eventually affect their suitability for cell therapy applica- tions.Totestthishypothesis,DOTcellsexpandedfromCMVnegor CMVposdonorswereanalyzedbymultiparametricflowcytometry.At day 0, Vδ2neg γδ T cells from CMVneg donors were predominantly naïve(CD27+CD45RA+,74.0±18.8%)whereasCMVposdonorsdis- played a higher frequency of TEMRA Vδ2neg γδ T cells (CD27negCD45RA+, 41.2±19.8%). This previously described difference24waslostattheendoftheexpansion,withavastmajorityof CD27+CD45RAneg central memory cells obtained for both serotypes (88.1±9.2%forCMVnegdonorsand88.1±9.6%forCMVposdonors) (Fig.1fandFig.S1a).Importantly,acentralmemoryphenotypewas previouslyshowntobefavorableforthepersistenceandlong-term efficacyofinfusedTcelltherapies34,35.

Amoreextensivephenotypingwasthenperformed,comparing3 CMVneg and 3 CMVpos donors (Fig. 1g). Gating strategies are pre- sentedinFig.S1b,c,witharepresentativeanalysisofactivationmar- kers, and in Fig. S2, with a representative analysis of killer cell immunoglobulin-like receptors (KIRs). After expansion, Vδ2neg γδ Tcellsfullyexpressedactivationmarkers,butonlysmallpercentages ofcellsexpressedtheinhibitorycheckpointsCTLA-4,LAG-3andPD-1. Besides,TIGITexpressionwasnotimpactedbyinvitroculture,while expandedcellsbecamepositiveforTIM-3.Co-stimulatoryreceptors wereabsent(OX40,CD40L),scarcelyexpressed(CD28,CD30,4-1BB, CD8αα),orhomogeneouslyexpressed(LFA-1)afterexpansion,while theNKactivatingreceptorsDNAM-1,NKG2D,NKp30andNKp44were strongly up-regulated, as previously described30. Of note, although percentagesofLFA-1+cellswerenotmodifiedbyinvitroexpansion (Fig.1g),itsexpressionwasstronglyupregulatedonapercellbasis,as indicatedbyasignificantincreaseinmedianfluorescenceintensity (Fig.1h).Importantly,percentagesofcellspositiveforthecytotoxicity ligandsTRAILandFasLweremarkedlyincreased,similarlytoprevious observationsongranzymeBandperforin31,thusindicatinganovert cytotoxicphenotype.ItisnoteworthythatneitherCD16norKIRswere inducedbytheexpansionprotocol.Finally,weobservednosignificant impactoftheCMVserotypeinthisphenotypicanalysis,asillustrated for3CMVnegand3CMVposdonors(Fig.1gandstatisticalcompar- isons in Fig. S3). Altogether, we were able to manufacture a DOT productcomposedofstronglyactivatedVδ2negγδTcells,poisedfor effector (particularly cytotoxic) functions, regardless of the donor CMV serologicalstatus.

DOTcellsdisplayanti-CMVeffectorfunctions
AprimaryobjectiveofthisstudywastodeterminewhetherDOTcells canrespondtoCMVinfectioninvitro.TheCMVclinicalstrainTB42/E wasusedtosuccessfullyinfectthreedifferenttargetcellmodels,as evidenced by the presence of Immediate-Early (IE) CMV antigen in human foreskin fibroblasts, autologous monocyte-derived macrophagesandglomerularendothelialcells(Fig.S4a).Buildingon their effector phenotype (Fig. 1g), we first explored the cytotoxic potentialofDOTcells,bymeasuringtheexpressionofthedegranu- lationmarkerCD107afollowingmonocultureorcoculturewithmock- infectedorCMV-infectedfibroblastsandmacrophages(Fig.S4b,cand Fig.2a).CMVinfectionsignificantlyincreasedDOTcelldegranulation against fibroblasts, and modestly against macrophages, with never- thelessastrongsignificanceobservedrelativetoDOTcellsculturedin mediumalone(Fig.2a).

Next,wequantifiedthesecretionofmultiplecytokines,chemo- kinesandgrowthfactors(listedintheMethodssection)incoculture supernatants to identify a potential CMV-induced secretome. The cytokinesIFNγandIL-10,aswellasthechemokinesGM-CSF,IP-10and I-TAC,wereinducedbyCMVinfectionincocultures,withthestrongest inductionobservedforIFNγincocultureswithCMV-infectedmacro- phages(Fig.S4d).WedecidedtofurtherfocusonIFNγ,whichhasbeen extensively characterized as a pleiotropic cytokine that modulates both innate and adaptive antiviral immune responses and can also inhibitdirectlyCMVreplication36.FNγsecretioninresponsetoCMV- infectedfibroblastsandmacrophageswasextensivelyconfirmedina larger cohort of DOT cell donors (Fig. S4e). Notably, as previously observed37,IL-18additiontococulturespotentiatedIFNγsecretionin responsetoCMV.Indeed,inthepresenceofIL-18,IFNγsecretionwas stronglyinducedbyCMV-infectionoffibroblasts,macrophages,but alsoendothelialcells(Fig.2b).

Importantly,CMV-inducedIFNγsecretionwasindependentofthe donor’sCMVserologicalstatus(means±SDof2.07±2.05×103pg/mL forCMVposdonorsversus2.18±1.18×103pg/mLforCMVnegdonors, P=0.52)(Fig.2c).Inaddition,DOTcellssecretedIFNγinresponseto twodistinctCMVstrains(TB42/EandMerlin)butnotinresponseto herpessimplexvirus(HSV)orvaricellazostervirus(VZV)infections (Fig.2d).TheseresultsshowastrikingspecificityofDOTcellreactivity toCMVinfection,consistentwithpreviousstudiesonVδ2negγδTcell clonesorcelllines22,24,25,38.

Finally, we examined whether degranulation, IFNγ secretion or other putative CMV-induced effector functions could be correlated with control of CMV spreading by DOT cells in vitro. Using a CMV dissemination assay (described in Fig. 2e, gating strategy shown in Fig.S4f),wefoundthatDOTcellsindeedinhibitedCMVpropagationin an effector-to-target (E:T) ratio-dependent manner (Fig. 2f). Alto- gether,theseresultsdemonstratethatDOTcellsarecytotoxic,secrete IFNγinresponsetoCMVinfection,andareabletocontrolCMVdis- seminationinvitro.

DOTcellsdonotneedtheirTCRtorecognizeCMV-infectedcells
The development of DOT cells as a therapeutic product requires a detailedunderstandingofthemolecularmechanismsunderlyingCMV sensingbythesecells.UsingaTranswellsystem,wefirstobservedthat direct contact between DOT cells and fibroblasts was required for CMV-inducedIFNγsecretion(Fig.3a).WethentestedwhichDOTcell receptors were involved in their reactivity against CMV-infected fibroblasts. We initially investigated the importance of the γδTCR, basedonpreviousevidencethatVδ2negγδTcellsisolatedfromCMV- infectedSOTRrecognizeCMV-infectedfibroblastsinaTCR-dependent manner25,38–40. In contrast to these observations, adding a Vδ1 TCR blocking antibody to cocultures did not impact IFNγ secretion by DOTcellsinresponsetoCMV,asshownforeightindependentdonors in Fig. 3b. However, as this result could have been explained by an incompleteVδ1TCRblocking(seeFig.S5a),weundertooktoknock outtheγδTCRofDOTcellsobtainedfromthreeindependentdonors. Knockout (KO) yielded >90% γδTCRneg cells in all three donors, as determinedbyTCRflowcytometrystaining(Fig.3c).KOefficiencywas alsoverifiedbySangerelectropherogramanalysesfordonors51and 84,with90%and96%ofeditingrespectively(givenbytheICEanalysis tool).WethussuccessfullygeneratedviableγδTcellslackingγδTCR expression,thatwefurthersortedtoobtain>99%ofγδTCRnegcells. γδTCRKOcellsshowedsimilarviabilitytocontrolDOTcellsinfunc- tionalassays(Fig.S5bandS5c)andnolongersecretedIFNγinresponse toanagonistanti-γδTCRantibody,whichvalidatedtheKOfunctionally (Fig. S5d). Nevertheless, γδTCR KO cell response to phorbol 12- myristate13-acetate(PMA)andionomycinstimulationwasmaintained (Fig. S5d). This stimulation, commonly used as a positive control, bypasses TCR signaling by directly activating downstream signaling pathways.

BasalIFNγsecretion byDOTcellscultured alone orin thepre- senceofmock-infectedfibroblastswascompletelyabrogatedinthe absenceoftheγδTCR(Fig.S5e).Incontrolcells(γδTCR+)basalIFNγ secretionismostlikelyexplainedbyremainingTCRsignalingfollow- ingitsstimulationbytheanti-CD3antibodyusedtoexpandDOTcells. InthepresenceofCMV-infectedfibroblasts,IFNγsecretionbyγδTCR KO cells was clearly reduced when compared to control γδ T cells (Fig.S5e),butwasstillquitesubstantial,andmuchhigherthaninthe presenceofmock-infectedfibroblasts,asbestillustratedwiththefold differencesshowninFig.3d.Altogether,thesedatashowthatDOT- cellsarestillabletorecognizeCMV-inducedsignalsoninfectedcellsin theabsenceofγδTCR.ThisTCR-independentrecognitionisconsistent withthehighlypolyclonalrepertoireofDOTcells.

LFA-1isrequiredforCMVsensingbyDOTcells
Because the NK activating receptors NKp30, NKp44, NKG2D and DNAM-1 were upregulated after expansion (Fig. 1g and h) and have previously been implicated in DOT cell response against tumor cells30,31,33,41,wenexttestedwhethertheycontributetoCMVrecogni- tion.BlockingthesereceptorsincocultureswithCMV-infectedfibro- blasts had no significant impact on IFNγ secretion (Fig. 3e). This suggeststhat,instarkcontrasttotheirkeyroleintumorcellrecog- nition,theseNKcellreceptorsarenotinvolvedintheanti-CMVreac- tivityofDOTcells.

Since DOT cells express high LFA-1 levels (Fig. 1h), and CMV- infectionoffibroblastsstronglyincreasesICAM-1surfaceexpression39, weinvestigatedwhetherthisaxisplaysaroleinCMVsensing,aspre- viouslyobservedwithVδ2negγδTcellclones39.DOTcellscouldindeed secrete IFNγ after stimulation with an anti-LFA-1 agonist antibody (Fig.3f),orstimulationwithitsactivatingligandICAM-1(Fig.3g),ina dosedependentmanner.Importantly,incoculturewithCMV-infected fibroblasts,blockadeofLFA-1orICAM-1systematicallyreducedIFNγ secretionbyDOTcellsexpandedfromsixindependentdonors,with thestrongesteffectobservedwhenbothwereblockedsimultaneously (Fig.3h).WecouldnotassessthefunctionalimpactofLFA-1knockout, as repeated attempts failed to generate LFA-1neg DOT cells, likely becauseofhighmRNAorproteinstability.Altogether,stimulationand blockingexperimentssuggestthatLFA-1contributestoDOTcellanti- CMVreactivity.

ExpandedmouseγδTcellscontrolCMVinfectioninvivo
WenextwantedtoassesstheefficacyofanadoptiveγδTcelltherapy totreatCMVinfectioninaninvivosetting.However,CMVtropism being species-specific, mice can only be infected by murine CMV (MCMV),whereashumanDOTcellscanonlyrecognizehumanCMV- infectedcells.WethereforedevelopedamethodrecapitulatingγδT cellexpansionfrommouseγδTsplenocytesinvitro,andusedthese expandedcellsasanadoptivetherapytotreatMCMV-infectedmice. WeadaptedthehumanDOTprotocol(seeMethodssection)toamplify γδ T cells from 9 MCMV-naïve (MCMVneg) and 9 MCMV-infected (MCMVpos)C57BL/6Jwildtypemice(Fig.4a–d).After7daysofcul- ture, we obtained a mean±SD of 2.8±1.2×107 γδ T cells from MCMVnegmice,comparedto2.5±1.1×107cellsfromMCMVposmice (Fig. 4b), with corresponding γδ T cell fold increases in culture of 1.15±0.49×102and1.16±0.46×102,respectively(Fig.4c).Astrongly enrichedcellularproductwasobtained,asγδTcellsrepresented 84.4±6.5% and 82.3±8.9% of expanded cells for MCMVneg and MCMVposmice,respectively(Fig.4d).Alltheseparametersdidnot significantlydifferbetweenthetwogroups(two-tailedMann–Whitney test,γδTcellabsolutenumbersP=0.65,foldincreasesP=0.80,and percentagesP=0.88,respectively).InlinewiththehumanDOTpro- duct,amajorityofcentralmemoryγδTcells(CD62L+CD44+)was obtainedafterexpansion:57.2±8.2%and54.7±4.9forMCMVnegand MCMVposmicerespectively(Fig.4e,representativedotplotsshownin Fig.S7a).PhenotypingofmurineγδTcellsbeforeandafterexpansion showedanincreaseinactivation(CD25),alackofimmunecheckpoints suchasPD-1orTIM-3,andanupregulationofNKactivatingreceptors (DNAM-1,NKG2D),cytotoxicityreceptors(FasLandTRAIL),aswellas LFA-1,independentlyofpreviousCMVpriming(Fig.4fandstatistical analysis in Fig. S6). Altogether, we were able to generate a cellular productofexpandedmurineγδTcellssimilarlyasandphenocopying humanDOTcells,regardlessoftheMCMVstatusofthedonormouse.

Wenextevaluatedwhetherinvitro-expandedmurineγδTcells couldprotectimmunodeficientRag−/−γc−/−micefromMCMV-induced mortality(Fig.4g).AdoptivetransferofmurineγδTcellsexpanded from MCMVneg (n=22) and MCMVpos (n=13) mice significantly improvedthesurvivalofMCMV-infectedRag−/−γc−/−recipients,com- pared to RPMI-treated controls (n=18) (P=0.0001 and P<0.0001 respectively,log-rankMantel-Coxtest).Thiseffectwasindependentof theMCMVstatusofdonormice(P=0.0837)(Fig.4h).Inparallel,we were able to detect the presence of transferred γδ T cells in the spleens,liversandlungsofsurvivingmiceatendpoint(n=11analyzed from5MCMVnegand6MCMVposdonors,Fig.4iandgatingstrategy illustrated in Fig. S7b), where they displayed a TEM phenotype as illustratedonFig.S7c.Inaddition,thismodelgivesustheopportunity toevaluatevirusdisseminationindifferentorgans.Survivingmicethat had received γδ T cells (n=7 analyzed; from 4 MCMVneg and 3 MCMVpos donors) exhibited significantly lower viral loads in the spleen, liver and lung, than those that succumbed to infection (P<0.0001, two-tailed Mann–Whitney test; Fig. S7d). To directly test whether γδ T cell adoptive transfer can control MCMV viral loadin organs,we transferred γδT cellsexpanded fromMCMVpos (n=5)orMCMVneg(n=5)C57BL/6JdonormiceintoMCMV-infected Rag−/−γc−/− mice and compared viral load to RPMI-treated controls (n=5)atday14postinfection.BothγδTcelltreatmentgroupshad significantlyreducedviralloadsinallthreeorgansrelativetocontrols (Fig.4j).Together,thesefindingsdemonstratethatadoptivelytrans- ferredγδTcellscanmigratetoMCMV-infectedorgans,controlviral replication, and protect immunodeficient mice from lethal MCMV infection.

AutologousCMV-reactiveDOTcellscanbeobtainedfromSOTR
Both autologous and allogeneic virus-specific T cell therapies have been or are currently being tested in phase I trials in SOTR15,17,42 (CTRN12621000323820) or in a phase III trial in HSCTR (TRACE study: NCT04832607). Among these two strategies, the autologous approach is particularly attractive in SOTR to avoid the risk of allosensitization deleterious to the transplant43. However, whether functional and CMV-reactive DOT cells can be expanded from immunosuppressedtransplantrecipientsremainstobeevaluated.In ordertoassessthefeasibilityofanautologousapproach,peripheral blood mononuclear cells (PBMCs) were obtained from eight D+R- KTR (see Table 1) with recurrent and refractory CMV infections, among which five exhibited an antiviral drug resistance (UL97 mutation) requiring Foscavir or Maribavir treatment. Despite lymphopenia,DOTcellswereexpandedfromalloftheseeightcritical patients with, at day 21 of culture, a mean±SD of 1.01±1.04×108 Vδ2negγδTcells,afoldincreaseof0.72±1.75×104andapercentage incultureof83.68±8.17%(Fig.5a–c).Importantly,theseeightcellu- lar products secreted IFNγ in response to CMV infection (Fig. 5d). Altogether, these results show that CMV-reactive DOT cells can be obtained from SOTR to serve as autologous adoptive cell therapy.

