Hormone- and antibody-mediated activation of the thyrotropin receptor.

Thyroid-stimulating hormone (TSH), through activation of its G-protein-coupled thyrotropin receptor (TSHR), controls the synthesis of thyroid hormone-an essential metabolic hormone1-3. Aberrant signalling of TSHR by autoantibodies causes Graves' disease (hyperthyroidism) and hypothyroidism, both of which affect millions of patients worldwide4. Here we report the active structures of TSHR with TSH and the activating autoantibody M225, both bound to the allosteric agonist ML-1096, as well as an inactivated TSHR structure with the inhibitory antibody K1-707. Both TSH and M22 push the extracellular domain (ECD) of TSHR into an upright active conformation. By contrast, K1-70 blocks TSH binding and cannot push the ECD into the upright conformation. Comparisons of the active and inactivated structures of TSHR with those of the luteinizing hormone/choriogonadotropin receptor (LHCGR) reveal a universal activation mechanism of glycoprotein hormone receptors, in which a conserved ten-residue fragment (P10) from the hinge C-terminal loop mediates ECD interactions with the TSHR transmembrane domain8. One notable feature is that there are more than 15 cholesterols surrounding TSHR, supporting its preferential location in lipid rafts9. These structures also highlight a similar ECD-push mechanism for TSH and autoantibody M22 to activate TSHR, therefore providing the molecular basis for Graves' disease.

Autoantibody mimicry of hormone action at the thyrotropin receptor.

Thyroid hormones are vital in metabolism, growth and development1. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)2. In patients with Graves' disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity3. How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves' disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains.

TSH-TSHR axis promotes tumor immune evasion.

BACKGROUND: Hormones are identified as key biological variables in tumor immunity. However, previous researches mainly focused on the immune effect of steroid hormones, while the roles that thyroid-stimulating hormone (TSH) played in the antitumor response were far from clear. METHODS: The source of TSH was determined using single-cell transcriptomic, histologic, quantitative PCR, and ELISA analysis. The influence of TSH on tumor proliferation, invasion, and immune evasion was evaluated in multiple cell lines of thyroid cancer, glioma, and breast cancer. Then transcriptomic sequencing and cellular experiments were used to identify signaling pathways. TSH receptor (TSHR) inhibitor was injected into homograft mouse tumor models with or without anti-programmed cell death protein-1 antibody. RESULTS: Monocyte-derived dendritic cells (moDCs) highly expressed TSHα and TSHß2 and were the primary source of TSH in the tumor microenvironment. TSH released by moDCs promoted proliferation and invasion of tumors with high TSHR expressions, such as thyroid cancers and glioma. TSH also induced tumor programmed death-ligand 1 (PD-L1) expression through the TSHR-AC-PKA-JNK-c-JUN pathway. TSHR inhibitors reversed tumor immune evasion by inhibiting PD-L1 expression in tumor and myeloid cells and enhancing Teff activation. CONCLUSIONS: TSH-TSHR axis promotes tumor evasion in thyroid cancers and glioma. TSH suppression therapy is an effective therapeutic strategy for combination in immune checkpoint blockades.

Clinical value of functional thyrotropin receptor antibodies in Serbian patients with Graves' orbitopathy.

PURPOSE: Thyrotropin receptor autoantibodies (TSH-R-Ab) are heterogeneous in their biological function and play a significant role in the pathophysiology of both Graves' disease and Graves' orbitopathy (GO). The clinical significance and utility of determining functional TSH-R-Ab in a Serbian collective were evaluated. METHODS: 91 consecutive patients with GO were included in this study. Total TSH-R-Ab concentration, referred to as TSH-R binding inhibitory immunoglobulins (TBII) was detected using a competitive-binding immunoassay. Stimulating and blocking TSH-R-Ab (TSAb and TBAb) were measured with cell-based bioassays. RESULTS: Stimulating TSAb activity and TBII positivity were detected in 85 of 91 (93.4%) and 65 of 91 (71.4%) patients with GO (P < 0.001). Blocking TBAb activity was observed in only one patient who expressed dual stimulating and blocking TSH-R-Ab activity. The sensitivity rates for differentiating between clinically active versus inactive and mild versus moderate-to-severe GO were 100% and 100% for TSAb, respectively. In contrast, these were 82% and 87% only for TBII. Seven of eight (87.5%) and one of eight (12.5%) euthyroid patients with GO were TSAb and TBII positive, respectively (P < 0.031). TSAb serum levels significantly predicted GO activity compared to TBII (odds ratio, OR, 95%CI: 3.908, 95%CI 1.615-9.457, P = 0.003; versus 2.133, 0.904-5.032, P = 0.084, univariate analysis; and OR 4.341, 95%CI 1.609-11.707, P = 0.004; versus 2.337, 0.889-6.145, P = 0.085 multivariate analysis). CONCLUSION: Stimulating TSAb are highly prevalent in patients with GO and show superior clinical characteristics and predictive potential compared to the traditionally used TBII.

