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Depletion of CD4⁺CD25⁺ Regulatory T Cells Promotes a Tumor-Specific Immune Response in Pancreas Cancer–Bearing Mice

¹ Department of Surgery, Washington University School of Medicine, Box 8109, 660 S. Euclid Ave., St. Louis, Missouri 63110, USA
² Department of Surgery, Divisions of General Surgery and Surgical Research, University of Basel, Spitalstrasse 21, 4031 Basel, Switzerland
³ Alvin J. Siteman Cancer Center, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, Missouri 63110, USA

Correspondence: Address correspondence and reprint requests to: David C. Linehan, MD; E-mail: linehand{at}wudosis.wustl.edu.

	ABSTRACT

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Background: Pancreas cancer–bearing mice have an increased prevalence of immunosuppressive CD4⁺CD25⁺ regulatory T cells (T_reg). Depletion of T_reg results in smaller tumors and prolonged host survival. The objective of this study was to evaluate the tumor-specific immune response after depletion of T_reg alone or in combination with a cancer vaccine.

Methods: Four groups of C57BL/6 mice were challenged with pancreas adenocarcinoma cells (Pan02). The mice received four combinations of antibody-mediated T_reg depletion and whole tumor cell vaccination: (1) no treatment, (2) T_reg depletion only, (3) vaccination only, or (4) T_reg depletion and vaccination. Splenocytes and lymphocytes from tumor-draining lymph nodes were analyzed for tumor-specific release of interferon by enzyme-linked immunosorbent spot assay.

Results: In T_reg-depleted and vaccinated mice, a strong statistical trend toward smaller tumors (P = .05) and longer survival (P = .054) was found compared with untreated mice. T_reg-depleted mice showed significantly more tumor-specific cells than undepleted mice (P = .02). The number of tumor-specific cells was significantly higher in tumor-draining lymph nodes than in the spleen (P = .002). Similarly, significantly more tumor-specific cells were found in spleens of T_reg-depleted and vaccinated mice than in vaccinated-only mice (P = .009).

Conclusions: Depletion of T_reg alone or in combination with a whole tumor cell vaccine promotes a tumor-specific immune response. Thus, strategies incorporating T_reg depletion might improve the efficacy of cancer vaccines.

Key Words: Tumor immunity • Regulatory T cells • Cancer vaccine • Pancreas cancer • Animal study

	INTRODUCTION

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Regulatory T cells (T_reg) actively suppress immune responses. They maintain immune homeostasis in humans and in mice by controlling the peripheral tolerance to self antigens, therefore protecting the host against autoimmune diseases.^1,2 A multitude of autoimmune diseases such as thyroiditis,³ insulitis,⁴ gastritis,⁵ and oophoritis⁶ can be triggered by depletion of T_reg. Conversely, the adoptive transfer of T_reg inhibited development of colitis in a murine model of inflammatory bowel disease.⁷ T_reg are activated through their T-cell receptor in an antigen-specific manner. After activation, however, they can inhibit immune effector cells in an antigen-unspecific way.⁸ They suppress proliferation of activated CD4⁺, CD8⁺, and natural killer T cells and inhibit interferon (IFN)- and interleukin (IL)-2 production by mechanisms requiring cell-to-cell contact.^9,10 Both murine^11,12 and human¹³ T_reg secrete the immunosuppressive cytokines transforming growth factor (TGF)-ß and IL-10 but secrete only low levels of IFN-.

T_reg have been described as CD4⁺CD25⁺ cells, thus making it difficult to distinguish T_reg from activated CD4⁺ T cells, which also express the -chain of the IL-2 receptor, CD25. Recently, Foxp3, a gene that encodes the forkhead/winged helix transcription factor, scurfin, has been found to be specifically expressed by T_reg and to program their development and function.¹⁴ However, until strategies to target Foxp3 and to shut down its function are established, isolation and depletion of T_reg mainly depend on such relatively unspecific targets as surface CD25 and intracellular cytotoxic T lymphocyte–associated antigen (CTLA)-4.

