Extracellular Domain Of TSH Receptor Research Articles

Practical applications of studies on the TSH receptor and TSH receptor autoantibodies

Studies on the TSH receptor (TSHR) have numerous practical applications in vitro and in vivo. For example human monoclonal autoantibodies (MAbs) to the TSHR are useful reagents for in vitro diagnostics. Measurement of TSHR autoantibodies (TRAbs) is helpful in diagnosis and management of autoimmune thyroid disease. Currently available highly sensitive and specific assays to measure TRAbs use the human TSHR MAb M22 instead of the TSH. Furthermore, preparations of the human TSHR MAb M22 are useful as the World Health Organisation International Standard for thyroid stimulating antibody and for calibration of the assays for measuring TRAbs. Preparations of thermostabilised TSHR extracellular domain have recently become available and this is likely to have an impact on improvements in specificity testing for TRAb assays. In addition the stable TSHR preparations have practical application for specific immunoadsorption of patient serum TRAbs. Human TSHR MAbs also have promising prospects as new therapeutics. Autoantibodies with TSHR antagonistic activities are “natural” inhibitors of TSHR stimulation and are expected to be helpful in controlling TSHR activity in patients with Graves’ disease, Graves’ ophthalmopathy and thyroid cancer.

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.

Non-autoimmune subclinical hypothyroidism due to a mutation in TSH receptor: report on two brothers

Subclinical hypothyroidism (SH) is a condition characterized by a mild persistent thyroid failure. The main cause is represented by autoimmune thyroiditis, but mutations in genes encoding proteins involved in TSH pathway are thought to be responsible for SH, particularly in cases arising in familial settings. Patients with the syndrome of TSH unresponsiveness may have compensated or overt hypothyroidism with a wide spectrum of clinical and morphological alterations depending on the degree of impairment of TSH-receptor (TSH-R) function. We describe the case of two brothers with non autoimmune SH carrying the same heterozygous mutation in the extracellular domain of TSH-R and presenting with different clinical, biochemical and morphological features. The first one had only a slight persistent elevation of TSH, a normal thyroid ultrasound and did never require l- thyroxine (L-T4) replacement treatment. The second one had a neonatal persistent moderate TSH levels increase associated with a thyroid gland hypoplasia and was treated with L-T4 since the first months of life.These two cases support the recent association of TSH-R mutations inheritance as an autosomal dominant pattern with variable expressivity and suggest that the decision to start replacement therapy in patients with persistent SH due to TSH resistance should be individualized.

Severe congenital non-autoimmune hyperthyroidism associated to a mutation in the extracellular domain of thyrotropin receptor gene

Activating mutations in the TSH Receptor (TSHR) gene have been identified as the molecular basis for congenital non-autoimmune hyperthyroidism. We describe the clinical findings and molecular characterization in a girl who presented severe non-autoimmune hyperthyroidism since birth, born to a mother with autoimmune thyroid disease. She was treated with methylmercaptoimidazol and β-blockers, but remained hyperthyroid and required total thyroidectomy. To characterize the presence of an activating mutation, the whole coding sequence and intron-exon boundaries of TSHR gene were analyzed. The patient was heterozygous for p.Ser281Asn mutation and p.Asp727Glu polymorphism. This recurrent mutation, p.Ser281Asn, characterized in vitro by increased basal production of cAMP, is the unique germline activating gene variant described so far in the extracellular domain of TSH receptor. Interestingly, the patient's mother presented hyperthyroidism but without any TSHR gene activating mutation. Although congenital non-autoimmune hyperthyroidism is a rare condition, it should be investigated when severe disease persists, even in a newborn from an autoimmune hyperthyroid mother, in order to differentiate it from the more common congenital autoimmune disease.

