This information is current as of July 5, Kutlu G. Elpek, Chantale Lacelle, Narendra P. Singh, Esma S. Yolcu and Haval Shirwan

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1 This information is current as of July 5, CD4 + CD25 + T Regulatory Cells Dominate Multiple Immune Evasion Mechanisms in Early but Not Late Phases of Tumor Development in a B Cell Lymphoma Model Kutlu G. Elpek, Chantale Lacelle, Narendra P. Singh, Esma S. Yolcu and Haval Shirwan J Immunol 2007; 178: ; ; doi: /jimmunol References Subscription Permissions Alerts This article cites 58 articles, 31 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on July 5, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2007 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology CD4 CD25 T Regulatory Cells Dominate Multiple Immune Evasion Mechanisms in Early but Not Late Phases of Tumor Development in a B Cell Lymphoma Model 1 Kutlu G. Elpek, Chantale Lacelle, Narendra P. Singh, Esma S. Yolcu, and Haval Shirwan 2 Tumors use a complex set of direct and indirect mechanisms to evade the immune system. Naturally arising CD4 CD25 FoxP3 T regulatory (Treg) cells have been implicated recently in tumor immune escape mechanism, but the relative contribution of these cells to overall tumor progression compared with other immune evasion mechanisms remains to be elucidated. Using the A20 B cell lymphoma as a transplantable tumor model, we demonstrate that this tumor employs multiple direct (expression of immunoinhibitory molecule PD-L1, IDO, and IL-10, and lack of expression of CD80 costimulatory molecule) and indirect (downregulation of APC function and induction of Treg cells) immune evasion mechanisms. Importantly, Treg cells served as the dominant immune escape mechanism early in tumor progression because the physical elimination of these cells before tumor challenge resulted in tumor-free survival in 70% of mice, whereas their depletion in animals with established tumors had no therapeutic effect. Therefore, our data suggest that Treg cells may serve as an important therapeutic target for patients with early stages of cancer and that more vigorous combinatorial approaches simultaneously targeting multiple immune evasion as well as immunosurveillance mechanisms for the generation of a productive immune response against tumor may be required for effective immunotherapy in patients with advanced disease. The Journal of Immunology, 2007, 178: Institute for Cellular Therapeutics and Department of Microbiology and Immunology, University of Louisville, Louisville, KY Received for publication August 2, Accepted for publication March 19, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was funded in parts by grants from Kentucky Lung Cancer Research Program, National Institutes of Health/National Cancer Institute Grant R43 CA109866, and the Commonwealth of Kentucky Research Challenge Trust Fund. 2 Address correspondence and reprint requests to Dr. Haval Shirwan, Institute for Cellular Therapeutics, 570 South Preston Street, Donald Baxter Biomedical Building, Suite 404E, University of Louisville, Louisville, KY address: haval.shirwan@louisville.edu Adaptive immune responses mediated by T cells serve as critical components of immunosurveillance mechanisms against cancer (1). T cells respond to cancerous cells expressing tumor-associated Ags, which are either mutated or over/aberrantly expressed self proteins or proteins derived from oncogenic viruses (2). However, the recognition of tumor-associated Ags by TCRs (signal 1) is not sufficient for the generation of a productive T cell response against cancer. Costimulation via CD28/B7 or other costimulatory receptor/ligand interactions (signal 2) is also required for the initiation of the response (3). After initiation, a productive immune response is maintained by signal 3 in the form of cytokines and chemokines produced as a result of reciprocal T cell and APC activation (4). Tumors have evolved various direct and indirect mechanisms to evade the immune system (1, 5). These mechanisms include the following: 1) lack of signal 1, arising from either the inefficient display of MHC/tumor Ag bimolecular complexes on tumor cells, defects in the transduction of this signal, or expression of MHC homologues, MHC class I chain-related, that inhibit NK cells expressing NKG2 inhibitory receptors; 2) absence of signal 2 originating from the lack of costimulatory molecules on tumor cells or tumor expression of coinhibitory molecules; 3) tumor-mediated suppression of immune responses through the secretion of antiinflammatory molecules, such as TGF- and IL-10, induction of anergy in tumor-reactive T cells, physical elimination of effector T cells via apoptosis, or induction of naturally occurring CD4 CD25 FoxP3 T regulatory (Treg) 3 cells and tolerogenic APCs; and 4) regulation of immunity by the tumor stroma. Accumulating evidence suggests that multiple immune evasion mechanisms may simultaneously operate in patients with advanced disease (6). However, the importance of each of these mechanisms to immune evasion and their temporal cross-regulation in the course of tumor progression remain to be elucidated. CD4 CD25 FoxP3 Treg cells have recently been the focus of intense studies due to their critical roles in tolerance to self Ags and regulation of immune responses to infections and transplantation Ags (7). A series of recent studies also provided evidence for the involvement of these cells in immune evasion mechanisms used by tumors (8 11). A positive correlation between the increased numbers of Treg cells and tumor progression in experimental as well as clinical settings provided the first indirect evidence that these cells may play an important role in tumor immune evasion (8, 9, 11). Direct evidence for such a role has been provided recently by studies demonstrating that physical elimination of Treg cells before tumor challenge resulted in tumor rejection in some animal tumor models (12 15). However, it is unknown whether elimination of Treg cells in established tumor models has any therapeutic efficacy and whether Treg cells serve as the common denominator of immune escape mechanisms used by all tumors or they may play a more dominant role in tumor settings, in which a limited number of immune evasion mechanisms are operative. Treg cells have been shown to mediate their suppressive function through cell-to-cell contact as well as via soluble mediators, such as anti-inflammatory cytokines IL-10 and TGF- (16 18). 3 Abbreviations used in this paper: Treg, T regulatory; 1-MT, 1-methyl-D-tryptophan; DC, dendritic cell; DP, double positive; HPRT, hypoxanthine phosphoribosyltransferase; SP, single positive; Teff, T effector. Copyright 2007 by The American Association of Immunologists, Inc /07/$2.00

