T-cell dependence of TCR-plasmid–induced tumor protection. (A) Plasmid-immunized mice (1 μg TCR-β + 6 ng β-gal/shot) received anti-CD4 (□) or anti-CD8 (○) depleting antibodies 2 days before subcutaneous tumor challenge with 10⁵ MBL-2 cells. Shown are average tumor sizes (n = 8 mice/group) with SE. (B) Survival of TCR-β plus β-gal–immunized mice challenged with MBL-2 tumor cells after (“acute”) depletion of effector CD4 or CD8 cells. (C) To examine the requirement for codelivery of tumor-antigen plasmid and helper plasmid, mice (n = 8/group) were immunized with gold particles carrying both plasmids (), with a mixture of gold particles separately coated with the 2 plasmids () or by delivering the 2 plasmids to 2 separate sites on the mice (). (D) Tumor challenge resulted in epitope spreading. Splenocytes from TCR-β–plasmid immunized challenge survivors (ie, tumor free at least 3 weeks after challenge with MBL-2 cells subcutaneously) or splenocytes from pcDNA-immunized but not tumor-challenged mice were tested in ELIspot assays for their antigen specificity. Target cells (MC-38) were pulsed with gag-peptide, or a gag-negative, TCR-mismatched T-cell lymphoma (C6VL) was used to detect challenge-induced gag-specific T cells. Shown are IFN-γ–ELIspot results from individual mice (average of triplicate wells).