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CD137 Agonist Therapy Can Reprogram

Regulatory T Cells into Cytotoxic CD4 + T
Cells with Antitumor Activity
This information is current as Ilseyar Akhmetzyanova, Gennadiy Zelinskyy, Elisabeth
of December 18, 2015. Littwitz-Salomon, Anna Malyshkina, Kirsten K. Dietze,
Hendrik Streeck, Sven Brandau and Ulf Dittmer
J Immunol 2016; 196:484-492; Prepublished online 25
November 2015;

Downloaded from http://www.jimmunol.org/ at AZ Library on December 18, 2015
doi: 10.4049/jimmunol.1403039
http://www.jimmunol.org/content/196/1/484

Supplementary http://www.jimmunol.org/content/suppl/2015/11/25/jimmunol.140303
Material 9.DCSupplemental.html
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in this FBL-3 tumor model. Because the majority of animal model has been the stimulation of the TNFR superfamily cytotoxic antitumor immunity is mediated by CD8+ T cell re- member CD137 (4-1BB). 45122 Essen. and ‡ Department of Otorhinolaryngology of the University Hospital Essen. Institute for Virology.org/ at AZ Library on December 18. C57BL/6. 12).* Elisabeth Littwitz-Salomon. which is involved in T cell activation sponse. University induced tumor cell line of C57BL/6 (B6) origin.de Tregs suppress effective antitumor CD4+ T cell responses and The online version of this article contains supplemental material. tration of an agonist CD137 Ab into mice lacking CD8+ T cells Copyright Ó 2015 by The American Association of Immunologists.00 promoted effective cytotoxic antitumor CD4+ T cell responses and www. Germany. 2014.* Kirsten K. 196: 484–492. when their suppression by Tregs was abrogated Nrp1. Inc. Accepted for publication October 30. a previous article. we used the highly immunogenic Friend virus–induced FBL-3 tumor as a model to study the mechanisms of immunological tumor control by CD4+ T cells in the course of CD137 (4-1BB) agonist immunotherapy in the absence of a CD8 T cell response. This cytotoxicity of CD4 Several strategies have been developed to increase the antitumor T cells was dependent on the regulatory T-box transcription factor immune responses including reversion of T cell exhaustion with Eomesodermin (Eomes) and mediated by KLRG-1+ terminally agonistic Abs such as PD1 or CTLA4 (1). mouse epithelial cell. Neuropilin 1. it remains unknown how much cytotoxic CD4+ T cells and function (4) including expansion. Virchowstrasse 179. control and clearance of tumor growth is often impaired in cancer patients. and cytokine actually contribute to tumor rejection in such immune studies and production of effector T cells (3. 2015 agonist resulted in complete FBL-3 tumor regression in CD8+ T cell–deficient mice. s.1.* Hendrik Streeck. MHCII. Melanoma. namely. enhanced tumor-specific cytotoxicity latory receptors (3).* Anna Malyshkina. 10).org/cgi/doi/10. dittmer@uni-due. the prominently expressed on CD8+ T cells than CD4+ T cells. Ulf Dittmer.jimmunol. our data show that tumor-induced Foxp3+CD4+ T cells can be reprogrammed into cytotoxic effector cells upon therapeutic costimulatory signaling and restore antitumor immunity. 2016. we made use of a murine retrovirus- Hospital in Essen. Foxp3 Eomes double-positive CD4+ T cells were capable of eliminating immunogenic virus-induced tumor cells in vivo. Eomes.4049/jimmunol. we show that adminis- T cell. tivity as previously suggested (8). Interestingly. DT.* Gennadiy Zelinskyy. and increasing antitumor activity by targeting costimu. CD4+ T cells into cytotoxic effectors (6).‡ and Ulf Dittmer* Recent successes in immune therapeutic strategies aimed to improve control over tumor growth have sparked hope that long-lived control of cancer through stimulation of the immune system can be possible. Curran et al. we reported that. In addition. strated that. The cytotoxic Abbreviations used in this article: B6. anti-CD137 treatment also drove differentiation of leading to uninhibited growth. by anti2CD137 treatment. regulatory because of Treg depletion. In this work. However.c. (6) nicely showed tumor-specific T cell responses by ex vivo expansion and reinfu. most significance of regulatory T cells (Tregs) after anti-CD137 therapy immunotherapy studies have focused on the impact of anti-CD137 has not been well defined and remains contradictory. 45122 Essen. ptc. FV. subsequent regression caused by effector CD8+ T cells (11. CD4+ T cells could fully compensate for the antitumor effect of sodermin. 5). FBL-3 of Duisburg-Essen.jimmunol. that does not produce infectious virus. Eome. a subset of CD4+Foxp3+ regu- latory T cells was reprogrammed to eliminate immunogenic virus-induced tumor cells in response to CD137 agonist treatment. increasing frequency of differentiated effector T cells (6). the underlying immunological mechanisms that are induced by immunotherapeutic strategies are not well understood. Germany. and death. Thus. 0022-1767/15/$30. MTEC.1403039 . but expresses highly im- 2015. The Journal of Immunology CD137 Agonist Therapy Can Reprogram Regulatory T Cells into Cytotoxic CD4+ T Cells with Antitumor Activity Ilseyar Akhmetzyanova. nTreg. MHC class II. Essen. FBL-3 is a Friend virus (FV)–transformed tumor cell line Received for publication December 4. We demonstrate that treatment with a CD137 Downloaded from http://www. munogenic FV Ags on the cell surface (9. natural Treg. diphtheria toxin.091. University of Duisburg. Dietze. Because on CD8+ T cell immunity (4). posttumor challenge. CD137 signaling enhanced the production of proinflammatory cytokines and cytotoxic molecules in tumor-specific CD4+ T cells. it is unknown whether activation of Tregs through CD137 may drive increased suppressive Treg ac- *Institute for Virology of the University Hospital in Essen. implantation of This work was supported by a grant from the Deutsche Forschungsgemeinschaft FBL-3 cells into B6 mice results in local tumor growth with (TRR60 Project B4) and from the Wilhelm Sander-Stiftung project 2014. but also on CD4+Foxp3+Tregs (7). †Institute for Medical Biology of the University To address these questions. One approach with promising results in an and in turn promoted tumor immunity. In Address correspondence and reprint requests to Prof. University of Duisburg-Essen. In this study. generated sion (2).† Sven Brandau. These cells expressed markers characteristic for Th cells (CD154) and produced the cytokine TNF-a or the T-box transcriptional factor Eomesodermin and granzyme B without loss of Foxp3 expression. metastasis. Germany. 45147 Essen. CD8+ T cells. that effector KLRG-1+Eomes+ CD4+ and CD8+ T cells. thereby contributed toward tumor progression (13). Germany cells. Treg. Because CD137 is more in particular after treatment with CD137 agonist. E-mail address: ulf. CD137 is not only expressed on activated effector T cells. 45147 Essen. Friend virus. survival. in addition to improved CD8+ T cell responses in B16 D espite robust anti-tumor immunity. The Journal of Immunology. a recent study demon. However.

