Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2006 May 28.
Published in final edited form as: Hum Gene Ther. 2003 Nov 1;14(16):1511–1524. doi: 10.1089/104303403322495025

Interleukin-7 Gene-Modified Dendritic Cells Reduce Pulmonary Tumor Burden in Spontaneous Murine Bronchoalveolar Cell Carcinoma

SHERVEN SHARMA 1,2,3,✉, RAJ K BATRA 1,2,3, SEOK CHUL YANG 1, SVEN HILLINGER 1, LI ZHU 1,2,3, KIMBERLY ATIANZAR 1, ROBERT M STRIETER 1, KAREN RIEDL 1, MIN HUANG 1,2,3, STEVEN M DUBINETT 1,2,3
PMCID: PMC1471881  NIHMSID: NIHMS9440  PMID: 14577913

Abstract

The antitumor efficiency of dendritic cells transduced with an adenovirus vector expressing interleukin (IL)- 7 (DC-AdIL-7) was evaluated in a murine model of spontaneous bronchoalveolar cell carcinoma. These transgenic mice (CC-10 TAg), expressing the SV40 large T antigen under the Clara cell promoter, develop bilateral multifocal pulmonary adenocarcinomas and die at 4 months as a result of progressive pulmonary tumor burden. Injection of DC-AdIL-7 in the axillary lymph node region (ALNR) weekly for 3 weeks led to a marked reduction in tumor burden with extensive lymphocytic infiltration of the tumors and enhanced survival. The antitumor responses were accompanied by the enhanced elaboration of interferon (IFN)-γ and IL-12 as well as an increase in the antiangiogenic chemokines, IFN-γ–inducible protein 10 (IP-10/CXCL10) and monokine induced by IFN-γ (MIG/CXCL9). In contrast, production of the immunosuppressive mediators IL-10, transforming growth factor (TGF)-β, prostaglandin E2 (PGE2), and the proangiogenic modulator vascular endothelial growth factor (VEGF) decreased in response to DC-AdIL-7 treatment. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for further evaluation of DC-AdIL-7 in regulation of tumor immunity and its use in lung cancer genetic immunotherapy.

OVERVIEW SUMMARY

In our previous studies, intratumoral administration of adenoviral interleukin (IL)-7–transduced dendritic cells (DC-AdIL- 7) resulted in specific systemic antitumor immune responses that led to complete tumor regression and long-term immunity. The intratumoral administration of gene-modified dendritic cells (DC) resulted in effective trafficking of transferred DCs to regional lymph nodes. To determine if direct administration of DC-AdIL-7 in the axillary lymph node region (ALNR) would mediate a significant response against pulmonary tumor, we tested the antitumor efficacy of DC-AdIL-7 in a spontaneous murine lung cancer model. In the CC-10 TAg mice, adenocarcinomas develop in an organ-specific manner and, compared to transplantable tumors, the pulmonary tumors in these mice more closely resemble human lung cancer. In this study we demonstrate that ALNR injection of DC-AdIL-7 mediates potent antitumor responses in vivo leading to a significant reduction in tumor burden.

INTRODUCTION

The central importance of functional host professional antigen-presenting cells (APCs) in the immune response against cancer has been well defined (Huang et al., 1994). Dendritic cells (DCs) are highly specialized professional APCs with potent capacity to capture, process, and present antigen to T cells (Banchereau and Steinman, 1998). Tumor cells interfere with host DC maturation and function (Gabrilovich et al., 1996; Kobie et al., 2003; Sharma et al., 2003). Thus, to circumvent tumor-mediated inhibition of DC maturation and function in vivo, DCs that have undergone cytokine-stimulated maturation ex vivo have been utilized in clinical trials and murine cancer models (Zitvogel et al., 1996; Ribas et al., 1997; Sharma et al., 1997; Miller et al., 1998; Nestle et al., 1998; Thurner et al., 1999; Dallal and Lotze, 2000; Banchereau et al., 2001a,b; Schuler et al., 2003;). Antigen-specific cytotoxic T-lymphocyte (CTL) responses have been achieved utilizing ex vivo antigen-pulsed DCs (Mayordomo et al., 1995; Boczkowski et al., 1996; Celluzzi et al., 1996; Zitvogel et al., 1996; Nestle et al., 1998; Thurner et al., 1999; Banchereau et al., 2001a; Schuler et al., 2003; Su et al., 2003). In addition, novel tumor antigen delivery systems utilizing cytokine gene-transduced tumor cells and DCs (Sharma et al., 1997; Miller et al., 1998) or fusion of tumor cells with DCs have resulted in induction of antitumor immunity (Gong et al., 1997; Celluzzi and Falo, 1998; Kugler et al., 2000; Parkhurst et al., 2003). Delivery of tumor antigens by ex vivo stimulated DCs has been shown to be superior to purified peptides in avoiding CTL tolerization (Toes et al., 1998). Vaccination with multiple tumor antigens may be superior to the use of a single epitope (Toes et al., 1997; Fields et al., 1998) and these responses can be further enhanced by the coadministration of immune-potentiating cytokines (Sharma et al., 1997; Miller et al., 1998; Kirk et al., 2001).

We have demonstrated previously that interleukin (IL)-7–transduced tumor cells administered intratumorally in conjunction with DCs elicit potent antitumor responses in a murine model of established lung cancer (Sharma et al., 1997). IL-7 delivery to the tumor site may be beneficial because it enhances antigen-specific T-cell cytotoxicity (Kos and Mullbacher, 1993) and synergizes with IL-12 in the induction of T-cell proliferation, cytotoxicity, and interferon (IFN)-γ release (Mehrotra et al., 1995). We have shown that IL-7 gene transfer in non-small–cell lung cancer results in modification of tumor phenotype (Sharma et al., 1996). In addition to its direct effects on tumor cells, IL-7 has the capacity to enhance both CTL generation and longevity (Lynch and Miller, 1994; Akashi et al., 1997). IL-7 also downregulates both macrophage and tumor production of transforming growth factor (TGF)-β and thus may serve to limit tumor-induced immune suppression (Dubinett et al., 1993; Miller et al., 1993). TGF-β has been shown to inhibit the antigen-presenting functions and antitumor activity of DC vaccines (Kobie et al., 2003). In addition to its capacity to downregulate TGF-β production, IL-7 can also limit TGF-β signaling (Huang et al., 2002). However, despite these beneficial effects of IL-7, maximal antitumor benefit was demonstrated when bone marrow-derived DCs were administered intratumorally in conjunction with IL-7 gene-modified autologous tumor (Sharma et al., 1997). Based on these studies we performed experiments utilizing transplantable murine lung cancer models in which we demonstrated that intratumoral administration of adenoviral IL-7–transduced dendritic cells (DC-AdIL-7) results in specific systemic antitumor immune responses that lead to complete tumor regression and long-term immunity (Miller et al., 2000). The intratumoral administration of gene-modified DC resulted in effective trafficking of transferred DCs to regional lymph nodes. To determine if direct administration of DC-AdIL-7 in the axillary lymph node region (ALNR) would mediate significant responses against pulmonary tumor, we tested the antitumor efficacy of intranodal injection of DC-AdIL-7 in a spontaneous murine lung cancer model. In the CC-10 TAg mice, adenocarcinomas develop in an organ-specific manner, and in comparison to transplantable tumors, more closely represent the clinical setting encountered in patients with lung cancer. Here we demonstrate for the first time that ALNR injection of DC-AdIL-7 mediates potent antitumor responses in vivo leading to a significant reduction in pulmonary tumor burden.

