Adoptive Cell Therapy for Lymphoma with CD4 T Cells Depleted of

Therapeutics, Targets, and Chemical Biology

Adoptive Cell Therapy for Lymphoma with CD4 T CellsDepleted of CD137-Expressing Regulatory T Cells

Matthew J. Goldstein, Holbrook E. Kohrt, Roch Houot, Bindu Varghese, Jack T. Lin,Erica Swanson, and Ronald Levy

AbstractAdoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer.

However, T-cell therapy may be limited by the cotransfer of regulatory T cells (Treg). Here, we exploredthis hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells whileexcluding Tregs. In a murine model of B-cell lymphoma, only CD137negCD44hi CD4 T cells infiltrated tumor sitesand provided protection. Conversely, the population of CD137posCD44hi CD4 T cells consisted primarily ofactivated Tregs. Notably, this CD137pos Treg population persisted following adoptive transfer and maintainedexpression of FoxP3 as well as CD137. Moreover, in vitro these CD137pos cells suppressed the proliferation ofeffector cells in a contact-dependentmanner, and in vivo adding theCD137posCD44hi CD4 cells to CD137negCD44hi

CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined apopulation of activated Tregs that greatly limited antitumor immune responses. Consistent with observations inthe murine model, human lymphoma biopsies also contained a population of CD137pos CD4 T cells that werepredominantly CD25posFoxP3pos Tregs. In conclusion, our findings identify 2 surface markers that can be used tofacilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory Treg population. Cancer Res;72(5); 1239–47. �2012 AACR.

IntroductionImmunotherapy for cancer has included nonspecific stim-

ulation of the immune system, active immunization withtumor-specific antigens, and adoptive cell therapy—thetransfer of tumor-specific T cells. We have investigatedadoptive cell therapy for treating lymphoma (1, 2). Histor-ically, adoptive cell therapy for cancer has focused onisolating and expanding populations of T cells that havedirect cytotoxic effects on tumors (3–6). We use activeimmunization to generate antitumor T cells in vivo andtransfer these T cells into lymphodepleted recipient mice.The efficacy of this maneuver is impressive and cures largetumors (1, 2). Specifically, our early work showed that CD8T-cell antitumor immunity can be induced by the combi-nation of cytotoxic chemotherapy with local, intratumoralinjection of CpG (1, 7). Recently, we have extended the use ofCpG as an immunotherapy by exposing tumor B cells to CpGex vivo and subsequently injecting them into the host as awhole-tumor cell vaccine (2). This approach obviates the

need for an accessible, injectable, tumor site. Importantly,vaccination with such CpG-loaded tumor B cells induces atumor-specific CD4 and not CD8 T-cell response. Relativelysmall numbers of these CD4 T cells were sufficient to curelarge and established lymphoma tumors.

This and other recent reports have suggested that CD4 Tcells can be effective in adoptive immunotherapy (2, 8–11).However, a relevant concern in using CD4 T cells for adoptivetherapy is the potential for cotransfer of regulatory T cells(Treg). Here, we have identified 2 surface markers—CD44 andCD137—that can be used to isolate a population of antitumorCD4 T cells while excluding a population of inhibitory Tregs.Adoptive transfer of CD137negCD44hi CD4 T cells providedsignificant protection from B-cell lymphoma. CD137posCD44hi

CD4 T cells were predominantly Tregs and suppressed botheffector cell proliferation and antitumor activity.

Materials and MethodsReagents

CpG 1826 with sequence 50-TCCATGACGTTCCTGACGTTwas provided by Coley Pharmaceutical Group (Ottawa,Canada). Fluorescein isothiocyanate–conjugated CpG 1826was purchased from InvivoGen. The following monoclo-nal antibodies (mAb) were used for flow cytometry: anti-mouse CD3, anti-mouse CD4, anti-mouse CD8, anti-mouseCD44, anti-mouse CD137, anti-mouse IFN-g , anti-mouse CD25,anti-mouse FoxP3, anti-mouse CD62L, anti-mouse CCR7, anti-mouseCD103, anti-mouseCD69, anti-mouseGITR, anti-mouseThy1.1, anti-mouse CD45.1, anti-human CD3, anti-humanCD4, anti-human CD8, anti-human CD45RO, anti-human

