Chun-Rong Chen

The thyrotropin receptor autoantigen in Graves disease is the culprit as well as the victim

Graves disease, a common organ-specific autoimmune disease affecting humans, differs from all other autoimmune diseases in being associated with target organ hyperfunction rather than organ damage. Clinical thyrotoxicosis is directly caused by autoantibodies that activate the thyrotropin receptor (TSHR). The etiology of Graves disease is multifactorial, with nongenetic factors playing an important role. Of the latter, there is the intriguing possibility that the molecular structure of the target antigen contributes to the development of thyroid-stimulatory autoantibodies (TSAbs). Among the glycoprotein hormone receptors, only the TSHR undergoes intramolecular cleavage into disulfide-linked subunits with consequent shedding of some of the extracellular, autoantibody-binding A subunits. Functional autoantibodies do not arise to the noncleaving glycoprotein hormone receptors. Recently, TSAbs were found to preferentially recognize shed, rather than attached, A subunits. Here we use a new adenovirus-mediated animal model of Graves disease to show that goiter and hyperthyroidism occur to a much greater extent when the adenovirus expresses the free A subunit as opposed to a genetically modified TSHR that cleaves minimally into subunits. These data show that shed A subunits induce or amplify the immune response leading to hyperthyroidism and provide new insight into the etiology of Graves disease.

Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor.

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAbs) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAbs]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAbs, but not for TBAbs, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.

The Thyrotropin Receptor Hinge Region Is Not Simply a Scaffold for the Leucine-Rich Domain but Contributes to Ligand Binding and Signal Transduction

The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Delta) in the TSH receptor (TSHR) hinge region (residues 277-418). TSHRDelta287-366, Delta287-371, Delta287-376, and Delta287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRDelta287-384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371-384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRDelta287-371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371-384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.

Antibodies to Thyroid Peroxidase Arise Spontaneously with Age in NOD.H-2h4 Mice and Appear after Thyroglobulin Antibodies

Hashimotos thyroiditis, a common autoimmune disease, is associated with autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). TPO, unlike abundant and easily purified Tg, is rarely investigated as an autoantigen in animals. We asked whether antibodies (Abs) develop to both TPO and Tg in thyroiditis that is induced (C57BL/6 and DBA/1 mice) or arises spontaneously (NOD.H-2h4 mice). Screening for TPOAbs was performed by flow cytometry using mouse TPO-expressing eukaryotic cells. Sera were also tested for binding to purified mouse Tg and human TPO. The antibody data were compared with the extent of thyroiditis. Immunization with mouse TPO adenovirus broke self-tolerance to this protein in C57BL/6 mice, but thyroiditis was minimal and TgAbs were absent. In DBA/1 mice with extensive granulomatous thyroiditis induced by Tg immunization, TPOAbs were virtually absent despite high levels of TgAbs. In contrast, antibodies to mouse TPO, with minimal cross-reactivity with human TPO, arose spontaneously in older (7-12 months) NOD.H-2h4 mice. Unexpectedly, TgAbs preceded TPOAbs, a time course paralleled in relatives of probands with juvenile Hashimotos thyroiditis. These findings demonstrate a novel aspect of murine and human thyroid autoimmunity, namely breaking B cell self-tolerance occurs first for Tg and subsequently for TPO.

Immune Deviation Away from Th1 in Interferon-γ Knockout Mice Does Not Enhance TSH Receptor Antibody Production after Naked DNA Vaccination

TSH receptor (TSHR) DNA vaccination induces high TSHR antibody levels in BALB/c mice housed in a conventional facility. However, under pathogen-free conditions, we observed a Th1 cellular response to TSHR antigen characterized by interferon-γ (IFNγ) production. In the present study we investigated the effect on TSHR DNA vaccination of diverting the cytokine milieu away from Th1 using 1) IFNγ knockout BALB/c mice, and 2) wild-type mice covaccinated with DNA for the TSHR and for IFNγ/receptor-Fc protein that prevents IFNγ from binding to its receptor. Neither approach enhanced TSHR antibody levels, although splenocyte IFNγ production in response to TSHR antigen was absent (IFNγ knockouts) or reduced (IFNγ receptor-Fc). Moreover, production of IL-2, another Th1 cytokine, but not Th2 cytokines, indicated that neither strategy overcame the Th1 bias of im DNA vaccination. Importantly, splenocyte production of IFNγ and IL-2 provides a sensitive detection system for TSHR-specific T cells. Unexpectedly, higher TSH...

Identification of Key Amino Acid Residues in a Thyrotropin Receptor Monoclonal Antibody Epitope Provides Insight into Its Inverse Agonist and Antagonist Properties

CS-17 is a murine monoclonal antibody to the human TSH receptor (TSHR) with both inverse agonist and antagonist properties. Thus, in the absence of ligand, CS-17 reduces constitutive TSHR cAMP generation and also competes for TSH binding to the receptor. The present data indicate that for both of these functions, the monovalent CS-17 Fab (50 kDa) behaves identically to the intact, divalent IgG molecule (150 kDa). The surprising observation that CS-17 competes for TSH binding to the human but not porcine TSHR enabled identification of a number of amino acids in its epitope. Replacement of only three human TSHR residues (Y195, Q235, and S243) with the homologous porcine TSHR residues totally abolishes CS-17 binding as detected by flow cytometry. TSH binding is unaffected. Of these residues, Y195 is most important, with Q235 and S243 contributing to CS-17 binding to a much lesser degree. The functional effects of CS-17 IgG and Fab on constitutive cAMP generation by porcinized human TSHR confirm the CS-17 binding data. The location of TSHR amino acid residues Y195, Q235, and S243 deduced from the crystal structure of the FSH receptor leucine-rich domain provides valuable insight into the CS-17 and TSH binding sites. Whereas hormone ligands bind primarily to the concave surface of the leucine-rich domains, a major portion of the CS-17 epitope lies on the opposite convex surface with a minor component in close proximity to known TSH binding residues.

