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.
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.
Bernardeau
Karine
K
INSERM U601, Institut de Biologie, and Université Nantes, Nantes, France.
Gouard
Sébastien
S
David
Gaëlle
G
Ruellan
Anne-Lise
AL
Devys
Anne
A
Barbet
Jacques
J
Bonneville
Marc
M
Chérel
Michel
M
Davodeau
François
F
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex / Q000276:immunology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D016827:CD8 Antigens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology*; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D016827:CD8 Antigens; D015789:HLA-A2 Antigen; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins; D011948:Receptors, Antigen, T-Cell