DOTcellsremainfunctionalunderimmunosuppression
AnotherimportantrequirementisthatinfusedDOTcellspersistand expandinSOTRdespiteimmunosuppressivetreatments.Totestthis, DOTcellsexpandedfromhealthydonorsweresubsequentlycultured inthepresenceofIL-15anddifferentimmunosuppressivedrugsclas- sically used in SOTR (two calcineurin inhibitors: tacrolimus and cyclosporin,themTORinhibitoreverolimusandtheantiproliferative drugmycophenolatemofetil).Aftersevendays,theirproliferationwas, asexpected,completelyinhibitedbymycophenolatemofetil(MMF), but was maintained in the presence of tacrolimus, cyclosporin and everolimus (Fig. 5e, f). These results were confirmed after longer treatments of 15 days, by analyzing DOT cell counts and viability (Fig.5g,h).Toevaluatetheirfunctionality,DOTcellswerecocultured during24hwithCMV-infectedfibroblastsinthepresenceofthesame immunosuppressivedrugs.CMV-inducedIFNγsecretionwasentirely suppressedbyMMF,butwasmaintainedinthepresenceoftacrolimus, cyclosporinandeverolimusalone(Fig.5i).Inadditiontotheimmu- nosuppressive drugs tested above, glucocorticoids are a standard component of the immunosuppressive regimen after SOT. Impor- tantly,treatmentwiththeglucocorticoidmethylprednisoloneatthe concentrationtypicallyusedinthemaintenancetreatmentofSOTR (around10ng/ml,12hafteringestion44)didnotinhibitDOTcellpro- liferation (Fig. S8a, b) or their IFNγ secretion in response to CMV- infected fibroblasts (Fig. S8c). In summary, these data show that DOT cells are maintained in vitro and respond to CMV despite the presence ofimmunosuppressivedrugs,stronglysupportingthefea- sibilityofaDOTcelltherapyinSOTR.

Methods
Studyapprovalanddesign
Bloodsamplesfromhealthydonorsandkidneytransplantrecipients wereobtainedafterwritteninformedconsent,inaccordancewiththe DeclarationofHelsinki.KTRwereincludedintheSPARCKLINGstudy (NCT03339661),approvedbytheBordeauxUniversityHospitalethics committee CHUBX 2016/40, or the HORUS study (NCT05701228), approved by the Bordeaux University Hospital ethics committee CHUBX2022/10. As CMVinfectioncanaffectallsexesandgendersand sexisnotaknownriskfactor,wedidnotperformsex-orgender-based analyses,andfindingsapplytoindividualsofallsexesandgenders.Sex andgenderwerenotconsideredinthedesignoftheSPARCKLINGand HORUSstudies,andwereself-reported.Besides,theinvivoprotocols described in this study were approved by a local ethics committee (BordeauxUniversity,committeen°2016092917471799).

DOTcellexpansion
Blood samples were obtained from healthy volunteers (50mL), recruitedatthelocalbloodbank(EtablissementFrançaisduSang),or fromKTR(20mL).CMVserologiesfromhealthydonorsandKTRwere determined by enzyme-linked immunosorbent assay (ELISA) upon request. When determined, the CMV serotype of healthy donors is providedasasourcedatafileandindicatedinthecorrespondingfig- ures. PBMCs were isolated using lymphocyte separation medium (EurobioScientific),andstainedwithanantibodydirectedagainstthe αβTCR,coupledtobiotin(MiltenyiBiotec,cloneREA652,diluted1:50), followedbyanti-biotinmicrobeads(MiltenyiBiotec).αβTcellswere removedfromPBMCsbymagnetic-activatedcellsortingandremain- ingPBMCswereculturedfor21daystoexpandVδ2negγδTcells,fol- lowingtheDOTcellprotocol30,31.CellswereculturedinOpTmizer™ CTS™serum-freemedium(GIBCO,LifeTechnologies)supplemented with 10% human serum, 1% Penicillin-Streptomycin (PS), and 2mM L-glutamine(expansionmedium),ataconcentrationof160,000cells/ mLinthepresenceofthefollowingrecombinanthumancytokines(all fromPeprotech):100ng/mLinterleukin4(IL-4),70ng/mLinterferonγ (IFNγ),7ng/mLIL-21,15ng/mLIL-1βandanactivatingantibodydirec- tedagainstCD3(1µg/mL,cloneOKT-3,Biolegend).Atdays6,11and16 ofculture,expansionmediumwasrenewedandsupplementedwith 70ng/mLIL-15(days6,11,16),13ng/mLIL-21(day6),70ng/mLIFNγ (day16),and2µg/mLanti-CD3(days6,11,16).

DOTcellcultureinthepresenceofimmunosuppressivedrugs
Afterinvitroexpansion,DOTcellswerecountedandresuspendedat 1×106cells/mLinthepresenceof100ng/mLIL-15andthefollowing immunosuppressivedrugs:tacrolimus(7ng/mL),cyclosporin(75ng/ mL),everolimus(0.5nM),MMF(1µg/mL),ormethylprednisolone(1,10 or 100ng/mL). When indicated, DOT cells were labeled with 5µM carboxyfluorescein succinimidyl ester (CFSE) or CellTrace™ Violet (CTV), following the CellTrace™ Cell Proliferation Kit instructions (Thermofisher Scientific). After 7 days, the culture medium was renewed with fresh IL-15 and immunosuppressive drugs. After 7 to 15days,DOTcellswereharvestedandviablecellswereanalyzedfor viabilityby4′,6-diamidino-2-phenylindole(DAPI)staining,orforpro- liferationbasedonCFSEorCTVdilution.Proliferationindexeswere calculatedusingtheproliferationtoolinFlowJoV10.5(TreeStar).

DOTcellphenotypingbyflowcytometry
Cellswereharvestedatdays0,11and21ofexpansion,andstainedfor flow cytometry analysis38. Single cell suspensions were first washed twice in staining buffer: phosphate-buffered saline (PBS) supple- mentedwith0.5%bovine serumalbumin (BSA)and2mMethylene- diaminetetraacetic acid (EDTA), with centrifugations at 300×g for 5min. Cells wereresuspended in staining buffer and stained with a viability dye (FixableViabilityStain780,BDBiosciencesor Viobility 405/520FixableDye,MiltenyiBiotec)for10minatroomtemperature inthedark,washedtwice,andsubsequentlyresuspendedinstaining bufferinthepresenceofthefollowingantibodies,for20minat4°Cin thedark.Forcellculturefollow-up(atdays0,11and21):antibodies directedagainstCD3,CD27,CD45RA,γδTCR,αβTCR,Vδ1TCRandVδ2 TCR were used. For characterization of cell populations among expandedcells,antibodiesdirectedagainstCD3,γδTCR,Vδ1TCR,Vδ2 TCR,Vδ3TCR,CD14,CD16,CD19andCD56wereused.Forextensive phenotyping at days 0 and 21, cells were stained with antibodies directedagainstthepopulationmarkersCD3,γδTCR,Vδ1TCR,Vδ2 TCR;theactivationmarkersCD25,CD69,CD71,HLA-DR;theimmune checkpointsCTLA-4,LAG-3,PD-1,TIGITandTIM-3;theco-stimulatory receptors CD8αα, CD11a-CD18 (LFA-1), CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD154 (CD40L); the NK activating receptorsCD16,CD226(DNAM-1),NKG2D,NKp30,NKp44,NKp46;the KIRsKIR2DL1(CD158a),KIR2DL1/S1(CD158a/h),KIR2DL2/L3(CD158b), KIR3DL1(CD158e1),KIR3DL1/S1(CD158e1/e2),KIR2DS4(CD158i);and thecytotoxicitymarkersTRAILandFasL.Afterantibodystaining,cells werewashedtwiceinstainingbufferandanalyzedonaCantoIIcyt- ometer(BDBiosciences)forcellculturefollowup,oronaLSRIIFor- tessacytometer(BDBiosciences)forotherpanels.Datawereanalyzed withFlowJoV10.5.HeatmaprepresentationwascreatedwiththeMeV (MultipleExperimentViewer)software.Dataregardingantibodiesare availableinSupplementaryTable1.

CRISPR-Cas9knockoutinDOTcells
Two guide RNAs (gRNAs) were designed using the CRISPOR algorythm62,63, targeting the T cell receptor delta constant (TRDC) gene, coding for the constant region of the γδTCR δ chain: gRNA1 TGGAACAAATGTCGCTTGTC and gRNA2 AAGTA- CAATGCTGTCAAGCT.gRNAswereselectedbasedontheirhighspe- cificityscores,ascalculatedbyCRISPORusingthecuttingfrequency determination(CFD)specificitymetric64,65,andbyinspectionofpre- dictedoff-targetsites.BothgRNAshadCFDspecificityscoresabove90 (95and92,respectively),withnopredictedoff-targetexonicsequen- ces bearing fewer than four mismatches. Detailed information regardinggRNAcharacteristicsisavailableinSupplementaryTable2.
After expansion, DOT cells were rested for 24h in expansion mediumsupplementedwith70ng/mLIL-15,inordertoallowcom- plete surface expression of the TCR. CRISPR-Cas9 knockout was performed by Cas9/gRNA ribonucleoprotein transfection of DOT cells66. crRNA and tracrRNA oligonucleotides were first annealedatequimolarconcentrationsbyheatingat95°Cfor5min. Ribonucleoproteincomplexeswereformedbyincubatingduplexed gRNAs(100µM)withCas9(62µM)ata1:1volumeratiofor10minat roomtemperature.TheAlt-RTMS.p.HiFiCas9NucleaseV3wasused (engineeredforreducedoff-targetactivity).DOTcellswerewashed inPBS,resuspendedin20µLofsupplementedNucleofectorsolution (Lonza, V4XP-3032) and mixed with 5µL of ribonucleoprotein complexand1µLofAlt-RTMCRISPR-Cas9ElectroporationEnhancer (100µM).Cellswereelectroporatedusinga4D-Nucleofectorsystem (Lonza)withtheEH115-P3program.Immediatelyafterelectropora- tion,cellswererecoveredintheirpre-warmedexpansionmedium supplemented with 70ng/mL IL-15. As a control, DOT cells were transfectedwitharibonucleoproteinincorporatinganon-targeting gRNA (Alt-RTM CRISPR-Cas9 Negative Control crRNA). All CRISPR reagentswerepurchasedfromIDT.Knockoutefficiencywasasses- sed 96h after transfection by flow cytometry, using antibodies directed againstCD3,γδTCR,Vδ1TCRandVδ2TCR.Knockout efficiency was also verified by the analysis of Sanger electro- pherograms, using the ICE v2 CRISPR Analysis Tool (Synthego) whichgivesapercentageofediting.RemainingγδTCR+cellswere subsequentlydepletedusingaFACSARIA(BDBiosciences)inorder toachieve>99%purity.Aftersorting,DOTcellswererestedover- nightandusedforfunctionalassays.

Targetcellcultures
Humanforeskinfibroblasts(HFF)wereprovidedbyDr.Hamid-Reza Rezvani (INSERM, U1035, Bordeaux). They were maintained in Dul- becco’sModifiedEagleMedium(DMEM)supplementedwith10%fetal bovineserum(FBS),2mML-glutamineand1%PS(completeDMEM), and used up to passage 18. Conditionally immortalized glomerular endothelialcells(CiGEnC)wereculturedincompleteEndothelialCell Growth Medium MV2 (Promocell) supplemented with 1% PS, maintained at33°Cand differentiated at37°C for 5 days67 prior to CMV infection. Autologous human monocytes were obtained from healthy donors’ PBMCs. PBMCs were stained with human CD14 MicroBeads (Miltenyi Biotec) and positively sorted by magnetic- activated cell sorting. Sorted cells were stained with a viability dye (FVS780)andanantibodydirectedagainstCD14(CD14-Vioblue,Mil- tenyiBiotec)toverifymonocytepurity.Monocyteswereseededatan initialconcentrationof500,000cells/mLanddifferentiatedintoM1 macrophages for 6 days, in Roswell Park Memorial Institute (RPMI) 1640mediumsupplementedwith10%FBS,2mML-glutamine,1%PS, and 10ng/mL recombinanthumangranulocyte-macrophage colony- stimulatingfactor(GM-CSF,Peprotech).Additional5ng/mLGM-CSF wasaddedtothecultureafter3days.Allcelllineswereregularlytested formycoplasmacontamination.

PreparationofCMV,HSVandVZVstocks
Fivevirusstrainswereusedforinvitroexperiments:theCMVclinical strain TB42/E was a gift from Prof. Dr. Christian Sinzger (Ulm Uni- versity, Germany), CMVclinicalstrains Merlin and Merlin UL32-GFP werekindgiftsfromDr.CeriFielding(CardiffUniversity,UK).Herpes simplexvirus-1(HSV-1)KOSstrainandVaricellaZosterVirus(VZV)OKA strainwerekindgiftsfromDr.SoniaBurrel(VirologyUnit,PitiéSal- pétrièreUniversity Hospital, France). CMVstockswereprepared by infectingHFFimmortalizedbyhumantelomerase(HFFTERT)atalow multiplicityofinfection(MOI)for2–3weeks.When100%cytopathic effect was observed, supernatants were harvested, centrifuged at 300×g for 10min and stored at −80°C. HSV and VZV stocks were preparedbyinfectingMRC5cells(obtainedfromEurobioScientific). When 50–100% cytopathic effect was observed, supernatants were harvestedanddirectlyfrozenat−80°C,in50%FBSforHSVandin10% dimethyl sulfoxide (DMSO), 20% FBS for VZV. CMV, HSV and VZV stocksweretitratedbyplaqueassaysonHFF68,using2%Avicel(Sigma- Aldrich).

CMV,HSVandVZVinfections
HFFweremock-infectedorinfectedbyCMVataMOIof0.01,byVZVat aMOIof0.01,andbyHSVataMOIof1incompleteDMEM.CiGEnCand macrophages were infected by CMV strain TB42/E at a MOI of 1 in completeDMEM.Afterovernightincubation,infectionmediumwas replacedbyfreshcompleteDMEM.HFFinfectionbyCMV,HSVand VZVweremonitoredbyevaluatingcytopathiceffectsbymicroscopy. Additionally,percentages of CMV-infected HFF,CiGEnCand macro- phageswerequantifiedbyflowcytometry:mock-infectedorinfected cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer’s instructions and stained with an antibody directed against the human CMV IE antigen (Merck Millipore). HFF were infectedbyCMVandVZVfor5days,andbyHSVfor2days,inorder toreach50–100%ofcellsshowingcytopathiceffectsatthestartof functionalassays.CiGEnCandmacrophageswereinfectedbyCMVfor 2days,inordertomaximizethe%ofCMV-IEcells,beforefunctional assays.

Coculturesandreactivityassays
After 21 days of expansion, DOT cells were washed and rested for 2daysinexpansionmediumsupplementedwith70ng/mLIL-15before functional assays. DOT cells were cocultured with CMV-infected or mock-infected target cells in 96-well plates,atan effector to target (E:T) ratio of 1:1, in complete RPMI 1640 medium. For experiments measuringIFNγsecretion,IL-18(50ng/mL)wasaddedtococultures whenspecifiedinthefigurelegendsandpanels.
To study the impact of immunosuppressive drugs, tacrolimus (7ng/mL), cyclosporin (75ng/mL), everolimus (0.5nM), MMF (1µg/ mL) or methylprednisolone (1,10or 100ng/mL) were added tothe coculturemedium.
Forreceptorblocking,DOTcellswerepreincubatedfor30minat roomtemperaturewithneutralizingantibodiesdirectedagainstVδ1 TCR,DNAM-1,NKG2D,NKp30,NKp44,LFA-1,oracontrolmouseIgG1k antibody.Whenindicated,targetfibroblastswerepreincubatedwith an antibodydirected againstICAM-1ora control mouse IgG1kanti- body.Cellsweresubsequentlywashedbeforecoculture.Forantibody- mediatedactivation,DOTcellswereincubatedwithcoatedantibodies directed against CD3 (clone OKT-3, Biolegend), LFA-1 (clone 25.3, BeckmanCoulter)oracontrolmouseIgG1kantibody.Informationon antibodies used for blocking and stimulation is availablein Supple- mentaryTable3.Whenindicated,DOTcellswereactivatedwithdif- ferent concentrations of coated recombinant human ICAM-1 FC Chimera(R&DSystems,catalognumber720-IC-050),orwith25ng/mL PMA(Sigma,catalognumberP8139)and1µg/mLionomycin(Sigma, catalognumberA23187).
Transwell experiments were performed in 24-well plates. DOTcellswereincubatedininserts(0.4µmporepolyestermembrane from Corning) and target fibroblasts in the lower chambers. Cocul- tureswereperformedasdescribedabove.
After 24h, supernatants were harvested for ELISA and LEGEN- Dplexassays.Whenindicated,cellswerealsoharvested,stainedwith 1µg/mL DAPI (Thermofisher Scientific, catalog number D1306) and percentagesofviablecells(DAPIneg)werequantifiedbyflowcytometry onaCantoIIcytometer(BDBiosciences).