Evaluation of analytic and clinical performance of two immunoassays for detecting thyroid-stimulating receptor antibody in the diagnosis of Graves' disease.

OBJECTIVE: To evaluate the analytical and clinical performance of two immunoassays for diagnosis of Graves' disease (GD), the Immulite thyroid-stimulating immunoglobulin (TSI), and Elecsys Anti-TSH receptor (TSHR) assay. METHODS: Precision and analytical measurement range were assessed using pooled samples of patients. The comparison between the two methods was evaluated using 579 clinical samples, and receiver operating characteristic (ROC) curves were drawn using the final diagnosis as reference. Clinical sensitivity and specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the two tests. RESULTS: The repeatability and intermediate imprecision coefficient of variation (CV%) of the TSI assay were 3.8% and 4.1% at 0.95 IU/L, and 3.5% and3.6% at 19.5 IU/L, respectively. The assays were linear over a range 0.27-38.5 IU/L. There was a high correlation between the quantitative results of the two methods (correlation coefficient r = 0.930). The cut-off value obtained by ROC analysis for TSI assay was 0.7 IU/L with sensitivity of 93.7% and specificity of 85.1%. An overall qualitative agreement of 91.5% between two methods was observed. Among 44 patients with discordant qualitative results, the TSI assay provided more satisfactory results consistent with clinical diagnoses. CONCLUSION: The TSI assay showed excellent analytical performance and provided a high PPV for GD.

Non-Conventional Clinical Uses of TSH Receptor Antibodies: The Case of Chronic Autoimmune Thyroiditis.

Anti TSH receptor antibodies (TSHrAb) are a family of antibodies with different activity, some of them stimulating thyroid function (TSAb), others with blocking properties (TBAb), it is a common finding that antibodies with different function might coexist in the same patient and can modulate the function of the thyroid. However, most of the labs routinely detect all antibodies binding to the TSH receptor (TRAb, i.e. TSH-receptor antibodies detected by binding assay without definition of functional property). Classical use of TSHr-Ab assay is in Graves' disease where they are tested for diagnostic and prognostic issues; however, they can be used in specific settings of chronic autoimmune thyroiditis (CAT) as well. Aim of the present paper is to highlight these conditions where detection of TSHr-Ab can be of clinical relevance. Prevalence of TSHrAb is different in in the 2 main form of CAT, i.e. classical Hashimoto's thyroiditis and in atrophic thyroiditis, where TBAb play a major role. Simultaneous presence of both TSAb and TBAb in the serum of the same patient might have clinical implication and cause the shift from hyperthyroidism to hypothyroidism and vice versa. Evaluation of TRAb is recommended in case of patients with Thyroid Associated Orbitopathy not associated with hyperthyroidism. At present, however, the most relevant recommendation for the use of TRAb assay is in patients with CAT secondary to a known agent; in particular, after treatment with alemtuzumab for multiple sclerosis. In conclusion, the routine use of anti-TSH receptor antibodies (either TRAb or TSAb/TBAb) assay cannot be suggested at the present for diagnosis/follow up of patients affected by CAT; there are, however, several conditions where their detection can be clinically relevant.

Regulatory B Cells Involvement in Autoimmune Phenomena Occurring in Pediatric Graves' Disease Patients.