TNF-a (BV510. Between 1 and 3 3 106 CD4+GFP2 cells and 1–5 3 105 CD4+GFP+ and DEREG (14) mice that were between 8 and 10 wk old at day 0 of the cells were transferred into CD45. CD45. mice received i. started at day 0 and carried out every other day for the tumor growth Nucleated lymph node cells were incubated with allophycocyanin-labeled analysis until mice were killed because of the progressive tumor growth. RM4-5). gp39.p. between the different parameters were performed testing with the Kruskal– ment depleted . anti-CD137 treatment converted a subpopulation of Foxp3+ acquired on an LSRII flow cytometer (Becton Dickinson) from 300. T-bet (PeCy7. Strikingly. The CD4+ T cells were depleted with the same efficacy by i. 3 cells were maintained in complete RPMI-1640 medium (Invitrogen. and injected tumor cell lines TexRed. mice were sacrificed and draining lymph CFSE+ signal. The treatment depleted .p. lavage was performed with 10 ml PBS to obtain cells. we determined the greatest longitudinal diameter FBL-3 cells were injected i. and anti-CD154 (PE. MD) supplemented with 10% FCS and 0. using B6 or DEREG mice. MHCII2/2. reveal an intriguing immunology of a unique subpopulation of Foxp3+CD4+ T cells that can adopt antitumor effector functions Cell sorting and adoptive cell transfer after potent costimulatory signaling.jimmunol. In addition. and TNF-a. fixed. This is only a slight delay of 7 d in tumor rejection .p. killed at day 6 postinoculation. lysis of targets. CD4+ T cell epitope (H19-Env. Statistical differences (p value) diphtheria toxin (DT) (Merck) diluted in endotoxin-free PBS.The Journal of Immunology 485 led to FBL-3 tumor elimination in the presence of Tregs. 1B). injection in 0. whereas only 40–50% of CD11c+CD8+ DCs were Statistical analyses depleted after this treatment (data not shown) (18). More. CD8. After that cells were labeled with mAbs specific for IL-2 T cells. Results Staining and flow cytometry CD137 agonist therapy in CD8+ T cell–depleted mice promotes FBL-3 tumor cell rejection through a CD4-dependent The Abs used for cell-surface staining were anti-CD4 (eFluor 605. into naive CD45. Intra- cellular granzyme B (monoclonal anti-human granzyme B Ab allophycocyanin.v. Life Technologies). cells. cervical. mice (length) and the greatest transverse diameter (width). Depletion was tained from the National Institutes of Health Tetramer Facility (Atlanta). we injected DEREG mice i. every other day from day 0.97% of the CD4+ T cells expressing fluorescent protein Wallis one-way ANOVA on ranks and Newman–Keuls multiple comparison (GFP) in DEREG mice. The cells were then starting at the time point of the FBL-3 tumor infusion to CD8+ washed. Data were over. After 6 d posttumor and i. 1B11). FJK-16s). Tumor target lysis assay was 100 ml PBS through a 27-gauge needle on day 0. To verify tumor volume performed using 2 3 105 FBL-3 cells/mouse labeled with 10 mM CFSE. permeabilized.v. as well as unloaded cells from CD45.5 ml experiments. injection of donor cells. by external caliper. MP6-XT22). To determine intracellular production of IFN-g. mesenteric.1 recipients by i.95% pure populations of CD4+GFP+ or CD4+GFP2 Experiments were done using sex. were completely rejected after on average 22 d of tumor challenge eBio4B10). For Treg conversion experiments. and IL-2.95% of the CD8+ T cells in lymph nodes. the costimulation of CD4+ T cells with CD137 agonist (eFluor 450. on the right flank in together with the target cell suspension.3) (19) used in vivo tests. 2.5 ml supernatant fluid Statistical analyses and graphical presentations were computed with obtained from hybridoma cell culture YTS 191. a 3 b. restored anti–FBL-3 tumor immunity.c. In addition. Cells were challenge (ptc). anti-CD11b (BV650. tracellular staining using the Foxp3 staining kit (eBioscience). peptide-loaded CFSE-labeled lymphocyte targets (Vybrant CFDA SE Cell Gaithersburg.1 (PeCy7. we stimulated cells from lymph nodes in the presence of animals had to be euthanized on average 15 d after tumor infusion. I-Ab tetramers for 3 h at 37˚C and later stained with anti-CD4 and anti– and three times (on days 0. Dosing per injection was 100 mg adminis- tered i. Statistical differences (p value) between two mAb (17). on the right (eBioscience) to exclude dead cells. Naive cells from MHCII2/2 mice were either Downloaded from http://www. 22). JES6-5H4).c.1 mice. and incubated with Fc blocking anti-mouse T cell–depleted mice. on day 6 ptc. XMG1. The anti-CD137 (LOB 12. nodes (inguinal) were extracted for further analysis. Twenty hours after i. Indeed. 2F1) (Biolegend). (21) FBL-3 is an FV-induced tumor cell line derived from a B6 mouse (15). 1A).5]). EPLTSLTPRCNTAWNRLKL) were ob- bridoma cell line 169. mice were sacrificed and draining lymph nodes (inguinal) washed once.p. SolA15) expression were detected by in. 2 mg/ml of the CD28 Ab and 2 mg/ml brefeldin A for 5 h at 37˚C. Cells were then sorted on a FACSAria (BD Mice Biosciences) to . The mice were sacrificed 48 h later. Mouse epithelial cells (MTECs) are transformed mouse tonsil epithelial cells. In vivo cytotoxicity Cell lines In vivo cytotoxicity assay was performed as described by Barber et al. IFN-g (FITC. similar to previous studies (11. and Ki67 (PeCy7. To address the question whether CD137 agonist treatment can alter A20).v. The analyses were done using the Tregs into tumor cell–killing effector CD4+ T cells.2). and CD43. anti–Mac-1 [WT. where a is half of length and b is half of width.05 were determined to be significant. KLRG-1 (BV421. with 1 mg groups were performed using an unpaired t test. all p values #0. Viability Dye (eBioscience). allophycocyanin. Tetramer staining In vivo cell depletion and CD137 agonist treatment MHC class II (MHCII) tetramers loaded with I-Ab–restricted FV-specific In brief. we found no tumor control in mice depleted for CD8+ T cells. Invitrogen) was performed as described previously (20).1 mice as a control population. and CD4+ T cell effector function and restore sufficient antitumor Neuropilin1 (Nrp1. 1 mg DT was added A total of 1 3 107 FBL-3 tumor cells were injected s. FBL. all groups of mice received i.1 producing CD4-specific GraphPad Prism version 6. CD40Ligand. MR1). M17/4). anti-CD11a (PE. labeled with CFSE and pulsed with peptide or labeled with CellTrace MTECs were grown in E media containing DMEM as previously described Violet (Invitrogen) without peptide incubation to confirm tumor-specific (16). The cells were then stained for surface expression of CD4. and axillary) of naive DEREG mice using CD4+ selection (Miltenyi Biotec) according to the Materials and Methods manufacturer’s instructions. The treat. Mice were housed in specific pathogen-free conditions and PBS on day 0 after FBL-3 challenge and Ab administration. In this work. injection of 0. Eight days ptc. Foxp3 (PE. the mice were sacrificed and in vivo killing activity was quantified Tumor challenge in single-cell suspensions from the draining lymph node of each tumor- bearing mouse. Five days ptc. mechanism anti-CD43 (PerCP. A total of 1 3 106 MTECs were implanted s. treated in accordance with institutional guidelines. and We next administered a CD137 agonist Ab every second day permeabilized with Cytofix/Cytoperm solution (BD).p. Eomes (PerCP-eFluor710. CD45. which do not carry viral proteins (HPV2) (16).4 producing CD8a-specific mAb (17). immunity in the absence of CD8+ T cell mediated control. 