Authors' Affiliation: Department of Medicine, Stanford University Schoolof Medicine, Stanford, California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Ronald Levy, Chief, Division of Oncology, 269Campus Drive, CCSR 1105, Stanford, CA 94305. Phone: 650-725-6452;Fax: 650-736-1454; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3375

�2012 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst January 9, 2012; DOI: 10.1158/0008-5472.CAN-11-3375

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CD137, anti-human CD25, anti-human FoxP3, and isotype con-trol. Antibodies were purchased from either Becton Dickinson(BD) Biosciences or eBioscience.

Cell lines and miceH11 is a pre-B cell line in the C57BL/6 background generated

as follows: primary bone marrow cells were isolated fromC57BL/6 mice and infected with the retrovirus vectorMSCV-neo/p190Bcr-Abl, which carries the oncogene Bcr-Abl(12; a gift from Drs. M. Cleary and K. Smith, Stanford School ofMedicine). Tumor cells were cultured in complete RPMI 1640(cRPMI) medium (Invitrogen Life Technologies) containing10% fetal calf serum (Thermo Scientific), 100 U/mL penicillin,100 mg/mL streptomycin (both from Invitrogen Life Techno-logies), and 50 mmol/L 2-ME (Sigma-Aldrich). This cell line waspreviously used in studies from our laboratory (2) and wastested for uniformity of cell-surface markers (B220posCD43neg

IgMnegIgDneg) by fluorescence-activated cell sorting (FACS).Six- to 8-week-old female Thy1.1, CD45.1, and wild-typeC57BL/6J mice were purchased from Jackson Laboratories.All studieswere approved by the StanfordAdministrative Panelon Laboratory Animal Care.

Tumor inoculation and animal studiesH11 tumor cells were incubated in the presence of 3 mg/mL

of CpG at 37�C and 5% CO2. As shown previously, CpG is takenup by H11 cells during this incubation (2). After 24 hours, cellswere vigorouslywashed 3 timeswithwash buffer to remove anyunbound, residual CpG. H11 cells that were loaded with CpG(CpG/H11) were irradiated at 50 Gy and used to vaccinateC57BL/6 donormice s.c. for 5 consecutive days. Onday 13, bonemarrow and splenocytes of donor mice were transferred by i.v.injection into irradiated C57BL/6 recipient mice (9.5Gy TBI,Phillips x-ray unit, 250 kV, 15mA) along with 1� 106 irradiatedCpG/H11 tumor cells as a posttransplant "booster" vaccinethat was prepared in the same fashion as above (1). In studiesusing congenic donors, transferred bone marrow was fromwild-type, noncongenic mice to allow precise tracking oftransplanted donor cells. Recipient mice were challenged withH11 tumor cells s.c. at a dose of 1� 107 cells in 50 mL of serum-free RPMI on day 16. Tumor growth was monitored by calipermeasurement (Fig. 1A).

In vitro assaysIFN-g production assay. Peripheral blood mononuclear

cells (PBMC) were collected from tail vein, anticoagulatedwith 2 mmol/L ethylenediaminetetraacetic acid in PBS, thendiluted 1:1 with Dextran T500 (Pharmacosmos) 2% in PBSand incubated at 37�C for 45 minutes to precipitate red cells.Leukocyte-containing supernatant was removed and centri-fuged, and remaining red cells were lysed with ammoniumchloride potassium buffer (Quality Biological). PBMCs werethen cocultured with 1 � 106 irradiated H11 cells for 24hours with 0.5 mg anti-mouse CD28mAb (BD PharMingen)and in the presence of monensin (Golgistop; BD Biosciences)for the last 5 hours at 37�C and 5% CO2. Intracellular IFN-gexpression was assessed with BD Cytofix/Cytoperm Plus Kitper instructions.

