IFN- γ ELISPOT analysis . (A) IFN-γ- stimulated D5lacZ tumor cells were used as stimulators in ELISPOT assays. Up-regulation of MHC class I (H2-kb) and class II (I-Ab) was confirmed by FACS. Isotypic controls (solid lines), MHC expression in the absence of IFN-γ (filled grey histograms), and MHC expression of D5lacZ tumor cells that had been treated for 72 hours with IFN-γ at 1,000 U/ml of media (dotted lines) are presented in our FACS analyses. (B) Frequencies of IFN-γ-secreting CD4+ and CD8+ T-effector cells in response to D5lacZ tumor stimulation. CD4+ and CD8+ T cells were isolated from spleens that were harvested from naïve mice (naïve), mice treated with control mAb and DC/D5lacZ fusion cells (Y13+FC), mice treated with PC61 mAb and fusion cells (PC61+FC), and mice treated with systemic IL-12 and fusion cells (IL-12+FC). IFN-γ secreting cells were enumerated without stimulation (−) or after stimulation with D5lacZ cells (stimulators). 2.5×105 T cells (responders) per well were stimulated at a responder to stimulator ratio of 5:1. ELISPOT assays were performed in triplicates and averaged data of 3 independent experiments are shown.