Mechanism of the Ad-sig-E7/ecdCD40L vector-induced suppression of the growth of E7-positive TC-1 tumor cells in C57BL/6 mice. (A) Resistance to the subcutaneous growth of 5 × 10⁵ E7-positive TC-1 cancer cells in mice after 2 injections with 1 × 10⁸ pfu of the Ad-sig-E7/ecdCD40L vector 7 days apart. (▪) Ad-sig-E7/ecdCD40L. (□) Ad-sig-ecdCD40L. (▴) Ad-E7/wtCD40L. () Ad-wtCD40L. (B) Survival of mice vaccinated with Ad-sig-E7/ecdCD40L vectors. The following vectors were injected into C57BL/6 mice, after which the E7-positive TC-1 cancer cells were injected into the subcutaneous spaces of the mice: bold continuous line, mice treated with 2 subcutaneous injections 7 days apart of 1 × 10⁸ pfu of the Ad-sig-E7/ecdCD40L vector; thin continuous line, mice treated with subcutaneous injections of the Ad-wtCD40L vector; broken thin line, control mice, which were not treated with vector injections. (C) Comparison of the effects of 2 subcutaneous injections of 1 × 10⁸ pfu of the Ad-sig-E7/ecdCD40L vector on the in vivo growth of the E7-positive TC-1 cells (♦) and the E7-negative EL-4 cell line (▦). Sizes of the subcutaneous tumors were estimated by measuring with calipers in 2 separate orthogonal directions and then calculating the volume assuming an elliptical shape. (D) Use of tetramers to measure the level of E7-specific CD8⁺ T cells in the spleens of Ad-sig-E7/ecd/CD40L vector-immunized C57BL/6 mice. Spleen cells were harvested 10 days after the completion of 2 subcutaneous injections 7 days apart with 1 × 10⁸ pfu of vectors Ad-sig-E7/ecdCD40L, Ad-E7/wtCD40L, Ad-wtCD40L, and Ad-sig-ecdCD40L. T cells were then analyzed for the percentage of E749-57 peptide-specific CD8⁺ T-cell lymphocytes by H-2D^b tetramer staining. (E) ELISPOT assay shows increase in the level of IFN-α-secreting cells in the spleen cells of mice injected subcutaneously twice (7 days apart) with 1 × 10⁸ pfu Ad-sig-E7/ecdCD40 vector. Mice were injected twice with the following vectors: Ad-sig-E7/ecdCD40L, Ad-sig-ecdCD40L, Ad-E7/wtCD40L, and Ad-wtCD40L. Splenic T cells taken from the mice 1 week later were analyzed by ELISPOT assay for the presence of IFN-γ. (F) Increase in the level of E7-specific CTLs in the spleens of Ad-sig-e7/ecdCD40L-injected mice. Mice were injected subcutaneously twice (7 days apart) with 1 × 10⁸ pfu of vectors Ad-sig-E7/ecdCD40L, Ad-E7/wtCD40L, Ad-sig-ecdCD40L, Ad-wtCD40L, and control (no vector injection). T cells were harvested from the spleens of the test mice 1 week after the second adenoviral vector injection and were restimulated in vitro with TC-1. After 7 days, restimulated effector cells (spleen cells exposed to TC-1 cells in vitro) were mixed at varying ratios with TC-1 (E7-positive) and EL-4 (E7-negative) target cells. Then the LDH released from the target cells was measured. No LDH was detectable from any of the mixtures of EL-4 and the restimulated effector cells isolated from the vaccinated mice, whereas significant levels of LDH were released from the TC-1 target cells when they were mixed with the restimulated effector cells isolated from the mice vaccinated with the Ad-sig-E7/ecdCD40L vector.