Graves's disease is the most common type of autoimmune hyperthyroidism. Numerous studies indicate different factors contributing to the onset of the disease. Despite years of research, the exact pathomechanism of Graves' disease still remains unresolved, especially in the context of immune response. B cells can play a dual role in autoimmune reactions, on the one hand, as a source of autoantibody mainly targeted in the thyroid hormone receptor (TSHR) and, on the other, by suppressing the activity of proinflammatory cells (as regulatory B cells). To date, data on the contribution of Bregs in Graves' pathomechanism, especially in children, are scarce. Here, we investigated the frequencies of Bregs before and during a methimazole therapy approach. We reported higher Foxp3+ and IL-10+ Breg levels with CD38- phenotype and reduced numbers of CD38 + Foxp3 + IL-10+ in pediatric Graves' patients. In addition, selected Breg subsets were found to correlate with TSH and TRAb levels significantly. Noteworthy, certain subpopulations of Bregs were demonstrated as prognostic factors for methimazole therapy outcome. Our data demonstrate the crucial role of Bregs and their potential use as a biomarker in Graves' disease management.

Predicting the Course of Graves' Orbitopathy Using Serially Measured TSH-Receptor Autoantibodies by Automated Binding Immunoassays and the Functional Bioassay.

The aim of the study was to investigate the use of serial measurements of TSH-receptor autoantibodies (TRAb) with the newest available assay technology to predict the course of Graves' Orbitopathy (GO) during the first 24 months from disease onset. Serial serum samples from patients with GO (103 mild/135 severe) were collected between 2007 and 2017 and retrospectively analyzed. The course of GO were classified into mild/severe 12 months after manifestation (severe: NOSPECS≥5; mild<5). TRAb were measured with automated binding immunoassays (IU/l): TRAb Elecsys (Cobas, Roche), TRAb bridge assay (IMMULITE, Siemens), and a cell-based bioassay (percent of specimen to reference ratio - SRR%) (Thyretain, Quidel). Variable cut off levels of measured TRAb were calculated at specificity of 90% from receiver operator curve (ROC) analysis for several timepoints during the course of GO. To select one: 5-8 months after first GO symptoms, which is the timepoint for usual referals for treatment mild course could be predicted at cut offs of 1.5 IU/l (Elecsys), 0.8 IU/l (Immulite) and 402% SRR (Thyretain) and the risc of severe course has to be anticipated if TRAb are above 11.6 IU/l (Elecsys), 6.5 IU/l (Thyretain), and 714% SRR (Thyretain). The Thyretain bioassay showed the highest diagnostic sensitivity (using the commercial cut off's) over the entire follow up period. TRAb measurements during the 24-month follow up of GO provide added value to the GO clinical activity and severity scores and should be used especially in the event of an unclear decision-taking situation with regard to therapy.

Identification of Two Different Phenotypes of Patients with Amiodarone-Induced Thyrotoxicosis and Positive Thyrotropin Receptor Antibody Tests.

Introduction: Serum thyrotropin (TSH) receptor antibodies (TRAbs) are occasionally found in patients with amiodarone-induced thyrotoxicosis (AIT), and usually point to a diagnosis of type 1 AIT (AIT1) due to Graves' disease (GD). However, the TRAb role and function in AIT have not been clarified. Methods: A retrospective cohort study of 309 AIT patients followed at a single academic center over a 30-year period. AIT TRAb-positive patients (n = 21, 7% of all cases) constituted the study group; control groups consisted of type 2 AIT (AIT2) TRAb-negative patients (n = 233), and 100 non-AIT patients with GD. Clinical and biochemical data at diagnosis and during the course of disease were compared. Histological samples of patients who had total thyroidectomy were reviewed. Stored serum samples were used for a functional assay of TRAb class G immunoglobulins (IgGs) in Chinese hamster ovary (CHO) cells stably transfected with complementary DNA encoding for the TSH receptor. Results: TRAb-positive patients were grouped according to color flow Doppler sonography, radioactive iodine thyroid uptake, and duration of amiodarone therapy before thyrotoxicosis in type 1 (n = 9, 43%; TRAb1) or type 2 (n = 12, 57%; TRAb2) AIT. TRAb1 patients had clinical and biochemical features indistinguishable from GD controls, and were responsive to methimazole. Conversely, TRAb2 patients had clinical features similar to AIT2 controls, and were responsive to glucocorticoids, but not to methimazole. The CHO cell functional assay demonstrated that TRAb1 IgGs had a stimulatory effect on cyclic AMP production, which was absent in TRAb2 IgGs. Pathology in TRAb1 showed hyperplastic thyroid follicles and mild lymphocyte infiltration, reflecting thyroid stimulation. On the contrary, TRAb2 samples revealed follicle destruction, macrophage infiltration, and sometimes fibrosis, consistent with a destructive process. Conclusions: Almost 60% of TRAb-positive AIT patients had a destructive thyroiditis. TRAb-positive tests in AIT patients do thus not necessarily imply a diagnosis of GD and AIT1, and should be evaluated in the clinical and biochemical setting of each AIT patient and confirmed by measuring thyroid-stimulating immunoglobulins.

