Combined cardiotoxin and DNA vaccine elicited potent, T cell–dependent, tumor antigen–specific immunity. (A) Splenocytes pooled from 5 BALB/c mice were injected intramuscularly with MCP3-sFv plasmid DNA with or without cardiotoxin as in Figure 1, or cardiotoxin alone. Ten days earlier they were stimulated in vitro with bone marrow–derived DCs pulsed with 5 μg/mL MHC class I–binding A20 idiotype epitope peptide²³  for 5 days. The stimulated cells (2 × 10⁵/well) were plated with either the peptide or irradiated A20 tumor cells at a 5:1 T cell/stimulator ratio,²⁴  respectively, and analyzed for INFγ production by ELISPOT after 48 hours. Differences between groups were analyzed using the Student t test (**P < .01; *P < .05). The error bars represent SEM. (B-D) In vivo T-cell depletion was achieved by intraperitoneal injection of 200 μg anti-CD8 mAb (clone 2.43 alone, CD8 depletion) or with 200 μg anti-CD4 mAb (clone GK1.5, CD4/CD8 depletion) according to the schedule in panel D. T-cell depletion was performed on mice vaccinated with cardiotoxin plus MCP3-sFv DNA vaccine as in Figure 1 (10 BALB/c mice per group). Controls received rat IgG instead of T-cell depletion antibodies. After confirming the efficiency of T-cell depletion, which was determined by the presence of CD8 and CD4 T cells in the peripheral blood samples (C), all the mice were challenged with 2 × 10⁵ A20 tumor cells as shown in Figure 1 and followed for survival for 80 days. The data represent 2 independent experiments.