575. Dendritic Cells Modified Using an Adenovirus Expressing Murine IL-12 Inhibits Development of Tumors in a Mouse Colon Cancer Model
Authors: Yoshio Sakai, Brian J. Morrison, John E. Janik, John C. Morris
Journal: Molecular Therapy (2004)

DCs are specialized antigen presenting cells that generate immune responses by presenting antigen epitopes in the context of MHC. DCs also secrete cytokines that direct the immune response. Interleukin-12 (IL-12) is important for directing immune system toward a cytolytic T cell response. We studied the effect of vaccination with DCs modified to express the murine IL-12 gene using a recombinant adenovirus. DCs were generated from bone marrow of C57Bl/6 mice in media supplemented with GM-CSF. Recombinant adenoviral vectors, Ad.mIL-12 expressing the p35/p40 subunits of murine IL-12 and a control vector, Ad.null, were generated in 293 cells. DCs were infected with Ad.mIL-12 at various MOIs and IL-12 secretion was measured by ELISA. Splenocytes (Spc) from naive C57Bl/6 mice were mixed with irradiated DCs infected with Ad.mIL-12 (DCAd.mIL-12) or Ad.null (DCAd.null) and in the presence or absence of anti-CD4 or anti-CD8 antibodies. Spc proliferation was assessed by [3H]-thymidine incorporation and IFN-γ production was measured. Groups of C57Bl/6 or IFN-γ −/− knockout C57Bl/6 mice were injected weekly with 1×106 DCAd.mIL-12, DCAd.null or with unmodified DCs for 2 weeks. A week after the last vaccination, mice were injected with 1×105 MC38.KRas murine colon carcinoma cells expressing mutant human K-Ras (G12D). Some groups of DCAd.mIL-12 treated mice received injections of anti-CD4 or anti-CD8 antibodies beginning either two days before initiation of vaccine or before tumor challenge. Tumor and Spc were obtained from each group of mice 21 days after injection of tumor. Tissues were examined for CD4 and CD8 cells, and for IFN-γ expression by in situ hybridization. Spc were co-cultured with irradiated MC38.KRas for 5 days and IFN-γ and IL-4 concentrations were measured by ELISA. After infection with Ad.mIL-12 (MOI = 10 and 30), mIL-12 p70 1628 and 3826 pg/mL/105 cells was measured, respectively. Spc stimulated with DCAd.mIL-12 had 2-fold greater [3H]-thymidine incorporation than controls. Anti-CD4 antibody, but not anti-CD8-antibody abolished this stimulation. DCAd.mIL-12 co-cultured with Spc produced 10-fold higher concentrations of IFN-γ than Spc stimulated with 300 ng of recombinant murine IL-12. When mice were challenged with MC38.KRas cells, inhibition of tumor growth was seen in the DCAd.mIL-12 vaccinated group but not the controls. Sixty percent of DCAd.mIL-12 treated mice were free of tumor at 77 days, while less than 20% of mice in the control groups tumor-free. Infiltration of tumors by CD4+ cells and expression of IFN-γ was seen in DCAd.mIL-12 treated mice. When Spc from mice treated with DCAd.mIL-12, DCAd.null or unmodified DC were mixed with MC38.KRas in vitro, supernatant contained 120, 35, 0.7 ng/ml of IFN-γ and 17.3, 8.9, 42.3 pg/ml of IL-4, respectively. Protection by DCAd.mIL-12 was abolished by depletion of CD4+ cells, but not by depletion of CD8+ cells. DCAd.mIL-12 vaccination was ineffective at inhibiting tumor growth in IFN-γ −/− knockout mice. [Conclusion] DCs modified with Ad.mIL-12 successfully protected C57Bl/6 mice from challenge with MC38KRas tumor cells. CD4+ T cells and secretion of IFN-γ appear to be important for the effectiveness of DCAd.mIL-12 vaccination.