Discoveries in Thyroid Autoimmunity in the Past Century.

This review on the 100th anniversary of the American Thyroid Association summarizes the remarkable progress attained during the past century regarding the pathogenesis and treatment of thyroid autoimmune diseases. Indeed, the general concept of autoimmune diseases in humans was established 70 years ago by thyroid investigators. Graves' disease is a paradigm for the rare occurrence of how autoimmunity can cause disease by stimulating rather than destroying an organ system. Therapeutic advances in the mid 20th century involving administration of thyroid hormones, thionamide drugs, and radioiodine have been hugely beneficial for human health. However, these approaches can only treat, but not cure, thyroid autoimmunity. Investigation of these diseases is facilitated by the identification of a limited number of specific autoantigens, whose molecular cloning has provided much information on their structure. This knowledge has led to highly sensitive and specific diagnostic tests, provided insight into novel aspects regarding the pathogenesis of thyroid autoimmunity, and has opened avenues for the development of new therapeutic agents. Immunotherapy for a cure as opposed to therapy of Graves' disease and Hashimoto's thyroiditis remains the holy grail for the 21st century.

Peri-tumor administration of controlled release anti-CTLA-4 synergizes with systemic anti-PD-1 to induce systemic antitumor immunity while sparing autoimmune toxicity.

Combination immunotherapy targeting the PD-1 and CTLA-4 checkpoint inhibitor pathways provides substantial clinical benefit in patients with advanced-stage cancer but at the risk of dose-limiting inflammatory and autoimmune toxicity. The delicate balance that exists between unleashing tumor killing and promoting systemic autoimmune toxicity represents a major clinical challenge. We hypothesized that targeting anti-CTLA-4 so that it perfuses tumor-draining lymph nodes would provide a significant therapeutic advantage and developed an injectable hydrogel with controlled antibody release characteristics for this purpose. Injection of hydrogel-encapsulated anti-CTLA-4 at a peri-tumor location (MC-38 tumor model) produced dose-dependent antitumor responses and survival that exceeded those by anti-CTLA-4 alone (p < 0.05). Responses to 100 µg of targeted anti-CTLA-4 also equaled or exceeded those observed with a series of systemic injections delivering 600 µg (p < 0.05). While preserving antitumor activity, this approach resulted in serum anti-CTLA-4 exposure (area under the curve) that averaged only 1/16th of that measured with systemic therapy. Consistent with the marked differences in systemic exposure, systemic anti-CTLA-4 stimulated the onset of autoimmune thyroiditis in iodide-exposed NOD.H-2h4 mice, as measured by anti-thyroglobulin antibody titer, while hydrogel-encapsulated anti-CTLA-4 had a minimal effect (p ≤ 0.01). At the same time, this targeted low-dose anti-CTLA-4 approach synergized well with systemic anti-PD-1 to control tumor growth and resulted in a high frequency of complete responders that were immune to tumor re-challenge at a distant site. We conclude that targeted and controlled delivery of low-dose anti-CTLA-4 has the potential to improve the benefit-risk ratio associated with combination checkpoint inhibitor therapy.

A transgenic mouse that spontaneously develops pathogenic TSH receptor antibodies will facilitate study of antigen-specific immunotherapy for human Graves' disease.