3 The Journal of Immunology 6841 These cytokines further provide a positive feedback loop for immune suppressive mechanisms by generating inducible Treg cells from CD4 CD25 T effector (Teff) cells (19). This positive feedback loop in a tumor setting may further be amplified by CTLA-4 on Treg cells engaging CD80/86 costimulatory molecules on dendritic cells (DCs), leading to the functional differentiation of DCs into suppressive cells expressing IDO (20, 21). IDO DCs may in turn perpetuate the suppressive milieu by further contributing to the generation of inducible or naturally occurring Treg cells. Recruitment and/or induction of Treg cells, as a bridge between anti-inflammatory cytokines and tolerogenic APCs, may be one of the major immune evasion mechanisms used by tumor cells. Therefore, elucidating the role of Treg cells in tumor progression, particularly their relative contribution to immune evasion mechanisms, may have important implications in the design of effective immunotherapeutic approaches against cancer. A20 B cell lymphoma was used as an aggressive tumor model to delineate the role of Treg cells in tumor development and progression. In this study, we demonstrate that tumor growth in this model was associated with a series of simultaneously operating direct and indirect immune evasion mechanisms. In particular, animals with tumors had increased percentages of intratumoral and systemic Treg cells, high levels of intratumoral and systemic IL- 10, moderate level of intratumoral TGF-, and APCs with altered function. Furthermore, A20 cells expressed the coinhibitory molecule PD-L1, anti-inflammatory cytokine IL-10, and immunomodulatory IDO, and lacked the expression of the costimulatory molecule CD80. Importantly, Treg cells played a dominant role in early tumor progression because in vivo depletion of these cells using an Ab to CD25 before tumor challenge resulted in tumorfree survival in 70% of the animals. In marked contrast, depletion of these cells in tumor-bearing animals had no beneficial effect. These findings were further corroborated in an adoptive transfer model in which the protective effect of ex vivo expanded tumorspecific Teff cells was blunted by ex vivo expanded Treg cells from tumor-bearing animals. The implication of these findings for strategies targeting Treg cells for immunotherapy in cancer patients is discussed. Materials and Methods Mice Male BALB/c and C57BL/6 mice aged 6 8 wk were purchased from The Jackson Laboratory or bred in our animal facility at the University of Louisville. Animals were provided food and water ad libitum and housed under specific pathogen-free conditions. Animals bearing tumors were euthanized when tumors reached a size of 20 mm in diameter or earlier if tumors ulcerated or animal showed sign of discomfort. All animals were cared for in accordance with institutional and National Institutes of Health guidelines. Abs and reagents Fluorochrome-conjugated Abs (CD4 allophycocyanin, CD8 PerCP, CD25 PE, CD19 allophycocyanin, IL-10 PE, H2Kd PE, I-A/I-E PE, PD-L1 PE, CD80 FITC, CD40 FITC, and CD86 allophycocyanin) and isotype controls were purchased from BD Pharmingen and ebioscience. Intracellular FoxP3 staining kit was purchased from ebioscience. Mouse IL-10 ELISA kit was purchased from BioSource International, and the assay was performed according to the manufacturer s protocol. Hybridoma for anti-mouse CD25 (PC61) and anti-mouse CD4 (GK1.5) Ab were gifts from S. Ildstad (Institute for Cellular Therapeutics and Department of Microbiology and Immunology, University of Louisville, Louisville, KY). The PC61 Ab was produced and purified in the laboratory of N. Egilmez (Brown Cancer Center, University of Louisville). Anti-mouse IL-10 (JES5-2A5) and isotype controls were purchased from Bioexpress. The 1-methyl-D-tryptophan (1-MT) was purchased from Sigma-Aldrich and prepared in 0.1 N NaOH (ph 8). Flow cytometry and cell sorting For phenotyping and sorting, spleens, lymph nodes, and tumors were processed into single-cell suspension, and cells were labeled with fluorochrome-conjugated Abs. Intracellular cytokine staining was performed as described (22). Briefly, single-cell suspensions of tumor cells were cultured in MLR medium supplemented with 1 l/ml GolgiPlug (BD Pharmingen) for 2 h. Cells were then stained with allophycocyanin-conjugated antimouse CD19 Ab, fixed with 4% paraformaldehyde, and stained with PEconjugated anti-mouse IL-10 or isotype control in permeabilization buffer containing saponin. Intracellular FoxP3 staining was performed using the anti-mouse/rat FoxP3 staining kit, according to the manufacturer s protocol (ebioscience). Flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using CellQuest (BD Biosciences) and FlowJo (Tree Star) softwares. CD4, CD4 CD25, and CD4 CD25 cells were sorted using a FACSVantage cell sorter (BD Biosciences). Sorted cells were reanalyzed by flow cytometry and found to be 95% pure. Tumor models and depletion studies A20 B lymphoma cell line was purchased from the American Type Culture Collection and cultured in DMEM supplemented with 10% FBS, 2 mm L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin at 37 C in a humidified 5% CO 2 incubator. To establish