Structural aspects of human leukocyte antigen class I epitopes detected by human monoclonal antibodies Rene J. Duquesnoy a, *, Marilyn Marrari a , Arend Mulder b , Frans H.J. Claas b , Justin Mostecki c , Ivan Balazs c a Division of Transplantation Pathology and Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA b Department of Immunohematology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands c Gen-Probe Transplant Diagnostics, Stamford, CT 06902, USA ARTICLE INFO Article history: Received 5 October 2011 Accepted 30 November 2011 Available online 8 December 2011 Keywords: HLA epitope Eplet HLAMatchmaker Antibody Nonself-self paradigm of epitope immunogenicity ABSTRACT This study addresses the concept that human leukocyte antigen (HLA) class I–specific alloantibodies are specific for epitopes that correspond to HLAMatchmaker-defined eplets. Eplets are essential parts of so-called structural epitopes that make contact with the 6 complementarity determining regions of an antibody. From published molecular models of crystallized protein antigen–antibody complexes, we have calculated that contact residues on structural HLA epitopes should reside within a 15-Å radius of a mismatched eplet. This study addresses the structural basis of high-frequency HLA class I epitopes reacting with human monoclonal antibodies (mAbs) derived from women sensitized during pregnancy. All mAbs were tested in Luminex assays with single HLA allele panels. The HLAMatchmaker algorithm was used to determine their specificity in context with eplet sharing between the immunizing allele and antibody-reactive alleles. To assess the autoreactive B cell origin of these antibodies, we have applied the recently developed nonself–self paradigm of epitope immunogenicity to analyze residue differences between the immunizer and the alleles of the antibody producer. A total of 9 mAbs were specific for epitopes associated with the 41T, 80NRG, 163LW, 69AA, or 80ERILR eplets. In each case, the immunizing allele had within 15 Å of the mismatched eplet, no residue differences with 1 of the alleles of the antibody producer. This observation is consistent with the concept that these mAbs originated from B cells with self HLA immunoglobulin receptors. Eplet-carrying alleles exhibited different levels of reactivity, which, when compared with the immunizing allele, ranged from high to intermediate to very low. In many cases, lower reactivities were associated with differences from self to nonself residues in surface locations within 15 Å of the specific eplet. Apparently, such locations may serve as critical contact sites for the antibody. In other cases, other residue differences did not appear to affect binding with the antibody, suggesting that these locations do not play a major role in antibody binding. For these mAbs we did not obtain convincing evidence that residue differences in hidden positions below the molecular surface had significant effects on antibody binding. These findings have increased our understand- ing of the structural basis of the immunogenicity and antigenicity of HLA class I epitopes and provide a basis for interpreting HLA antibody reactivity patterns in Luminex assays with single alleles.  2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. 1. Introduction Human leukocyte antigen (HLA) antibodies are significant risk factors for transplant rejection and failure and it has become evi- dent that such antibodies are specific for epitopes rather than antigens. Epitopes are important not only for identifying acceptable mismatches for sensitized patients but also for a better under- standing of the sensitization process induced by an HLA mismatch. Certain epitopes are located on 1 or a few HLA antigens, whereas others are shared by large groups of HLA antigens. Antibodies to high-frequency epitopes are generally responsible for the broad serum reactivity in highly sensitized patients. With a few excep- tions, such as the Bw4 and Bw6 epitopes, there is little structural information about high-frequency epitopes and how HLA mis- matches elicit specific antibody responses. The analysis of reactivity patterns of human monoclonal anti- bodies (mAbs) with single HLA class I allele panels offers an attrac- tive approach to identify HLA epitopes. Detailed information about the HLA molecular structure and amino acid sequence differences between reactive and nonreactive alleles has provided a basis for a structural characterization of HLA epitopes. Interpretations of mAb reactivity patterns should be based on current concepts about the molecular structure of the antigen– antibody interface. Each antibody makes contact with an antigen * Corresponding author. E-mail address: Duquesnoyr@upmc.edu (R.J. Duquesnoy). Human Immunology 73 (2012) 267-277 Contents lists available at SciVerse ScienceDirect 0198-8859/$36.00  2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.11.011