Crucial role of TNF-alpha in CD8 T cell-mediated elimination of 3LL-A9 Lewis lung carcinoma cells in vivo.

The role of perforin, IFN-gamma, and TNF-alpha in anti-tumor CD8 T cell immunity was examined in a new tumor model using a CD8 T cell epitope (GP33) derived from lymphocytic choriomeningitis virus as a tumor-associated Ag. In contrast with parental 3LL-A9 (A9) Lewis lung carcinoma cells that progressively grow in C57BL/6 mice, s.c. injection of GP33-transfected A9GP33 tumor cells induced a protective GP33-specific CD8 T cell response that led to complete tumor cell elimination. Tumor regression was dependent on perforin, IFN-gamma, or TNF-alpha, because A9GP33 tumors developed in mice deficient in one of these genes. A9GP33 tumors arising in perforin- and IFN-gamma-deficient mice represented GP33 Ag-loss variants, demonstrating that GP33-specific CD8 T cells from these mice were able to exert an Ag selection pressure. In contrast, tumor cells growing in TNF-alpha knock-out mice still expressed the tumor-associated GP33 peptide despite the presence of activated GP33-specific CD8 T cells. These findings provide evidence for a crucial role of TNF-alpha in A9 tumor cell elimination by CD8 T cells in vivo.

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo.

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.

Cytokine gene transfer in cancer therapy.

New strategies based on gene transfer technology are employed in cancer therapy. Cytokines are polypeptides involved in immunity and inflammation, and essentially control the magnitude of the immune response. Genetically modified tumor cells releasing various cytokines have been shown to enhance tumor immunogenicity and to induce the regression of preexisting tumors. In some instances, immunological memory has been generated to resist the subsequent challenge with unmodified, parental tumor cells. Cytokine gene transfer into antitumor effector cells, as well as antigen presenting cells, is also being investigated to augment antitumor immune responses.

Cloning and sequence analysis of the immediate promoter region and cDNA of porcine granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage. Recombinant G-CSF (rG-CSF) is routinely used in the prevention of chemotherapy-induced neutropenia and in the setting of bone marrow transplantation. Chronic idiopathic and congenital neutropenic disorders also show improvement after rG-CSF injections. Applications of either rG-CSF or G-CSF gene transfected cells into mice give rise to leukocytosis, which can be measured easily. This makes G-CSF a versatile tool for studying systemic effects of therapeutic proteins delivered by genetically modified cells in vivo. Although the biological activity of G-CSF is not species-specific, studies on long-term expression would require the use of species-identical proteins in order to avoid host immune reactions against the foreign gene product. Because of the physiological and immunological similarity of pigs and human, the pig has become an important large-animal model for biomedical research. We have therefore cloned porcine G-CSF cDNA from RNA isolated from pig PBLs. Pig G-CSF is a 195-amino-acid polypeptide that shares a high degree of homology to human (78%), murine (71%) as well as rat (68%) G-CSF. In contrast to human and murine, but not to rat G-CSF, a different ATG translation start codon is used, resulting in a shorter, but still functional signal sequence.

Paracrine stimulation of keratinocytes in vitro and continuous delivery of epidermal growth factor to wounds in vivo by genetically modified fibroblasts transfected with a novel chimeric construct.

BACKGROUND: Growth factors play an important role in tissue repair. While the effectiveness of growth factor therapy in animal wound healing models and limited human clinical trials has been demonstrated, the ideal method for their administration to the wound remains unclear. Experimental data suggest that the continuous presence in the early stages of wound repair is beneficial. MATERIALS AND METHODS: We have constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene is fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS: Clonally selected human fibroblasts transfected with this construct secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF can be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 pg/ml to 140 pg/ml on day 7. No EGF was found in the wound at day 14. CONCLUSIONS: A single application of irradiated EGF genetransfected fibroblasts to wounds can thus continuously deliver the transgene in vivo and could be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.

NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line.

We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design.

Collagen as matrix for neo-organ formation by gene-transfected fibroblasts.