0.and age-matched B6.5 ml supernatant fluid obtained from hy.1. Our results FACSDiva software (Becton Dickinson) and the FlowJo software (Tree Star). were produced by BioXCell.v.000 lymphocyte-gated events per sample. FAB566A. 2015 streptomycin. and Fc block anti-mouse CD16/CD32 (93) (eBioscience). in some experiments.org/ at AZ Library on December 18. (Fig. R&D Systems). CD4+ T cells were isolated from spleens and lymph nodes (inguinal. Tumor size based on received sorted CD4+GFP+ cells from either anti-CD8 or anti-CD8+anti- caliper measurements was calculated by formula: tumor area (cm2) = p 3 CD137 tumor-bearing or naive mice. resuspended in buffer containing fixable viability dye were resected. and analyzed by flow cytometry for flank of B6 mice. Dan11mag). Dead cells depleted mice of CD8+ T cells and determined FBL-3 tumor for cell-surface and intracellular staining were excluded by using Fixable progression (Fig. we first conjugated. 4) for the experiments where mice were Mac-1 for 10 min at room temperature.5% penicillin/ Tracer Kit. To deplete Tregs. despite the absence of CD8+ CD16/CD32.000– 500.

significant expansion of Foxp3+ Tregs comprising up to 30% of all depleted. 2E). we next depleted mice CD8+ T cell–depleted tumor-bearing mice. Anti-CD137 treatment induces tumor-specific cytotoxic CD137 signaling elicits activation.c. We first lyze the antitumor immune response after CD137 treatment. activation markers CD43 and CD11a (p = 0. we next investigated whether anti-CD137 treatment CD8+anti-CD137–treated mice (Supplemental Fig. We tional factors T-bet and Eomes (Fig. anti- restore antitumor immunity in the presence of CD137 costimu.0003. Fig. and anti-CD137 treatment (C). 3C). we also found a significant increase of recently IFN-g. 2A).0001. proliferated Tregs identified by the expression of Ki67 (p = 0. As expected. 3B) and activated Tregs identified by the expression of the showed significantly enhanced expression of the T-box transcrip. after anti-CD137 injection into CD8-depleted mice. and depletion of CD8+ T cells. Collectively. which has been linked to cytotoxic activity of CD8+ T cells (24). quency was unchanged in CD8+ T cell–depleted mice. The dotted line in (A) represents mean and error of tumor growth of seven tumor-bearing mice. and therefore suggesting that CD4+ T cells may have acquired cytotoxic activity analyzed the expression of Nrp1. confirming that we indeed measured indicating that this is a T cell–mediated effect. which have been also found increased expression of the maturation marker KLRG- suggested to control the Th1 helper versus cytotoxic function of 1 on Tregs. anti-CD137–treated mice Fig. we found that Eomes+CD4+ T cells vascular endothelial growth factor family expressed on nTregs in the treated mice showed substantially higher expression of the (25). responses that are capable of controlling FBL-3 tumor cell growth. 1C). we found not only more antitumor CD4+ T cells in CD8. and CD4+ T cells that can kill FBL-3 cells differentiation of natural Tregs To understand the underlying immunobiology of the described In a previous study we have demonstrated that antitumor effects phenomenon.30% target cell with a CD137 agonist Ab during FBL-3 tumor challenge killing (Fig. However. The effects of no depletion (injected with PBS) are presented by dotted line. 1A). 2015 during FBL-3 tumor challenge. An in vivo cytotoxicity significant expansion of tumor-specific Tregs (tetramerII+) was FIGURE 1. We next investigated the MHCII tetramers loaded with the H-2I-Ab–restricted CD4+ T cell impact of CD137 agonist on the phenotype and function of Tregs epitope FV H19-Env (23). and tumor size was measured.0001. 1A). 3D). Fig. In contrast. TNF-a. in the absence of CD8+ T cells.486 CONVERSION OF REGULATORY T CELLS BY CD137 compared with only FBL-3–challenged mice (see dotted line in assay with MHCII-FV peptide-loaded target cells revealed that Fig. When peptide-loaded targets from MHCII2/2 (Fig.jimmunol.017. Furthermore. more polyfunctional. CD4+ T cells usually express only little and NK cells (Fig. with 1 3 107 FBL-3 cells (1 3 107). Interestingly. producing all three proinflammatory cytokines 3A). we found a CD137. Thus. we next characterized differences in the antitumor of cytotoxic CD4+ T cells in CD8+ T cell–depleted animals are CD4+ T cell responses in mice depleted for CD8+ T cells com. influences the otherwise suppressive Treg response. proliferation. 2B. Notably. CD4+ T cells and treated with anti-CD137 as described in Materials and Methods. anti-CD137–treated mice.org/ at AZ Library on December 18. Mice were depleted for their CD8+. whereas the additional not only of CD8+ T cells but also CD4+ T cells and treated them anti-CD137 treatment resulted in an average of . a receptor for ligands of the after CD137 signaling. the CD4+ T cells in CD8-depleted. and depletion of CD8+ T cells is presented by solid line (A). B6 mice were injected s. Because we found that anti-CD137 treatment Downloaded from http://www. demonstrate that. Indeed. CD137 therapy induces strong helper and cytotoxic CD4+ T cell lation. . Fig. our data MHCII–restricted CD4+ T cell killing in our assay. killing activity anymore. indicating draining lymph nodes was Tregs on day 6 ptc and that this fre- that CD137 signaling enhances antitumor CD4+ T cell immunity. To validate the CD4+ T cell–mediated control of tumor CD4+ T cell killing was at the detection limit of the assay in only growth in CD8+ T cell–depleted animals. Using intracellular cytokine staining after stimulation with anti. there was no difference drives CD4+ T cell–mediated cytotoxicity and restores antitumor in total CD4+ T cell frequencies between anti-CD8– and anti. Interestingly. but also that these cells were CD4+ T cells in the tumor-draining lymph nodes (p = 0. To ana. As previously described (13). At 6 d ptc. we significantly more tetramer+ CD4+ T cells than only CD8+-depleted found that only 13% of the total CD4+ T cell population in mice that did not receive treatment (p = 0. 2A). and the specificity of the expanding Tregs. 1B–D). depletion of CD4+ and CD8+ T cells led to mice were used for the in vivo cytotoxicity assay. CD4+ T cells can the data indicate that. †Mice were euthanized because of progressive tumor growth. CD4+ T cells. Influence of different cell populations and anti-CD137 therapy on tumor formation. anti-CD137–treated mice had during FBL-3 tumor rejection. despite the presence of an anti-CD137 Ab. We next addressed whether the expanded Eomes because cytotoxicity is mostly mediated by CD8+ T cells. 2C). and IL-2 (Supplemental Fig. Tregs were natural Tregs (nTregs) or induced Tregs. in the absence of CD8+ T cells. depletion of CD8+ T cells and anti-CD137 treatment (B). we confirmed expression of CD137 on Tregs of FBL-3–challenged quantified the population of tumor-specific effector CD4+ T cells mice and found CD137 expression on a subpopulation of activated by staining lymphocytes from tumor-draining lymph nodes with (CD43+) Tregs (Supplemental Fig. immunity. 2D). no effector molecule granzyme B (Fig. we found no a loss of tumor control. strictly regulated and suppressed by Tregs impairing antitumor pared with those that also received CD137 agonist treatment immunity (13). Each solid line represents tumor progression in an individual mouse.