Treg suppressionassay. CD137pos andCD137neg cell popu-lations were isolated by FACS and mixed at various ratios in a96-well roundbottomplate. Cellswere stimulated inwith equalnumbers of polystyrene latex beads (Interfacial Dynamics)coated with 1.0 mg/mL anti-CD3 (145-2C11; eBioscience) and0.5 mg/mL anti-CD28 (37.51; eBioscience). Cells were pulsedwith 1 mCi of methyl-[3H]thymidine (Amersham Biosciences)for 6 hours during the last 72 hours of stimulation andharvested onto filters (Wallac). Filters were wetted with Beta-plate scintillation fluid (PerkinElmer) and counts per minuteread on a 1205 Betaplate liquid scintillation counter (Wallac).

Flow cytometryCells were surface stained in wash buffer (PBS, 1% FBS, and

0.01% sodium azide), fixed in 2% paraformaldehyde and ana-lyzed by flow cytometry on a BD FACS Calibur, LSR II, or FACSAria System. Data were analyzed with Cytobank (13). Flow-cytometric cell sorting was used to purify T-cell subsets fromsplenocytes of CpG/H11-vaccinated mice.

Primary human lymphoma specimensTumor specimens were obtained with informed consent in

accordance with the Declaration of Helsinki and with approvalby Stanford University's Administrative Panels on HumanSubjects inMedical Research. Samples were transferred direct-ly from the operating room to the laboratory and used for thepreparation of viable, sterile single-cell suspensions. Lymphnode tissue was disaggregated, filtered through a metal sieve,washed, resuspended in media composed of 90% FBS(HyClone) and 10% dimethyl sulfoxide (Sigma), frozen slowlyin the vapor phase of liquid nitrogen inmultiple cryotubes, andstored in liquid nitrogen. PBMCs fromhealthy individuals wereisolated with density gradient separation Ficoll-Paque PLUS(Amersham Biosciences) and subsequently stained with anti-bodies from Becton Dickinson or eBioscience as above.

Statistical analysisPrism software (GraphPad) was used to analyze tumor

growth and determine statistical significance of differencesbetween groups by applying an unpaired Student t test. P < 0.05were considered significant.

ResultsVaccine-induced CD4 T cells are necessary and sufficientfor tumor rejection

We have developed a model for adoptive cell therapy oflymphoma whereby antitumor T cells are generated in vivothrough vaccination with a CpG-loaded whole-cell vaccine(CpG/H11; ref. 2). These vaccine-induced cells are tumorspecific and cure large and established tumors when isolatedand transferred into lethally irradiated, syngeneic recipientmice (2). We investigated whether a specific subset of CD4 Tcells was tumor reactive. CD4 or CD8 T-cell subsets werenegatively isolated from CpG/H11-vaccinated, Thy1.1 con-genic, donor mice. These purified subsets were transferredinto lethally irradiated, Thy1.2 congenic recipients. Three daysposttransfer, recipients were challenged s.c. with 1 � 107 H11

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Cancer Res; 72(5) March 1, 2012 Cancer Research1240




tumor cells (Fig. 1A). Purified CD4 T cells from vaccinateddonors were sufficient to protect 80% of recipient mice formore than 50 days (Fig. 1B). Conversely, purified CD8 T cellswere comparable with splenocytes from unvaccinated donorsand had no effect on tumor growth rate.We investigated the mechanism for CD4-mediated tumor

cell killing. As we described previously, the H11 tumor cell lineis MHC class II negative, therefore CD4 T cells cannot killtumor cells directly (2). We tested the role of other effector cellpopulations by treating recipient mice with purified CD4 T

cells plus a depleting antibody for natural killer (NK) cells (anti-NK1.1), CD8 T cells (anti-CD8), or a blocking antibody againstIFN-g (anti–IFN-g). Depletion of NK cells and CD8 T cells wereconfirmed by flow cytometry. Three days posttransfer, recipi-ents were challenged s.c. with 1 � 107 H11 tumor cells.Depletion of NK cells had no effect on tumor rejection.However, depletion of CD8 T cells resulted in late relapseswith tumors beginning to grow 20 days after tumor challenge.Blockade of IFN-g eliminated the antitumor effect in 6 of 10recipient mice (Fig. 1C). Reconstitution of the endogenous,

Figure 1. Vaccine-induced CD4 Tcells are necessary and sufficient fortumor rejection. A, vaccination andadoptive therapy schema. B,recipient mice received purified CD4or CD8 T cells from vaccinated donormice or complete splenocytes fromunvaccinated donor mice. Arrowindicates day of tumor challenge. C,recipient mice received purified CD4T cells as above and were treatedwith depleting antibodies. Recipientmice were followed for tumor growth(n; 10 mice per group). D, PBLs wereisolated from recipient mice 10 daysafter transfer and assayed for IFN-gproduction. Plots are representativeof 3 independent samples per group.N/A, only T cells of the transferredsubset were present in recipientmice.

