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

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

Cancer Therapeutics, Targets, and Chemical Biology Research

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

Abstract Adoptive 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 explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while neg hi ﬁ excluding Tregs. In a murine model of B-cell lymphoma, only CD137 CD44 CD4 T cells in ltrated tumor sites and provided protection. Conversely, the population of CD137posCD44hi CD4 T cells consisted primarily of pos activated Tregs. Notably, this CD137 Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells deﬁned a

population of activated Tregs that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137pos CD4 T cells that were pos pos ﬁ predominantly CD25 FoxP3 Tregs. In conclusion, our ndings identify 2 surface markers that can be used to Cancer Res; facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory Treg population. 72(5); 1239–47. 2012 AACR.

Introduction need for an accessible, injectable, tumor site. Importantly, Immunotherapy for cancer has included nonspecificstim- vaccination with such CpG-loaded tumor B cells induces a fi ulation of the immune system, active immunization with tumor-speci c CD4 and not CD8 T-cell response. Relatively fi tumor-specific antigens, and adoptive cell therapy—the small numbers of these CD4 T cells were suf cienttocure transfer of tumor-specific T cells. We have investigated large and established lymphoma tumors. adoptive cell therapy for treating lymphoma (1, 2). Histor- This and other recent reports have suggested that CD4 T – ically, adoptive cell therapy for cancer has focused on cells can be effective in adoptive immunotherapy (2, 8 11). isolating and expanding populations of T cells that have However, a relevant concern in using CD4 T cells for adoptive direct cytotoxic effects on tumors (3–6). We use active therapy is the potential for cotransfer of regulatory T cells fi — immunization to generate antitumor T cells in vivo and (Treg). Here, we have identi ed 2 surface markers CD44 and — transfer these T cells into lymphodepleted recipient mice. CD137 that can be used to isolate a population of antitumor fi CD4 T cells while excluding a population of inhibitory Tregs. The ef cacy of this maneuver is impressive and cures large neg hi tumors (1, 2). Specifically, our early work showed that CD8 Adoptive transfer of CD137 CD44 CD4 T cells provided fi pos hi T-cell antitumor immunity can be induced by the combi- signi cant protection from B-cell lymphoma. CD137 CD44 nation of cytotoxic chemotherapy with local, intratumoral CD4 T cells were predominantly Tregs and suppressed both injection of CpG (1, 7). Recently, we have extended the use of effector cell proliferation and antitumor activity. CpG as an immunotherapy by exposing tumor B cells to CpG ex vivo and subsequently injecting them into the host as a Materials and Methods whole-tumor cell vaccine (2). This approach obviates the Reagents CpG 1826 with sequence 50-TCCATGACGTTCCTGACGTT was provided by Coley Pharmaceutical Group (Ottawa, – Authors' Affiliation: Department of Medicine, Stanford University School Canada). Fluorescein isothiocyanate conjugated CpG 1826 of Medicine, Stanford, California was purchased from InvivoGen. The following monoclo- fl Note: Supplementary data for this article are available at Cancer Research nal antibodies (mAb) were used for ow cytometry: anti- Online (http://cancerres.aacrjournals.org/). mouse CD3, anti-mouse CD4, anti-mouse CD8, anti-mouse Corresponding Author: Ronald Levy, Chief, Division of Oncology, 269 CD44, anti-mouse CD137, anti-mouse IFN-g, anti-mouse CD25, Campus Drive, CCSR 1105, Stanford, CA 94305. Phone: 650-725-6452; anti-mouse FoxP3, anti-mouse CD62L, anti-mouse CCR7, anti- Fax: 650-736-1454; E-mail: [email protected] mouse CD103, anti-mouse CD69, anti-mouse GITR, anti-mouse doi: 10.1158/0008-5472.CAN-11-3375 Thy1.1, anti-mouse CD45.1, anti-human CD3, anti-human 2012 American Association for Cancer Research. CD4, anti-human CD8, anti-human CD45RO, anti-human