Graves' hyperthyroidism can be treated but not cured. Antigen-specific immunotherapy would accomplish this goal, for which purpose an animal model is an invaluable tool. Two types of animal models are available. First, pathogenic TSHR antibodies (TSHRAb) can be induced by injecting mice with fibroblasts co-expressing the human TSHR (hTSHR) and MHC class II, or in mammals using plasmid or adenovirus vectors encoding the hTSHR or its A-subunit. Second, a mouse model that spontaneously develops pathogenic TSHRAb resembling those in human disease was recently described. This outcome was accomplished by transgenic intrathyroidal expression of the hTSHR A-subunit in NOD.H2h4 mice that are genetically predisposed to develop thyroiditis but, without the transgene, do not generate TSHRAb. Recently, novel approaches to antigen-specific immunotherapy have been tested, primarily in the induced model, by injecting TSHR A-subunit protein or cyclic TSHR peptides. T-cell tolerance has also been induced in "humanized" HLA-DR3 mice by injecting synthetic peptides predicted in silico to mimic naturally processed TSHR T-cell epitopes. Indeed, a phase 1 study based on the latter approach has been conducted in humans. In the spontaneous model (hTSHR/NOD.H2h mice), injection of soluble or nanoparticle-bearing hTSHR A-subunits had the unwanted effect of exacerbating pathogenic TSHRAb levels. A promising avenue for tolerance induction, successful in other conditions and yet to be tested with the TSHR, involves encapsulating the antigen. In conclusion, these studies provide insight into the potential outcome of immunotherapeutic approaches and emphasize the importance of a spontaneous model to test future novel, antigen-specific immunotherapies for Graves' disease.

A Mouse Thyrotropin Receptor A-Subunit Transgene Expressed in Thyroiditis-Prone Mice May Provide Insight into Why Graves' Disease Only Occurs in Humans.

Background: Graves' disease, caused by autoantibodies that activate the thyrotropin (TSH) receptor (TSHR), has only been reported in humans. Thyroiditis-prone NOD.H2h4 mice develop autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO) but not to the TSHR. Evidence supports the importance of the shed TSHR A-subunit in the initiation and/or amplification of the autoimmune response to the holoreceptor. Cells expressing the gene for the isolated A-subunit secrete A-subunit protein, a surrogate for holoreceptor A-subunit shedding. NOD.H2h4 mice with the human TSHR A-subunit targeted to the thyroid (a "self" antigen in such transgenic (Tgic) animals), unlike their wild-type (wt) siblings, spontaneously develop pathogenic TSHR antibodies to the human-TSH holoreceptor. These autoantibodies do not recognize the endogenous mouse-TSH holoreceptor and do not cause hyperthyroidism. Methods: We have now generated NOD.H2h4 mice with the mouse-TSHR A-subunit transgene targeted to the thyroid. Tgic mice and wt littermates were compared for intrathyroidal expression of the mouse A-subunit. Sera from six-month-old mice were tested for the presence of autoantibodies to Tg and TPO as well as for pathogenic TSHR antibodies (TSH binding inhibition, bioassay for thyroid stimulating antibodies) and nonpathogenic TSHR antibodies (ELISA). Results: Expression of the mouse TSHR A-subunit transgene in the thyroid was confirmed by real-time polymerase chain reaction in the Tgics and had no effect on the spontaneous development of autoantibodies to Tg or TPO. However, unlike the same NOD.H2h4 strain with the human-TSHR A-subunit target to the thyroid, mice expressing intrathyroidal mouse-TSHR A subunit failed to develop either pathogenic or nonpathogenic TSHR antibodies. The mouse TSHR A-subunit differs from the human TSHR A-subunit in terms of its amino acid sequence and has one less glycosylation site than the human TSHR A-subunit. Conclusions: Multiple genetic and environmental factors contribute to the pathogenesis of Graves' disease. The present study suggests that the TSHR A-subunit structure (possibly including posttranslational modification such as glycosylation) may explain, in part, why Graves' disease only develops in humans.

Nanoparticles Bearing TSH Receptor Protein and a Tolerogenic Molecule Do Not Induce Immune Tolerance but Exacerbate Thyroid Autoimmunity in hTSHR/NOD.H2h4 Mice.