Representative contour plot of Eomes and GzmB expression (D) in different treatment of mice. This (Fig. with 1 3 107 FBL-3 cells and treated with anti-CD8 or anti-CD8+anti-CD137 as described in Materials and Methods. Foxp3+ CD4+ Treg CD4+ effector T cell balance. Supplemental Fig. Mean percentages of killing in an in vivo CTL assay (E) (described in Materials and Methods). CD8-depleted treated mice acquired the expression of CD154 whereas no granzyme B production was found in the cells of mice (CD40L).jimmunol. Statistically significant differences between the groups are given (*p . CD8-depleted mice (Supplemental effector T cell response. treatment may have altered or impaired the function of Tregs. 4D). 2B) and were therefore poten.c. We next addressed whether anti-CD137 treatment was also supported by the observation that Foxp3+ Tregs expanded also reprograms Tregs to a cytotoxic phenotype. tumor-bearing mice induced strong activation. Thus. of the Foxp3+ CD4+ T cells coexpressed Eomes after anti-CD137 proliferation. with 1 3 107 FBL-3 cells and treated with anti-CD8 or anti-CD8+anti-CD137 as described in Materials and Methods. Most of the Foxp3+ Eomes+ A subset of Tregs acquires helper and cytotoxic CD4+ T cell CD4+ T cells in the tumor-draining lymph node also expressed the functions after anti-CD137 treatment Treg activation marker Helios (data not shown). both populations of target cells were cotransferred i. draining lymph nodes were analyzed for expression of different molecules by flow cytometry. Anti-CD137 treatment programs tumor-specific cytotoxic CD4+ T cell differentiation and enhances killing of FBL-3 tumor cells. Target cells from the spleen and lymph nodes of the naive donor mice were either labeled with CFSE and pulsed with the specific CD4 epitope peptide to be tested or were obtained from CD45. we made the significant expansion of Foxp3+ Eomes+ CD4+ T cells in the contradictory observation of an increase in the antitumor CD4+ thymus of FBL-3–challenged.1+. we asked whether CD137 T cells started to produce granzyme B after anti-CD137 therapy. Tregs were excluded from analysis by intracellular marker Foxp3. lymphocytes were isolated from the draining lymph nodes and analyzed by flow cytometry to determine the percentage of remaining target cells that are either CFSE+ or CD45. In brief. but no ex. we used cells from MHCII2/2 as donor cells and labeled with either CFSE or CellTrace Violet. To verify tumor-specific killing.005. At day 6 ptc. Within the tumor-draining lymph nodes. CD137 expression on thymic nTregs was in these cells and found a significant increase in the expression found on ∼7. of Eomes in Foxp3+CD4+ T cells in anti-CD137–treated mice depleted mice (Supplemental Fig. Flow cytometry was used to determine the numbers of CD4+ T cells expressing intracellular transcription factors T-bet (B) and Eomes (C). 4E). B6 mice were inoculated s. and the mean numbers are indicated by a line. 3E). found (data not shown).0005). 4C). . whereas almost none of these double-positive cells were found before Ab treatment (Fig.05. 2D). Data in (D) are representative of at least three independent experiments. 4A). injection.c. To address this apparent disconnect in the Fig. tumor-bearing B6 mice were inoculated s. from the control groups (Fig. anti- Even though we found that CD137 agonist treatment significantly CD137 treatment also had an effect on thymic nTregs. The number of granzyme B–ex- Tregs from tumor-draining lymph nodes of anti-CD137–treated. pressing cells and the expression levels significantly increased. At day 5 after tumor inoculation. which was measured in direct ex vivo staining experiments Using intracellular cytokine staining. Cells from MHCII2/2 mice were transferred to the group treated with anti-CD8+anti-CD137. expression of TNF-a in Foxp3+CD154+CD4+ T cells. All tetramer+ T cells expressed cell-surface activation markers CD43 and CD11a. anti-CD137 treatment challenged with FBL-3 tumor cells (Fig. 4F. 4B. ***p . in the same amount into treated tumor-bearing mice. Twenty hours later. Indeed. CD8. which is critical for T cell–mediated help (Fig. and for MHCII2/2 targets either CFSE+ or CellTrace Violet+. compared with animals depleted only for CD8+ T cells and tial targets for anti-CD137 therapy.v. **p . Remarkably. Each dot represents an individual mouse. and differentiation of a subset of nTregs. CD137 treatment expressed Nrp1 (Fig. we found that anti-CD137 treatment induced significant no granzyme production in Tregs was found in tumor-challenged. Dif- ferences between the two groups were analyzed by using one-way ANOVA test. 0.The Journal of Immunology 487 Downloaded from http://www. CD8-depleted mice that did not receive anti-CD137 Ab treatment.1 naive mice and not pulsed. 0. but the majority of Tregs expanding after pression of other proinflammatory cytokines (IL-2 or IFN-g). The experiment was repeated three times with comparable results. Numbers of leukemia-specific CD4+TetII+ T cells reactive with I-Ab MHCII tetramers specific for FV-Env epitope are shown (A). suggesting that suggesting a major alteration of the functional Treg phenotype CD137 treatment mostly affected thymic-derived nTregs.org/ at AZ Library on December 18. 2C).5% of all Foxp3+ cells in tumor-challenged. 2015 FIGURE 2. 0. we found that a subset of without in vitro restimulation. Because Moreover. We therefore in the thymus of anti-CD8+anti-CD137–treated tumor-bearing quantified the expression of the T-box transcriptional factor Eomes mice (data not shown). up to 14% of CD8-depleted. It induced a augmented Treg responses in tumor-bearing mice.

