Antibodies that stimulate the thyrotropin receptor (TSHR), the cause of Graves’ hyperthyroidism, only develop in humans. TSHR antibodies can be induced in mice by immunization but studying pathogenesis and therapeutic intervention requires a model without immunization. Spontaneous, iodine-accelerated, thyroid autoimmunity develops in NOD.H2h4 mice associated with thyroglobulin and thyroid-peroxidase, but not TSHR, antibodies. We hypothesized that transferring the human (h)TSHR A-subunit to NOD.H2h4 mice would result in loss of tolerance to this protein. BALB/c hTSHR A-subunit mice were bred to NOD.H2h4 mice and transgenic offspring were repeatedly backcrossed to NOD.H2h4 mice. All offspring developed antibodies to thyroglobulin and thyroid-peroxidase. However, only TSHR-transgenic NOD.H2h4 mice (TSHR/NOD.H2h4) developed pathogenic TSHR antibodies as detected using clinical Graves’ disease assays. As in humans, TSHR/NOD.H2h4 females were more prone than males to developing pathogenic TSHR antibodies. Fortunately, in view of the confounding effect of excess thyroid hormone on immune responses, spontaneously arising pathogenic (h)TSHR antibodies cross-react poorly with the mouse TSHR and do not cause thyrotoxicosis. In summary, the TSHR/NOD.H2h4 mouse strain develops spontaneous, iodine-accelerated, pathogenic TSHR antibodies in females, providing a unique model to investigate disease pathogenesis and test novel TSHR-antigen specific immunotherapies aimed at curing Graves’ disease in humans.

Graves’ hyperthyroidism, a common autoimmune disease caused by pathogenic autoantibodies to the thyrotropin (TSH) receptor, can be treated but not cured. This single autoantigenic target makes Graves’ disease a prime candidate for antigen-specific immunotherapy. Previously, in an induced mouse model, injecting TSHR A-subunit protein attenuated hyperthyroidism by diverting pathogenic TSHR antibodies to a non-functional variety. Here we explored the possibility of a similar diversion in a mouse model that spontaneously develops pathogenic TSHR autoantibodies, NOD.H2h4 mice with the human(h) TSHR A-subunit transgene expressed in the thyroid and (shown here) the thymus. We hypothesized that such diversion would occur following injection of “inactive” hTSHR A-subunit protein recognized only by non-pathogenic (not pathogenic) TSHR antibodies. Surprisingly, rather than attenuating the pre-existing pathogenic TSHR level, in TSHR/NOD.H2h4 mice inactive hTSHR antigen injected without adjuvant enhanced the levels of pathogenic TSH-binding inhibition (TBI) and thyroid stimulating antibodies, as well as non-pathogenic antibodies detected by ELISA. This effect was TSHR-specific as spontaneously occurring autoantibodies to thyroglobulin and thyroid peroxidase were unaffected. As controls, non-transgenic NOD.H2h4 mice similarly injected with inactive hTSHR A-subunit protein unexpectedly developed TSHR antibodies, but only of the non-pathogenic variety detected by ELISA. Our observations highlight critical differences between induced and spontaneous mouse models of Graves’ disease with implications for potential immunotherapy in humans. In hTSHR/NOD.H2h4 mice with ongoing disease, injecting inactive hTSHR A-subunit protein fails to divert the autoantibody response to a non-pathogenic form. Indeed, such therapy is likely to enhance pathogenic antibody production and exacerbate Graves’ disease in humans.

NOD.H2k and NOD.H2h4 mice carry the MHC class II molecule I-Ak associated with susceptibility to experimentally-induced thyroiditis. Dietary iodine enhanced spontaneous thyroid autoimmunity, well known in NOD.H2h4 mice, has not been investigated in NOD.H2k mice. We compared NOD.H2h4 and NOD.H2k strains for thyroiditis and autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) without or with dietary sodium iodide (NaI) for up to 32 weeks. TgAb levels were significantly higher in NOD.H2h4 than NOD.H2k mice on NaI and TPOAb developed in NOD.H2h4 but not NOD.H2k mice. DNA exome analysis revealed, in addition to the differences in the chromosome (Chr) 17 MHC regions, that NOD.H2k and particularly NOD.H2h4 mice have substantial non-MHC parental DNA. KEGG pathway-analysis highlighted thyroid autoimmunity and immune-response genes on Chr 17 but not on Chr 7 and 15 parental B10.A4R DNA. Studies of parental strains provided no evidence for non-MHC gene contributions. The exon 10 thyroglobulin haplotype, associated with experimentally-induced thyroiditis, is absent in NOD.H2h4 and NOD.H2k mice and is not a marker for spontaneous murine thyroid autoimmunity. In conclusion, the absence of I-E is a likely explanation for the difference between NOD.H2h4 and NOD.H2k mice in TgAb levels and, as in humans, autoantibody spreading to TPO.