activating and propagating polyclonal gamma delta t cells with broad speci city … · cancer...

Cancer Therapy: PreclinicalSee related commentary by D�echanet-Merville, p. 5573

Activating and Propagating Polyclonal Gamma Delta T Cellswith Broad Specificity for Malignancies

Drew C. Deniger1,5, Sourindra N. Maiti1, Tiejuan Mi1, Kirsten C. Switzer1, Vijaya Ramachandran2,Lenka V. Hurton1,5, Sonny Ang1, Simon Olivares1, Brian A. Rabinovich1, M. Helen Huls1, Dean A. Lee1,5,Robert C. Bast Jr3, Richard E. Champlin4, and Laurence J.N. Cooper1,5

AbstractPurpose: To activate and propagate populations of gd T cells expressing polyclonal repertoire of g and d

T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved.

ExperimentalDesign:Clinical-grade artificial antigen-presenting cells (aAPC) derived fromK562 tumor

cells were used as irradiated feeders to activate and expand human gd T cells to clinical scale. These cells weretested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing.

Results: gd T-cell proliferation was dependent upon CD137L expression on aAPC and addition ofexogenous IL2 and IL21. Propagated gd T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3,TRDV5, TRDV7, and TRDV8with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9�A1, TRGV10�A1, and TRGV11TCR chains. IFNg production by Vd1, Vd2, and Vd1negVd2neg subsets was inhibited by pan-TCRgd antibodywhen added to cocultures of polyclonal gd T cells and tumor cell lines. Polyclonal gd T cells killed acute andchronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic

normal B cells. Blocking antibodies demonstrated that polyclonal gd T cells mediated tumor cell lysisthrough combination of DNAM1, NKG2D, and TCRgd. The adoptive transfer of activated and propagatedgd T cells expressing polyclonal versus defined Vd TCR chains imparted a hierarchy (poly-clonal>Vd1>Vd1negVd2neg>Vd2) of survival of mice with ovarian cancer xenografts.Conclusions: Polyclonal gd T cells can be activated and propagated with clinical-grade aAPCs and

demonstrate broad antitumor activities, which will facilitate the implementation of gd T-cell cancerimmunotherapies in humans. Clin Cancer Res; 20(22); 5708–19. �2014 AACR.

IntroductionHuman gd T cells exhibit an endogenous ability to

specifically kill tumors and hold promise for adoptiveimmunotherapy. They have innate and adaptive qualitiesexhibiting a range of effector functions, including cytolysisupon cell contact (1, 2). Recognition and subsequent killingof tumor is achieved upon ligation of antigens to hetero-dimers of g and d T-cell receptor (TCR) chains. The humanTCR variable (V) region defines 14 unique Vg alleles(TRGV), 3 unique Vd alleles (TRDV1, TRDV2, and TRDV3),and 5 Vd alleles that share a common nomenclature with

Va alleles (TRDV4/TRAV14, TRDV5/TRAV29, TRDV6/TRAV23, TRDV7/TRAV36, and TRDV8/TRAV38-2; ref. 3).T cells expressing TCRa/TCRb heterodimers composeapproximately 95% of peripheral blood T cells and recog-nize peptides in the context of MHC (4). In contrast, TCRgdligands are recognized independent of MHC and these cellsare infrequent (1%–5% of T cells) in peripheral blood(1, 5, 6). Many conserved ligands for TCRgd are present oncancer cells, thus an approach to propagating these T cellsfrom small starting numbers while maintaining a polyclon-al repertoire of gd TCRs has appeal for human application.

Clinical trials highlight the therapeutic potential of gd Tcells, but numeric expansion is needed for adoptive immu-notherapy because they circulate at low frequencies inperipheral blood. Methods to propagate ab T cells, for e.g., using interleukin-2 (IL2) and/or antibody cross-linkingCD3, cannot sustain proliferation of gd T cells (7, 8).Aminobisphosphonates, for e.g., zoledronic acid, havebeenused to initiate a proliferative signal in gd T cells (5, 9), butonly one lineage of gd T cells, expressing Vg9Vd2 TCR, canbe reliably expanded by zoledronic acid. The adoptivetransfer of Vg9Vd2 T cells has yielded clinical responses forinvestigational treatment of solid and hematologic cancers(10–14). Furthermore, long-term remission of leukemiaamong recipients of haploidentical ab T-cell–depleted

Authors' Affiliations: Departments of 1Pediatrics, 2Cancer Biology,3Experimental Therapeutics, and 4Stem Cell Transplantation and CellularTherapy, University of TexasMD Anderson Cancer Center; and 5Universityof Texas Graduate School of Biomedical Sciences at Houston, Houston,Texas

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Laurence J.N. Cooper, Department of Pediatrics,University of TexasMDAndersonCancerCenter, Unit 907, 1515HolcombeBlvd., Houston, TX 77030. Phone: 713-563-3208; Fax: 713-792-9832;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-3451

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(22) November 15, 20145708

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hematopoietic stem cell transplantation (HSCT) correlatedwith increased frequency of engrafted donor-derived Vd1cells (8, 15–17).However, direct administration of Vd1 cellsor other non-Vg9Vd2 cell lineages has yet to be performed.In addition, no reports to date have described the therapeu-tic impact of Vd1negVd2neg cells in cancer immunotherapyand this subset has not been directly compared with T cellsexpressing Vd1 and Vd2 TCRs. Thus, there are significantgaps in the knowledge and human application of non-Vg9Vd2 lineages.Given that gd T cells have endogenous anticancer

