Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease

Abstract

Context: The TSH receptor (TSHR) A-subunit shed from the cell surface contributes to the induction and/or affinity maturation of pathogenic TSHR autoantibodies in Graves' disease.

Objective: This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation.

Design: The isolated TSHR A-subunit generated by transfected mammalian cells exists in two forms; one (active) is recognized only by Graves' TSHR autoantibodies, the second (inactive) is recognized only by mouse monoclonal antibody (mAb) 3BD10. Recent evidence suggests that both Graves' TSHR autoantibodies and mAb 3BD10 recognize the A-subunit monomer. Therefore, if the A-subunit monomer is an immunogen, Graves' sera should have antibodies to both active and inactive A-subunits. Conversely, restriction of TSHR autoantibodies to active A-subunits would be evidence of a role for shed A-subunit multimers, not monomers, in the pathogenesis of Graves' disease. Therefore, we tested a panel of Graves' sera for their relative recognition of active and inactive A-subunits.

Results: Of 34 sera from unselected Graves' patients, 28 were unequivocally positive in a clinical TSH binding inhibition assay. None of the latter sera, as well as 8/9 sera from control individuals, recognized inactive A-subunits on ELISA. In contrast to Graves' sera, antibodies induced in mice, not by shedding from the TSHR holoreceptor, but by immunization with adenovirus expressing the free human A-subunit, were directed to both the active and inactive A-subunit forms.

Conclusions: The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers.

Figure 1.

The TSHR A-subunit monomer cannot represent both forms (active and inactive) of purified A-subunits. The crystal structure of a Fab for human monoclonal TSAb M22 (red) in complex with the major component of TSHR A-subunit (amino acids 22–260; green) has been solved (6) (protein data base 3GO4), as has the crystal structure of a Fab for mouse monoclonal antibody (mAb) 3BD10 (blue/violet) generated to the human A-subunit (7) (protein data base 4QT5). TSAb M22 specifically recognizes the active, not the inactive, A-subunit form (8, 9). Conversely, mAb 3BD10 only recognizes inactive A-subunits (5). Guided by amino acids contributing to its epitope, the 3BD10 Fab can be docked in silico with the M22/TSHR structure (7). Because both M22 and 3BD10 can simultaneously bind to the TSHR A-subunit monomer, the latter cannot explain the reciprocally exclusive TSAb and 3BD10 binding that is observed experimentally (5).

Figure 2.

Pathogenic autoantibodies in Graves' sera do not recognize 'inactive' TSHR A-subunits. Pathogenic autoantibodies in Graves' sera detected in the TSH binding inhibition (TBI) assay are neutralized by active, not inactive, A-subunits (5). A panel of 34 unselected Graves' sera (A) and nine sera from individuals without a history of autoimmune thyroid disease (B) were tested for TBI activity. TBI activity was clearly present in most samples, with only two being unequivocally negative (<10% TSH binding inhibition; horizontal dashed line). Four sera provided values in the borderline range (10–15% TSH binding inhibition). C, serum from a mouse immunized with adenovirus expressing the human TSHR A-subunit. The same sera were tested by ELISA for antibodies to purified, inactive A-subunits; 34 Graves' sera (D) and 9 control sera (E). The threshold for ELISA positivity (OD 0.1) is indicated by the horizontal dashed line. The arrows in Panels A and D identify Graves' serum No. 29 that was TBI negative, but positive by ELISA for inactive A-subunits. F, The same serum as in Panel C included as a positive control (using an antimouse IgG second antibody).

Figure 3.

Pathogenic TSHR antibodies induced in mice by TSHR A-subunit DNA immunization also recognize inactive A-subunits. Sera from 29 CXB recombinant inbred mice from a previous study (11) were randomly selected for TBI positivity following immunization with adenovirus coding for the human TSHR A-subunit (A). TBI values for sera from six mice injected with control adenovirus are shown in Panel B. Values above the dashed horizontal line (15% TSH binding inhibition) indicate unequivocal TBI positivity. The same sera were tested by ELISA for antibodies to purified, inactive A-subunits (C and D). The threshold for ELISA positivity (OD 0.1) is indicated by the horizontal dashed line.