Insulin-Like Growth Factor Pathway and the Thyroid.

The insulin-like growth factor (IGF) pathway comprises two activating ligands (IGF-I and IGF-II), two cell-surface receptors (IGF-IR and IGF-IIR), six IGF binding proteins (IGFBP) and nine IGFBP related proteins. IGF-I and the IGF-IR share substantial structural and functional similarities to those of insulin and its receptor. IGF-I plays important regulatory roles in the development, growth, and function of many human tissues. Its pathway intersects with those mediating the actions of many cytokines, growth factors and hormones. Among these, IGFs impact the thyroid and the hormones that it generates. Further, thyroid hormones and thyrotropin (TSH) can influence the biological effects of growth hormone and IGF-I on target tissues. The consequences of this two-way interplay can be far-reaching on many metabolic and immunologic processes. Specifically, IGF-I supports normal function, volume and hormone synthesis of the thyroid gland. Some of these effects are mediated through enhancement of sensitivity to the actions of TSH while others may be independent of pituitary function. IGF-I also participates in pathological conditions of the thyroid, including benign enlargement and tumorigenesis, such as those occurring in acromegaly. With regard to Graves' disease (GD) and the periocular process frequently associated with it, namely thyroid-associated ophthalmopathy (TAO), IGF-IR has been found overexpressed in orbital connective tissues, T and B cells in GD and TAO. Autoantibodies of the IgG class are generated in patients with GD that bind to IGF-IR and initiate the signaling from the TSHR/IGF-IR physical and functional protein complex. Further, inhibition of IGF-IR with monoclonal antibody inhibitors can attenuate signaling from either TSHR or IGF-IR. Based on those findings, the development of teprotumumab, a ß-arrestin biased agonist as a therapeutic has resulted in the first medication approved by the US FDA for the treatment of TAO. Teprotumumab is now in wide clinical use in North America.

Blocking the Thyrotropin Receptor with K1-70 in a Patient with Follicular Thyroid Cancer, Graves' Disease, and Graves' Ophthalmopathy.

Background: We report the therapeutic use of K1-70™, a thyrotropin receptor (TSHR) antagonist monoclonal antibody, in a patient with follicular thyroid cancer (FTC), Graves' disease (GD), and Graves' ophthalmopathy (GO). Methods: A 51-year-old female patient, who smoked, presented in October 2014 with FTC complicated by GD, high levels of TSHR autoantibodies with high thyroid stimulating antibody (TSAb) activity, and severe GO. K1-70 was administered at 3 weekly intervals with the dose adjusted to block TSAb activity. Her cancer was managed with lenvatinib and radioiodine therapy. Results: Following initiation of K1-70 therapy, TSAb activity measured in serum decreased and GO (proptosis and inflammation) improved. On K1-70 monotherapy during the pause in lenvatinib, several metastatic lesions stabilized while others showed progression attenuation compared with that before lenvatinib therapy. Conclusions: These observations suggest that blocking TSHR stimulation with K1-70 can be an effective treatment for GO and may also benefit select patients with FTC and GD.

A cyclic peptide significantly improves thyroid function, thyrotropin-receptor antibodies and orbital mucine /collagen content in a long-term Graves' disease mouse model.