BACKGROUND: Genetically modified cells have been used in several animal models for the in vivo delivery of therapeutic proteins. One of the problems encountered is early cell death after the implantation of cells. MATERIALS AND METHODS: To improve the survival of implanted cells, we have developed a system in which transfected fibroblasts are seeded onto biodegradable collagen matrices and transplanted into animals after several days of in vitro culture. Since G-CSF is widely used clinically to accelerate reconstitution of hematopoiesis after cancer chemotherapy, it was chosen to investigate in vivo delivery by transfected fibroblasts. RESULTS: Expression of the human G-CSF gene is maintained by transfected cells when grown on collagen scaffolds in vitro. After the i.p. implantation of collagen matrices seeded with G-CSF gene transfected fibroblasts, G-CSF serum levels could be detected for more than 2 weeks. Histological analysis of matrices explanted on day 31 and demonstration of in vitro G-CSF production reveals that genetically modified cells can survive on these implants in vivo. Large areas of the collagen are degraded and substituted with a network of endogenous argyrophilic fibers. Also ingrowth of blood vessels into the matrices is observed leading to the formation of "neo-organ' like structures. CONCLUSIONS: Biodegradable collagen matrices can serve as scaffolds for survival of transfected fibroblasts in vivo.

A phase-I clinical study of autologous tumor cells plus interleukin-2-gene-transfected allogeneic fibroblasts as a vaccine in patients with cancer.

Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL-2-secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST 6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL-2 per 10(6) cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL-2 and Neo(r). Fifteen patients with refractory malignant tumors received 3-4 injections of irradiated KMST6.14 and autologous tumor cells in a phase-I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3+ T cells with numbers of CD4+ helper cells exceeding those of CD8+ cytotoxic T cells (CTL). CD8+ T-cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal-cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8+ T-cell clones established from the vaccination site of 1 of 2 renal-cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal-cell carcinoma cells. In conclusion, a vaccine composed of IL-2 gene-transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti-tumor T-cell responses in vivo without major side-effects. Malignant melanoma and renal-cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials.

Processing of tumor tissues for vaccination with autologous tumor cells.

Vaccination with gene-transfected tumor cells has recently been proposed as a new strategy in the immunotherapy of cancer. Since autologous tumor cells provide an optimal antigen profile, the possibility of generating single cell suspensions from renal cell carcinoma (RCC), malignant melanoma (MM), colon carcinoma (CC), and non-small-cell lung cancer (NSCLC) biopsies was investigated. One hundred and seventy-four tumor biopsies were processed by mechanic and enzymatic dissociation, yielding 1-2 x 10(6) cells/g tumor (median), irrespective of tumor type. Primary tumor cell cultures (PTCC) of > or = 10(7) cells were established from 29 of 86 (34%) RCC, 14 of 38 (37%) MM, 11 of 23 (48%) NSCLC and 4 of 27 (15%) CC specimens. The amount of non-tumor cells, as assessed by morphology and immunocytology, was generally low (< 30%) in RCC (35 of 41) and MM (11 of 17), while it exceeded 60% in 8 of 11 PTCC from NSCLC and 3 of 11 CC. A high tumor cell yield was obtained in biopsies with a high degree of vascularization and in the virtual absence of necrosis. Thus, PTCC > or = 10(7) cells were obtained in 73% of MM with a high degree of vascularization and in 22% of MM with a low degree of vascularization (p < 0.007). Long-term tumor cell cultures exceeding 20 passages were established in 24 of 86 (18%) RCC, 7 of 38 (18%) MM and 3 of 27 (11%) CC, while successful implantation in nude mice was achieved in 8 of 20 RCC and 5 of 10 MM. Thus, under the conditions described, > or = 10(7) primary tumor cells of high purity could be generated from about one third of RCC and MM biopsies, while the success rate increased to > 50 and > 70%, respectively, in samples with a high degree of vascularization generated by an optimized biopsy technique excluding necrotic parts.

Systemic hematological effects of granulocyte colony-stimulating factor produced by irradiated gene-transfected fibroblasts.

Although long-term expression of therapeutic molecules is necessary for the treatment of permanent deficiencies, short-term expression of therapeutic molecules inducing local or systemic effects is preferable in clinical situations where temporary substitution is the goal. One such clinical setting is the administration of hematopoietic growth factors in cancer chemotherapy-induced myelosuppression. Several plasmid vectors containing the human granulocyte colony-stimulating factor (G-CSF) gene under transcriptional control of different regulatory elements were constructed. In vitro production of G-CSF by nonvirally transfected murine fibroblast clones initially increased after lethal irradiation and was detectable for at least 12 days. We also demonstrate that a single injection of irradiated G-CSF-secreting fibroblasts leads to accelerated hematopoietic recovery and mobilization of committed peripheral blood progenitor cells equivalent to that achieved by twice daily s.c. administration of high doses of recombinant human G-CSF. Using dicistronic vectors, high levels of G-CSF secretion were also obtained in human fibroblasts.

In vitro differentiation of CD34+ hematopoietic progenitor cells toward distinct dendritic cell subsets of the birbeck granule and MIIC-positive Langerhans cell and the interdigitating dendritic cell type.

We have demonstrated recently that Birbeck granule-positive Langerhans cells (LC) can be derived from CD34+ peripheral blood progenitor cells in the presence of a seven-cytokine cocktail (CC7-7). Here, we show that the sequential use of early-acting hematopoietic growth factors, stem cell factor, interleukin (IL)-3, and IL-6, followed on day 8 by differentiation in the two-factor combination IL-4 plus granulocytemacrophage colony-stimulating factor (GM-CSF) (CC4GM) is more efficient and allows the cells to be arrested in the LC stage for more than 1 week while continuous maturation occurs in CC7-7. Maturation of LC to interdigitating dendritic cells (DC) could specifically be induced within 60 hours by addition of tumor necrosis factor-alpha (20 ng/mL) or lipopolysaccharide (100 ng/mL). Using LC that had been enriched to greater than 90% CD1a+ cells by an immunoaffinity column, we were able to define clear-cut differences between LC and DC that corroborate data of the respective cells derived from epithelial borders (LC) or from lymph nodes (LN) and spleen (DC). Thus, molecules and functions involved in antigen (AG) uptake and processing were highly expressed in LC, while those involved in AG presentation were at maximum in DC. LC were CD1a+2 DR+2, CD23+, CD36+, CD80-, CD86-, and CD25-, while DC were CD1a+/- DR+3, CD23-, CD36-, CD80+, CD86+2, and CD25+, CD40 and CD32 were moderately expressed and nearly unchanged on maturation, in contrast to monocyte-derived DC. Macropinocytosis of fluorescein isothiocyanate-dextran was dominant in LC, as were multilamellar major histocompatibility complex (MHC) class II compartments (MIICs), which were detected by electron microscopy. The functional dichotomy of these cell types was finally supported by testing the AG-presenting cell function for tetanus toxoid to primed autologous T-cell lines, which was optimal when cells were loaded with AG as LC and subsequently induced to become DC.

Systematic evaluation of chimeric marker genes on dicistronic transcription units for regulated expression of transgenes in vitro and in vivo.

Plasmid expression vectors combining human cytokine cDNAs and selectable marker genes on dicistronic transcription units were functionally characterized in vitro and in vivo. The internal ribosome entry sequence (IRES) of encephalomyocarditis virus mediated cap-independent translation of the downstream cistron. After cationic lipofection of cells with a dicistronic construct containing the Neor gene downstream of a human interleukin-2 (IL-2) cDNA, all G418-resistant clones secreted high amounts of IL-2. Reversal of the order of the cDNAs was associated with less efficient transgene expression and represented no advantage in comparison to separate expression cassettes. To combine direct in vitro selection of expression with in vivo elimination of cytokine-secreting cells, an improved chimeric cDNA of the Neor and herpes simplex virus (HSV) thymidine kinase (TK) genes was constructed and shown to confer sensitivity to ganciclovir concentrations that can be achieved in human patients. This chimeric marker was coupled on dicistronic constructs with a granulocyte colony-stimulating factor (G-CSF) cDNA as a molecule with easily detectable bioactivity in vivo. Subcutaneous implantation of pCMV.GCSF.ires TK/NEO-transfected CMS-5 cells into syngeneic BALB/c mice resulted in excessive leukocytosis and progressively growing tumors. Treatment with ganciclovir led to normalization of leukocyte counts in all animals, whereas complete regression of tumors was observed in only 3/5 mice. Hypermethylation of the transfected promoter was demonstrated in both ganciclovir-resistant tumors. Thus, transcription units combining selectable markers and genes of interest allow selection of high producer cells in vitro and efficient elimination of transgene-expressing cells in vivo. However, cells that hypermethylate transfected genes to terminate gene expression in vivo may escape conditional ablation.

Delineation of the dendritic cell lineage by generating large numbers of Birbeck granule-positive Langerhans cells from human peripheral blood progenitor cells in vitro.

It is well established by in vivo and in vitro studies that dendritic cells (DCs) originate from hematopoietic progenitor cells. However, the presumed intermediate of Birbeck granule (BG)+ Langerhans cells (LCs) has not been detected in cultures derived from bone marrow or peripheral blood progenitor cells (PBPCs), thus contrasting with the data obtained with cord blood. We show here that large numbers of BG+ LCs can be generated from human CD34+ PBPCs in vitro, when granulocyte-macrophage colony-stimulating factor and interleukin-4, potent promotors of LC/DC differentiation, are combined with a cocktail of early acting hematopoietic growth factors. LCs were found to emerge from CD33+CD11b+CD14- progenitor cells that they share with the monocytic lineage. During culture, these cells exhibited a sequence of dramatic morphologic changes, starting with a major increase in granularity followed by an increase in size herein exceeding that of all peripheral blood cells. At the same time, CD1a and major histocompatibility complex class II expression were upregulated and virtually all CD1a++ cells were BG+ by electron microscopy. With prolonged culture, CD1a was downregulated on a major population of cells, paralleled by a loss of BG and an increase of CD4, CD25, and CD80 expression that may correspond to the maturation of epidermal LC in vitro. However, these cells were consistently CD5- and did not exhibit changes in the CD45-isoform expression during culture. The availability of large numbers of these highly purified BG+ LCs and mature DCs allows for specific analysis of these subpopulations and provides a source of potent antigen-presenting cells from individual patients for vaccination protocols against infectious or tumor-associated antigens.

Human tumor vaccines and genetic engineering of tumors with cytokine and histocompatibility genes to enhance immunogenicity.

Genetically engineered tumor cells can be used as vaccines in order to stimulate an immune response. To date, tumor cells have been modified in vitro so that they secrete cytokines or express histocompatibility molecules that they naturally fail to express. These tumor cells differ in the types of immune responses they induce and in whether the responses have local or systemic efficacy. Many questions have been raised during the past year, including whether allogeneic or autologous tumor cells should be employed and whether there may be a risk of inducing autoimmune disease along with the antitumor response. Nevertheless, because of the paucity of available therapies for patients with advanced cancer, investigators must attempt to refine the approaches used in order to minimize patient risk while maximizing tumor cell destruction.

Cytokine therapy with gene-transfected cells: single injection of irradiated granulocyte-macrophage colony-stimulating factor-transduced cells accelerates hematopoietic recovery after cytotoxic chemotherapy in mice.

Development of cell-based delivery systems that can release therapeutic levels of hematopoietic growth factors into the systemic circulation would facilitate treatment of patients requiring cytokine therapy. In this study, we have investigated the potential of granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, irradiated syngeneic murine cells to accelerate hematopoietic recovery after cytotoxic chemotherapy. As a model, CMS-5 fibrosarcoma cells, were transduced with a retroviral vector containing the murine GM-CSF cDNA. Transduced cells secreted 38 ng GM-CSF/10(6) cells in 24 hours. After irradiation, in vitro GM-CSF production initially increased up to fivefold and was measurable for about 2 weeks. One and 2 days after injection of irradiated, GM-CSF-secreting CMS-5 cells (N2/CMVGM-CSF/CMS5 # 6 cells) into mice, GM-CSF serum levels of 405 +/- 58 pg/mL and 183 +/- 36 pg/mL were measured, respectively. Serum levels were comparable with levels detected 3 hours after injection of 100 ng recombinant murine GM-CSF (rmGM-CSF) subcutaneously (90 pg/mL). Injection of N2/CMVGM-CSF/CMS5 # 6 cells in cyclophosphamide-treated mice was as effective in accelerating neutrophil recovery as twice daily subcutaneous injections of rmGM-CSF. These data suggest that irradiated hematopoietic growth factor-secreting cells might offer an alternative to parenteral injections of recombinant cytokines in the treatment of neutropenic patients.

Immunotherapy of bladder cancer with cytokine gene-modified tumor vaccines.

This study explored the use of cytokine gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology and responds to treatment in a manner similar to that of its human counterpart. In a previous study we have shown that interleukin 2 (IL-2)-secreting, irradiated, MBT-2 cell preparations were capable of curing animals from orthotopically established tumors and engendered protective immunological memory in the cured animals. In this study we have compared the effectiveness of several cytokines and found that while IL-1 alpha, IL-1 beta, and gamma-interferon were only weakly effective in the therapeutic vaccination protocol, granulocyte-macrophage colony-stimulating factor was almost as effective as but not superior to IL-2, as reported previously for another tumor model system. Induction of cytotoxic T-lymphocyte correlated only poorly with the therapeutic benefit of the cytokine gene-modified tumor cell preparations, questioning its prognostic value for the development of improved genetically modified tumor vaccines.