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

Abstract

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.

Use of Single Point Mutations in Domain I of β2-Glycoprotein I to Determine Fine Antigenic Specificity of Antiphospholipid Autoantibodies

“…A similar restriction of autoantibodies to a single immunodominant region has been shown for other autoantigens, such as thyroid peroxidase (25). Given that potential epitopes would be expected to be found over much of the protein (26), this restriction of autoantibody repertoire found in both our study and another suggests that generation of autoantibody-producing B cell clones is influenced by other factors in addition to the availability of epitopes.…”

Proportion of Antibodies to the A and B Immunodominant Regions of Thyroid Peroxidase in Graves and Hashimoto Disease

“…[11,12,19,20] We have also estimated the reaction of sera from 143 patients with thyroid autoimmune diseases and positive for the presence of TPO autoantibodies with reduced TPO. Only 14 (9.8%) of tested sera reacted with reduced TPO in ELISA experiments at serum dilution 1/50.…”

Antibodies to Acetylcholine Receptor in Parous Women with Myasthenia: Evidence for Immunization by Fetal Antigen

“…Because the dominant VH3-07 used by AMC-M2 was paired with so many unrelated light chains (both and ), its VH probably makes the major contribution to binding specificity, as has frequently been observed with other antibodies (Collet et al, 1992; reviewed in Dorrington and Tanford, 1970;Guo et al, 1999;Weigert and Potter, 1977;Winter et al, 1994). With AMC-M6, by contrast, the recurring usage of highly mutated and clonally related light chains in exclusive combination with the dominant VH3-21 suggests that they also have a major influence on specificity (as reviewed by Weigert and Potter, 1977).…”

“…The VH3 germline genes of our two clonal families are both commonly used by normal blood B cells , as is the V1 02/12 that predominated in AMC-M6 and recurred in AMC-M2 Foster et al, 1997). A PCR bias seems very unlikely because the five nonspecific Fabs isolated from our AMC-M2 VH/V library did not include the VH3-07, VH3-21, or V1 02/12 genes and VH3 genes are not overrepresented in Fabs cloned, using the same primers, from other MG patients against other antigens (Y Wong, N Willcox, and A Vincent, unpublished observations) or from autoimmune thyroid tissue (Chazenbalk et al, 1993;Guo et al, 1999).…”