Mechanism of the suppressive effect of the Ad-sig-ecdhMUC1/ecdCD40L vector on induction of the immune suppression of the growth of the LL2/LL1hMUC1 cells in hMUC1.Tg mice. (A) Subcutaneous injection of the ecdhMUC1/ecdCD40L protein does not induce suppression of the growth of hMUC1-positive cells, which is equivalent to that seen with 2 subcutaneous injections of 1 × 10⁸ pfu Ad-sig-ecdhMUC1/ecdCD40L vector. Five hundred thousand LL2/LL1hMUC1 cells were injected subcutaneously into the hMUC1.Tg mice. Two days after injection of the tumor cells, the ecdhMUC1/ecdCD40L protein was injected subcutaneously into hMUC1.Tg mice. (○) No protein injection. (♦) Ad-sig-ecdhMUC1/ecdCD40L vector. (▴) Two injections of the ecdhMUC1/ecdCD40L protein. (▪) One injection of the ecdhMUC1/ecdCD40L protein. (B) CD4⁺-depleted T cells from hMUC1 transgenic mice after 2 subcutaneous injections of 1 × 10⁸ pfu Ad-sigecdhMUC1/ecdCD40L vector secrete increased levels of IFN-γ. CD8⁺ T cells were isolated from hMUC1.Tg mice that had been vaccinated twice with the Ad-sig-ecdhMUC1/ecdCD40L vector or with the Ad-sig-ecdhMUC-1 vector or that had been unvaccinated (labeled as control). Seven days after vaccination, CD8⁺ cells were harvested from the spleens of the test animals and were incubated for 24 hours. The supernatant medium was analyzed for IFN-γ levels. (C) Cytotoxicity of CTLs from hMUC1.Tg transgenic mice after 2 subcutaneous injections (7 days apart) of 1 × 10⁸ pfu of Ad-sig-ecdhMUC1/ecdCD40L vector against LL2/LL1-MUC1 hMUC1-positive cancer cells or against LL2/LL1 cancer cells negative for the hMUC1 antigen. CD8⁺ T-cell lymphocytes were isolated from the spleens of hMUC1.Tg mice 1 week after vaccination with the Ad-sig-ecdhMUC1/ecdCD40L vector. Cells were restimulated in vitro with the LL2/LL1hMUC1 cell line for 5 days (♦) or the LL2/LL1 cell line (▪). CD8⁺ T-cell lymphocytes were also isolated from the spleens of hMUC1.Tg mice 1 week after vaccination with the Ad-sig-ecdhMUC1 vector, which was then stimulated in vitro with the LL2/LL1hMUC1 cell line (▴). Different effector/target ratios (20:1, 10:1, and 5:1) were used. The LDH released from each of these cell mixtures (ordinate) was then measured. (D) Phosphorylation of the ERK1/ERK2 proliferation pathway in CD8 T cells from hMUC1 transgenic mice after stimulation with bone marrow-derived DCs infected with the Ad-sig-ecdhMUC1/ecdCD40L vector. CD8 T cells were isolated by CD4 depletion from the spleen cells of hMUC1.Tg mice 1 week after the completion of 2 subcutaneous injections (1 week apart) with the Ad-sig-ecdhMUC1/ecdCD40L vector (i) or from mice that were not vaccinated (ii). DCs that had been infected with the Ad-sig-ecdhMUC1/ecdCD40L vector were then mixed in a 1:1 ratio with the restimulated CD8⁺ T cells. Proteins were isolated from these mixtures 0, 5, 15, and 45 minutes later and were separated using SDS-PAGE, transferred by Western blot analysis to a filter, and analyzed for phosphorylation of the p44 and p42 mitogen-activated kinase proteins using the New England BioLabs kit for phosphorylated proteins. The blot for the vaccinated mice is shown in panel i, and the blot for the unvaccinated mice is shown in panel ii.