CD4 T-cell Subsets in Adoptive Cell Therapy for Lymphoma

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host CD8 T cell compartment can be observed as early as 10days posttransplant (Supplementary Fig. S1), these resultssuggest that CD4-mediated IFN-g production is a critical earlymediator in inducing a host CD8 T-cell response that isultimately necessary for lasting tumor rejection.

Antitumor immune responses in the recipient mice wereanalyzed 15 days after transfer. Phenotypic fidelity of thetransferred T-cell subsets was confirmed and donor-derivedcells (identified by expression of the Thy1.1marker) weremorethan 99% pure populations of either CD4 or CD8 T cells(Supplementary Fig. S2). Previously, we showed that CD4antitumor immune responses were tumor specific (2). Here,we evaluated by in vitro IFN-g production assay whether tumorreactivity was limited to donor cells. Whole peripheral bloodlymphocytes (PBL) from recipient mice were collected, placedin coculture with irradiated tumor cells, and IFN-g productionwas measured by intracellular flow cytometry. Using theThy1.1 marker, we were able to distinguish whether donor-derived recipient cells (Thy1.1pos) or reconstituted recipientcells (Thy1.1neg) were producing IFN-g . At day 15, only a subsetof donor-derived CD4 T cells produced IFN-g : 5.1% of Thy1.1pos

CD4 T cells were IFN-gpos (Fig. 1D). Conversely, only 0.6% ofdonor-derived (Thy1.1pos) CD8 T cells were IFN-gpos.

Because it has been reported that homeostatic proliferationinduces a memory phenotype in transferred T cells (14, 15), weassayed for CD44 expression on tumor-reactive CD4 and CD8T cells in an in vitro IFN-g production assay. CD44, thehyaluronic acid receptor, identifies antigen-experienced CD4T cells (16). In recipients transplanted with vaccine-inducedT cells, there was evident increased expression of CD44,specifically among the IFN-g producing, donor T cells (Fig.1D). This suggested that expression of CD44might serve as onepotentialmarker for the identification and eventual isolation ofa tumor-reactive subset of CD4 T cells.

CD137posCD44hi CD4 T cells are expanded in vaccinateddonor mice

We sought to identify the CD4 T cell subset necessary formediating antitumor immune responses. We compared vac-cinated and unvaccinated donor mice for expression of mem-ory and activation markers including CD44, CD62L, and CCR7(14–18). CD44 expression was increased on CD4 T cells invaccinated mice. However, we observed no differencesbetween vaccinated and unvaccinated mice in any of the othermarkers or combinations thereof (Supplementary Fig. S3).

When comparing expression of CD137 in vaccinated andunvaccinated donor mice, we observed a distinct increase inthe expression of CD137 on CD4 T cells in vaccinated animals(Fig. 2). The CD137pos population was predominantly withinthe CD44hi subset, similar to IFN-g–producing cells shownin Fig. 1. Based on these observations, we initially hypothesizedthat CD137 expression on CD4 T cells in vaccinated mice mayidentify a tumor reactive population. To further support thishypothesis, studies have shown that CD137 is an activationmarker on many immune cell types including CD4 and CD8 Tcells, B cells, NK cells, NK-T cells, monocytes, neutrophils, anddendritic cells (19). On CD8 T cells, binding of CD137 leads toproliferation, cytokine production, functional maturation, and

prolonged survival (19). Recent work from our group andothers has shown that agonistic mAbs against CD137 (4-1BB)can provoke CD8 T-cell antitumor responses capable of erad-icating established tumors in a range of murine tumor models(20–22).