tumors, live A20 cells were resuspended in 200 l of PBS and s.c. injected into the right back flank of naive syngeneic BALB/c mice. Tumor growth was monitored three times per week using calipers. Average tumor size was calculated by measuring two perpendicular diameters. For depletion studies, tumorbearing animals were injected with 50 g of anti-mouse CD4 (GK1.5) intratumorally or 300 g of anti-mouse CD25 (PC61) i.v. twice at a 7-day interval as soon as the tumors were palpable. A third group of animals was treated with concurrent injection of 300 g of PC61 i.v. and 50 g of GK1.5 intratumorally when tumors were palpable, followed by intratumoral injection of the same dose of GK1.5 mab 7 days later. For predepletion studies, 300 g of PC61 was injected i.v. 4 days before the tumor challenge. PBS was used as a control. Depletion of regulatory T cells was confirmed by flow cytometry analysis of blood samples at various times after the administration of the Ab using the FoxP3 staining kit (ebioscience). For IL-10 neutralization, one group of animals received 1 mg of anti-mouse IL-10 (JES5-2A5) mab i.p. twice with a 1-wk interval starting 1 day after tumor challenge. This treatment regimen was adopted from a previously published study in tumor setting demonstrating therapeutic utility of blocking IL-10 in vivo (23 25). In addition to anti-il-10 Ab, a second group of animals also received anti- CD25 mab i.v. when tumors were palpable. Isotype Abs were used as control. RT-PCR RNA was isolated from the spleen, lymph nodes, and/or tumors using TRI reagent (Molecular Research Center) and cdna generated by reverse transcription. PCR for cytokines and hypoxanthine phosphoribosyltransferase (HPRT) were performed, as previously described (26). PCR for FoxP3 (35 cycles) and IDO (40 cycles) were performed using primers for FoxP3 (forward, 5 -CAG CTG CCT ACA GTG CCC CTA G; reverse, 5 -CAT TTG CCA GCA GTG GGT AG) and IDO (forward, 5 -GAT GTG GGC TTT GCT CTA CC; reverse, 5 -TTC TTC CAG TTT GCC AGG AC). Mixed lymphocyte reactions Standard 5-day MLR assays were performed using lymph node cells from naive BALB/c mice ( /well) as responders and irradiated (2000 cgy) splenocytes from naive C57BL/6 mice ( /well) as stimulators. To test the suppressive effect of sera from tumor-bearing animals on MLR, cultures were supplemented with various amounts (1 10 l/well) of sera collected from naive, tumor-bearing, and tumor-free animals. Cells were pulsed with [ 3 H]thymidine during the last 16 h of the 5-day culture, and harvested on a Tomtec Harvester 96 (Tomtec) for quantification of incorporated thymidine. Results were expressed as mean cpm of triplicate wells. In some experiments, splenocytes from naive C57BL/6 mice were labeled with CFSE (Molecular Probes), and used as responder against APCs from naive and tumor-bearing animals. Briefly, cells were washed with PBS, and then incubated in 4 ml of 2.5 M CFSE/ cells for 7 min at room temperature. Cells were incubated with FBS for 1 min, and washed twice with PBS. To test the function of APCs, splenocytes from naive and tumor-bearing animals were processed into single cells and panned on plastic dishes. Irradiated (2,000 cgy) adherent cells ( /well) were then used as stimulators in MLR assays, whereas splenocytes ( /well) from naive C57BL/6 were used as responders. In some assays, irradiated (10,000 cgy) A20 cells were used as stimulators in 4-day cultures against CFSE-labeled C57BL/6 splenocytes at a 1:4 ratio in the presence or absence of 200 M 1-MT. Proliferation was assessed using flow cytometry.

4 6842 ROLE OF CD4 CD25 Treg CELLS IN CANCER IMMUNE EVASION FIGURE 1. CD4 CD25 Treg cells increase as a function of tumor size. BALB/c mice were inoculated s.c. in the right flank with live A20 cells. Cells harvested from tumor, spleen, and draining lymph nodes of animals with small ( mm, n 4), medium ( mm, n 6), and large ( mm, n 5) tumors analyzed for phenotype and function. Cells from naive animals (n 3) served as controls. A, Percentage of CD4 (top panel), CD8 (middle panel), and CD4 CD25 T cells (bottom panel) in various tissues., p 0.05 as compared with percentages of respective groups. B, FoxP3 expression in intratumoral CD4 CD25 T cells from a medium size tumor using anti-foxp3 Ab in flow cytometry (gray filled histogram represents the isotype control). C, Suppression assay. Spleen and lymph node cells of tumor-bearing (Tumor-DP) and tumor-free (TFree-DP) animals were cultured with irradiated A20 cells in the presence of IL-2 for 4 5 days. CD4 CD25 DP T cells were then sorted by flow cytometry and used in coculture experiments with sorted CD4 CD25 SP T cells from naive animals at 1:1 ratio in a standard anti-cd3 stimulation and [ 3 H]thymidine incorporation-based assay. Sorted DP T cells from naive BALB/c mice (DP) were used as controls. Suppression and adoptive transfer assays For ex vivo activation, spleen and/or lymph nodes were harvested from naive, tumor-bearing, and tumor-free animals; processed into single-cell suspensions; and cultured in a 2:1 ratio with irradiated (10,000 cgy) A20 cells in MLR medium supplemented with 50 U/ml IL-2 (Roche) for 4 5 days. For suppression assays, CD4 CD25 (single-positive (SP)) and CD4 CD25 (double-positive (DP)) cells were sorted by flow cytometry. DP cells from naive, tumor-bearing, and tumor-free cultures were used as suppressors for SP responder cells from naive BALB/c mice. The 3-day suppression assay was performed in the presence of SP and/or DP cells/ well, 0.5 g/ml anti-mouse CD3 (clone 145-2C11) Ab (BD Pharmingen), and irradiated (2,000 cgy) splenocytes from naive BALB/c mice. For adoptive transfer studies, lymphocytes from tumor-free animals were cultured for 4 days in the presence of 50 U/ml IL-2 and irradiated A20 (10,000 cgy), and then purified using Lympholyte-M, according to the manufacturer s protocol (Cedarlane Laboratories). Cells from tumor-free animal cultures ( ) were mixed with live A20 cells and injected s.c. with or without DP cells ( ) sorted from ex vivo activation cultures of tumor-bearing animals. Tumor growth was monitored, as described above. Results Increased percentages of CD4 CD25 FoxP3 Treg cells in tumor-bearing animals CD4 CD25 FoxP3 Treg cells are critical to peripheral tolerance to self Ags and have been implicated in immune evasion mechanisms by tumors (7, 10). To study the contribution of Treg cells in tumor progression, we choose A20 B lymphoma as a model system. This tumor cell line is derived from a spontaneous reticulum cell neoplasm in BALB/c mice and is highly tumorogenic and poorly immunogenic (27, 28). Four groups of mice were used in this study, as follows: naive animals (n 3) and animals with small tumors ( mm, n 4), medium tumors ( mm, n 6), and large tumors ( mm, n 5). Spleen, draining lymph nodes, and tumors were harvested and analyzed for the presence of Treg cells using Abs to various cell surface markers in flow cytometry. There was a decrease in the percentages of CD4 and CD8 cells in the spleen, lymph nodes, and tumors as tumor sized increased (Fig. 1A, top and center panels). These differences were significant when the spleen and lymph node cells from large tumor-bearing animals were compared with those of naive and small tumor-bearing animals. In marked contrast, we found an increase in the percentage of CD4 CD25 T cells in the spleen, lymph nodes, and tumors as tumor size increased; animals with large tumors had significantly higher percentages of Treg cells in their spleen and lymph nodes as compared with naive and small tumor-bearing animals (Fig. 1A, bottom panel). The regulatory nature of these cells was confirmed by two independent lines of evidence. First, these cells expressed the signature transcriptional factor FoxP3 as determined by intracellular staining with FoxP3 Ab (Fig. 1B) and RT-PCR (data not shown). Second, CD4 CD25 T cells from tumor-bearing animals suppressed the proliferation of CD4 CD25 Teff cells in vitro. In this assay, lymphocytes were harvested from medium size tumor-bearing animals or tumor-free animals that had undergone successful immunotherapy with CD80-decorated cells (29). These cells were then cocultured with irradiated A20 cells as the relevant source of tumor-associated Ags in the presence of exogenous IL-2 for 4 5 days. CD4 CD25 DP T cells were sorted from both cultures using flow cytometry, and their regulatory function was tested in coculture experiments with sorted naive CD4 CD25 SP T cells in a standard CD3 stimulation assay. DP T cells sorted from cultures of tumor-bearing animals were anergic, and as such failed to respond to CD3 stimulation. However, these cells potently inhibited the proliferation of SP naive T cells (Fig. 1C). In marked contrast, DP cells from cultures of tumor-free animals responded to CD3 stimulation and did not inhibit the proliferative response of SP T cells. Taken together, these data demonstrate that CD4 CD25 T cells expanded in lymphocyte cultures of tumor-bearing animals are Treg cells, but not Teff cells that up-regulated CD25 in response to irradiated A20 cells. CD4 CD25 Treg cells facilitate the growth of A20 tumors We next tested the role of these ex vivo expanded Treg cells in tumor immune evasion mechanisms using an adoptive transfer model.

5 The Journal of Immunology 6843 FIGURE 2. CD4 CD25 Treg cells facilitate the growth of A20 tumors in an adoptive transfer model. Splenocytes and lymph node cells were harvested from BALB/c mice bearing medium to large tumors and tumorfree animals having undergone successful immunotherapy with irradiated cells decorated with chimeric CD80 costimulatory molecule. Cells were stimulated in vitro with irradiated A20 cells as the source of tumor Ags in the presence of IL-2 for 5 days. Live cells harvested from cultures of tumor-free animals using Lympholyte-M were directly used in adoptive transfer experiments, whereas those from cultures of tumor-bearing animals were sorted by flow cytometry to isolate CD4 CD25 T cells. Adoptive transfer of ex vivo activated lymphocytes from tumor-free (TFree) animals into naive secondary animals simultaneously challenged with a lethal dose of live A20 cells s.c. (n 6). One group of animals was coadoptively transferred with CD4 CD25 T cells (Tumor-DP) sorted from cultures of tumor-bearing animals (n 6). Animals inoculated with live A20 alone served as controls (n 5). Adoptive transfer of total cells from ex vivo stimulated lymphocytes from cultures of mice that had undergone successful immunotherapy with CD80-decorated A20 cells (29) into mice simultaneously challenged with a lethal dose of A20 cells resulted in 50% tumor-free survival during a 100-day observation period (Fig. 2). However, this protective effect was totally abolished by coadoptive transfer of flow-sorted CD4 CD25 T cells from cultures of tumor-bearing animals. These results further confirm that CD4 CD25 T cells accumulating in tumor-bearing animals as a function of tumor growth are Treg cells, and that these cells play a critical role in tumor evasion mechanisms in this model when present at early stages of tumor progression. Early, but not late, depletion of CD4 CD25 Treg cells during tumor progression results in tumor-free survival It was demonstrated recently by Yu et al. (30) that systemic depletion of Treg cells using the PC61 Ab against CD25 or intratumoral depletion using a low dose anti-cd4 Ab resulted in complete eradication of large tumors in a fibrosarcoma model. We, therefore, adapted this protocol and demonstrated that i.v. injection of 300 g of PC61 Ab twice at a 7-day interval into mice with palpable tumors had no effect on tumor growth (Fig. 3A), albeit successful depletion of Treg cells in the periphery and within the tumor, as confirmed using Abs to FoxP3 and CD25 (Fig. 3B). Indeed, Treg cells were absent in the blood of these animals for 7 days and only recovered to one-half of their original levels by day 14 following Ab treatment. FIGURE 3. CD4 CD25 Treg cells are critical to the initial