BACKGROUND: BALB/c mice which received long-term immunizations of adenovirus (Ad) expressing thyrotropin receptor A-subunits (TSHR) developed stable Graves' disease (GD). TSHR-derived cyclic peptide 19 (P19) was identified as effective therapy in this model. METHODS: In Ad-TSHR mice, we investigated shorter disease intervals up to 4 months for histological alterations of the orbits, fine tuning of anti-TSHR antibodies (Ab) and free thyroxine (fT4) hormone levels by using novel detection methods in an independent laboratory. Therapy (0.3 mg/kg P19 or vehicle) was given intravenously after the fourth Ad-TSHR immunization (week 11) and continued until week 19. RESULTS: Thyrotropin binding inhibitory immunoglobulins (TBII, bridge immunoassay), blocking (TBAb) and stimulating (TSAb) TSHR-Ab (both cell-based bioassays) and serum levels of fT4 were significantly elevated at week 11 in Ad-TSHR-immunized mice versus none in control mice. For the first time, TSAb, TBAb, and thyroperoxidase-Ab were detected in 17 of 19, 12/19 and 6/19 Ad-TSHR immunized mice, respectively at week 21. Also, for the first time, this study showed that P19 treatment markedly reduced serum TBII (p < 0.0001), serum fT4 (p = 0.02), and acidic mucins and collagen content in the orbital tissue of Ad-TSHR-immunized mice. CONCLUSION: P19 significantly improved thyroid function, confirming previous results in an independent second laboratory. A relevant shift of anti-TSHR antibody subpopulations in response to P19 therapy may help explain its immunological effects. Moreover, P19 exerted a beneficial effect on mucine and collagen content of orbital tissue. Hence, P19 offers a potential novel therapeutic approach for GD and associated orbitopathy.

Combination Model of Thyrotrophin Receptor Antibody and Volumetric Orbital Apex Crowding Index as an Indicator of Dysthyroid Optic Neuropathy.

BACKGROUND: Dysthyroid optic neuropathy (DON) is one of the most serious vision-threatening complications of thyroid eye disease (TED); however, accurate and established diagnostic tools for DON are yet lacking. The present study was aimed at identifying new diagnostic factors for the accurate diagnosis of DON. METHODS: This retrospective cross-sectional study included 25 TED patients (50 eyes) with enlarged extraocular muscles, no previous anti-inflammatory therapy, and the absence of other vision-affecting diseases between May 2017 and August 2019. Baseline data, such as gender, age, ophthalmological history, thyroid disease and management, TED history including clinical features, management, and long-term results, ophthalmological examinations, serology examinations, and single-photon emission computed tomography/computed tomography (SPECT/CT) results, were extracted. The diagnostic criteria were as follows: (1) best-corrected visual acuity (BCVA) loss coexisting with either of the following-increased latency or reduction of amplitude on visual evoked potential (VEP), impaired color vision, visual field defects, contrast sensitivity impairment, and optic disk swelling-and (2) Barrett's index ≥ 60% in CT. Univariate and multivariate logistic regression analyses assessed the differences in age, gender, eyes, medical history, clinical activity, thyroid hormone and antibodies, uptake ratio (UR) of extraocular muscles in SPECT/CT, and volumetric orbital apex crowding index (VACI) using the generalized estimation equation. Consequently, the receiver operating characteristic curve (ROC) of the significant factors was constructed. RESULTS: Univariate analysis revealed significant differences in the clinical activity, free triiodothyronine (FT3), free thyroxine (FT4), thyrotrophin receptor antibody (TRAb) levels, the UR of superior and medial rectus, and VACI between DON and TED (without DON) groups. Multivariate regression analysis revealed that TRAb and VACI were significantly different. ROC analysis showed that the univariate models of TRAb or VACI and the multivariate model were effective indicators of DON, while the multivariate model had the highest area under the ROC curve. CONCLUSION: A combination of TRAb and VACI is an effective indicator for DON.

Relationship between autoimmune thyroid disease and nephropathy: A clinicopathological study.