Degranulationassays
DOT cells were labeled with 0.5µM CFSE, following the CellTrace™ CFSECellProliferationKitinstructions(ThermofisherScientific)and subsequently cocultured for 16h with target cells. An anti-CD107a antibody,0.67µL/mLmonensin(BDGolgiStop,BDBiosciences)and 1µg/mLbrefeldinA(Sigma)wereaddedtothecoculturemediumfor the last 4h. Cells were then collected, stained with a viability dye (FVS780),antibodiesdirectedagainstγδTCRandVδ2TCR,andana- lyzedonaLSRIIFortessacytometer(BDBiosciences).

ControlofCMVdisseminationbyDOTcells
TomeasureCMVdisseminationinvitro,fibroblastswereseededin24- wellplatesand,whenconfluent,infectedwiththeCMVstrainMerlin UL32-GFPatalowMOI(0.005).24hafterinfection,DOTcellswere addedtofibroblastsatdifferentE:Tratios.Infectedfibroblastswithout DOT cells were used as a positive control and mock-infected fibro- blastsasanegativecontrol.After6daysofcoculture,fibroblastswere harvestedusingAccutasecelldetachmentsolution(Sigma),washed, andthepercentageofCMV-infectedfibroblastswasmeasuredbyflow cytometry(%ofGFP+cells)onaCantoIIcytometer(BDBiosciences). The control of CMV dissemination by DOT cells was measured by comparingpercentagesofGFP+cellstothecontrolconditionwithout effectorcells.

ELISAandLEGENDplexassays
Cellculturesupernatants were analyzed for IFNγ concentrations by ELISA,using the human IFNγ ELISA development kit (Mabtech) and according to manufacturer’s instructions. Additionally, cell culture supernatantswereanalyzedusingLEGENDplexassays(Biolegend)for theconcentrationofthefollowingcytokines,chemokinesandgrowth factors: IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, CXCL10 (IP-10), CCL2 (MCP-1), CXCL8 (IL-8), IFNγ, TGF-β1, TNFα, (LEGENDplex Human EssentialImmune ResponsePanel,Biolegend),CCL3(MIP-1α),CCL4 (MIP-1β),CCL5(RANTES),CCL11(Eotaxin),CCL17(TARC),CCL20(MIP- 3α), CXCL1 (GROα), CXCL5 (ENA-78), CXCL9 (MIG), CXCL11 (I-TAC) (LEGENDplex Human Proinflammatory Chemokines Mix and Match Subpanel), Angiopoietin-2, EGF, FGF-basic, G-CSF, GM-CSF, M-CSF, PDGF-AA,PDGF-BB,VEGF(LEGENDplexHumanGrowthFactorMixand Match Subpanel). LEGENDplex assays were performed according to manufacturer’sinstructions.

Mice
C57BL/6J and BALBcBy/J mice were purchased from Charles River LaboratoriesandRag−/−γc−/−micewereobtainedfromtheCentrede Distribution, Typage et Archivage animal (CDTA), Orleans, France. Micewerebredandmaintainedunderspecificpathogen-freecondi- tionsinanappropriatebiosafetylevel2facility(BordeauxUniversity, approvaln°B33-063-916).Micewerehousedundercontrolledcondi- tions(21°C,55–65%humidity,12h/12hlight/darkcycle).Experiments wereperformedon8–12weeks-oldmice,inaccordancewithgovern- mental guidelines and european ethical regulations. The protocols describedinthisstudywerenotablyapprovedbyanethicscommittee (BordeauxUniversity,committeen°2016092917471799).

MouseγδTcellexpansionandphenotyping
TheMCMVSmithstrainwasobtainedfromtheAmericanTypeCulture Collection(ATCCVR-194)andusedtoinfectBALB/cByJmiceforvirus propagation. 2–3 weeks after MCMV infection, salivaryglands were collectedfrominfectedBALB/cByJmicetogeneratetheMCMVstock solutionusedforallmouseexperiments.Virustitersweredetermined bystandardplaqueformingassayonmonolayersofmouseembryonic fibroblasts.C57BL/6Jwild-typemicewereinfectedbyintraperitoneal (i.p.) injection with 2×104 plaque-forming units (PFU) of MCMV,or mock-infected. Three weeks after infection, spleens were harvested from mock-infected (MCMV-negative) or MCMV-infected (MCMV- positive)mice.Splenocyteswereisolatedthrough cellstrainers and magnetically-depletedofαβTcellsandNKcells.Depletionwasper- formedbystainingwithbiotin-coupledantibodies(anti-TCRβ,clone REA318, diluted 1:50 and anti-NKp46, clone 29A1.4.9, diluted 1:10, MiltenyiBiotec)followedbyanti-biotinmicrobeads(MiltenyiBiotec) andmagneticsorting.Sortedcellswereseededat200,000cells/well inround-bottom96-wellplates,inRPMI1640supplementedwith10% FBS, 1% PS, 2mM L-glutamine, 1% 4-(2-hydroxyethyl)-1-piper- azineethanesulfonic acid (HEPES) and 50uM β-Mercaptoethanol (mouse γδ T cell expansion medium). Expansion medium was sup- plemented with an ultra-LEAF™ purified anti-mouse CD3ε Antibody (1µg/mL, clone 145-2C11, Biolegend) and the following cytokines: recombinant murine IL-1β (15ng/mL), recombinant human IL-15 (20ng/mL), recombinant murine IL-21 (7ng/mL) and recombinant murineIFNγ(70ng/mL),allfromPeprotech.After6daysofexpansion, cellswereharvested,washed,andrestedovernightbeforeflowcyto- metryanalysisoradoptivetransfer.Restingwasperformedinmouse γδTcellexpansionmediumsupplementedwith20ng/mLIL-15.The phenotypes of αβ and NK-depleted splenocytes (day 0) as well as invitroexpandedγδTcells(day7)wereanalyzedbyflowcytometry. Briefly, 2×106 cells were stained with a viability dye (FVS780) and antibodiesdirectedagainstmouseCD3,γδTCR,CD11a(LFA-1),CD25, CD44,CD62L,CD178(FasL),CD226(DNAM-1),CD253(TRAIL),CD279 (PD-1),CD314(NKG2D)andCD366(TIM-3)(SupplementaryTable1). CellswereanalyzedonanAuroracytometer(Cytek)anddataanalyzed withFlowJov10.8.

MouseγδTcelladoptivetransferinaMCMVinfectionmodel
Rag−/−γc−/− mice received either 106 γδ T cells (expanded from MCMVneg or MCMVpos C57/BL6J mice) or RPMI 1640 medium, by retro-orbitalintravenousinjectionatday0andday7.Onedaypost adoptivetransfer(day1),micewereinfectedwithMCMV(Smithstrain, 2×102 PFU, i.p.). Infected mice were monitored twice per week for clinicalsignsofinfection(weightloss,roundback,tip-toewalkingand/ or absence ofhaircare) and sacrificed upon reaching humane end- points,asdefinedinaccordancewiththeethicscommittee.Attheend oftheexperiments,spleen,liverandlungsweresampledforviralload measurementandprocessedtoanalyzeγδTcellinfiltration.Forthe latteranalysis,spleensweredissociatedona70µmcellstrainerfol- lowedbyredbloodcelllysisinACKlysisbuffer(A1049201,Thermo- fisherScientific)andwashesbeforesplenocytestaining.Lungswere digestedat37°Cduring40mininRPMI1640supplementedwith1mg/ mLcollagenaseIand50µg/mLDNAseI(Sigma),thenpassedthrough 70µm cell strainers. Livers were directly dissociated on 70µm cell strainers. Afterredbloodcelllysis,lungandlivercellsuspensionswere separatedbyadiscontinuous40/80%Percollgradient(accordingto manufacturer’sinstructions,SigmacatalognumberP4937).Immune cellswerethenharvestedandstainedwithaviabilitydye(FVS780)and antibodies directed against mouse CD3, γδTCR, CD44 and CD62L. Cells were analyzed on a Canto II (BD) and data was analyzed on FlowJov10.8.

Viralloadquantifications
ViralDNAwasextractedfromorgansusingtheQIAwaveDNABlood& Tissueskit(Qiagen).LysiswasperformedwithTissueLyserII(Qiagen). DNA was quantified using DeNovix Nanodrop (Thermofisher Scien- tific) and 300ng to 500ng DNA was used for PCR amplification. ReactionswerepreparedusingGoTaqqPCRMasterMix(Promega)and MCMV-specificprimerstargetingglycoproteinB(gB)(forwardprimer: GGTAAGGCGTGGACTAGCGAT; reverse primer: CTAGCTGTTT- TAACGCGCGG). Viral loads were quantified using standard curves preparedwith101to107copiesofplasmidscomprisinga517bpfrag- mentofDNAencodingMCMVgB28.

