Characterization of the murine immune response to the murine TSH receptor ectodomain: induction of hypothyroidism and TSH receptor antibodies

Abstract

Abstract The thyrotropin receptor (TSHR) is the major autoantigen of human Graves' disease. In order to define the antigenicity of the TSHR in a defined model, we examined the immune response of BALB/c mice to immunization with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25–50 μg of insect cell expressed, purified and refolded, mTSHR-ecd in alum adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. Control mice received wild-type baculovirus-infected insect cell protein lysate, in a similar way. After 28 days, murine serum contained high titres of antibodies specific to mTSH-ecd and their titres continued to increase over 90 days. Antibody epitope mapping, using 26 peptides spanning the human TSHR-ecd, showed that a variety of regions of the ectodomain were antigenic. The earliest epitope included aa 22–41, but later two regions of reactivity were noted clustered towards the mid portion and carboxyl terminus of the ectodomain. The murine TSHR autoantibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of blocking the native TSHR. We showed that these TSHR antibodies acted, in vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with the hTSHR. Hence, the antibody response to mTSHR-ecd was potentially antagonistic in its influence on the TSHR. Assessment of thyroid function in the immunized mice showed a fall in serum total T3 by 90 days and markedly elevated murine TSH levels (from 64.0 to 239.6 ng/ml), confirming the onset of thyroid failure. However, thyroid histology remained grossly normal. These data demonstrate that mTSHR-ecd is a potent antigen with three major immunogenic regions. The induced mTSHR-Abs blocked TSH action in vivo and reduced murine thyroid function.