CD137 identifies a population of activated Tregs

We characterized the population of CD137pos CD4 T cellsfurther and analyzed whole splenocytes from vaccinated andunvaccinated donor mice for expression of CD44, CD137, andFoxP3. As expected, FoxP3 staining identified the naturalphysiologic ratio of approximately 5% to 15% of CD4 T cellsas Tregs in donor mice (23). Surprisingly, in vaccinated mice,CD137 expression marked more than 65% of this Treg popu-lation. Conversely, in unvaccinated donors or donors vacci-nated with CpG alone, CD137 identified less than 35% of Tregs

(Fig. 3A and Supplementary Fig. S4). Therefore, in the contextof our vaccination, CD137 identifies an expanded subset ofFoxP3pos CD4 T cells.

We compared CD137pos and CD137neg Tregs from vaccinateddonors for expression of traditional Treg markers includingCD25, GITR, and CD103 as well as memory/activation markersincluding CD44, CD62L, and CD69 (Fig. 3B). Compared withCD137neg Tregs, the CD137

pos subset is distinct in its expressionof CD69 (high), CD44 (high), and CD62L (low). High CD69expression suggests that these cells are more activatedthan their CD137neg counterparts. The phenotype ofCD44hiCD62Llow is characteristic of an effector memorypopulation.

CD137posCD44hi and CD137negCD44hi CD4 T cellsmaintain expression of both CD137 and FoxP3 afteradoptive transfer

As they seem to have an effector memory phenotype, weasked whether CD137posCD44hi Tregs persisted following adop-tive transfer andwhether theymaintained expression of CD137and FoxP3. With FACS, we isolated CD137posCD44hi andCD137negCD44hi CD4 T cells from vaccinated, congenic(CD45.1pos) donors to purity of greater than 98% (Fig. 4A). Wetransferred these purified populations into lethally irradiated

Figure 2. CD137posCD44hi CD4 T cells are expanded in vaccinated donormice. Vaccination carried out as described. CD4 T cells from vaccinatedor unvaccinated donor mice were analyzed for expression of CD44 andCD137. Plots are representative of 3 independent samples per group.

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recipients. Ten days after transfer, recipient mice were sacri-ficed and splenocytes were analyzed for the presence of T cellsexpressing the congenicmarker, CD45.1. Both CD137posCD44hi

and CD137negCD44hi donor cells were present in the spleensof recipient mice. Furthermore, each transferred subsetretained its original phenotype: both CD137posCD44hi andCD137negCD44hi donor cells maintained their original expres-sion levels of both CD137 and FoxP3 (Fig. 4B).

CD137posCD44hi CD4 T cells are present in spleen but donot home to tumor sitesWe investigated whether CD137posCD44hi CD4 T cells were

capable of infiltrating sites of tumor. CD137posCD44hi and

CD137negCD44hi CD4 T cells were isolated from vaccinated,congenic (CD45.1pos) donors by FACS and adoptively trans-ferred into lethally irradiated recipients. Three days posttrans-fer, recipientswere challenged s.c. with 1� 107H11 tumor cells.Approximately 7 days after transfer, tumor size averagedapproximately 0.5 cm2 in both groups. Mice were sacrificedand both spleen and tumor analyzed for T-cell infiltration.Transferred donor cells were identified by surface expressionof the congenic marker, CD45.1.

T-cell infiltration of tumors was markedly different inmice that received CD137pos versus CD137neg subsets.CD137posCD44hi cells made up only 14.4% of total tumor-infiltrating T cells, whereas CD137negCD44hi cells made up

Figure 3. CD137 identifies apopulation of activated Tregs. A, CD4T cells from vaccinated orunvaccinated donor mice wereanalyzed for expression of CD137and FoxP3. B, CD137pos andCD137neg FoxP3pos T cells fromvaccinated donor mice werecompared for expression of CD25,CD44, CD62L, CD103, GITR, andCD69. Plots are representative of 3independent samples per group.