The administration of CD137 agonist therapy led to the T cell epitope peptides that are expressed on FBL-3 tumor cells) delay in tumor growth compared with nontreated animals (data not target cells were injected i. 5A). B6 mice were inoculated s. DT administration (Fig. In PBS control mice. depleted for CD8+ T cells. 0.005. This FBL-3 cell killing was significantly reprogrammed Tregs are involved in the elimination of FBL-3 higher than in groups that received Foxp3+ cells either from . centage of Eomes+ cells among the Foxp3+ T cells (around 3%) mediated a potent in vivo killing activity (Fig.0005). draining lymph nodes were analyzed for expression of different molecules by flow cytometry.org/ at AZ Library on December 18. as well as the 20 h later. The FBL-3 cells were even cytotoxic killer cells. up to 90% of the tumor cells were Foxp3+ T cells after CD137 treatment prompted us to ask whether eliminated (Fig. Cell tracer-labeled peptide-loaded (FV CD4+ die (16). Forty-eight hours later. the total population of anti- frequency of Eomes+Foxp3+ T cells among the CD4+ T cells CD137–stimulated CD4+ T cells. This effect of CD137 stimulation could transferred into the peritoneal cavity of naive mice together with be demonstrated in different tumor models and was most efficient enriched donor Foxp3+ CD4+ T cells from tumor-challenged mice in the absence of CD8+ T cells. To investigate whether the effect of the anti-CD137 which express a DT receptor under the control of the Foxp3 Ab on Tregs is intrinsic or requires other cell populations. and the activation of Tregs was analyzed by surface expression of CD11a and CD43 (double positive) (C). 5A). This was important because we wanted to study the the transferred Foxp3+ T cells differentiated into Eomes-expressing immediate effect of the Foxp3+ cells on target cells and not the cells (Supplemental Fig. 4G). The Ab treatment of MTEC-challenged mice significantly day 5 ptc. suggesting that the effect of the anti. Anti-CD137 stimulates nTreg activation. Statistically significant differences between the groups are given (**p . either left untreated. performed a series of in vivo killing experiments in DEREG mice. The proliferation of Tregs was measured by the intracellular expression of Ki67 (B). 5B). A subpopulation of not shown). 1.jimmunol. In this model. Each dot represents an individual mouse. i. secondary effect that a more sustained Treg depletion has on ef- CD137 Ab on Tregs was induced by intrinsic signaling. fector cells.c. The maturation capacity was detected by surface expression of KLRG-1 (D). administration of DT allowed to rapidly (within performed an adoptive transfer of purified Foxp3+ cells from hours) and selectively delete . Total numbers of CD4+ Foxp3+ T cells and the percentages of total CD4+ T cells expressing Foxp3 are shown (A). we reisolated and CD137-stimulated Foxp3+CD4+ T cells mediate tumor cell quantified the remaining FBL-3 cells by a peritoneal lavage. At day 6 ptc. We reprogram a subpopulation of the Tregs to become cytotoxic ef. similar results were obtained in the MTEC tumor mors. these results demonstrate that CD137 signaling confirmed in another in vivo cytotoxicity assay. killing activity of CD137-stimulated Foxp3+ CD4+ T cells was Taken together. Strikingly. 0. DEREG mice were challenged with tu- Interestingly. 2D. the subpopulation of Eomes+ Foxp3+ cells did not restricted killing of peptide-loaded targets (Figs. the tumor cells are not controlled by tumor induce the population of Foxp3+ CD4+ T cells with the cytotoxic immunity. The percentages of Tregs in draining lymph nodes were analyzed for expression surface Nrp1 in naive and treated tumor-bearing mice (E).v. when no CD8 + T cells were depleted ablation because of i. activity was significantly decreased after specific Foxp3+ cell CD137 injection.v. tional T cells and the Foxp3+ CD4+ T cells that expressed Eomes. this was similar to what we found in the FBL-3 model after anti. The per. treated with anti-CD8 alone. fector T cell responses. and mice implanted with MTECs develop tumors and effector phenotype. suggesting a (Supplemental Fig. most likely including conven- compared with only MTEC-challenged mice (Fig. in which we di- reprograms subsets of Tregs into cytokine-expressing Th cells or rectly used FBL-3 tumor cells as targets. 4B). 3C). and treated with anti-CD137 to model. or treated with anti-CD8+anti-CD137. This seem to contribute to FBL-3 tumor rejection (data not shown). 2015 FIGURE 3. we promoter. However. Differences between the two groups were analyzed by using one-way ANOVA test. we conclude that only the CD137 costimulatory signal was able tumor cells and restore tumor immunity as shown in Fig. simultaneously with DT or PBS on shown). and the in vivo killing of the targets was determined increased the percentages of total Foxp3+ T cells. with 1 3 107 FBL-3 cells and treated with anti-CD8 or anti-CD8+anti- CD137 as described in Materials and Methods. ***p . as long as potent CD8+ T cells potent contribution of the converted Tregs to the total MHCII- were present. In killing mice that received Foxp3+ CD4+ T cells from anti-CD8+anti- The surprising alteration of the effector cell characteristics of CD4+ CD137–treated animals.v. The experiment was repeated three times with comparable results. 5A).95% of Foxp3+CD4+ T cells (data DEREG mice into CD137-treated RAG mice.488 CONVERSION OF REGULATORY T CELLS BY CD137 Downloaded from http://www.