Statisticsandreproducibility
Analyses were performed using conventional statistical methods. Wilcoxonmatched-pairssigned rank test, two-tailed Mann–Whitney test,pairedorunpairedt-tests,Welch’st-tests,ordinaryorrepeated- measures one-way ANOVA with Dunnett’s or Tukey’s multiple com- parisons were used when appropriate. Kaplan-Meier survival curves were analyzed with log-rank (Mantel-Cox) tests. P<0.05 was con- sideredstatisticallysignificant.
Toaddressexperimentalvariations,allinvitroexperimentswere performedinduplicatesortriplicatesofculture.Thisinformationis indicatedinthecorrespondingfigurelegends.Toaddressbiological variations, all in vitro experiments were repeated on independent healthydonorsorpatients.Thisinformationisindicatedinthecor- responding figure legends. Each analysis presented in this study is basedonaminimumofthreeindependentexperimentsperformedon independentdonorsorpatients.
Samplesizesforexperimentsinvolvinghealthyhumandonors werenotdeterminedusingstatisticalpowercalculations,butwere insteadbasedonpriorexperienceandexperimentalobservationsto ensuredatarobustnessandreproducibility.Forinvivoexperiments, samplesizesweredeterminedupfrontbasedonpriorexperimental datatodetectmeaningfuldifferenceswhilecomplyingwithethical guidelines.Forexperimentsinvolvingpatientsamples,samplesizes weredeterminedbythenumberofeligibleparticipantsthatcould berecruitedduringthestudyperiod.Samplesizesforeachexperi- ment are provided in the corresponding figure legends. No datawereexcludedfromtheanalysis.Miceusedforinvivoexperi- mentswererandomlyassignedtothedifferentgroups.Thefollow- up of MCMV-infected mice after γδ T cell adoptive transfer was performed blinded to the conditions of the experiment. Blindingwasnotapplicabletoinvitroexperiments,inwhichwell- controlled quantitative measurement methods were used to excludebias.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript by Marsères et al, a new cell therapy is proposed to combat the important issue of post-transplantation\nrefractory CMV infection in donor organ recipients. Especially CMV-naïve recipients receiving a transplant from CMV-\nseropositive donors are at high risk of developing refractory CMV infection, ultimately leading to diminished transplant\nsurvival outcomes. Adoptive transfer of CMV-specific αβ T cells has previously demonstrated benefits for graft survival, but\nthis cell population is challenging to obtain from CMV-naïve recipients and would require HLA-matching in an allogeneic\nsetting. To circumvent these challenges, adoptive transfer of HLA-independent γδ T cells is proposed, of which specifically\nthe Vδ2neg γδ T cell population has demonstrated anti-CMV immune response, also called “Delta One T” or DOT cells.\nFirst, the authors demonstrate the suitability of a scalable production method for clinical-grade DOT cell expansion, capable\nof expanding DOT cells both from CMV seropositive and seronegative healthy donors with comparable effector\ncharacteristics. The authors demonstrate that CMV-mediated IFN-γ induction is dependent on LFA-1 and independent of the\nγδTCR presence on the DOT cells. Subsequently, the authors use a murine CMV infection model to recapitulate the human\nsituation in an in vivo setting. Finally, the authors demonstrate DOT cell expansion is possible from CMV-naïve receivers of\nCMV-seropositive donor kidneys under immunosuppressive conditions, while maintaining CMV-induced IFN-γ secretion in\nthese cells. Altogether, the authors suggest a robust proof of feasibility for DOT cell therapy in kidney transplant recipients is\ndemonstrated with the evidence presented in the manuscript.\nWe have thoroughly reviewed the presented evidence, and although in general the level of evidence provided support the\nauthors’ conclusions, we do have some concerns that need to be addressed before we can provide a positive opinion on\naccepting the manuscript in a high-level journal such as Nature Communications. Our concerns are illustrated below.\nGeneral considerations and points for improvement\n1. Throughout the manuscript, the authors present experiments using different numbers of donors per experiment, and\ndonors with different CMV-serological status. It is often unclear from the figure whether the same donors are used as in the\nfigure before, and which criteria are used to select a specific donor (e.g. figure 3 B: 4 donors, 3C: 2 donors – are the 2 in C\nalso represented in B, and if so, why are these 2 selected over the other 2 used in B?). Although the authors demonstrate\ncomparability of DOT cell characteristics obtained from CMV naïve vs. CMV seropositive healthy donors in the first\ncharacterization experiments, in later experiments other characteristics are interrogated in more detail, in which differences\nbetween the serological status can become more apparent. By not disclosing the origin of the cells used in experiments after\nfigure 1, these (likely minor) differences may be overlooked. Altogether, in our opinion the interpretability of the presented\ndata would improve by disclosing this missing information.\n2. In the materials and methods it is mentioned that, unless stated otherwise, IL-18 is used in the co-culture experiments. This creates confusion when going back and forth between main figures and supplementals, with the need to constantly re-\nread the legends searching for a mentions of IL-18 absence before being able to compare figures. In our opinion, adding a\nsimple ‘IL-18+’ or ‘IL18-’ label in the figures would improve readability and interpretability.\n3. Many abbreviations and acronyms are used, which are not consistently explained (sometimes in the main text, sometimes\nonly in the figure legend). We urge the authors to critically go through the main text and explain abbreviations/acronyms\nappropriately. Highlighting (bold letters) and using a glossary would increase readability, if this is allowed in the Nature\nComms format.\n4. The figure panels (especially the flow cytometry gates panels) are too small to read comfortably from an A4 printed paper\nformat. We strongly advise to consider using larger panels.\n5. The authors address the risks of off-target reactivity in the discussion. What is the expected side effect profile in humans,\nthis should be considered. More data are needed, not only mock infected cell lines (which also do not induce ‘zero’\nresponse) and a reference to an ongoing clinical study in leukemia patients. The SOT patient with an ongoing infection\n(activated stage, possibly other viruses) and the TCR independent mechanism as shown is this manuscript should be taken\ninto account and when possible other targets should be included in the experiments.\nSpecific considerations\nMethods section: no mentioning of off target scores of the used sgRNAs for the KO model. Was this check performed? How\nelse have off-target editing effects been excluded? Please explain.\nLine 375: Please elaborate on how these donors are selected and is this enough to make the conclusion in line 390-391?\nFigure 1E: include CMV status (donor 1-2 = CMV neg, donor 3-4 = CMV pos)\nFigure 1F: In the text the terms central memory or effector cells are used, in the legend within the figure this is translated to\nCD27 and CD45RA status. Provide a label in the figure for the central memory or effector population as well for clarity. Also,\nSD are shown, but no statistical analyses are provided.\nFigure 1F is associated with figure 5E (where a similar analysis is performed in the murine CMV in vivo model). We suggest\nto model the 2 figures in a similar way so they can be compared side by side and similarities in orthologous cell populations\nare immediately apparent.\nLine 409-410: more elaborate explanation of why authors chose to focus on IFN-γ is needed. They refer to a paper, but a\nshort explanation of why this is the most interesting factor (compared to the other targets in the CMV-induced secretome)\nwould be appreciated.\nFigure S3A: contains endothelial cells, effect of CMV infection is minimal. Please comment on why this is (and also why in\nS3D they don’t seem to react to CMV induction without IL-18, but with IL-18, suddenly IFN-g is increased).\nFigure S3B: FMO of infected cells is missing. Can the addition of these cells lead to an increase in the CD107a fluorescence\nchannel? These data should be added.\nLine 412: IL-18 is used in most co-cultures (please add in the figure panel e.g. in S3D when it is used and when not), but\nhow does this relate to an expected effect in vivo in a human? As it seems IL-18 is needed to increase functionality of the gd\nT-cells. Would you expect to see the same effects as observed in vitro, when in an in vivo situation you cannot co-stimulate\nwith IL-18? Please comment on that.\nFigure S3C: no statistical analyses. Different secretome factors are shown (with various efficiency of induction by CMV\ninfection). IFN-γ was selected as the most important one. Was this based on statistical significance?\nFigure 2D: please explain the use of HSV and VZV in more detail. Why are they used as controls? Are there controls used to\nshow successful infection, what is their infection rate? Infection and expression of specific antigen (IE-1) is shown for CMV-\ninfected cells, but not for HSV and VZV-infected cells. Is this the correct infection model these viruses? A bit of background\nis needed. And also are these still considered the right controls considering that later in the paper is shown that the cells do\nnot require TCR engagement.\nFigure 2 figure legend ‘Mock-infected fibroblasts were used to set the GFP-positive gate’: please show the gating used in the\nsupplements.\nLine 439-441: ‘addition of a Vδ1 TCR blocking….’ Could this be because the effect is mainly through Vδ3 then? Please\ncomment.\nLine 443: ‘two distinct donors’ – why are these donors selected and what makes them ‘distinct’ (in what respect? CMV\nserology?). What is their CMV Serological status?\nLine 446: ‘edition’   is ‘editing’ meant here?\nLine 450: explain briefly what the expected effect is of PMA ionomycin (used as a general inductor, this may be common\nknowledge in the field but requires a short explanation for the non-hematological experts reading this paper.\nLine 452: remanent = remnant or better even ‘remaining’ ?\nFigure 3A: Here fold increase is depicted while in the rest of the figure pg/ml IFNg production is depicted. It makes it easier\nto read these figures when the axes are comparable.\nFigure 3B: lacks a positive control for the blocking effect of αVδ1\nFigure 3D (and also in other figure panels where similar antibodies are used): it is unclear whether antibodies used are\ninhibitory or inducing. Please improve the colour scheme in a way to clearly show the expected effect, so it is clearly visible\nin the figure.\nFigure 3G: ‘representative’ donor – only 1 donor is shown so this is an N=1 figure, while donor variability can be important\nhere.\nLine 472: it is not entirely convincing from the presented data that the main response is through LFA-1. We do not agree with\nthe word ‘mainly’, difference between ICAM and LFA-1 is not convincing.\nLine 475: edition = editing?\nFigure 4D+E(bottom): because the KO of donor 2 is less efficient, there is a large spread in IFN induction in donor 2 and ns\ndifference between mock and CMV cocultured cells, so we are effectively looking at data from only 1 donor that is\nconclusive. Based on an N=1 we cannot draw these strong conclusions. Additional data is needed here.\nLine 481 - 482: Not enough data here to conclude it is a key-mediator (see comment above).\nFigure 5E: see remark for Fig 1E. Mirror these 2 so you can compare human and mouse.\nFigure 5F: conclusion on LFA-1 cannot be drawn. For human, MdFI is measured in figure 1H to show LFA-1 is induced (in\nheat map this is comparable for day 0 and day 21. In 5F there is no remarkable difference in LFA-1 between day 0 and day\n7. We cannot assume without the MdFI that the effect is the same as in human, so it cannot be said that (line 507) LFA-1\nexpression is upregulated.\nFigure 5G: 2 doses of cells given   how does this compare to the dosing regimen in human? Also: unclear label, in the top it says C57BL/6J +/- MCMV, is that the serological status? Because below it says ‘MCMV i.p.’ which is infection time point.\nThe top reads as if the mice are administered the cell therapy and simultaneously receive the MCMV virus or not. Please\nchange to less confusing label.\nFigure 5H: the lethality differences are not very convincing. Plus there seems to be a difference between MCMVpos and neg\n(this is ns, but the numbers per group are different), any comments on this?\nFigure 5I: this is a confusing experiment. It tells us that in mice that are still alive, the viral load in the organs is lower than in\nmice that were lethally infected. The mock treated mice (with RPMI) all had a high titer in the organs and were lethally\ninfected. But of the treated mice, some still end up in the lethally infected groups with high viral load. Ergo, there seems to be\na trend but you cannot draw a causative conclusion that the lower viral load is because of the cell therapy.\nFigure 5J: Please comment on what the ‘effector memory population’ would be. In the text it is said the transferred T cells\nhad a ‘TEM phenotype’ (line 521-522) but TEM is characterized as CD62L-, CD44- in the figure legend. The majority of cells\nis CD62L-,CD44+.\nLine 524-525: this is a strong conclusion which is not entirely supported by the presented data mainly in figure 5H and I. In\naddition, it should be discussed whether this is the right mouse model to makes these conclusions.\nTitle of last results paragraph (p24): we do not agree that the DOT cells ‘meet cell therapy requirements’. This is a\ngeneralization. While the DOT cells may meet some requirements, the data presented does not unequivocally show it meets\nall the cell therapy requirements.\nLine 544: please explain why it is necessary to add IL-15 to these cultures. Is IL-15 needed to make the cells less vulnerable\nfor the immune suppressive drugs? And how does this relate to the in vivo situation?\nFigure 6F and H: please comment on the very low proliferation index observed (high contrast with the DOT expansion\nprotocol from healthy donors) and the relatively low amounts of live cells? H also doesn’t have statistics.\nFigure 6I: what is the dotted line representing? Mock+DOT as a baseline? Please explain?\nFigure legend for 6 on p. 44. Inconsistent representation of ‘donors’ vs. ‘experiments’. Sometimes donors are mentioned,\nsometimes it’s ‘representative experiments’ but are these then from same donor? Please make this consistent and clear how\nmany donors (patients or healthy) are used per experiment.", "reviewer_2": "The authors present interesting data regarding an adoptive gamma-delta T cell based approach using only 50 ml of patient blood to tackle CMV disease which are of significance to the field, however to increase rigor, the amount of donors should be increased to show be more convincing.\nI would recommend the following adjustments:\n- l. 370: The numbers/percentages of TEMRA should be mentioned.\n- At least 6 donors should be shown for each experiment, preferentially 3 females and 3 males, technical replicates are less\nrelevant, they can be used to calculate a medium/median for each donor\n- The appropriateness of the mouse experiment should be further discussed regarding differences of humans and mice\ngamma-delta T cells.\n- l. 501: All data should be shown either in the supplement or main figures, not showing data is not acceptable.\n- l. 528: Is there supporting evidence for this claim, especially having in mind the TRACE study?\n- l.532: Actually, there are many reports showing successful expansion of autologous T cell products from kidney\ntransplanted patients, please show evidence or adjust the statement (e.g. 10.3389/fimmu.2019.01148).\n- l. 542: What was the rationale to exclude glucocorticoids from the study, although they are standard immunosuppressants\nused after SOT? Please add data on the effect of glucocorticoids on the cell product.\n- Please add data on the killing capacity of patient derived T cell products regarding killing of the CMV+ fibroblasts.", "reviewer_3": "The paper by Marsères et al. asks whether the long-established capacity of human non-Vδ2+ T cells to respond to CMV-\ninfected cells (and, at the same time, tumor cells) can be exploited as a cell therapy using the DOT cell technique to\npropagate and enrich for Vδ1+ T cells. Overall, the results seem encouraging and provide a basis for considering a clinical\ntrial to limit CMV infection or reactivation in immunosuppressed transplant recipients. This is a major unmet need although\nanalogous clinical approaches using αβ T cells are further advanced. For the most part, the experiments are logical and\nseem well-executed, but there are areas where the authors’ interpretations need to be placed very clearly in the context of\nDOT cells since they may not apply to human γδ T cells in vivo. Indeed, this is an immunotherapy paper rather than a paper\nabout human immunology, which is fine, so long as the two are not confused.\nMain points:\nFigure 1: this shows that DOT cells can be expanded equally well from CMV-sero(+) donor and CMV-sero(-) donors, thereby\nshowing that an initial status of terminally differentiated effector non-V 2+ cells in CMV-sero(+) donors does not prevent the\ncells expanding and adopting a phenotype more like activated central memory cells. However, a major advantage of γδ T\ncells for immunotherapy is their lack of MHC restriction and potential to be used in the allogeneic setting, in which case, a\nset of CMV-sero(-) donors would presumably be the main source. Hence, why is it important to know that cells from CMV-\nsero(+) donors can be expanded? The authors might argue that this is because autologous cells may be required for solid-\norgan recipients, but for that setting, which is a major unmet need, the authors need to be much more clear about the\nadvantages over autologous αβ T cells.\nFigure 2: this shows that DOT cells from sero(+) or sero(-) donors limit CMV dissemination in vitro. However, the data do not\nseem as simple as the text describes. First, there is rather marginal activation of cytolysis by CMV-infected fibroblasts\ncompared to medium alone, whereas the strong activation of cytolysis against infected macrophages is barely greater than\nthat against mock-infected macrophages. For putative DOT-immunotherapy the fibroblast data might question efficacy,\nwhereas the macrophage data might question safety. The IFNγ data seem more convincing.\nThe specificity for CMV versus other viruses is striking, but this needs to be considered in the context of Fig 3.\nFig 3: this shows that the TCR is not required for targeting CMV-infected cells. Note, the abstract states: “DOT cells\nefficiently controlled CMV dissemination in vitro. This antiviral effect was independent of the γδTCR”. This seems very\nsurprising given the wealth of evidence from this team that the TCRs of human non-Vδ2 T cells recognise self-antigens\nupregulated on CMV-infected cells. Furthermore, it was reasonable to assume that such TCR reactivities would account for\nCMV-specificity (above). So, do the data hold up? The use of a blocking antibody is problematic because without a clear\ncontrol to prove otherwise, it is common for anti-TCR antibodies, even those that can block, to cross-link TCRs, thereby\nclouding the interpretation. Therefore, the logical experiment is to delete the TCR, which is accomplished. Here the problem\nmay be that (according to the Methods) the TCR deletion was validated over a 4-day period after which cells were rested\novernight, and then used in the experiments. Prior to this, the cells had constant TCR stimulation for over two weeks and\nprobably therefore carry a legacy of this exposure in terms of downstream signalling effectors and epigenetic imprinting. This\ncould explain why the evident reduction in IFN  response (Fig 3E) is not complete. The hypothesis that TCR signalling is\nrequired could explain why there was no impact of blocking any NKRs (Fig 3G) and seems to be supported by the data in\nFig 4 (below). Also, the argument that the high background of IFNγ in DOT cells responding to mock-transfected cells\nprobably relates to a legacy of continued TCR signalling does not seem consistent with there being no background IFNγ in\ncells stimulated with isotype antibodies as a control for anti-CD3 (compare Fig 3D and 3E).