stages of tumor progression. A, Tumor growth in BALB/c mice inoculated with of live A20 cells s.c. and injected with 300 g of anti-cd25 Ab PC61 (n 5) or PBS (n 5) i.v. twice at a 7-day interval when tumors were palpable. B, Confirmation of Treg cell depletion in mice injected i.v. with a single dose of 300 g of PC61 Ab. Depletion was shown in peripheral blood using FoxP3 Ab (top panel) and within tumor using an anti-cd25 mab (clone 7D4) that recognizes a different epitope than PC61 Ab used for CD25 depletion (bottom panel). C, Tumor growth in mice subjected to various Ab treatments. A group of animals (n 5) was injected intratumorally with 50 g of anti-cd4 Ab GK1.5 when tumors were palpable, followed by a second injection 7 days later (GK1.5). A second group of animals (n 5) was injected i.v. with 300 g and intratumorally with 50 g of PC61 when tumors were palpable. Seven days later, these animals received one dose of intratumoral injection of 50 g of GK1.5 (PC61 GK1.5). Arrows indicate the time points at which PC61 and GK1.5 were injected. D, Tumor growth in BALB/c mice (n 10) subjected to systemic depletion by a single injection of 300 g of PC61 Ab i.v. 4 days before A20 challenge. Tumor growth in animals injected with PBS served as controls (n 5).

6 6844 ROLE OF CD4 CD25 Treg CELLS IN CANCER IMMUNE EVASION A20 B lymphoma cells express an altered pattern of costimulatory molecules Our data demonstrating that Treg cells serve as an important immune evasion mechanism early in the progression of A20 tumors suggested that other immune evasion mechanisms either induced by Treg cells or independent of these cells may operate in late stages of tumor growth. We, therefore, characterized A20 cells for the expression of various cell surface molecules involved in immune responses. This tumor cell line expressed normal levels of both MHC class I and class II involved in the transduction of signal 1 (Fig. 4A) and high levels of CD40 and CD86 and low levels of 4-1BBL costimulatory molecules involved in the transduction of signal 2 (data not shown). Consistent with our previous observation, the CD80 costimulatory molecule was not detected on the surface of A20 cells. The lack of CD80 is highly significant because we and others have demonstrated that expression of this molecule via genetic manipulations or cell surface protein displays results in high immunogenicity and tumor cell rejection in syngeneic BALB/c mice (29, 31, 32). Importantly, A20 cells expressed high levels of coinhibitory molecule PD-L1, which has been implicated in tumor immune evasion mechanisms as well as tolerance in various animal models (33). This pattern of MHC and costimulatory molecule expression was similar between cultured A20 cells and cells extracted from tumors (data not shown). FIGURE 4. A20 B lymphoma cells show altered expression of costimulatory molecules. A20 cells were stained with Abs against various molecules involved in the transduction of signal 1 (A) and signal 2 (B), and analyzed using multiparameter flow cytometry. Because the lack of a protective response may be due to the elimination of activated CD25 Teff cells rather than the depletion of Treg cells, we followed the protocol of Yu et al. (30), who demonstrated that intratumoral injection of one dose of 40 g of anti-cd4 Ab (GK1.5) resulted in the eradication of established fibrosarcoma tumors. In marked contrast, intratumoral injection of 50 g of GK1.5 Ab twice at a 7-day interval had no effect on tumor growth in our A20 model (Fig. 3C). Furthermore, concurrent systemic and intratumoral injection of PC61 when tumors were palpable, followed by an intratumoral GK1.5 injection 7 days later did not change the kinetics of tumor growth (Fig. 3C). We next assessed whether systemic pretreatment of mice with PC61 has any effect on the growth of A20 tumors. Intravenous injection of 300 g of PC61 Ab 4 days before tumor inoculation resulted in tumor-free survival in 70% of mice over a 60-day observation period (Fig. 3D). In contrast, all animals without Ab treatment were euthanized within 35 days due to large tumor burden. Importantly, tumor-free animals did not develop tumors when rechallenged with a lethal dose of live A20 60 days after the initial tumor inoculation (data not shown), suggesting that elimination of Treg cells not only leads to the rejection of tumors, but also allows for the generation of a memory response that protects against recurrences. Taken together, these data demonstrate that physical elimination of Treg cells early in tumor progression has a therapeutic effect, whereas depletion of these cells in animals with established tumors has no effect on tumor growth. Intratumoral and systemic expression of IL-10 Cytokines play important roles in the regulation of adaptive as well as innate immune responses to tumors. In particular, Treg cell development and function are regulated by cytokines, such as IL-2, IL-10, and TGF- (18, 34). We, therefore, characterized the cytokine expression pattern within A20 tumors using RT-PCR with particular focus on Th1 (IL-2, IFN- ) and Th2/Treg (IL-2, IL-10, TGF- ) cytokines (26). A20 tumors were found to express low levels of IL-2, IL-4, and IFN- ; moderate levels of TGF- ; and high levels of IL-10 (Fig. 5A). The high level of intratumoral IL-10 expression was rather striking and suggested that it may be expressed by the tumor itself in addition to tumor-infiltrating lymphocytes. To test this hypothesis, total RNA isolated from cultured A20 cells was subjected to RT- PCR analysis. A20 cells expressed high levels of IL-10 transcripts (Fig. 5B, top panel) with minimal or undetectable levels of transcripts for other cytokines (data not shown). The expression of IL-10 was further confirmed at the protein level by intracellular cytokine staining (Fig. 5B, bottom panel). Importantly, A20 cells freshly extracted from tumors expressed higher levels of IL-10 protein than A20 cell line maintained in culture (44 vs 10%; Fig. 5B, bottom panel). These data suggest