Taken together. injection. 0. fect of these CD4+Foxp3+ Eomes+ T cells was demonstrated in the . 0.c.org/ at AZ Library on December 18. Differences between the two groups were analyzed by using one-way ANOVA test. to our knowledge. B6 mice were inoculated s. Flow cytometry was used to determine the percentages of CD4+ T cells expressing intracellular transcription factors Foxp3 and Eomes+ (G). We demonstrate that.2+ cells were from model to study the mechanisms of immunological tumor control DEREG mice and distinguished based on their GFP expression) in the course of CD137 costimulatory immunotherapy and lack of were injected into CD45. 5B). Interestingly. with 1 3 107 FBL-3 cells and treated with anti-CD8 or anti-CD8+anti-CD137 as described in Materials and Methods (A–F).05. 2015 FIGURE 4. responding to anti-CD137 therapy.005. After 6 d of CD8+ T cells. 5C).p. 5C). cytotoxic CD4+ T cells can restore antitumor the donor cells were isolated from the tumor-draining lymph node immunity after systemic CD137 activation. The appearance of Foxp3+CD4+ T cells with cytotoxic activity Discussion against tumor cells after anti-CD137 therapy may be caused by Despite advances in the development of immune therapeutic Tregs converting into cytotoxic effectors or rather. Differences between the two groups were analyzed by using t test. Every second day some mice were treated with anti-CD137 by i. nontreated or only anti-CD8–treated tumor-bearing mice (Fig. we were able to demonstrate recipient mice (Fig. At day 6 ptc. Interestingly. The percentages of total conCD4+ T cells expressing Eomes are shown in the lower right squares (E). The experiment was repeated three times with comparable results. (G) B6 mice were injected with 1 3 106 mouse tonsil epithelial cells s. indicating that indeed Tregs can be that under certain circumstances Tregs can turn on the cytolytic reprogrammed into effector CD4+ T cells after CD137 signaling program and gain cytotoxic effector functions. The numbers of CD154-expressing (A) and TNF-a–producing (B) Tregs are shown. this and analyzed for Foxp3 and Eomes expression. we performed an adoptive transfer experiment well understood.1 mice that were challenged with FBL-3 proper CD8+ T cell responses. Thus.The Journal of Immunology 489 Downloaded from http://www. **p . CD4+Foxp3+ T cells producing GzmB and MFI for GzmB in Foxp3+ T cells (F) in different treatment of mice are shown. in the absence cells and treated with anti-CD8+ and anti-CD137 Abs. Statistically significant differences between the groups are given (*p .c. ***p .0005). cytotoxic CD4+ strategies to combat cancer. Tregs upregulate effector-like and helper-like phenotypes after anti-CD137 treatment. However. none tumor clearance was partially due to a subset of CD4+Foxp3+ of the conventional CD4+ T cells (Foxp32 donor cells) that expressed T cells that were reprogrammed to eliminate immunogenic virus- Eomes also expressed Foxp3. Foxp3+ Tregs from draining lymph nodes were analyzed for different characteristics. a subset of Tregs obtained drives a subpopulation of nTregs into cytotoxic effector T cells that an effector phenotype of cytotoxic CD4+ T cells and was able to significantly contribute to FBL-3 tumor cell elimination. Animals were euthanized at day 8 ptc and draining lymph nodes were isolated. To test the changes occurring under immune-modulating therapies are not both possibilities. The antitumor ef- (Fig. 14% of the transferred Tregs induced tumor cells in response to CD137 agonist treatment. we used an FV-induced FBL-3 tumor where Foxp3+ or Foxp32 donor cells (CD45. In this study. the underlying immunobiology and T cells starting to express Foxp3 after CD137 signaling. (Foxp3+ donor cells) additionally induced Eomes expression in the for the first time. eliminate FBL-3 tumor cells in vivo.jimmunol. in the lower dorsal quadrant near the spine (G). The percentages of total Foxp3+ T cells expressing Eomes are shown in the upper right squares (E). Representative dot plots of TNF-a+CD154+ cells gated on CD4+Foxp3+ T cells (C) in different treatment of mice are shown. these data suggest that anti-CD137 treatment Thus. Numbers of CD4+ Foxp3+ T cells positive for tran- scription factor Eomes (D) and representative dot plots of Eomes+Foxp3+ cells gated on CD4+ T cells (E). 0.

ever. Previously we demonstrated that tumor-specific as costimulatory receptors for CD4+ and CD8+ T cells. in many tumor entities and tumor models. 2015 FIGURE 5. The results depict mean percentages of FBL-3 cells killing calculated according to control.p. and others. In addition.1+. A targeted activation of tumor-specific CD4+ T cells by possible immunotherapy target (26).org/ at AZ Library on December 18. only FBL-3–challenged mice. CD45. It has previously been reported that the cytotoxic potential of moted effective antitumor immune responses (27).12CD45. demonstrating that treat. therefore also be induced by competing with CD137 ligand gation. an tumor-specific CD4+ killer T cells depends on transcription factor anti-CD137 Ab that provides additional costimulatory signal to Eomes expression (6).05). whereas gray bars correspond to the mice additionally treated with DT. with sorted CD4+GFP2 or CD4+GFP+ cells from naive DEREG mice and treated with anti-CD8+anti-CD137 as described in Materials and Methods (C).v. because a subpopulation of Foxp3+ fect of the Ab. FBL-3 tumor-bearing DEREG mice were depleted for their CD8+ T cells and treated with anti-CD137. to antitumor immunity and immune control of tumor progression. 0. At day 5 after tumor inoculation. All experiments were repeated at least two times with comparable results. intracellular expression of Foxp3 and Eomes on transferred CD45.1 mice were challenged with FBl-3 cells and additionally transferred i. Each dot represents an individual mouse. The white bars correspond to the FBL-3–challenged and anti-CD8+anti-CD137–treated group. Among CD4+ T cells apart from being helper cells can substitute for the various TNFR family members including CD134 (OX-40).490 CONVERSION OF REGULATORY T CELLS BY CD137 Downloaded from http://www. which likely the effect of anti-CD137 Ab on Tregs. both populations of target cells together with DT were cotransferred i. mice received sorted CD4+GFP+ cells from either anti-CD8 or anti-CD8+anti-CD137 tumor-bearing or naive mice. Naive CD45.v. Statistically significant differences between the groups are given (*p . Functional plasticity of Tregs after anti-CD137 leads to FBL-3 tumor elimination. How- RAG mice turned on the expression of Eomes (Supplemental Fig. this is very unlikely for a gain-of-function ef- CD137 signaling in Tregs.1 mice received i.jimmunol. CD137 was the first identified as a (13).p. Mean percentages of killing in an in vivo CTL assay (A) (described in Materials and Methods). In addition. indicating that no other lymphocyte population is needed for exhausted and impaired to control tumor spread. it was demonstrated that Eomes cells was used. Target cells from the spleen and lymph nodes of the naive donor mice were either labeled with CFSE and pulsed with the specific CD4 epitope peptide to be tested or naive cells from CD45. but whether this also ligand binding. It remains unknown whether this Ab blocks natural plays an important role in initiating granzyme production in . However. lymphocytes were isolated from the draining lymph nodes and analyzed by flow cytometry to determine the percentage of remaining target cells that are either CFSE+ or CD45. injection of FBL-3 cells labeled with CFSE (B).1 mice were not pulsed. This is supported by our findings that a Previous studies have demonstrated that in the FBL-3 tumor subpopulation of Foxp3+ T cells transferred into CD137-treated model CD8+ T cells are essential to control tumor growth. CD27. highly immunogenic FBL-3 tumor model. Twenty hours later. Part of the biological effect of the Ab could applies to less immunogenic tumors is an area of future investi. Mice were sacrificed 48 h later and i. lavage was performed. ment of tumor-bearing mice with CD137 agonist therapy pro. CD8+ T cells are 4B). Six days ptc. cells expresses CD137. as the one that we describe in this article. The cytolytic program might be directly induced by binding. contributes to the often-observed limited efficacy of immuno- The TNFR family members have been found to play major roles therapy (28). function of CD8+ T cells and efficiently control tumor growth CD357 (GITR). in the same amount into treated DEREG tumor-bearing mice. The efficacy of its ligation CD137 costimulatory Ab might therefore substantially contribute was shown in different tumor studies. In this work.2+ cells was detected. Differences between the two groups were analyzed by using one-way ANOVA test.

