Rarity of autoantibodies to a major autoantigen, thyroid peroxidase, that interact with denatured antigen or with epitopes outside the immunodominantregion

The nature of the autoantibody repertoire to the dominant autoantigen in hu man autoimmune thyroid disease is controversial. There is evidence that aut oantibodies to thyroid peroxidase (TPO) interact with overlapping conformat ional epitopes in an immunodominant region and binding to denatured (DN) pr otein is decreased. Contrary data demonstrate TPO autoantibody reactivity w ith DN-TPO or polypeptide fragments. However, none of the TPO-specific, hum an monoclonal autoantibodies isolated to date preferentially recognize dena tured autoantigen. We therefore searched an immunoglobulin gene phage displ ay library for human autoantibodies that bind TPO denatured by reduction an d alkylation (DN-TPO). Thyroid-infiltrating B cells from a typical TPO auto antibody-positive patient were the source of mRNA for library construction. Surprisingly, the library enriched after panning on DN-TPO, as well as a p anel of individual clones, preferentially bound native (N)-TPO. Of 13 clone s selected using DN-TPO or N-TPO, 12 clones recognized the TPO immunodomina nt region. Moreover, regardless of selection with N-TPO or DN-TPO, their he avy and light chains were encoded by similar VDJ and V-kappa combinations. One clone (DN4), isolated using DN-TPO, did not interact with the TPO immun odominant region and its H chain derives from a different V-H gene. Althoug h DN4 binds specifically to TPO, its affinity is low, unlike the high affin ities of other human TPO autoantibodies. In conclusion, human monoclonal au toantibodies that preferentially recognize denatured TPO could not be isola ted from an immunoglobulin gene library despite selection with denatured pr otein. Our findings demonstrate the bias of the human B cell repertoire tow ards recognition of an immunodominant region on the conformationally intact form of a major thyroid autoantigen.