Figure 4. CD137posCD44hi and CD137negCD44hi CD4 T cells maintain expression of both CD137 and FoxP3 after adoptive transfer. Vaccination and adoptivetransfer were carried out as described. A, sort strategy and results for isolation of CD137posCD44hi and CD137negCD44hi CD4 T cells from vaccinatedCD45.1pos donor mice. CD25 and FoxP3 expression were used to characterize the Treg phenotype of these 2 subpopulations. B, CD137pos and CD137neg

subpopulations were adoptively transferred into CD45.1neg recipients. Seven days after transfer, recipient mice were sacrificed. In recipients, transferreddonor cells were identified by expression of CD45.1. CD45.1pos cells were analyzed by flow cytometry for expression of CD137 and FoxP3. Plots arerepresentative of 3 individual mice per group.






57.6% of total tumor-infiltrating T cells (Fig. 5A).CD137negCD44hi cells infiltrate sites of tumor and facilitatetumor rejection while CD137posCD44hi cells do not. However,both subsets of donor-derived CD4 T cells were similarlypresent in recipient spleens. In mice receiving CD137posCD44hi

cells, 14.5% of splenic T cells were of donor origin (Fig. 5B, left).Similarly, in mice receiving CD137negCD44hi cells, 18.8% ofsplenic T cells were of donor origin (Fig. 5B, right). Recipientbone marrow and lymph node compartments were not stud-ied. These findings suggest that the suppressive effect of theCD137pos population takes place in the spleen or other lym-phocyte compartments but not in the tumor itself.

CD137posCD44hi CD4 T cells have Treg function andsuppress the antitumor activity of CD137negCD44hi

effector cellsWe sought to confirm the suppressive function of

CD137posCD44hi CD4 T cells both in vitro and in vivo.CD137posCD44hi andCD137negCD44hi CD4T cellswere isolatedby FACS from vaccinated donors to greater than 98% purity.We cocultured CD137posCD44hi and CD137negCD44hi CD4

T cells at ratios of 0:1, 1:1, 1:2, 1:4, and 1:8 (CD137pos:CD137neg)and stimulated them with bead-bound anti-CD3/anti-CD28.CD137posCD44hi cells inhibited the proliferation ofCD137negCD44hi cells in a dose-dependent manner (Fig. 6A).To address whether this suppressive effect required cell–cellcontact, we carried out a similar assay but separatedCD137posCD44hi and CD137negCD44hi cells across a Transwell.The suppressive effect of CD137posCD44hi cells was eliminatedin the absence of cell–cell contact. In coculture across theTranswell, proliferation of CD137negCD44hi cells was similar tocontrols (Fig. 6B).

Next, we tested whether CD137posCD44hi cells could sup-press the antitumor response mediated by CD137negCD44hi

cells. Previously, when we transferred whole CD4 T cells weobserved impressive tumor rejection (2; Fig. 1). In this bulk CD4population, the ratio of CD137negCD44hi to CD137posCD44hi

was approximately 3:1. To determine whether CD137posCD44hi

could suppress antitumor immune responses in vivo, weisolated CD4T cells by FACS from vaccinated donors to greaterthan 98% purity. Either CD137neg cells alone (2 � 106 permouse) or a combination of CD137neg (2� 106 per mouse) andCD137pos (4 � 106 per mouse) cells at a ratio of 1:2 wereadoptively transferred into lethally irradiated recipients. Alethal challenge of 1 � 107 H11 tumor cells was given s.c. onday 3 following transfer. CD137negCD44hi cells were sufficientto protect 100% of recipient mice from lethal tumor challenge.When CD137posCD44hi cells we cotransferred, this antitumoreffect was blocked and 0% of recipient mice survived longerthan 20 days (Fig. 6C). Lower numbers of CD137posCD44hi cellsdid not inhibit tumor rejection. On day 15 after transfer, PBLsfrom recipient mice were collected and placed in coculturewith irradiated tumor cells for 24 hours. IFN-g production wasmeasured by intracellular flow cytometry. In mice receivingCD137pos and CD137negCD44hi cells, only 0.9% of CD4 T cellsproduced IFN-g (Fig. 6D). Conversely, in recipient mice receiv-ing CD137negCD44hi cells alone, 4.7% of CD4 T cells producedIFN-g .

