Antibody-mediated suppression of grafted lymphoma. IV. Influence of time of tumor residency in vivo and tumor size upon the effectiveness of suppression by syngeneic antibody.

In the suppression of the growth of a mouse lymphoma 6C3HED by antibody, the effectiveness of antibody in suppressing growing or established tumor cells and comparable number of freshly injected tumor cells is quantitatively similar. The effectiveness of antibody diminishes markedly when the number of tumor cells per mouse reaches the level of 10(6) due to the development of a macrophage shortage. At the 10(5) tumor cells level, antibody-mediated suppression takes place in an optimal manner and between 10(5) and 10(4) tumor cell numbers, the amount of antibody required to suppress 50% of the tumor cells is directly proportional to the number of tumor cells suppressed.

Genetic analysis of the human tumor necrosis factor alpha/cachectin promoter region in a macrophage cell line.

The 615-bp 5' flanking region of the human TNF-alpha/cachectin gene was isolated and ligated to the luciferase reporter gene. In addition, a series of truncated promoter constructs was generated by exonuclease III digestion. The promoter activity of these constructs was studied in a transient transfection system using the TNF-alpha-producing U937 cell line. Full-length and truncated TNF promoter constructions extending from -615 to -95 bp relative to the transcription start site (TSS) could be induced by phorbol esters. A construct truncated to within 36 bp of the TSS (and within 11 bp of the TATAA box) was inactive. Therefore, the phorbol ester responsive is localized in the TNF/cachectin promoter to a relatively short region proximal to the TATAA box.

Immunotherapy of cancer with antibody.

A long-term suppression of a transplanted solid tumor that has been growing in a syngeneic animal can be achieved by the administration of antibody against the tumor. The susceptibility of such growing tumor cells to antibody treatment is similar to that of a comparable number of freshly injected tumor cells.

In vivo trafficking of adoptively transferred interleukin-2 expanded tumor-infiltrating lymphocytes and peripheral blood lymphocytes. Results of a double gene marking trial.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and IL-2 appears to produce dramatic regressions in patients with metastatic melanoma and renal cancer. However, the in vivo mechanism of TIL function is not known. We conducted an UCLA Human Subject Protection Committee, Recombinant DNA Advisory Committee, and FDA-approved clinical trial using genetically-marked TIL to test the hypothesis that these cells have unique, tumor-specific in vivo trafficking patterns. TIL and PBL (as a control effector cell population) were isolated and expanded in parallel in vitro in IL-2-containing medium for 4-6 wk. During the expansion, TIL and PBL were separately transduced with the amphotropic retroviral vectors LNL6 and G1Na. Transduced TIL and PBL were coinfused into patients and their respective numbers measured in tumor, peripheral blood, and normal tissues; integrated provirus could be quantitated and distinguished by DNA PCR. Nine patients were treated (six melanoma, three renal) and received between 4.5 x 10(8) and 1.24 x 10(10) total cells. Both "marked" TIL and PBL could be detected circulating in the peripheral blood, in some patients for up to 99 d after infusion. Marked TIL and/or PBL could be detected in tumor biopsies in six of nine patients as early as day 6 and as late as day 99 after infusion. No convincing pattern of preferential trafficking of TIL vs. PBL to tumor was noted. Moreover, concurrent biopsies of muscle, fat, and skin demonstrated the presence of TIL/PBL in comparable or greater numbers than in tumor in five patients. The results of this double gene marking trial provide interesting insights into the life span and trafficking of adoptively transferred lymphocytes, but do not support the hypothesis that TIL specifically traffic to tumor deposits.

Down-regulation of murine fibrosarcoma transforming growth factor-beta 1 expression by interleukin 7.