MATERIALS AND METHODS

CC10 TAg mice

The transgenic CC-10 TAg mice, in which the SV40 large TAg is expressed under control of the murine Clara cell-specific promoter, CC-10, were used in these studies (Magdaleno et al., 1997). All of the mice expressing the transgene developed diffuse bilateral bronchoalveolar carcinoma. Tumor was evident bilaterally by microscopic examination as early as 4 weeks of age. After 3 months of age, the bronchoalveolar pattern of tumor growth coalesced to form multiple bilateral tumor nodules throughout both lungs. The CC-10 TAg transgenic mice had an average life span of 4 months. Extrathoracic metastases were not noted. Breeding pairs for these mice were generously provided by Francesco J. DeMayo (Baylor College of Medicine, Houston, TX). Transgenic mice were bred at the VA Greater Los Angeles Healthcare System vivarium and maintained in the animal research facility. All studies were in compliance with the Institutional Review Board. Before each experiment using the CC-10 TAg transgenic mice, presence of the transgene was confirmed by polymerase chain reaction (PCR) of tail biopsies. The 5′ primer sequence was SM19-TAG: 5′-TGGACCTTCTAGGTCTTGAAAGG-3′, and the 3′ primer sequence was SM36-TAG: 5′-AGGCATTCCACCACTGCTCCCATT-3′. The size of the resulting PCR fragment is 650 bp and the amplification products were visualized against molecular weight standards on a 1.5% agarose gel stained with ethidium bromide. All of the experiments used pathogen-free CC-10 TAg transgenic mice beginning at 4–5 weeks of age.

Isolation and propagation of bone marrow-derived DCs

DCs were isolated from bone marrow and incubated with lymphocyte- and macrophage-depleting antibodies (CD45R, anti-B cell; TIB 229, anti-Ia; TIB 150, anti-CD8; and TIB 207, anti-CD4; all obtained from the ATCC, Manassas, VA) and rabbit serum complement (Sigma, St. Louis, MO) for 1 hr. Cells were washed and then incubated overnight to allow contaminating macrophages to adhere, and nonadherent DCs were harvested and cultured in vitro for 6 days with murine granulocyte macrophage colony-stimulating factor (GM-CSF; 2 ng/ml) and IL-4 (20 ng/ml; R&D Systems, Minneapolis, MN) as previously described (Sharma et al., 1997). Consistent with previous studies from our laboratory as well as others (Inaba et al., 1992; Sharma et al., 1997; Fields et al., 1998; Miller et al., 1998) dendritic cells characterized by flow cytometry were found to have high-level expression of B7-1, B7-2, CD11c, major histocompatibility complex (MHC) II and MHC I. These cells were found to be 90% DCs as defined by coexpression of these cell surface antigens (data not shown).

Fibroblast culture

Fibroblast cultures were established from FVB mouse skin. Skin from FVB mice was isolated and cut into small pieces, digested in trypsin for 1 hr, washed in phosphate-buffered saline (PBS) three times, and plated in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F11 containing bovine insulin (10 μg/ml), 15 mM HEPES buffer (GIBCO, Gaithersburg, MD), human transferrin (25 μg/ml), human high-density lipoprotein (20 μg/ml), human platelet-derived growth factor (1 U/ml), and mouse epidermal growth factor (100 ng/ml) (Sigma). Monolayer cells were trypsinized to obtain single cell suspension for adenoviral transduction.

Transduction of dendritic cells and fibroblasts with adenoviral 5 vector

The adenoviral construct (AdIL-7) is an E1-deleted, replication- deficient adenoviral type 5 vector (Ad5) encoding human IL-7 cDNA (Arthur et al., 1997). The control vector (AdRR5) did not contain the human IL-7 cDNA. The IL-7 cDNA was inserted into the former E1 site and driven by the cytomegalovirus (CMV) promoter–enhancer. To optimize the multiplicity of infection (MOI) for IL-7 production, in vitro propagated DCs were transduced on day 6, in RPMI containing 2% fetal bovine serum (FBS), for 2 hr with AdIL-7 (DC-AdIL- 7) at MOIs of 10:1, 20:1, 50:1, and 100:1. IL-7 protein concentrations in transduced DC or fibroblast supernatants were determined by IL-7–specific enzyme-linked immunosorbent assay (ELISA) as previously described (Sharma et al., 1996). IL-7 protein concentrations in transduced DC or fibroblast supernatants were assessed in vitro from day 1 to day 17. Transduced DCs and fibroblasts produced IL-7 for up to 17 days in culture. Efficiency of transduction of DCs by adenoviral type 5 vector was assessed with an Ad5LacZ vector. DCs (5 × 106) were transduced with Ad5LacZ vector (MOIs of 100:1 and 20:1) in vitro. The DCs were incubated with the virus for 2 hr and washed prior to 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining 24 hr posttransduction (Arthur et al., 1997). For in vivo injection, an MOI of 20:1 was selected on the basis of an efficiency of transduction of 50% and optimal IL-7 protein production posttransduction. At an MOI of 20:1, DC-AdIL-7 secreted 8 ng/ml per 106 cells per 24 hr. Similarly, fibroblasts were transduced at an MOI of 20:1, with similar transduction efficiency. AdIL-7–transduced fibroblasts secreted IL-7 at 7.5 ng/ml per 106 cells per 24 hr. DCs and fibroblasts were washed with PBS three times prior to injection. DCs and fibroblasts were also transduced with the control vector at an MOI of 20:1. As additional controls, mice received DCs that did not undergo transduction. These cells are referred to as unmodified DCs in the description of the results.

Therapeutic model in CC10 Tag mice

Beginning at 4–5 weeks of age, CC-10 TAg transgenic mice were injected in the ALNR with one of the following treatments once per week for 3 weeks: (1) diluent; (2) DC-AdIL-7; (3) empty control adenoviral vector-transduced DCs (DC-AdCV); (4) unmodified DCs; (5) fibroblasts transduced with AdIL-7 (Fib-AdIL-7); (6) unmodified DCs plus recombinant IL-7 (5 ng); (7) recombinant IL-7 (5 ng) alone; (8) AdIL-7 (109 plaque-forming units [pfu]); and (9) AdCV (109 pfu). For groups receiving DCs, a total of 1 × 106 DCs in 100 μl volume was administered per injection. The specific activity of recombinant IL-7 was 5.6 × 107 U/mg protein (Sterling Winthrop, Malvern, PA). The endotoxin level reported by the manufacturer was less than 0.1 ng/μg of IL-7. The dose of recombinant IL-7 selected corresponded to the amount of IL-7 secreted after DC transduction. At 4 months, mice were sacrificed, and lungs were isolated for quantification of tumor surface area. Tumor burden was assessed by microscopic examination of hematoxylin and eosin (H&E)-stained sections with a calibrated graticule (a 1- cm2 grid subdivided into 100 1-mm2 squares). A grid square with tumor occupying more than 50% of its area was scored as positive, and the total number of positive squares was determined as described previously (Sharma et al., 1999). Ten separate fields from four histologic sections of the lungs from eight mice per group were examined under high-power (20× objective). Ten mice from each group were not sacrificed so that survival could be assessed.

Cytokine determination from tumor nodules and spleens

The cytokine profiles in tumors, lymph nodes, and spleens were determined in the treatment groups as described previously (Sharma et al., 1999). Non-necrotic tumors were harvested, cut into small pieces, homogenized and passed through a sieve (Bellco, Vineland, NJ). Cytokines and chemokines were determined from lung tissue homogenates and expressed as picograms per milligram of total protein. Total protein in the homogenates was determined with a Bradford kit from Sigma. Spleens and lymph nodes were harvested, teased apart, red blood cell count (RBC)-depleted with ddH2O, and brought to tonicity with 1 × PBS. After a 24-hr culture period, tumor nodule, splenocyte, and lymph node supernatant production of IL-10, IL-12, GM-CSF, IFN-γ, TGF-β, vascular endothelial growth factor (VEGF), MIG/CXCL9, and IP-10/CXCL10 were determined by ELISA and prostaglandin E2 (PGE2) by enzyme immunoassay (EIA) and results are expressed as picograms per milliliter per 106 cells.

