Cytotoxic T-cell response in DNA immunized mice detected by IFN-γ-ELISPOT assay. Cellular immune responses after four DNA vaccinations are shown. Six groups of mice (5 per group) were immunized with HPV16 L1 DNA on days 0, 14, 28 and 98 either by tattoo or intramuscular delivery without any adjuvant, in combination with prior application of cardiotoxin 5 days before the first immunization or in mixture with mouse GM-CSF DNA (1:1). A control group of three mice was tattooed with mouse GM-CSF DNA. Splenocytes were isolated 9 days after the last DNA immunization and examined in 4 serial dilutions in the IFN-γ-ELISPOT assay. The representative numbers of spots reflecting IFN-γ-producing cells per 250,000 splenocytes are shown. Splenocytes were stimulated non-specifically with mitogen or specifically with the L1 peptide (aa 165–173). Non-stimulated splenocytes were used as negative controls.