Transgenic NOD.H2h4 mice that express the human (h) TSHR A-subunit in the thyroid gland spontaneously develop pathogenic TSHR autoantibodies resembling those in patients with Graves disease. Nanoparticles coupled to recombinant hTSHR A-subunit protein and a tolerogenic molecule (ligand for the endogenous aryl-hydrocarbon receptor; ITE) were injected i.p. four times at weekly intervals into hTSHR/NOD.H2h4 mice with the goal of blocking TSHR Ab development. Unexpectedly, in transgenic mice, injecting TSHR A-subunit-ITE nanoparticles (not ITE-nanoparticles or buffer) accelerated and enhanced the development of pathogenic TSHR Abs measured by inhibition of TSH binding to the TSHR. Nonpathogenic TSHR Abs (ELISA) were enhanced in transgenics and induced in wild-type littermates. Serendipitously, these findings have important implications for disease pathogenesis: development of Graves TSHR Abs is limited by the availability of A-subunit protein, which is shed from membrane bound TSHR, expressed at low levels in the thyroid. The enhanced TSHR Ab response following injected TSHR A-subunit protein-nanoparticles is reminiscent of the transient increase in pathogenic TSHR Abs following the release of thyroid autoantigens after radio-iodine therapy in Graves patients. However, in the hTSHR/NOD.H2h4 model, enhancement is specific for TSHR Abs, with Abs to thyroglobulin and thyroid peroxidase remaining unchanged. In conclusion, despite the inclusion of a tolerogenic molecule, injected nanoparticles coated with TSHR A-subunit protein enhanced and accelerated development of pathogenic TSHR Abs in hTSHR/NOD. NOD.H2h4 These findings emphasize the need for sufficient TSHR A-subunit protein to activate the immune system and the generation of stimulatory TSHR Abs in genetically predisposed individuals.

Thyroid Hemiagenesis in a Thyroiditis Prone Mouse Strain.

BACKGROUND: Thyroid hemiagenesis, a rare congenital condition detected by ultrasound screening of the neck, is usually not manifested clinically in humans. This condition has been reported in mice with hypothyroidism associated with induced deficiency in paired box 8 and NK2 homeobox 1, sonic hedgehog, or T-box 1. Unexpectedly, we observed thyroid hemiagenesis in NOD.H2h4 mice, an unusual strain that spontaneously develops iodide enhanced thyroid autoimmunity but remains euthyroid. OBJECTIVES AND METHODS: First, to compare mice with thyroid hemiagenesis versus bilobed littermates for serum T4, autoantibodies to thyroglobulin (ELISA) and thyroid peroxidase (TPO; flow cytometry with eukaryotic cells expressing mouse TPO), gross anatomy, and thyroid histology; second, to estimate the percentage of mice with thyroid hemiagenesis in the NOD.H2h4 mice we have studied over 6 years. RESULTS: Thyroid hemiagenesis was observed in 3 of 1,025 NOD.H2h4 mice (2 females, 1 male; 0.3$). Two instances of hemiagenesis were in wild-type females and one in a transgenic male expressing the human TSHR A-subunit in the thyroid. Two mice had very large unilobed glands, as in some human cases with this condition. Thyroid lymphocytic infiltration, serum T4, and the levels of thyroid autoantibodies were similar in mice with thyroid hemiagenesis and bilobed littermates. CONCLUSIONS: Unlike hypothyroidism associated with hemiagenesis in transcription factor knockout mice, hemiagenesis in euthyroid NOD.H2h4 mice occurs spontaneously and is phenotypically similar to that occasionally observed in humans.

Reflections on Thyroid Autoimmunity: A Personal Overview from the Past into the Future.

