Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors.

Antiangiogenic gene therapy is a promising strategy for cancer treatment, which generally requires highly efficient delivery systems. To date, success of this strategy has depended almost exclusively on the delivery of high titers of viral vectors, which can result in effective transgene expression. However, their cytotoxicity and immunogenicity are a major concern for clinical applications. Recent advances in delivery efficiency of naked DNA could potentially meet the requirement for both high transgene expression and minimal side effects. To investigate whether naked DNA can be used for antiangiogenic cancer therapy, an expression plasmid was generated that encodes a soluble form of fetal liver kinase-1 (Flk-1) gene, a receptor for vascular endothelial growth factor (VEGF). Hydrodynamic injection of this plasmid resulted in close to 0.1 mg/ml of soluble Flk-1 protein in mouse serum and blocked VEGF-driven angiogenesis in matrigel in vivo. The same delivery significantly suppressed the growth of two different pre-existing subcutaneous tumors, Renca renal cell carcinoma and 3LL lung carcinoma. CD31 immunohistochemistry revealed that the tumor-associated angiogenesis was also highly attenuated in soluble Flk-1-treated mice. Thus, expression of genes by hydrodynamics-based gene delivery of naked DNA appears to be a promising approach for antiangiogenic cancer gene therapy.

Constitutive expression of functional CD40 on mouse renal cancer cells: induction of Fas and Fas-mediated killing by CD40L.

CD40, a member of the TNF receptor superfamily, is expressed on B cells, dendritic cells, and some tumor cells, including melanoma and bladder carcinoma. In this study, we report that both mouse and human renal carcinoma cells (RCC) also constitutively express functional CD40. Treatment of mouse RCC with CD40L induced strong expression of genes and proteins for ICAM-1 and Fas, and this expression was further enhanced by combining CD40L with IFN-gamma. Similar effects were demonstrated using an agonist anti-CD40 antibody. The increased levels of Fas expression on RCC after treatment with CD40L plus IFN-gamma resulted in potent killing by either FasL-positive effector cells or agonistic anti-Fas antibody. The combination of CD40L plus IFN-gamma also significantly enhanced killing of RCC by tumor-specific CTL lines. Our results demonstrate that constitutively expressed CD40 is functionally active and may provide a molecular target for the development of new approaches to the treatment of RCC.

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer.

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.

Hematopoietic switch from lymphoid to granulocytic development in 3LL tumor-bearing mice.

A significant splenomegaly and lymphadenopathy develops during the progressive growth of Lewis Lung (3LL) tumors in mice. Enlarged spleen and lymph nodes occur because of a pronounced increase in granulocytes in these organs. This granulocytosis in spleen and lymph node was not simply due to recruitment of granulocytes from peripheral blood to spleen and lymph nodes, but also a result of development and/or differentiation of granulocytes from the bone marrow. There was a marked increase in development of myeloid lineage cells, whereas lymphoid populations including T cells and B cells, were dramatically decreased in bone marrow and peripheral blood of 3LL tumor-bearing mice. These data demonstrate that host hematopoiesis shifts from lymphoid to granulocytic development in the 3LL tumor-bearing mice. Interestingly, a somatic mutation of N-Ras gene was found in 3LL tumor cells at codon 61, suggesting that mutated N-Ras may contribute to induction of granulocytosis in 3LL tumor-bearing mice.

Induction of transplantable mouse renal cell cancers by streptozotocin: in vivo growth, metastases, and angiogenic phenotype.

Interleukin-2-based regimens of biological therapy have shown some clinical promise for the treatment of kidney cancer in humans, although the mechanisms responsible for tumor regression occurring in these patients remain unclear. Preclinical insight into these mechanisms is limited by a paucity of orthotopic animal models of kidney cancer. We have used streptozotocin, an antibiotic and diabetogenic nitrosamine compound derived from Streptomyces achromogenes to induce new kidney tumors in BALB/c mice. Single or multiple doses of streptozotocin induced kidney tumors in up to 25% of mice by 50-90 weeks of age, with up to 18% characterized as renal cell carcinomas (RCCs). Several transplantable lines were obtained from the RCCs, and one of these lines was subsequently cloned. The initial tumor isolates and sublines were histologically reconfirmed to be RCCs, and all grew progressively but slowly (mean survival times, 57 to >100 days) in vivo after intrarenal implant. None of the primary isolates or sublines revealed mutations in either the VHL or Ras genes, although karyotype analysis and chromosome painting revealed the consistent presence of a submetacentric chromosome resulting from the fusion of chromosomes 16 and 19. Biological characterization of these tumors revealed several features analogous to the growth of human kidney cancers, including a propensity for the formation of lung metastases and high vascularity. This hypervascularity is evident by both gross and microscopic analysis and correlates with the expression of several proangiogenic genes. Overall, the features of orthotopic transplantability, slower in vivo growth (relative to the rapid growth rates of other transplantable mouse kidney tumors), propensity for lung metastases, and hypervascularity may make these tumors valuable models for the study of new therapeutic strategies based on antineovascular agents and antitumor cytokines.