In human lymphoma-involved lymph nodes, CD137identifies a population of activated Tregs

Recent studies have identified cell surface markers thatdefine a discrete subset of CD25posFoxP3pos Tregs preferentiallyexpanded in patients with cancer (24). We investigated wheth-er our observations about CD137 in a murine tumor modelwere consistent in the human tumor samples. In tumor-bear-ing mice, we observed that a large percentage of CD137pos CD4T cells were present in tumor-involved spleen. This CD137pos

population was overwhelmingly CD25posFoxP3pos Tregs

(Fig. 7A).We examined human lymphomabiopsies for whetherCD137 similarly served as a marker for CD25posFoxP3pos Tregs.Follicular lymphoma (FL), chronic lymphocytic leukemia(CLL), and mantle cell lymphoma (MCL) were analyzed byflow cytometry. A representative flow plot shows the gatingstrategy used to define these populations (Fig. 7B). CD137pos

CD45ROpos CD4 T cells were present in FL (mean ¼ 5.14% �SEM 0.34%) and MCL (mean ¼ 8.24% � SEM 1.68%) (Fig. 7C,left). As in the murine system, this CD137pos population waspredominantly CD25posFoxP3pos (FL: mean ¼ 68.65% � SEM

Figure 5. CD137posCD44hi CD4 T cells are present in spleen but do nothome to tumor sites. CD137pos and CD137neg populations were isolatedby FACS from vaccinated, CD45.1pos donors and adoptively transferredinto tumor challenged, lethally irradiated CD45.1neg recipients. Spleenand tumor were analyzed for total T-cell populations by surface CD3 andCD4 expression. Transferred donor cells were identified by surfaceexpression of the congenic marker CD45.1. Tissues were analyzed byflow cytometry from recipients of either CD137pos or CD137neg

populations. Plots showing tumor-infiltrating CD3pos T cells from tumorbiopsies (A) and splenicCD3pos T cells (B). All plots are representative of 5individual mice per group.

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3.28%; CLL: mean ¼ 54.73% � SEM 5.70%; Fig. 7C, right).Therefore, CD137 may serve as an effective marker forCD25posFoxP3pos Tregs in human lymphoma patients as well.

DiscussionOur results indicate that 2 markers—CD44 and CD137—can

be used to isolate a population of antitumor CD4 T cells whileeffectively excluding Tregs. Here, we show that (i)CD137posCD44hi expression defines a population of activatedCD4 Tregs that can suppress the proliferation of effector cells ina contact-dependent manner, (ii) the CD137posCD44hi Treg

population persists following adoptive transfer and can inhibittumor rejection in vivo, and (iii) vaccination with CpG-acti-vated lymphoma induced CD137negCD44hi CD4 T cells cantransfer protection from B-cell lymphoma tumor challenge.Rosenberg and colleagues have shown impressive clinical

outcomes using ex vivo generated T cells in adoptive immu-notherapy for metastatic melanoma (3–5). Our prior workestablished that a CpG-loaded whole-cell vaccine inducedantitumor CD4 T cells that were both necessary and sufficientto adoptively transfer antitumor immunity. To date, the field ofadoptive cell therapy has focused primarily on CD8 CTLs(3, 5, 6). However, important roles for CD4 T cells in antitumor

immunity have also been shown (8–11). Hunder and colleaguesshowed durable clinical remissions in melanoma patientstreated with ex vivo expanded, antigen-specific CD4 T cells(8). Recently, studies by Xie and colleagues (10) and Quezadaand colleagues (9) have shown in a TCR transgenic model thatnaive tumor-specific CD4 T cells are able to induce regressionof established tumors in lymphopenic hosts. Our findingssupport this prior body of work and suggest that antitumorCD4 T cells can be induced in vivo, isolated, and used inadoptive immunotherapy of established lymphoma tumors.Our results suggest that these antitumor CD4 T cells produceIFN-g and recruit a long-lasting antitumor response mediatedby host CD8 T cells. Importantly, our work does not rule outother sources of IFN-g including, but not limited to, NKT cells.

