DNA vaccination induces WT1-specific IFN-γ–secreting T cells in HHD mice. HHD mice were vaccinated with (A) p.DOM-WT1.37 (n = 7), (B) p.DOM-WT1.126 (n = 8), (C) p.DOM-WT1.235 (n = 12), or (D) p.DOM (n = 20) and were boosted 28 days later with the same vaccine delivered using in vivo electroporation. On day 36, splenic lymphocytes were harvested by density centrifugation of spleen cells and the frequency of specific T cells assessed by IFN-γ ELISpot following a brief incubation alone (no pep), with an irrelevant peptide (irr pep; 10−6 M) with p30 peptide (10−6 M) or with the relevant WT1 peptide (10−6 M or 10−8 M). Data are expressed as the number of spot-forming cells (SFCs) per million lymphocytes and are a combination of 2 experiments with similar results; group means are represented by a horizontal bar. Responses were considered significant if the frequency of IFN-γ–secreting cells was more than double the frequency detected in wells without peptide. (E) Lymphocytes from mice vaccinated with p.DOM-WT1.37 (n = 4) or p.DOM-WT1.126 (n = 9) were incubated with a range of peptide concentrations and the frequency of specific cells assessed by ELISpot analysis as before. Data are shown as the mean percentage of the maximum obtained for each mouse with the SEM indicated. A nonlinear line of best fit was plotted using GraphPad Prism 4 software. (F) HHD mice were injected with DNA vaccine (p.DOM-WT1.126; DNA) or with peptide vaccine (WT1.126 peptide in IFA mixed with PADRE peptide: peptide). Lymphocytes were harvested on day 10 and the frequency of vaccine-specific T cells assessed by IFN-γ ELISpot following a brief incubation alone (no pep), with the Th peptides p30 or PADRE (10−6 M) or with the WT1.126 peptide (10−6 M). Group means are represented by a horizontal bar.