activity, such as against K562 cells (8, 18), we hypothe-sized that malignant cells would serve as a cellular sub-strate to propagate polyclonal gd T cells. K562 cells havebeen genetically modified to function as artificial antigen-presenting cells (aAPC) to ex vivo activate and numericallyexpand ab T cells and NK cells (19–23). We determinedthat IL2, IL21, and g-irradiated K562-derived aAPCs (des-ignated clone #4, genetically modified to coexpress CD19,CD64, CD86, CD137L, and a membrane-bound muteinof IL15 (mIL15); used in selected clinical trials at MDAnderson Cancer Center, Houston, TX) can sustain theproliferation of gd T cells with polyclonal TCR repertoire.Polyclonal gd T cells exhibited broad tumor reactivity anddisplayed a multivalent response to tumors as evidencedby the ability of separated Vd subpopulations to kill andsecrete cytokine against the same tumor target. Further-more, killing by polyclonal populations was multifacto-rial being mediated through DNAM1, NKG2D, andTCRgd. Tumor xenografts were eliminated by both poly-clonal and distinct gd T-cell subsets, and mice treated withpolyclonal gd T cells had superior survival. Given theavailability of aAPCs as a clinical reagent, trials can, forthe first time, evaluate polyclonal populations of gd Tcells as a cancer immunotherapy.

Materials and MethodsCell lines

HCT-116, Kasumi-3, and K562 were acquired fromATCC. Jurkat was purchased fromDeutsche Sammlung vonMikroorganismen und Zellkulturen. cALL-2 and RCH-ACVwere gifts from Dr. Jeff Tyner (Oregon Health & ScienceUniversity, Portland,OR). BxPC-3,MiaPaCa-2, and Su8686(pancreatic cancer) were donated by Dr. Vijaya Ramachan-dran (MD Anderson Cancer Center). A2780, CAOV3,EFO21, EFO27, Hey, IGROV1, OAW42, OC314, OVCAR3,and UPN251 (ovarian cancer) were provided by Dr. RobertC. Bast, Jr. (MD Anderson Cancer Center, Houston, TX).Identities of all cell lines were confirmed by STR DNAFingerprinting at MD Anderson Cancer Center’s "Charac-terized Cell Line Core" and cells were used within 6monthsof authentication.

Propagation of gd T cellsPeripheral blood mononuclear cells (PBMC) and umbil-

ical cord blood (UCB) were isolated from healthy volun-teers by Ficoll-Hypaque (GE Healthcare) after informedconsent (24). Thawed PBMCs (108) were initially treatedwithCD56microbeads (cat#130-050-401,Miltenyi Biotec)and separated on LS columns (cat# 130-042-401, MiltenyiBiotec) to deplete NK cells from cultures because theyproliferate on aAPCs (23) and would contaminate thepurity of the gd T-cell product. Unlabeled cells from CD56depletion sorting were then labeled with TCRg/dþ T-cellisolation kit (cat# 130-092-892,Miltenyi Biotec) andplacedon LS columns to separate gd T cells in the unlabeledfraction from other cells attached to magnet. gd T cellswere cocultured at a ratio of one T cell to two g-irradiated(100 Gy) aAPCs (clone #4) in presence of exogenous IL2(Aldesleukin; Novartis; 50 IU/mL), and IL21 (cat#AF20021; Peprotech; 30 ng/mL) in complete media (CM;RPMI, 10% FBS, 1% Glutamax). Cells were serially resti-mulated with addition of g-irradiated aAPCs every 7 daysfor 2 to 5weeks in presence of soluble cytokines,whichwereadded three times per week beginning the day of aAPC add-ition. K562 were genetically modified to function as aAPCs(clone #4) as previously described (25, 26). Validation ofcoexpression of CD19, CD64, CD86, CD137L, and eGFP(IL15 peptide fused in frame to IgG4 Fc stalk and coexpressedwith eGFP) on aAPC clone #4 was performed before addi-tion to T-cell cultures (25). Fluorescence-activated cell sorting(FACS) was used to isolate Vd1 (TCRd1þTCRd2neg), Vd2(TCRd1negTCRd2þ), and Vd1negVd2neg (TCRd1negTCRd2neg)populations, which were stimulated twice as above withaAPC clone #4, phenotyped, and used for functional assays.gd T cells from UCB were isolated by FACS from thawedmononuclear cells using anti-TCRgd and anti-CD3 mono-clonal antibodies (mAb) and were stimulated for 5 weeks onaAPCs/cytokines as per PBMCs.

Abundance and identity of mRNA molecules by DTEAAt designated times after coculture on aAPCs, T cells were

lysed at a ratio of 160 mL RLT buffer (Qiagen) per 106 cellsand frozen at �80�C. RNA lysates were thawed and

Translational Relevancegd T cells have anticancer activity, but only one subset,

Vg9Vd2, has been harnessed for immunotherapy. Ourstudy establishes that artificial antigen-presenting cells(aAPC), IL2, and IL21 can activate and propagate gd Tcells with polyclonal T-cell receptor repertoire to clinicalscale. The heterogeneous population of gd T cells pro-duced from ex vivo culture secreted proinflammatorycytokines, lysed a broad range of malignancies, andimproved survival in an ovarian cancer xenograft model.Given that gd T cells are not thought to recognize ligandsin the context ofMHC, there is limited risk of graft versushost disease in an allogeneic setting. Thus, third party gdT cells from an unrelated (healthy) donor could beproduced in bulk and be administered as an off-the-shelf investigational therapy for hematologic and solidtumors. The aAPCs are already available as a clinicalreagent, which will facilitate the human application ofpolyclonal gd T cells.

Polyclonal Gamma Delta Therapy for Cancer

www.aacrjournals.org Clin Cancer Res; 20(22) November 15, 2014 5709




immediately analyzed using nCounter Analysis System(NanoString Technologies) with "designer TCR expressionarray" (DTEA), as previously described (27, 28). DTEA datawere normalized to both spiked positive control RNA andhousekeeping genes (ACTB, G6PD, OAZ1, POLR1B,POLR2A, RPL27, RPS13, and TBP). Spiked positive controlnormalization factor was calculated from the average ofsums for all samples divided by the sum of counts for anindividual sample. Spiked positive control normalizationfactor was calculated from the average of geometric meansfor all samples divided by the geometric mean for anindividual sample. Normalized counts were reported.