the existence of positive feedback mechanism(s) within the tumor microenvironment contributing to the higher expression of IL-10. IL-10 secretion was systemic, as determined using ELISA. There was significantly higher level of circulating IL-10 in the sera of tumor-bearing animals as compared with that of naive and tumor-free animals vaccinated with CD80-decorated A20 cells (Fig. 5C). IL-10 was also present in the culture medium of A20 cells, suggesting that these cells are likely to be the main source of IL-10 and use this molecule as a means of immune evasion. Indeed, sera from tumor-bearing animals inhibited BALB/c T cell response to irradiated C57BL/6 splenocytes in in vitro proliferative responses. There was a significant inhibition of T cell proliferation in cultures supplemented with 1 l of sera from tumor-bearing animals as compared with cultures containing sera from naive or tumor-free animals (Fig. 5D). The proliferative response was significantly recovered using a blocking Ab (JES5-2A5) against IL-10 (data not shown). To demonstrate whether IL-10 neutralization in vivo has any therapeutic effect, a group of animals was challenged with A20 cells and subjected to i.v. treatment twice with 1 mg of clone JES5-2A5 at weekly intervals starting 1 day posttumor challenge. This treatment regimen had no effect on tumor growth (Fig. 5E). A combination treatment that involved both anti-il-10 and anti- CD25 Abs also had no effect on the normal kinetics of tumor growth. Although the in vivo neutralization experiments do not provide a direct role for IL-10 in the progression of A20 tumor, they cannot rule out a possible role for this cytokine in tumor immune evasion. This is consistent with our in vitro experiments as well as various reports implicating IL-10 in tumor immune evasion (24, 35, 36). A20 cells express high levels of IL-10 and may use this cytokine as a means of immune evasion mechanisms by affecting various arms of the immune system, including Treg cells and DCs (16, 18).

7 The Journal of Immunology 6845 FIGURE 5. A20 cells express high levels of intratumoral and systemic IL-10. A, Expression of intratumoral cytokines. RT-PCR of total RNA obtained from tumor cells using primer sets specific for the indicated cytokines and HPRT as an internal control. Data are presented as the ratio of cytokines to HPRT. B, Expression of IL-10 transcripts in cultured A20 cells using RT-PCR (top panel). Expression of A20 cells from tumors and cultures using intracellular IL-10 staining on CD19-gated cells in flow cytometry (bottom panel; gray filled, isotype control; black line, IL-10). C, ELISA showing IL-10 levels in sera from naive, tumor-bearing (Tumor), tumor-free (TFree) immunotherapy animals, and A20 culture supernatants. D, Effect of sera from the same groups of animals (C) on allogeneic proliferative responses. Naive BALB/c lymph node cells were used as responders to irradiated C57BL/6 splenocytes, and cultures were supplemented with 1 l/well serum from the indicated animals (top panel). E, Tumor growth in mice treated with 1 mg of neutralizing anti-il-10 Ab (JES5-2A5)/animal injected twice at weekly intervals (n 6) starting 1 day after tumor challenge. Another group of animals (n 5) was treated with both anti-il-10 and anti-cd25 Abs. Animals treated with isotype Abs served as controls (n 5). Downward arrows show the injection time for anti-il-10 mab, whereas the upward arrow shows the time of injection for anti-cd25 Ab. A20 cells and APCs from tumor-bearing animals serve as poor stimulators of allogeneic responses Altered function of APCs has been shown to be an important tumor immune evasion mechanism (37, 38). In particular, it has been demonstrated recently that a subpopulation of DCs expressing IDO plays a critical role in tumor escape from immune destruction (19 21). IDO may also be expressed by tumor cells and exhibit a direct effect on T cells (39). We, therefore, tested the expression of IDO in A20 cells and within the tumor using RT-PCR. A20 cell line as well as freshly extracted tumors expressed high levels of IDO transcripts (Fig. 6A). To test whether IDO expression plays a role in the Ag-presenting function of A20 cells, we performed CFSEbased MLR using C57BL/6 lymphocytes as responders against irradiated A20 cells. A20 cells generated a moderate alloreactive proliferative response, which was further enhanced by the addition of the IDO inhibitor 1-MT into the culture medium (49 vs 61%; Fig. 6B). To test whether A20 tumor-bearing animals have APCs with altered function, irradiated splenocytes from naive and tumor-bearing animals were used as stimulators for CFSE-labeled splenocytes from naive C57BL/6 mice. There was minimal T cell proliferative response to splenocytes from tumor-bearing animals as compared with those from naive animals (9.3 vs 54.4%; Fig. 6C). Unlike A20 cultures, the addition of 1-MT did not restore the proliferative response. This observation suggests that either IDO does not play a major role or this effect is subdominant to other more tolerogenic mechanisms regulating APC function in tumor-bearing animals. Taken together, these data demonstrate that A20 tumors may evade the immune system by directly expressing IDO and/or indirectly via regulating the function of host APCs. Discussion Tumor development and progression is a complex process that involves various genetic alterations in the tumor as well as tumor immune evasion via a complex set of molecular and cellular mechanisms that may operate simultaneously or sequentially in the course of tumor progression. The critical role of the immune system in immunosurveillance against tumors served as the basis of intense efforts over the last few decades to develop effective immunotherapeutic approaches against cancer. Although some of these approaches demonstrated efficacy in preclinical transplantable tumor models in preventive as well as therapeutic settings, their translation to clinical settings was met with little or no success (40). This may be due to the existence of various immune evasion mechanisms in individuals with advanced tumors and the inability of current therapeutic approaches to override these immunoregulatory mechanisms while generating new antitumor responses and/or up-regulating the existing ones. CD4 CD25 FoxP3 Treg cells have emerged recently as important players in tumor immune evasion mechanisms. Increase in the number of Treg cells as a function of tumor growth has been shown in various animal models as well as clinical settings (8, 9, 11, 30), suggesting that these cells may serve as a common immune evasion mechanism. Treg cells may, therefore, serve as an