IFN-gamma and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy.

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

Antibodies to CD40 induce a lethal cytokine cascade after syngeneic bone marrow transplantation.

CD40 stimulation, by either antibody or ligand, has been shown to inhibit the growth of a variety of neoplastic cells, both in vivo and in vitro. In this study, we assessed the effects of CD40 stimulation using a murine agonistic CD40 monoclonal antibody (MoAb) (FGK115) or a soluble recombinant murine CD40 ligand (srmCD40L) in both lethally irradiated and nonirradiated BALB/c mice. Toxicity after CD40 stimulation was not observed in nonirradiated animals receiving up to 100 microg of the agonist anti-CD40 MoAb. However, as little as 10 microg of the agonistic anti-CD40 MoAb induced acute toxicity resulting in 100% morbidity of lethally irradiated animals by 4 days after irradiation. Histological evaluation of animals receiving anti-CD40 MoAb revealed severe intestinal lesions with disruption of the villi, goblet cell depletion, and crypt hyperplasia of the small intestine, colon, and cecum. Delaying the administration of anti-CD40 MoAb or reducing the amount of irradiation given resulted in increased survival and less severe lesions. Analysis of serum cytokine levels in lethally irradiated mice receiving agonistic anti-CD40 showed a marked increase of interferon (IFN)-gamma. Lethally irradiated IFN-gamma knockout mice given the agonistic anti-CD40 MoAb demonstrated significant increases in survival and minimal gut lesions compared with wild-type mice receiving the same regimen, suggesting that IFN-gamma plays a major role in this toxic reaction. These results indicate that CD40 stimulation using agonistic antibodies following lethal irradiation leads to a fatal, cytokine-induced disease affecting the intestine.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to interferon gamma-dependent natural killer cell protection from tumor metastasis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-gamma production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, alpha-galactosylceramide (alpha-GalCer), a powerful inducer of NKT cell IFN-gamma and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-gamma-deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and alpha-GalCer were strictly IFN-gamma dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-gamma-mediated antimetastatic effects of IL-12 and alpha-GalCer.

Anoikis and metastatic potential of cloudman S91 melanoma cells.

Anoikis is a form of apoptosis induced in normal cells as a result of loss of their adhesion to substrate. In the present study, we have tested whether tumor cells are also sensitive to anoikis and whether selection of tumor cells for resistance to anoikis could increase their metastatic ability. In vitro cultured Cloudman S91 melanoma cells are strongly adherent to the plastic. Prevention of their adherence by rocking or by covering culture plates with polyhydroxyethylmethacrylate resulted in induction of anoikis and death of almost all cells. Their death was prevented in the presence of caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone. To select anoikis-resistant cells, S91 cells floating in the culture medium were sequentially isolated and transferred for seven generations. As a result, a new subline of S91 cells capable of growing in free cell suspension was selected. These S91 nonadherent (S91Nadh) cells were completely resistant to anoikis and manifested higher metastatic ability than S91Adh cells. Anoikis resistance of S91Nadh cells was not attributable to their resistance to other apoptotic signals in vitro, and they showed no increase in their survival in vivo in the lungs after i.v. inoculation. Increased metastatic potential of the anoikis-resistant S91Nadh cells was associated with various phenotypic changes, including increased proliferation and loss of VLA-4 integrin expression because of down-regulation of the VLA-49alpha (CLD49d) gene. In parallel, they showed a reduction in homotypic aggregation and binding to endothelial cells, increased Matrigel invasiveness, and decreased matrix metalloproteinase-2 and matrix metalloproteinase-9 activity that paralleled up-regulation of the TIMP-1 gene. S91Nadh cells also manifested changes in cell surface carbohydrates, such as appearance of alpha-galactosyl epitopes as a result of up-regulation of the alpha1,3-galactosyltransferase gene and concomitant reduction in cell membrane sialylation. Thus, selection of S91 melanoma cells for anoikis resistance resulted in an increase in their metastatic potential in parallel with multiple alterations in their phenotypic properties.

Primary hepatocytes from mice treated with IL-2/IL-12 produce T cell chemoattractant activity that is dependent on monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2).