Flow cytometryCells were phenotyped with antibodies detailed in Sup-

plementary Table S1. Gating strategy is displayed inSupplementary Fig. S1. Samples were acquired on FACSCalibur (BD Biosciences) and analyzed with FlowJo soft-ware (version 7.6.3).

Cytokine production and cytolysis assaysExpression of cytokines was assessed by intracellular

staining and secretion of cytokines into tissue culture super-natants was evaluated by Luminex multiplex analysis.In vitro specific lysis was assessed using a standard 4-hourCRA, as previously described (25). Additional informationcan be found in the Supplementary Materials andMethods.

Mouse experimentsIn vivo antitumor efficacy was assessed in NSG mice

(NOD.Cg-PrkdcscidIl2rg tm1Wjl/SzJ; Jackson Laboratories).CAOV3 ovarian cancer cell line was transducedwith recom-binant lentivirus (Supplementary Fig. S2) encoding mKatered fluorescence protein (29) to identify transduced cellsand enhanced firefly luciferase (effLuc) for noninvasive bio-luminescence imaging (30). CAOV3-effLuc-mKate (clone1C2; 3 � 106 cells/mouse) tumors were established byintraperitoneal injection and mice were randomly distrib-uted into treatment groups. Eight days later (designated day0), a dose escalation regimen was initiated with gd T cellsand PBS (negative control) administered intraperitoneally.T-cell doses were 3 � 106, 6 � 106, 107, and 1.5 � 107 ondays 0, 7, 14, and 21, respectively. Noninvasive biolumi-nescence images (BLI) was performed during the course ofthe experiments to serially measure tumor burden ofCAOV3-effLuc-mKate following subcutaneous administra-tion of D-Luciferin (cat#122796, Caliper) as detected withIVIS-100 Imager (Caliper). BLI was analyzed using LivingImage software (version 2.50, Xenogen, Caliper).

ResultsEx vivo numeric expansion of gd T cells on aAPCsdepends on costimulation and cytokines

The adoptive transfer of gd T cells requires ex vivo prop-agation as starting numbers fromPBMCs are limiting (gatingon lymphocyte pool: 3.2% � 1.2%; mean � SD; n ¼ 4;Fig. 1A). gd T cells from PBMCs were isolated by "negative"paramagnetic bead selection and cocultured for 22 days

with weekly addition of g-irradiated K562-derived aAPCs(clone #4) in the presence of soluble recombinant IL2 andIL21 in alignment with protocols at MD Anderson CancerCenter (Houston, TX) for propagation of clinical-grade ab Tcells. This resulted in the outgrowth of a population of T cellshomogeneously coexpressing CD3 and TCRgd (97.9% �0.6%). NK cells (CD3negCD56þ) and ab T cells (TCRabþ)were absent from these cultures supporting the purity ofthe gd T-cell product. Populations of TCRd1þTCRd2neg,TCRd1negTCRd2þ, and TCRd1negTCRd2neg were detectedindicating that aAPCs, IL2, and IL21 supported polyclonalgd T-cell proliferation (Fig. 1A far right). Cells were activatedas marked by expression of CD38 (93.5% � 3.5%) andCD95 (99.7% � 0.1%; Supplementary Fig. S3). Thisapproach to propagation yielded >109 gd T cells from

could increase the number of ligands targeted by theengrafted cells and result in long-term engraftment in therecipient (31). However, the limited number of mononu-clear cells within a banked UCB unit curtails the number ofneonatal gd T cells directly available for adoptive transfer.Thus, we evaluated whether aAPCs could sustain prolifer-ation from small starting numbers of neonatal gd T cells.Fluorescence-activated cell sorting (FACS) was used toisolate 104 UCB-derived gd T cells (�0.01% of a typicalUCB unit) which were cocultured on aAPC clone #4 withIL2 and IL21. After 35 days, there was a 107-fold increase incell number, as an average of 1011 UCB-derived gd T cells(range: 6� 109–3� 1011; n¼ 5) were propagated from the104 initiating gd T cells (Supplementary Fig. S5A). Twoadditional stimulations were performed for gd T cellsderived fromUCBcomparedwithPBMCshighlighting theirpotential for proliferating to clinically appealing numbers.The propagated gd T-cell populations exhibited uniformcoexpression of CD3 and TCRgd and lacked TCRabþ T cellsor presence of CD3negCD56þ NK cells (Supplementary Fig.S5B–S5D). Collectively, these data demonstrate that aAPCclone #4 with IL2 and IL21 could sustain the ex vivoproliferation of gd T cells from a small starting populationof neonatal UCB.

Ex vivo activated and propagated gd T cells expresspolyclonal and defined TCRgd repertoireUpon establishing that gd T cells could numerically

expand on aAPCs and selected cytokines, we sought to

determine the TCR repertoire of the propagated cells. Beforenumeric expansion, resting gd T-cell repertoire followedTCRd2>TCRd1negTCRd2neg>TCRd1 by flow cytometry(Supplementary Fig. S6). However, the gd T-cell repertoirefollowed TCRd1>TCRd1negTCRd2neg>TCRd2 followingexpansion, suggesting that there was a proliferative advan-tage for Vd1 cells within polyclonal gd T-cell cultures. Tolook more in-depth at TCRgd diversity in aAPC-expandedgd T cells, we adapted a nonenzymatic digital multiplexassay used to quantify the TCR diversity in gd T cellsexpressing a CD19-specific chimeric antigen receptor (CAR;ref. 27) termed DTEA. After expansion (day 22), 4 of 8 Vdalleles (TRDV1, TRDV2-2, TRDV3, and TRDV8) weredetected in PBMC-derived gd T cells (Fig. 2A) and werecoexpressed with Vg alleles TRGV2, TRGV7, TRGV8 (twoprobes), TRGV9�A1, TRGV10�A1, and TRGV11�02 (Fig.2B). Similarly, a polyclonal assembly of Vd and Vg chainswas observed in gd T cells from UCB following expansion(days 34–35), albeit with reduced abundance of TRDV2-2,more TRGV2, and presence of TRGV3F, TRDV5, and TRDV7cells not seen fromPBMCs (Fig. 2C andD). Similar patternsof Vd andVgmRNAusagewere detected in PBMCs andUCBbefore and after expansion (Supplementary Fig. S7)although overall mRNA counts were fewer in the restingcells (day 0) relative to the activated gd T cells. Thus, aAPC-expanded gd T cells maintain a polyclonal TCR repertoirefrom both PBMCs and UCB.