8 6846 ROLE OF CD4 CD25 Treg CELLS IN CANCER IMMUNE EVASION FIGURE 6. A20 cells and APCs from tumor-bearing animals are poor stimulators of allogeneic responses. A, RT-PCR analysis of IDO expression in total tumor and A20 cells. B, Effect of IDO inhibitor 1-MT on the proliferative response of CFSE-labeled C57BL/6 T cell response against irradiated A20 cells. C, APCs from naive or tumor-bearing BALB/c mice were irradiated and used as stimulators for CFSE-labeled C57BL/6 lymph node cells, as in B. The 1-MT did not restore the proliferative response of naive C57BL/6 T cells against APCs harvested from tumor-bearing animals. important target for immunotherapeutic approaches against cancer. However, before such approaches are developed, it is paramount to delineate the relative contribution of these cells to overall tumor progression. In this study, we used A20 B cell lymphoma as an aggressive transplantable tumor model to delineate the role of Treg cells in tumor growth at various stages of tumor progression. Tumor-bearing animals showed increased percentages of Treg cells within the tumor, spleen, and tumor-draining lymph nodes as a function of tumor size. Importantly, almost all CD4 CD25 T cells within the tumor expressed FoxP3, suggesting that these were Treg cells, not newly activated Teff cells. It is presently unknown whether these cells were derived from the preferential infiltration of naturally occurring Treg cells into the tumor and/or from CD4 CD25 Teff cells converted into Treg cells in response to various cues within the tumor microenvironment. The latter notion is consistent with a recent study demonstrating that the majority of Treg cells in tumor-bearing mice were derived from CD4 CD25 Teff cells without a requirement for thymus or proliferation (41). The presence of high levels of IL-10 and TGF- within the tumor in our model may drive this process. It has been shown recently that Gr-1 CD115 immature myeloid suppressor cells accumulate within tumors and secrete IL-10 and TGF-, which play critical roles in the conversion of CD4 CD25 Teff cells into Treg cells (42). Direct evidence for the role of Treg cells in A20 evasion of the immune system in our model was provided by three lines of evidence. First, CD4 CD25 T cells sorted from cultures of lymphocytes harvested from tumor-bearing animals and stimulated ex vivo against A20 cells in the presence of IL-2 suppressed the proliferation of CD4 Teff cells in a CD3 stimulation assay. Second, these cells blocked the therapeutic efficacy of Teff cells, harvested from animals vaccinated with CD80-decorated A20 cells (29), when used in coadoptive transfer experiments. Third, consistent with several published studies (12 15), systemic treatment of mice with one dose of a depleting anti-cd25 mab 4 days before tumor challenge resulted in tumor-free survival in 70% of animals. Taken together, these data provide direct evidence for the critical role of Treg cells in tumor immune evasion mechanisms early in tumor progression and are consistent with a series of recent findings implicating these cells in tumor growth (10, 30, 41, 42). However, the physical elimination of Treg cells in established tumors had no therapeutic efficacy. Animals with palpable tumors injected with two doses of PC61 Ab i.v. at a 1-wk interval developed tumors at a similar rate to control animals. The lack of a therapeutic effect is not due to the inability of the Ab to deplete Treg cells, because we demonstrated that one dose of PC61 was sufficient to deplete Treg cells to background levels and these cells could only recover to 50% of their initial value in 2 wk. This is further consistent with other studies (12 15) and our results demonstrating that a single injection of this dose of Ab was effective in preventing tumor growth in 70% of the animals when used for pretreatment. The lack of a PC61 Ab therapeutic effect in animals with established tumors may be attributable to the fact that this Ab not only depletes Treg cells, but also newly activated Teff cells expressing CD25. However, this notion is inconsistent with the findings of Yu et al. (30), showing that systemic administration of one dose of PC61 Ab resulted in eradication of established tumors in a fibrosarcoma model, suggesting that this Ab may not deplete Teff cells. This may be due to the lack of sustained expression of CD25 on Teff cells in contrast to Treg cells that express high and sustained levels of CD25 (43). A more stringent regimen involving intratumoral treatment with depleting doses of an anti-cd4 mab singly or in combination with systemic and intratumoral injection of depleting doses of anti- CD25 mab also failed to alter the rate of tumor growth. In contrast to our study, this regimen resulted in the eradication of established tumors in the aforementioned fibrosarcoma model (30), providing further evidence for the lack of a therapeutic effect of Treg cell depletion in late stage tumors in our A20 model. This fibrosarcoma tumor, to our knowledge, is the only model in which the depletion of Treg cells using CD25 Ab alone could result in the eradication of established tumors. Although the source of the discrepancy between our study and that of Yu et al. (30) is presently unknown, the aggressive nature of the A20 tumor model and the use of multiple immune evasion mechanisms by A20 may provide an explanation. Indeed, our data imply that A20 tumors may use several direct (lack of CD80 costimulatory molecule, high level PD-L1 coinhibitory molecule, antiinflammatory cytokine IL-10, and immunoregulatory IDO enzyme) as well as indirect immune evasion mechanisms, such as Treg cells and APCs with altered immune functions, to evade immune destruction. The importance of the lack of CD80 costimulation as an important immune evasion mechanism has already been demonstrated by studies using genetically manipulated A20 cells expressing CD80 as vaccine (29, 31, 32). We have also shown that A20 cells

