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The Journal of Immunology, 2008, 181, 3690 -3697
Copyright © 2008 by The American Association of Immunologists, Inc.
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Expression of a Soluble TGF-β Receptor by Tumor Cells Enhances Dendritic Cell/Tumor Fusion Vaccine Efficacy1

Min Zhang, Bradford E. Berndt, Jian-Jun Chen and John Y. Kao2

Division of Gastroenterology, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-β limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-β on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-β receptor type II fused to the Fc region of human IgM (Adv-TGF-β-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-β receptor (TGF-β-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-β-R (4T1+Adv-TGF-β-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-β-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-β-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-β is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-β-R fusion vaccine induced fewer CD4+CD25+Foxp3+ Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4+CD25+ Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-β. We conclude that the blockade of tumor-derived TGF-β reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The use of tumors as immunogens to induce antitumor immunity continues to be a major area of cancer research. The dendritic cell (DC)3-based antitumor vaccine has emerged as a promising cancer immunotherapy with proven clinical efficacy (1, 2, 3, 4, 5). Phase III DC-based immunotherapy for hormone-refractory metastatic prostate cancer showed efficacy and safety, and the U.S. Food and Drug Administration is currently reviewing the data for its clinical use. Other trials, however, have reported a limited clinical response leading to early termination of the studies (6).

One possible explanation for the limited clinical response is that whole-tumor substrate may contain immunosuppressive factors that reduce vaccine efficacy. We have shown that tumor-derived TGF-β inhibits the efficacy of the DC/tumor fusion vaccine, and this effect appears to occur in the microenvironment where DCs and T cells interact (7). Furthermore, we have found that tumor-bearing hosts have the propensity to skew the DC-induced response toward IL-10 production by T cells (8). Because TGF-β is crucial for the development of regulatory T cells (Tregs), which produce IL-10, this represents another potential immune escape mechanism. Indeed, recent studies of Tregs indicate that this cell population is a significant obstacle for the induction of robust antitumor immunity (9). These data suggest that tumor-derived TGF-β represents a critical target for intervention. However, because TGF-β has other important physiological properties (e.g., wound healing), a systemic blockade strategy is not feasible. In the present study we designed a strategy to block TGF-β signaling in the microenvironment of the DC-T cell interaction using a soluble form of TGF-β receptor (TGF-β-R).

An adenovirus encoding human TGF-β-R type II fused with the Fc region of human IgM was used to neutralize the inhibitory effect of tumor-derived TGF-β on DC priming of antitumor immunity. Expression of TGF-β-R was detected in 4T1 tumor cultures after transfection using a multiplicity of infection (MOI) of 300, but not in DC cultures. Injection of irradiated 4T1 cells expressing TGF-β-R resulted in the induction of antitumor immunity. Moreover, a fusion vaccine using DCs and 4T1 cells expressing TGF-β-R showed an enhanced antitumor response and, in vitro, induced fewer Tregs. These data suggest that neutralization of tumor-derived TGF-β using an adenovirus expressing a soluble form of TGF-β-R is an effective strategy to enhance the efficacy of DC-based vaccines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Specific-pathogen-free female BALB/c mice aged 8–10 wk were purchased from The Jackson Laboratory and housed in the animal maintenance facility at the University of Michigan Health System. Experiments were conducted on mice aged 10–14 wk. All animal experiments were approved by the University Committee on Use and Care of Animals at the University of Michigan.

Media and cytokines

Complete medium (CM) consisted of RPMI 1640 with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The following recombinant cytokines (R&D Systems) were diluted in CM: mouse GM-CSF (10 ng/ml) and mouse IL-4 (10 ng/ml).

Tumor cell lines

Murine breast carcinoma (4T1), murine renal cell carcinoma (RenCa), and mink lung cells (Mv1Lu) were obtained from American Type Culture Collection and murine colon cancer cell lines (CT26-neo, CT26-TGF-β; Refs. 7 , 10) were propagated in CM. Additional culture supernatants used for this study were from other mouse tumor cell lines: B16F10 (melanoma), P815 (lymphoblast-like mastocytoma), EL4 (lymphoma), MCA205 (fibrosarcoma), and control 3T3 (fibroblasts).

Modified ELISA to detect TGF-β-R

A human TGF-β ELISA kit (R&D Systems) was modified to detect TGF-β-R. The concentration of TGF-β-R was estimated by measuring the amount of TGF-β bound to TGF-β-R (see Fig. 1A). Standard 96-well plates were coated with 1 µl/ml goat anti-human IgM Abs (Cappel-Organon Teknika) and stored overnight in a moist chamber at 4°C. The plates were washed with PBS, blocked with 10% FCS for 2 h at room temperature, and washed with PBS. Supernatants from transfected DCs or tumor cells were added and the cells were incubated at room temperature for 2 h. After a PBS wash, human TGF-β (8 ng/ml) was added and the cells were incubated for 1 h and then washed. The subsequent steps followed the manufacturer’s instructions, beginning with the addition of biotinylated anti-human TGF-β followed by HRP and substrates.


