IFN-gamma MODULATES TUMOR-SPECIFIC IMMUNE RESPONSE INDUCED EITHER BY GENETICALLY MODIFIED TUMOR CELLS OR DENDRITIC CELLS LOADED WITH POST-APOPTOTIC TUMORS IN A MOUSE MODEL OF PROSTATE CANCER

The mouse prostatic adenocarcinoma tumorigenic cell line TRAMP-C2 represents a suitable animal model to study the role of Major Histocompatibility Class-I (MHC-I) molecules expression in protection against tumor development and progression in vivo. In this cell line, MHC-I expression decreases after time of in vitro cell culture, but it can be restored by treatment with IFN-g. We have transfected TRAMP-C2 cells with the cDNA of the co-stimulatory molecule B7-1. TRAMP-C2/B7 transfectants showed impaired growth in vivo, but they did not elicit a protective response against TRAMP-C2 parental tumor, unless after treatment with IFN-g prior to injection. IFN-g is an inducer of some components of the MHC-I antigen processing and presentation machinery (APM). IFN-g is also an antagonist of the immunosuppressant activity of TGF-b, largely produced by TRAMP-C2. Thus, immunization with TRAMP-C2/B7 conferred a cytotoxic T cell (CTL)-dependent protection against TRAMP-C2-derived tumors in function of the IFN-g-mediated fine-tuned modulation of either APM expression or TGF-b signaling. IFN-g-treatment of TRAMP-C2 cells resulted in an increased number of tumor-free animals and improvement of the overall survival, if compared with mice immunized or challenged with cells not treated with IFN-γ. We then attempted to deliver IFN-g to TRAMP-C2-tumor growth site by means of genetically engineered mesenchymal stromal cells (MSCs) secreting IFN-g. We demonstrated that MSCs/IFN-γ reached the tumor mass and, in combination with the immunization with TRAMP-C2/B7 treated with IFN-γ, the results matched those obtained with IFN-g-treated TRAMP-C2 cells, with an increase of tumor protection by the IFN-g secreted by tumor-infiltrating MSCs. In order to make these results better translatable to clinics, we explored methods of immunization using wild type (w.t.) TRAMP-C2 necrotic cells, instead of alive transfected cells, made “palatable” for phagocytosis by dendritic cells (DCs), by means of hyperthermia treatment. We tested the effects of hyperthermic treatment at 56°C, estimating the percentage of necrotic and apoptotic cells after the treatment, and we have observed that the treatment at this temperature could induce necrosis and consequent release of danger associated molecular pattern (DAMP), with high capacity to activate DCs, according to current literature. We then investigated the capacity of syngeneic DCs to phagocyte the necrotic cells: the hyperthermia treatment at 56°C induced a high percentage of phagocytosed cells by DCs, suggesting their maturation and capacity to activate a tumor-specific CTL immune response. To validate this hypothesis, we moved on with in vivo experiments: we delivered subcutaneously (s.c.) either necrotic tumor cells to mice relying on activation of resident DCs, or activated DCs loaded with necrotic tumor cells in vitro. The results obtained demonstrated that immunization with necrotic tumor cells washed after heat treatment did not protect against challenge with viable TRAMP-C2 tumor cells. On the contrary, immunizations with necrotic tumor cells unwashed after heat treatment and with activated DCs loaded with necrotic cells were both protective, but at different levels: unwashed necrotic cells protected only against TRAMP-C2 treated with IFN-g, whilst DCs loaded with necrotic cells showed partial protection (50%) also against untreated TRAMP-C2 . Activation of resident DCs at inoculation site is probably more complicated and delicate than in vitro. In addition, when injecting the suspension of necrotic tumor cells containing the released DAMPs, it is conceivable that not all the material is addressed to resident DCs. More experiments are needed to improve activation of resident DCs with injection of necrotic cells in suspension with DAMPs.