ABSTRACT: The association of nephropathy with autoimmune thyroid disease (AITD) has been reported previously. However, there is limited information on the relationship between thyroid autoantibodies and nephropathy. A retrospective study was conducted using the medical records of 246 patients with nephropathy, 82 of whom had concurrent AITD. General characteristics, thyroid function, autoantibodies, and the pathological types of nephropathy were analyzed. Immunohistochemistry was used to detect the thyroglobulin antibody (TG-Ab) and thyroid peroxidase antibody (TPO-Ab) in the kidneys. We found nephropathy patients with AITD exhibited higher serum levels of TPO-Ab, TG-Ab, thyroid-stimulating hormone receptor antibody (TR-Ab), and immunoglobulin G (IgG) (P < .05). Compared with the nephropathy without AITD group, the nephropathy with AITD group exhibited higher proportions of membranous nephropathy (MN) and focal segmental glomerulosclerosis (FSGS), and relatively lower proportions of mesangial proliferative glomerulonephritis (MsPGN) and minimal change nephropathy (MCN) (P = .005). TPO-Ab and TG-Ab levels in the kidney were more prevalent in nephropathy patients with AITD than those without AITD (P = .015 and P = .026, respectively). Subgroup analysis demonstrated that serum levels of thyroid stimulating hormone (TSH), TG-Ab, TPO-Ab, immunoglobulin M (IgM), and IgG in the MN group were significantly higher, whereas the levels of free thyroxine (FT4) and estimated glomerular filtration rate (eGFR) were lower, as compared with MN with Hashimoto thyroiditis (HT) group (P < .05). TPO-Ab and TG-Ab expression levels in the kidneys were more prevalent in the MN group than in the MN with HT group (P = .034). The expression levels of FT4, TG-Ab, TPO-Ab, and thyroid-stimulating hormone receptor antibody (TSHR-Ab) in the serum were significantly higher in the MN group than in the MN with Graves disease (GD) group (P < .05). The expression of TPO-Ab in the kidneys was more prevalent in the MN group than in the MN with GD group (P = .011). In sum, the expressions of TPO-Ab and TG-Ab were more prevalent in the kidneys of patients with nephropathy and AITD. Our findings indicate that TPO-Ab and TG-Ab may play a role in the development of AITD-related nephropathy.

Cytokines as Targets of Novel Therapies for Graves' Ophthalmopathy.

Graves' disease (GD) is an organ-specific autoimmune disorder of the thyroid, which is characterized by circulating TSH-receptor (TSH-R) stimulating antibodies (TSAb), leading to hyperthyroidism. Graves' ophthalmopathy (GO) is one of GD extra-thyroidal manifestations associated with the presence of TSAb, and insulin-like growth factor-1 receptor (IGF-1R) autoantibodies, that interact with orbital fibroblasts. Cytokines are elevated in autoimmune (i.e., IL-18, IL-6) and non-autoimmune hyperthyroidism (i.e., TNF-α, IL-8, IL-6), and this could be associated with the chronic effects of thyroid hormone increase. A prevalent Th1-immune response (not related to the hyperthyroidism per se, but to the autoimmune process) is reported in the immune-pathogenesis of GD and GO; Th1-chemokines (CXCL9, CXCL10, CXCL11) and the (C-X-C)R3 receptor are crucial in this process. In patients with active GO, corticosteroids, or intravenous immunoglobulins, decrease inflammation and orbital congestion, and are considered first-line therapies. The more deepened understanding of GO pathophysiology has led to different immune-modulant treatments. Cytokines, TSH-R, and IGF-1R (on the surface of B and T lymphocytes, and fibroblasts), and chemokines implicated in the autoimmune process, are possible targets of novel therapies. Drugs that target cytokines (etanercept, tocilizumab, infliximab, adalimumab) have been tested in GO, with encouraging results. The chimeric monoclonal antibody directed against CD20, RTX, reduces B lymphocytes, cytokines and the released autoantibodies. A multicenter, randomized, placebo-controlled, double-masked trial has investigated the human monoclonal blocking antibody directed against IGF-1R, teprotumumab, reporting its effectiveness in GO. In conclusion, large, controlled and randomized studies are needed to evaluate new possible targeted therapies for GO.