Unfortunately, T_reg that protect the host against autoimmune diseases may also suppress spontaneous and cancer vaccine–induced antitumor immune responses in patients.^15–18 The suppressive effect of T_reg on the activation of antitumor effector cells has also been demonstrated in various murine tumor models.^19–22 Injections of a monoclonal anti-CD25 antibody led to rejection of different murine tumors,^23–25 thus underlining the key role T_reg are playing in blocking the immune response against cancer.

We previously reported that patients with breast or pancreas adenocarcinoma have an increased prevalence of T_reg in the peripheral blood and in the tumor microenvironment,²⁶ a finding that has been confirmed by several groups and for different cancers.^15–17 We recently provided evidence that the progressive growth of tumor upregulates the prevalence of T_reg in a murine pancreas cancer model.²⁷ Intraperitoneal injection of anti-CD25 antibodies effectively depleted T_reg in the spleen and tumor-draining lymph nodes (TDLNs) up to 3 weeks after the last injection. Subsequently, tumor-bearing T_reg-depleted mice showed slower tumor growth and prolonged survival.²⁷

The objective of this study was to evaluate the tumor-specific immune response after depletion of T_reg alone or in combination with a whole tumor cell vaccine. The experimental data outlined in this article provide evidence that depletion of T_reg or its combination with a cancer vaccine promotes a tumor-specific immune response.

	METHODS

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Animals and Tumor Cell Lines
Eight-week-old female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed under pathogen-free conditions. All experimental protocols were approved by the institutional Animal Studies Committee, and all experiments were subsequently conducted in compliance with the institutional guidelines for the care and use of research animals.

The poorly immunogenic mouse pancreas adenocarcinoma cell line, Pan02 (National Cancer Institute, Frederick, MD), was cultured in RPMI-1640 medium (Cellgro, Herndon, VA) supplemented with 10% (v/v) fetal bovine serum (Valley Biomedical, Winchester, VA), 1% penicillin/streptomycin (Cellgro), 1% glutamine (Cellgro), and 1% 4-(2-hydroxyethyl)-1-pipe-razineethanesulfonic acid (Cellgro).

T_reg Depletion and Vaccination
C57BL/6 mice were divided into four groups. Each group was challenged with 2.5 x 10⁵ viable Pan02 cells on day 0 (injected subcutaneously in the left inner thigh). The mice received one of the following combinations of T_reg depletion and vaccination: group 1, no T_reg depletion and no vaccination; group 2, T_reg depletion only (no vaccination); group 3, vaccination only (no T_reg depletion); and group 4, T_reg depletion and vaccination. As in our previous study,²⁷ mice were T_reg depleted with three weekly intraperitoneal injections of 100 µg of anti-CD25 monoclonal antibodies (clone PC61, unlabeled rat anti-mouse CD25 immunoglobulin [Ig]G without sodium azide and low endotoxin; BD Pharmingen Biosciences, San Diego, CA) starting on day –1. Control groups received intraperitoneal injections of 100 µg of rat IgG_2b isotype control (BD Pharmingen) at identical time points (Fig. 1). The vaccine, consisting of 1 x 10⁶ lethally irradiated Pan02 cells (10,000 rads) mixed with 20 ng of recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF; Biosource, Camarillo, CA), was injected subcutaneously in the left inner thigh three times at weekly intervals on days –21, –14, and –7 (Fig. 1).

View larger version (8K): [in this window] [in a new window]   FIG. 1. Schedule for vaccination (vacc), regulatory T cell depletion (depl), and tumor implantation.

Transcutaneous Tumor Measurement
Twenty C57BL/6 mice were divided into four groups of five mice each and received combinations of T_reg depletion and vaccination as outlined previously. The two largest axes of the tumor were measured transcutaneously at weekly intervals by using a Vernier caliper, and the tumor volume was calculated as (major axis x minor axis x .5236).²⁸ Average tumor volumes are given; error bars indicate the standard error of the mean.