The IFN-gamma-inducible proteins monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-gamma-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-gamma. In addition to depending on IFN-gamma, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-gamma R alpha and beta-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-gamma contain increased chemotactic activity for enriched human and mouse CD3(+) T cells, as well as mouse DX5(+) NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.

Gastrointestinal cells of IL-7 receptor null mice exhibit increased sensitivity to irradiation.

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.

Complete regression of established spontaneous mammary carcinoma and the therapeutic prevention of genetically programmed neoplastic transition by IL-12/pulse IL-2: induction of local T cell infiltration, Fas/Fas ligand gene expression, and mammary epithelial apoptosis.

Using a novel transgenic mouse model of spontaneous mammary carcinoma, we show here that the IL-12/pulse IL-2 combination can induce rapid and complete regression of well-established autochthonous tumor in a setting where the host immune system has been conditioned by the full dynamic process of neoplastic progression and tumorigenesis. Further, this regimen inhibits neovascularization of established mammary tumors, and does so in conjunction with potent local induction of genes encoding the IFN-gamma- and TNF-alpha-inducible antiangiogenic chemokines IFN-inducible protein 10 and monokine induced by IFN-gamma. In contrast to untreated juvenile C3(1)TAg mice in which histologically normal mammary epithelium predictably undergoes progressive hyperplasia, atypical changes, and ultimately transition to overt carcinoma, the current studies also demonstrate a unique preventative therapeutic role for IL-12/pulse IL-2. In juvenile mice, early administration of IL-12/pulse IL-2 markedly limits the expected genetically programmed neoplastic transition within the mammary epithelium and does so in conjunction with enhancement of constitutive Fas and pronounced induction of local Fas ligand gene expression, T cell infiltration, and induction of apoptosis within the mammary epithelium. These events occur in the absence of a durable Ag-specific memory response. Thus, this novel model system demonstrates that the potent therapeutic activity of the IL-12/pulse IL-2 combination rapidly engages potent apoptotic and antiangiogenic mechanisms that remain active during the delivery of IL-12/pulse IL-2. The results also demonstrate that these mechanisms are active against established tumor as well as developing preneoplastic lesions.

Opposing roles of interferon-gamma on CD4+ T cell-mediated graft-versus-host disease: effects of conditioning.

Although alloreactive T cells are required for the induction of graft-versus-host disease (GVHD), other factors can influence outcome in murine models of the disease. Lethal total body irradiation (TBI) conditioning regimens followed by reconstitution with allogeneic lymphohematopoietic cells results in the generation of donor anti-host cytotoxic T lymphocyte (CTL)-mediated solid organ (gut, liver, skin) destruction. In contrast, donor anti-host CTL-mediated hematopoietic failure is the primary cause of morbidity following sublethal TBI. To determine the role of interferon (IFN)-gamma in graft-versus-host reactions against hematopoietic and solid organ targets, we used IFN-gamma knockout mice as donors in both lethal TBI and bone marrow transplantation (BMT) rescue and sublethal TBI models. In this report, we show that CD4+ T cells from IFN-gamma knockout (KO) mice resulted in accelerated GVHD after lethal TBI/BMT using a single major histocompatibility class II mismatch model. In marked contrast, the use of these same IFN-gamma KO CD4+ donor cells in combination with sublethal TBI significantly ameliorated GVHD-associated mortality. In these recipients, severe anemia, bone marrow aplasia, and intestinal lesions were observed in the presence but not the absence of donor-derived IFN-gamma. Administration of anti-IFN-gamma antibodies to sublethally irradiated recipients of wild-type donor cells confirmed the role of IFN-gamma depletion in CD4+ T cell-mediated GVHD. In conclusion, the extent of conditioning markedly affects the role of IFN-gamma in GVHD lesions mediated by CD4+ T cells. In models using sublethal TBI, the absence of IFN-gamma is protective from GVHD, whereas in lethal TBI situations, the loss is deleterious.

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells.

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.

Regulation and antimetastatic functions of liver-associated natural killer cells.

The liver is a complex organ composed of hepatic parenchymal cells and a variety of non-parenchymal cells that consist of endothelial cells, Kupffer cells, and several subsets of resident lymphocytes, including natural killer (NK), T, and NK1.1+/CD3+ (NK/T) cells. The regulation of these various lymphoid subpopulations and their relative contributions to antiviral, antitumor and pathogenic inflammatory responses in the liver remain topics of much interest. Studies from our laboratory have shown that various immune stimulants and cytokines can augment liver-associated NK activity at least partially through the mobilization of NK cells from the bone marrow to the liver. The mobilization process can be dependent on the induction of interferon (IFN)-gamma and/or tumor necrosis factor-alpha and on very late activation antigen-4/vascular cell adhesion molecule-1 interaction. The induction of IFN-gamma by cytokines such as interleukin (IL)-12 also rapidly triggers the induction of chemokine genes in parenchymal cells that may contribute to the localization of NK and T cells. Both IL-2 and IL-12 trigger changes in the number and functions of liver-associated leukocyte subsets, and induce antimetastatic effects that are likely mediated through several direct and indirect mechanisms. The overall goal of these studies is to understand the interactions and functions of liver-associated NK1.1+ cells in the context of innate and adaptive immune responses to neoplasia.

