Evidence that shed thyrotropin receptor A subunits drive affinity maturation of autoantibodies causing Graves' disease.
BACKGROUND
In Graves' disease, thyroid-stimulating antibodies (TSAb) activate the TSH receptor (TSHR) causing hyperthyroidism. Serum polyclonal TSAb are difficult to study because of their extremely low serum levels.


OBJECTIVE
Our objective was to determine whether monoclonal TSAb possess characteristics previously reported for polyclonal autoantibodies in Graves' sera.


METHODS
We studied monoclonal TSAb from three laboratories: six generated from mice with induced hyperthyroidism; and one, M22, a human autoantibody obtained from Graves' B cells.


RESULTS
All TSAb with one exception were potent activators of TSHR-mediated cAMP generation, with relatively similar half-maximal stimulatory concentrations. Like polyclonal autoantibodies, monoclonal TSAb were largely neutralized by conformationally "active" (but not "inactive") recombinant TSHR A subunits (the N-terminal cleavage product of the TSHR). Chimeric substitutions of TSHR amino acids 25-30 (the extreme N terminus after removal of the 21 residue signal peptide) abrogated the binding and function of all monoclonal TSAb but with one antibody (TSAb4) revealing a nonidentical epitope. Remarkably, these residues are uninvolved in the M22 epitope determined by x-ray analysis. Finally, flow-cytometric dose-response analyses, not previously possible with polyclonal TSAb, revealed that all monoclonal TSAb, human and murine, bound with lower affinity to their in vivo target, the TSH-holoreceptor, than to the isolated TSHR ectodomain.


CONCLUSIONS
TSAb function does not require antibodies with identical epitopes, and human autoantibody M22 may, therefore, not represent the full epitopic repertoire of polyclonal TSAb in Graves' disease. Most important, we provide strong evidence that the shed ectodomain (primarily the A subunit) is the primary antigen driving affinity maturation of TSAb producing B cells.
Mizutori
Yumiko
Y
Autoimmune Disease Unit, Cedars-Sinai Research Institute and David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90048, USA.
Chen
Chun-Rong
CR
Latrofa
Francesco
F
McLachlan
Sandra M
SM
Rapoport
Basil
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000502:physiology; D000915:Antibody Affinity; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006111:Graves Disease / Q000209:etiology* / Q000276:immunology; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating / Q000276:immunology / Q000502:physiology*; D011487:Protein Conformation; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000502:physiology*
D000911:Antibodies, Monoclonal; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin