title: HLAMatchmaker-based analysis of human monoclonal antibody reactivity demonstrates the importance of an additional contact site for specific recognition of triplet-defined epitopes.
abstract: Five HLA-A3 reactive human monoclonal antibodies (mAbs) originating from a parous woman were screened against HLA-typed panels by means of complement-dependent lymphocytotoxicity, high-definition ELISA, and flow cytometry with single antigen beads. Antibody reactivity profiles were compared with triplet amino acid sequence polymorphisms identified by HLAMatchmaker, and a three-dimensional structural modeling program (Cn3D of the National Center for Biotechnology Information) was used to determine the topography of epitopes recognized by each mAb. These mAbs originated from a woman who during pregnancy developed antibodies to the paternal HLA-A3 antigen of her child. Each mAb was specific for one mismatched triplet on HLA-A3, and the reactivity patterns of these IgM-type mAbs were practically the same in lymphocytotoxicity and antigen-binding assays. One mAb was specific for 163dT, a unique triplet present only on A3. The other mAbs reacted with 62Qe, 142mI, or 144tKr; these triplets are present on different groups of HLA-A alleles, some of which, however, did not react. Topographic modeling of triplet-defined epitopes identified clusters of polymorphic surface residues that were shared between reactive alleles. These clusters may serve as primary contact sites for the specificity-determining complementarity-determining region (CDR) loops of antibody. The reactivity with these mAbs required also the presence of self-sequence elsewhere on the HLA molecular surface as a critical secondary contact site for antibody, likely through another CDR loop. For instance, the reactivity of the 62Qe-specific mAb required the presence of a glycine residue in position 56 and the reactivity of the 142mI-specific mAb required the presence of the GTLRG sequence in positions 79-83. Conversely, there were many other amino acid differences between the mAb-reactive alleles and HLA-A3 that did not prevent antibody binding. For instance, the 62Qe-specific mAb-reactive alleles had 35 and the 142mI-reactive alleles had 50 of such "permissive" residue differences. An HLAMatchmaker-based analysis of the reactivity of human mAbs will increase our understanding of the structural definition of HLA epitopes and their reactivity with alloantibodies.
authors.affiliation: University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA. Duquesnoyr@msx.upmc.edu
authors.forename: René J
authors.initials: RJ
authors.lastname: Duquesnoy
authors.forename: Arend
authors.initials: A
authors.lastname: Mulder
authors.forename: Medhat
authors.initials: M
authors.lastname: Askar
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernandez-Vina
authors.forename: Frans H J
authors.initials: FH
authors.lastname: Claas
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D015234:HLA-A Antigens / Q000235:genetics / Q000276:immunology*; D015791:HLA-A3 Antigen / Q000235:genetics / Q000276:immunology; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D007518:Isoantibodies / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic / Q000276:immunology; D011247:Pregnancy; D017386:Sequence Homology, Amino Acid
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D015234:HLA-A Antigens; C442141:HLA-A*03:01 antigen; D015791:HLA-A3 Antigen; D007075:Immunoglobulin M; D007518:Isoantibodies