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pos neg CD137, anti-human CD25, anti-human FoxP3, and isotype con- Treg suppression assay. CD137 and CD137 cell popu- trol. Antibodies were purchased from either Becton Dickinson lations were isolated by FACS and mixed at various ratios in a (BD) Biosciences or eBioscience. 96-well round bottom plate. Cells were stimulated in with equal numbers of polystyrene latex beads (Interfacial Dynamics) Cell lines and mice coated with 1.0 mg/mL anti-CD3 (145-2C11; eBioscience) and H11 is a pre-B cell line in the C57BL/6 background generated 0.5 mg/mL anti-CD28 (37.51; eBioscience). Cells were pulsed as follows: primary bone marrow cells were isolated from with 1 mCi of methyl-[3H]thymidine (Amersham Biosciences) C57BL/6 mice and infected with the retrovirus vector for 6 hours during the last 72 hours of stimulation and MSCV-neo/p190Bcr-Abl, which carries the oncogene Bcr-Abl harvested onto filters (Wallac). Filters were wetted with Beta- (12; a gift from Drs. M. Cleary and K. Smith, Stanford School of plate scintillation fluid (PerkinElmer) and counts per minute Medicine). Tumor cells were cultured in complete RPMI 1640 read on a 1205 Betaplate liquid scintillation counter (Wallac). (cRPMI) medium (Invitrogen Life Technologies) containing 10% fetal calf serum (Thermo Scientific), 100 U/mL penicillin, Flow cytometry 100 mg/mL streptomycin (both from Invitrogen Life Techno- Cells were surface stained in wash buffer (PBS, 1% FBS, and logies), and 50 mmol/L 2-ME (Sigma-Aldrich). This cell line was 0.01% sodium azide), fixed in 2% paraformaldehyde and ana- previously used in studies from our laboratory (2) and was lyzed by flow cytometry on a BD FACS Calibur, LSR II, or FACS pos neg tested for uniformity of cell-surface markers (B220 CD43 Aria System. Data were analyzed with Cytobank (13). Flow- neg neg IgM IgD )byfluorescence-activated cell sorting (FACS). cytometric cell sorting was used to purify T-cell subsets from Six- to 8-week-old female Thy1.1, CD45.1, and wild-type splenocytes of CpG/H11-vaccinated mice. C57BL/6J mice were purchased from Jackson Laboratories. All studies were approved by the Stanford Administrative Panel Primary human lymphoma specimens on Laboratory Animal Care. Tumor specimens were obtained with informed consent in accordance with the Declaration of Helsinki and with approval Tumor inoculation and animal studies by Stanford University's Administrative Panels on Human H11 tumor cells were incubated in the presence of 3 mg/mL Subjects in Medical Research. Samples were transferred direct- of CpG at 37 C and 5% CO2. As shown previously, CpG is taken ly from the operating room to the laboratory and used for the up by H11 cells during this incubation (2). After 24 hours, cells preparation of viable, sterile single-cell suspensions. Lymph were vigorously washed 3 times with wash buffer to remove any node tissue was disaggregated, filtered through a metal sieve, unbound, residual CpG. H11 cells that were loaded with CpG washed, resuspended in media composed of 90% FBS (CpG/H11) were irradiated at 50 Gy and used to vaccinate (HyClone) and 10% dimethyl sulfoxide (Sigma), frozen slowly C57BL/6 donor mice s.c. for 5 consecutive days. On day 13, bone in the vapor phase of liquid nitrogen in multiple cryotubes, and marrow and splenocytes of donor mice were transferred by i.v. stored in liquid nitrogen. PBMCs from healthy individuals were injection into irradiated C57BL/6 recipient mice (9.5Gy TBI, isolated with density gradient separation Ficoll-Paque PLUS 6 Phillips x-ray unit, 250 kV, 15 mA) along with 1 10 irradiated (Amersham Biosciences) and subsequently stained with anti- CpG/H11 tumor cells as a posttransplant "booster" vaccine bodies from Becton Dickinson or eBioscience as above. that was prepared in the same fashion as above (1). In studies using congenic donors, transferred bone marrow was from Statistical analysis wild-type, noncongenic mice to allow precise tracking of Prism software (GraphPad) was used to analyze tumor transplanted donor cells. Recipient mice were challenged with growth and determine statistical significance of differences H11 tumor cells s.c. at a dose of 1 107 cells in 50 mL of serum- between groups by applying an unpaired Student t test. P < 0.05 free RPMI on day 16. Tumor growth was monitored by caliper were considered significant. measurement (Fig. 1A).