After investigating thyroid autoimmunity for more than 40 years, we present a personal perspective on the field. Despite effective therapies for Graves' hyperthyroidism and Hashimoto's thyroiditis, cures are elusive. Novel forms of therapy are being developed, such as small molecule inhibitors of the TSH receptor (TSHR), but cure will require immunotherapy. This goal requires advances in understanding the pathogenesis of thyroid autoimmunity, the 'keys' for which are the thyroid antigens themselves. Presently, however, greater investigative focus is on non-thyroid specific immune cell types and molecules. Thyroid autoantigens are the drivers of the autoimmune response, a prime example being the TSHR. In our view, the TSHR is the culprit as well as the victim in Graves' disease because of its unique structure. Unlike the closely related gonadotropin receptors, the TSHR cleaves into subunits and there is strong evidence that its shed extracellular A-subunit, not the holoreceptor, is the major antigen driving pathogenic thyroid stimulating autoantibodies (TSAb) development. There is no Graves' disease of the gonads. Studies of potential antigen-specific immunotherapies require an animal model. Such models have been developed in which TSAb can be induced or, more importantly, arise spontaneously. Not appreciated until recently by thyroid investigators is that B cell surface autoantibodies are highly efficient 'antigen receptors' and the epitope to which an autoantibody binds influences antigen processing and which peptide is presented to T cells. These animal models and recombinant human autoantibodies cloned from Graves' and Hashimoto's B cells (plasma cells) are available for study by future generations.

Structure of a Thyrotropin Receptor Monoclonal Antibody Variable Region Provides Insight into Potential Mechanisms for its Inverse Agonist Activity.

BACKGROUND: The high constitutive, or ligand-independent, activity of the thyrotropin receptor (TSHR) is of clinical importance in some thyroid conditions, particularly well-differentiated thyroid carcinoma remnants following incomplete ablative therapy (surgery and radioiodine). Under these conditions, even total suppression of TSH by thyroid hormone administration does not fully reduce TSHR activity, a driver of thyrocyte growth. METHODS: CS-17 is a murine monoclonal antibody that has inverse agonist activity in that it suppresses TSHR constitutive activity. This study crystallized the CS-17 Fab and determined its atomic structure at a resolution of 3.4 Å. RESULTS: In silico docking of this structure to that of the TSHR extracellular domain was accomplished by targeting to TSHR residue tyrosine 195 (Y195) known to contribute to the CS-17 epitope. High affinity interaction between these two molecules, primarily by the CS-17 immunoglobulin heavy chain, was validated by energetic analysis (KD of 8.7 × 10-11 M), as well as by previously obtained data on a number of individual TSHR amino acids in three regions whose mutagenesis reduced CS-17 binding as detected by flow cytometry. CONCLUSIONS: Structural insight at atomic resolution of a TSHR antibody with inverse agonist activity opens the way for the development of a molecule with therapeutic potential, particularly in thyroid carcinoma. For this purpose, CS-17 will require "humanization" by substitution of its constant region (Fc component). In addition, with its epitope defined, the CS-17 affinity can be increased further by mutagenesis of selected amino acids in its heavy- and light-chain complementarity determining regions.

Aberrant Iodine Autoregulation Induces Hypothyroidism in a Mouse Strain in the Absence of Thyroid Autoimmunity.

We investigated factors underlying the varying effects of a high dietary iodide intake on serum T4 levels in a wide spectrum of mouse strains, including thyroiditis-susceptible NOD.H2h4, NOD.H2k, and NOD mice, as well as other strains (BALB/c, C57BL/6, NOD.Lc7, and B10.A4R) not previously investigated. Mice were maintained for up to 8 months on control or iodide-supplemented water (NaI 0.05%). On iodized water, serum T4 was reduced in BALB/c (males and females) in association with colloid goiters but was not significantly changed in mice that developed thyroiditis, namely NOD.H2h4 (males and females) or male NOD.H2k mice. Neither goiters nor decreased T4 developed in C57BL/6, NOD, NOD.Lc7, or B10.A4R female mice. In further studies, we focused on males in the BALB/c and NOD.H2h4 strains that demonstrated a large divergence in the T4 response to excess iodide. Excess iodide ingestion increased serum TSH levels to the same extent in both strains, yet thyroidal sodium iodide symporter (NIS) messenger RNA (mRNA) levels (quantitative polymerase chain reaction) revealed greatly divergent responses. NOD.H2h4 mice that remained euthyroid displayed a physiological NIS iodine autoregulatory response, whereas NIS mRNA was inappropriately elevated in BALB/c mice that became hypothyroid. Thus, autoimmune thyroiditis-prone NOD.H2h4 mice adapted normally to a high iodide intake, presumably by escape from the Wolff-Chaikoff block. In contrast, BALB/c mice that did not spontaneously develop thyroiditis failed to escape from this block and became hypothyroid. These data in mice may provide insight into the mechanism by which iodide-induced hypothyroidism occurs in some humans without an underlying thyroid disorder.

