Assessment of CD8 involvement in T cell clone avidity by direct measurement of HLA-A2/Mage3 complex density using a high-affinity TCR like monoclonal antibody

Eur J Immunol. 2005 Oct;35(10):2864-75. doi: 10.1002/eji.200526307.


Peptide affinity for MHC molecules determines the number of MHC/peptide complexes stabilized at the cell surface in in vitro tests or in vaccination protocols. We isolated a high affinity monoclonal antibody specific for the HLA-A2/Mage3 complex that enables an equilibrium binding assay to be performed on T2 cell line loaded with a range of Mage3 peptides. Binding of Mage3 to the HLA-A2 molecule can be modeled by a standard receptor-ligand interaction characterized by an affinity constant. This model enables the measurement of the affinity of other immunogenic peptides for HLA-A2 by a competition test and the calculation of the density of complexes stabilized at the T2 cell surface for all peptide concentrations. Quantification of the HLA-A2/Mage3 complexes at target cell surfaces was used to estimate the number of complexes required to reach cytotoxicity ED50 of human T cell clones sorted from an unprimed repertoire. We confirm with this antibody the direct relationship between clone avidity and TCR affinity, and the moderate contribution of the CD8 co-receptor in the reinforcement of TCR-MHC/peptide contact. Nevertheless, CD8 plays a critical role in the amplification of the specific signal to establish an efficient T cell response at low specific complex densities found in physiological situations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / immunology
  • Antibody Affinity
  • Antigen-Antibody Complex / immunology
  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / immunology*
  • CD8 Antigens / immunology*
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • HLA-A2 Antigen / immunology*
  • Humans
  • Lymphocyte Activation / immunology*
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / immunology*
  • Protein Binding
  • Receptors, Antigen, T-Cell / immunology
  • T-Lymphocytes / immunology*


  • Antibodies, Monoclonal
  • Antigen-Antibody Complex
  • Antigens, Neoplasm
  • CD8 Antigens
  • HLA-A2 Antigen
  • MAGEA3 protein, human
  • Neoplasm Proteins
  • Receptors, Antigen, T-Cell