Figure 1
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  		FIGURE 1. A soluble form of TGF-β-R expressed by stably transfected CT26-TGF-β-TGF-β-R tumor cells was shown to bind TGF-β and reduce TGF-β inhibitory activity. A, A modified ELISA was used to detect TGF-β-R. Note that the binding of TGF-β-R to TGF-β is required for positive detection (measured by OD). B, Modified TGF-β-R ELISA standard curve. As a recombinant TGF-β-R standard solution was commercially unavailable we used the supernatant of an overnight culture as the standard solution: 4T1+Adv-TGF-β-R (1 x 106 cells per ml, concentration 4000x). Using this relative concentration of TGF-β-R we demonstrated the linearity of serial dilutions and maintained quality control of the modified TGF-β-R ELISA. C, The production of TGF-β-R in the culture supernatant of CT26-neo, CT26-TGF-β, and CT26-TGF-β-TGF-β-R. Confluent culture supernatants of CT26-neo, CT26-TGF-β, and CT26-TGF-β-TGF-β-R were measured using the modified ELISA (n = 3). D, TGF-β-R inhibited the effect of tumor-derived TGF-β on Mv1Lu cell proliferation. Mv1Lu cells were cocultured for 48 h with PBS, TGF-β (10 ng/ml), and supernatants of confluent cell culture of CT26-neo, CT26-TGF-β, and CT26-TGF-β–TGF-β-R. Cell proliferation was measured by tritiated thymidine incorporation and expressed as cpm. The addition of TGF-β or supernatant from CT26-TGF-β culture inhibited Mv1Lu cell growth (positive controls), whereas supernatant from CT26-neo culture did not (negative control). Supernatant from CT26-TGF-β-TGF-β-R culture partially reversed the inhibition by TGF-β indicating neutralizing activity of TGF-β-R (n = 3). *, p < 0.05, **, p < 0.01, ***, p < 0.001.

 
Alternatively, the direct measurement of TGF-β-R (a human TGF-β-R type II fused with the Fc region of human IgM) was measured by human IgM ELISA (see Fig. 2A). Standard 96-well plates were coated 1 µl/ml with goat anti-human IgM Abs (Cappel-Organon Teknika) and stored overnight in a moist chamber at 4°C. The plates were washed with PBS, blocked with 10% FCS for 2 h at room temperature, and washed with PBS. Supernatants from transfected DCs or tumor cells were added, and the cells were incubated at room temperature for 2 h. After a PBS wash, alkaline phosphatase-conjugated anti-human IgM Abs (Cappel-Organon Teknika) were added for 1 h and followed by the addition of alkaline phosphatase substrates (Sigma Fast p-nitrophenyl phosphate tablets; Sigma-Aldrich).


Figure 2
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  		FIGURE 2. 4T1 cells but not DCs were transfectable with an adenoviral vector encoding a soluble form of human TGF-β receptor (Adv-TGF-β-R). A, A diagram of the human IgM ELISA used to measure TGF-β-R secretion. B, A standard curve of the human IgM ELISA. Similar to Fig. 1B, we used the supernatant of an overnight culture as the standard solution: 4T1+Adv-TGF-β-R (1 x 106 cells per ml, concentration 4000x). Using this relative concentration of TGF-β-R we demonstrated the linearity of serial dilutions and maintained quality control of this modified TGF-β-R ELISA. C, Permissive cell line 293T, 4T1 cells, and BALB/c bone marrow-derived DCs were transfected with Adv-TGF-β-R or control Adv-LacZ. Supernatants were collected after 48 h to measure TGF-β-R production using the human IgM ELISA. Medium alone and supernatants from 293T cells, 4T1 cells, and DCs (6 x 105 cells per 3 ml) served as controls. TGF-β-R expression was detected in Adv-TGF-β-R transfected 293T and 4T1 cells but not DCs (n = 2–3). D and E, Preventive vaccine efficacy was compared (tumor incidence, D; tumor volume, E) between Adv-TGF-β-R transfected DC/4T1 fusion cells (DC+Adv-TGF-β-R/4T1) and Adv-LacZ transfected DC/4T1 fusion cells (DC+Adv-LacZ/4T1). Naive BALB/c mice (n = 15 per group, three separate experiments) were immunized (i.p.) on days 0 and 14 and then challenged with wild-type 4T1 implantation. Tumor incidence and volume for each mouse were recorded. **, p < 0.01, ***, p < 0.001.