We sought to validate these mRNA data by sorting poly-clonal populations with TCRd-specific antibodies and

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Figure 1. Sustained proliferation of polyclonal PBMC-derived gd T cells on g-irradiated aAPCs in the presence of soluble IL2 and IL21. A, frequency of gd T cellsbefore (day 0) and after (day 22) coculture on g-irradiated aAPCs, IL2, and IL21 where expression of CD3, CD56, TCRab, TCRgd, TCRd1, and TCRd2is shown at day 22 of coculture. One of 7 representative donors is shown. Quadrant frequencies (percentage) within flow plots are displayed in top rightcorners. B, inferred cell counts of polyclonal gd T cells are displayed calculated on the basis of weekly yields and relative fold changes, where threearrows represent addition of aAPCs. Black line, mean � SD (n¼ 4) pooled from 2 independent experiments and each gray line is an individual donor. C, foldincrease over 9 days of gd T cells cocultured with IL2 and IL21 along with aAPC expressing membrane-bound IL15 (mIL15), CD86, and/or CD137L.Data, mean � SD (n ¼ 3) pooled from two independent experiments and each shape represents an individual donor. Two-way ANOVA with Bonferroniposttestswas used for statistical analysis. �,P < 0.05; ��,P < 0.01. D, fold increase over 9 days of gd T cells coculturedwith aAPCs (clone #4) in the presence ofeither soluble recombinant IL2 and/or IL21. Data are mean � SD (n ¼ 3) pooled from two independent experiments where each shape represents anindividual donor. Two-way ANOVA with Bonferroni post tests was used for statistical analysis. �, P < 0.05.






repeatingDTEAon isolatedcultures.Thereareonly twoTCRd-specific mAbs commercially available and they identifiedthree discrete Vd populations (Vd1: TCRd1þTCRd2neg, Vd2:TCRd1negTCRd2þ, and Vd1negVd2neg: TCRd1negTCRd2neg)within aAPC-expanded gd T cells from PBMCs (Fig. 1A) andUCB (Supplementary Fig. S8) with abundance follow-ing Vd1>Vd1negVd2neg>Vd2. FACS-isolated subsets fromPBMC-derived gd T-cell pools were propagated with clone#4 as discrete populations and maintained their identity asassessed by expression of TCRd isotypes (Fig. 3A). Each of theseparated subsets could be identified by a pan-specific TCRgdantibody confirming that these cells were indeed gd T cells(Fig. 3B). Furthermore, each population could be differenti-ated based on pan-TCRgd antibodymean fluorescence inten-sity (MFI) where Vd2, Vd1negVd2neg, and Vd1 T cells corre-sponded to the TCRgdlow (43 � 9; mean � SD; n ¼ 4),TCRgdintermediate (168� 40), and TCRgdhi (236� 56) group-ings, respectively. No differences in proliferation kinetics on

aAPCs were observed between isolated Vd-sorted subsets(Fig. 3C) indicating that the observed inversion of Vd1and Vd2 frequencies in polyclonal cultures before versusafter expansion was not due to a proliferative defect inone of the subsets. DTEA demonstrated that isolated andpropagated Vd1, Vd2, and Vd1negVd2neg subpopulationswere homogeneous populations as they predominantlyexpressed TRDV1, TRDV2-2, and TRDV3 mRNA speciesat 261 � 35, 3,910 � 611, and 5,559 � 1119 absolutecounts, respectively (Fig. 3D). Therefore, there werefewer TRDV1 mRNA species expressed by Vd1 cellsrelative to the TRDV2-2 expressed by Vd2 cells andTRDV3 expressed by Vd1negVd2neg cells. Moreover, thesedata indicated that the relatively low counts observed forTRDV1 in polyclonal populations with a preponderanceof TCRd1þ cells was not a defect in DTEA detection butrather a product of fewer total mRNA transcripts relativeto other Vd species. Given the wide range of mRNA

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Deniger et al.

Clin Cancer Res; 20(22) November 15, 2014 Clinical Cancer Research5712




transcript quantities for each allele, DTEA was not usefulfor calculation of relative frequencies of Vd subsets inpolyclonal populations but rather was indicative ofpresence or absence of a particular gd T-cell subset.Expression of other Vd2 alleles (TRDV2-1 and TRDV2-1F) was absent from polyclonal gd T cells (Fig. 2A) andeach of the sorted subsets (data not shown). Smallamounts of TRDV4, TRDV5, TRDV6, and TRDV7 mRNAspecies were detected in the three subsets of T cellssorted for Vd expression (Supplementary Fig. S9).TRDV8 mRNA was exclusively present in sortedVd1negVd2neg cells and these T cells are likely the maincontributors of TRDV8 in bulk gd T cells. The same VgmRNA present in polyclonal cultures was detected in Vd-sorted cultures (Supplementary Fig. S10). Furthermore,Vd1 and Vd1negVd2neg were not different (P ¼ 0.419;two-way ANOVA) but Vd2 was different to both Vd1 (P <0.0001) and Vd1negVd2neg (P < 0.0001) in Vg usage.Collectively, these results confirmed DTEA from unsort-ed cultures and strongly supported the polyclonal TCRgdexpression on gd T cells activated to proliferate by aAPCsand cytokines.