Cytokine ELISA

Cytokine protein concentrations from tumor nodules, spleens and lymph nodes were determined by ELISA as described previously (Sharma et al., 1997). The plates were read at 490 nm with a Molecular Devices Microplate Reader (Sunnyvale, CA). The recombinant cytokines used as standards in the assay were obtained from PharMingen (San Diego, CA). IL-12 (Biosource, Camarillo, CA) and VEGF (Oncogene Research Products, Cambridge, MA) quantities were determined using kits according to the manufacturer’s instructions. MIG/CXCL9 and IP-10/CXCL10 were quantified using a modification of a double-ligand method as described previously (Standiford et al., 1990). The MIG/CXCL9 and IP-10/CXCL10 antibodies and protein were obtained from R&D. For the TGF-β ELISA measurements, samples were acidified and hence the active form of TGF-β was measured. The sensitivities of the IL-10, GM-CSF, IFN-γ, TGF-β, MIG/CXCL9, and IP-10/CXCL10 ELISA were 15 pg/ml. For IL-12 and VEGF the ELISA sensitivities were 5 pg/ml.

PGE2 EIA

PGE2 concentrations were determined using a kit from Cayman Chemical Co. (Ann Arbor, MI) according to the manufacturer’s instructions as described previously (Huang et al., 1998). The EIA plates were read by a Molecular Devices Microplate reader.

Statistical analysis

Groups of 6–10 mice were used. All experiments were repeated at least twice, and the DC-AdIL-7 group was repeated three times. Statistical analyses of the data was performed using the Kruskal-Wallis one-way analysis of variance on ranks, followed by multiple pairwise comparisons according to Dunn’s method. Significance at the p < 0.05 level is denoted.

RESULTS

DC-AdIL-7 mediates potent antitumor responses in a murine model of spontaneous bronchoalveolar carcinoma

We evaluated the antitumor efficacy of DC-AdIL-7 in a spontaneous bronchoalveolar cell carcinoma model in transgenic mice in which the SV40 large TAg is expressed under control of the murine Clara cell-specific promoter, CC-10 (Magdaleno et al., 1997). Mice expressing the transgene develop diffuse bilateral bronchoalveolar carcinoma and have an average life span of 4 months. Beginning at 4–5 weeks of age, CC-10 TAg transgenic mice were injected in the ALNR with the following treatments weekly for 3 weeks: (1) diluent; (2) DC-AdIL-7; (3) DC-AdCV; (4) unmodified DCs; (5) fibroblasts transduced with AdIL-7 (Fib-AdIL-7); (6) unmodified DCs plus recombinant IL-7 (5 ng); (7) recombinant IL-7 (5 ng); (8) AdIL-7 (109 pfu); and (9) AdCV (109 pfu). At 4 months when the control mice started to succumb because of progressive lung tumor growth, mice were sacrificed in all of the treatment groups, and lungs were isolated and paraffin embedded. H&E staining of paraffin-embedded lung tumor sections from control-treated mice revealed large tumor masses throughout both lungs with minimal lymphocytic infiltration (Fig. 1A and 1B). Compared to diluent-treated controls there was a decrease in the tumor burden in the following treatment groups: DC (1.6-fold), DC-AdCV (1.5-fold); Fib-AdIL-7 (1.9-fold); unmodified DCs plus recombinant IL-7 (1.9-fold); recombinant IL- 7 (1.7-fold); AdIL-7 (1.4-fold), and DC-AdIL-7 (10-fold) (Fig. 1C). Although E1-deleted adenoviral vectors have been reported to elicit antiviral CTL responses (Yang et al., 1994), we found that therapy with DC-AdCV was not more effective than unmodified DCs in reducing tumor burden in the CC-10 TAg mice because both of these groups had a 1.5-fold decrease in tumor burden (Fig. 1A and 1C). Similarly, injection of recombinant IL-7, DC plus recombinant IL-7 or AdIL-7 in CC-10 TAg did not improve the antitumor therapeutic efficacy in this model. In contrast, mice treated with DC-AdIL-7 had a greater than 10-fold (p < 0.001) reduction in pulmonary tumor burden compared to diluent-treated mice. These findings suggest that IL-7 is a critical element for optimal responses and must be secreted by DC for effective therapy in this model system (Fig. 1A and 1C). To determine the importance of DCs as a vehicle for IL-7 delivery, AdIL-7–transduced fibroblasts were administered in the ALNR. Although they produced similar levels of IL-7, AdIL-7–transduced fibroblasts were not as effective as DC-AdIL-7 in reducing tumor burden in the CC-10 TAg mice (Fig. 1A and 1C). Whereas diluent-treated control mice revealed large tumor masses throughout both lungs with minimal lymphocytic infiltration, DC-AdIL-7–treated mice had significantly smaller tumor nodules with extensive lymphocyte infiltration. DC-AdIL-7 treatment prolonged survival; median survival was 18 ± 2 weeks for control-treated mice, whereas mice treated with DC-AdIL-7 had a median survival of more than 40 ± 2 weeks (p < 0.001). All other therapies resulted in a median survival of 26 ± 2 weeks (Fig. 1D).

FIG. 1.

FIG. 1

FIG. 1

FIG. 1

Open in a new tab

A, B, and C: DC-AdIL-7 mediates potent antitumor responses in a murine model of spontaneous lung cancer. Beginning at 4–5 weeks of age, CC-10 TAg transgenic mice were injected in the axillary lymph node region (ALNR) with the following treatments once per week for 3 weeks. Treatment groups included: (1) diluent; (2) DC-AdIL-7; (3) empty control adenoviral vector-transduced DCs (DC-AdCV); (4) unmodified dendritic cells (DCs); (5) fibroblasts transduced with AdIL-7 (Fib-AdIL-7); (6) unmodified DCs plus recombinant interleukin (IL)-7 (5 ng); (7) recombinant IL-7 (5 ng); (8) AdIL-7 (109 plaque-forming units [pfu]); and (9) AdCV (109 pfu). A: Hematoxylin and eosin (H&E) staining of paraffin-embedded lung tumor sections from control-treated mice evidenced large tumor masses throughout both lungs without detectable lymphocytic infiltration. Compared to diluent-treated controls there was a decrease in the tumor burden in the following treatment groups: DC, DC-AdCV; Fib-AdIL-7; unmodified DCs plus recombinant IL-7; recombinant IL-7; AdIL-7. However, compared to diluent control group and other therapies, the DC-AdIL-7 group evidenced extensive lymphocytic infiltration with marked reduction in tumor burden (Magnifications, 1×, 40×, and 100×). B: Arrows depict tumor (*1) and infiltrate (*2). (Magnifications, 400×). C: Tumor burden was quantified within the lung by microscopy of H&E-stained paraffin-embedded sections. Although all treatment groups showed a decrease in tumor burden, DC-AdIL-7 treatment led to the greatest reduction (p < 0.001) compared to diluent-treated control (C) (n = 8 mice/group). D: Survival was prolonged in the DC-AdIL-7 treatment group (p < 0.05 compared to the various treatment groups and p < 0.001 as compared to diluent-treated control).