\nFig 4: this claims to show that LFA1 is important for the response to CMV-infected cells. This would be consistent with the\nTCR engaging CMV-infected targets and activating LFA1 via inside-out signalling, thereby enhancing intimate effector-\ntarget cell contacts. One can hypothesise that in TCRk.o. cells, residual signalling events following exposure to chronic TCR\nsignalling are sufficient to activate LFA1. Indeed, if the TCR was not involved, and none of the NKRs examined in Fig 3G is\ninvolved, it is very challenging to understand how activating LFA1 alone could induce a response from T cells, since it is at\nbest a co-stimulator and primarily an adhesion molecule. It is perhaps important to note that if the TCR were not required, the\nauthors would be obliged to explain why transfer of γδ T cells would be preferable to transferring NK cells. In fact, the\nresponse of donor 2 LFA1k.o. cells (Fig 4E) is interpreted as negative, but it looks equally as strong as the responses of\ndonors 1 and 2 TCRk.o. cells (Fig 3E) which were scored as positive. In sum, it seems highly likely to this reviewer that\nhuman Vδ1 cells require the TCR to engage CMV-infected cells and that they then activate LFA1. In DOT cells, LFA1 is\nalready activated by chronic TCR signalling and can collaborate with residual TCR signals in TCR k.o. cells. This is an\nunusual situation that should not be confused with human immunology.\nFig 5: this shows that murine γδ T cells produced via a DOT-type culture can be transferred to recipient lymphocyte, ILC, and\nNK cell deficient mice, wherein they become activated and distribute into multiple organs to limit CMV in vivo. This is a\nproof-of-principle; one suspects that NK cells might have achieved the same outcome.\nFig 6: this shows that the DOT protocol can be applied to kidney transplant recipients and that the cells may be functional in\nthe presence of common immunosuppressants that would be the milieu for an adoptive therapy. This is encouraging\nalthough no evidence is given for how the cells remain functional in the immunosuppressants.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Reply: First, we would like to thank the reviewer for summarizing the main findings of our\nstudy, and for their careful reviewing of the manuscript. All the comments were\nthoroughly considered, and in most cases, complementary experiments were conducted\nin response. These eSorts have substantially enhanced the quality of our results.\nGeneral considerations and points for improvement:\n1. Throughout the manuscript, the authors present experiments using diSerent numbers\nof donors per experiment, and donors with diSerent CMV-serological status. It is often\nunclear from the figure whether the same donors are used as in the figure before, and\nwhich criteria are used to select a specific donor (e.g. figure 3 B: 4 donors, 3C: 2 donors\n– are the 2 in C also represented in B, and if so, why are these 2 selected over the other 2\nused in B?). Although the authors demonstrate comparability of DOT cell characteristics\nobtained from CMV naïve vs. CMV seropositive healthy donors in the first\ncharacterization experiments, in later experiments other characteristics are\ninterrogated in more detail, in which diSerences between the serological status can\nbecome more apparent. By not disclosing the origin of the cells used in experiments\nafter figure 1, these (likely minor) diSerences may be overlooked. Altogether, in our\nopinion the interpretability of the presented data would improve by disclosing this\nmissing information.\nReply: Thank you for this fair suggestion. Because the number of DOT cells that can be\nexpanded from one donor is limited, we generally used diSerent donors for diSerent\nassays. However, in some cases, it was possible to use the same donor in diSerent\nassays. Therefore, in agreement with the reviewer and to clarify this point, we have now\nidentified in the revised manuscript each donor with a specific number, which is\nindicated either directly in the corresponding figures or in the source data file linked to\neach figure. Throughout the revised manuscript, we have also systematically specified\nthe CMV-serotype of each donor when determined. This was done either by clearly\nindicating the CMV-serotypes in the figure labels or legends, or by providing a color-code\nfor CMVneg, CMVpos and Not Determined. Healthy donor CMV serotypes are also\nprovided as a source data file. These clarifications confirm that healthy donor CMV\nserostatus does not aSect DOT cell expansion, phenotype and anti-CMV reactivity.\nOf note, CMV serology must be specifically requested from our local blood bank. For this\nreason, it was not systematically determined for all donors in the later phases of the\nproject, after having established that CMV serostatus does not impact DOT cell\nexpansion, phenotype and anti-CMV reactivity.\n2. In the materials and methods it is mentioned that, unless stated otherwise, IL-18 is\nused in the co-culture experiments. This creates confusion when going back and forth\nbetween main figures and supplementals, with the need to constantly re-read the\nlegends searching for a mentions of IL-18 absence before being able to compare figures.\nIn our opinion, adding a simple ‘IL-18+’ or ‘IL18-’ label in the figures would improve\nreadability and interpretability.\nReply: We have now clearly indicated in the legends of Figures 2, 3, and 5 which\ncocultures were performed in the presence of 50 ng/mL IL-18. We have additionally\nadded a +IL-18 label in these Figures.\n3. Many abbreviations and acronyms are used, which are not consistently explained\n(sometimes in the main text, sometimes only in the figure legend). We urge the authors\nto critically go through the main text and explain abbreviations/acronyms appropriately.\nHighlighting (bold letters) and using a glossary would increase readability, if this is\nallowed in the Nature Comms format.\nReply: We carefully checked and defined all abbreviations. However, Nature\nCommunications does not allow to highlight abbreviations and does not recommend to\nprovide a glossary.\n4. The figure panels (especially the flow cytometry gates panels) are too small to read\ncomfortably from an A4 printed paper format. We strongly advise to consider using\nlarger panels.\nReply: Following the reviewer’s recommendation, we enlarged Figure panels, especially\nthe flow cytometry gate panels in Figure 1, Supplementary Figure 1 and\nSupplementary Figure 2.\n5. The authors address the risks of oS-target reactivity in the discussion. What is the\nexpected side eSect profile in humans, this should be considered. More data are\nneeded, not only mock infected cell lines (which also do not induce ‘zero’ response) and\na reference to an ongoing clinical study in leukemia patients. The SOT patient with an\nongoing infection (activated stage, possibly other viruses) and the TCR independent\nmechanism as shown is this manuscript should be taken into account and when\npossible other targets should be included in the experiments.\nReply: In the initial studies describing the generation of DOT cells (PMID: 27307596;\nPMID: 30894378), their absence of reactivity was shown against healthy alpha-beta T\ncells, B cells, NK cells and other leukocyte populations. In addition, in our study, we show\nminimal DOT cell reactivity against healthy primary fibroblasts and endothelial cells. We\nbelieve that it is already quite a good demonstration that DOT cells are at low risk of oS-\ntarget reactivity. We do not think that adding other targets will add much to the\ndemonstration. As a matter of fact, DOT cells have been approved in a clinical trial\n(NCT05886491), which will be a more relevant way to assess the safety of this product.\nThis important point mentioned by the reviewer is addressed in the discussion of the\nrevised manuscript.\nSpecific considerations:\n6. Methods section: no mentioning of oS target scores of the used sgRNAs for the KO\nmodel. Was this check performed? How else have oS-target editing eSects been\nexcluded? Please explain.\nReply: OS-target scores of gRNAs have indeed been evaluated. gRNA were designed\nusing the CRISPOR algorythm (described in Haeussler et al. Genome Biology 2016, DOI:\nhttps://doi.org/10.1186/s13059-016-1012-2) using specificity as the main criterion. This\nspecificity was assessed: (i) by the CFD specifity scores (Doench et al., Nature\nBiotechnology 2016, doi.org/10.1038/nbt.3437; Tycko et al., Nature Communications\n2019, doi: 10.1038/s41467-019-11955-7) which is an accurate score ranging from 0 to\n100, with 100 being the highest specificity and (ii) by looking closely at the potential\nnumber of oS-target exons.\nCharacteristics of each gRNA used in this study according to CRISPOR:\n___________________________________________________________________\nGene –gRNA CFD specificity score Number of exonic sequences Number of exonic sequences\nwith 3 mismatches with 4 mismatches\nTDRC1-gRNA1 95 0 5\nTDRC1-gRNA2 92 0 4\nITGAL1-gRNA1 93 0 3\nITGAL1-gRNA2 93 1 4\n__________________________________________________________________________________________\nFurthermore, all the experiments were carried out using a high specificity Cas9 protein\nfrom IDT : Alt-R™ S.p. HiFi Cas9 Nuclease V3, which has been engineered to\ndramatically reduce oS-target eSects (Vakulskas et al, Nat. Med. 2018, doi:\n10.1038/s41591-018-0137-0).\nThis information has been added to the Methods section (Section: CRISPR-Cas9\nknockout in DOT cells).\n7. Line 375: Please elaborate on how these donors are selected and is this enough to\nmake the conclusion in line 390-391?\nThe reviewer refers to the following conclusion: “Finally, we observed no impact of the\nCMV serotype in this phenotypic analysis, as illustrated for 3 CMVneg and 3 CMVpos\ndonors”.\nReply: No specific selection criteria were applied to include these donors, apart from\ntheir CMV-serotype. To further support the conclusions of these experiments, we\nperformed a statistical comparison between CMVneg and CMVpos donors after DOT cell\nexpansion. These analyses have now been added to the revised manuscript as\nSupplementary Figure 3. We compared either the percentage of positive cells for each\nmarker (Fig. S3A-E), or the median fluorescence intensity of DNAM-1, NKG2D and LFA-1\n(for which >90% of Vd2neg gd T cells are positive after expansion, Fig. S3F). These analyses\nconfirm that the CMV serotype does not significantly impact DOT cell phenotypes after\nexpansion. Of note, as KIRs were not expressed by DOT cells after expansion, no\nstatistical analysis comparing CMVneg and CMVpos was performed for these markers.\n8. Figure 1E: include CMV status (donor 1-2 = CMV neg, donor 3-4 = CMV pos)\nReply: The CMV status of the donors is now indicated. In addition, to strengthen these\ndata, we phenotyped 2 additional CMVpos donors. Figure 1E now includes 3 CMVneg\ndonors and 3 CMVpos donors. For each cell population, the percentages are not\nsignificantly diSerent between CMVneg and CMVpos donors (unpaired t test, P values\ndescribed in the results section).\n9. Figure 1F: In the text the terms central memory or eSector cells are used, in the\nlegend within the figure this is translated to CD27 and CD45RA status. Provide a label in\nthe figure for the central memory or eSector population as well for clarity. Also, SD are\nshown, but no statistical analyses are provided.\nReply: A label for each population has been added in Figure 1F and the legend has been\nclarified to indicate naïve: CD45RA+ CD27+; T central memory (TCM): CD45RA- CD27+;\nT eSector memory (TEM): CD45RA- CD27-; T eSector memory re-expressing CD45RA\n(TEMRA): CD45RA+ CD27-. Statistical analyses (unpaired t test for each population) are\nnow provided and described in the figure legend of the revised manuscript.\n10. Figure 1F is associated with figure 5E (where a similar analysis is performed in the\nmurine CMV in vivo model). We suggest to model the 2 figures in a similar way so they\ncan be compared side by side and similarities in orthologous cell populations are\nimmediately apparent.\nReply: Figure 1F and Figure 4E of the revised manuscript (previously Figure 5E) are now\npresented identically following the reviewer’s suggestion. In particular, naïve, TCM and\nTEM populations are clearly labelled in both figures. A statistical analysis was also added\nto Figure 4E to mirror Figure 1F.\n11. Line 409-410: more elaborate explanation of why authors chose to focus on IFN-γ is\nneeded. They refer to a paper, but a short explanation of why this is the most interesting\nfactor (compared to the other targets in the CMV-induced secretome) would be\nappreciated.\nReply: Since its initial description as a soluble macromolecule “interfering” with viral\nreplication (PMID: 17533668), IFNγ has been extensively characterized as a pleiotropic\ncytokine that modulates both innate and adaptive antiviral immune responses and can\nalso directly inhibit virus replication (as reviewed for instance in PMID: 30402328). In\nprevious studies from our group, IFNg has consistentely been the best read-out of gd T\ncell reactivity against CMV-infected cells (PMID:22180442; PMID: 3262235; PMID:\n34725108) and we have shown that the ability of activated gd T cell supernatants to\ncontrol virus replication is mediated by IFNγ (PMID: 22180442). These clarifications have\nbeen added to the Results section ‘DOT cells display anti-CMV eSector functions’.\n12. Figure S3A: contains endothelial cells, eSect of CMV infection is minimal. Please\ncomment on why this is (and also why in S3D they don’t seem to react to CMV induction\nwithout IL-18, but with IL-18, suddenly IFN-g is increased).\nReply: Infection of endothelial cells by CMV in vitro has been described to be far less\neSicient than for fibroblasts, when the CMV strain used is propagated in fibroblasts as\nperformed in our study (see for instance PMID: 2550582). The low percentage of infected\nendothelial cells shown in Figure S4A of the revised manuscript was therefore expected.\nAs a consequence, the reactivity of DOT cells is expected to be less important against\ninfected endothelial cells compared to infected fibroblasts, as shown in Figure S4D of\nthe revised manuscript. When IL-18 is added to co-cultures (Fig. 2B), it potentiates CMV-\ninduced IFNg production (as described in Guerville et al., Oncoimmunol 2015; PMID:\n26155394). In cocultures with endothelial cells, IL-18 therefore reveals a reactivity\notherwise invisible.\n13. Figure S3B: FMO of infected cells is missing. Can the addition of these cells lead to\nan increase in the CD107a fluorescence channel? These data should be added.\nReply: We would like to clarify here that the CD107a stainings were performed on DOT\ncells , not on infected or uninfected target cells. In the revised manuscript, a label was\nadded in Figure S4B (previously S3B) indicating “gated on Vd2neg gd T cells” to clarify. If\nthe reviewer meant the FMO of DOT cells cultured with infected or uninfected cells, those\nare similar as shown below (Figure R1). The addition of target cells (mock or CMV-\ninfected) to DOT cells does not induce any detectable change in their size or\nautofluorescence, and therefore in the % of cells in the CD107a-positive gate in FMO\ncontrols.\nFigure R1: Related to Figure S4B. Fluorescence Minus One (FMO) controls are shown\nfrom one representative experiment (DOT donor 62) in the diSerent coculture conditions.\nDot plots were gated on Vd2neg gd T cells.\n14. Line 412: IL-18 is used in most co-cultures (please add in the figure panel e.g. in S3D\nwhen it is used and when not), but how does this relate to an expected eSect in vivo in a\nhuman? As it seems IL-18 is needed to increase functionality of the gd T-cells. Would\nyou expect to see the same eSects as observed in vitro, when in an in vivo situation you\ncannot co-stimulate with IL-18? Please comment on that.\na701DC\nOMF\nDonor 62 - Gated on Vδ2neg γδ T cells:\nDOT DOT + HFF Mock DOT + HFF CMV DOT + Mφ Mock DOT + Mφ CMV\n0.09 0.09 0.16 0.01 0.09\nVδ2 TCR\nReply: We have now clarified when IL-18 is used in the diSerent figures (see response to\ncomment 2 above). Regarding the expected eSect in patients, IL-18 has been detected at\nhigh level in the sera of renal transplant recipients during CMV-infection, concomitantly\nwith elevated levels of IFNg (PMID: 20632887). In an experimental model of CMV infection\nin mice, IL-18 has been shown to be produced by dendritic cells in infected spleens\n(PMID: 39134803). In addition, we have previously shown that IL-18 can be produced by\nCMV-infected human veinous endothelial cells (HUVEC, PMID: 26155394). It is therefore\nreasonable to expect that IL-18 produced in vivo will help DOT cells to control CMV.\nThese comments have been included in the discussion section of the revised manuscript.\n15. Figure S3C: no statistical analyses. DiSerent secretome factors are shown (with\nvarious eSiciency of induction by CMV infection). IFN-γ was selected as the most\nimportant one. Was this based on statistical significance?\nReply: In agreement with the reviewer’s comment, we have added a statistical analysis\non the results presented in Figure S4C (previously S3C), showing that both IFNγ and GM-\nCSF are significantly induced when DOT cells were co-cultured with CMV-infected cells\n(compared to DOT cells cultured alone). This induction was particularly striking for IFNg\nafter coculture with CMV-infected macrophages. Because of the well-established\nfunction of IFNg against viral replication (see response to comment 11), by contrast to\nGM-CSF, we then focused on IFNg.\n16. Figure 2D: please explain the use of HSV and VZV in more detail. Why are they used\nas controls? Are there controls used to show successful infection, what is their\ninfection rate? Infection and expression of specific antigen (IE-1) is shown for CMV-\ninfected cells, but not for HSV and VZV-infected cells. Is this the correct infection model\nthese viruses? A bit of background is needed. And also are these still considered the\nright controls considering that later in the paper is shown that the cells do not require\nTCR engagement.\nReply: HSV and VZV are used as controls because they belong to the same family of\nHerpesviridae as CMV and share genetic similarities (PMID: 2552898). They also can be\neasily compared to CMV in vitro since they can readily infect fibroblasts, which are the\nprototypic target cells used for in vitro infection by the three viruses. We have already\nshown in previously published studies (Halary et al., JEM 2005 PMID: 15897274; Kaminski\net al., JID 2021 PMID: 32622351) that HSV-1 and VZV-infected fibroblasts do not activate\nVd2neg gd T cells. This is consistant with the absence of gd T cell responses in kidney\ntransplant recipients infected with HSV or VZV, by contrats to patients infected with CMV\n(Dechanet et al., JID 1999 PMID: 9841815; Kaminski et al., JID 2021 PMID: 32622351). In\nvitro, successful infection can be determined by observing the cytopathic eSects of these\n3 lytic viruses by microscopy. We then used cultures with the same rates of cytopathic\neSect (in between 50% and 100%) to compare DOT cell reactivities.