IFNg produced in response to tumors is dependent onTCRgd

A multiplex analysis of cytokines and chemokines wasperformed to determine whether aAPC-propagated gd Tcells might foster a proinflammatory response in a tumormicroenvironment (Fig. 4A). The Th1-associated cytokinesIFNg and TNFa were secreted in abundance by gd T cellsupon exposure to leukocyte-activated cocktail (LAC; PMAand ionomycin for nonspecific mitogenic stimulation), inaddition to small amounts of IL2 and IL12 p70. In contrast,no significant production of the Th2-associated cytokinesIL4, IL5, and IL13was observed from LAC-treated gd T cells,but there was a small increase in IL10 production overbaseline. Similarly, Th17-associated cytokines IL1RA, IL6,and IL17 were secreted at low levels by LAC-treated gd Tcells. The chemokines CCL3, CCL4, CCL5, andCXCL8weredetected in abundance. Minor contributions of non-gd Tcells in the culture that could have been activated by LAC tosecrete cytokines could not be ruled out, but given that thecells tested were 97.9% � 0.6% CD3þTCRgdþ these dataindicate that itwas activationof gd T cells that led to a largelyproinflammatory response. IFNg was the most responsive

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Figure 3. Sustainedproliferation of PBMC-derived Vd T-cell subsets expandedon g-irradiated aAPCs/IL2/IL21. After two7-day stimulationswith aAPCs (clone#4) and IL2/IL21, the bulk population of gd T cells was separated into Vd1, Vd2, and Vd1negVd2neg subsets by FACS based on staining of T cells defined asTCRd1þTCRd2neg, TCRd1negTCRd2þ, and TCRd1negTCRd2neg, respectively. A, expression of TCRd1 and TCRd2 chains on Vd1, Vd2, and Vd1negVd2neg

subsets of gd Tcells (from left to right) after 15 daysof numeric expansion on aAPCsandcytokines as isolated groups.Oneof 4 representative donors is shownpooled from two independent experiments. Quadrant frequencies (percentage) within flow plots are displayed in top right corners. Frequency ofTCRd1þTCRd2neg (open bars), TCRd1negTCRd2þ (black bars), and TCRd1negTCRd2neg (gray bars) cell surface protein expression in subsets of gd T cells after15 days numeric expansion on aAPCs and cytokines as isolated groups. Data are mean � SD (n ¼ 4) pooled from two independent experiments.B, flow cytometry plots of CD3 and TCRgd expression in Vd1, Vd2, and Vd1negVd2neg subsets (from left to right). Mean fluorescence intensity (MFI) ofTCRgd staining in Vd1, Vd2, and Vd1negVd2neg T-cell subsets where each shape represents a different donor and data are mean � SD (n ¼ 4) pooledfrom two independent experiments. C, proliferation of each isolated Vd subset stimulated twicewith aAPCclone #4 (arrows) in presence of cytokines and totalcell counts are displayed. Data are mean � SD (n ¼ 4) pooled from 2 independent experiments. D, DTEA was used to identify and measure abundanceofmRNA species coding for TRDV1, TRDV2-2, and TRDV3 (from left to right) in gd T-cell subpopulations after 15 days of proliferation on aAPCs and cytokinesas separated subsets. Box and whiskers plots display 25% and 75% percentiles where lines represent maximum, mean, and minimum from top to bottom(n ¼ 4). Student paired, two-tailed t tests were undertaken for statistical analyses between groups. ��, P < 0.01; ��, P < 0.001.






of all the assessed cytokines and was chosen to measureresponses ofVd subsets to tumor cells (Fig. 4B).Coculture ofpolyclonal aAPC-propagated/activated gd T cells with can-cer cells resulted in a hierarchy of IFNg production follow-ing Vd2>Vd1>Vd1negVd2neg as shown byMFI of 855� 475,242� 178, and 194� 182 (mean� SD; n¼ 4), respectively.IFNg production by Vd1, Vd2, and Vd1negVd2neg subsetswas inhibited by pan-TCRgd antibody when added to gdT-cell/tumor cocultures indicating that response to thetumor in each subset was dependent upon activationthrough TCRgd (Fig. 4C). This observation supported thepremise that a single cancer cell could be targeted bydiscrete gd TCRs. Thus, a multivalent proinflammatoryresponse to the tumor cell was achieved by polyclonal gdT cells.

Polyclonal gd T cells lyse a broad range of tumor cellsthrough combination of DNAM1, NKG2D, and TCRgd

After establishing that propagated gd T cells could beactivated to produce proinflammatory cytokines, we exam-ined their ability to specifically lyse a panel of tumor celllines. Polyclonal gd T cells demonstrated a range of cytolysisagainst solid and hematologic cancer cell lines without aclear preference towards a particular tumor histology orgrade (Fig. 5 and Supplementary Fig. S11). We previouslyestablished that B-cell acute lymphoblastic leukemia (ALL)cell line NALM-6 was largely resistant to lysis by gd T cells,

which required a CD19-specific CAR to acquire significantkilling capability (27). In this study, it was also observedthat autologous and allogeneic normal B cells were sparedfrom cytolysis (Fig. 5A), and that B-ALL cell line cALL-2 andmurine T-cell lymphoma cell line EL4 were lysed poorly bypolyclonal gd T cells, which indicated that some cells wereresistant and/or not recognized by polyclonal gd T cells. Incontrast, T-ALL cell line Jurkat and B-ALL cell lines RCH-ACV were both killed efficiently by polyclonal gd T cells(Fig. 5B), indicating that gd T cells could be used to targetsome B-cell and T-cell malignancies. Kasumi-3 is a CD33þ

