ALLOREACTIVE MONOCLONAL-ANTIBODIES SELECT K-D MOLECULES WITH DIFFERENT PEPTIDE PROFILES

As a part of our continuing effort to study the antigenic structure of class I molecules, we have undertaken two types of studies, First, we have studied the capacity of five different K-d-reactive mAbs to reco gnize a panel of 25 site-directed mutants of the H-2K(d) molecule. Bot h the gain and the decrease in Ab binding resulted from a single amino acid substitution at different positions. All mutations that increase the binding of the tested mAbs are located on the alpha-helices, indi cating that the replacement of an Ig-contacting surface residue with a charged or polar side chain by a short one generally favors Ab bindin g. Mutation of two alpha-helix-situated residues, 58 and 166, complete ly abolished the binding of one mAb (Tu191.7.1), indicating that these two residues contribute to the antigenic determinant defined by this mAb, The overwhelming majority of mutations that diminished Ab binding concerns residues buried within the Ag binding groove, suggesting the possibility of peptide contribution to serologic epitopes defined by alloreactive Abs, We have addressed this issue by comparison of the re pertoire of peptides eluted from K-d molecules precipitated by differe nt K-d-reactive mAbs, The results reveal that the two-dimensional prof ile obtained with one (F35.119.18) of the alloreactive mAbs is clearly different, The use of 21 single amino acid variants of a K-d-restrict ed 10-mer peptide allowed us to identify the residue of the bound pept ide contributing to the epitope recognized by this mAb, Thus, we have shown that at least in some instances, changes induced in the MHC mole cules by the binding of distinct peptides can be recognized as alterat ions in serologic determinants expressed on the class I molecules.