In vitro assays Results IFN-g production assay. Peripheral blood mononuclear Vaccine-induced CD4 T cells are necessary and sufficient cells (PBMC) were collected from tail vein, anticoagulated for tumor rejection with 2 mmol/L ethylenediaminetetraacetic acid in PBS, then We have developed a model for adoptive cell therapy of diluted 1:1 with Dextran T500 (Pharmacosmos) 2% in PBS lymphoma whereby antitumor T cells are generated in vivo and incubated at 37C for 45 minutes to precipitate red cells. through vaccination with a CpG-loaded whole-cell vaccine Leukocyte-containing supernatant was removed and centri- (CpG/H11; ref. 2). These vaccine-induced cells are tumor fuged,andremainingredcellswerelysedwithammonium specific and cure large and established tumors when isolated chloride potassium buffer (Quality Biological). PBMCs were and transferred into lethally irradiated, syngeneic recipient then cocultured with 1 106 irradiated H11 cells for 24 mice (2). We investigated whether a specific subset of CD4 T hours with 0.5 mg anti-mouse CD28mAb (BD PharMingen) cells was tumor reactive. CD4 or CD8 T-cell subsets were and in the presence of monensin (Golgistop; BD Biosciences) negatively isolated from CpG/H11-vaccinated, Thy1.1 con- fi for the last 5 hours at 37 Cand5%CO2. Intracellular IFN-g genic, donor mice. These puri ed subsets were transferred expression was assessed with BD Cytofix/Cytoperm Plus Kit into lethally irradiated, Thy1.2 congenic recipients. Three days per instructions. posttransfer, recipients were challenged s.c. with 1 107 H11

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Figure 1. Vaccine-induced CD4 T cells are necessary and sufficient for tumor rejection. A, vaccination and adoptive therapy schema. B, recipient mice received purified CD4 or CD8 T cells from vaccinated donor mice or complete splenocytes from unvaccinated donor mice. Arrow indicates day of tumor challenge. C, recipient mice received purified CD4 T cells as above and were treated with depleting antibodies. Recipient mice were followed for tumor growth (n; 10 mice per group). D, PBLs were isolated from recipient mice 10 days after transfer and assayed for IFN-g production. Plots are representative of 3 independent samples per group. N/A, only T cells of the transferred subset were present in recipient mice.

tumor cells (Fig. 1A). Purified CD4 T cells from vaccinated cells plus a depleting antibody for natural killer (NK) cells (anti- donors were sufficient to protect 80% of recipient mice for NK1.1), CD8 T cells (anti-CD8), or a blocking antibody against more than 50 days (Fig. 1B). Conversely, purified CD8 T cells IFN-g (anti–IFN-g). Depletion of NK cells and CD8 T cells were were comparable with splenocytes from unvaccinated donors confirmed by flow cytometry. Three days posttransfer, recipi- and had no effect on tumor growth rate. ents were challenged s.c. with 1 107 H11 tumor cells. We investigated the mechanism for CD4-mediated tumor Depletion of NK cells had no effect on tumor rejection. cell killing. As we described previously, the H11 tumor cell line However, depletion of CD8 T cells resulted in late relapses is MHC class II negative, therefore CD4 T cells cannot kill with tumors beginning to grow 20 days after tumor challenge. tumor cells directly (2). We tested the role of other effector cell Blockade of IFN-g eliminated the antitumor effect in 6 of 10 populations by treating recipient mice with purified CD4 T recipient mice (Fig. 1C). Reconstitution of the endogenous,