CD34þ undifferentiated leukemia cell line that was lysedat intermediate levels by gd T cells. Chronic myelogenousleukemia (CML) cell line K562 and K562-derived clone#4aAPCs were killed by polyclonal gd T cells, which corrob-orated the notion that these cells could serve as a prolifer-ative substrate. Pancreatic cancer cell lines BxPc-3, Mia-PaCa-2, and Su8686, were lysed by gd T cells, as was thecolon carcinoma cell line HCT-116 (Fig. 5C). Ovarian celllines were killed by polyclonal gd T cells in the followingorder of decreasing sensitivity: CAOV3 > EFO21 >UPN251> IGROV1 >OC314 >Hey > A2780 >OVCAR3 >OAW42 >EFO27. Each of the separated Vd subsets lysed hematologic(Jurkat and K562) and solid (OC314 and CAOV3) tumorcell lines, which showed that polyclonal gd T cells coulddirect a multivalent response against common targets (Sup-plementary Fig. S12). The strength of cytolysis followed the

Figure 4. Dependence on TCRgd for IFNg secretion in response to tumor cells. At day 22 of coculture on g-irradiated aAPCs (clone #4) with IL2 and IL21, T cellswere incubated with media (mock) or leukocyte activation cocktail (LAC; PMA/Ionomycin) for 6 hours at 37�C. Tissue culture supernatants were interrogatedusing 27-Plex Luminex array to detect presence of (A) Th1, Th2, and Th17 cytokines and selected chemokines (from left to right). Data are mean � SDpooled from4donors in two independent experimentswhere eachdonor had triplicate experimentalwells pooledbeforemultiplexanalysis. Student one-tailedt test performed for statistical analysis between mock and LAC groups. �, P < 0.05; ��, P < 0.01; and ��, P < 0.001. B, polyclonal gd T cells were incubatedfor 1 hour before and during 6-hour tumor cell coculture with normal mouse serum or neutralizing TCRgd antibody (clone IM). Cells were stained forTCRd1, TCRd2, CD3, and IFNg to gate T-cell subsets and assess IFNg production. Comparisons of histograms detailing Vd1, Vd2, and Vd1negVd2neg gates(from left to right) cocultured with CAOV3 ovarian cancer cells and treated with serum (open) or TCRgd (shaded). Numbers next to histograms are MFI.Flow plots are representative of 1 of 3 peripheral blood donors cocultured with CAOV3 cells in 2 independent experiments. C, percent inhibition ofIFNg secretion in response toCAOV3cellswascalculated for eachVd T-cell subset basedon the followingequation: Inhibition (%)¼100�100� [(MFITUMORþT CELL � MFIT CELL ONLY)TCRgd/(MFITUMOR þ T CELL � MFIT CELL ONLY)Serum]. Data are mean � SD (n ¼ 3) pooled from two independent experiments.

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hierarchy of TCR usage (Vd2>Vd1negVd2neg>Vd1) that wasconsistent with the premise that a propensity to betriggered for effector function would increase with T-celldifferentiation (Supplementary Fig. S13). Lysis by poly-clonal populations was apparently not due to one specificVd subtype but rather from contributions of multiple gdT-cell subsets, because it was observed that (i) a numberof tumor cell lines were equivalently killed by polyclonalgd T cells containing different frequencies of Vd1, Vd2,and Vd1negVd2neg cells and (ii) a polyclonal populationwas not identified with dominant cytolysis. We alsosought to determine which surface molecules wereresponsible for cytolysis by blocking immunoreceptors

with antibodies (Fig. 5D). Our experimental approachalso took into account that gd T cells coexpress DNAM1(97.7% � 0.9%; mean � SD; n ¼ 4) and NKG2D (40.1%� 16.5%) which can activate both T cells and NK cells forkilling (32, 33). Addition of individual antibodies did notreduce lysis, except for TCRgd in 2 of 3 cell lines tested. Incontrast, a pool of antibodies binding NKG2D, DNAM1,TCRgd resulted in significant inhibition, in a dose-depen-dent manner, of gd T-cell–mediated cytolysis against all 3targets. Collectively, these data established that ex vivo-propagated gd T cells have broad antitumor capabilitieslikely mediated by activation though DNAM1, NKG2D,and TCRgd.

Figure5. Specific lysis of tumor cell panel bypolyclonal gd Tcells. A–C, standard4-hourCRAwasperformedwith increasingeffector (polyclonal gd Tcells; eachshape represents a different donor) to target (E:T) ratios against (A) healthyB cells froman allogeneic donor (one of four representative donors); B, hematologictumor cell lines derived from B-ALL: RCH-ACV, T-ALL: Jurkat, and CML: K562; C, solid tumor cell lines derived from pancreatic cancer: BxPc-3, coloncancer: HCT-116, and ovarian cancer: OC314 and CAOV3. Data are mean � SD (n ¼ 3 wells/assay) from two independent experiments. D, neutralizingantibodies to NKG2D (squares), DNAM1 (triangles), TCRgd (inverted triangles), or a pool (diamonds) of all three antibodies were used to block killing ofJurkat (left), IGROV1 (middle), or OC314 (right) tumor targets antibodies at 0.3, 1, and 3 mg/mL and an E:T ratio of 12:1 in standard 4-hour CRA. Normalmouse serum (circles) served as control for addition of antibody and wells without antibody were used for normalization purposes. Specific lysis wasnormalized towellswithout antibody to yield relative cytolysis as definedby: Relative cytolysis (%)¼ (Specific Lysis)With Antibody/(SpecificLysis)Without Antibody�100. Data are mean � SD (n ¼ 4 donors) from triplicates pooled and normalized from two independent experiments. Repeated-measures two-way ANOVAwas used for statistical analysis between antibody treatments. ��, P < 0.01; ��, P < 0.001.