Variable Effects of Dietary Selenium in Mice That Spontaneously Develop a Spectrum of Thyroid Autoantibodies.

Selenium (Se) is a critical element in thyroid function, and variable dietary Se intake influences immunity. Consequently, dietary Se could influence development of thyroid autoimmunity and provide an adjunct to treat autoimmune thyroid dysfunction. Nonobese diabetic (NOD).H2h4 mice spontaneously develop autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). This mouse strain expressing a human thyroid-stimulating hormone receptor (TSHR) A-subunit transgene in the thyroid also develops pathogenic TSHR autoantibodies. In this report, we investigated whether dietary Se influences these immune processes. Male and female wild-type and transgenic NOD.H2h4 mice were maintained on normal-, low-, or high-Se (0.1, 0, or 1.0 mg/kg) rodent diets. After 4 months, Se serum levels were extremely low or significantly increased on 0 or 1.0 mg/kg Se, respectively. Varying Se intake affected Tg antibody (TgAb) levels after 2 (but not 4) months; conversely, TPO antibody (TPOAb) levels were altered by dietary Se after 4 (but not 2) months. These data correspond to the earlier development of TgAb than TPOAb in NOD.H2h4 mice. In males, TgAb levels were enhanced by high Se and in females by low Se intake. Se intake had no effect on pathogenic TSHR autoantibodies in TSHR transgenic NOD.H2h4 females. In conclusion, in susceptible NOD.H2h4 mice, we found no evidence that a higher dietary Se intake ameliorates thyroid autoimmunity by reducing autoantibodies to Tg, TPO, or the TSHR. Instead, our finding that low dietary Se potentiates the development of autoantibodies to Tg and TPO in females is consistent with reports in humans of an increased prevalence of autoimmune thyroiditis in low-Se regions.

Mechanisms of Autoantibody-Induced Pathology.

Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves' disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein.

Genes Outside the Major Histocompatibility Complex Locus Are Linked to the Development of Thyroid Autoantibodies and Thyroiditis in NOD.H2h4 Mice.

Thyroiditis and autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) develop spontaneously in NOD.H2h4 mice, a phenotype enhanced by dietary iodine. NOD.H2h4 mice were derived by introducing the major histocompatibility class (MHC) molecule I-Ak from B10.A(4R) mice to nonobese diabetic (NOD) mice. Apart from I-Ak, the genes responsible for the NOD.H2h4 phenotype are unknown. Extending serendipitous observations from crossing BALB/c to NOD.H2h4 mice, thyroid autoimmunity was investigated in both genders of the F1, F2, and the second-generation backcross of F1 to NOD.H2h4 (N2). Medium-density linkage analysis was performed on thyroid autoimmunity traits in F2 and N2 progeny. TgAb develop before TPOAb and were measured after 8 and 16 weeks of iodide exposure; TPOAb and thyroiditis were studied at 16 weeks. TgAb, TPOAb, and thyroiditis, absent in BALB/c and F1 mice, developed in most NOD.H2h4 and in more N2 than F2 progeny. No linkages were observed in F2 progeny, probably because of the small number of autoantibody-positive mice. In N2 progeny (equal numbers of males and females), a chromosome 17 locus is linked to thyroiditis and TgAb and is suggestively linked to TPOAb. This locus includes MHC region genes from B10.A(4R) mice (such as I-Ak and Tnf, the latter involved in thyrocyte apoptosis) and genes from NOD mice such as Satb1, which most likely plays a role in immune tolerance. In conclusion, MHC and non-MHC genes, encoded within the chromosome 17 locus from both B10.A(4R) and NOD strains, are most likely responsible for the Hashimoto disease-like phenotype of NOD.H2h4 mice.