DC-AdIL-7 therapy promotes type 1 cytokine and antiangiogenic chemokine release and a decline in the immunosuppressive cytokines TGF-β and VEGF in CC-10 TAg mice

Based on previous reports indicating that tumor progression can be modified by host cytokine profiles (Alleva et al., 1994; Rohrer and Coggin, 1995), we measured the cytokine production from tumor sites and spleen therapy. Lungs, spleens, and lymph nodes were evaluated for the presence of VEGF, IL-10, IFN-γ GM-CSF, IL-12, MIG/CXCL9, IP-10/CXCL10, and TGF-β by ELISA and for PGE2 by EIA. Compared to diluent-treated controls treatment groups receiving DC, DC-AdCV, DC plus rIL-7, and AdIL-7 had a modest yet significant increase in type 1 cytokines (IFN-γ, IL-12) and antiangiogenic chemokines (IP-10/CXCL10, MIG/CXCL9) with a concomitant decrease in the immunosuppressive mediators (PGE2, VEGF, TGF-β) at the tumor sites. In comparison to diluent and the other treatment groups, DC-AdIL-7 produced the most significant increases in type 1 cytokines and antiangiogenic chemokines and the most substantial decline in the pulmonary production of immunosuppressive mediators. Compared to lungs from the diluent- treated group, CC-10 TAg mice treated with DC-AdIL-7 had significant reductions in VEGF (3-fold, p < 0.05), TGF-β (2-fold, p < 0.05), IL-10 (6-fold, p < 0.01) and PGE2 (32-fold, p < 0.001) but an increase in IFN-γ (15-fold, p < 0.001), IP- 10/CXCL10 (2-fold, p < 0.05), IL-12 (3-fold, p < 0.05), and MIG/CXCL9 (3-fold, p < 0.05) (Fig. 2A, 2B, and 2C). Similar cytokine patterns were also observed in the spleens of DC-AdIL-7–treated mice. Compared to the diluent-treated group, splenocytes from DC-AdIL-7–treated CC-10 TAg mice revealed reduced levels of PGE2 (5-fold, p < 0.05), IL-10 (2- fold, p < 0.05), and VEGF (3-fold, p < 0.05) but an increase in GM-CSF (23-fold, p < 0.001), IFN-γ (8-fold, p < 0.01), IL- 12 (3-fold, p < 0.05), MIG/CXCL9 (4-fold, p < 0.05), and IP- 10/CXCL10 (4-fold, p < 0.05) (Fig. 3A, 3B, and 3C).

FIG. 2.

FIG. 2

Open in a new tab

A–C: DC-AdIL-7 therapy promotes type 1 cytokine and antiangiogenic chemokine release and a decline in the immunosuppressive cytokines transforming growth facor (TGF)-β and vascular endothelial growth factor (VEGF) in the lungs of CC-10 TAg mice. After axillary lymph node region (ALNR) injection of (1) diluent; (2) DC-AdIL-7; (3) empty control adenoviral vector-transduced DCs (DC-AdCV); (4) unmodified dendritic cells (DCs); (5) fibroblasts transduced with AdIL-7 (Fib-AdIL-7); (6) unmodified DCs plus recombinant interleukin (IL)-7 (5 ng); (7) recombinant IL-7 (5 ng); (8) AdIL-7 (109 plaque forming units [pfu]); and (9) AdCV (109 pfu), pulmonary cytokine profiles in CC-10 TAg mice were determined and compared to those in diluent-treated tumor-bearing control mice. Cytokine and prostaglandin E2 (PGE2) from the lung tissue are expressed as picograms per milligram of total protein. Compared to lungs from diluent-treated CC-10 tumor- bearing mice, CC-10 mice treated with DC-AdIL-7 had significant reductions in IL-10, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, and PGE2 but a significant increase in interferon (IFN)-γ, IL-12, IP-10/CXCL10 and MIG/CXCL9 (A, B, and C). Values given reflect mean ± standard error (SE) for six mice per group. *p < 0.05 compared to diluent-treated control; ♦ p < 0.05 compared to the DC-AdIL-7 treatment group.

FIG. 3.

FIG. 3

Open in a new tab

A–C: DC-AdIL-7 therapy promotes type 1 cytokine and antiangiogenic chemokine release systemically in CC-10 TAg mice. After axillary lymph node region (ALNR) injection of: (1) diluent; (2) DC-AdIL-7; (3) empty control adenoviral vector-transduced DCs (DC-AdCV); (4) unmodified DCs; (5) Fibroblasts transduced with AdIL-7 (Fib-AdIL-7); 6) unmodified dendritic cells (DCs) plus recombinant interleukin (IL)-7 (5 ng); (7) recombinant IL-7 (5 ng); (8) AdIL-7 (109 plaque-forming units [pfu]); and (9) AdCV (109 pfu), spleen cytokine profiles in CC-10 TAg mice were determined and compared to those in diluent-treated tumor bearing control mice. Cytokine and prostaglandin E2 (PGE2) from the spleen are expressed as picograms per milliliter per 106 cells. Compared to diluent-treated CC-10 TAg mice, splenocytes from DC-AdIL- 7–treated CC-10 mice had reduced levels of PGE2 and vascular endothelial growth factor (VEGF) but significant increases in granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-γ, IL-12, MIG/CXCL9, and IP-10/CXCL10 (A, B, and C). Values given reflect mean ± standard error (SE) for six mice per group. *p < 0.05 compared to diluent-treated control; ♦ p < 0.05 compared to the DC-AdIL-7 treatment group.

DISCUSSION

In an attempt to stimulate specific antitumor immunity, experimental models and clinical studies are currently evaluating the potent antigen-presenting capacity of DCs combined with single or multiple tumor antigen epitopes (Schuler et al., 2003). However, the problems in utilizing tumor antigen-based immunization strategies include: (1) the potential induction of tolerance (Dhodapkar and Steinman, 2002); (2) the inability to utilize repeated dosing because of vector-associated neutralization (Rahman et al., 2001); and (3) the limitation of therapy to patients whose tumors express defined specific tumor antigens in the context of the correct HLA phenotype (Dubinett et al., 2000).

We and others have previously described a therapeutic paradigm that overcomes these deficits by intratumoral administration of cytokine gene-modified DCs (Melero et al., 1999; Kirk and Mule, 2000; Miller et al., 2000; Shimizu et al., 2001). This antitumor DC-based therapy exploits the professional APC as an effective vehicle for cytokine delivery and presentation of multiple tumor antigens in situ. In earlier studies, genetic immunotherapy administered by the intratumoral route led to augmentation of antitumor reactivity (Sharma et al., 1997; Miller et al., 1998; Gambotto et al., 1999). The DC-based intratumoral delivery of the immunopotentiating cytokine IL-7 resulted in potent systemic, specific antitumor immune responses that resulted in eradication of established murine lung cancers and heightened immunogenicity (Miller et al., 2000).

In the models reported previously, the antitumor efficacy of DC-AdIL-7 was determined using transplantable murine or human tumors propagated at subcutaneous sites. We embarked on the current studies to determine the antitumor properties of DC-AdIL-7 in a clinically relevant model of lung cancer in which adenocarcinomas develop in an organ-specific manner. Transgenic mice expressing SV40 large TAg transgene under the control of the murine Clara cell-specific promoter, CC-10, develop diffuse bilateral bronchoalveolar carcinoma, and have an average life span of 4 months (Magdaleno et al., 1997).

In previous studies, intratumoral injection of DC-AdGFP demonstrated the capacity of DCs to migrate to regional and contralateral lymph nodes as well as the spleen (Miller et al., 2000). Trafficking of the gene-modified DCs from the tumor to lymph node sites brings both potent antigen presentation and IL-7 in proximity to primary immune effector cells, thus augmenting systemic antitumor reactivity. Based on trafficking of gene-modified DCs to the regional lymph nodes after intratumoral administration observed in our previous studies, we hypothesized that injection of DC-AdIL-7 in the ALNR would lead to antitumor responses. The antitumor activity of DC-AdIL- 7 was determined in the spontaneous model for lung cancer by injecting DC-AdIL-7 into the ALNR of the transgenic mice. Our rationale for injecting DC-AdIL-7 in the lymph node region was to provide for nodal delivery of IL-7 at the immediate cellular interface of the intimate interactions between DCs and lymphocyte effectors where they can prime specific antitumor immune responses. The efficacy of lymph node injection with immune stimulators for the treatment of cancer has been demonstrated in recent studies; vaccination with tumor cell-DC hybrids in the lymph node region led to regression of human metastatic renal cell carcinoma (Kugler et al., 2000). In many clinical situations access to lymph node sites for injection may also be more readily achievable than intratumoral administration. The ALNR was chosen as a site for injection because of ease of repetitive administration, rather than any documented drainage pattern of tumor cells from the lung. It is quite probable that any secondary antigen-presenting site may be an effective route for induction of therapeutic antitumor responses; however, this hypothesis needs to be tested in future experiments. Our results show that this approach is effective in generating systemic antitumor responses. DC-AdIL-7 injected in the axillary lymph node regions of the CC-10 TAg mice evidenced potent antitumor responses with reduced tumor burden and a survival benefit compared to CC-10 TAg mice receiving diluent control injections. Unlike DCs, fibroblasts were not effective vehicles for IL-7. We interpret these results as an indication that DCs are necessary for maximal antitumor efficacy.