Analysis of the Tumor-Specific Immune Response by IFN- Enzyme-Linked Immunosorbent Spot Assay
For functional studies, C57BL/6 mice were again divided into four groups of mice and received combinations of T_reg depletion and vaccination as out-lined previously. Mice were sacrificed at given time points, and spleens and TDLNs (left inguinal and periaortic) were harvested. After single-cell suspensions were made, tumor-specific cytokine release by splenocytes or lymphocytes from TDLNs was assessed with a commercially available murine IFN- enzyme-linked immunosorbent spot assay (ELI-SPOT; Biosource). In precoated wells, 200,000 effector cells per well were cocultured at 37°C with 100,000 Pan02 cells per well or with medium only. Every sample was run in triplicate. The Pan02 tumor cells were pretreated with IFN- 250 U/mL for 24 hours to upregulate major histocompatibility complex class I expression and underwent three freeze-thaw cycles between liquid nitrogen and room temperature to reduce their adherence to the wells. After 19 hours of coculture, the plate was developed according to the manufacturer’s protocol, and the spots were counted under a dissection microscope at x40 magnification. Results are given as the average of triplicate samples (number of spot-forming cells per 10⁵ plated cells) corrected for the individual medium background. Error bars indicate the standard error of the mean.

Statistics
Survival data were depicted as Kaplan-Meier curves and were compared by using the log-rank test. Tumor volumes from transcutaneous measurements were analyzed by Mann-Whitney U-test. ELISPOT results were analyzed by Student’s t-test. All tests were two sided. P < .05 was considered significant.

	RESULTS

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T_reg Depletion and Vaccination Delays Tumor Growth and Improves Survival
Forty C57BL/6 mice were divided into 4 groups of 10 mice each and received combinations of T_reg depletion and vaccination as outlined in Fig. 1. All mice were implanted with viable Pan02 tumor at day 0 and followed up for survival. The median survival time for the respective groups was as follows: no T_reg depletion and no vaccination, 72.5 days (range, 56–80 days); T_reg depletion only, 69.5 days (range, 59–86 days); vaccination only, 71.0 days (range, 58–76 days); and T_reg depletion and vaccination, 77.5 days (range, 61–101 days). In T_reg-depleted and vaccinated mice, a statistical trend toward longer survival (P = .054) was found compared with untreated mice (Fig. 2A). Additionally, transcutaneous measurements of the tumor volume were performed at weekly intervals in 20 mice (4 groups of 5 mice each). T_reg-depleted and vaccinated mice showed a strong statistical trend toward smaller tumors compared with the control group (P = .05; Fig. 2B).

View larger version (22K): [in this window] [in a new window]   FIG. 2. In regulatory T cell (Treg)-depleted and vaccinated mice, a strong statistical trend toward smaller tumors and longer survival was found compared with untreated mice. Mice were divided into four groups: no treatment (no depl, no vacc), Treg depletion (depl only), vaccination (vacc only), or a combination of Treg depletion and vaccination (depl + vacc). All mice were challenged with viable pancreas cancer cells, Pan02, implanted in the left hind limb. For survival experiments, animals were allowed to die spontaneously or were sacrificed once the largest tumor diameter reached 2 cm. Either death event is depicted in the Kaplan-Meier curves. Treg-depleted and vaccinated mice had an improved survival (P = .054; log-rank test) as compared with untreated mice (A). Additionally, tumor size was measured transcutaneously at weekly intervals, and tumor volumes were calculated. Shown is the average tumor volume per group; error bars indicate the standard error of the mean. Treg-depleted and vaccinated mice had smaller tumors (P = .05; Mann-Whitney U-test) than untreated mice (B).

