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 cytokin e 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 (mlL-2pMX) and mouse IL-12 (mlL-12pMX) genes. Supernatants from 293 ce lls transfected with pMX retroviral vectors were harvested and used to tran sduce mouse lin bone marrow (BM) progenitor cells. After 48 h co-culture wi th pseudotype retrovirus, BM cells were cultured for 12 days in the presenc e of mGM-CSF, mSCF and mTNF-alpha to obtain a DC-enriched fraction. Flow cy tometric 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 ma rker, DEC205. About 60% of cells sorted for DEC205 also expressed GFP. The supernatants of DC-mlL-2 and DC-mlL-12 cultured for 48 h contained 5.2 +/- 0.15 and 33.9 +/- 2.6 ng cytokine protein per milliliter, respectively. Int ratumoral injection of DC-mlL-2 or DC-mlL-12 on days 8 and 15 after the int radermal injection of 1 x 10(5) B16F10 cells, resulted in a significant red uction in tumor size by day 21, as compared with mice treated with unmodifi ed DC or DC-GFP. Longer term analysis as assessed at day 42 revealed that B 16 tumor-bearing mice treated with cytokine gene-modified DC survived signi ficantly longer than mice from other groups. Spleen cells obtained from DC- treated mice were specifically sensitized for the generation of CTL by subs equent 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.