TEL-AML1 cannot prime CD8+ T cells unless an anchor modification is introduced, and exhibits very low HLA-A*0201 binding affinity. (A) ABabDII mice were immunized subcutaneously with the native TEL-AML1 peptide (top row) or the anchor-modified TEL-AML1-9V (bottom row), mixed with CpG oligodeoxynucleotides, PBS and IFA. They were boosted twice, and splenocytes and draining LNs were isolated 10-14 days later. The cells were in vitro restimulated overnight with 10−6M corresponding peptide (TEL-AML1 or TEL-AML1-9V), with an irrelevant peptide - anchor-modified Melan-A/MART-1, or with anti-CD3/CD28 antibodies. 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 (IFN-γ+ cells residing in the upper right). For the native peptide, 1 representative of 5 analyzed mice is shown; for the anchor-modified, 1 of 3 is shown. (B) ABabDII mice were immunized with the anchor-modified TEL-AML1-9V, and pooled spleen and LN cells were restimulated either with the anchor-modified, the native, the irrelevant peptide or anti-CD3/CD28 antibodies as described in panel A. One representative of 3 mice is shown. (C) T2 cell assay. Cells were either coated with TEL-AML1, TEL-AML1-9V or Melan-A/MART-1 at the final concentration 10−5M, or left uncoated. On the next day, they were stained for HLA-A*0201 and analyzed by flow cytometry. The graph shows changes in MFI during 6 hours and FI of each peptide. One of 2 experiments with similar results is shown.