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host CD8 T cell compartment can be observed as early as 10 days posttransplant (Supplementary Fig. S1), these results suggest that CD4-mediated IFN-g production is a critical early mediator in inducing a host CD8 T-cell response that is ultimately necessary for lasting tumor rejection. Antitumor immune responses in the recipient mice were analyzed 15 days after transfer. Phenotypic fidelity of the transferred T-cell subsets was confirmed and donor-derived cells (identified by expression of the Thy1.1 marker) were more than 99% pure populations of either CD4 or CD8 T cells (Supplementary Fig. S2). Previously, we showed that CD4 antitumor immune responses were tumor specific (2). Here, we evaluated by in vitro IFN-g production assay whether tumor reactivity was limited to donor cells. Whole peripheral blood Figure 2. CD137posCD44hi CD4 T cells are expanded in vaccinated donor lymphocytes (PBL) from recipient mice were collected, placed mice. Vaccination carried out as described. CD4 T cells from vaccinated or unvaccinated donor mice were analyzed for expression of CD44 and in coculture with irradiated tumor cells, and IFN-g production CD137. Plots are representative of 3 independent samples per group. was measured by intracellular flow cytometry. Using the Thy1.1 marker, we were able to distinguish whether donor- derived recipient cells (Thy1.1pos) or reconstituted recipient prolonged survival (19). Recent work from our group and cells (Thy1.1neg) were producing IFN-g. At day 15, only a subset others has shown that agonistic mAbs against CD137 (4-1BB) of donor-derived CD4 T cells produced IFN-g: 5.1% of Thy1.1pos can provoke CD8 T-cell antitumor responses capable of erad- CD4 T cells were IFN-gpos (Fig. 1D). Conversely, only 0.6% of icating established tumors in a range of murine tumor models donor-derived (Thy1.1pos) CD8 T cells were IFN-gpos. (20–22). Because it has been reported that homeostatic proliferation induces a memory phenotype in transferred T cells (14, 15), we CD137 identifies a population of activated Tregs assayed for CD44 expression on tumor-reactive CD4 and CD8 We characterized the population of CD137pos CD4 T cells T cells in an in vitro IFN-g production assay. CD44, the further and analyzed whole splenocytes from vaccinated and hyaluronic acid receptor, identifies antigen-experienced CD4 unvaccinated donor mice for expression of CD44, CD137, and T cells (16). In recipients transplanted with vaccine-induced FoxP3. As expected, FoxP3 staining identified the natural T cells, there was evident increased expression of CD44, physiologic ratio of approximately 5% to 15% of CD4 T cells fi speci cally among the IFN-g producing, donor T cells (Fig. as Tregs in donor mice (23). Surprisingly, in vaccinated mice, 1D). This suggested that expression of CD44 might serve as one CD137 expression marked more than 65% of this Treg popu- potential marker for the identification and eventual isolation of lation. Conversely, in unvaccinated donors or donors vacci- a tumor-reactive subset of CD4 T cells. nated with CpG alone, CD137 identified less than 35% of Tregs (Fig. 3A and Supplementary Fig. S4). Therefore, in the context CD137posCD44hi CD4 T cells are expanded in vaccinated of our vaccination, CD137 identifies an expanded subset of donor mice FoxP3pos CD4 T cells. pos neg We sought to identify the CD4 T cell subset necessary for We compared CD137 and CD137 Tregs from vaccinated mediating antitumor immune responses. We compared vac- donors for expression of traditional Treg markers including cinated and unvaccinated donor mice for expression of mem- CD25, GITR, and CD103 as well as memory/activation markers ory and activation markers including CD44, CD62L, and CCR7 including CD44, CD62L, and CD69 (Fig. 3B). Compared with neg pos (14–18). CD44 expression was increased on CD4 T cells in CD137 Tregs, the CD137 subset is distinct in its expression vaccinated mice. However, we observed no differences of CD69 (high), CD44 (high), and CD62L (low). High CD69 between vaccinated and unvaccinated mice in any of the other expression suggests that these cells are more activated markers or combinations thereof (Supplementary Fig. S3). than their CD137neg counterparts. The phenotype of When comparing expression of CD137 in vaccinated and CD44hiCD62Llow is characteristic of an effector memory unvaccinated donor mice, we observed a distinct increase in population. the expression of CD137 on CD4 T cells in vaccinated animals (Fig. 2). The CD137pos population was predominantly within CD137posCD44hi and CD137negCD44hi CD4 T cells the CD44hi subset, similar to IFN-g–producing cells shown maintain expression of both CD137 and FoxP3 after in Fig. 1. Based on these observations, we initially hypothesized adoptive transfer that CD137 expression on CD4 T cells in vaccinated mice may As they seem to have an effector memory phenotype, we pos hi identify a tumor reactive population. To further support this asked whether CD137 CD44 Tregs persisted following adop- hypothesis, studies have shown that CD137 is an activation tive transfer and whether they maintained expression of CD137 marker on many immune cell types including CD4 and CD8 T and FoxP3. With FACS, we isolated CD137posCD44hi and cells, B cells, NK cells, NK-T cells, monocytes, neutrophils, and CD137negCD44hi CD4 T cells from vaccinated, congenic dendritic cells (19). On CD8 T cells, binding of CD137 leads to (CD45.1pos) donors to purity of greater than 98% (Fig. 4A). We proliferation, cytokine production, functional maturation, and transferred these purified populations into lethally irradiated