Thyroid Autoantibodies Display both "Original Antigenic Sin" and Epitope Spreading.

Evidence for original antigenic sin in spontaneous thyroid autoimmunity is revealed by autoantibody interactions with immunodominant regions on thyroid autoantigens, thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyrotropin receptor (TSHR) A-subunit. In contrast, antibodies induced by immunization of rabbits or mice recognize diverse epitopes. Recognition of immunodominant regions persists despite fluctuations in autoantibody levels following treatment or over time. The enhancement of spontaneously arising pathogenic TSHR antibodies in transgenic human thyrotropin receptor/NOD.H2h4 mice by injecting a non-pathogenic form of TSHR A-subunit protein also provides evidence for original antigenic sin. From other studies, antigen presentation by B cells, not dendritic cells, is likely responsible for original antigenic sin. Recognition of restricted epitopes on the large glycosylated thyroid autoantigens (60-kDa A-subunit, 100-kDa TPO, and 600-kDa Tg) facilitates exploring the amino acid locations in the immunodominant regions. Epitope spreading has also been revealed by autoantibodies in thyroid autoimmunity. In humans, and in mice that spontaneously develop autoimmunity to all three thyroid autoantigens, autoantibodies develop first to Tg and later to TPO and the TSHR A-subunit. The pattern of intermolecular epitope spreading is related in part to the thyroidal content of Tg, TPO and TSHR A-subunit and to the molecular sizes of these proteins. Importantly, the epitope spreading pattern provides a rationale for future antigen-specific manipulation to block the development of all thyroid autoantibodies by inducing tolerance to Tg, first in the autoantigen cascade. Because of its abundance, Tg may be the autoantigen of choice to explore antigen-specific treatment, preventing the development of pathogenic TSHR antibodies.

Critical Differences between Induced and Spontaneous Mouse Models of Graves' Disease with Implications for Antigen-Specific Immunotherapy in Humans.

Graves' hyperthyroidism, a common autoimmune disease caused by pathogenic autoantibodies to the thyrotropin (TSH) receptor (TSHR), can be treated but not cured. This single autoantigenic target makes Graves' disease a prime candidate for Ag-specific immunotherapy. Previously, in an induced mouse model, injecting TSHR A-subunit protein attenuated hyperthyroidism by diverting pathogenic TSHR Abs to a nonfunctional variety. In this study, 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 (hTSHR) A-subunit transgene expressed in the thyroid and (shown in this article) the thymus. We hypothesized that such diversion would occur after injection of "inactive" hTSHR A-subunit protein recognized only by nonpathogenic (not pathogenic) TSHR Abs. Surprisingly, rather than attenuating the pre-existing pathogenic TSHR level, in TSHR/NOD.H2h4 mice inactive hTSHR Ag injected without adjuvant enhanced the levels of pathogenic TSH-binding inhibition and thyroid-stimulating Abs, as well as nonpathogenic Abs detected by ELISA. This effect was TSHR specific because spontaneously occurring autoantibodies to thyroglobulin and thyroid peroxidase were unaffected. As controls, nontransgenic NOD.H2h4 mice similarly injected with inactive hTSHR A-subunit protein unexpectedly developed TSHR Abs, but only of the nonpathogenic 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 nonpathogenic form. Indeed, such therapy is likely to enhance pathogenic Ab production and exacerbate Graves' disease in humans.

Withdrawn: TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective.

