title: Proteosomal degradation of BCR/ABL protein can generate an HLA-A*0301-restricted peptide, but high-avidity T cells recognizing this leukemia-specific antigen were not demonstrated.
abstract: OBJECTIVE
  Cytotoxic T-lymphocytes (CTL) have been generated in vitro against chronic myeloid leukemia (CML)-associated BCR/ABL-specific peptides. We analyzed the existence of high-avidity T cells recognizing endogenously processed BCR/ABL-specific proteins.
  
  
  METHODS
  We performed binding studies of BCR/ABL-specific peptides, proteosomal digestion of BCR/ABL breakpoint overlapping protein, mass spectrometry of eluates from HLA-*0301-transduced K562 cells, and tried to isolate peptide-specific T-cells using tetramers.
  
  
  RESULTS
  We confirmed the binding of the BCR/ABL-specific peptides KQSSKALQR to HLA-A*0301 and GFKQSSKAL to HLA-B*0801. Proteasomal digestion showed cleavage sites leading to KQSSKALQR but not to GFKQSSKAL. Using mass spectrometry KQSSKALQR could not be detected in the eluates from HLA-A*0301-transduced K562 cells. We attempted to induce BCR/ABL-specific CTL lines from 4 healthy donors using dendritic cells pulsed with KQSSKALQR and performed single cell sorting to isolate tetramer-positive T cells. None of 31 generated clones showed BCR/ABL-specific cytotoxicity. Isolation of tetramer-positive cells from peripheral blood of relapsed CML patients after allogeneic transplantation treated with donor lymphocyte infusion resulted in 38 T-cell clones which did not show peptide-specific cytotoxicity.
  
  
  CONCLUSIONS
  We provide evidence that BCR/ABL protein processing can lead to KQSSKALQR peptide binding to HLA-A*0301. However, KQSSKALQR could not be detected in HLA-A*0301-transduced K562 cells, and KQSSKALQR could not be demonstrated to induce high-avidity BCR/ABL-specific CTL.
authors.affiliation: Dept. of Hematology, Leiden, University Medical Center, PO Box 9600, 2300 RC, The Netherlands. posthuma@rdgg.nl
authors.forename: Eduardus F M
authors.initials: EF
authors.lastname: Posthuma
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: van Bergen
authors.forename: Michel G D
authors.initials: MG
authors.lastname: Kester
authors.forename: Roelof A
authors.initials: RA
authors.lastname: de Paus
authors.forename: Peter A
authors.initials: PA
authors.lastname: van Veelen
authors.forename: Arnoud H
authors.initials: AH
authors.lastname: de Ru
authors.forename: Jan-Wouter
authors.initials: JW
authors.lastname: Drijfhout
authors.forename: Ellie G A
authors.initials: EG
authors.lastname: Lurvink
authors.forename: Roel
authors.initials: R
authors.lastname: Willemze
authors.forename: J H Frederik
authors.initials: JH
authors.lastname: Falkenburg
mesh_terms: D000595:Amino Acid Sequence; D017951:Antigen Presentation; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D002999:Clone Cells / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology; D016044:Fusion Proteins, bcr-abl / Q000276:immunology* / Q000378:metabolism; D015234:HLA-A Antigens / Q000276:immunology*; D015791:HLA-A3 Antigen; D015235:HLA-B Antigens / Q000276:immunology*; D015795:HLA-B8 Antigen; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000276:immunology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D037182:T-Cell Antigen Receptor Specificity
chemical_list: D000951:Antigens, Neoplasm; D015234:HLA-A Antigens; C442141:HLA-A*03:01 antigen; D015791:HLA-A3 Antigen; D015235:HLA-B Antigens; C509422:HLA-B*08:01 antigen; D015795:HLA-B8 Antigen; D010446:Peptide Fragments; D016044:Fusion Proteins, bcr-abl; D046988:Proteasome Endopeptidase Complex