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Figure 3. CD137 identiﬁes a population of activated Tregs. A, CD4 T cells from vaccinated or unvaccinated donor mice were analyzed for expression of CD137 and FoxP3. B, CD137pos and CD137neg FoxP3pos T cells from vaccinated donor mice were compared for expression of CD25, CD44, CD62L, CD103, GITR, and CD69. Plots are representative of 3 independent samples per group.

recipients. Ten days after transfer, recipient mice were sacri- CD137negCD44hi CD4 T cells were isolated from vaccinated, ficed and splenocytes were analyzed for the presence of T cells congenic (CD45.1pos) donors by FACS and adoptively trans- expressing the congenic marker, CD45.1. Both CD137posCD44hi ferred into lethally irradiated recipients. Three days posttrans- and CD137negCD44hi donor cells were present in the spleens fer, recipients were challenged s.c. with 1 107 H11 tumor cells. of recipient mice. Furthermore, each transferred subset Approximately 7 days after transfer, tumor size averaged retained its original phenotype: both CD137posCD44hi and approximately 0.5 cm2 in both groups. Mice were sacrificed CD137negCD44hi donor cells maintained their original expres- and both spleen and tumor analyzed for T-cell infiltration. sion levels of both CD137 and FoxP3 (Fig. 4B). Transferred donor cells were identified by surface expression of the congenic marker, CD45.1. CD137posCD44hi CD4 T cells are present in spleen but do T-cell infiltration of tumors was markedly different in not home to tumor sites mice that received CD137pos versus CD137neg subsets. We investigated whether CD137posCD44hi CD4 T cells were CD137posCD44hi cells made up only 14.4% of total tumor- capable of infiltrating sites of tumor. CD137posCD44hi and infiltrating T cells, whereas CD137negCD44hi cells made up

Figure 4. CD137posCD44hi and CD137negCD44hi CD4 T cells maintain expression of both CD137 and FoxP3 after adoptive transfer. Vaccination and adoptive transfer were carried out as described. A, sort strategy and results for isolation of CD137posCD44hi and CD137negCD44hi CD4 T cells from vaccinated pos pos neg CD45.1 donor mice. CD25 and FoxP3 expression were used to characterize the Treg phenotype of these 2 subpopulations. B, CD137 and CD137 subpopulations were adoptively transferred into CD45.1neg recipients. Seven days after transfer, recipient mice were sacrificed. In recipients, transferred donor cells were identified by expression of CD45.1. CD45.1pos cells were analyzed by flow cytometry for expression of CD137 and FoxP3. Plots are representative of 3 individual mice per group.

