Evaluating Tumor-Specific IFN-γ Immunity

To assess tumor-specific IFN-γ-producing immune cell populations, IFN-γ ELISPOT assays were performed. Six days following last Ad injection, spleens were harvested aseptically from mice, and unicellular splenocytes prepared as previously described [19 (link)]. Prepared spleen cells (2.5 × 10⁵) were co-cultured with pre-irradiated 4T1 (5.0 × 10³ cells; 5 Gy) for 24 hr in the presence of recombinant mouse IL-2 (100 units/mL). IFN-γ ELISPOT assays were performed according to the manufacturer's specifications for IFN-γ ELISPOT kits (BD Biosciences). Colored spots were quantitatively analyzed by a computer-based immunospot system (AID Elispot Reader System, Version 3.4, Autoimmun Diagnostika GmbH, Strassberg, Germany).