 
TGF-β activity assay

Mv1Lu mink lung cells were grown for 48 h in 96-well plates at 5 x 104 cells per well. Tritiated thymidine (1 µCi/well; Amersham Biosciences) was added to each well and the cells were incubated for 16 h. At completion, the cells were harvested and the radioactivity was measured using a scintillation counter and reported as the mean cpm ± SEM from duplicate samples.

Generation of bone marrow-derived DCs

Erythrocyte-depleted murine bone marrow cells obtained from BALB/c mouse femurs were cultured in CM supplemented with GM-CSF (10 ng/ml) and IL-4 (10 ng/ml) at 1 x 106 cells/ml (6). The cytokines were replenished on day 3. On day 6, nonadherent DCs were harvested by vigorous pipetting and enriched by gradient centrifugation using the density gradient medium OptiPrep (Sigma-Aldrich). In brief, 11.5% OptiPrep was overlaid with day 6-nonadherent cultured bone marrow cells, and the mixture was centrifuged at 1650 rpm for 15 min. The cell layer at the upper interface of the 11.5% Optiprep was collected and washed twice with RPMI 1640 and cultured in CM supplemented with GM-CSF (10 ng/ml).

Transfection of tumor cells with Adv-TGF-β-R or Adv-LacZ

The tumor cells were suspended in 2% FBS RPMI 1640. Adv-TGF-β-R or Adv-LacZ was added, and the cells were incubated for 1 h at room temperature and then for 48 h at 37°C. We tested a range of MOIs from 150 to 300, adenovirus to target cell ratios of 9–18 x 107 PFU to 6 x 105 cells. TGF-β-R expression was measured by the modified ELISA as described above. The transfected tumor cells were harvested and used for immunotherapy.

DC/tumor fusion cell preparation

Bone marrow-derived DCs were fused with tumor cells at a DC to tumor cell ratio of 2:1 using 50% polyethylene glycol (m.w. of 1500; Roche Diagnostics; Ref. 11). In brief, the DC/tumor cell suspension was washed twice with RPMI 1640 prewarmed to 37°C. PEG (50%, 1 ml) was added over 1 min and the suspension was stirred gently for 1–2 min. Prewarmed RPMI 1640 (1 ml) was then added over 1 min and the suspension was stirred. An additional 3 ml of RPMI 1640 was added over 3 min, after which 10 ml of RPMI 1640 was added slowly. After a 5-min incubation at 37°C, the resultant cell mixture was pelleted and grown overnight in CM with GM-CSF.

Animal studies

Prevention model. Mice were immunized s.c. with irradiated (12,000 rad) 2 x 106 4T1+Adv-TGF-β-R or 4T1+Adv-LacZ or i.p. with irradiated (16,000 rad) DC/4T1+Adv-TGF-β-R or DC/4T1+Adv-LacZ on days 0, 7, and 14. On day 21, the mice were challenged with a s.c. injection of 2 x 104 4T1 cells in the right flank.

Therapeutic model. 4T1 (2 x 104 cells) or RenCa (5 x 105 cells) were injected s.c. into the right flank on days 0, 3, and 7, followed by an i.p. injection of irradiated (16,000 rad) DC/4T1+Adv-TGF-β-R or DC/RenCa+Adv-TGF-β-R (with their respective tumor+Adv-LacZ controls; 2 x 106 DC/tumor fusion cells per injection) every 3 days for a total of 3 injections. Tumor size was determined biweekly by calculating tumor volume. Tumor volume = (b2 x a)/2 where a represents the longest diameter and b the shortest diameter.

Tumor-specific CTL assay and inhibition of CTL killing of 4T1 cells

CD8+ CTL cells isolated using the mouse CD8+ T cell isolation kit (Miltenyi Biotec) were stimulated with irradiated 4T1 (12,000 rad) at a CTL to tumor cell ratio of 20:1 in CM supplemented with 0.1% 2-mercaptoethanol for 5 days. IL-2 (20 U/ml) was added on day 2. Target cells (104 4T1 cells) were incubated overnight at 37°C at a T cell to tumor cell ratio of 20:1. The supernatant from each well was harvested, and the percentage of tumor killing was determined using CytoTox 96 (Promega). For Treg inhibition of CTL activity, CD4+CD25+ Tregs isolated using the mouse CD4+CD25+ Regulatory T cell Isolation kit (Miltenyi Biotec) were added at different CTL to Treg ratios.