The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with lossofaC-peptideregion. The potential pathophysiological importance of TSHR cleavage into A-and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling. (Endocrine Reviews 37: 114-134, 2016).

TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective.

The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling.

Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease.

CONTEXT: The TSH receptor (TSHR) A-subunit shed from the cell surface contributes to the induction and/or affinity maturation of pathogenic TSHR autoantibodies in Graves' disease. OBJECTIVE: This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation. DESIGN: The isolated TSHR A-subunit generated by transfected mammalian cells exists in two forms; one (active) is recognized only by Graves' TSHR autoantibodies, the second (inactive) is recognized only by mouse monoclonal antibody (mAb) 3BD10. Recent evidence suggests that both Graves' TSHR autoantibodies and mAb 3BD10 recognize the A-subunit monomer. Therefore, if the A-subunit monomer is an immunogen, Graves' sera should have antibodies to both active and inactive A-subunits. Conversely, restriction of TSHR autoantibodies to active A-subunits would be evidence of a role for shed A-subunit multimers, not monomers, in the pathogenesis of Graves' disease. Therefore, we tested a panel of Graves' sera for their relative recognition of active and inactive A-subunits. RESULTS: Of 34 sera from unselected Graves' patients, 28 were unequivocally positive in a clinical TSH binding inhibition assay. None of the latter sera, as well as 8/9 sera from control individuals, recognized inactive A-subunits on ELISA. In contrast to Graves' sera, antibodies induced in mice, not by shedding from the TSHR holoreceptor, but by immunization with adenovirus expressing the free human A-subunit, were directed to both the active and inactive A-subunit forms. CONCLUSIONS: The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers.

Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells.

The TSH receptor (TSHR) extracellular domain (ECD) comprises a N-terminal leucine-rich repeat domain and an hinge region (HR), the latter contributing to ligand binding and critical for receptor activation. The crystal structure of the leucine-rich repeat domain component has been solved, but previous attempts to generate conformationally intact complete ECD or the isolated HR component for structural analysis have failed. The TSHR HR contains a C-peptide segment that is removed during spontaneous TSHR intramolecular cleavage into disulfide linked A- and B-subunits. We hypothesized that deletion of the redundant C-peptide would overcome the obstacle to generating conformationally intact TSHR ECD protein. Indeed, lacking the C-peptide region, the TSHR ECD (termed ECD-D1) and the isolated HR (termed HR-D1) were secreted into medium of insect cells infected with baculoviruses coding for these modified proteins. The identities of TSHR ECD-D1 and HR-D1 were confirmed by ELISA and immunoblotting using TSHR-specific monoclonal antibodies. The TSHR-ECD-D1 in conditioned medium was folded correctly, as demonstrated by its ability to inhibit radiolabeled TSH binding to the TSH holoreceptor. The TSHR ECD-D1 purification was accomplished in a single step using a TSHR monoclonal antibody affinity column, whereas the HR-D1 required a multistep protocol with a low yield. In conclusion, we report a novel approach to generate the TSHR ECD, as well as the isolated HR in insect cells, the former in sufficient amounts for structural studies. However, such studies will require previous complexing of the ECD with a ligand such as TSH or a thyroid-stimulating antibody.

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.H2(h4) mice associated with thyroglobulin and thyroid-peroxidase, but not TSHR, Abs. We hypothesized that transferring the human TSHR A-subunit to NOD.H2(h4) mice would result in loss of tolerance to this protein. BALB/c human TSHR A-subunit mice were bred to NOD.H2(h4) mice, and transgenic offspring were repeatedly backcrossed to NOD.H2(h4) mice. All offspring developed Abs to thyroglobulin and thyroid-peroxidase. However, only TSHR-transgenic NOD.H2(h4) mice (TSHR/NOD.H2(h4)) developed pathogenic TSHR Abs as detected using clinical Graves' disease assays. As in humans, TSHR/NOD.H2(h4) 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.H2(h4) 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.