We previously found specific enhanced splenocyte release of IFN-γ and GM-CSF after treatment with DC-AdIL-7 (Miller et al., 2000). The specific cytokine release and intracellular cytokine analysis data from in vitro studies were consistent with the role of the DC-AdIL-7 in antitumor responses in vivo (Miller et al., 2000). Consistent with these findings, the cytokine production from tumor sites and spleens in the CC-10 TAg mice was altered as a result of DC-AdIL-7 therapy. The following cytokines were measured: VEGF, IL-10, PGE2, TGF-β, IFN-γ, GM-CSF, IL-12, MIG/CXCL9, and IP-10/CXCL10 (Figs. 2 and 3). The production of these cytokines was evaluated for the following reasons: the tumor site has been documented to be an abundant source of PGE2, VEGF, IL-10, and TGF-β, and these mediators have been shown to suppress immune responses (Gabrilovich et al., 1996; Huang et al., 1998; Bellone et al., 1999). VEGF, PGE2, and TGF-β promote angiogenesis (Ferrara, 1995; Fajardo et al., 1996; Tsujii et al., 1998). Antibodies to VEGF, TGF-β, PGE2, and IL-10 have the capacity to suppress tumor growth in in vivo model systems. VEGF has also been shown to interfere with DC maturation (Gabrilovich et al., 1996). Both IL-10 and TGF-β are immune inhibitory cytokines that may potently suppress antigen presentation and antagonize CTL generation and macrophage activation (Bellone et al., 1999; Sharma et al., 1999). Although at higher pharmacologic concentrations IL-10 may cause tumor reduction, physiologic concentrations of this cytokine suppress antitumor responses (Halak et al., 1999; Sharma et al., 1999; Sun et al., 1999; Stolina et al., 2000; Yang and Lattime, 2003). Prior to DC-AdIL-7 treatment in the transgenic tumor-bearing mice, the levels of the immunosuppressive proteins VEGF, PGE2, and TGF-β were elevated compared to the levels in normal control mice (data not shown). There was no such increase in IL-10 levels. Similarly there were not significant alterations in IL-4 and IL-5 accompanying DC-AdIL-7 therapy (data not shown). DC-AdIL-7–treated CC-10 TAg mice showed significant reductions in VEGF, TGF-β, IL-10, and PGE2. The decrease in immunosuppressive cytokines was not limited to the lung but was also evident systemically. Thus, possible benefits of a DC-AdIL- 7–mediated decrease in these cytokines include promotion of antigen presentation and CTL generation (Bellone et al., 1999; Sharma et al., 1999), as well as a limitation of angiogenesis (Ferrara, 1995; Fajardo et al., 1996; Tsujii et al., 1998). In future experiments, we will address the significance of the decrease in immunosuppressive factors in response to DC-AdIL-7 therapy.

Successful immunotherapy shifts tumor-specific T-cell responses from a type 2 to a type 1 cytokine profile (Hu et al., 1998). Responses depend on IL-12 and IFN-γ to mediate a range of biologic effects, which facilitate anticancer immunity. IL-12, produced by macrophages (Trinchieri, 1998) and DC (Johnson and Sayles, 1997), plays a key role in the induction of cellular immune responses (Ma et al., 1997). IL-12 mediates potent antitumor effects that are the result of several actions involving the induction of CTL, type 1-mediated immune responses, and natural killer activation (Trinchieri, 1998), as well as the impairment of tumor vascularization (Voest et al., 1995). IP-10/CXCL10, and MIG/CXCL9 are CXC chemokines that chemoattract activated T cells expressing the CXCR3 chemokine receptor (Loetscher et al., 1996). Both IP-10/CXCL10 and MIG/CXCL9 are known to have potent antitumor and antiangiogenic properties (Brunda et al., 1993; Luster and Leder, 1993; Arenberg et al., 1996; Sgadari et al., 1997). The lungs of DC-AdIL-7–treated CC-10 TAg mice revealed significant increases in IFN-γ, IL-12, IP-10/CXCL10, MIG/CXCL9, and GM-CSF. MIG/CXCL9 and IP-10/CXCL10 are potent angiostatic factors that are induced by IFN-γ (Strieter et al., 1995; Arenberg et al., 1996; Tannenbaum et al., 1998) and may be responsible in part for the tumor reduction in CC-10 TAg mice following DC-AdIL-7 administration. Because MIG/CXCL9 and IP-10/CXCL10 have antiangiogenic effects (Brunda et al., 1993; Luster and Leder, 1993; Arenberg et al., 1996; Sgadari et al., 1997), the tumor reductions observed in this model may be attributable to T-cell–dependent immunity as well as participation by T cells secreting IFN-γ and inhibiting angiogenesis (Tannenbaum et al., 1998). Hence, an increase in IFN-γ at the tumor site of DC-AdIL-7–treated mice could explain the relative increases in IP-10/CXCL10 and MIG/CXCL9. Both MIG/CXCL9 and IP- 10/CXCL10 are chemotactic for stimulated CXCR3-expressing T lymphocytes that could additionally amplify IFN-γ at the tumor site (Farber, 1997). An increase in GM-CSF in DC-AdIL- 7–treated mice could enhance DC maturation and antigen presentation (Banchereau and Steinman, 1998). We postulate that the antitumor responses seen in this model are T-cell–dependent. However, additional studies are necessary to precisely define the T-cell subsets and the host cytokines that are critical to the DC-AdIL-7–mediated antitumor response.

Taken together, the current study indicates that DC-AdIL-7 injected in the ALNR in this spontaneous lung cancer model leads to the generation of systemic antitumor responses. Host APC are critical for the cross-presentation of tumor antigens (Huang et al., 1994; Banchereau and Steinman, 1998). However, tumors have the capacity to limit APC maturation, function, and infiltration of the tumor site (Gabrilovich et al., 1996; Kobie et al., 2003; Sharma et al., 2003). The current model system overcomes this detriment by activating DC precursors ex vivo in GM-CSF and IL-4. This allows DC propagation to occur in an environment conducive to full activation without interference from deleterious tumor-derived products. Although DCs have been implicated in promoting tolerance in chronic inflammatory states (Kronin et al., 1996), tolerance induction may occur when DCs are in a low state of activation or maturation, resulting in decreased lymph node recruitment of mature DCs (Sallusto et al., 1995) or the generation of T-regulatory cells (Roncarolo et al., 2001; Dhodapkar and Steinman, 2002). Thus the use of activated DC in the ALNR may help recruit host DC that are already primed with tumor antigens to these sites and help initiate and/or maintain antitumor immune responses. Hence, the antitumor properties of DC-AdIL-7 may be attributable to the stimulation of host antigen-presenting functions by IL-12 as well as antiangiogenic activities mediated via IFN-γ induction of IP-10/CXCL10 and MIG/CXCL9. Secretion of IL-7 by DCs at the nodal sites may also increase the longevity of the activated T cells (Lynch and Miller, 1994; Akashi et al., 1997). Additional studies will be required to delineate the importance of each of these cytokines in DC-AdIL-7–mediated antitumor responses and the precise role of the DCs in this model await definition in further studies. The potent antitumor properties demonstrated in this model of spontaneous bronchoalveolar carcinoma provide a strong rationale for additional evaluation of DC-AdIL-7 in the regulation of tumor immunity.

