Role of Self-Tolerance and Chronic Stimulation in the Long-Term Persistence of Adenovirus-Induced Thyrotropin Receptor Antibodies in Wild-Type and Transgenic Mice

Abstract

Graves'-like disease, reflected by thyrotropin receptor (TSHR) antibodies and hyperthyroidism in some mouse strains, can be induced by immunization with adenovirus-expressing DNA for the human TSHR or its A-subunit. The conventional approach involves two or three adenovirus injections at 3-week intervals and euthanasia 10 weeks after the first injection. To investigate TSHR antibody persistence in mice with differing degrees of self-tolerance to the TSHR A-subunit, we studied the effect of delaying euthanasia until 20 weeks after the initial immunization. Wild-type (WT) mice and transgenic (tg) mice expressing low intrathyroidal levels of the human TSHR A-subunit were immunized with A-subunit-adenovirus on two occasions; a second group of mice was immunized on three occasions. Sera obtained 4, 10, and 20 weeks (euthanasia) after the initial immunization were tested for thyrotropin (TSH) binding inhibition (TBI), antibody binding to TSHR A-subunit protein-coated enzyme-linked immunosorbent assay (ELISA) plates, and thyroid stimulating antibody activity (TSAb; cyclic adenosine monophosphate [cAMP] generation). Serum thyroxine (T4) and thyroid histology were studied at euthanasia. THE majority of WT mice retained high TSHR antibody levels measured by TBI or ELISA at euthanasia but only about 50% were TSAb positive. Low-expressor tgs exhibited self-tolerance, with fewer mice positive by TBI or ELISA and antibody levels were lower than in WT littermates. In WT mice, antibody persistence was similar after two or three immunizations; for tgs, only mice immunized three times had detectable TSAb at 20 weeks. Unlike our previous observations of hyperthyroidism in WT mice examined 4 or 10 weeks after immunization, all mice were euthyroid at 20 weeks. Our findings for induced TSHR antibodies in mice, similar to data for human thyroid autoantibodies, indicate that the parameters that contribute to the concentration of the antibody and thereby play a critical role in long-term persistence of TSHR antibodies are the degree of self-tolerance to the TSHR and chronic stimulation.