In vitro Treg induction experiments

Irradiated (16,000 rad) DC/tumor fusion cells were cocultured with naive syngeneic BALB/c splenocytes (1 x 104 cells) for 72 h in an MLR. The percentage of CD4+CD25+Foxp3+ Tregs/CD4+ T cells was determined using the Mouse Regulatory T cell Staining kit (eBioscience) and measured by FACS.

In vivo Treg induction experiments

BALB/c mice were immunized with DC/4T1, DC/4T1+Adv-LacZ, or DC/4T1+Adv-TGF-β-R on days 7 and 14. Splenocytes were isolated on day 21 and the percentages of CD4+CD25+Foxp3+ Tregs were determined using the Mouse Regulatory T cell Staining kit (eBioscience) and FACS.

ELISA

Supernatants of DCs and tumor cells were collected and stored immediately at –20°C until further use. Bioactive TGF-β secretion was determined by ELISA (R&D Systems). The supernatants were acidified according to the manufacturer’s instructions.

Statistical analysis

Statistical significance was determined by unpaired t test or by survival curve (Kaplan and Meier) using commercially available software (PRISM; GraphPad). p < 0.05 was considered statistically significant (*, p < 0.05, **, p < 0.01, ***, p < 0.001).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
A soluble form of TGF-β-R expressed by stably transfected CT26-TGF-β-TGF-β-R tumor cells was shown to bind TGF-β and to reduce TGF-β inhibitory activity

We have previously shown that tumor-derived TGF-β significantly reduces the ability of DC/tumor fusion cells to prime antitumor immunity in vivo (7). To determine whether neutralization of TGF-β enhances the efficacy of the DC/tumor fusion vaccine, we constructed a soluble form of TGF-β-R by fusing human TGF-β-R type II to the Fc portion of human IgM. Human TGF-β-R type II was previously shown to cross-react with mouse TGF-β (12). This construct in a retroviral vector was used to stably transfect the CT26-TGF-β subline (7), which constitutively expresses the active form of TGF-β (12). To determine the ability of TGF-β-R to capture TGF-β, we designed an in vitro assay that borrowed reagents from a TGF-β ELISA kit (R&D Systems). As shown in Fig. 1A, TGF-β-R is captured via the Fc component of human IgM by coated anti-human IgM Abs. If TGF-β-R is able to bind to the added TGF-β, the subsequent addition of biotinylated anti-TGF-β Abs and HRP/substrates will allow quantification of TGF-β-R in a colorimetric fashion as measured by a standard ELISA. This modified ELISA estimates the TGF-β-R concentration by measuring the amount of TGF-β bound to TGF-β-R. To demonstrate the linearity of this ELISA, we generated a standard curve with increasing concentrations of TGF-β-R (Fig. 1B). Fig. 1C shows TGF-β-R expression by CT26-TGF-β-TGF-β-R, but not by CT26-neo nor by the CT26-TGF-β subline.

To ensure that the binding of TGF-β by TGF-β-R would neutralize TGF-β activity, we performed a standard TGF-β activity assay using Mv1Lu mink lung cells. TGF-β inhibits the growth of Mv1Lu cells in a dose-dependent fashion, thus enabling the determination of TGF-β activity. The supernatant from CT26-TGF-β cells inhibited the growth of Mv1Lu cells, whereas the supernatant from CT26-TGF-β-TGF-β-R cells prevented the complete inhibition of Mv1Lu cell growth, indicating the neutralizing ability of the TGF-β-R expressed by tumor cells (Fig. 1D).

Tumor cells but not DCs are transfectable with Adv-TGF-β-R at MOI 300

In studying the effect of TGF-β-R on the DC/tumor fusion vaccines, the delivery of the gene encoding TGF-β-R can target either DCs or tumor fusion partners. We generated an adenovirus vector encoding TGF-β-R (Adv-TGF-β-R) and a control adenovirus encoding the LacZ gene (Adv-LacZ) and compared the transfectability of bone marrow-derived DCs and tumor cells to that of 293T cells, which are a permissible host for viral transfection. We tested a range of MOIs from 150 to 300 and found the optimal MOI to be 300 for 4T1 cells and RenCa cells despite the inability to transfect DCs. To measure the level of TGF-β-R in the supernatant of each transfectant culture, we chose a more simplified approach by detecting human IgM using a standard human IgM ELISA (see Fig. 2A and Materials and Methods). The standard curve for the human IgM ELISA was shown on Fig. 2B with a linear OD range of 0.2–2.0. As shown in Fig. 2C, we found that, unlike 293T and 4T1 cells, DCs are not transfectable with Adv-TGF-β-R at an MOI of 300. To further confirm this observation, the efficacy of the DC/tumor fusion vaccine generated with DCs transfected with Adv-TGF-β-R and 4T1 (DC+Adv-TGF-β-R/4T1) was compared with the efficacy of DC/fusion vaccine generated with DCs transfected with Adv-LacZ and 4T1 (DC+Adv-LacZ/4T1). Consistent with the lack of TGF-β-R expression by DC+Adv-TGF-β-R, we found no significant difference in the preventive vaccine efficacy between the two groups, indicating that Adv-TGF-β-R is not able to transfect bone marrow-derived DCs (Figs. 2D and 2E). We did observe that DCs transfected with either Adv-LacZ or Adv-TGF-β-R then fused with 4T1 (DC+Adv-LacZ or DC+Adv-TGF-β-R/4T1) appeared to have enhanced vaccine efficacy compared with vaccines generated using DCs that were not transfected with adenovirus (DC/4T1). This may be due to an enhanced DC activation after adenovirus transfection as the use of more activated DCs in vaccine protocols have been shown to augment the efficacy of DC vaccines (13, 14).