BACKGROUND: Cytokine genes encode proteins that modulate immune system responses. Modification of tumor cells by the introduction of cytokine genes has been used as a strategy to augment host immunity. Interleukin 7 (IL-7) gene transfer enhances the immune response to tumor cells and can result in tumor regression. Transforming growth factor-beta 1 (TGF-beta 1) is a potent immunosuppressive cytokine produced by many tumors. We have previously reported that recombinant IL-7 decreases the expression of TGF-beta 1 by murine macrophages. PURPOSE: This study investigates the inhibition of tumor-derived TGF-beta 1 production as a possible mechanism for the enhanced antitumor immunity that accompanies IL-7 gene transfer. METHODS: A fibrosarcoma cell line (FSA-JmIL-7) genetically modified to produce IL-7 was used to evaluate the effects of IL-7 on tumor production of TGF-beta 1. The control cell line (FSA-Jneo) originated from the same parental fibrosarcoma cell line (FSA) and was produced by transduction with the same retroviral vector without the IL-7 gene. FSA-Jneo and FSA-JmIL-7 tumor cells were evaluated for the expression of TGF-beta 1 messenger RNA (mRNA). To determine if the observed change in TGF-beta 1 mRNA was associated with an alteration in protein secretion, we compared supernatants from tumor cell cultures for TGF-beta 1 production. Specific anti-TGF-beta 1 monoclonal antibody (MAb) was used to confirm the role of TGF-beta 1 in these assays. RESULTS: Compared with FSA parental and FSA-Jneo cells, FSA-JmIL-7 cells expressed TGF-beta 1 mRNA at a lower level. Compared with supernatants from FSA-Jneo cells, FSA-JmIL-7 supernatants contained consistently lower levels of TGF-beta 1 activity (P < .05). In addition, FSA-Jneo supernatants suppressed lymphocyte proliferation to a significantly greater degree than supernatants from FSA-JmIL-7 cells (P < .05). Studies with anti-TGF-beta 1 MAb added to the supernatants confirmed the role of TGF-beta 1 in inhibition of lymphocyte proliferation. CONCLUSION: These findings suggest that IL-7 gene transfer inhibits the production of TGF-beta 1 by tumor cells and thus may enhance the efficacy of the host's antitumor immune response. IMPLICATION: The regulation of endogenous tumor-derived cytokines in response to cytokine gene transfer may contribute to altered immune responses in the tumor microenvironment and thus may be an important additional parameter to assess in gene therapy.

Influences of interleukins 2 and 4 on tumor necrosis factor production by murine mononuclear phagocytes.

Administration of recombinant human interleukin 2 (IL-2) to mice gave rise to peritoneal macrophages and blood monocytes that were primed to produce large amounts of tumor necrosis factor (TNF). Macrophages from IL-2-treated athymic mice responded less well than those from euthymic mice. In addition to its in vivo priming effect, IL-2 was able to directly stimulate TNF production in vitro by purified monocytes. Macrophages responded to IL-2 generally less well than monocytes both in vitro and in vivo. In contrast to IL-2, recombinant murine interleukin 4 (IL-4) down-regulated TNF synthesis by macrophages. In vitro pretreatment of macrophages with IL-4 largely abolished their ability to synthesize TNF in response to IL-2 or lipopolysaccharide. Also, administration of IL-4 to mice blocked the ability of IL-2 and lipopolysaccharide to prime macrophages in vivo for TNF production. Overall, these results demonstrate that IL-2 and IL-4 can act antagonistically to regulate TNF production by macrophages. In spite of its down-regulatory action on TNF production, IL-4 was unable to protect mice against the lethal toxic effects of lipopolysaccharide or IL-2.

Generation of CD8+ and CD4+ T-cell response to dendritic cells genetically engineered to express the MART-1/Melan-A gene.

Both CD8+ and CD4+ T cells have demonstrated roles in antitumor immune response in many animal tumor systems. In many human tumor systems, although abundant literature exists on the evidence of tumor antigen-specific CD8+ CTL response, only limited information is available on tumor antigen-specific CD4+ T-cell response. Using the MART-1/Melan-A (MART-1) antigen system as a prototype human tumor-associated antigen (TAA)- and dendritic cell (DC)-based MART-1 antigen presentation system (i.e., DCs transduced with an adenoviral vector-based construct carrying the MART-1 gene), we explored, in vitro, the feasibility of generating both CD8+ and CD4+ T-cell responses in the same individual. Here, we show that autologous DCs from both HLA-A2-positive melanoma patients and normal healthy individuals that are transduced with an adenoviral vector containing the MART-1 antigen are capable of inducing both MART-1-specific CD8+ and CD4+ T cells in in vitro coculture. After several rounds of stimulation, both the CD4+ and CD8+ T cells synthesized IFN-gamma when they were specifically stimulated. The CD8+ T cells generated in such cocultures also recognized the MART-1(27-35) peptide, AAGIGILTV, in 4-h cytotoxicity assays. These observations, therefore, suggest that Th1-type responses can be generated, in vitro, by stimulation with DCs that are genetically modified to express a TAA. Although the outcome of this type of genetically engineered DC-based stimulation may vary from system to system, this type of in vitro antigen presentation may be very useful in more comprehensive analyses of CD4+ T-cell response to defined TAAs, and such genetically engineered autologous DCs might be better candidates to serve as surrogate cancer vaccines.

