MON-429 The TSH Receptor Antibody Reactome Determines Thyroid and Retroorbital Responsiveness

Abstract Disclosure: P. xiang: None. R. Latif: None. T.F. Davies: None. The relative role of the different TSH receptor antibodies generated in patients with Graves’ disease remains unclear. Several animal models of Graves' disease (GD) and thyroid eye disease (TED) have been reported in response to immunization with different forms of TSH receptor (TSHR) antigen and should have helped clarify this immunopathology. However, these animal models remain insufficiently robust and continue to fall short in fully replicating the complexities of the human disease with its mixture of stimulating, blocking and neutral TSH receptor antibodies. In this study, we further evaluated a mouse model of GD and the role of TSHR antibodies of differing bioactivity. Balb/c mice were immunized with an IM injection of an adenovirus expressing residues 1-289 of the human TSHR (Ad-TSHR 289) (absent the hinge region) 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) were evaluated using flow cytometry and a luciferase based cell bioassay. To further assess the induced changes we also carried out thyroid gland ultrasound and histology, and for eye changes we performed MRI scans along with histological examination of the eyes at 30 weeks. Out 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 detectable TSAb of low levels when compared to that obtained with TSH ligand and control TSAb. Seven additional mice showed detectable TBAb only and only 2 mice had detectable linear antibodies to the TSHR. Five hyperthyroid mice showed eye signs, including conjunctival redness and eyelid thickening. MRI scans and histological staining revealed mild enlargement of retrobulbar adipose tissue and retro-orbital muscle accompanied by a macrophage infiltration. Hyperthyroidism was only found in 9/16 (56%) of mice despite all mice expressing TSHR antibodies. The TSAbs detected were of a low level and in their presence we could not detect TBAb in these mice although such blocking antibodies may have developed simultaneously explaining the low stimulating bioactivity. In support of this interpretation, additional mice developed detectable TBAb only - highly suggestive of their dampening any TSAb response and preventing the onset of hyperthyroidism. These multiple antibodies to TSHR antigen (the reactome) have to be considered in all immunization mouse models of GD since they are the ultimate determination of the thyroid response. Presentation: Monday, July 14, 2025