Immunization with irradiated TGF-β-R expressing 4T1 cells slowed the growth of wild-type 4T1 cells in vivo

One possible mechanism of tumor immune escape is the effect of tumor-derived TGF-β on immune surveillance. We first determined whether most tumor cells produce TGF-β. As shown in Fig. 3A, B16F10 (melanoma), P815 (lymphoblast-like mastocytoma), EL4 (lymphoma), MCA205 (fibrosarcoma), 4T1 (breast adenocarcinoma), and RenCa (renal cell carcinoma) produced similar or higher levels of TGF-β compared with 3T3 (fibroblast) control cells. To test the hypothesis that tumor-derived TGF-β is responsible for tumor immune escape, 4T1 cells were transfected with Adv-TGF-β-R and then implanted to determine whether TGF-β neutralization by TGF-β-R enhances the ability of the host immune system to recognize 4T1 cells. We observed that the implantation of irradiated (nonproliferative) 4T1+Adv-TGF-β-R cells, compared with irradiated 4T1+Adv-LacZ cells, lowered tumor incidence and slowed growth rate after tumor challenge with wild-type 4T1 (Fig. 3B and 3C), indicating that tumor-derived TGF-β contributed to the failure of the host’s immune system to limit tumor growth.


Figure 3
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  		FIGURE 3. Immunization with irradiated TGF-β-R expressing 4T1 (4T1+Adv-TGF-β-R) slowed the growth of wild-type 4T1 cells in vivo. A, The expression of TGF-β by solid tumors. Tumor cells (5 x 105 cells/3 ml) were grown in culture medium for 72 h. Supernatants and medium alone were each assessed for TGF-β by the human IgM ELISA. All tumor cells tested expressed TGF-β (n = 2–4). B and C, Irradiated 4T1+Adv-TGF-β-R cells induced antitumor immunity. The 4T1, 4T1+Adv-LacZ, and 4T1+Adv-TGF-β-R were irradiated (12,000 rad) before being injected (s.c.) into the left flank of BALB/c mice on days 0 and 14. Tumor challenge with wild-type 4T1 followed on day 21 (n = 5–10 mice per group). Tumor incidence (B) and tumor volume (C) were recorded. Expression of TGF-β-R by 4T1 cells enhanced the host immune recognition of the tumor cells. *, p < 0.05 compared with irrad. 4T1+Adv-LacZ, **, p < 0.01 compared with irrad. 4T1+Adv-LacZ.

 
The enhanced efficacy of the DC/tumor fusion vaccine generated with 4T1 cells expressing TGF-β-R

Our previous investigations showed that tumor-derived TGF-β could significantly hinder the antitumor immunity induced by the DC/tumor fusion vaccine (7). Therefore we speculated that neutralization of tumor-derived TGF-β by TGF-β-R during the priming of effector T cells might enhance the efficacy of the vaccine. DC/tumor fusion vaccines were generated with 4T1+Adv-LacZ or 4T1+Adv-TGF-β-R (designated as DC/4T1+Adv-LacZ or DC/4T1+Adv-TGF-β-R). We showed that TGF-β was detected in the supernatants of the overnight cultures of DC/4T1 and DC/4T1+Adv-LacZ but not DC/4T1+Adv-TGF-β-R, indicating the neutralizing effect of TGF-β-R (Fig. 4A). In vivo, the enhanced efficacy of the DC/4T1+Adv-TGF-β-R fusion vaccine was shown by the significant delay in tumor growth after tumor challenge with wild-type 4T1 cells in both the preventive (Fig. 4B) and the therapeutic models (Fig. 4C). These data indicated that TGF-β neutralization in the microenvironment where DCs and T cells interact resulted in an enhanced antitumor immunity.