alpha-Fetoprotein-specific tumor immunity induced by plasmid prime-adenovirus boost genetic vaccination.

alpha-Fetoprotein (AFP) is a potential target for immunotherapy in hepatocellular carcinoma; both the murine and human T-cell repertoires can recognize AFP-derived epitopes in the context of the MHC. Protective immunity can be generated with AFP-engineered dendritic cell-based vaccines. We now report a DNA-based immunization strategy using a prime-boost approach: coadministration of plasmid DNA encoding murine AFP and murine granulocyte-macrophage colony-stimulating factor followed by boosting with an AFP-expressing nonreplicating adenoviral vector. This immunization strategy can elicit a high frequency of Th1-type AFP-specific cells leading to tumor protective immunity in mice at levels comparable with AFP-engineered dendritic cells. This cell-free mode of immunization is better suited for large-scale vaccine efforts for patients with hepatocellular carcinoma.

Genetic modification of a murine fibrosarcoma to produce interleukin 7 stimulates host cell infiltration and tumor immunity.

Retroviral-mediated gene transfer was used to introduce and express the gene for murine interleukin 7 (IL-7) in a fibrosarcoma tumor (FSA). The tumorigenicity of these genetically modified FSA cells was greatly decreased in immunologically intact syngeneic mice but was unaltered in T-cell-deprived mice. IL-7-infected tumors that did grow in intact animals from large size inocula did so slowly and had a high incidence of spontaneous regression. Furthermore, mice that had rejected tumors became specifically immune to challenge with uninfected parental tumor cells. IL-7-infected FSA growing in intact mice were heavily infiltrated with host T-cells that were presumably responsible for slow growth and tumor regression, and tumor cells were in the minority. Fluorescence-activated cell sorter analysis showed that there was a 530% increase in T-cells in IL-7-infected FSA compared with control tumors. CD8+ T-cells were particularly elevated, but CD4+ lymphocytes were also increased in number, as were eosinophils and basophils. The CD4+:CD8+ ratio in IL-7-infected FSA was 1:1.7 in comparison to 1:0.6 in control tumors. Lymphocytes isolated from IL-7-producing tumors had greatly enhanced cytotoxicity towards uninfected, parental FSA cells. Killing of non-cross-reacting fibrosarcoma line was also increased but to a much lesser extent. Injection of recombinant human IL-7 directly into established FSA tumors slowed their growth and, in a significant number of instances, caused complete regression. Mice that had rejected tumor became specifically immune. The dose that was needed for this effect was, however, somewhat large: 20 micrograms twice daily for 10 days. This result contrasts with the efficacy of IL-7 gene infection in stimulating responses to the same tumor. These considerations make IL-7 a good candidate for tumor-directed cytokine gene therapy.

Generation of human T-cell responses to an HLA-A2.1-restricted peptide epitope derived from alpha-fetoprotein.

Alpha-fetoprotein (AFP) is often derepressed in human hepatocellular carcinoma. Peptide fragments of AFP presented in the context of major histocompatibility molecules could serve as potential recognition targets by CD8 T cells, provided these lymphocytes were not clonally deleted in ontogeny. We therefore wished to determine whether the human T-cell repertoire could recognize AFP-derived peptide epitopes in the context of a common class I allele, HLA-A2.1. Dendritic cells genetically engineered to express AFP were capable of generating AFP-specific T-cell responses in autologous human lymphocyte cultures and in HLA-A2.1/Kb transgenic mice. These T cells recognize a 9-mer peptide derived from the AFP protein hAFP(542-550) (GVALQTMKQ). Identified as a potential A2.1-restricted peptide epitope from a computer analysis of the AFP sequence, hAFP(542-550) proved to have low binding affinity to A2.1, but slow off-kinetics. AFP-specific CTL- and IFN-gamma-producing cells recognize hAFP(542-550)-pulsed targets. Conversely, hAFP(542-550) peptide-generated T cells from both human lymphocyte cultures and A2.1/Kb transgenic mice recognized AFP-transfected targets in both cytotoxicity assays and cytokine release assays. These lines of evidence clearly demonstrate that AFP-reactive clones have not been deleted from the human T-cell repertoire and identify one immunodominant A2.1-restricted epitope. These findings also clearly establish AFP as a potential target for T-cell-based immunotherapy.

