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The long-term goal of this study is to develop autologus tumor cells transduced for production of GMCSF and IL-12 as vaccines for immunotherapy of malignant melanoma and other cancers. For our study, autologus primary melanoma cell lines were established from fresh surgical specimens of patients. The primary cell lines were transduced with lentiviral vectors for production of GMCSF or IL-12. In order to learn how the transduced autologus tmor cells prime peripheral T cells of patients, we studied the effect of the transduced tumor cells (4x10e3) inactivated with 100 Gy gamma ray on production of cytokines (IFN-gamma, IL-2, IL-10, IL-4, IL-5 and TNF-alpha) and proliferation by autologus T cells (1x10e5) in the presence of monocytes (6x10e4) with or without tumor cell lysate (2x10e4) in a final volume of 200 ul media. The cytokines were measured by immunoassays at 48 and 96 hours after initiation of incubation. The irradiated autologus tumor cells (4x10e3) transduced for GMCSF or IL-12 continued to produce the respective cytokine at a rate of 5 ng/day or 1.2 ng/day. The production could last for more than 2 weeks ex vivo after gamma irradiation. The results of our study showed that tumor lysate by itself can prime autologus T cells to produce IL-10 in the presence of monocytes. The production of IL-10 at 48 hours was enhanced by 5, 15, 8 and 11 folds with presence of autologus melanoma cells without transduction, GMCSF-melanoma cells, IL-12 melanoma cells and GMCSF+IL12 melanoma cells, respectively. The amounts of IL-10 were reduced to between 15% and 30% at 96 hours. The amounts of IFN-gamma continue to increase from 48 hour to 96 hours only in the presence of autologus tumor cells producing IL-12. The presence of tumor lysate and GMCSF significantly enhanced the production of IFN-gamma. The production of IL-2 was detected after incubation for 96 hours, and was enhanced with presence of irradiated autologus tumor cells producing IL-12 and/or GMCSF. Tumor lysate had no effect on production of IL-2. Production of IL4 and IL-5 were not detectible. The assay of BrdU incorporation showed proliferation in incubations with presence of irradiated autologus tumor cells. Similar results were noted for different patients. Our findings support that autologus tumor cells producing GMCSF and IL-12 are effective for priming T cells to produce type 1 cytokines, and may be used as vaccines to induce cellular immune response. The effect of tumor lysate to stimulate the production of IL-10 could adversely affect the induction of cellular immune response and raises the question on the benefit of including tumor lysate for immunotherapy.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]