Established ovarian cancer xenografts are eliminatedby adoptive transfer of gd T cells

To test whether polyclonal gd T cells were effective intargeting and killing tumors in vivo, we created a xenograftmodel for ovarian cancer in immunocompromised mice.NSG mice were injected intraperitoneally with CAOV3-effLuc-mKate ovarian cancer cells and then randomizedinto five treatment groups. After 8 days of tumor engraft-ment, either PBS (vehicle/mock), Vd1, Vd2, Vd1negVd2neg,or polyclonal gd T cells were administered in escalatingdoses (Fig. 6). Tumor burden and biodistribution wereserially measured by noninvasive BLI. Established tumorscontinued to grow in vehicle-treated mice, but tumor bio-burden was significantly reduced (P � 0.001) in micereceiving gd T-cell treatments at day 72, relative to theirinitial tumor burden (Fig. 6A and B). Adoptive transfer ofpolyclonal gd T cells, Vd1, and Vd1negVd2neg T cells signif-icantly (P� 0.01), andVd2 almost significantly (P¼0.055),increased long-term survival compared with mock-treated

mice. This corresponded to overall survival following poly-clonal>Vd1>Vd1negVd2neg>Vd2 (Fig. 6C). This is the firsttime that three Vd subsets have been compared for theirability to target tumor in vivo and is the first display of in vivoantitumor activity by Vd1negVd2neg cells. In sum, activatedand propagated gd T cells were effective in treating cancerin vivo and thus represent an attractive approach to adoptiveimmunotherapy.

DiscussionThis study establishes our aAPC clone #4 as a cellular

platform for the sustained proliferation ofmultiple gd T-cellpopulations that demonstrate extensive reactivity againsthematologic and solid malignancies. T cells expressingdefined Vd TCRs have been associated with clinicalresponses against cancer. For example, the Vd1 subsetcorrelated with complete responses observed in patientswith ALL and acute myelogenous leukemia (AML) after ab

Figure 6. In vivo clearance of ovarian cancer upon adoptive transfer of polyclonal gd T cells and gd T-cell subsets propagated/activated on aAPCs with IL2and IL21. CAOV3-effLuc-mKate tumor cells were injected into NSG mice at day -8 and engrafted until day 0 when treatment was started with eitherPBS (vehicle/mock) or gd T cells. Four T-cell doses were administered in weekly escalating doses. A, BLI images at day 0 (top) or day 72 (bottom) in PBS, Vd1,Vd2, Vd1negVd2neg, and polyclonal gd T-cell treatment groups. Images are representative of 6 to 14 mice from two independent experiments. B, BLImeasurements ofmice at day 0 (white) and day 72 (gray) pooled from two independent experiments. Box andwhiskers plots display 25%and75%percentileswhere lines represent maximum, mean, and minimum from top to bottom (n ¼ 6–14). Student paired, two-tailed t tests were used for statistical analysisbetween time points. C, overall survival of mice treated with PBS (dashed), polyclonal (black), Vd1 (red), Vd2 (blue), or Vd1negVd2neg (green) gd T cells.Log-rank (Mantel–Cox) test was used to calculate P values. �, P < 0.05; ��, P < 0.01; and ��, P � 0.001.

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T-cell–depleted haploidentical HSCT (15–17). Vd1 cellswere also shown to kill glioblastoma independent of cyto-megalovirus (CMV) status (34). However, Vd1 cells havenot been directly administered. Our data establish that suchcells could mediate antitumor immunity and supports theadoptive transfer Vd1 T cells for cancer therapy. In contrastto Vd1 and Vd1negVd2neg cells, T cells expressing Vd2 TCRhave been directly infused and elicited responses againstsolid andhematologic tumors (9, 35). Little is known aboutVd1negVd2neg T cells, but these lymphocytes have displayedrecognition of the nonclassical MHC molecule CD1d withcorresponding NKT-like functions and have also been cor-related with immunity to HIV and CMV (36–39). Ourresults are the first to directly show that Vd1negVd2neg cellsexhibit antitumor activities, and given their propensity toengage both viruses and cancer the add-back of this subsetcould especially benefit immunocompromised cancerpatients. Because aAPCs with IL2 and IL21 can propagatepolyclonal gd T cells, mAbs can now be raised against Vd3,Vd5, Vd7, and Vd8 isotypes to help elucidate their potentialroles in clearance of pathogens and cancer. In aggregate, ourdata support the adoptive transfer of gd T cells thatmaintainexpression of multiple Vd TCR types as investigationaltreatment for cancer.The molecules on aAPCs that activate gd T cells for

numeric expansion are not well known. K562-derivedaAPCs express endogenous MHC class-I chain-related pro-tein A and B (MICA/B) which are ligands for both Vd1 andNKG2D (6, 40). Indeed, NKG2D was observed on poly-clonal gd T cells that also predominantly expressed Vd1 TCR(Fig. 1A). Polyclonal gd T cells also demonstrate expressionfor activating receptors typically found onNK cells (NKp30,NKp44, and NKp46; collectively expressed at 26% � 7%),and future studies will examine their contribution to gd T-cell effector function. Somemalignant cells were recognizedpoorly by gd T cells, for e.g., EL4, EFO27, OAW42, cALL-2,and NALM-6, which provides an opportunity to furtherinterrogate the mechanism by which gd T cells recognizeand kill tumor cells. Given that inhibition of cytolysis wasmaximized by neutralizing DNAM1, NKG2D, and TCRgdreceptors simultaneously, it may be that sensitivity of atumor cell resides on the expression of ligand combinationsthat can bind these receptors. Two ligands recognized byVd2 TCR are surface mitochondrial F1-ATPase and phos-pho-antigens, both of which are found in K562 cells (41,42). Enhanced responses of T cells expressing Vg9Vd2 wereobserved when K562 cells were treated with aminobispho-sphonates (41) and a similar strategy could be employedupon coculture with aAPC clone #4 to increase the abun-dance of T cells bearing Vd2 TCR (18). Future studies willevaluate additional TCRgd ligands that naturally occur inthese aAPCs.We enforced expression of costimulatory molecules to

ascertain and improve the capability of K562-derived aAPCsto propagate gd T cells expressing a diversity of TCR. Indeed,CD137L was the dominant costimulatory proliferative sig-nal on aAPCs for expansion of gd T cells with broad tumorreactivity (Fig. 1C), and its receptor, CD137, has been used