\nInterestingly, and as mentioned by the reviewer, even though the activation of DOT cells\nby CMV-infected cells is TCR-independent, it remains specific of CMV when compared\nto VZV and HSV, indicating that the induction of other molecules than a TCRligand can be\nspecific of one virus. Alternatively, VZV and HSV could encode unique immune evasion\nmechanisms absent during CMV infection.\n17. Figure 2 figure legend ‘Mock-infected fibroblasts were used to set the GFP-positive\ngate’: please show the gating used in the supplements.\nReply: The gating strategy used to obtain these results is now shown in Figure S4E and\nmentioned in the results section of the revised manuscript.\n18. Line 439-441: ‘addition of a Vδ1 TCR blocking….’ Could this be because the eSect is\nmainly through Vδ3 then? Please comment.\nReply: We agree with the reviewer’s comment: the use of an anti-Vd1 blocking antibody\ndoes not preclude a remaining response by Vd3 T cells, even though they represent a\nminor population in the DOT cell product, as shown in Figure 1E. However, TCR knockout\nexperiments (shown in Figure 3C and D and Figure S5 of the revised manuscript) in which\nthe TCRd constant region shared by all gd T cells is targeted, clearly showed a gdTCR-\nindependent IFNg secretion in response to CMV.\n19. Line 443: ‘two distinct donors’ – why are these donors selected and what makes\nthem ‘distinct’ (in what respect? CMV serology?). What is their CMV Serological status?\nReply: As mentioned above, donors were not selected based on specific characteristics\nbut were freshly amplified specifically to perform these knockout experiment. The term\n“distinct” was used to indicate that the knockout experiments were performed on two\nindependent donors. To avoid ambiguity, we have also replaced “distinct” with\n“independent” when referring to donors in the revised manuscript. Additionally, the CMV\nserological status of the three donors used for TCR knockout experiments is now\nindicated in the Figure 3 legend of the revised manuscript.\n20. Line 446: ‘edition’ à is ‘editing’ meant here?\nReply: ‘Edition’ has now been replaced by ‘editing’ in the text.\n21. Line 450: explain briefly what the expected eSect is of PMA ionomycin (used as a\ngeneral inductor, this may be common knowledge in the field but requires a short\nexplanation for the non-hematological experts reading this paper.\nReply: Stimulation with PMA and ionomycin is a commonly used positive control to\ninduce a polyclonal T cell activation. PMA notably induces protein kinase C activation,\nwhile ionomycin is a calcium ionophore triggering calcium-dependent signaling\npathways. Altogether, stimulation with these two compounds bypasses the TCR complex\nto activate several intracellular signaling pathways. We have added a summary of these\nexplanations in the revised manuscript (Results section “DOT cells do not need their TCR\nto recognize CMV-infected cells”).\n22. Line 452: remanent = remnant or better even ‘remaining’ ?\nReply: The correction has been made in the text.\n23. Figure 3A: Here fold increase is depicted while in the rest of the figure pg/ml IFNg\nproduction is depicted. It makes it easier to read these figures when the axes are\ncomparable\nReply: We chose to plot fold increases in Figure 3A because the basal production of IFNg\nby DOT cells (in the absence of mock-infected or CMV-infected fibroblasts) can vary\ngreatly from one donor to another, which was particularly visible in this experiment. To\nillustrate this, please find below the corresponding raw data, where IFNg production is\nshown in pg/ml (Figure R2). This important variation makes it diSicult to compare\n“transwell” and “contact” in the conditions where DOT cells are co-cultured with CMV-\ninfected fibroblasts. For this reason, we propose to keep fold increases in Figure 3A.\nFigure R2 (related to Figure 3A). DOT cells were cultured alone or in the presence of mock\nor CMV-infected fibroblasts, either in contact or separated by a Transwell system, in the\npresence of IL-18. IFNg secretion is shown for three experiments performed on\nindependent donors. Each datapoint represents the mean of culture triplicates. ns\nP>0.05, paired t-test.\n24. Figure 3B: lacks a positive control for the blocking eSect of αVδ1\nReply: We thank the reviewer for this important comment. As a positive control for\nassessing the blocking eSicacy of the Vδ1 antibody, we stimulated DOT cells obtained\nfrom four independent donors with a plate-bound anti-CD3 antibody (clone OKT-3),\nwhich is known to robustly induce IFN-γ secretion in these cells. Prior to stimulation, DOT\ncells were pre-incubated with either an isotype control antibody or the Vδ1-blocking\nantibody. Pre-treatment with the Vδ1-blocking antibody resulted in a significant\nreduction in IFN-γ production compared to the isotype control. These results are\npresented in Figure R3 below. Additionally, to strengthen the results shown in Figure 3B,\nwe repeated the experiment, increasing the sample size to n = 8 donors.\nFigure R3: Assessment of the blocking eSicacy of the Vδ1 antibody.\nDOT cells were pre-incubated with either an isotype control antibody or an anti-Vδ1\nblocking antibody prior to stimulation with plate-bound anti-CD3 (clone OKT-3) in the\npresence of IL-18 for 24h. IFNγ secretion was measured. Each datapoint represents the\nmean of technical triplicates from an independent donor (n = 4). Statistical comparisons\nwere performed using ratio paired t tests.\n25. Figure 3D (and also in other figure panels where similar antibodies are used): it is\nunclear whether antibodies used are inhibitory or inducing. Please improve the colour\nscheme in a way to clearly show the expected eSect, so it is clearly visible in the figure.\nReply: We have now directly indicated in Fig. 3B, Fig. 3E, Fig. 3F, Fig. 3H and Fig. S5A\n(previously Figure 3D) whether the antibodies used were activating or blocking\nantibodies. This information is now also indicated in the corresponding figure legends.\n26. Figure 3G: ‘representative’ donor – only 1 donor is shown so this is an N=1 figure,\nwhile donor variability can be important here.\nReply: We thank the reviewer for this important comment. We fully agree that donor\nvariability is a critical consideration. To address this, we repeated this experiment on\nthree additional donors and now show data from n=6 donors in total. In the revised Figure\n3E (previously Figure 3G) one dot corresponds to the average of culture triplicates from\none donor. In this expanded dataset the same conclusion can be drawn as in the initial\ndata set: blocking antibodies directed against NKp30, NKp44, NKG2D, or DNAM-1 do not\nsignificantly reduce IFNγ production by DOT cells in response to CMV-infected\nfibroblasts, as compared to isotype control conditions. These results show that these\nactivating NK receptors are not individually required for CMV-induced IFNγ production in\nDOT cells.\n27. Line 472: it is not entirely convincing from the presented data that the main\nresponse is through LFA-1. We do not agree with the word ‘mainly’, diSerence between\nICAM and LFA-1 is not convincing.\nReply: As suggested, we have deleted the word ‘mainly’ and rephrased this sentence. In\nthe revised manuscript, we have now written: “Importantly, in coculture with CMV-\ninfected fibroblasts, blockade of LFA-1 or ICAM-1 systematically reduced IFNγ secretion\nby DOT cells expanded from six independent donors, with the strongest eSect observed\nwhen both were blocked simultaneously”.\n28. Line 475: edition = editing?\nReply: ‘Edition’ has now been replaced by ‘editing’ in the text.\n29. Figure 4D+E(bottom): because the KO of donor 2 is less eSicient, there is a large\nspread in IFN induction in donor 2 and ns diSerence between mock and CMV\ncocultured cells, so we are eSectively looking at data from only 1 donor that is\nconclusive. Based on an N=1 we cannot draw these strong conclusions. Additional data\nis needed here.\nReply: We appreciate this important comment and fully agree that conclusions drawn\nfrom a single donor with eSicient knockout are insuSicient. To address this comment, we\nattempted LFA-1 knockout in DOT cells from three additional donors. Knockout eSiciency\nwas assessed by flow cytometry (Figure R4).\nUnfortunately, these three attempts to obtain a full knockout failed (as shown in Figure\nR4 below), as LFA-1 expression remained high following editing. We also attempted to\nenrich LFA1-negative cells by cell sorting, but in all cases, we were unable to recover a\nsuSicient number of viable cells for downstream functional assays. Given these\ntechnical limitations, we have decided to remove the initial LFA-1 knockout data from the\nmanuscript. Instead, conclusions about the involvement of LFA-1 in CMV-specific\nreactivity are now based on functional experiments using anti ICAM1 and LFA-1 blocking\nantibodies, now performed in six independent DOT donors in total. These results\nconsistently support a role for LFA-1 in DOT cell anti-CMV reactivity.\nWe have revised Figure 3 and Figure 4 accordingly. The two figures are now merged in\none. In the revised manuscript, we have mentioned this technical limitation in the Results\nsection. We have also revised our interpretation and discussion of this mechanism\naccordingly (in the Discussion section).\nFigure R4: DOT cells from three independent donors (donors R6, R7 and R8) were\nknocked out for LFA-1 (ITGAL gene) and KO eSiciency was verified by flow cytometry. LFA-\n1 staining is shown for control (orange), LFA-1 knockout (green) and FMO (grey)\nconditions.\n30. Line 481 - 482: Not enough data here to conclude it is a key-mediator (see comment\nabove).\nReply: We agree with the reviewer. While blocking experiments (now confirmed in three\nadditional donors) support a role for LFA-1, we were unable to validate this through LFA-\n1 knockout due to technical limitations. Accordingly, we have revised the manuscript text\nto adopt a more cautious interpretation: “Altogether, stimulation and blocking\nexperiments suggest that LFA-1 contributes to DOT cell anti-CMV reactivity”.\n31. Figure 5E: see remark for Fig 1E. Mirror these 2 so you can compare human and\nmouse.\nReply: Figure 1F and Figure 4E (previously Figure 5E) of the revised manuscript are now\npresented identically.\n32. Figure 5F: conclusion on LFA-1 cannot be drawn. For human, MdFI is measured in\nfigure 1H to show LFA-1 is induced (in heat map this is comparable for day 0 and day 21.\nIn 5F there is no remarkable diSerence in LFA-1 between day 0 and day 7. We cannot\nassume without the MdFI that the eSect is the same as in human, so it cannot be said that\n(line 507) LFA-1 expression is upregulated.\nReply: In order to provide a more systematic statistical analysis of the data presented in\nFig. 4F (previously Fig. 5F), we compared the % of mouse gd T cells positive for each\nmarker (Fig. S6A of the revised manuscript) as well as the MdFI of each marker on gd T\ncells (Fig. S6B of the revised manuscript). Notably, the comparisons of LFA-1 % and MdFI\nat day 0 and day 7 show a significant increase. These analyses have now been added to\nthe revised manuscript as Supplementary Figure 6, to support the following conlusions\n“Phenotyping of murine γδ T cells before and after amplification showed an increase in\nactivation (CD25), a lack of immune-checkpoint inhibitors such as PD-1 or TIM-3, and an\nupregulation of NK activating receptors (DNAM-1, NKG2D), cytotoxicity receptors (FasL\nand TRAIL), as well as LFA-1 expression, independently of previous CMV priming”.\n33. Figure 5G: 2 doses of cells given à how does this compare to the dosing regimen in\nhuman? Also: unclear label, in the top it says C57BL/6J +/- MCMV, is that the serological\nstatus? Because below it says ‘MCMV i.p.’ which is infection time point. The top reads as\nif the mice are administered the cell therapy and simultaneously receive the MCMV\nvirus or not. Please change to less confusing label.\nReply: Although a γδ T cell therapy has not yet been tested in humans to treat CMV\ninfection, we can compare the numbers of mouse γδ T cells administered in our in vivo\nexperiments (Figure 4 of the revised manuscript) to the numbers of infused CMV-specific\nCD8⁺ T cells in Smith et al. (Clin Infect Dis 2019 DOI: 10.1093/cid/ciy549) and Khoury et\nal. (Am J Transplant 2024, DOI: 10.1016/j.ajt.2024.04.009). These two pioneering studies\nevaluated CMV-specific CD8⁺ T cell adoptive transfer to treat CMV infection in solid-\norgan transplant recipients.\nIn Khoury et al., patients received a median of two infusions of virus-specific T cells at a\ndose of 50 × 10⁶/m² body surface area. In Smith et al., patients received up to six doses\nof in vitro-expanded T cells at 10–20 × 106/m² every two weeks.\nIn our experiments, Rag−/−γc−/− mice received 10⁶ γδ T cells on day 0 and day 7. Given\ntheir average body surface area of 0.007 m², this equates to 143 × 10⁶/m² per dose. This\nmeans the mice received approximately three times the maximal dose used in Khoury et\nal., and 2.4 times the maximal dose used in Smith et al. Thus, the γδ T cell dose\nadministered to the mice falls within the range of those used in these two human trials.\nWe have also modified the labels and legends in Fig. 4G of the revised manuscript\n(previously Fig. 5G) to improve clarity.\n34. Figure 5H: the lethality diSerences are not very convincing. Plus there seems to be a\ndiSerence between MCMVpos and neg (this is ns, but the numbers per group are\ndiSerent), any comments on this?\nReply: In the first version of the manuscript, the diSerences in mouse numbers between\ngroups resulted from pooling three independent experiments, each with varying numbers\nof mice. To strengthen our findings and increase group sizes, we conducted an additional\nexperiment and incorporated the results into the existing dataset (Figure 4H of the\nrevised manuscript, previously Fig.5H). We now present data from 13 Rag-/-γc-/- mice\nthat received γδ T cells from MCMVpos C57BL/6J mice (+7), 22 mice that received γδ T\ncells from MCMVneg C57BL/6J mice (+5), and 18 control mice (+3). Our results confirm\nthat the transfer of γδ T cells from both MCMVpos and MCMVneg mice significantly\nimproved the survival of MCMV-infected Rag-/-γc-/- mice compared to the RPMI control\n(P < 0.0001 and P = 0.0001, respectively; log-rank Mantel-Cox test), with no significant\ndiSerence between MCMV status of donor mice (P = 0.0837).\n35. Figure 5I: this is a confusing experiment. It tells us that in mice that are still alive, the\nviral load in the organs is lower than in mice that were lethally infected. The mock\ntreated mice (with RPMI) all had a high titer in the organs and were lethally infected. But\nof the treated mice, some still end up in the lethally infected groups with high viral load.\nErgo, there seems to be a trend but you cannot draw a causative conclusion that the\nlower viral load is because of the cell therapy.\nReply: We appreciate this insightful comment. We agree that Fig. 5I shows a correlation\n(mice that survived MCMV infection had lower viral loads in the spleen, liver, and lungs\ncompared to those that succumbed) but does not allow to draw a causative conclusion.\nIn the revised manuscript this figure is now positionned as Fig. S7A.\nTo formally test whether γδ T cell adoptive transfer can control MCMV viral load in\ninfected mice, we performed an additional experiment using the same protocol as in the\nprevious Fig. 5I. MCMV-infected Rag-/-γc-/- mice were adoptively transferred with γδ T\ncells amplified from MCMVpos (n=5) or MCMVneg (n=5) C57BL/6J donor mice, or with\nRPMI as a control (n=5). All mice were sacrificed at day 14 post MCMV infection to allow\ndirect comparison of viral loads. This experiment showed that the transfer of γδ T cells\namplified from both MCMVpos and MCMVneg donor mice significantly reduced viral\nloads in the spleen, liver, and lungs compared to RPMI-treated controls, demonstrating\nthat γδ T cell adoptive transfer induces a decrease in viral load. This new experiment is\nnow included in the revised manuscript as Figure 4J.\n36. Figure 5J: Please comment on what the ‘eSector memory population’ would be. In\nthe text it is said the transferred T cells had a ‘TEM phenotype’ (line 521-522) but TEM is\ncharacterized as CD62L-, CD44- in the figure legend. The majority of cells is CD62L-\n,CD44+.\nReply: Thank you for this valuable comment. There was an error in the legend of this\nfigure: TEM cells are CD62L⁻ CD44⁺, not CD44⁻. We have corrected the legend of Figure\n4E (previously Figure 5E) accordingly.\n37. Line 524-525: this is a strong conclusion which is not entirely supported by the\npresented data mainly in figure 5H and I. In addition, it should be discussed whether\nthis is the right mouse model to makes these conclusions.\nReply: Please refer to our responses to comments 34 and 35, where new experiments\nhave been carried out to support these conclusions. We have also revised our conclusion\nas follows: “Together, these findings demonstrate that adoptively transferred γδ T cells\ncan migrate to MCMV-infected organs, control viral replication, and protect\nimmunodeficient mice from lethal MCMV infection”.\nThe main advantage of this mouse model of MCMV infection is its similarity to human\nCMV pathogenesis. It induces a systemic disease, as observed in humans, and is widely\nused, making it an appropriate model for testing new cell therapies. However, its primary\nlimitation is that it does not allow testing of human DOT cells. Nevertheless, we\naddressed this limitation by generating murine “DOT-like” cells that exhibit the same\ncentral memory phenotype as human DOT cells and shared their anti-CMV activity. In the\nfuture, humanized CMV mouse models could provide additional insight into the eSicacy\nof human DOT cells in vivo.\nThese additional points have been incorporated into the discussion section of the revised\nmanuscript.\n38. Title of last results paragraph (p24): we do not agree that the DOT cells ‘meet cell\ntherapy requirements’. This is a generalization. While the DOT cells may meet some\nrequirements, the data presented does not unequivocally show it meets all the cell\ntherapy requirements.\nReply: This is a fair comment. To take it into account, we have split the final section of\nthe Results into two parts with more appropriate titles.\n- The first paragraph, which includes Table 1 and Fig. 5A-D, is now titled “Autologous\nCMV-reactive DOT cells can be obtained from SOTR”.\n- The second paragraph, which includes Fig. 5E-I and Fig. S8, is now titled “DOT cells\nremain functional under immunosuppression”.\n39. Line 544: please explain why it is necessary to add IL-15 to these cultures. Is IL-15\nneeded to make the cells less vulnerable for the immune suppressive drugs? And how\ndoes this relate to the in vivo situation?\nReply: IL-15 is essential to support the survival of DOT cells both in vitro and in vivo (in\nNSG mice), as demonstrated in previous studies (Mensurado et al., Blood 2024 PMID:\n38437507; Blanco-Dominguez et al., Nat Cancer 2025 PMID: 40240620). Following\nexpansion, DOT cells are systematically rested in complete medium supplemented with\nIL-15 prior to their use in functional assays, including the proliferation and reactivity\nassays in the presence of immunosuppressive drugs shown in Figure 5 of the revised\nmanuscript (described in the Methods section).\nRegarding the in vivo relevance, several studies have shown that IL-15 levels increase\nduring CMV infection. For instance, increased IL-15 mRNA levels have been observed in\nthe bone marrow of CMV-seropositive individuals compared to CMV-seronegative\nindividuals (Pangrazzi et al., Front Immunol 2017, PMID: 28674537). Additionally, ICU\npatients exhibit higher levels of IL-15 before CMV reactivation, compared to controls\n(Chiche et al., Crit Care Med 2012, PMID: 22971588). Based on this evidence, we can\nspeculate that the production of IL-15 during CMV infection may similarly support DOT\ncell survival in vivo.