Rarity of autoantibodies to a major autoantigen, thyroid peroxidase, that interact with denatured antigen or with epitopes outside the immunodominant region.
The nature of the autoantibody repertoire to the dominant autoantigen in human autoimmune thyroid disease is controversial. There is evidence that autoantibodies to thyroid peroxidase (TPO) interact with overlapping conformational epitopes in an immunodominant region and binding to denatured (DN) protein is decreased. Contrary data demonstrate TPO autoantibody reactivity with DN-TPO or polypeptide fragments. However, none of the TPO-specific, human monoclonal autoantibodies isolated to date preferentially recognize denatured autoantigen. We therefore searched an immunoglobulin gene phage display library for human autoantibodies that bind TPO denatured by reduction and alkylation (DN-TPO). Thyroid-infiltrating B cells from a typical TPO autoantibody-positive patient were the source of mRNA for library construction. Surprisingly, the library enriched after panning on DN-TPO, as well as a panel of individual clones, preferentially bound native (N)-TPO. Of 13 clones selected using DN-TPO or N-TPO, 12 clones recognized the TPO immunodominant region. Moreover, regardless of selection with N-TPO or DN-TPO, their heavy and light chains were encoded by similar VDJ and Vkappa combinations. One clone (DN4), isolated using DN-TPO, did not interact with the TPO immunodominant region and its H chain derives from a different VH gene. Although DN4 binds specifically to TPO, its affinity is low, unlike the high affinities of other human TPO autoantibodies. In conclusion, human monoclonal autoantibodies that preferentially recognize denatured TPO could not be isolated from an immunoglobulin gene library despite selection with denatured protein. Our findings demonstrate the bias of the human B cell repertoire towards recognition of an immunodominant region on the conformationally intact form of a major thyroid autoantigen.
Autoimmune Disease Unit, Cedars-Sinai Research Institute and the University of California, Los Angeles School of Medicine, Los Angeles, CA, USA.
J
J
Guo
Y
Y
Wang
J C
JC
Jaume
B
B
Rapoport
S M
SM
McLachlan
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007453:Iodide Peroxidase / Q000737:chemistry / Q000276:immunology*; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000276:immunology; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D016056:Immunodominant Epitopes; D007140:Immunoglobulin Fab Fragments; D033862:Iron-Binding Proteins; D011993:Recombinant Fusion Proteins; C056626:thyroid microsomal antibodies; C056658:TPO protein, human; D007453:Iodide Peroxidase