The Thyrotropin Receptor Antibody Reactome Determines Thyroid and Retro-orbital Responsiveness

Background: The roles of different thyrotropin (TSH) receptor (TSHR) antibodies in Graves' disease (GD) and thyroid eye disease (TED) remain unclear, and animal models have been used to try and clarify. While several models have been developed using different TSHR antigens, they have failed to robustly replicate the complexities of human disease, regardless of mouse strain, immunization method, or TSHR antigen used, and often overlook the complete TSHR reactome. In this study, we evaluated a mouse model of GD, focusing on TSHR antibodies with different bioactivities. Methods: Female Balb/c mice were immunized intramuscularly with an adenovirus expressing residues 1-289 of the human TSHR (Ad-TSHR 289) or control vector (Ad-Lacz) with 10 injections at 3-week intervals. Thyroid function was assessed by total thyroxine (T4) and TSH levels. The presence of TSHR binding antibodies as well as stimulating TSHR antibodies (TSAb) and TSH-blocking TSHR antibodies (TBAb) was evaluated using flow cytometry and a transcriptional-based luciferase cell bioassay. We also conducted thyroid ultrasound and histology, micro-magnetic resonance imaging (micro-MRI) for orbital changes, and histological analysis of orbital tissue after 30 weeks of immunization to assess immunopathological changes. Results: Out of a total of 16 mice, 9 became hyperthyroid-characterized by decreased TSH levels, increased T4 levels, and diffuse enlargement of the thyroid glands. All mice developed TSHR antibodies when assessed by flow cytometry. 8/9 of the hyperthyroid mice had TSAb but mostly at low levels, while 7 showed only TBAb but without hypothyroidism. Only 2 mice had detectable linear antibodies. Five hyperthyroid mice showed eye signs, including conjunctival redness and eyelid thickening. Micro-MRI and histology revealed mild retrobulbar adipose and muscle enlargement with macrophage infiltration. Conclusions: Hyperthyroidism occurred in 56% (9/16) of mice, despite all developing TSHR antibodies. The detected TSAbs were of low-level despite their high levels by flow cytometry, suggesting that the simultaneous presence of TBAbs may explain the weak stimulating activity. In the mice with TBAbs, there was no hypothyroidism, suggesting they were not highly effective due to simultaneous TSAb activity and may indeed have prevented hyperthyroidism. These findings highlight the importance of considering the full TSHR antibody reactome in GD mouse models, as it ultimately determines thyroid outcomes.