Acknowledgments

This work was supported by the National Institutes of Health Grant R01 CA78654, CA85686, P50 CA90388, Medical Research Funds from the Department of Veterans Affairs, The Research Enhancement Award Program in Cancer Gene Medicine, and the Tobacco-Related Disease Research Program of the University of California. We thank Jeff Lin and Lawrence Hsu for their assistance with the statistical analyses of the data.

References

  1. AKASHI K, KONDO M, VON FREEDEN-JEFFRY U, MURRAY R, WEISSMAN IL. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell. 1997;89:1033–1041. doi: 10.1016/s0092-8674(00)80291-3. [DOI] [PubMed] [Google Scholar]
  2. ALLEVA DG, BURGER CJ, ELGERT KD. Tumor-induced regulation of suppressor macrophage nitric oxide and TNF-alpha production: Role of tumor-derived IL-10, TGF-beta and prostaglandin E2. J Immunol. 1994;153:1674–186. [PubMed] [Google Scholar]
  3. ARENBERG DA, KUNKEL SL, POLVERINI PJ, MORRIS SB, BURDICK MD, GLASS MC, TAUB DT, IANNETTONI MD, WHYTE RI, STRIETER RM. Interferon-gamma-inducible protein 10 (IP-10/CXCL10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med. 1996;184:981–992. doi: 10.1084/jem.184.3.981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. ARTHUR J, BUTTERFIELD L, ROTH M, BUI L, KIERTSCHER S, LAU R, DUBINETT S, GLASPY J, MCBRIDE W, ECONOMOU J. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther. 1997;4:17–25. [PubMed] [Google Scholar]
  5. BANCHEREAU J, STEINMAN RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. doi: 10.1038/32588. [DOI] [PubMed] [Google Scholar]
  6. BANCHEREAU J, PALUCKA AK, DHODAPKAR M, BURKEHOLDER S, TAQUET N, ROLLAND A, TAQUET S, COQUERY S, WITTKOWSKI KM, BHARDWAJ N, PINEIRO L, STEINMAN R, FAY J. Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor- derived dendritic cell vaccine. Cancer Res. 2001a;61:6451–6458. [PubMed] [Google Scholar]
  7. BANCHEREAU J, SCHULER-THURNER B, PALUCKA AK, SCHULER G. Dendritic cells as vectors for therapy. Cell. 2001b;106:271–274. doi: 10.1016/s0092-8674(01)00448-2. [DOI] [PubMed] [Google Scholar]
  8. BELLONE G, TURLETTI A, ARTUSIO E, MARESCHI K, CARBONE A, TIBAUDI D, ROBECCHI A, EMANUELLI G, RODECK U. Tumor-associated transforming growth factor-beta and interleukin-10 contribute to a systemic Th2 immune phenotype in pancreatic carcinoma patients. Am J Pathol. 1999;155:537–547. doi: 10.1016/s0002-9440(10)65149-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. BOCZKOWSKI D, NAIR SK, SNYDER D, GILBOA E. Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med. 1996;184:465–472. doi: 10.1084/jem.184.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. BRUNDA MJ, LUISTRO L, WARRIER RR, WRIGHT RB, HUBBARD BR, MURPHY M, WOLF SF, GATELY MK. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med. 1993;178:1223–1230. doi: 10.1084/jem.178.4.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. CELLUZZI C, FALO LD., Jr Cutting edge: Physical interaction between dendritic cells and tumor cells result in an immunogen that induces protective and therapeutic tumor rejection. J Immunol. 1998;160:3081–3085. [PubMed] [Google Scholar]
  12. CELLUZZI CM, MAYORDOMO JI, STORKUS WJ, LOTZE MT, FALO LD., Jr Peptide-pulsed dendritic cells induce antigen-specific, CTL-mediated protective tumor immunity. J Exp Med. 1996;183:283–287. doi: 10.1084/jem.183.1.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DALLAL RM, LOTZE MT. The dendritic cell and human cancer vaccines. Curr Opin Immunol. 2000;12:583–588. doi: 10.1016/s0952-7915(00)00146-1. [DOI] [PubMed] [Google Scholar]
  14. DHODAPKAR MV, STEINMAN RM. Antigen-bearing immature dendritic cells induce peptide-specific CD8(+) regulatory T cells in vivo in humans. Blood. 2002;100:174–177. doi: 10.1182/blood.v100.1.174. [DOI] [PubMed] [Google Scholar]
  15. DUBINETT SM, HUANG M, DHANANI S, WANG J, BEROIZA T. Down-regulation of macrophage transforming growth factor-β messenger RNA expression by interleukin-7. J Immunol. 1993;151:6670–6680. [PubMed] [Google Scholar]
  16. DUBINETT SM, BATRA RK, MILLER PW, SHARMA S. Tumor antigens in thoracic malignancy. Am J Respir Cell Mol Biol. 2000;22:524–527. doi: 10.1165/ajrcmb.22.5.f186. [DOI] [PubMed] [Google Scholar]
  17. FAJARDO LF, PRIONAS SD, KWAN HH, KOWALSKI J, ALLISON AC. Transforming growth factor beta1 induces angiogenesis in vivo with a threshold pattern. Lab Invest. 1996;74:600–608. [PubMed] [Google Scholar]
  18. FARBER JM. MIG/CXCL9 and IP-10/CXCL10: CXC chemokines that target lymphocytes. J Leukoc Biol. 1997;61:246–257. [PubMed] [Google Scholar]
  19. FERRARA N. The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res Treat. 1995;36:127–137. doi: 10.1007/BF00666035. [DOI] [PubMed] [Google Scholar]
  20. FIELDS RC, SHIMIZU K, MULE JJ. Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc Natl Acad Sci USA. 1998;95:9482–9487. doi: 10.1073/pnas.95.16.9482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. GABRILOVICH DI, CHEN HL, GIRGIS KR, CUNNINGHAM HT, MENY GM, NADAF S, KAVANAUGH D, CARBONE D. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat Med. 1996;2:1096–1103. doi: 10.1038/nm1096-1096. [DOI] [PubMed] [Google Scholar]
  22. GAMBOTTO A, TUTING T, MCVEY D, KOVESDI I, TAHARA H, LOTZE M, ROBBINS P. Induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12. Cancer Gene Ther. 1999;6:45–53. doi: 10.1038/sj.cgt.7700013. [DOI] [PubMed] [Google Scholar]
  23. GONG J, CHEN D, KASHIWABA M, KUFE D. Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nat Med. 1997;3:558–561. doi: 10.1038/nm0597-558. [DOI] [PubMed] [Google Scholar]
  24. HALAK BK, MAGUIRE HC, Jr, LATTIME EC. Tumor- induced interleukin-10 inhibits type 1 immune responses directed at a tumor antigen as well as a non-tumor antigen present at the tumor site. Cancer Res. 1999;59:911–917. [PubMed] [Google Scholar]
  25. HU HM, URBA WJ, FOX BA. Gene-modified tumor vaccine with therapeutic potential shifts tumor-specific T cell response from a type 2 to a type 1 cytokine profile. J Immunol. 1998;161:3033–3041. [PubMed] [Google Scholar]
  26. HUANG AYC, GOLUMBEK P, AHMADZADEH M, JAFFEE E, PARDOLL D, LEVITSKY H. Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science. 1994;264:961–965. doi: 10.1126/science.7513904. [DOI] [PubMed] [Google Scholar]
  27. HUANG M, STOLINA M, SHARMA S, MAO J, ZHU L, MILLER P, WOLLMAN J, HERSCHMAN H, DUBINETT S. Non-small cell lung cancer cyclooxygenase-2-dependent regulation of cytokine balance in lymphocytes and macrophages: Up-regulation of interleukin 10 and down-regulation of interleukin 12 production. Cancer Res. 1998;58:1208–1216. [PubMed] [Google Scholar]