Figure 4
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  		FIGURE 4. DC/tumor fusion vaccine generated using 4T1 cells that express TGF-β-R enhanced its vaccine efficacy. DC/tumor fusion vaccines were generated using BALB/c bone marrow-derived DCs and 4T1+Adv-TGF-β-R or 4T1+Adv-LacZ (DC/4T1+Adv-TGF-β-R or DC/4T1+Adv-LacZ). A, TGF-β content of the DC-only supernatant or the postfusion supernatants of overnight cultures of DC/4T1, DC/4T1+Adv-LacZ, or DC/4T1+Adv-TGF-β-R. Medium alone served as a negative control. B, Prevention model (Vac->Vac->Tumor): DC/4T1, DC/4T1+Adv-LacZ, or DC/4T1+Adv-TGF-β-R fusion cells were injected (s.c.) into BALB/c mice on days 0 and 14. Tumor challenge with wild-type 4T1 followed on day 21. *, p < 0.05 compared with DC/4T1+Adv-LacZ, #, p < 0.05 compared with DC/4T1. C, Therapeutic model (Tumor->Vac->Vac->Vac): wild-type 4T1 cells were implanted in the right flank on day 0. DC/4T1, DC/4T1+Adv-LacZ, or DC/4T1+Adv-TGF-β-R fusion cells were injected (i.p.) on days 3, 7, and 10. Mice without vaccination (No vaccine group) served as a negative control. Tumor volumes were recorded. Data shown are from three separate experiments. DC/4T1+Adv-TGF-β-R fusion vaccine showed increased vaccine efficacy compared with DC/4T1+Adv-LacZ fusion vaccine (n = 15 mice per group, except n = 5 mice in the DC/4T1 group). **, p < 0.01 compared with DC/4T1+Adv-LacZ.

 
The DC/tumor fusion vaccine generated with 4T1+Adv-TGF-β-R induced fewer CD4+CD25+Foxp3+ Tregs compared with the control vaccines

TGF-β signaling is essential for Treg induction (15). One potential mechanism of the enhanced antitumor immunity observed with the DC/4T1+Adv-TGF-β-R fusion vaccine is a reduction in the percentage of Tregs induced. In an MLR with naive syngeneic splenocytes and DC/tumor fusion cells, the magnitude of the Treg response induced by DC/4T1+Adv-TGF-β-R fusion cells was lower than that induced by DC/4T1+Adv-LacZ fusion cells, as determined by the percentage of CD4+CD25+Foxp3+ Tregs (Fig. 5A). This finding was confirmed in vivo by demonstrating fewer splenic Tregs in mice immunized with DC/4T1+Adv-TGF-β-R compared with mice immunized with DC/4T1 or DC/4T1+Adv-LacZ fusion cells (Fig. 5B and 5C). We also provided evidence for the role of CD4+CD25+ Tregs in this vaccination model by showing that CD4+CD25+ Tregs isolated from DC/4T1+Adv-LacZ-immunized mice were capable of suppressing CD8+ CTLs derived from DC/4T1+Adv-TGF-β-R-immunized mice (Fig. 5D). Thus, these data indicate that the neutralization of TGF-β by TGF-β-R may enhance antitumor immunity by lowering the magnitude of the Treg response, leading to enhanced effector T cell function.


Figure 5
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  		FIGURE 5. The DC/tumor fusion vaccine generated with 4T1+Adv-TGF-β-R induced a lower percentage of CD4+CD25+Foxp3+ Tregs compared with controls. Naive BALB/c splenocytes were mixed with DC/4T1+Adv-LacZ or 4T1+Adv-TGF-β-R fusion cells and cultured for 72 h. The percentage of CD4+CD25+Foxp3+ T cells was determined by FACS. A, Representative dot plots gated on live cells (left panels). Percentages of CD4+CD25+Foxp3+ T cells from three separate experiments (right panel). *, p < 0.05. B and C, In vivo confirmation that 4T1+Adv-TGF-β-R induced fewer CD4+CD25+Foxp3+ regulatory T cells. Mice (n = 5) were immunized with 2 x 106 DC/4T1, DC/4T1+Adv-LacZ, or 4T1+Adv-TGF-β-R fusion cells on days 0 and 14. On day 21, the percentage of CD4+CD25+Foxp3+/CD4+ T cells was determined by FACS. DC/4T1+Adv-TGF-β-R fusion cells induced a Treg response of lower magnitude compared with that induced by DC/4T1 or DC/4T1+Adv-LacZ fusion cells. *, p < 0.05. D, Inhibitory effect CD4+CD25+Foxp3+ Tregs on CD8+ CTL killing of 4T1 cells. CD8+ T cells isolated from the spleen of DC/4T1+Adv-TGF-β-R immunized mice were stimulated ex vivo for 5 days and used in a standard CTL assay in 96-well plates. A CTL to target cell ratio of 20:1 was used for all wells. CD4+CD25+ Tregs (isolated from DC/4T1+Adv-LacZ mice) were then added at the indicated effector to Treg ratios. Tregs isolated from DC/4T1+Adv-LacZ mice inhibited CTL killing of 4T1 cells (n = 5 mice per group). *, p < 0.05.