CD40 cross-linking bypasses the absolute requirement for CD4 T cells during immunization with melanoma antigen gene-modified dendritic cells.

Genetic immunization of mice with dendritic cells (DCs) engineered to express a melanoma antigen generates antigen-specific, MHC-restricted, CD4-dependent protective immune responses. We wanted to determine the role of CD4 cells and CD40 ligation of MART-1 gene-modified DC in an animal model of immunotherapy for murine melanoma. CD4 knock-out (CD4KO) or antibody-depleted mice were immunized with DC adenovirally transduced with the MART-1 gene (AdVMART1/DC) with or without CD40 cross-linking. Tumor protection was absent in CD4-depleted mice, but protection was reestablished when the CD40 receptor was engaged using three different constructs. Transduction of DCs with vectors expressing the Th1 cytokines interleukin (IL)-2, IL-7, or IL-12 could not reproduce the CD40-mediated maturation signal in this model. CD8 T-cell depletion in CD4KO mice immunized with CD40-ligated DCs abrogated the protective response. Pooled analysis of CD40 cross-linking of AdVMART1/DC administered to wild-type C57BL/6 mice revealed an overall enhancement of antitumor immunity. However, this effect was inconsistent between replicate studies. In conclusion, maturation of AdVMART1-transduced DCs through the CD40 ligation pathway can promote a protective CD8 T-cell-mediated immunity that is independent of CD4 T-cell help.

Genetic immunization for the melanoma antigen MART-1/Melan-A using recombinant adenovirus-transduced murine dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells that process and present antigenic peptides and are capable of generating potent T-cell immunity. A murine tumor model was developed to evaluate methods of genetic immunization to the human MART-1/Melan-A (MART-1) melanoma antigen. A poorly immunogenic murine fibrosarcoma line (NFSA) was stably transfected with the MART-1 gene. This transfected tumor [NFSA(MART1)] grows progressively in C3Hf/Kam/Sed (H-2k) mice. Partial protection against a challenge with NFSA(MART1) could be achieved with i.m. injections of a MART-1 expression plasmid or with systemic administration of an adenovirus vector expressing MART-1. However, superior protection was achieved when granulocyte macrophage colony-stimulating factor/interleukin-4-differentiated murine DCs transduced with an adenovirus vector expressing MART-1 were used for immunization. Both partial and complete protection could be achieved with i.v. administration of MART-1-engineered DCs. Splenocytes from immunized mice contained MHC class 1-restricted CTLs specific for MART-1. This preclinical model of genetic immunization supports a therapeutic strategy for human melanoma.

Immune deviation and Fas-mediated deletion limit antitumor activity after multiple dendritic cell vaccinations in mice.

Genetic immunization with a single injection of dendritic cells (DCs) expressing a model melanoma antigen generates antigen-specific, MHC-restricted, protective immune responses. After initiating the immune response, additional vaccinations may increase the protection or conversely downregulate the immune response. Groups of mice were vaccinated several times with DCs transduced with the MART-1 gene, and the anti-tumor protection was compared with that of mice receiving a single vaccination. C3H mice had poorer protection from a syngeneic MART-1-expressing tumor challenge with multiple vaccinations. This was accompanied by lower levels of splenic CTL effectors and a shift from a type 1 to a type 2 cytokine profile. On the contrary, multiple vaccinations in C57BL/6 mice generated greater in vivo antitumor protection with no decrease in splenic CTLs and no cytokine shift. Antiadenoviral humoral or cellular immune responses did not seem to contribute to these effects. When studies were performed in Fas receptor-negative C3H.(lpr) mice, the adverse effect of multiple vaccinations disappeared, and there was no cytokine shift pattern. In conclusion, C3H mice but not C57BL/6 mice receiving multiple vaccinations with DCs expressing the MART-1 tumor antigen show decreased protection associated with deviation from a type 1 to a type 2 cytokine response attributable to a Fas-receptor mediated clearance of antigen-specific IFN-gamma-producing cells.

p53 selective and nonselective replication of an E1B-deleted adenovirus in hepatocellular carcinoma.