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57.6% of total tumor-infiltrating T cells (Fig. 5A). T cells at ratios of 0:1, 1:1, 1:2, 1:4, and 1:8 (CD137pos:CD137neg) CD137negCD44hi cells infiltrate sites of tumor and facilitate and stimulated them with bead-bound anti-CD3/anti-CD28. tumor rejection while CD137posCD44hi cells do not. However, CD137posCD44hi cells inhibited the proliferation of both subsets of donor-derived CD4 T cells were similarly CD137negCD44hi cells in a dose-dependent manner (Fig. 6A). present in recipient spleens. In mice receiving CD137posCD44hi To address whether this suppressive effect required cell–cell cells, 14.5% of splenic T cells were of donor origin (Fig. 5B, left). contact, we carried out a similar assay but separated Similarly, in mice receiving CD137negCD44hi cells, 18.8% of CD137posCD44hi and CD137negCD44hi cells across a Transwell. splenic T cells were of donor origin (Fig. 5B, right). Recipient The suppressive effect of CD137posCD44hi cells was eliminated bone marrow and lymph node compartments were not stud- in the absence of cell–cell contact. In coculture across the ied. These findings suggest that the suppressive effect of the Transwell, proliferation of CD137negCD44hi cells was similar to CD137pos population takes place in the spleen or other lym- controls (Fig. 6B). phocyte compartments but not in the tumor itself. Next, we tested whether CD137posCD44hi cells could suppress the antitumor response mediated by CD137negCD44hi

pos hi cells. Previously, when we transferred whole CD4 T cells we CD137 CD44 CD4 T cells have Treg function and neg hi observed impressive tumor rejection (2; Fig. 1). In this bulk CD4 suppress the antitumor activity of CD137 CD44 neg hi pos hi population, the ratio of CD137 CD44 to CD137 CD44 effector cells pos hi was approximately 3:1. To determine whether CD137 CD44 We sought to confirm the suppressive function of could suppress antitumor immune responses in vivo,we CD137posCD44hi CD4 T cells both in vitro and in vivo. pos hi neg hi isolated CD4 T cells by FACS from vaccinated donors to greater CD137 CD44 and CD137 CD44 CD4 T cells were isolated neg 6 than 98% purity. Either CD137 cells alone (2 10 per by FACS from vaccinated donors to greater than 98% purity. neg 6 pos hi neg hi mouse) or a combination of CD137 (2 10 per mouse) and We cocultured CD137 CD44 and CD137 CD44 CD4 pos 6 CD137 (4 10 per mouse) cells at a ratio of 1:2 were adoptively transferred into lethally irradiated recipients. A lethal challenge of 1 107 H11 tumor cells was given s.c. on day 3 following transfer. CD137negCD44hi cells were sufficient to protect 100% of recipient mice from lethal tumor challenge. When CD137posCD44hi cells we cotransferred, this antitumor effect was blocked and 0% of recipient mice survived longer than 20 days (Fig. 6C). Lower numbers of CD137posCD44hi cells did not inhibit tumor rejection. On day 15 after transfer, PBLs from recipient mice were collected and placed in coculture with irradiated tumor cells for 24 hours. IFN-g production was measured by intracellular flow cytometry. In mice receiving CD137pos and CD137negCD44hi cells, only 0.9% of CD4 T cells produced IFN-g (Fig. 6D). Conversely, in recipient mice receiving CD137negCD44hi cells alone, 4.7% of CD4 T cells produced IFN-g.

