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#34-OR
THE IMPACT OF PEPTIDES ON THE RECOGNITION OF HLA CLASS I MOLECULES BY HUMAN HLA ANTIBODIES.
Arend Mulder PhD , Chantal Eijsink MT , Michel Kester MT , Marrie J. Kardol, MT , Marry E.I. Franke-van Dijk, MT , Jan Wouter Drijfhout PhD , Frits Koning PhD , Ilias I.N. Doxiadis, PhD and Frans H.J. Claas, PhD . Leiden Netherlands, Leiden University Medical Center, 2300RC, Immunohaematology and Bloodtransfusion and Leiden Netherlands, Leiden University Medical Center, 2300RC, Experimental Haematology .
MHC class I molecules expressed on cell surfaces are composed of heavy chain, β2-microglobulin and a vast array of peptides that are the products of processing of endogenous proteins. Lacking evidence to the contrary, the assumption is generally made that serum HLA antibodies recognize HLA-class I molecules regardless of the nature of the peptide bound. We generated a panel of 11 human monoclonal HLA-antibodies (HumAbs) that contain HLA-A2 in their CDC defined specificity pattern, but recognize different epitopes on HLA-A2. Flow cytometry (FC) on an HLA-A2 transfected K562 cells revealed differential binding within the panel of HumAbs. As differences in signal intensity did not correlate with the epitope recognized nor with the HumAb isotype, we suspected that some HumAbs recognize subsets of HLA-A2 molecules. To substantiate this, we created a series (n=12) of recombinant monomeric HLA-A2 molecules, each containing a different peptide, and developed ELISAs in which these monomers constituted the ligands for HumAb binding. Here, HumAbs again showed different binding characteristics, ranging from reactivity to one or to all of the HLA-A2 monomers. Intermediate FC staining correlated with a restricted pattern of monomer binding. Subjecting one of the peptides to ala scanning, and incorporating these mutant peptides into monomers, we further probed the influence of individual amino acid positions on HumAb binding. Thus, while it is known that peptides are necessary for synthesis and the proper conformation of the HLA class I molecule, they play decisive roles in shaping serological epitopes that may or may not be recognized by human HLA antibodies.