and therefore counteracting antitumor seem to affect T cell responses. 29–31). H. Klussman. proliferation. Moreover. Montalvo.. 2006. D. 229: 126–144. graft rejection (35). shown that triggering the CD134 pathway impairs the immuno- Generally the role of Tregs is an immunosuppressive regulation suppressive function of Tregs and results in increased lethality of activated CD8+ T cells and CD4+ T cells. C. Foxp3+CD4+ T cells also showed Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration. has shown that the CD4+ T cell compartment. It has been T cells. P. K. K. Lehman. Gajewski. W. Inter. supporting the functional change of the Foxp3+CD4+ T cells. Interestingly. Zhang.. However. Siadak. It has been shown that in draining immunotherapy of cancer (43). PD-1 and CTLA- without loss of Foxp3. makes the interpretation of the data somewhat complicated. Downloaded from http://www. Choi et al. Med. In this work. and J. However. Chalupny. 29). K. the subpopulation of granzyme B+ Foxp3+ CD137 can be constitutively expressed on CD4+Foxp3+ Tregs cells seems to acquire an effector rather than regulatory pheno- (7. CD4+ T cells. CD137 signaling can obviously induce a killed inflammatory effector T cells (39). Curran. the CD154+Foxp3+ The authors have no financial conflicts of interest. In. 5. Tritchler. Sci. J. A. similar to CD8+ T cells. in which Eomes. H. S. However. Immunol. tumors (34). K. CD137 stimulation. Tregs can undergo reprogram. We found the emergence of a Treg sub. These results provide indirect evidence that the Foxp3+ T cell population was smaller than in CD8-depleted mice Eomes+CD4+ T cells may not mediate immunosuppressive effects (Supplemental Fig. 2. in this study.’s study (29) was performed by staining for CD25 recognition was absent. F. surface expression (29). main function was clearly immune suppressive because they totoxic function. R. Allison. CD134 Abs protects mice from tumor progression by inhibiting This phenotype includes a helper-like role and has been observed the suppressive function of Tregs (42). Brown. Tregs upregulated in these studies. E. pressing Eomes could also be found in another tumor model Combination therapy with anti-CTL antigen-4 and anti-4-1BB antibodies en- (Fig. Moreover. However. Zheng. Abdessalam. 66: 7276–7284. Chang. M. this novel population of CD4+Foxp3+ T cells coex. we showed that upon anti-CD137 therapy. However. Driessens. Loebbermann et al. In contrast with our study. T. In our present work. we found that CD137 ligation on Tregs increases not only typic characterization is based on intracellular markers. Foxp3 expression be determined in future studies. F. We show in this study that they kill tumor cells but do not suppressive Treg activity. Tregs that contained ∼15% of Foxp3+ Eomes+ cells was slightly duction of CD4+ T cells that express Foxp3 and at the same time less suppressive for the proliferation of effector CD8+ T cells than TNF-a and granzyme B by CD137 Ab was also observed in the a Treg population without this subpopulation (Supplemental Fig. including Tregs. A. This is indeed very inter- in different models. and Y. In a recent study by Sharma et al. ming and upregulate CD154 (37). May. In line with this study. 186: 47–55. and cytokine production. CD154 is an important functional in vivo. F. 2009. After anti- cytotoxic activity of these cells. ventional T cells cannot be directly tested because their pheno- estingly. 2D). (Eomes/granzyme B). initiates the cy. Emswiler. we provide evi- by the converted Tregs was lost. Proc. 2010. J. Montalvo. A. CD4+ T cell subset expressed the proinflammatory cytokine TNF-a. Kocak. is CD134. change their functional capability and turn them into cytotoxic 3. Costimulatory and coinhibitory that in response to specific inflammatory signals. or esting data for developing new concepts in tumor immune therapy. Kline.. Jarjoura. . Lute. in that study. et al. and J. Jr. Loo. Shuford. PLoS increased expression of markers delineating a cytotoxic program One 6: e19499. In particular. Al-Shamkhani. Exp. with anti-CD134 Abs or other costimulatory molecules has to flammatory cytokines. tolytic molecules in Tregs and converts these cells into Foxp3+ tained Foxp3 expression but additionally increased expression of cytotoxic killer cells that contribute to Ag-specific tumor rejection markers of Th cells (CD154). P. the reprograming of Tregs that an increased tumor-specific killing by CD4+ T cells after anti. Tregs rather main. and T. Rev.. W. (29) on conventional T cells and are therefore distinct from the Treg showed that CD137 ligation on Tregs inhibited their suppressive population described by Loebbermann et al. 4- population that expresses the cytotoxic transcription factor Eomes 1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead while maintaining Foxp3 identity after anti-CD137 treatment. Acad. there is a in mice with graft-versus-host disease (41). The use of a nonspecific Treg marker such as Another member of the TNFR superfamily that is transiently CD25. J. G. Thus. Because agonistic Abs to CD137 are in clinical trials for mediator of T cell help (36). Thus. T. USA 107: whether Foxp3+CD4+ T cells can undergo conversion that will 4275–4280. P.jimmunol. W. D. such as proin. M. although the size of the cytotoxic CD4+ 4A). Natl. transcriptional program in CD4+ T cells that results in potent the authors did not perform costimulatory treatment. into cytotoxic Eomes+CD4+ T cells was accompanied by gran- CD137 therapy correlates with levels of Eomes and granzyme B in zyme B production (Fig.. 4G) and in chronically FV-infected mice after anti-CD137 hances cancer immunity and reduces autoimmunity. 2015 presence of CD8+ T cells. Their suppressive effect on con- CD4+ T cell responses. M. X. J. (39). 3). Foxp3+CD4+ receptors in anti-tumor immunity. W. Their cells. these cells were redirected by bispe- manipulate their functions (31). 1997. P. Interestingly. suggesting a complete reversion dence that CD137 stimulation can induce the production of cy- of Tregs into Th cells. Raecho. the plasticity of Tregs during treatment genes characteristic for a Th cell phenotype. Allison. H. we show in this article therapy (data not shown). 4. A. it has not been addressed so far 4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. 2011. which do the size of the Treg population but also drives alterations of Treg not allow isolation as life cells. (38) it has also been demonstrated References 1. Kim. D. T cells were transformed into biologically important helper cells. a population of total functions to effector cells in the absence of CD8+ T cells. Addey et al. Exten. to the amplification in vivo of cytotoxic T cell responses. CD154 was expressed on Disclosures a subset of Foxp3+CD4+ T cells. Cancer Res. Yagita. is currently unknown (5. However. information about their possible mode of action. However. A. (34) demonstrated but the phenotype of Tregs has not been investigated in detail that. in the murine urothelial carcinoma model.The Journal of Immunology 491 T cells (24). 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005. Differences between the two groups were analyzed by using one-way ANOVA test. per million cells 300000 200000 100000 0 Naïve CD8 CD8+CD137 B C D Numbers of CD4+Foxp3. . B6 mice were inoculated s.0005). At day 6 ptc draining lymph nodes were analyzed for expression of different molecules by flow cytometry.c. Statistically significant differences between the groups are given in the figures (**P˂0.CD154+IL-2+ ** *** ** 25000 ** 50000 *** 15000 *** per million cells per million cells per million cells 20000 40000 15000 10000 30000 10000 20000 5000 5000 10000 0 0 0 Naïve CD8 CD8+CD137 Naïve CD8 CD8+CD137 Naïve CD8 CD8+CD137 Supplementary figure 1. (A) Total numbers of CD4 + T cells are shown. αCD137 stimulates CD4+ T cells with the helper phenotype. with 1×107 FBL-3 cells and treated with αCD8 or αCD8+αCD137 as described in methods. ***P˂0. TNF-α (C) and IL-2 (D) are shown. Total numbers of CD4+ T cells expressing cytokines IFN-γ (B). The experiment was repeated three to five times with comparable results.CD154+TNF- + IFN-γ TNF-α IL-2 Numbers of CD4+Foxp3-CD154+IFN- + Numbers of CD4+Foxp3.s. Supplementary figure 1 A CD4+ Numbers of CD4+ T cells 400000 n.