\n40. Figure 6F and H: please comment on the very low proliferation index observed (high\ncontrast with the DOT expansion protocol from healthy donors) and the relatively low\namounts of live cells? H also doesn’t have statistics.\nReply: The aim of this experiment was to evaluate the survival and proliferation of DOT\ncells in vitro, in the presence of immunosuppressive drugs. For this, we measured their\nproliferation capacity and viability in culture during 15 days, after the end of their\namplification. Once amplified, their proliferation index was expected to be lower than\nduring their amplification phase.\nThe number of living cells did not exceed 50% at the end of the culture, indicating a\ncertain degree of cell death. Nevertheless, their proliferation capacity still allowed for the\nrenewal of a suSicient number of DOT cells to maintain the DOT cell population at day 15\nat the same level as on day 0 (see Fig. 5G of the revised manuscript).\nFurthermore, we would like to highlight that for the experiments presented in panels E-\nF-G-H-I, we used DOT cells amplified from healthy donors.\nIn summary, their ability to persist for 15 days in vitro suggests that DOT cells could\nsurvive in vivo for days or weeks after infusion. This was confirmed in our mouse model,\nwhere in vitro amplified gd T cells cells were found in infected organs more than 15 days\nafter infusion.\nFig. 5H now shows P values (determined by repeated-measures one-way ANOVA).\n41. Figure 6I: what is the dotted line representing? Mock+DOT as a baseline? Please\nexplain?\nReply: The dotted line indeed represented Mock+DOT at baseline but did not provide any\nrelevant additional information to interprete this figure. As a result, we have removed the\ndotted line to avoid any confusion in the interpretation of the figure.\n42. Figure legend for 6 on p. 44. Inconsistent representation of ‘donors’ vs.\n‘experiments’. Sometimes donors are mentioned, sometimes it’s ‘representative\nexperiments’ but are these then from same donor? Please make this consistent and\nclear how many donors (patients or healthy) are used per experiment.\nReply: We thank the reviewer for pointing this out. We have now clarified the number of\ndonors used in each panel of the figure. Specifically, for panels A to D, DOT cells were\namplified from 8 kidney transplant recipients. For panels E to I, DOT cells were amplified\nfrom healthy donors. 4 donors were used for panels E and F, 3 donors for panels G and H,\nand 5 donors for panel I.\nIn the revised manuscript, Figure 6 has been renumbered as Figure 5.\nReviewer #2 (Remarks to the Author):\n1. The authors present interesting data regarding an adoptive gamma-delta T cell based\napproach using only 50 ml of patient blood to tackle CMV disease which are of\nsignificance to the field, however to increase rigor, the amount of donors should be\nincreased to show be more convincing.\nReply: We thank the reviewer for this insightful comment. In response, we have increased\nthe number of donors analyzed to strengthen the robustness of our findings. Specifically:\n- Fig. 1E: this analysis is now performed on DOT cells from n=6 donors (three\nCMVneg and three CMVpos) instead of n=4 previously.\n- Fig. 3B: this analysis is now performed on DOT cells from n=8 donors (previously\nn=4).\n- Fig. 3C and 3D: gdTCR knockout experiments were performed on an additional\nDOT donor, for a total of n=3.\n- Fig. 3E: we now present an analysis on n=6 donors, instead of one representative\ndonor shown previously.\n- Fig. 3H: ICAM1 and LFA1 blocking experiments were performed on three\nadditional DOT donors, for a total of n=6.\n- Fig. 5A-D: DOT cells were amplified from three additional kidney transplant\nrecipients, for a total of n=8. In Fig. 5D, all 8 patients are now represented in the\nsame graph (each datapoint corresponds to the mean of culture triplicates for one\npatient).\nAcross the revised manuscript, each analysis is now based on a minimum of n=3 healthy\ndonors or patients. Technical replicates are no longer represented in figure panels, with\nthe exception of Fig. 3D and Fig. S5 (gdTCR knockout experiments), where each donor is\nshown on an individual graph.\n2. I would recommend the following adjustments:\n- l. 370: The numbers/percentages of TEMRA should be mentioned.\nReply: We have now included the relevant percentages in the revised Results section:\n- In CMV-seronegative donors, naïve cells represented 74.0 ± 18.8% of Vd2neg gd T\ncells at day 0.\n- In CMV-seropositive donors, TEMRA cells represented 41.2 ± 19.8% of Vd2neg gd T\ncells at day 0.\n- At day 21, DOT cells from both CMV-seronegative and CMV-seropositive donors\nwere predominantly central memory cells (88.1 ± 9.2% and 88.1 ± 9.6% of\nVd2neg gd T cells respectively).\n3. - At least 6 donors should be shown for each experiment, preferentially 3 females and\n3 males, technical replicates are less relevant, they can be used to calculate a\nmedium/median for each donor\nReply: We appreciate the reviewer’s suggestion to strengthen the robustness of our study\nby increasing donor numbers and considering sex balance.\nWhile we were not able to include six donors for every experiment, we have increased\ndonor numbers throughout the manuscript wherever possible (as detailed in our\nresponse to comment 1). All analyses are now performed on a minimum of three\nindependent donors, and all analyses presented in Figure 1 and 2 were performed on a\nminimum of 6 donors. Regarding donor sex, we regret that we do not have access to this\ninformation for the healthy donor samples used in this study. Regarding kidney transplant\nrecipients, the sex of the patients is now included in Table 1 of the revised manuscript.\nWe agree with the reviewer that technical replicates are less informative than biological\nreplicates, and we have revised the presentation of our data accordingly: individual data\npoints now represent means for each donor, and technical replicates are no longer\nshown in the figures (except in Fig. 3D and Fig. S5, where each donor is presented\nseparately).\nWe hope that this expanded dataset addresses the reviewer’s concerns, as it enhances\nthe robustness of our findings.\n4. The appropriateness of the mouse experiment should be further discussed regarding\ndiSerences of humans and mice gamma-delta T cells.\nReply: The main advantage of this mouse model of MCMV infection is its similarity to\nhuman CMV pathogenesis. It induces a systemic disease, as observed in humans, and is\nwidely used, making it an appropriate model for testing new cell therapies. Furthermore\nwe have previously shown that MCMV induces long-term expansion of murine γδ T cells\nthat protect mice from the infection, very similarly to what can be observed in human\nCMV infection (PMID: 25747674 ; PMID: 38976755). MCMV is then precisely an\ninteresting model to make parallels between human and mouse γδ T cells. However, the\nprimary limitation of this MCMV model is that it does not allow testing of human DOT\ncells. Nevertheless, we addressed this limitation by generating murine “DOT-like” cells\nthat exhibited the same central memory phenotype as human DOT cells and shared their\nanti-CMV activity. In the future, humanized CMV mouse models could provide additional\ninsight into the eSicacy of human DOT cells in vivo. We have incorporated these\ncomments into the discussion section.\n5. l. 501: All data should be shown either in the supplement or main figures, not showing\ndata is not acceptable.\nReply: We agree with the reviewer that all data described in the manuscript should be\nshown. In order to simplify the lecture for the reader, we have deleted this sentence\nfrom the manuscript.\n6. l. 528: Is there supporting evidence for this claim, especially having in mind the\nTRACE study?\nReply: Both autologous and allogeneic virus-specific cell therapies have been or are\ncurrently being tested in phase I trials in SOTR (PMID: 29982441; PMID: 38643944;\nACTRN12621000323820) or following allogeneic stem cell transplantation (TRACE study,\nNCT04832607). We agree that there is currently no direct evidence that “an allogeneic T-\ncell therapy would eventually be rejected by the host immune system, thus limiting its\nlong-term eSicacy”. This was merely a hypothesis, and we have removed this sentence.\n7. l.532: Actually, there are many reports showing successful expansion of autologous T\ncell products from kidney transplanted patients, please show evidence or adjust the\nstatement (e.g. 10.3389/fimmu.2019.01148).\nReply: We thank the reviewer for this fair comment, but there is currently no data showing\nsuccessful expansion of autologous gd T cells from solid organ transplant recipients\nreceiving immunosupressants. To address this comment, we have adjusted the\nintroduction of this paragraph and added references regarding the successful expansion\nof autologous ab T cell products from solid organ transplant recipients as follows:\n“Both autologous and allogeneic virus-specific T cell therapies have been or are currently\nbeing tested in phase I trials in SOTR15,17,42 (CTRN12621000323820) or in a phase III trial\nin HSCTR (TRACE study: NCT04832607). Among these two strategies, the autologous\napproach is particularly attractive in SOTR to avoid the risk of allosensitization\ndeleterious to the transplant43. However, whether functional and CMV-reactive DOT cells\ncan be amplified from immunosuppressed transplant recipients remains to be\nevaluated.”\n8. l. 542: What was the rationale to exclude glucocorticoids from the study, although\nthey are standard immunosuppressants used after SOT? Please add data on the eSect\nof glucocorticoids on the cell product.\nReply: As requested, we have tested the impact of glucocorticoids (methylprednisolone)\non DOT cell proliferation and anti-CMV reactivity in vitro. At the concentrations used in\nthe maintenance treatment of SOTR (at around 10 ng/ml 12 hours after the ingestion)\n(PMID: 9797792), methylprednisolone did not inhibit the persistence and reactivity of\nDOT cells against CMV-infected fibroblasts. These new data are now presented in\nSupplementary Figure 8 of the revised manuscript.\n9. Please add data on the killing capacity of patient derived T cell products regarding\nkilling of the CMV+ fibroblasts.\nReply: Demonstrating the killing capacity of DOT cells against CMV-infected fibroblasts\nis technically challenging. Indeed, CMV infection is lytic and eventually leads to\nfibroblast death independently of T cell cytotoxicity. 5 days of infection, when DOT cells\nare cocultured with fibroblasts, is the limit to keep infected cells reasonably alive, making\nit technically unfeasible to distinguish additional killing mediated by DOT cells. Moreover,\nkilling infected cells is not necessarily the most important function of gd T cells in the\ncontext of CMV infection. Secreting IFNg, which has potent anti-viral functions and\ninhibits CMV replication is also critical to control the virus propagation.\nReviewer #3 (Remarks to the Author):\nThe paper by Marsères et al. asks whether the long-established capacity of human non-\nVδ2+ T cells to respond to CMV-infected cells (and, at the same time, tumor cells) can\nbe exploited as a cell therapy using the DOT cell technique to propagate and enrich for\nVδ1+ T cells. Overall, the results seem encouraging and provide a basis for considering\na clinical trial to limit CMV infection or reactivation in immunosuppressed transplant\nrecipients. This is a major unmet need although analogous clinical approaches using αβ\nT cells are further advanced. For the most part, the experiments are logical and seem\nwell-executed, but there are areas where the authors’ interpretations need to be placed\nvery clearly in the context of DOT cells since they may not apply to human γδ T cells in\nvivo. Indeed, this is an immunotherapy paper rather than a paper about human\nimmunology, which is fine, so long as the two are not confused.\nMain points:\n1. Figure 1: this shows that DOT cells can be expanded equally well from CMV-sero(+)\ndonor and CMV-sero(-) donors, thereby showing that an initial status of terminally\ndiSerentiated eSector non-Vd2+ cells in CMV-sero(+) donors does not prevent the cells\nexpanding and adopting a phenotype more like activated central memory cells.\nHowever, a major advantage of γδ T cells for immunotherapy is their lack of MHC\nrestriction and potential to be used in the allogeneic setting, in which case, a set of\nCMV-sero(-) donors would presumably be the main source. Hence, why is it important\nto know that cells from CMV-sero(+) donors can be expanded? The authors might argue\nthat this is because autologous cells may be required for solid-organ recipients, but for\nthat setting, which is a major unmet need, the authors need to be much more clear\nabout the advantages over autologous αβ T cells.\nReply: We thank the reviewer for this comment. As correctly pointed out, the relevance\nof expanding DOT cells from CMV-seropositive donors depends on the therapeutic\nsetting (allogeneic or autologous). At this stage, both allogeneic and autologous DOT cell\ntherapies can be considered.\nIn the allogeneic setting, access to a broad and diverse donor pool is essential. CMV\nprevalence exceeds 50% in most western countries, reaching for instance over 70% in\nSpain and Portugal (PMID: 38275940). Demonstrating that DOT cells can be eSiciently\nexpanded from CMV-seropositive donors ensures broad donor availability, making it\nfeasible to manufacture a diverse and partially HLA-matched donor bank. This could\nreduce the risk of allosensitization and more specifically the occurrence of donor-\nspecific antibodies in transplant recipients.\nIn the autologous setting, refractory CMV infections typically occur in CMV-seronegative\npatients (transplanted with an organ coming from a CMV-seropositive donor) whose\nadaptive immune response to CMV is lacking (PMID: 26054538). In this context,\ngenerating DOT cells from the γδ T cell pool of these patients may oSer an advantage:\nnaïve γδ T cells can be expanded ex vivo into functional and CMV-reactive DOT cells,\nwhereas it is challenging to obtain CMV-specific memory αβ T cells from naïve cells\n(PMID: 25925682).\nThese points are now included in the Discussion section of the revised manuscript.\n2. Figure 2: this shows that DOT cells from sero(+) or sero(-) donors limit CMV\ndissemination in vitro. However, the data do not seem as simple as the text describes.\nFirst, there is rather marginal activation of cytolysis by CMV-infected fibroblasts\ncompared to medium alone, whereas the strong activation of cytolysis against infected\nmacrophages is barely greater than that against mock-infected macrophages. For\nputative DOT-immunotherapy the fibroblast data might question eSicacy, whereas the\nmacrophage data might question safety. The IFNγ data seem more convincing.\nReply: We agree that IFNg secretion assays provide a more convincing indication of DOT\ncell anti-CMV reactivity compared to CD107a assays. However, we would like to argue\nthat the combined eSects of IFNg secretion and the limited, yet statistically significant,\ncytotoxic activity likely act synergistically to limit CMV dissemination, as illustrated in\nFigure 2F.\nRegarding the safety concern raised about macrophage targeting, we would like to clarify\nthat the macrophages used in these experiments are M1-like activated macrophages,\ngenerated by diSerentiating monocytes in the presence of GM-CSF for six days. These\nactivated macrophages diSer substantially from resting tissue macrophages, which\nmitigates potential safety issues raised by the reviewer. Importantly, this diSerentiation\nand activation step is necessary to render macrophages permissive to CMV infection and\nmay explain why they induce a basal activation of DOT cells. This notwithstanding,\ninfection by CMV significantly further increased DOT cell degranulation and IFNg\nsecretion, underscoring the capacity of CMV to activate DOT cells.\n3. The specificity for CMV versus other viruses is striking, but this needs to be\nconsidered in the context of Fig 3.\nFig 3: this shows that the TCR is not required for targeting CMV-infected cells. Note, the\nabstract states: “DOT cells eSiciently controlled CMV dissemination in vitro. This\nantiviral eSect was independent of the γδTCR”. This seems very surprising given the\nwealth of evidence from this team that the TCRs of human non-Vδ2 T cells recognise\nself-antigens upregulated on CMV-infected cells. Furthermore, it was reasonable to\nassume that such TCR reactivities would account for CMV-specificity (above).\nReply: This issue of human non-Vδ2 T cells specificity for CMV is undoubtly an interesting\nand striking point. This specificity is observed both in patients (only CMV infection can\ninduce non-Vg9Vδ2 T cell response and expansion, not HSV or VZV; Déchanet et al., JID\n1999; Pitard et al., Blood 2008; Kaminski et al., JID 2021) and in vitro (only CMV infection\ncan induce fibroblast ability to activate non-Vg9Vδ2 T cells; Halary et al., JEM 2005;\nKaminski et al., JID 2021). However, it is not clear at all whether TCR reactivities account\nfor CMV-specificity. We have for instance shown in one of our study (Willcox et al., Nat\nImmunol 2012) that the Vg4Vd5 T cell clone LES was recognizing EPCR, a constitutively\nexpressed ligand on fibroblasts. The specificity of this clone for CMV-infected cells was\ndependent on the strong induction of CD54/ICAM-1 expression by CMV. Hence, CMV\nspecificity is most probably due to its capacity to induce a multimolecular stress\nsignature on infected cells recognized not only by the TCR but also by co-stimulatory\nmolecules such as LFA-1. Why HSV-1 and VZV cannot induce the same signature is\ncurrently not resolved, but might rely on inhibitory signals preventing gd T cell activation.\nRegarding the TCR-independent CMV recognition by DOT cells, we are not as surprised\nas the reviewer. All our previous studies were conducted using non-Vδ2 T cell clones, or\nnon-Vδ2 T cells isolated from CMV-infected transplant patients (which repertoire is highly\nrestricted or even oligoclonal in some patients, Dechanet et al , JCI 1999). On the\ncontrary, the repertoire of DOT cells has been shown to be highly polyclonal (Di Lorenzo\net al., Cancer Immunol Res 2019, PMID: 30894378) supposedly because they expand\nfrom naïve cells. Then, it would have been more surprising to observe TCR-specificity of\na polyclonal population. Tumor cell recognition by DOT cells was also previously shown\nto be TCR-independent by the team of B Silva-Santos (Di Lorenzo et al., Cancer Immunol\nRes 2019).\n4. So, do the data hold up? The use of a blocking antibody is problematic because\nwithout a clear control to prove otherwise, it is common for anti-TCR antibodies, even\nthose that can block, to cross-link TCRs, thereby clouding the interpretation. Therefore,\nthe logical experiment is to delete the TCR, which is accomplished. Here the problem\nmay be that (according to the Methods) the TCR deletion was validated over a 4-day\nperiod after which cells were rested overnight, and then used in the experiments. Prior\nto this, the cells had constant TCR stimulation for over two weeks and probably\ntherefore carry a legacy of this exposure in terms of downstream signalling eSectors\nand epigenetic imprinting.