 
Enhanced therapeutic efficacy of the DC/tumor fusion vaccine by TGF-β neutralization was also demonstrated in tumor cells that produce high levels of TGF-β

To show that TGF-β-R expression is effective against other types of tumors, we studied RenCa tumor cells, in part because these cells produced a high level of TGF-β in our initial screen (Fig. 3A) and it was possible that a high level of tumor-derived TGF-β might overwhelm the capacity of TGF-β-R to impact vaccine efficacy. We showed that the transfectability of RenCa cells was similar to that of 4T1 cells (Fig. 6A). The secretion of TGF-β-R by RenCa+Adv-TGF-β-R cells effectively reduced TGF-β levels in the 4T1/RenCa+Adv-TGF-β-R cell culture supernatants (Fig. 6B). Immunotherapy with the DC/tumor fusion vaccine generated using RenCa+Adv-TGF-β-R (designated as DC/RenCa+Adv-TGF-β-R) resulted in a small but significant growth delay compared with immunotherapy with DC/RenCa+Adv-LacZ fusion vaccine in wild-type RenCa-tumor-bearing mice (Fig. 6C). Of note, one mouse in the control DC/RenCa+Adv-LacZ group was excluded from analysis due to unusual shrinkage of an implanted tumor on day 34 likely resulting from ischemic central necrosis. These data indicate that the TGF-β neutralization strategy with adenoviral TGF-β-R gene transfer is also effective in a RenCa model, which is known to produce high levels of TGF-β.


Figure 6
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  		FIGURE 6. Enhanced therapeutic efficacy of DC/tumor fusion vaccine by TGF-β neutralization was also demonstrated in RenCa tumor cells, which produce high levels of TGF-β. A, The expression of TGF-β-R was detected in RenCa+Adv-TGF-β-R culture supernatant by the human IgM ELISA (n = 2). B, TGF-β content of the DC only supernatant or the postfusion supernatants of overnight cultures of DC/RenCa, DC/RenCa+Adv-LacZ, or DC/RenCa+Adv-TGF-β-R. Medium alone served as a negative control. Therapeutic model (Tumor->Vac->Vac->Vac): RenCa cells were implanted on day 0 and DC/tumor fusion cells were injected on days 3, 7, and 10. Tumor volume was recorded. Data shown are from three separate experiments (n = 10 per group). *, p < 0.05 vs all other groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Tumor-derived TGF-β participates in tumor immune escape by suppressing the host’s CTL function, which is critical for tumor killing (16). We previously demonstrated the inhibitory effect of tumor-derived TGF-β on DC/tumor fusion vaccine efficacy (7). In the present study we provide evidence that neutralization of tumor-derived TGF-β using an adenoviral gene transfer strategy is effective in enhancing antitumor immunity. First, tumor-derived TGF-β was found to play a role in tumor immune escape (Fig. 3). Next, we showed that TGF-β-R expression by the tumor fusion partners significantly enhanced the efficacy of the DC/tumor fusion vaccine in preventing tumors in naive mice and in treating mice with tumors (Fig. 4). A possible mechanism for the improvement in vaccine efficacy is the lower magnitude of the Treg response (Fig. 5). Finally, the proposed strategy had some effect in RenCa cells, which produce high levels of TGF-β (Fig. 6).

TGF-β is a regulatory cytokine that has a direct effect on DCs. TGF-β treated myeloid DCs appear to be toleragenic rather than immunogenic, as they do not respond to stimuli and can protect mice from lethal LPS-induced inflammation (17). Furthermore, TGF-β treatment of myeloid DCs is capable of inducing Tregs (18). Our study further supports these regulatory properties of TGF-β in DCs and shows enhanced antitumor immunity and lower Treg priming when TGF-β is inhibited by TGF-β-R.