A concern in any CD4 T cell–based therapy is the potentialfor contamination by Tregs. This is particularly relevant to oursystem in which antitumor T cells are isolated following in vivoinduction. Hunder and colleagues addressed this issue byselecting tumor reactive clones and expanding them ex vivo.Quezada and colleagues combined CD4 adoptive transfer withblockade of CTLA-4 to inhibit the function of Tregs. Theseapproaches have led to impressive outcomes in human mel-anoma and mouse melanoma tumor models, respectively. Wehave defined a surface marker for a population of activated

Figure 6. CD137posCD44hi CD4T cells suppress the proliferationand antitumor activity ofCD137negCD44hi effector cells. Tregsuppression assays: CD137pos andCD137neg populations were isolatedfrom vaccinated mice by FACS. A,CD137pos cells were cocultured withpurified CD137neg cells at ratios of0:1, 1:1, 1:2, 1:4, and 1:8 (CD137jpos:CD137neg). Proliferation wasassessed by incorporation of 3H(cpm). All conditions were carried outin triplicate. B, CD137pos andCD137neg populations were platedacross a Transwell. C, CD137pos andCD137neg populations were isolatedand transferred into lethally irradiatedrecipient mice (n; 5 mice per group).Mice were followed for tumor growthand overall survival. Arrow indicatesdayof tumor challenge.D,PBLswereisolated from recipient mice 10 daysafter transfer and assayed for IFN-gproduction following 24-hourcoculture with irradiated tumor cells.Plots are representative of 3independent samples. ��, P < 0.001;��, P < 0.0001.






Tregs that can facilitate their removal prior to adoptive transferor allow their targeting with mAbs in the posttransplantrecipient. Specifically, CD137 and CD44 can be used to distin-guish antitumor CD4 T cells from Tregs. To our knowledge, thisis thefirst report of CD137 expression being used to distinguishthese 2 populations.

Surface marker identification of Tregs is not a novel pursuitand a number of studies have claimed to identify Tregs byexpression of surface markers including MHC-II, CD45RO/RA,CCR7, and ICOS (25–28). Most recently, Camisaschi andcolleagues (24) have observed that the expression of LAG-3on human CD4 T cells defines a discrete subset of CD25pos-

FoxP3posTregs that is preferentially expanded in patients withcancer. Although LAG-3 was not included in our evaluationof human lymphoma biopsies, we anticipate that both CD137

and LAG-3 may be complementary in identifying tumor-reac-tive Tregs.

In summary, the work reported here shows that CD137expression on CD4 T cells identifies a population of activatedTregs that suppress proliferation and antitumor immuneresponses. Importantly, the combination of cell surfacemarkersCD44 and CD137 can be used to isolate a population of anti-tumor CD4 T cells while excluding a large, activated subset ofTregs for the purpose of adoptive immunotherapy of lymphoma

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank K. Sachen and D. Czerwinski for their review of this

manuscript.

Figure 7. CD137 identifies apopulation of activated, Tregs inhuman lymphoma. A, splenocytesfrom a 10-day tumor-bearingmouse were analyzed by flowcytometry forCD44,CD137,CD25,and FoxP3 expression. Plots arerepresentative of 3 independentanimals. B and C, human tumorbiopsies and PBMCs wereanalyzed by flow cyotmetry forCD45RO, CD137, CD25, andFoxP3 expression. B, arepresentative plot of a FL tumorbiopsy showing the gatinghierarchy. C, summary plots of flowcytometry analyses.

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Grant SupportThis work was supported by grants from the NIH (grants CA33399 and

CA34233). M.J. Goldstein is supported by the Ruth L. Kirschstein NationalResearch Service Award (F30HL103193). R. Levy is an American Cancer SocietyClinical Research Professor.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received October 12, 2011; revised December 8, 2011; accepted December 29,2011; published OnlineFirst January 9, 2012.

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2012;72:1239-1247. Published OnlineFirst January 9, 2012.Cancer Res Matthew J. Goldstein, Holbrook E. Kohrt, Roch Houot, et al. CD137-Expressing Regulatory T CellsAdoptive Cell Therapy for Lymphoma with CD4 T Cells Depleted of

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