View larger version (26K): [in this window] [in a new window]   FIG. 3. Regulatory T cell (Treg) depletion results in an increase of tumor-specific effectors. Tumor-bearing mice received no treatment (no depl, no vacc) or Treg depletion (depl only) by three weekly injections of anti-CD25 antibody. At weeks 3 and 6, splenocytes (spleno) and tumor-draining lymph node–derived lymphocytes (TDLN) were harvested and tested for tumor-specific interferon release by enzyme-linked immunosorbent spot assay. For Treg-depleted mice, this analysis was performed weekly. Shown is the average number of spot-forming cells (SFCs) per 100,000 cells; error bars indicate the standard error of the mean. Data were analyzed by Student’s t-test. Treg-depleted mice showed significantly more tumor-specific splenocytes and TDLN-derived lymphocytes than nondepleted mice at week 6 (A). Additionally, significantly more tumor-specific lymphocytes were found in the TDLN than in the spleen at weeks 4 and 5 (B). n.s., not significant.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Regulatory T cell (Treg) depletion enhances the tumor- specific response in splenocytes after vaccination. Tumor-bearing mice received vaccination with a whole-cell cancer vaccine (vacc only) or a combination of Treg depletion and vaccination (depl + vacc). Control mice received injections of immunoglobulin G isotype antibodies diluted in Hanks’ balanced salt solution. At weeks 3 and 6, splenocytes (spleno) and tumor-draining lymph node–derived lymphocytes (TDLN) were harvested and tested for tumor-specific interferon release by enzyme-linked immunosorbent spot assay. Treg depletion enhances the tumor-specific response in the spleen after vaccination, because vaccinated and depleted mice showed significantly (t-test) more tumor-specific splenocytes at week 6 than vaccinated but nondepleted mice. n.s., not significant; SFC, spot-forming cell.

	DISCUSSION

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T_reg depletion, used without vaccination, led to an increase of tumor-specific effectors in the spleen and the TDLNs compared with undepleted mice (Fig. 3A). This effect was still significant at week 6, i.e., 4 weeks after the last depletion. This usually is a moment in the course of the disease when T_reg quickly start to repopulate, particularly in tumor-bearing mice.²⁷ In this study, significantly more tumor-specific effectors were found in the tumor microenvironment (TDLN) than in the spleen at several points in time (Fig. 3B). Because this is the region of intensive tumor antigen presentation and recognition, but also the region found to show the highest prevalence of T_reg in undepleted mice,²⁷ T_reg depletion as the sole treatment seems to have its most distinct effect in the tumor microenvironment. These data are supported by our recent findings suggesting that transforming growth factor ß secreted by Pan02 promotes the induction of Foxp3 and, thereby, the regulatory phenotype of T_reg.²⁹

In vaccinated mice, tumor-specific effectors were similarly able to proliferate, as evidenced by an increase of the number of tumor-specific cells from weeks 3 through 6. This effect was most prominent in the spleen; this is in line with findings by Dranoff et al.,³⁰ who vaccinated mice with different irradiated, GM-CSF–secreting tumor cells and induced an increased systemic immunity. Additional T_reg depletion further enhanced the tumor-specific response in splenocytes after vaccination in our experiments; T_reg-depleted and vaccinated mice showed significantly more tumor-specific splenocytes at week 6 than mice that were vaccinated only (Fig. 4). However, the numbers of tumor-specific effectors in the TDLNs were roughly equal between the two groups. This finding is similar to data by Sutmuller et al.²⁵ Mice challenged with B16 melanoma cells were vaccinated with irradiated, GM-CSF–secreting B16 cells, and T_reg were depleted with a single injection of anti-CD25 antibodies. Additionally, CTLA-4 was blocked by anti–CTLA-4 antibodies. The authors found an overall increase of tyrosinase-related protein 2–specific IFN-–producing cytotoxic T lymphocytes (CTLs). However, this effect was mostly seen in the spleen, where the number of tyrosinase-related protein 2–specific CTLs almost tripled, whereas it increased only by approximately 80% in the TDLN. Because our ELISPOT analysis was performed only at weeks 3 and 6, subtle changes or a peak synergistic effect between these time points might have been missed.

