The only proposed T-cell epitope derived from the TEL-AML1 translocation is not naturally processed by Jelena Popović, Liang-Ping Li, Peter Michael Kloetzel, Matthias Leisegang, Wolfgang Uckert, and Thomas Blankenstein Blood Volume 118(4):946-954 July 28, 2011 ©2011 by American Society of Hematology

TEL-AML1 cannot prime CD8+ T cells unless an anchor modification is introduced, and exhibits very low HLA-A*0201 binding affinity. 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. Jelena Popović et al. Blood 2011;118:946-954 ©2011 by American Society of Hematology

The anchor-modification is needed to induce effector CTLs capable of degranulation. The anchor-modification is needed to induce effector CTLs capable of degranulation. ABabDII mice were immunized subcutaneously with (A) the native peptide TEL-AML1 or (B) the anchor-modified peptide TEL-AML1-9V, mixed with CpG oligodeoxynucleotides, PBS and IFA. They were boosted, and splenocytes and draining LNs were isolated 10 days later. The cells were in vitro restimulated for 6 hours with 10−6M specific peptides (TEL-AML1 or TEL-AML1-9V), with an irrelevant peptide (NY-BR-1), or with anti-CD3/CD28 antibodies, as indicated. Prestaining for CD107a was performed by including anti-CD107a antibodies during the whole period of stimulation, with the addition of monensin. The cells were subsequently stained for CD3 and CD8 and analyzed by flow cytometry. Events shown are gated on CD3+CD8+ lymphocytes; numbers indicate percentages of cells in the respective quadrants. One representative of 5 (A) and 1 of 6 (B) mice are shown, from 2 independent experiments with similar results. Jelena Popović et al. Blood 2011;118:946-954 ©2011 by American Society of Hematology

Elongated peptides comprising the TEL-AML1 epitope cannot evoke CD8+ T-cell responses. Elongated peptides comprising the TEL-AML1 epitope cannot evoke CD8+ T-cell responses. ABabDII mice were immunized subcutaneously in the tail base with 30-mer peptides, comprising either (A) the native TEL-AML1 sequence, (B) the anchor-modified TEL-AML1-9V sequence, or (C) the anchor-modified gp100, mixed with CpG oligodeoxynucleotides, PBS, and IFA. In the cases of the TEL-AML1 native and anchor-modified peptide, some of the mice were additionally boosted after 2 weeks with the corresponding peptides. Spleen and draining LNs were isolated 10 days after the immunization, or boosting. The cells were in vitro restimulated overnight either with 10−6M corresponding nonamer peptide (TEL-AML1, TEL-AML1-9V, or gp100), an irrelevant peptide (NY-BR-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. In the case of gp100 one representative of 3 analyzed mice is shown, and in the cases of the TEL-AML1 anchor-modified, as well as the native peptide, 1 representative of 6 analyzed mice is shown. Jelena Popović et al. Blood 2011;118:946-954 ©2011 by American Society of Hematology

The TEL-AML1 peptide is not naturally processed. 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. Jelena Popović et al. Blood 2011;118:946-954 ©2011 by American Society of Hematology

In vitro digestion of synthetic peptides with human proteasomes. In vitro digestion of synthetic peptides with human proteasomes. (A) Digestion of the peptide TEL-AML1-319. Predominant cleavage products and cleavage sites are shown. Synthetic peptides were incubated until 50% of the peptide substrate was turned over by purified LCL (lymphoblastoid cell lines) 20S proteasomes, which mainly expresses immunoproteasomes. Peptide fragments are detected by LC-MS/MS (reversed phase liquid chromatography coupled online with tandem mass spectrometry) using a normal triple method. Arrows of various thickness are proportional to the relative cleavage intensity. Note the large number of destructive cleavages within the epitope (in red) and the complete absence of a potentially functional N-terminal cleavage. (B) Generation of the LLO-296-304 CD8+ T-cell epitope by digestion of synthetic polypeptide LLO-291 derived from bacterial listeriolysin O. The experiment was performed as described in panel A. The dominant relevant cleavage products and the MHC class I ligand Val296-Leu304 (in red) as well as the N-terminally elongated epitope precursor peptide thereof (in green) are shown. Arrows indicate major and minor cleavage sites. Note the strong cleavage behind the C-terminal Leu304 residue, resulting in the predominant generation of the epitope. Cleavage within the epitope does not effect the overall predominance of Val296-Leu304 peptide generation. Numbers designate amino acid residue positions in the corresponding proteins (TEL-AML1 and listeriolysin O). Jelena Popović et al. Blood 2011;118:946-954 ©2011 by American Society of Hematology