An E1B gene-attenuated adenovirus (dl1520) has been proposed to have a selective cytolytic activity in cancer cells with a mutation or deletion in the p53 tumor suppressor gene (p53-null), a defect present in almost half of human hepatocellular carcinomas (HCCs). In this study, the in vitro and in vivo antitumor activity of dl1520 was investigated focusing on two human HCC cell lines, a p53-wild type (p53-wt) cell line and a p53-null cell line. dl1520 was tested for in vitro cytopathic effects and viral replication in the human HCC cell lines Hep3B (p53-null) and HepG2 (p53-wt). The in vivo antitumor effects of dl1520 were investigated in tumors grown s.c. in a severe combined immunodeficient mouse model. In addition, the combination of dl1520 infection with systemic chemotherapy was assessed in these tumor xenografts. At low multiplicities of infection, dl1520 had an apparent p53-dependent in vitro viral growth in HCC cell lines. At higher multiplicities of infection, dl1520 viral replication was independent of the p53 status of the target cells. In vivo, dl1520 significantly retarded the growth of the p53-null Hep3B xenografts, an effect augmented by the addition of cisplatin. However, complete tumor regressions were rare, and most tumors eventually grew progressively. dl1520 had no effect on the in vivo growth of the p53-wt HepG2 cells, with or without cisplatin treatment. The E1B-deleted adenoviral vector dl1520 has an apparent p53-dependent effect in HCC cell lines. However, this effect is lost at higher viral doses and only induces partial tumor regressions without tumor cures in a human HCC xenograft model.

Alpha-fetoprotein-specific genetic immunotherapy for hepatocellular carcinoma.

The majority of human hepatocellular carcinomas overexpress alpha-fetoprotein (AFP). Two genetic immunization strategies were used to determine whether AFP could serve as a target for T-cell immune responses. Dendritic cells engineered to express AFP produced potent T-cell responses in mice, as evidenced by the generation of AFP-specific CTLs, cytokine-producing T cells, and protective immunity. AFP plasmid-based immunization generated less potent responses. These novel observations demonstrate that this oncofetal antigen can serve as an effective tumor rejection antigen. This provides a rational, gene therapy-based strategy for this disease, which is responsible for the largest number of cancer-related deaths worldwide.

Adenovirus-interleukin-12-mediated tumor regression in a murine hepatocellular carcinoma model is not dependent on CD1-restricted natural killer T cells.

The cytokine interleukin-12 (IL-12) has shown potent antitumor activity in several tumor models. Recently, natural killer (NK) T cells have been proposed to mediate the antitumor effects of IL-12. In this study, the antitumor response of IL-12 was investigated in a gene therapeutic model against s.c. growing mouse hepatocellular carcinomas using an adenoviral vector expressing murine IL-12 (AdVmIL-12). An adenoviral-based system was chosen because of the ability of adenoviruses to transduce dividing and nondividing cells and because of their high transduction efficiencies. Our goals were to examine the efficacy of AdVmIL-12 in a hepatocellular carcinoma model and to investigate the mechanism of the AdVmIL-12-mediated antitumor response with specific interest in the role of NK T cells. Our studies demonstrate that intratumoral AdVmIL-12-mediated regression of s.c. hepatocellular tumors is associated with rapid antitumor responses. AdVmIL-12 treatment was associated with an immune cellular infiltrate consisting of CD4 and CD8 T lymphocytes, macrophages, NK cells, and NK T cells. Antibody ablation of CD4 and CD8 T cells and use of NK cell-defective beige mice failed to abrogate the response to AdVmIL-12. Studies in T-cell- and B-cell-deficient severe combined immunodeficient and recombinase activating gene-2-deficient mice and T-cell-, B-cell-, and NK cell-defective severe combined immunodeficient/beige mice also failed to abrogate this response. AdVmIL-12 retained potent antitumor activity in mice with specific genetic defects in immune cellular cytotoxicity (perforin knockout mice) and costimulation (CD28 knockout mice). Use of mice with specific NK T cell deficiencies, Valpha14 T-cell receptor and CD1 knockout mice, also failed to abrogate the response to AdVmIL-12. Histological and immunohistochemical studies of AdVmIL-12-treated tumors showed extensive inhibition of neovascularization and a marked decrease in factor VIII-stained endothelial cells. Our studies indicate that the antitumor response of AdVmIL-12 is independent of direct cytotoxic cellular immunity (specifically, the function of NK T cells) and suggest that the initial mechanisms of AdVmIL-12-mediated tumor regression involve inhibition of angiogenesis.

Interleukin-4 downregulates interleukin-6 production in human peripheral blood mononuclear cells.