An alternative explanation of the aforementioned observation might be that our immunization and depletion schedule was not optimal. Because T_reg typically start to increase in prevalence as the tumo grows, we adhered to our previously described T_reg depletion protocol:²⁷ anti-CD25 antibody injection occurred 1 day before the initial tumor challenge and then two more times at weekly intervals. Not to interfere with these injections, we chose to administer the vaccination in this experiment before tumor challenge and antibody-mediated T_reg depletion. By doing so, however, some CD4⁺ and CD8⁺ T cells, activated by the vaccine and therefore expressing the CD25 antigen, might have been eliminated by the antibodies, as previously shown by Sutmuller et al.²⁵ Additionally, Dranoff et al.³⁰ showed that both CD4⁺ and CD8⁺ T cells were required for effective vaccination with a GM-CSF–secreting whole-cell cancer vaccine. Elimination of some activated T cells might therefore have mitigated the overall effect of our vaccine. We speculate that this effect was more prominent in the tumor microenvironment than systemically. This problem might be overcome in the future when more specific techniques to deplete T_reg are available. Alternatively, several strategies are currently being tested to block or overcome T_reg-mediated suppression, such as through anti–CTLA-4–or anti-glucocorticoid–induced tumor necrosis factor receptor antibodies.^31,32

Our experimental setting did not evaluate changes in the general cell-dependent immune response over time (by, e.g., analyzing the immune response against a number of different tumor cell lines). Thus, it is possible that the observed increased number of tumor-specific immune effectors after T_reg depletion resulted from a selective, tumor-directed, or generalized increase in the cell-dependent immune response. Because we do not know which antigens are recognized by the immune effectors, we cannot conclusively state which mechanism is at play. Similarly, Sutmuller et al.²⁵ observed an increase of tumor-specific CTLs after T_reg depletion and vaccination in a murine melanoma model; however, they did not provide data on the development of the general immune response. In light of a recent clinical study³³ in which considerable numbers of patients showed grade 3 and 4 autoimmune effects after repeated T_reg blockade by human anti–CTLA-4 anti-bodies, the evaluation of the general immune response (and of autoimmune effects, in particular) will be of paramount importance for further studies including T_reg depletion. Recently, Dr. William Hawkins in our department initiated studies in which mice are immunized against the pancreas cancer antigen, mesothelin, before a challenge with syngeneic mesothelin-expressing Pan02 tumor cells. Approximately 40% of the mice are protected against tumor development, and this suggests that mesothelin is the target antigen expressed on Pan02 tumor cells (unpublished data). In collaboration with Dr. Hawkins, we plan to further address how modulation of T_reg suppression promotes antitumor immunity.

In conclusion, these findings provide proof of principle that depletion of CD4⁺CD25⁺ T_reg used as a sole treatment or in combination with a whole tumor cell vaccine promotes a tumor-specific immune response in pancreas cancer–bearing mice. Synergistic effects between the two modalities were noted. However, they may have been mitigated by suboptimal timing of the treatments and the partially unspecific nature of the antibody-mediated depletion. Thus, strategies incorporating T_reg depletion or blockade might improve the efficacy of cancer vaccines.

	ACKNOWLEDGMENTS

 
Supported in part by National Institutes of Health grants K08 CA87018-01 (D.C.L.) and T32 CA09621-11 (T.J.E.), as well as by grants from the Swiss National Science Foundation (81BE-067988) and the Regional Cancer League of Basel, Switzerland (C.T.V.)

	FOOTNOTES

 
Presented at the 57th Annual Cancer Symposium of the Society of Surgical Oncology, New York, New York, March 18–21, 2004.

Received for publication October 17, 2005. Accepted for publication February 13, 2006.

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