NOD.H-2h4 mice are genetically predisposed to thyroid autoimmunity and spontaneously develop autoantibodies to thyroglobulin (TgAb) and thyroiditis. Iodide administration enhances TgAb levels and the incidence and severity of thyroiditis. Using these mice, we investigated the interactions between thyrotropin receptor (TSHR) antibodies induced by vaccination and spontaneous or iodide-enhanced thyroid autoimmunity (thyroiditis and TgAb). Mice were immunized with adenovirus expressing the TSHR

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... bunit (or control adenovirus). Thyroid antibodies, histology and serum thyroxine levels were compared in animals on a regular diet or on a high iodide diet (0.05% NaI-supplemented water). Thyroiditis severity and TgAb levels were enhanced by iodide administration and were independent of the type of adenovirus used for immunization. In contrast, TSHR antibodies measured by TSH binding inhibition (TBI), thyroid stimulating activity (TSAb) and TSH blocking activity (TBAb), were induced in the majority of animals immunized with TSHR (but not control) adenovirus and were unaffected by dietary iodide. The NOD.2h4 strain was less susceptible than BALB/c or BALB/k mice to TSHRadenovirus induced hyperthyroidism. Nevertheless, hyperthyroidism developed in approximately one third of TSHR-adenovirus injected NOD.2h4 mice. This hyperthyroidism was suppressed by a high iodide diet, probably by a non-immune mechanism. The fact that inducing an immune response to the TSHR had no effect on thyroiditis raises the possibility that the TSHR may not be the target involved in the variable thyroiditis component in some humans with Graves' disease. Graves' thyroid glands typically have lymphocytic infiltration, though to a lesser degree than in Hashimoto's thyroiditis. Therefore, TPO autoantibodies in Graves' disease may reflect an underlying process of thyroid inflammation. However, hyperthyroidism in Graves' disease indicates that thyroid stimulation by TSHR autoantibodies exceeds any thyroid damage associated with the autoimmune response to TPO and perhaps also to Tg. In this context, it is noteworthy that changes from hypothyroidism in Hashimoto's thyroiditis to hyperthyroid Graves' disease (16), or the reverse (17), have been reported. Recently, we found that injecting adenovirus expressing the TSHR A-subunit, particularly 4 at low doses, optimized the induction of Graves' hyperthyroidism in BALB/c mice (18, 19) . However, despite goiter and follicular hyperplasia, no thyroid lymphocytic infiltration was evident. Because NOD.H-2h4 mice are prone to develop thyroiditis, we hypothesized that an immune response to the TSHR induced by TSHR A-subunit adenovirus immunization in these mice would lead to thyroid lymphocytic infiltration, even without iodide administration. Also, because of the known effects of dietary iodide on this mouse strain, we compared the outcome of TSHRadenovirus immunization in animals with or without iodide supplementation in their drinking water. METHODS AND MATERIALS Mice. NOD.H-2h4 mice were derived by crossing NOD mice with B10.A(4R) mice and backcrossing to obtain animals on the NOD background with the B10.A(4R) MHC haplotype (I-A k ). The breeding stock was provided by Dr. L. Wicker (Merck Laboratories, Rahway, NJ) and the strain was subsequently bred at the University of Missouri, Columbia. From 7-8 weeks of age, half the mice (all females) were maintained on 0.05% NaI in their drinking water until they were euthanized. All animal studies were approved by the local institutional animal committee and were performed in accordance with the highest standards of care in a pathogen-free facility. TSHR adenovirus immunization. Construction and purification of adenoviruses expressing TSHR amino acid residues 1-289 (A-subunit) and β-galactosidase (control) have been described previously (12, 18). In brief, TSHR A-subunit adenovirus (abbreviated TSHR-Ad) and control adenovirus (Con-Ad) were propagated in HEK293 cells (American Type Culture Collection, Manassas, VA), purified by CsCl density gradient centrifugation, and viral particle concentration was determined by measuring the absorbance at 260 nm (20). All viruses used in this study were from the same preparation, stored in aliquots at -80 o C. Mice on regular drinking water or on water supplemented with 0.05% NaI (18 animals in 17. Tamai H, Kasagi K, Takaichi Y, Takamatsu J, Komaki G, Matsubayashi S, Konishi J, Kuma K, Kumagai LF, Nagataki S 1989 Development of spontaneous hypothyroidism in patients with Graves' disease treated with antithyroidal drugs: clinical, immunological, and histological findings in 26 patients.