title: A panel of unique HLA-A2 mutant molecules define epitopes recognized by HLA-A2-specific antibodies and cytotoxic T lymphocytes.
abstract: HLA-A2.1 and HLA-A2.3, which differ from one another at residues 149, 152, and 156, can be distinguished by the mAb CR11-351 and many allogeneic and xenogeneic CTL. Site-directed mutagenesis was used to incorporate several different amino acid substitutions at each of these positions in HLA-A2.1 to evaluate their relative importance to serologic and CTL-defined epitopes. Recognition by mAb CR11-351 was completely lost when Thr but not Pro was substituted for Ala149. A model to explain this result based on the 3-dimensional structure of HLA-A2.1 is presented. In screening eight other mAb, only the substitutions of Pro for Val152 or Gly for Leu156 led to the loss of mAb binding. Because other non-conservative substitutions at these same positions had no effect, these results suggest that the loss of serologic epitopes is in many cases due to a more indirect effect on molecular conformation. Specificity analysis using 28 HLA-A2.1-specific alloreactive and xenoreactive CTL clones showed 19 distinct patterns of recognition. The epitopes recognized by alloreactive CTL clones demonstrated a pronounced effect by all substitutions at residue 152, including the very conservation substitution of Ala for Val. Overall, the most disruptive substitution at amino acid residue 152 was Pro, followed by Glu, Gln, and then Ala. In contrast, substitutions at 156 had little or no effect on allogeneic CTL recognition, and most clones tolerated either Gly, Ser, or Trp at this position. Similar results were seen using a panel of murine HLA-A2.1-specific CTL clones, except that substitutions at position 156 had a greater effect. The most disruptive substitution was Trp, followed by Ser and then Gly. In addition, when assessed on the entire panel of CTL, the effects of Glu and Gln substitutions at position 152 demonstrated that the introduction of a charge difference is no more disruptive than a comparable change in side chain structure that does not alter charge. Taken together, these results indicate that the effect of amino acid replacements at positions 152 and 156 on CTL-defined epitopes depends strongly on the nature of the substitution. Thus, considerable caution must be exercised in evaluating the significance of particular positions on the basis of single mutations. Nonetheless, the more extensive analysis conducted here indicates that there are differences among residues in the class I Ag "binding pocket," with residue 152 playing a relatively more important role in formation of allogeneic CTL-defined epitopes than residue 156.
authors: Department of Microbiology, University of Virginia, Charlottesville 22908.
authors: K T
authors: KT
authors: Hogan
authors: C
authors: C
authors: Clayberger
authors: E J
authors: EJ
authors: Bernhard
authors: S F
authors: SF
authors: Walk
authors: J P
authors: JP
authors: Ridge
authors: P
authors: P
authors: Parham
authors: A M
authors: AM
authors: Krensky
authors: V H
authors: VH
authors: Engelhard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D003601:Cytotoxicity Tests, Immunologic; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D015234:HLA-A Antigens / Q000032:analysis / Q000235:genetics*; D015789:HLA-A2 Antigen; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D015234:HLA-A Antigens; D015789:HLA-A2 Antigen