  28. HUANG M, SHARMA S, ZHU LX, KEANE MP, LUO J, ZHANG L, BURDICK MD, LIN YQ, DOHADWALA M, GARDNER B, BATRA RK, STRIETER RM, DUBINETT SM. IL-7 inhibits fibroblast TGF-beta production and signaling in pulmonary fibrosis. J Clin Invest. 2002;109:931–937. doi: 10.1172/JCI14685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. INABA K, INABA M, ROMANI N, AYA H, DEGUCHI M, IKEHARA S, MURAMATSU S, STEINMAN RM. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophages colony-stimulated factor. J Exp Med. 1992;176:1693–1702. doi: 10.1084/jem.176.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. JOHNSON LL, SAYLES P. Interleukin-12, dendritic cells and the initiation of of host-protective mechanisms against Toxoplasma gondii. J Exp Med. 1997;186:1799–1802. doi: 10.1084/jem.186.11.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. KIRK CJ, HARTIGAN-O’CONNOR D, NICKOLOFF BJ, CHAMBERLAIN JS, GIEDLIN M, AUKERMAN L, MULE JJ. T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: Augmentation of dendritic cell-based immunotherapy. Cancer Res. 2001;61:2062–2070. [PubMed] [Google Scholar]
  32. KIRK CJ, MULE JJ. Gene-modified dendritic cells for use in tumor vaccines. Hum Gene Ther. 2000;11:797–806. doi: 10.1089/10430340050015419. [DOI] [PubMed] [Google Scholar]
  33. KOBIE JJ, WU RS, KURT RA, LOU S, ADELMAN MK, WHITESELL LJ, RAMANATHAPURAM LV, ARTEAGA CL, AKPORIAYE ET. Transforming growth factor beta inhibits the antigen-presenting functions and antitumor activity of dendritic cell vaccines. Cancer Res. 2003;63:1860–1864. [PubMed] [Google Scholar]
  34. KOS FJ, MULLBACHER A. IL-2-independent activity of IL-7 in the generation of secondary antigen-specific cytotoxic T cell responses in vitro. J Immunol. 1993;150:387–393. [PubMed] [Google Scholar]
  35. KRONIN V, WINKEL K, SUSS G, KELSO A, HEATH W, KIRBERG J, VON BOEHMER H, SHORTMAN K. A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production. J Immunol. 1996;157:3819–3827. [PubMed] [Google Scholar]
  36. KUGLER A, STUHLER G, WALDEN P, ZOLLER G, ZOBYWALSKI A, BROSSART P, TREFZER U, ULLRICH S, MULLER CA, BECKER V, GROSS AJ, HEMMERLEIN B, KANZ L, MULLER GA, RINGERT RH. Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids. Nat Med. 2000;6:332–6. doi: 10.1038/73193. [DOI] [PubMed] [Google Scholar]
  37. LOETSCHER M, GERBER B, LOETSCHER P, JONES SA, PIALI L, CLARK-LEWIS I, BAGGIOLINI M, MOSER B. Chemokine receptor specific for IP10 and MIG/CXCL9: Structure, function, and expression in activated T-lymphocytes. J Exp Med. 1996;184:963–969. doi: 10.1084/jem.184.3.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. LUSTER AD, LEDER P. IP-10/CXCL10, a CXC chemokine, elicits a potent thymus-dependent anti-tumor response in vivo. J Exp Med. 1993;178:1057–1065. doi: 10.1084/jem.178.3.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. LYNCH DH, MILLER RE. Interleukin 7 promotes long-term in vitro growth of antitumor cytotoxic T lymphocytes with immunotherapeutic efficacy in vivo. J Exp Med. 1994;179:31–42. doi: 10.1084/jem.179.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. MA X, ASTE-AMEZAGA M, GRI G, GEROSA F, TRINCHIERI G. Immunomodulatory functions and molecular regulation of IL-12. Chem Immunol. 1997;68:1–22. doi: 10.1159/000058687. [DOI] [PubMed] [Google Scholar]
  41. MAGDALENO S, WANG G, MIRELES V, RAY M, FINEGOLD M, DEMAYO F. Cyclin-dependent kinase inhibitor expression in pulmonary Clara cells transformed with SV40 large T antigen in transgenic mice. Cell Growth Differ. 1997;8:145–155. [PubMed] [Google Scholar]
  42. MAYORDOMO JI, ZORINA T, STORKUS WJ, ZITVOGEL L, CELLUZZI C, FALO LD, MELIEF CJ, ILDSTAD ST, KAST WM, DELEO AB, LOTZE MT. Bone marrow- derived dendritic cells pulsed with synthetic tumor peptides elicit protective and therapeutic antitumor immunity. Nat Med. 1995;1:1297–1302. doi: 10.1038/nm1295-1297. [DOI] [PubMed] [Google Scholar]
  43. MEHROTRA PT, GRANT AJ, SIEGEL JP. Synergistic effects of IL-7 and IL-12 on human T cell activation. J Immunol. 1995;154:5093–6102. [PubMed] [Google Scholar]
  44. MELERO I, DUARTE M, RUIZ J, SANGRO B, GALOFRE J, MAZZOLINI G, BUSTOS M, QIAN C, PRIETO J. Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas. Gene Ther. 1999;6:1779–1784. doi: 10.1038/sj.gt.3301010. [DOI] [PubMed] [Google Scholar]
  45. MILLER AR, MCBRIDE WH, DUBINETT SM, DOUGHERTY GJ, THACKER JD, SHAU H, KOHN DB, MOEN RC, WALKER MJ, CHIU R, SCHUCK BL, ROSENBLATT JA, HUANG M, DHANANI S, RHOADES K, ECONOMOU JS. Transduction of human melanoma cell lines with the human interleukin-7 gene using retroviral-mediated gene transfer: Comparison of immunologic properties with interleukin-2. Blood. 1993;82:3686–3694. [PubMed] [Google Scholar]
  46. MILLER PW, SHARMA S, STOLINA M, CHEN K, ZHU L, PAUL RW, DUBINETT SM. Dendritic cells augment granulocyte-macrophage colony-stimulating factor (GM-CSF)/herpes simplex virus thymidine kinase-mediated gene therapy of lung cancer. Cancer Gene Ther. 1998;5:380–589. [PubMed] [Google Scholar]
  47. MILLER PW, SHARMA S, STOLINA M, BUTTERFIELD LH, LUO J, LIN Y, DOHADWALA M, BATRA RK, WU L, ECONOMOU JS, DUBINETT SM. Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication. Hum Gene Ther. 2000;11:53–65. doi: 10.1089/10430340050016157. [DOI] [PubMed] [Google Scholar]
  48. NESTLE FO, ALIJAGIC S, GILLIET M, SUN Y, GRABBE S, DUMMER R, BURG G, SCHADENDORF D. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998;4:328–332. doi: 10.1038/nm0398-328. [DOI] [PubMed] [Google Scholar]
  49. PARKHURST MR, DEPAN C, RILEY JP, ROSENBERG SA, SHU S. Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules. J Immunol. 2003;170:5317–5325. doi: 10.4049/jimmunol.170.10.5317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. RAHMAN A, TSAI V, GOUDREAU A, SHINODA JY, WEN SF, RAMACHANDRA M, RALSTON R, MANEVAL D, LAFACE D, SHABRAM P. Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy. Mol Ther. 2001;3:768–778. doi: 10.1006/mthe.2001.0316. [DOI] [PubMed] [Google Scholar]
  51. RIBAS A, BUTTERFIELD L, MCBRIDE W, JILANI S, BUI L, VOLLMER C, LAU R, DISSETTE V, HU B, CHEN A, GLASPY J, ECONOMOU J. Genetic immunization for the melanoma antigen MART-1/Melan-A using recombinant adenovirus- transduced murine dendritic cells. Cancer Res. 1997;57:2865–2869. [PubMed] [Google Scholar]
  52. ROHRER JW, COGGIN JH., Jr CD8 T cell clones inhibit antitumor T cell function by secreting IL-10. J Immunol. 1995;155:5719–5727. [PubMed] [Google Scholar]
  53. RONCAROLO MG, LEVINGS MK, TRAVERSARI C. Differentiation of T regulatory cells by immature dendritic cells. J Exp Med. 2001;193:F5–9. doi: 10.1084/jem.193.2.f5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. SALLUSTO F, CELLA M, DANIELI C, LANZAVECCHIA A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: Downregulation by cytokines and bacterial products. J Exp Med. 1995;182:389–400. doi: 10.1084/jem.182.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. SCHULER G, SCHULER-THURNER B, STEINMAN RM. The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol. 2003;15:138–147. doi: 10.1016/s0952-7915(03)00015-3. [DOI] [PubMed] [Google Scholar]
  56. SGADARI C, FARBER JM, ANGIOLILLO AL, LIAO F, TERUYA-FELDSTEIN J, BURD PR, YAO L, GUPTA G, KANEGANE C, TOSATO G. MIG/CXCL9, the monokine induced by interferon-gamma, promotes tumor necrosis in vivo. Blood. 1997;89:2635–2643. [PubMed] [Google Scholar]
  57. SHARMA S, WANG J, HUANG M, PAUL R, LEE P, MCBRIDE W, ECONOMOU J, ROTH M, KIERTSCHER S, DUBINETT S. Interleukin-7 gene transfer in non-small cell lung cancer decreases tumor proliferation, modifies cell surface molecule expression, and enhances antitumor reactivity. Cancer Gene Ther. 1996;3:302–313. [PubMed] [Google Scholar]
  58. SHARMA S, MILLER P, STOLINA M, ZHU L, HUANG M, PAUL R, DUBINETT S. Multi-component gene therapy vaccines for lung cancer: Effective eradication of established murine tumors in vivo with Interleukin 7/herpes simplex thymidine kinase-transduced autologous tumor and ex vivo-activated dendritic cells. Gene Ther. 1997;4:1361–1370. doi: 10.1038/sj.gt.3300531. [DOI] [PubMed] [Google Scholar]
  59. SHARMA S, STOLINA M, LIN Y, GARDNER B, MILLER PW, KRONENBERG M, DUBINETT SM. T cell-derived IL-10 promotes lung cancer growth by suppressing both T cell and APC function. J Immunol. 1999;163:5020–5028. [PubMed] [Google Scholar]
  60. SHARMA S, STOLINA M, YANG SC, BARATELLI F, LIN JF, ATIANZAR K, LUO J, ZHU L, LIN Y, HUANG M, DOHADWALA M, BATRA RK, DUBINETT SM. Tumor cyclooxygenase 2-dependent suppression of dendritic cell function. Clin Cancer Res. 2003;9:961–968. [PubMed] [Google Scholar]
  61. SHIMIZU T, BERHANU A, REDLINGER RE, Jr, WATKINS S, LOTZE MT, BARKSDALE EM., Jr Interleukin-12–transduced dendritic cells induce regression of established murine neuroblastoma. J Pediatr Surg. 2001;36:1285–1292. doi: 10.1053/jpsu.2001.25796. [DOI] [PubMed] [Google Scholar]
  62. STANDIFORD TJ, KUNKEL SL, BASHA MA, CHENSUE SW, LYNCH JP, III, TOEWS GB, WESTWICK J, STRIETER RM. Interleukin-8 gene expression by a pulmonary epithelial cell line: A model for cytokine networks in the lung. J Clin Invest. 1990;86:1945–1953. doi: 10.1172/JCI114928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. STOLINA M, SHARMA S, LIN Y, DOHADWALA M, GARDNER B, LUO J, ZHU L, KRONENBERG M, MILLER PW, PORTANOVA J, LEE JC, DUBINETT SM. Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immunol. 2000;164:361–370. doi: 10.4049/jimmunol.164.1.361. [DOI] [PubMed] [Google Scholar]
  64. STRIETER RM, KUNKEL SL, ARENBERG DA, BURDICK MD, POLVERINI PJ. Interferon gamma-inducible protein 10 (IP-10/CXCL10), a member of the C-X-C chemokine family, is an inhibitor of angiogenesis. Biochem Biophys Res Commun. 1995;210:51–57. doi: 10.1006/bbrc.1995.1626. [DOI] [PubMed] [Google Scholar]
  65. SU Z, DANNULL J, HEISER A, YANCEY D, PRUITT S, MADDEN J, COLEMAN D, NIEDZWIECKI D, GILBOA E, VIEWEG J. Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells. Cancer Res. 2003;63:2127–2133. [PubMed] [Google Scholar]
  66. SUN H, JACKSON M, KUNDU N, FULTON A. Interleukin 10 gene transfer activates interferon gamma and the interferon gamma inducible genes Gbp-1/Mag-1 and MIG/CXCL9-1 in mammary tumors. Int J Cancer. 1999;80:624–629. doi: 10.1002/(sici)1097-0215(19990209)80:4<624::aid-ijc23>3.0.co;2-9. [DOI] [PubMed] [Google Scholar]
  67. TANNENBAUM CS, TUBBS R, ARMSTRONG D, FINKE JH, BUKOWSKI RM, HAMILTON TA. The CXC chemokines IP-10/CXCL10 and MIG/CXCL9 are necessary for IL-12–mediated regression of the mouse RENCA tumor. J Immunol. 1998;161:927–932. [PubMed] [Google Scholar]
  68. THURNER B, HAENDLE I, RODER C, DIECKMANN D, KEIKAVOUSSI P, JONULEIT H, BENDER A, MACZEK C, SCHREINER D, VON DEN DRIESCH P, BROCKER EB, STEINMAN RM, ENK A, KAMPGEN E, SCHULER G. Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med. 1999;190:1669–1678. doi: 10.1084/jem.190.11.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. TOES R, VAN DER VOORT E, SCHOENBERGER S, DRIJF HOUT J, VAN BLOOIS L, STORM G, KAST W, OFFRINGA R, MELIEF C. Enhancement of tumor outgrowth through CTL tolerization after peptide vaccination is avoided by peptide presentation on dendritic cells. J Immunol. 1998;160:4449–4456. [PubMed] [Google Scholar]
  70. TOES RE, HOEBEN RC, VAN DER VOORT EI, RESSING ME, VAN DER EB AJ, MELIEF CJ, OFFRINGA R. Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion. Proc Natl Acad Sci USA. 1997;94:14660–14665. doi: 10.1073/pnas.94.26.14660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. TRINCHIERI G. Interleukin-12: A cytokine at the interface of inflammation and immunity. Adv Immunol. 1998;70:83–243. doi: 10.1016/s0065-2776(08)60387-9. [DOI] [PubMed] [Google Scholar]
  72. TSUJII M, KAWANO S, TSUJI S, SAWAOKA H, HORI M, DUBOIS RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell. 1998;93:705–716. doi: 10.1016/s0092-8674(00)81433-6. [DOI] [PubMed] [Google Scholar]
  73. VOEST EE, KENYON BM, O’REILLY MS, TRUITT G, D’AMATO RJ, FOLKMAN J. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst. 1995;87:581–586. doi: 10.1093/jnci/87.8.581. [DOI] [PubMed] [Google Scholar]
  74. YANG AS, LATTIME EC. Tumor-induced interleukin 10 suppresses the ability of splenic dendritic cells to stimulate CD4 and CD8 T-cell responses. Cancer Res. 2003;63:2150–2157. [PubMed] [Google Scholar]
  75. YANG Y, NUNES FA, BERENCSI K, FURTH EE, GÖNCZÖL E, WILSON JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA. 1994;91:4407–4411. doi: 10.1073/pnas.91.10.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. ZITVOGEL L, MAYORDOMO JI, TJANDRAWAN T, DELEO AB, CLARKE MR, LOTZE MT, STORKUS WJ. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Med. 1996;183:87–97. doi: 10.1084/jem.183.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES