This study provides evidence that there is no deficit in T regulatory cells during active GD, or during the months post therapy.

Background: Hyperthyroidism of Graves' disease is caused by auto-antibodies to human thyrotropin receptor (hTSH-R). To elucidate important T-cell epitopes in TSH-R, we studied three models of immunity to TSH-R in mice. Methods: Mice transgenic for histocompatibility leukocyte antigen DR3 or DR2 were immunized with cDNA for hTSH-R-extracellular domain (hTSH-R-ECD), or hTSH-R-ECD protein, or hTSH-R peptide epitopes. Proliferative responses of immunized splenocytes to epitopes derived from the hTSH-ECD sequence, anti-TSH-R antibody responses, serum thyroxine and TSH, and thyroid histology were recorded. Results: DR3 mice responded to genomic immunization with proliferative responses to several epitopes, which increased in intensity and spread to include more epitopes, during a 6-week immunization program. DR2 transgenic mice developed weak proliferative responses. Both types of mice developed anti-TSH-R antibodies measured by enzyme-linked immunosorbent assay or TSH-binding inhibition assay in 16-60% of animals. There was evidence of weak thyroid stimulation in one group of animals. Immunization of DR3 transgenic mice to hTSH-R-ECD protein induced a striking response to an epitope with sequence ISRIYVSIDVTLQQLES (aa78-94). Immunization to peptides derived from the TSH-R-ECD sequence (including aa78-94) caused strong responses to the epitopes, and development of immune responses to several other nonoverlapping epitopes within the hTSH sequence (epitope spreading) and antibodies reacting with hTSH-R. This implies that immunization with hTSH-R epitopes produced immunity to mouse TSH-R. Conclusion: T-cell and B-cell responses to genetic immunization differ in DR3 and DR2 transgenic mice, and there is less genetic control of antibody than of T-cell responses. During both genomic and peptide epitope immunization there was evidence of epitope spreading during the immunization. Several functionally important epitopes are evident, especially aa78-94. However, if similar progressive epitope recruitment occurs in human disease, epitope-based therapy will be difficult to achieve.

Sex steroid hormones have been reported to be modulators that augment or suppress immune functions. Applying estrogen to chick embryos has been reported to influence antibody production after hatching, suggesting that estrogen acts on B cell differentiation and proliferation in the bursa of chick embryos. We previously reported the presence of estrogen receptor a (ERa) in the bursa during the late period of embryogenesis. In the present study we examined the time course of ERa expression in the bursa of chick embryos at the late period of embryogenesis by ERa-messenger RNA (mRNA) expression analysis by reverse-transcription-polymerase chain reaction (RT-PCR) using primers for chicken ERa, and immunohistochemistry using an anti-ER antibody. The quantity of ERa-mRNA expressed, estimated from the relative densities of the ERa RT-PCR products to those of b-actin, changed with time during the late period of embryogenesis (day 10 to day 21). ERa-mRNA expression was observed at all ages examined in the present experiment. The expression increased between day 10 and day 15 of embryogenesis and then the value was decreased between day 15 and day 21 of embryogenesis. The numbers of ER-positive cells in the bursa also changed with time during the late period of embryogenesis (day 14 to day 18). ER-positive cells showed the highest level on day 14 of embryogenesis, and then the value declined. ER-positive cells were observed in lymphoid follicular cells, stromal cells and epithelial cells, and the density of ER-positive cells was highest in epithelial cells among the three cell components of the bursa. The high level of ER expression in the bursa of Fabricius (BF) of chick embryos at the late period of embryogenesis suggests that this stage of embryogenesis is critical in B cell differentiation in the bursa in connection with estrogen effects on antibody production after hatching.