Several TGF-β blocking strategies to enhancing antitumor immunity have been reported. Forced expression of a nonfunctional (dominant-negative) TGF-β-R may enhance the ability of host immune system to reject tumors (19, 20, 21, 22, 23). Soluble dominant-negative TGF-β-Rs have also resulted in reduced mammary tumor cell viability and migratory potential (24); however, given the pleiotropic effect of TGF-β, such an approach may be toxic in humans. Our laboratory and others have shown the direct suppressive effect of TGF-β on DC antitumor vaccine efficacy (7, 25). Our current study examines the efficacy of neutralizing tumor-derived TGF-β in the microenvironment where DCs interact with host T cells, namely secondary lymphoid structures. The neutralization of TGF-β in vivo using tumors expressing TGF-β-R produced two interesting observations. First, whereas most tumor-derived Ags are poorly immunogenic (26), we found that the presence of tumors in a microenvironment where TGF-β signaling was interrupted resulted in host recognition of the tumor. This indicates that tumors thrive in an environment where TGF-β signaling may restrict the ability of the host to prime an antitumor response. Secondly, our data indicate that TGF-β neutralization enhances the efficacy of the DC/tumor fusion vaccine, both in preventive and therapeutic models. Our prior demonstration that DC/tumor fusion vaccine was more efficacious than tumor Ag-pulsed DC vaccines (27) led us to speculate that the efficacy of the DC/tumor fusion vaccine could be further enhanced by interrupting TGF-β signaling using an adenoviral gene delivery strategy targeting DCs or their tumor fusion partners. We found that tumor cells but not DCs are transfectable using the adenoviral gene delivery system at an MOI of 300. We acknowledge that DCs have been successfully transfected at higher MOIs (28, 29); nevertheless, the results of our study show that the use of a surrogate target provides an alternative approach to the delivery of genes into DCs.

The recently described TGF-β-dependent Th17 lineage and its role in innate immunity has generated many questions regarding the possible role of TGF-β in antitumor immunity (30). Although the specific role of Th17 in antitumor immunity is unknown, a few reports have suggested a role in tumor prevention (31, 32). In our study, the neutralization of TGF-β resulted in an enhanced antitumor immunity, indicating that the dominant effect of TGF-β in a tumor-bearing host is one that favors tumor survival. Because it is theoretically possible that TGF-β-R may lower the Th17 response, future studies are needed to examine the effect of TGF-β neutralization strategies on the Th17 response in vivo.

In a study similar to the present study, Wang and colleagues showed that DCs genetically engineered to secrete dominant-negative TGF-β-R when pulsed with tumor lysate were more efficacious in generating an antitumor response compared with nonsecreting tumors in a mouse prostate adenocarcinoma model (31). Given our previous finding that the DC/tumor fusion approach is superior to tumor lysate-pulsed DC vaccines (27), we speculate that the DC/fusion vaccine generated using tumors transfected with Adv-TGF-β-R will be more effective than tumor lysate-pulsed DCs expressing dominant-negative TGF-β-Rs. More study is needed to compare the relative efficacy of these two strategies.

We speculate that the use of CpG- or LPS-activated DCs in combination with Adv-TGF-β-R transfected tumor may further enhance the effectiveness of DC/tumor fusion vaccines. We have previously shown (8) that the use of LPS-activated DCs did not enhance the therapeutic efficacy of the vaccine in tumor-bearing mice compared with the use of non-LPS-activated DCs, despite increased Th1 priming. The lack of enhanced therapeutic efficacy with LPS-activated DCs is not a lack of CTL response but the result of Treg inhibitory activity. Thus, we expect that the lower magnitude of the Treg response with the use of TGF-β-R may lead to an improvement in the therapeutic effect of CpG- or LPS-activated DC vaccines. Future studies are required to examine this hypothesis.

In summary, given the broad range of TGF-β-dependent processes governing immune homeostasis, it is critical to target a specific microenvironment if the interruption of TGF-β signaling is desired. We generated an adenoviral vector encoding TGF-β-R that binds to TGF-β and interrupts TGF-β function. We showed that neutralization of tumor-derived TGF-β in the microenvironment of DC-T cell priming is an effective strategy to enhance the efficacy of DC/tumor fusion vaccines. Our study also demonstrated a site-specific targeting of TGF-β blockade using DCs as a delivery vehicle.


   Acknowledgments
 
We appreciate Weiping Zou’s contribution in the preparation of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
We have no financial conflict of interest.


   Footnotes
 
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 supported by a grant from Department of Defense (to J.-J.C.) and by the Foundation for Digestive Health and Nutrition (to J.Y.K.). Back

2 Address correspondence and reprint requests to Dr. John Y. Kao, Division of Gastroenterology, Department of Internal Medicine, University of Michigan Health System, 6520A Medical Sciences Research Building I, Special Postal Code 5682, 1150 West Medical Center Drive, Ann Arbor, MI 48109. E-mail address: jykao{at}umich.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; Treg, regulatory T cell; TGF-β-R, TGF-β receptor; MOI, multiplicity of infection; CM, complete medium; RenCa, murine renal cell carcinoma. Back

Received for publication November 8, 2007. Accepted for publication July 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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