9 The Journal of Immunology decorated with a chimeric CD80 protein served as effective vaccine for the prevention of tumor growth (29). Although we did not specifically probe the role of PD-L1 coinhibitory molecule expressed by A20 in tumor growth, this molecule has been implicated in peripheral tolerance in various experimental settings, including tumors (44). Many tumors express PD-L1 on their surface, and blockade of this molecule results in susceptibility to immune destruction (33). PD-L1 interaction with PD-1 receptor may play an important role in the generation of Treg cells and/or regulation of their function in the A20 model. Signaling through PD-1 in Teff cells results in IL-10 production, and IL-10 has been shown to have a plethora of anti-inflammatory effects on the immune system, including generation of tolerogenic APCs as well as Treg cells or modulation of their function (45). Consistent with this notion is our observation that serum from tumor-bearing animals had significant levels of IL-10 and inhibited alloreactive responses ex vivo. The suppressive effect of serum could be neutralized using a mab against IL-10. However, we failed to demonstrate a significant role for IL-10 in tumor immune evasion in vivo because the treatment of tumor-challenged animals with the IL-10 mab singly or in combination with anti-cd25 mab had no effect on tumor growth. These data suggest that IL-10 in the A20 model may serve a complementary role to other immune evasion mechanisms, such as IDO, PD-L1, and Treg cells, rather than playing a dominant one. Alternatively, the in vivo blocking conditions tested in our study may not be sufficient to neutralize all the available IL-10 for a detectable effect. Although the amount of anti-il-10 mab used in this study was comparable to that used for the same clone in several studies with efficacy (24, 46, 47), the high levels of IL-10 secretion by A20 cells in our model may require a more aggressive treatment. There was an inverse correlation between the tumor size and the percentages of CD4 and CD8 T cells within the spleen, lymph nodes, and tumor. Although exact mechanisms are unknown, apoptotic molecules, such as PD-L1 (48) expressed by A20 cells or NO released by host cells, such as tumor-infiltrating macrophages (49), may play a role. Furthermore, we have observed metastases/ migration of A20 tumor cells into peripheral lymph tissues as a function of tumor growth (data not shown) that may also be responsible for the observed decrease in the percentages of T cells in these organs. These various immune evasion mechanisms may complement and/or augment the role of Treg cells in tumor progression. It has been shown that various immunoregulatory molecules, such as PD-L1, B7-H4, IL-10, TGF-, and IDO, mediate immune suppression in tumor environment (20, 21, 24, 33, 48, 50 53). These molecules may be constitutively expressed by tumor cells or be induced by tumor microenvironment for expression in tumor cells or APCs (20, 21, 24, 33, 48, 50 53). The coordinated expression of these immunoregulatory molecules within the tumor may set in motion a suppressive circuit involving various immune cells with regulatory functions. This notion is consistent with recent studies demonstrating the role of some of these immunoregulatory molecules in the generation, expansion, and/or function of CD11b IL- 4R or Gr-1 CD115 immature myeloid suppressor cells (42, 54) and CD4 CD25 FoxP3, CD4 CD25 FoxP3,orCD8 Treg cells (55). Therefore, tumors that use fewer of these immune evasion mechanisms may primarily rely on Treg cells for progression. However, those that use multiple immune evasion mechanisms, such as A20, may rely on Treg cells early when some of these immune evasion mechanisms are not fully developed. Once other mechanisms are induced and fully functional late in tumor progression, the lack/elimination of any one immune evasion mechanism, including Treg cells, may be compensated by others, leading to undetectable/minimal effect on tumor growth. This notion is consistent with our findings that in vivo blocking of IL-10 and physical depletion of Treg cells in established A20 tumors had no detectable effect on tumor growth. Our findings are further supported by clinical studies demonstrating that the ril-2 diphtheria toxin conjugate (ONTAK) is effective in reducing the number of Treg cells in the peripheral blood of patients with metastatic renal cell carcinoma without therapeutic efficacy (56). In addition, the therapeutic efficacy of Treg cell depletion in various preclinical models was shown to depend on the use of other adjuvant immunotherapeutic regimens, such as IL-12 gene therapy or DC vaccines (57, 58). These observations from animal studies and clinical trials reveal that therapeutic approaches only targeting Treg cells in cancer patients may fail or not be sufficient to reverse tumor growth depending on the cancer type. Treg cells may only be critical at early stages of tumor progression, and other immune evasion mechanisms that are related or unrelated to Treg cells may be the dominant factors at late stages of tumor progression. Therefore, immunotherapeutic approaches targeting Treg cells for physical and functional elimination in cancer patients need to consider the type and stage of tumor, and its associated immune evasion mechanisms for success. 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