\nReply: We fully agree with the reviewer’s interpretation. We had attempted to address this\npoint in the discussion, but our explanation may not have been suSiciently clear. We did\nnot mean that TCR signaling is not necessary for the overall DOT cell response. Rather,\nour data indicate that during the interaction with CMV-infected cells, TCR engagement is\nnot necessary for DOT cell activation, as evidenced by the robust response of TCR-\nknockout DOT cells to CMV-infected, compared to mock-infected targets (Fig. 3D and\nFig. S5B of the revised manuscript). In other words, the specific recognition of CMV-\ninfected cells by DOT cells does not require ligand recognition by the TCR. We propose\nthat the DOT cell expansion procedure, which involves constant TCR signalling, provides\nan essential initial TCR signalling (that could be considered as a priming), and that CMV-\ninfected cells then provide the costimulation, leading to a CMV-specific response.\nTo clarify this distinction in the manuscript, we have revised our wording by replacing\n“TCR-independent response” by “TCR-independent recognition” or by saying that TCR-\nengagement is not required during the interaction with CMV-infected cells.\n5. This could explain why the evident reduction in IFNg response (Fig 3E) is not\ncomplete. The hypothesis that TCR signalling is required could explain why there was no\nimpact of blocking any NKRs (Fig 3G) and seems to be supported by the data in Fig 4\n(below).\nAlso, the argument that the high background of IFNγ in DOT cells responding to mock-transfected cells\nprobably relates to a legacy of continued TCR signalling does not seem consistent with there being no background IFNγ in\ncells stimulated with isotype antibodies as a control for anti-CD3 (compare Fig 3D and 3E).\nReply: We respectfully disagree with this comment and would like to clarify the data as\nfollows:\n- In Fig. S5B of the revised manuscript (previously Fig. 3E), we can see that the\nbackground of IFNg production by control DOT cells is comparable between\n“medium” alone and “mock” conditions, indicating that coculture with mock-\ninfected fibroblasts does not induce a consistant additional background\nactivation in these 3 DOT donors.\n- In Fig. S5A of the revised manuscript (previously Fig. 3D), there is indeed\nbackground IFNg secretion in the “Isotype” control condition, but because of the\nscale, it is not visible on the graph. When the same data are plotted on the same\naxis (as shown in Figure R5 below), it becomes visible that IFNg background levels\nare in the same range in the “isotype” condition of Fig. S5A and in the “medium”\nand “mock” conditions of Fig. S5B.\nFigure R5: IFNg secretion by DOT cells from 3 independent donors (51, 84 and R6),\nincubated for 24h in medium alone, with an isotype control antibody, or with mock-\ninfected fibroblasts.\nThis basal secretion of IFNg is consistent with the “legacy of continued TCR signaling”\nargued by the reviewer and that we fully agree with. The abrogation of this IFNg production\nbackground in TCR-KO DOT cells allows to reveal the “true” CMV-specific response, as\nshown in the green histograms in Fig. S5B and Fig. 3D of the revised manuscript. Notably,\nthe fold increase in IFNγ production by TCR-KO DOT cells in response to CMV-infected\ncells exceeded 15-fold (Fig. 3D), suggesting that the TCR-independent signals induced\nby CMV-infected cells are very potent and may play a crucial role in their activity.\n6. Fig 4: this claims to show that LFA1 is important for the response to CMV-infected\ncells. This would be consistent with the TCR engaging CMV-infected targets and\nactivating LFA1 via inside-out signalling, thereby enhancing intimate eSector-target cell\ncontacts. One can hypothesise that in TCRk.o. cells, residual signalling events following\nexposure to chronic TCR signalling are suSicient to activate LFA1. Indeed, if the TCR was\nnot involved, and none of the NKRs examined in Fig 3G is involved, it is very challenging\nto understand how activating LFA1 alone could induce a response from T cells, since it\nis at best a co-stimulator and primarily an adhesion molecule.\nReply: We show in Fig. 3F of the revised manuscript that a recombinant ICAM-1-Fc protein\nor an agonist anti-LFA-1 antibody can activate DOT cells even when used alone. This\ndemonstrates that LFA-1 engagement, independently of TCR engagement, can activate\nDOT cells. LFA-1 engagement by CMV-infected cells might therefore be suSicient to\nactivate TCR-KO DOT cells. It is possible that this response requires a prior activation of\nthe gdTCR during DOT cell expansion, that would activate LFA-1 via inside-out signalling.\nWe however cannot exclude the involvement of other undetermined activating\nreceptor(s) during the co-culture with CMV-infected cells.\n7. It is perhaps important to note that if the TCR were not required, the authors would be\nobliged to explain why transfer of γδ T cells would be preferable to transferring NK cells.\nIn fact, the response of donor 2 LFA1k.o. cells (Fig 4E) is interpreted as negative, but it\nlooks equally as strong as the responses of donors 1 and 2 TCRk.o. cells (Fig 3E) which\nwere scored as positive.\nReply: Regarding the adoptive transfer of gd T cells versus NK cells, please see our\nresponse to comment 9 below.\nIt is diSicult to compare the response of DOT cells between Figure 3 and Figure 4 of the\noriginal manuscript, because they have been generated from diSerent donors. As the\nreviewer can appreciate in Fig. 2B, IFNg production is very variable from one DOT cell\nproduct to another.\nWe would like to mention here our reply to Reviewer 1 comment 29:\nReply: We appreciate this important comment and fully agree that conclusions drawn\nfrom a single donor with eSicient knockout are insuSicient. To address this comment, we\nattempted LFA-1 knockout in DOT cells from three additional donors. Knockout eSiciency\nwas assessed by flow cytometry (Figure R4).\nUnfortunately, these three attempts to obtain a full knockout failed (as shown in Figure\nR4 below), as LFA-1 expression remained high following editing. We also attempted to\nenrich LFA1-negative cells by cell sorting, but in all cases, we were unable to recover a\nsuSicient number of viable cells for downstream functional assays. Given these\ntechnical limitations, we have decided to remove the initial LFA-1 knockout data from the\nmanuscript. Instead, conclusions about the involvement of LFA-1 in CMV-specific\nreactivity are now based on functional experiments using anti ICAM1 and LFA-1 blocking\nantibodies, now performed in six independent DOT donors in total. These results\nconsistently support a role for LFA-1 in DOT cell anti-CMV reactivity.\nWe have revised Figure 3 and Figure 4 accordingly. The two figures are now merged in\none. In the revised manuscript, we have mentioned this technical limitation in the Results\nsection. We have also revised our interpretation and discussion of this mechanism\naccordingly (in the Discussion section).\n8. In sum, it seems highly likely to this reviewer that human Vδ1 cells require the TCR to\nengage CMV-infected cells and that they then activate LFA1. In DOT cells, LFA1 is\nalready activated by chronic TCR signalling and can collaborate with residual TCR\nsignals in TCR k.o. cells. This is an unusual situation that should not be confused with\nhuman immunology.\nReply: We thank the reviewer for this summary that fits perfectly with our own\ninterpretation. We agree that DOT cell activity cannot be compared to the physiological\nactivity of non-Vd2 T cells. As pointed out by the reviewer, our previous studies have\nconsistently shown that non-Vd2 T cells, including Vd1 T cells, recognize CMV-infected\ncells in a TCR-dependent manner (Halary et al., JEM 2005; Willcox et al., Nat Immunol\n2012; Kaminski et al., JID 2021).\nDOT cells represent a highly activated cell product, shaped by in vitro expansion and\nchronic TCR engagement, leading to a unique way of responding to CMV-infected cells.\nWhile this does not directly reflect the physiology of human non-Vd2 T cells, it is essential\nto dissect the functional requirements of DOT cells to develop them as a therapeutic\nproduct, which was the purpose of our study. We have clarified this point in the revised\nmanuscript (Results and Discussion sections).\n9. Fig 5: this shows that murine γδ T cells produced via a DOT-type culture can be\ntransferred to recipient lymphocyte, ILC, and NK cell deficient mice, wherein they\nbecome activated and distribute into multiple organs to limit CMV in vivo. This is a\nproof-of-principle; one suspects that NK cells might have achieved the same outcome.\nReply: We agree with the reviewer that our in vivo experiments using murine γδ T cells\ngenerated via a DOT-like protocol serve as a proof-of-principle.\nNK cells indeed have well-established antiviral functions, including against CMV,\neven\nthough the demonstrations have been mainly obtained in mice. However, there are at\nleast two reasons why DOT cells may oSer distinct advantages or complement NK-based\ntherapies:\n- We have shown that the DOT protocol allows for a robust and eSicient expansion\nof clinically relevant numbers of non-Vδ2 T cells from the peripheral blood of both\nhealthy individuals and immunosuppressed transplant recipients. In contrast,\ngenerating large-scale, functional NK cell products can be more variable and often\nrequires longer culture times or feeder-cell systems, which pose regulatory and\ntranslational challenges (PMID: 38443448; PMID: 40681797).\n- DOT cell harbour a unique landscape of receptors, notably lacking expression of\nNKG2A and KIRs (Fig. 1G and Figure R6 below). These receptors are classically\nexpressed by NK cells, contribute to their recognition of CMV-infected cells, and\nare common targets of CMV-induced immune evasion strategies\n(PMID: 12032324, PMID: 18069056, PMID: 29632329). Many evasion\nmechanisms have been selected by CMV to escape from NK cell control, which\nhas not been reported yet to our knowledge for gd T cells or DOT cells. In addition,\nas discussed above, TCR priming during the expansion phase most likely\nparticipates in the overall response of DOT cells to CMV, distinguishing them from\na NK cell product. Altogether, the molecular mechanisms underlying CMV\nrecognition by DOT and NK cells are likely distinct.\nFigure R6: NKG2A expression on DOT cells.\nDOT cells amplified from three independent donors (88, 97 and 98) were stained with\nantibodies directed against the populations markers CD3, gdTCR, Vd1 TCR and Vd2 TCR\nand the NK receptor NKG2A. Shown are percentages of NKG2A+ Vd2neg gd T cells.\nFluorescence Minus One controls were used to set positivity gates.\nFig 6: this shows that the DOT protocol can be applied to kidney transplant recipients\nand that the cells may be functional in the presence of common immunosuppressants\nthat would be the milieu for an adoptive therapy. This is encouraging although no\nevidence is given for how the cells remain functional in the immunosuppressants.\nReply: Cyclosporin and Tacrolimus are calcineurin inhibitors that block NFAT\ntranslocation and IL-2 production in T cells. However, the DOT protocol does not rely on\nIL-2. Instead, it relies on IL-15 as the critical cytokine during the second stage of\nexpansion and diSerentiation (day 7 to day 21 of expansion). Moreover, IL-15 has been\nshown to enhance DOT cell therapeutic eSicacy in vivo (Sanchez Martinez et al., J\nImmunother Cancer 2022, PMID: 36162920; Blanco-Dominguez et al., Nat Cancer 2025,\nPMID: 40240620). Importantly, IL-15 signalling is independent of the calcineurin-NFAT\npathway. Based on these observations, we hypothesize that DOT cells are more\ndependent on IL-15 than IL-2 signaling and are therefore resistant to the eSects of\ncalcineurin inhibitors.\nRegarding Everolimus, an mTOR inhibitor, we have previously shown that, at clinically\nrelevant concentrations such as used in a transplantation setting, everolimus does not\nprevent T cell proliferation nor response to CMV, and by contrast promotes T cell survival\nand prevents T cell dysfunctionality (Kaminski et al., JASN 2022, PMID: 34725108).\nConsistently, Everolimus has been associated with a reduced occurrence of CMV\ninfections in transplant recipients in several independent studies (PMID: 34990047;\nPMID: 31152476; PMID: 25988935).\nWe have summarized these points in the discussion section of the revised manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nPoint-by-point response\nReviewer #1 (Remarks to the Author):\nReviewer Response to Rebuttal Letter (Reviewer #1 + co-reviewer) :\nWe would like to begin by congratulating the authors on the work they have put into\nrevising their manuscript. The additional experiments, clarifications to the figures, and\nimprovements to the text have significantly enhanced the quality of the work. We\nespecially appreciate the consistency in which CMV status of the donor cells is now\nshown in the figures, providing transparency in the functional equivalence of the DOT\ncells expanded from diDerent donors. These thoughtful revisions demonstrate a strong\ncommitment to scientific rigor and communication, and we commend the authors for\ntheir eDorts.\nReply: We thank the reviewers for their kind comments.\nThat said, we would like to kindly request the authors to address a few remaining points:\n1. Inclusion of Reviewer-Specific Figures\nSeveral figures created specifically in response to reviewer comments (the CRISPOR\noutput data with sgRNA specificity scores, and specifically R1 and R3) provide valuable\ninsights and clarity. We believe these should be included in the supplemental data to\nensure transparency and to benefit future readers. Figure R1 addresses baseline gating\ndata often requested in the field for assay validation purposes. Furthermore, figure R3\nshows important data serving as a positive control for main figure 3B, without which the\nmain figure is not adequately interpretable.\nReply: All requested figures have now been included in the supplementary materials of\nthe revised manuscript. The gRNA characterization table (previously Figure R3) was\nincluded as Supplementary Table 2. Figure R1 (Fluorescence Minus One controls from\nthe diDerent culture conditions) was included as Supplementary Figure 4b. Finally,\nFigure R3 (positive control experiment for Vd1 TCR blocking) was included as\nSupplementary Figure 5a.\n2. Clarification on Specific Findings\nWe would appreciate further commentary on the following points:\n• Comment #14 and #39: While the authors have touched on the presence of IL-18 (in\nCMV-infected human endothelial cells) and IL-15 (in bone marrow of CMV-infected\nindividuals), a more detailed contextualization would strengthen the author’s views on\nwhat this means regarding the survival and activity of DOT cells in human patients. For\ninstance, which concentration of these cytokines is required for the activity/support of\nthese cells in vitro, and how does that correlate with expected levels in relevant tissues\nin a CMV-infected SOTR patient receiving the DOT therapy?\nReply: In our hands, the optimal concentration of IL-15 required to support DOT cell\nproliferation in vitro is 70 ng/mL, while the concentration of IL-18 needed in vitro to\npotentiate anti-CMV activity is 50 ng/mL. To the best of our knowledge, cytokine levels\n(including IL-18 and IL-15) have never been measured directly in CMV-infected human\ntissues. However, serum concentrations of IL-18 increase during CMV infection, reaching\nmedian levels of 2000 pg/mL (PMID: 34216086, reference previously included in the\nDiscussion, line 375). Taken together, while systemic IL-18 levels in patients are expected\nto be lower than the concentrations used in our in vitro assays, local microenvironments\n(tissue niches, infected organs) may achieve higher, functionally relevant cytokine\nconcentrations.\n• Figure R3 and main figure 3B: We do not fully agree with the author’s interpretation of\nthese results. Although from later experiments using functionally knocked-out γδTCR\ncells, it can be concluded that DOT cells can recognize CMV-infection in the absence of\nγδTCR, the data presented in R3 and 3B are not contributing to this finding. Specifically,\nin R3, the authors show the inhibitory eDect of the Vδ1 antibody, which is reducing IFNγ\ninduction by approximately 50% (and sometimes less). We appreciate the significant\nreduction compared to iso control, but the blocking eDect seems incomplete, and\nresidual δ1 signaling eDects cannot be excluded. Combined with the data presented in\nfigure 3B, where we see variability of DOT cell response to CMV infection in combination\nwith Vδ1 blocking antibody on the individual donor level, with most donors showing a\ndecrease in IFNγ secretion after Vδ1 blocking, and 2 donors showing an opposite eDect.\nGrouped together, these changes are not deemed statistically significant diDerent\n(P=0,051), however in the context of incomplete Vδ1 blocking and the large variability\nobserved, the validity of this conclusion can be debated (i.e. the lack of diDerence may\nbe because residual δ1 signaling is suDicient to maintain a certain level of IFNγ\nsecretion)\nReply: We agree that in Figure R3 (now Supplementary Figure 5A of the revised\nmanuscript) the blocking eDect is incomplete. As the reviewers point out, residual Vδ1\nTCR signaling could result from this incomplete blocking. We have therefore modified our\ninterpretation of these results and conclusions as follows (in the Results section, line\n182): « In contrast to these observations, adding a Vδ1 TCR blocking antibody to\ncocultures did not impact IFNg secretion by DOT cells in response to CMV, as shown for\neight independent donors in Fig. 3b. However, as this result could have been explained\nby an incomplete Vδ1 TCR blocking (see Figure. S5a), we undertook to knock out the\ngdTCR of DOT cells obtained from three independent donors”.\n• Figure 3H: This result raises an interesting observation that merits a brief discussion,\neven if speculative – now that the CMV status of the donor cells from which the DOTs are\nexpanded is made visible, it seems that a trend exists of higher IFNγ induction in CMV +\nIL-18 cocultured DOTs from CMV negative donors (ie: gray dots are predominantly in the\nupper values, while the red dots dominate the lower values). Do the authors wish to\ncomment on this interesting trend?\nReply: A trend is indeed visible in Fig. 3h. This trend is visible in the absence of any\nblocking, and remains consistent throughout blocking conditions. However in Fig. 2c, the\nimpact of the DOT cell donor CMV serotype on CMV-induced IFNg secretion was\nspecifically addressed in a larger cohort of donors (n=13 CMVneg and n=14 CMVpos).\nThis comparison showed no significant diDerence in between CMVneg and CMVpos\ndonors.\n• Figure 3G: We appreciate the added clarity of activating and blocking antibodies to\nentire figure 3. Specifically in figure 3G, the activating label underneath ICAM-1 is\nmissing. Please add the missing label for additional clarity.\nReply: We have clarified Fig. 3g to indicate that DOT cells were stimulated with\nrecombinant human ICAM-1, indeed activating LFA-1 on DOT cells. This was also\nclarified in the Results section (lines 215-216).\n• Lastly, we encountered these 2 minor points:\n1. the HSCTR acronym is explained in line 49, but used earlier without an explanation in\nline 31 and 33.\nReply: We have now defined the acronym HSCTR when first used in the revised\nmanuscript (line 31).\n2. We noticed that in the abstract and other places in the main text, ‘amplified’ is\nchanged to ‘expanded’, which fits better in the context of proliferating cells. The authors\nshould consider making this same change to line 65 and 117, where ‘amplified’ is still\nused.\nReply: We have now systematically replaced ‘amplified’ by ‘expanded’ when describing\nDOT cells in the revised manuscript.\nWe consider these points to be addressable, and we look forward to seeing them\nincorporated in the final version.\nOnce again, we thank the authors for their work and responsiveness throughout the\nreview process."}, {"round": 2, "reviewer_comments": {"reviewer_1": "We confirm that the authors have addressed all the points raised in our previous review.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter