Generation of antitumor response by IL-2-transduced JAWS II dendritic cells.

Antigen-loaded dendritic cells (DCs) are a promising tool for inducing a tumor-specific immune response. It seems probable that co-administration of those cells together with cytokine-transduced DCs can further increase effectiveness of the antitumor vaccine. The local production of IL-2 by genetically modified DCs may result in alteration of the unfavorable tumor environment causing immune response dysfunction. In the presented study murine DCs of an established JAWS II cell line were transduced with a retroviral vector carrying murine IL-2 gene (JAWS II/IL-2). JAWS II/IL-2 cells demonstrated slightly decreased tumor antigen (TAg) uptake capacities. However, this modification resulted in enhanced ability of the cells to migrate in vivo. The multiple injection of vaccines containing JAWS II/IL-2 cells caused MC38 tumor growth delay and prolonged mice survival. The immunological response was manifested as cytotoxic natural killer (NK) and T cell activation and tumor tissue infiltration by CD8(+) and CD4(+) cells, accompanied by increased IFN-γ production by spleen cells. These observations suggest that repeated peritumoral administration of IL-2-producing dendritic cells can inhibit tumor growth by intensification of CD8(+) and CD4(+) cells' influx into tumor tissue and further activation of the systemic antitumor response. It can be concluded that IL-2 transduced dendritic cells may be used as a potent adjuvant in antitumor immunotherapy.

Tumour antigen-loaded mouse dendritic cells maturing in the presence of inflammatory cytokines are potent activators of immune response in vitro but not in vivo.

The use of dendritic cells (DCs) loaded with tumour antigen is one of the most promising approaches to induce tumour-specific immune response. However, methods of the vaccine preparation have not yet been standardized. The purpose of the study was to analyse the anti-tumour efficacy of tumour antigen-loaded mouse bone marrow-derived dendritic cells (BM-DC/TAg) at different maturation stages. BM-DCs were loaded with MC38 colon carcinoma cell lysate (TAg) alone, to become partially differentiated, or were additionally stimulated with inflammatory cytokines such as TNF-alpha, IFN-gamma, or IL-12 to reach complete maturity. BM-DCs simultaneously stimulated with TAg and cytokines (especially IL-12 or IFN-gamma+IL-12) were in vitro more effective immune response activators than BM-DC/TAg cells. However, the highest anti-tumour effect in vivo was noted when mice were treated just with BM-DC/TAg. In a further study, the ability of IL-12 gene transduced BM-DCs (BM-DC/IL-12) to augment the immune response induced by BM-DC/TAg cells at different stages of maturation was examined. The highest anti-tumour effect was observed when partially differentiated BM-DC/TAg cells were injected simultaneously with BM-DC/IL-12 cells. The results suggest that partially differentiated BM-DC/TAg cells are more potent in evoking a strong anti-tumour response in vivo than mature BM-DCs. Moreover, the capacity of BM-DC/TAg cells for further differentiation and their sensitivity to factors secreted in vivo by the host or cells engineered to cytokine production seem to be of great importance.

Generation of anti-tumor response by JAWS II mouse dendritic cells transduced with murine interleukin 12 genes.

Murine dendritic cells (DCs) of the established JAWS II cell line were transduced with a retroviral vector carrying murine interleukin 12 (IL-12) genes (JAWS II/IL-12 cells). The JAWS II/IL-12 cells produced approximately 9-18 ng IL-12 protein/ml/5 x 10(5) cells/48 h and displayed an increased CD80 and CD86 expression as well as major histocompatibility complex antigen up-regulation. The JAWS II/IL-12 cells were used as a temporary source of IL-12 for the immunotherapy of C57BL/6 mice bearing transplantable murine colon carcinoma (MC38). The cell vaccines were administered according to different application schedules into the vicinity of subcutaneously growing palpable MC38 tumors. The JAWS II/IL-12 cells were delivered alone or in combination with JAWS II cells pulsed with MC38 tumor cell lysate (TAg) (JAWS II/TAg cells). The anti-tumor response was estimated as the tumor growth delay--the time (days) required for the tumor to reach a volume of 1 cm3 (TRV), and as the increase in animal life-span (ILS). Mice treated with three consecutive injections of JAWS II/IL-12 or JAWS II/TAg cells responded with moderate tumor growth delay (up to 6.5 and 9.5 days, respectively). After the administration of the JAWS II/IL-12 and JAWS II/TAg cell combination, the TRV was prolonged up to 12.5 days and there was a long-lasting tumor growth delay. Increasing the number of DC-based vaccines to four, resulted in the ILS extension of up to 87% over the control. A similar effect was observed when the vaccine containing the combination of both DC components was delivered prior to the three consecutive injections of JAWS II/IL-12 or JAWS II/TAg cells administered independently. The JAWS II/IL-12 cell vaccination of MC38 tumor-bearing mice was accompanied by an increased percentage of IFN-gamma-producing CD8+ spleen cells. Concluding, JAWS II DCs transduced with IL-12 genes could be used as an adjuvant vaccine for immuno- as well as combined immuno-chemotherapy of experimental tumors.

Tissue localization of tumor antigen-loaded mouse dendritic cells applied as an anti-tumor vaccine and their influence on immune response.

The recognition, internalization and intracellular processing of antigen are the main functions of dendritic cells (DCs). In the course of these processes, DCs differentiate and acquire the ability to produce cytokines responsible for polarization of the immunological response. Therefore, vaccination with tumor antigen-loaded DCs is one of the most promising approaches to induce tumor-specific immune response. The purpose of this study was to analyze the migratory abilities, from an injection site to tumor-draining lymph nodes (tLN), of DCs applied as an anti-tumor vaccine and their capacity for immune response activation. Mouse DCs of the established JAWS II cell line transduced with EGFP gene or ex vivo bone marrow-isolated DCs (BM-DCs) stained with intravital CFDA dye were loaded with MC38 colon carcinoma tumor lysate (TAg) and then administered peritumorally to MC38 tumor-bearing C57BL/6 mice. On the first, third, fifth and seventh days after injection the tumors, tLNs and spleens were examined. The TAg-loaded DCs migrated more effectively to the tLNs than did the unloaded control DCs; however, the majority of them remained in the tumor vicinity. Immunohistological analysis of the tumor tissues demonstrated that only TAg-loaded DCs activated an immune response. Seven days after DCs vaccine administration, numerous necrotic areas and some apoptotic bodies were observed in the tumor tissue. However, the anti-MC38 tumor cytotoxic activity of spleen and tLN cells from mice treated with both TAg-loaded and unloaded DCs reached a maximum on the fifth day after DC injection. Concluding, TAg-loaded DCs migrated more efficiently to tLNs and were more effective activators of local (but not systemic) cellular immune response than were unloaded DCs. We hypothesize that only the application of TAg-loaded DCs to tumor-bearing mice as an adjuvant supporting chemotherapy may activate a more effective anti-tumor response.

Optimization of a retroviral vector for transduction of human CD34 positive cells.

Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37 degrees C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.

[Do dendritic cells modified with retroviral vectors carrying cytokine genes become a therapeutic tool?]. / Czy komórki dendrytyczne modyfikowane wektorami retrowirusowymi z genami cytokin stana sie narzedziem terapeutycznym?

For many years, the application of various types of immunostimulators (among which cytokines have proved to play a crucial role) has been one of the ways to enhance anti-tumor immunity. Dendritic cells genetically modified towards cytokine production have been proposed as a potential therapeutic tool to enable restoration of the cytokine environment diminished by a growing tumor. Retroviral vectors have been the most often used carriers of cytokine genes. Therefore, optimization of the genetic procedure of inserting retroviral vectors into DCs has been widely studied largely in order to achieve better immunotherapeutic effects against tumor.