Differences between the two groups were analyzed by using one-way ANOVA test. Data are representative for at least 3 independent experiments. with 1×107 FBL-3 cells and treated with αCD8 or αCD8+αCD137 as described in methods. C. B.05). Data are representative for at least 3 independent experiments. B6 mice were treated as described above. Supplementary figure 2 Gated on GFP+CD4+ T cells B CD137+ %CD137+ /GFP+ CD4+ T cells A DEREG naive DEREG+FBL-3 DEREG +FBL-3+αCD8 15 3% 15% 31% 10 5 CD43 0 CD137 C D Gated on CD4+ T cells wt CD8 naive FBL-3+αCD8 FBL-3+αCD8+ αCD137 0. At day 6 post tumor challenge (ptc) draining lymph nodes were analyzed for expression CD43 and CD137 molecules by flow cytometry. At day 6 ptc the thymus was analyzed by flow cytometry. with 1×107 FBL-3 cells and some were treated with αCD137 and depleted for CD8+ T cells as described in methods.c. DEREG mice were inoculated s.5% 0. Representative dot plots of Foxp3 and GzmB co-expression in different group of mice are shown. Percentages of CD137 among CD4+GFP+ T cells in the thymus are shown. Surface expression of CD137 molecule on GFP+Foxp3+CD4+ T cells.c. Total numbers of CD4+ Foxp3+Eomes+T cells in the thymus are shown. with 1×107 FBL-3 cells and treated with αCD8 or αCD8+αCD137. GFP+ cells were analyzed for CD137 expression. B. DEREG mice were inoculated s. B6 mice were inoculated s. Representative dot plots of CD43 and CD137 co-expression in different group of mice are shown. . The experiment was repeated two times with comparable results. At day 6 ptc the thymus was analyzed by flow cytometry. Intracellular expression of GzmB molecule on CD4+ T cells. A.c. Foxp3+ cells were analyzed for expression of Eomes. D. Tregs in the thymus of tumor-bearing mice express CD137 and Eomes following CD137 agonist therapy. Statistically significant differences between the groups are given in the figures (*P˂0.7% 11% Foxp3 GzmB Supplementary figure 2.C.

(C) The frequencies of CD4+ Foxp3+ T cells positive for transcription factor Eomes. (B) The frequencies of CD154 expressing and TNF-α producing Foxp3+ T cells cells are shown. αCD137 converts Tregs in the presence of CD8+ T cells.0005. Statistically significant differences between the groups are given in the figures (*P˂0. * per million cells per million cells 4000 n.05. **P˂0. At day 6 post tumor challenge (ptc) draining lymph nodes were analyzed for expression of different molecules by flow cytometry. . The experiment was repeated one to two times with comparable results. with 1×107 FBL-3 cells and treated with αCD8 or αCD8+αCD137 as described in methods. Differences between the two groups were analyzed by using one- way ANOVA test.c.s. – not significant). (A) Percentages of Foxp3+ among CD4+ T cells.s. ***P˂0.s. 15000 * 3000 10000 2000 5000 1000 0 0 Naïve CD137 CD8+CD137 Naïve CD137 CD8+CD137 Supplementary figure 3.Supplementary figure 3 A % Foxp3+ B TNFα+ *** CD4 Foxp3+CD154+TNF + 40 *** %Foxp3+/CD4+ T cells ** 2500 * per million cells 30 2000 *** Numbers of 1500 20 1000 * 10 500 + 0 0 Naïve CD137 CD8+CD137 Naïve CD137 CD8+CD137 C D Eomes+ GzmB+ Numbers of CD4+Foxp3+Eomes+ Numbers of CD4+Foxp3+GzmB+ 5000 * 20000 *** n. n.005. B6 mice were inoculated s. (D) CD4+Foxp3+ T cells producing GzmB.

treated with αCD8 or αCD8+αCD137. whereas the control group received PBS. These cells were cultured with CD8+ T cells from FV TCRtgxCD45.1 mice incubated for 3 hrs with BM-derived DCs. FMO: fluorescence minus one control for Eomes. On day 3. loaded with the associated FV peptide at the indicated ratios. To compare the suppressive capacity of Tregs. A. . Foxp3+GFP+CD4+ T cells were sorted to >95% purity from naïve DEREG mice and were adoptively transferred into RAG mice. Histograms of Eomes expression of Foxp3+- gated cells are shown. There is no significant difference in the groups. On day 1 and 2 post transfer one group of mice received αCD137 treatment. In vitro suppression of antigen-specific CD8+ T cell-proliferation by Tregs.Supplementary figure 4 Gated on Foxp3+ T cells A % of proliferated CD8+ T cells Suppresssion of proliferation B 80 FMO for Eomes 60 transfer transfer + αCD137 Normalized to mode 40 20 0 no Tregs 1:2 1:1 2:1 no peptide CD8+ T : Tregs +FBL-3+CD8 +FBL-3+CD8 +CD137 Eomes Supplementary figure 4. B. 72 hrs later proliferation of CD8+ T cells based on Ki67 expression was measured by flow cytometry. In vivo stimulation of Foxp3+CD4+ T cells by αCD137 antibody in RAG mice. treated and untreated mice were sacrificed and transferred Foxp3+ T cells from the bone marrow were analyzed for transcription factor Eomes expression by flow cytometry. Foxp3/GFP+CD4+ T cells were sorted to >95% purity from DEREG mice.