to enrich tumor-reactive ab T cells following antigen expo-sure and presumably TCR stimulation (43–45). CD137wasnot expressed on resting gd T cells before expansion, sug-gesting that the importance of CD137L costimulation byaAPCs followed TCR stimulation by the aAPCs and expres-sion ofCD137on the gd T-cell surface. CD27þ andCD27neg

gd T cells have been shown to produce IFNg and IL17 (46),respectively; therefore, CD27 could be used as a marker forisolating gd T cells with a preferred cytokine output. ICOS-ligand in absence of CD86 was shown to polarize CD4þ abT cells to produce IL17 instead of IFNg (47), and currentstudies are investigating whether combinations of costimu-latorymolecules can selectively propagate cytokine-produc-ing subpopulations of gd T cells. Thus, the aAPC coculturesystem in the context of desired cytokines provides a clin-ically relevant methodology to tailor the type of therapeuticgd T cell produced for adoptive immunotherapy.

Our data have implications for the design and inter-pretation of clinical trials. Expression of IL15 was impor-tant for the maintenance of transferred gd T cells in vivo(48), supporting the use of IL15 on aAPCs, and futurestudies could inform on other molecules that could beintroduced to maximize the cell therapy product. Correl-ative studies are enhanced by our observation that TCRgdmAb can be used to readily distinguish the three (Vd1,Vd2, and Vd1negVd2neg) T-cell subsets based on MFI ofTCRgd expression (Fig. 3B). Given that gd T cells are notthought to recognize ligands in the context of MHC (17),there is potential to infuse allogeneic, including thirdparty, gd T cells in lymphodepleted hosts to achieve anantitumor effect while mitigating the risk of graft versushost disease. Restoration of lymphopoiesis may result ingraft rejection, but a therapeutic window could be estab-lished whereby tumors are directly killed by infused gd Tcells, which may result in desired bystander effects asconserved or neoantigens are presented to other lympho-cytes. Indeed, gd T cells have been shown to lyse cancercells, cross-present tumor-specific antigens to ab T cells,and license them to kill tumors (49, 50). The aAPC clone#4 has been produced as a master cell bank in compliancewith current good manufacturing practice and provides aclear path to generating clinical-grade gd T cells forhuman application. Human trials can now, for the firsttime, test the efficacy of adoptive transfer of T cells withpolyclonal TCRgd repertoire for treatment of solid andhematologic tumors.

Disclosure of Potential Conflicts of InterestD. Deniger has, along with L. Cooper, submitted a patent application,

through his institution, to the US patent office for the methods described inthis paper. L. Cooper reports receiving speakers’ bureau honoraria fromMiltenyi Biotec; has ownership interest (includingpatents) inAmerican StemCell and Sangamo Bioscience; and is a consultant/advisory board memberfor Ferring pharmaceuticals and GE Healthcare. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: D.C. Deniger, S. Maiti, R.E. Champlin, L.J.N.CooperDevelopment ofmethodology:D.C. Deniger, S.Maiti, M.H.Huls, D.A. Lee






Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.):D.C.Deniger, T.Mi, K. Switzer, V. Ramachandran,L.V. HurtonAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):D.C.Deniger, K. Switzer, S. Ang,D.A. Lee,R.C. Bast Jr, R.E. ChamplinWriting, review, and/or revision of the manuscript: D.C. Deniger,S.Maiti, B.A. Rabinovich,D.A. Lee, R.C. Bast Jr, R.E. Champlin, L.J.N. CooperAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Maiti, B.A. Rabinovich, R.E.ChamplinStudy supervision: R.C. Bast Jr, L.J.N. CooperProvided reagents: S. Olivares

AcknowledgmentsThe authors thank Dr. Carl June and colleagues (University of Pennsyl-

vania, Pittsburgh, PA) for help generating K562-derived aAPC clone #4 andDr. Perry Hackett (University of Minnesota, Minneapolis, MN) for hisassistancewith the SB system. The authors also thankDrs. GeorgeMcNamara(MD Anderson Cancer Center) and Eric Tran (National Cancer Institute)who edited the text. The ImmuneMonitoringCore Lab (IMCL;MDAndersonCancer Center) who performed Luminex assays and the flow cytometry corelab (MDAnderson Cancer Center) assisted with FACS facility. Cancer CenterSupport Grant (CCGS) supported STR DNA fingerprinting at MD AndersonCancer center. D.C.D. is an American Legion Auxiliary Fellow in CancerResearch (UT-GSBS-Houston), Andrew Sowell-Wade Huggins Scholar in

Cancer Research (Cancer Answers Foundation), and a Teal Pre-doctoralScholar (Department of Defense Ovarian Cancer Research Program).

Grant SupportThis work was supported by funding from Cancer Center Core Grant

(CA16672); RO1 (CA124782, CA120956, CA141303); R33 (CA116127);P01 (CA148600); S10RR026916; Albert J. Ward Foundation; BurroughsWellcome Fund; Cancer Prevention and Research Institute of Texas; CLLGlobal Research Foundation; Department of Defense; Estate of Noelan L.Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr, Fund forLeukemia Immunotherapy.; Fund for Leukemia Immunotherapy; Instituteof Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lym-phoma Research Foundation; Miller Foundation; Mr. Herb Simons; Mr. andMrs. Joe H. Scales; Mr. Thomas Scott; MD Anderson Cancer Center MoonShot; National Foundation for Cancer Research; Pediatric Cancer ResearchFoundation; Production Assistance for Cellular Therapies (PACT); Team-Connor; Thomas Scott; William Lawrence and Blanche Hughes Children’sFoundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 21, 2013; revised April 8, 2014; accepted April 27,2014; published OnlineFirst May 15, 2014.

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2014;20:5708-5719. Published OnlineFirst May 15, 2014.Clin Cancer Res Drew C. Deniger, Sourindra N. Maiti, Tiejuan Mi, et al. Broad Specificity for MalignanciesActivating and Propagating Polyclonal Gamma Delta T Cells with

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