In human lymphoma-involved lymph nodes, CD137 identifies a population of activated Tregs Recent studies have identified cell surface markers that pos pos define a discrete subset of CD25 FoxP3 Tregs preferentially expanded in patients with cancer (24). We investigated whether our observations about CD137 in a murine tumor model were consistent in the human tumor samples. In tumor-bearing mice, we observed that a large percentage of CD137pos CD4 T cells were present in tumor-involved spleen. This CD137pos pos pos population was overwhelmingly CD25 FoxP3 Tregs (Fig. 7A). We examined human lymphoma biopsies for whether Figure 5. CD137posCD44hi CD4 T cells are present in spleen but do not pos pos pos neg CD137 similarly served as a marker for CD25 FoxP3 Tregs. home to tumor sites. CD137 and CD137 populations were isolated Follicular lymphoma (FL), chronic lymphocytic leukemia by FACS from vaccinated, CD45.1pos donors and adoptively transferred into tumor challenged, lethally irradiated CD45.1neg recipients. Spleen (CLL), and mantle cell lymphoma (MCL) were analyzed by and tumor were analyzed for total T-cell populations by surface CD3 and flow cytometry. A representative flow plot shows the gating CD4 expression. Transferred donor cells were identified by surface strategy used to define these populations (Fig. 7B). CD137pos expression of the congenic marker CD45.1. Tissues were analyzed by pos ¼ fl pos neg CD45RO CD4 T cells were present in FL (mean 5.14% ow cytometry from recipients of either CD137 or CD137 ¼ populations. Plots showing tumor-infiltrating CD3pos T cells from tumor SEM 0.34%) and MCL (mean 8.24% SEM 1.68%) (Fig. 7C, pos biopsies (A) and splenic CD3pos T cells (B). All plots are representative of 5 left). As in the murine system, this CD137 population was individual mice per group. predominantly CD25posFoxP3pos (FL: mean ¼ 68.65% SEM

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Figure 6. CD137posCD44hi CD4 T cells suppress the proliferation and antitumor activity of neg hi CD137 CD44 effector cells. Treg suppression assays: CD137pos and CD137neg populations were isolated from vaccinated mice by FACS. A, CD137pos cells were cocultured with puriﬁed CD137neg cells at ratios of 0:1, 1:1, 1:2, 1:4, and 1:8 (CD137jpos: CD137neg). Proliferation was assessed by incorporation of 3H (cpm). All conditions were carried out in triplicate. B, CD137pos and CD137neg populations were plated across a Transwell. C, CD137pos and CD137neg populations were isolated and transferred into lethally irradiated recipient mice (n; 5 mice per group). Mice were followed for tumor growth and overall survival. Arrow indicates day of tumor challenge. D, PBLs were isolated from recipient mice 10 days after transfer and assayed for IFN-g production following 24-hour coculture with irradiated tumor cells. Plots are representative of 3 independent samples. , P < 0.001; , P < 0.0001.

3.28%; CLL: mean ¼ 54.73% SEM 5.70%; Fig. 7C, right). immunity have also been shown (8–11). Hunder and colleagues Therefore, CD137 may serve as an effective marker for showed durable clinical remissions in melanoma patients pos pos ex vivo fi CD25 FoxP3 Tregs in human lymphoma patients as well. treated with expanded, antigen-speci c CD4 T cells (8). Recently, studies by Xie and colleagues (10) and Quezada and colleagues (9) have shown in a TCR transgenic model that Discussion naive tumor-specific CD4 T cells are able to induce regression Our results indicate that 2 markers—CD44 and CD137—can of established tumors in lymphopenic hosts. Our findings be used to isolate a population of antitumor CD4 T cells while support this prior body of work and suggest that antitumor in vivo effectively excluding Tregs. Here, we show that (i) CD4 T cells can be induced , isolated, and used in CD137posCD44hi expression defines a population of activated adoptive immunotherapy of established lymphoma tumors. CD4 Tregs that can suppress the proliferation of effector cells in Our results suggest that these antitumor CD4 T cells produce pos hi a contact-dependent manner, (ii) the CD137 CD44 Treg IFN-g and recruit a long-lasting antitumor response mediated population persists following adoptive transfer and can inhibit by host CD8 T cells. Importantly, our work does not rule out tumor rejection in vivo, and (iii) vaccination with CpG-acti- other sources of IFN-g including, but not limited to, NKT cells. vated lymphoma induced CD137negCD44hi CD4 T cells can A concern in any CD4 T cell–based therapy is the potential transfer protection from B-cell lymphoma tumor challenge. for contamination by Tregs. This is particularly relevant to our Rosenberg and colleagues have shown impressive clinical system in which antitumor T cells are isolated following in vivo outcomes using ex vivo generated T cells in adoptive immu- induction. Hunder and colleagues addressed this issue by notherapy for metastatic melanoma (3–5). Our prior work selecting tumor reactive clones and expanding them ex vivo. established that a CpG-loaded whole-cell vaccine induced Quezada and colleagues combined CD4 adoptive transfer with antitumor CD4 T cells that were both necessary and sufficient blockade of CTLA-4 to inhibit the function of Tregs. These to adoptively transfer antitumor immunity. To date, the field of approaches have led to impressive outcomes in human mel- adoptive cell therapy has focused primarily on CD8 CTLs anoma and mouse melanoma tumor models, respectively. We (3, 5, 6). However, important roles for CD4 T cells in antitumor have defined a surface marker for a population of activated

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Figure 7. CD137 identifies a population of activated, Tregs in human lymphoma. A, splenocytes from a 10-day tumor-bearing mouse were analyzed by flow cytometry for CD44, CD137, CD25, and FoxP3 expression. Plots are representative of 3 independent animals. B and C, human tumor biopsies and PBMCs were analyzed by flow cyotmetry for CD45RO, CD137, CD25, and FoxP3 expression. B, a representative plot of a FL tumor biopsy showing the gating hierarchy. C, summary plots of flow cytometry analyses.

Tregs that can facilitate their removal prior to adoptive transfer and LAG-3 may be complementary in identifying tumor-reac- or allow their targeting with mAbs in the posttransplant tive Tregs. recipient. Specifically, CD137 and CD44 can be used to distin- In summary, the work reported here shows that CD137 guish antitumor CD4 T cells from Tregs. To our knowledge, this expression on CD4 T cells identifies a population of activated fi is the rst report of CD137 expression being used to distinguish Tregs that suppress proliferation and antitumor immune these 2 populations. responses. Importantly, the combination of cell surface markers Surface marker identification of Tregs is not a novel pursuit CD44 and CD137 can be used to isolate a population of anti- and a number of studies have claimed to identify Tregs by tumor CD4 T cells while excluding a large, activated subset of expression of surface markers including MHC-II, CD45RO/RA, Tregs for the purpose of adoptive immunotherapy of lymphoma CCR7, and ICOS (25–28). Most recently, Camisaschi and colleagues (24) have observed that the expression of LAG-3 Disclosure of Potential Conflicts of Interest on human CD4 T cells defines a discrete subset of CD25pos- No potential conflicts of interest were disclosed. FoxP3posT that is preferentially expanded in patients with regs Acknowledgments cancer. Although LAG-3 was not included in our evaluation The authors thank K. Sachen and D. Czerwinski for their review of this of human lymphoma biopsies, we anticipate that both CD137 manuscript.

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CD4 T-cell Subsets in Adoptive Cell Therapy for Lymphoma

Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this This work was supported by grants from the NIH (grants CA33399 and fact. CA34233). M.J. Goldstein is supported by the Ruth L. Kirschstein National Research Service Award (F30HL103193). R. Levy is an American Cancer Society Clinical Research Professor. The costs of publication of this article were defrayed in part by the Received October 12, 2011; revised December 8, 2011; accepted December 29, payment of page charges. This article must therefore be hereby marked 2011; published OnlineFirst January 9, 2012.

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www.aacrjournals.org Cancer Res; 72(5) March 1, 2012 1247

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

Matthew J. Goldstein, Holbrook E. Kohrt, Roch Houot, et al.

Cancer Res 2012;72:1239-1247. Published OnlineFirst January 9, 2012.

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