The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma.

The 5' flanking region of the C3(1) component of the rat prostate steroid binding protein (PSBP) has been used to successfully target the expression of the SV40 large T-antigen (Tag) to the epithelium of both the mammary and prostate glands resulting in models of mammary and prostate cancers which histologically resemble the human diseases. Atypia of the mammary ductal epithelium develops at about 8 weeks of age, progressing to mammary intraepithelial neoplasia (resembling human ductal carcinoma in situ [DCIS]) at about 12 weeks of age with the development of invasive carcinomas at about 16 weeks of age in 100% of female mice. The carcinomas share features to what has been classified in human breast cancer as infiltrating ductal carcinomas. All FVB/N female mice carrying the transgene develop mammary cancer with about a 15% incidence of lung metastases. Approximately 10% of older male mice develop anaplastic mammary carcinomas. Unlike many other transgenic models in which hormones and pregnancy are used to induce a mammary phenotype, C3(1)/Tag mice develop mammary tumors in the mammary epithelium of virgin animals without hormone supplementation or pregnancy. Although mammary tumor development appears hormone-responsive at early stages, invasive carcinomas are hormone-independent, which corresponds to the loss of estrogen receptor-alpha expression during tumor progression. Molecular and biologic factors related to mammary tumor progression can be studied in this model since lesions evolve over a predictable time course. Genomic alterations have been identified during tumor progression, including an amplification of the distal portion of chromosome 6 containing ki-ras and loss of heterozygosity (LOH) in other chromosomal regions. We have demonstrated that stage specific alterations in the expression of genes which are critical regulators of the cell cycle and apoptosis are functionally important in vivo. C3(1)/Tag mice appear useful for testing particular therapies since growth of the mammary tumors can be reduced using chemopreventive agents, cytokines, and an anti-angiogenesis agent.

Enhancement of antitumor immunity against B16 melanoma tumor using genetically modified dendritic cells to produce cytokines.

Dendritic cells (DC) that have been genetically modified to express cytokine genes may be novel tools for inducing antitumor immune responses. In the present study, the pMX retroviral vector was modified to express the mouse IL-2 (mIL-2pMX) and mouse IL-12 (mIL-12pMX) genes. Supernatants from 293 cells transfected with pMX retroviral vectors were harvested and used to transduce mouse lin- bone marrow (BM) progenitor cells. After 48 h co-culture with pseudotype retrovirus, BM cells were cultured for 12 days in the presence of mGM-CSF, mSCF and mTNF-alpha to obtain a DC-enriched fraction. Flow cytometric analysis showed that GFP protein expression in these cultures was 20-40% and that 40-50% of the cultured BM cells were positive for the DC marker, DEC205. About 60% of cells sorted for DEC205 also expressed GFP. The supernatants of DC-mIL-2 and DC-mIL-12 cultured for 48 h contained 5.2 +/- 0.15 and 33.9 +/- 2.6 ng cytokine protein per milliliter, respectively. Intratumoral injection of DC-mIL-2 or DC-mIL-12 on days 8 and 15 after the intradermal injection of 1 x 105 B16F10 cells, resulted in a significant reduction in tumor size by day 21, as compared with mice treated with unmodified DC or DC-GFP. Longer term analysis as assessed at day 42 revealed that B16 tumor-bearing mice treated with cytokine gene-modified DC survived significantly longer than mice from other groups. Spleen cells obtained from DC-treated mice were specifically sensitized for the generation of CTL by subsequent restimulation with gene-modified DC. These results suggested that DC genetically modified to express IL-2 or IL-12 can induce potent antitumor responses against well-established, poorly immunogenic B16F10 tumors. Gene Therapy (2000) 7, 2113-2121.

IFN-gamma-dependent delay of in vivo tumor progression by Fas overexpression on murine renal cancer cells.

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-gamma and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-gamma-/- mice, indicating that IFN-gamma is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.

Intradermal delivery of IL-12 naked DNA induces systemic NK cell activation and Th1 response in vivo that is independent of endogenous IL-12 production.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-gamma and IFN-gamma-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-gamma proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o- plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o- to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o- into BALB/c IL-12 p40-/- mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-gamma protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.