Enhanced Response to Mouse Thyroid-Stimulating Hormone (TSH) Receptor Immunization in TSH Receptor-Knockout Mice

Graves-like hyperthyroidism is induced in BALB/c mice by immunization with adenovirus expressing the human TSH receptor (TSHR) A-subunit (amino acids 1-289). However, because of nonidentity between the human and mouse TSHR ( approximately 87% amino acid homology), we compared the responses of mice immunized with adenoviruses expressing either the mouse or the human TSHR A-subunit. Wild-type (wt) BALB/c mice immunized with the mouse A-subunit developed neither TSHR antibodies (measured by flow cytometry) nor thyroid lymphocytic infiltration. However, wt C57BL/6 mice developed sparse intrathyroidal lymphocyte infiltration without antibody production. Depletion of naturally occurring regulatory CD4(+)CD25(+) T cells had little effect. These results indicate the inability to break tolerance to the mouse TSHR in wt mice. In contrast, TSHR knockout (KO) BALB/c mice generated mouse TSHR antibodies in response to mouse A-subunit immunization and augmented human TSHR antibody response to human A-subunit immunization. Thyroid-stimulating antibody titers measured in a functional bioassay were comparable in human A-subunit immunized wt mice and in TSHR KO mice immunized with either the mouse or human A-subunit. In conclusion, immune response to the mouse TSHR is readily induced in TSHR KO but not in wt mice. Only in the former does immunization with adenovirus expressing the mouse A-subunit generate antibodies capable of activating the mouse TSHR. TSHR KO mice are, therefore, of value for future studies dissecting the autoimmune response to the mouse TSHR.

A Unique Mouse Strain That Develops Spontaneous, Iodine-Accelerated, Pathogenic Antibodies to the Human Thyrotrophin Receptor

Abs that stimulate the thyrotropin receptor (TSHR), the cause of Graves’ hyperthyroidism, only develop in humans. TSHR Abs 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, Abs. We hypothesized that transferring the human TSHR A-subunit to NOD.H2h4 mice would result in loss of tolerance to this protein. BALB/c human TSHR A-subunit mice were bred to NOD.H2h4 mice, and transgenic offspring were repeatedly backcrossed to NOD.H2h4 mice. All offspring developed Abs to thyroglobulin and thyroid-peroxidase. However, only TSHR-transgenic NOD.H2h4 mice (TSHR/NOD.H2h4) developed pathogenic TSHR Abs as detected using clinical Graves’ disease assays. As in humans, TSHR/NOD.H2h4 female mice were more prone than male mice to developing pathogenic TSHR Abs. Fortunately, in view of the confounding effect of excess thyroid hormone on immune responses, spontaneously arising pathogenic human TSHR Abs 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 Abs in female mice, providing a unique model to investigate disease pathogenesis and test novel TSHR Ag-specific immunotherapies aimed at curing Graves’ disease in humans.

Evidence That the Thyroid-stimulating Hormone (TSH) Receptor Transmembrane Domain Influences Kinetics of TSH Binding to the Receptor Ectodomain

Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (koff) of prebound 125I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, 125I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH Kd values for these two receptor forms at 4 °C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.

A Monoclonal Antibody with Thyrotropin (TSH) Receptor Inverse Agonist and TSH Antagonist Activities Binds to the Receptor Hinge Region as Well as to the Leucine-Rich Domain

Monoclonal antibody CS-17 is a TSH receptor (TSHR) inverse agonist (suppresses constitutive activity) and a TSH antagonist. Elucidation of the CS-17 epitope will provide insight into TSHR structure and function. Present information on its epitope conflicts with recent data regarding another TSHR inverse agonist antibody. To characterize further the CS-17 epitope, we exploited the observation that CS-17 does not recognize a chimeric receptor with TSHR hinge region residues 261-289 replaced with homologous rat LH receptor residues (13 mismatches). We generated individual and double TSHR mutations corresponding to these mismatches. On flow cytometry, only T273L/R274V reduced CS-17 recognition. No mutation affected TSH-stimulated cAMP generation. Because the immunogen for CS-17 generation was highly glycosylated, we also investigated whether the glycan moiety at N198, topologically adjacent to Y195 (a previously identified epitopic component), could contribute to the CS-17 epitope. Elimination of this N-linked glycan (mutations of N198 and T200) abrogated CS-17 binding without altering TSH responsiveness. However, studies with tunicamycin suggested that these mutations affected CS-17 binding by altering the polypeptide backbone rather than eliminating the glycan moiety. TSHR residues N198 and T200, like Y195, are on the convex facet of the leucine-rich domain. In summary, the present data indicate that the discontinuous epitope of CS-17, a TSHR inverse agonist and TSH antagonist, includes a component in the hinge region as well as the convex surface of the TSHR leucine-rich domain. These findings expand our present concept of glycoprotein hormone binding and function.