We report that recombinant human interleukin-4 (IL-4) downregulates interleukin-6 (IL-6) production by human peripheral blood mononuclear cells (PBMC). PBMC were preincubated for up to 24 hr in the presence of IL-4 (100 U/ml) and then activated with lipopolysaccharide B Escherichia coli 026:B6 (LPS, 10 micrograms/ml), recombinant human tumor necrosis factor-alpha (TNF-alpha, 200 U/ml), or Concanavalin A (Con A, 10 micrograms/ml). Although all these signals induced IL-6 production, IL-4-treated cells produced significantly reduced levels of IL-6 protein. This effect was dose and time dependent. We conclude that IL-4 is a potent downregulatory modulator of IL-6 expression in human PBMC.

Fine specificity analysis of an HLA-A2.1-restricted immunodominant T cell epitope derived from human alpha-fetoprotein.

Human alpha-fetoprotein (AFP) is a potentially important target for the immunotherapy of hepatocellular carcinoma (HCC). AFP(542-550) (GVALQTMKQ) is one of several HLA-A2.1-restricted immunodominant AFP peptides that consistently generate AFP-specific T cell responses in human T cell cultures and in HLA-A2.1/K(b) transgenic (A2.1 tg) mice. We performed a fine specificity analysis of this nonamer to determine which amino acid side chains were critical for T cell priming and recognition. Using peptide-pulsed dendritic cells (DC) as an immunization strategy, we characterized the effects of AFP(542-550) amino acid substitutions on priming and recognition in A2.1 tg mice. Replacing the glutamine at anchor position 9 with a leucine enhanced MHC binding and AFP-specific T cell responses. Substitution of leucine at non-anchor position 4 with an alanine did not alter binding but greatly diminished T cell recognition. Computer-generated three-dimensional models provided the structural rationale for these observed effects in MHC binding and T cell responses resulted from the modifications in the AFP(542-550) sequence.

In vivo therapy of hepatocellular carcinoma with a tumor-specific adenoviral vector expressing interleukin-2.

A recombinant adenovirus (AdVAFP1-IL2) containing the murine alpha-fetoprotein (AFP) promoter was constructed to direct hepatocellular carcinoma (HCC)-specific expression of the human interleukin-2 (IL-2) gene. In vitro testing of AdVAFP1-IL2 showed HCC-specific IL-2 gene expression three to four orders of magnitude higher in AFP-producing HCC lines compared to non-AFP producing non-HCC lines. The in vivo efficacy and tumor specificity of AdVAFP1-IL2 was evaluated compared to AdVCMV-IL2 (in which the IL-2 gene is driven by the strong constitutive cytomegalovirus promoter) in the treatment of established human HCC (Hep 3B/Hep G2) xenografts growing in CB-17/SCID mice. Intratumoral injection of AdV resulted in growth retardation and regression in a majority of established hepatomas, but with a much wider therapeutic index and less systemic toxicity using the AFP vector. This study illustrates the superiority of using transcriptionally targeted recombinant AdV vectors in cytokine-based gene therapy.

Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication.

In two murine lung cancer models adenoviral interleukin 7-transduced dendritic cells (DC-AdIL-7) were administered intratumorally, resulting in complete tumor regression. Intratumoral DC-AdIL-7 therapy was as effective as DCs pulsed with specific tumor peptide antigens. Comparison with other intratumoral therapies including recombinant IL-7, AdIL-7 vector alone, unmodified DCs, IL-7-transduced fibroblasts, or DCs pulsed with tumor lysates revealed DC-AdIL-7 therapy to be superior in achieving antitumor responses and augmenting immunogenicity. Mice with complete tumor eradication as a result of either DC-AdIL-7 or AdIL-7 therapy were rechallenged with parental tumor cells 30 days or more after complete tumor eradication. All the DC-AdIL-7-treated mice completely rejected a secondary rechallenge, whereas the AdIL-7-treated mice had sustained antitumor effects in only 20-25% of the mice. DC-AdIL-7 therapy was more effective than AdIL-7 in achieving systemic antitumor responses and enhancing immunogenicity. After complete tumor eradication, those mice treated with DC-AdIL-7 evidenced significantly greater release of splenocyte GM-CSF and IFN-gamma than did controls or AdIL-7-treated mice. After intratumoral injection, gene-modified DCs trafficked from the tumor to lymph node sites and spleen. DCs were detected in nodal tissues for up to 7 days after intratumoral injection. We report that intratumoral DC-AdIL-7 leads to significant systemic immune responses and potent antitumor effects in murine lung cancer models.