Changes in Thyrotropin Receptor Antibody Levels Following Total Thyroidectomy or Radioiodine Therapy in Patients with Refractory Graves' Disease.

Background: The actions of thyrotropin-binding inhibitory immunoglobulins (TBIIs) against thyrotropin receptors in thyroid follicular cells have been studied as important etiological factors in Graves' disease (GD). The purpose of this study was to investigate changes in the TBII levels of patients undergoing total thyroidectomy (TTx) or radioactive iodine (RAI) therapy for GD refractory to antithyroid drugs (ATDs). Methods: We enrolled patients who underwent TTx or RAI for GD with previous ATD use between January 2011 and December 2017 at the Samsung Medical Center in Seoul, Korea. Thorough retrospective reviews of medical records were performed in 130 patients. Results: Patients with goiter, ophthalmopathy, high levels of TBIIs, and high doses of ATDs received TTx. Elderly patients with arrhythmia received RAI. We observed that TBII levels continued to decrease after TTx. On the contrary, TBIIs initially increased for 138 days (estimated median time) and then decreased slowly after RAI. A faster decline in TBII levels was observed in the TTx group than in the RAI group (p < 0.001). The estimated median time for TBIIs to decrease below 4.5 IU (3 × upper normal limit, which is known to be a risk factor for fetal hyperthyroidism) was 318 days in the TTx group and 659 days in the RAI group, respectively. In the RAI group, high levels of TBII (>4.5 IU/L) were present in 70 (82%) at 6 months, 57 (67%) at 1 year, and 3 (3%) at 2 years. In the TTx group, rapid decreases in TBII levels were observed in younger patients and those with lower baseline TBII levels. In the RAI group, smaller thyroid volume was correlated with more rapid decrease in TBII levels. Conclusions: The changes in TBII levels following TTx or RAI were different in patients with refractory GD. When deciding on TTx or RAI, this difference should be considered with patient age, severity of hyperthyroidism, goiter, ophthalmopathy, and future pregnancy plans (for young female patients).

Mechanisms That Underly T Cell Immunity in Graves' Orbitopathy.

Graves' orbitopathy (GO), also known as thyroid-associated ophthalmopathy, is the most common ocular abnormality of Graves' disease. It is a disfiguring, invalidating, and potentially blinding orbital disease mediated by an interlocking and complicated immune network. Self-reactive T cells directly against thyroid-stimulating hormone receptor-bearing orbital fibroblasts contribute to autoimmune inflammation and tissue remodeling in GO orbital connective tissues. To date, T helper (Th) 1 (cytotoxic leaning) and Th2 (antibody leaning) cell subsets and an emerging role of Th17 (fibrotic leaning) cells have been implicated in GO pathogenesis. The potential feedback loops between orbital native residential CD34- fibroblasts, CD34+ infiltrating fibrocytes, and effector T cells may affect the T cell subset bias and the skewed pattern of cytokine production in the orbit, thereby determining the outcomes of GO autoimmune reactions. Characterization of the T cell subsets that drive GO and the cytokines they express may significantly advance our understanding of orbital autoimmunity and the development of promising therapeutic strategies against pathological T cells.

Decreased Treg Cell and TCR Expansion Are Involved in Long-Lasting Graves' Disease.

