The TEL-AML1 peptide is not naturally processed. (A) Schematic representation of the 2 MP71 constructs. EGFP, ubiquitin (Ub) and either TEL-AML1 minigene or Melan-A/MART-1 constitute a single ORF. Site of cleavage by Ub-specific proteases is indicated by arrow. LTR indicates long terminal repeat; PRE, woodchuck hepatitis virus posttranscriptional regulatory element; and s, spacer peptide. (B) Levels of expression of TEL-AML1 and Melan-A/MART-1, measured by EGFP expression (left), and of HHD, measured after staining with HLA-A*0201 specific antibody (right) in the transduced cell lines. (C) ABabDII mice were immunized either with TEL-AML1-9V (top row) or Melan-A/MART-1 peptide (bottom row) as described in Figure 1 and boosted after 14 days. Ten days after the boost spleen and LNs were isolated, pooled and cocultured overnight either with the NIH-HHD cells loaded with the corresponding peptide, or with NIH-HHD endogenously expressing TEL-AML1 fusion region (NIH-HHD-TEL-AML1) or Melan-A/MART-1 (NIH-HHD-Melan-A/MART-1). In addition, they were cultured with NIH-HHD cells only. On the next day, the cells were stained for CD8, as well as for CD3 and IFN-γ intracellularly, and analyzed by flow cytometry. Events shown are gated on CD3⁺CD8⁺ lymphocytes; numbers indicate percentages of cells in the respective quadrants. In each case one representative of 3 analyzed mice is shown.