By the sixth back\cross stage, transgenic mice have more than 98% of the NOD

By the sixth back\cross stage, transgenic mice have more than 98% of the NOD.genome 24. in non\obese diabetic (NOD).recipients of the Hi\expressor Ginsenoside Rf TSHR A\subunit transgene. Data not published previously as a regression for Lo\expressor NOD.mice reported in Rapoport thyroid stimulating antibody (TSAb) (% control). Dotted lines indicate cut\off points for TBI and TS antibody. The patterned area shows that sera negative for TBI also lack TS antibody activity. Data not published previously as a regression for Lo\expressor non\obese diabetic (NOD).mice reported in Rapoport mice bearing a human TSHR A\subunit transgene, which is expressed at low levels in both the thyroid and thymus (Lo\expressor transgene). The present study tested recent evidence that high intrathymic TSHR expression protects against the development of pathogenic TSHR antibodies in humans. By successive back\crossing, we transferred to the NOD.background a human TSHR A\subunit transgene expressed at high levels in the thyroid and thymus (Hi\expressor transgene). In the sixth back\cross generation (>?98% NOD.genome), only transgenic offspring produced spontaneously immunoglobulin (Ig)G class non\pathogenic human TSHR A\subunit antibodies. In contrast, both transgenic and non\transgenic offspring developed antibodies to thyroglobulin and thyroid peroxidase. However, non\pathogenic human TSHR antibody levels in Hi\expressor offspring were lower than in Lo\expressor transgenic mice. Moreover, pathogenic TSHR antibodies, detected by inhibition of TSH binding to the TSHR, only developed in back\cross offspring bearing the Lo\expressor, but not the Hi\expressor, transgene. High low expression human TSHR A\subunit in the NOD.thymus was not explained by the transgene locations, namely chromosome 2 (127C147 Mb; Hi\expressor) and chromosome 1 (22.9C39.3 Mb; low expressor). Nevertheless, using thyroiditis\prone NOD.mice and two transgenic lines, our data support the association from human studies that low intrathymic TSHR expression is associated with susceptibility to developing pathogenic TSHR antibodies, while high intrathymic TSHR expression is protective. Keywords: autoimmunity, Graves’ disease, intrathymic transcription, thyroid autoantigens, tolerance, TSHR autoantibodies Introduction Graves’ hyperthyroidism, a common autoimmune disease in humans, is caused by autoantibodies that stimulate the thyrotrophin receptor (TSHR) 1, 2. Genes conferring susceptibility to Graves’ disease include single nucleotide polymorphorphisms (SNPs) in non\coding regions of the TSHR (reviewed in 3). TSHR genotypes responsible for JMS low intrathymic TSHR transcription levels are associated with susceptibility to Graves’ disease and, conversely, TSHR genotypes responsible for high intrathymic TSHR expression are protective 4. Moreover, a transcriptional repressor of interferon (IFN)\, a cytokine that can trigger thyroid autoimmunity, interacts with a Graves’ disease\associated TSHR SNP to reduce intrathymic TSHR expression 5. These observations provide insight into the breakdown in self\tolerance to the TSHR and TSHR autoantibody production Ginsenoside Rf in patients with Graves’ disease. Central tolerance is determined by T cell education: immature T cells that bind with high affinity to peptides derived from self\proteins expressed in the thymus undergo apoptosis and are deleted 6. Defects in central tolerance contribute to autoimmune susceptibility. For example, in autoimmune\prone non\obese diabetic (NOD) mice, the altered process of thymic education leads to an increased proportion of autoreactive T cells in peripheral lymphoid organs 7, 8, 9, 10. Moreover, manipulating the magnitude of thymic autoantigen expression in mice controls autoreactive T cell deletion and affects the development of autoimmune disease 11. T cells play a critical role in providing help to B cells for antibody production. In particular, defective central tolerance in mice lacking the autoimmune regulator gene is associated with the development of autoantibodies to a variety of autoantigens 12, 13. Indeed, in studying the association between TSHR SNPs and Graves’ hyperthyroidism, the selection criteria of individuals included, in addition to clinical parameters, the presence of TSHR antibodies in patients but not in controls 4. Previously, we generated two transgenic lines of BALB/c mice with the human TSHR A\subunit targeted to the thyroid gland 14. One line expresses very high, and the other much lower, intrathyroidal levels of the human TSHR A\subunit 15. Consistent with the intrathyroidal differences, intrathymic human being TSHR A\subunit transcripts were extremely high in the Hi there\expressor collection but similar to Ginsenoside Rf the levels of the endogenous mouse TSHR in the Lo\expressor collection 16. As might be expected, TSHR autoantibodies were induced readily in the Lo\expressors by immunization with moderate titres of human being TSHR A\subunit adenovirus. In contrast, inducing TSHR antibodies in Hi there\expressor mice required injection of extremely high\titre human being TSHR A\subunit adenovirus without or with TSHR A\subunit protein in adjuvant 16, 17. BALB/c mice do not develop pathogenic TSHR antibodies spontaneously and (like the TSHR human being A\subunit transgenic BALB/c mice) only do this after immunization with plasmid or adenovirus vectors expressing the TSHR or its A\subunit (examined in 18). Conversely,.