Clonal relationships between thyroid‐stimulating hormone receptor‐stimulating antibodies illustrate the effect of hypermutation on antibody function

Abstract

Summary Graves’ disease is characterized by production of agonist antibodies to the thyroid‐stimulating hormone receptor (TSHR), but knowledge of the genetic and somatic events leading to their aberrant production is limited. We describe the genetic analysis of two monoclonal antibodies (mAbs) with thyroid‐stimulating activity (TSAb) obtained from a single mouse with experimental Graves’ disease. The mAbs were class switched, but used the same rearrangement of immunoglobulin heavy chain, variable region (IGHV) and immunoglobulin light chain, variable region (IGLV) germline genes, implying a clonal relationship and derivation from a single precursor B‐cell clone. The IGHV‐region genes of the two mAbs underwent high degrees of somatic hypermutation by sharing numerous mutations before diverging, while the IGLV genes evolved separately. Interestingly, the mutations were present in both the complementarity‐determining regions (CDRs) and the framework regions. The cloned IGHV and IGLV genes were confirmed to have TSAb properties in experiments in which they were expressed as recombinant Fabs (rFabs). In other experiments, we swapped the IGLV genes with IGHV genes by constructing chimeric rFabs and showed that the chimeras retained TSAb activities, confirming the close functional relatedness of the V‐region genes. Importantly, the IGLV genes in chimeric rFabs had a dominant stimulatory effect at low concentrations, while the IGHV genes had a dominant effect at higher concentrations. Our findings demonstrate that, in experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V‐region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves’ disease.

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease

“…The lack of binding of rFab germline to TSHR was unlikely to be due to the integrity of the rFab preparations, because rFabs prepared from KSAb1 and KSAb2 continue to interact with TSHR as effectively as their IgG counterparts (30).We therefore conclude that the germline B cell Ab was not reactive with the target autoantigen, TSHR, and that hypermutation of the germline Ab was necessary to confer TSHR autoreactivity. Somatic hypermutation serves to increase affinity for an Ag after exposure.…”

“…KSAb1 and KSAb2 are thus equivalent to the TSAbs present in Graves' disease patients (11,12). Importantly, both KSAb1 and KSAb2 are derived from the same original precursor B cell as they use identical germline gene rearrangements, thus giving an opportunity to evaluate the Ag specificity of their germline precursor (30). This led to an important finding, described to our knowledge for the first time in Graves' disease, that the germline revertant of the two TSAbs, when expressed as rFab, does not bind TSHR.…”

“…1) into the bacterial expression vector pAK19 for rFab expression (30). The rFabs were purified by Ni-affinity chromatography and analyzed by SDS-PAGE with Coomassie Blue staining (Fig.…”

“…1). This construct was cloned into the expression vector pAK19 (30), for rFab expression (termed rFab germline). Both the H and L chain Ig genes were controlled by the alkaline phosphatase promoter, and expression was induced by phosphate starvation.…”

“…We have previously described two monoclonal TSAbs, KSAb1 (IgG 2b /k) and KSAb2 (IgG 2a /k), with strong agonist activity in the low nanogram range, developed from a single animal undergoing experimental Graves' disease (9,10). Detailed genetic analysis revealed both TSAbs to have undergone numerous somatic hypermutations (SHM) and to be clonally related, because they use the same germline Ig V region gene rearrangements (30). This gave an opportunity to study the Ag specificity of their germline Ig genes, which by extensive SHM give rise to TSAb activity with consequent pathogenesis.…”