Graves' disease (GD) is a T cell-mediated organ-specific autoimmune disorder. GD patients who have taken anti-thyroid drugs (ATDs) for more than 5 years with positive anti-thyroid stimulating hormone receptor autoantibodies value were defined as persistent GD (pGD). To develop novel immunotherapies for pGD, we investigated the role of T cells in the long-lasting phase of GD. Clinical characteristics were compared between the pGD and newly diagnosed GD (nGD) (N = 20 respectively). Flow cytometric analysis was utilized to determine the proportions of Treg and Th17 cells (pGD, N = 12; nGD, N = 14). T cell receptor sequencing (TCR-seq) and RNA sequencing (RNA-seq) were also performed (pGD, N = 13; nGD, N = 20). Flow cytometric analysis identified lower proportions of Th17 and Treg cells in pGD than in nGD (P = 0.0306 and P = 0.0223). TCR-seq analysis revealed a lower diversity (P = 0.0025) in pGD. Specifically, marked clonal expansion, represented by an increased percentage of top V-J recombination, was observed in pGD patients. Interestingly, pGD patients showed more public T cell clonotypes than nGD patients (2,741 versus 966). Meanwhile, RNA-seq analysis revealed upregulation of the inflammation and chemotaxis pathways in pGD. Specifically, the expression of pro-inflammatory and chemotactic genes (IL1B, IL13, IL8, and CCL4) was increased in pGD, whereas Th17 and Treg cells associated genes (RORC, CARD9, STAT5A, and SATB1) decreased in pGD. Additionally, TCR diversity was negatively correlated with the expression of pro-inflammatory or chemotactic genes (FASLG, IL18R1, CCL24, and CCL14). These results indicated that Treg dysregulation and the expansion of pathogenic T cell clones might be involved in the long-lasting phase of GD via upregulating chemotaxis or inflammation response. To improve the treatment of pGD patients, ATDs combined therapies, especially those aimed at improving Treg cell frequencies or targeting specific expanded pathogenic TCR clones, are worth exploring in the future.

Teprotumumab: The Dawn of Therapies in Moderate-to-Severe Thyroid-Associated Ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is a potentially sight-threatening ocular disease. About 3-5% of patients with TAO have severe disease with intense pain, inflammation, and sight-threatening corneal ulceration or compressive optic neuropathy. The current treatments of TAO are often suboptimal, mainly because the existing therapies do not target the pathogenesis of the disease. TAO mechanism is unclear. Ocular fibrocytes express relatively high levels of the functional TSH receptor (TSHR), and many indirect evidences support its participation. Over expression of insulin-like growth factor-1 receptor (IGF-IR) in fibroblasts, leading to inappropriate expression of inflammatory factors, production of hyaluronic acid and cell activation in orbital fibroblasts are also possible mechanisms. IGF-1R and TSHR form a physical and functional signaling complex. Inhibition of IGF-IR activity leads to the attenuation of signaling initiated at either receptor. Teprotumumab (TMB) is a human immunoglobulin G1 monoclonal antibody, binding to IGF-IR. Recently two TMB clinical trials had been implemented in TAO patients, indicating dramatic reductions in disease activity and severity, which approved its use for the treatment of TAO in the US. This review summarizes the treatments of TAO, focusing on the pathogenesis of IGF-1R in TAO and its application prospects.

Predicting the Relapse of Hyperthyroidism in Treated Graves' Disease with Orbitopathy by Serial Measurements of TSH-Receptor Autoantibodies.

The aim of this study was to investigate the potential of the new TSH-receptor antibody (TRAb) assays to predict remission or relapse of hyperthyroidism in patients with Graves' disease (GD) and Graves' orbitopathy (GO). TRAbs were measured retrospectively in sera from a cohort of GD patients with GO (n=117; remission n=38 and relapse n=79-Essen GO biobank) with automated binding immunoassays: TRAb Elecsys (Cobas Roche) and TRAb bridge assay (IMMULITE, Siemens), and the TSAb (thyroid stimulating Ab) cell-based bioassay (Thyretain, Quidel Corp.). To identify relapse risk/remission of hyperthyroidism patients were followed up at least 10 months after the end of antithyroid drug therapy (ATD) therapy. ROC plot analysis was performed to calculate cut-off levels of TRAb and TSAb for prediction of relapse and remission of hyperthyroidism. Cut-off serum levels are provided for timepoints around 3, 6, 10, and 15 months after the beginning of ATD. Repeated measurements of TRAb increase the rate of relapses predictions to 60% (Elecsys), 70% (IMMULITE), and 55% (Thyretain). Patients with remission have consistently TRAb levels below the cut off for relapse in repeated measurements. The cell-based bioassay was the most sensitive - and continued to be positive during follow up [at 15 months: 90% vs. 70% (IMMULITE) and 65% (Elecsys)]. Identification of relapsing hyperthyroidism is possible with automated immunoassays and cell-based bioassay especially with serial TRAb measurements during the course of ATD therapy. Patient who need eye surgery may profit from an early decision towards definitive treatment.