Crystal structure of the TSH receptor bound to a blocking type TSHR autoantibody

A complex of the TSH receptor extracellular domain (amino acids 22-260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1.9 Å resolution. K1-70 Fab binds to the concave surface of the TSHR leucine-rich domain (LRD) forming a large interface (2565 Å(2)) with an extensive network of ionic, polar and hydrophobic interactions. Mutation of TSHR or K1-70 residues showing strong interactions in the solved structure influenced the activity of K1-70, indicating that the binding detail observed in the complex reflects interactions of K1-70 with intact, functionally active TSHR. Unbound K1-70 Fab was prepared and crystallised to 2.22 Å resolution. Virtually no movement was observed in the atoms of K1-70 residues on the binding interface compared with unbound K1-70, consistent with 'lock and key' binding. The binding arrangements in the TSHR260-K1-70 Fab complex are similar to previously observed for the TSHR260-M22 Fab complex; however, K1-70 clasps the concave surface of the TSHR LRD in approximately the opposite orientation (rotated 155°) to M22. The blocking autoantibody K1-70 binds more N-terminally on the TSHR concave surface than either the stimulating autoantibody M22 or the hormone TSH, and this may reflect its different functional activity. The structure of TSHR260 in the TSHR260-K1-70 and TSHR260-M22 complexes show a root mean square deviation on all C(α) atoms of only 0.51 Å. These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.

An Inactivating Mutation within the First Extracellular Loop of the Thyrotropin Receptor Impedes Normal Posttranslational Maturation of the Extracellular Domain

The TSH receptor (TSHR), a member of the large family of G protein-coupled receptors, controls both function and growth of thyroid cells; hence, mutations of this receptor result in thyroid dysfunction. Here, we took advantage of the description of a new inactivating TSHR mutation (Q489H) in two brothers with hypothyroidism, to precise maturation, intracellular trafficking, exporting pathways, and activation mechanisms of this receptor. Functional characterization of the Q489H-TSHR in transiently transfected HEK293 cells showed cell surface expression, normal TSH binding affinity, and its inability to generate intracellular cAMP in response to TSH stimulation. Western blot analysis of the whole membrane proteins or proteins expressed at the cell surface showed that Q489H-TSHR expressed in HEK293 transfected cells are restricted to mannose-rich uncleaved receptor. Analysis of the export pathway toward cell surface indicated that both Q489H and wild-type receptors followed the same intracellular route to cell surface throughout endoplasmic reticulum and Golgi apparatus. This study shows that Q489H substitution impedes complete glycosylation of TSHR extracellular domain within the Golgi apparatus and that Q489H-TSHR expressed at the cell surface is unable to undergo intramolecular cleavage as well as to switch toward an active conformation under TSH stimulation. Altogether, our results show that 1) Q489H substitution within the first extracellular loop induces a misfolding of TSHR, blocking it into an inactive conformation and impeding complete glycosylation and intramolecular cleavage, and 2) a misfolded G protein-coupled receptor can bypass endoplasmic reticulum or Golgi apparatus quality control and reach the cell surface as an immature receptor.

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.

Suppression of Thyrotropin Receptor Constitutive Activity by a Monoclonal Antibody with Inverse Agonist Activity

TSH binding to the TSH receptor (TSHR) induces thyrocyte growth and proliferation primarily by activating the adenylyl cyclase signaling pathway. Relative to the other glycoprotein hormone receptors, the TSHR has considerable ligand-independent (constitutive) activity. We describe a TSHR monoclonal antibody (CS-17) with the previously unrecognized property of being an inverse agonist for TSHR constitutive activity. This property is retained, even when constitutive activity is extremely high consequent to diverse TSHR extracellular region mutations. A similar effect on an activating mutation at the base of the sixth transmembrane helix (not accessible to direct CS-17 contact) indicates that CS-17 is acting allosterically. Administered to mice in vivo, CS-17 reduces serum T(4) levels. The CS-17 epitope is conformational and a significant portion lies in the C-terminal region of the TSHR leucine-rich domain (residues 260-289). By interacting with the large TSHR extracellular domain, CS-17 is, to our knowledge, the first antibody reported to be an inverse agonist for a member of the G protein receptor superfamily. After humanization of its murine constant region, CS-17 has the potential to be an adjunctive therapeutic agent in athyreotic patients with residual well-differentiated thyroid carcinoma as well as pending definitive treatment in some selected hyperthyroidism states.

Thyrotropin Receptor Epitopes and Their Relation to Histocompatibility Leukocyte Antigen-DR Molecules in Graves’ Disease

Graves' disease (GD) is characterized by autoimmunity to the TSH receptor (TSHR). We sought to identify T cell epitopes in TSHR that initiate this immune response and their interaction with human histocompatibility leukocyte antigen (HLA) molecules predisposing to GD. We examined the affinity of 31 overlapping peptides spanning the TSHR extracellular domain for binding in vitro to five purified HLA-DR molecules; DRB1*0101 (DR1), DRB1*1501 (DR2), DRB1*0301 (DR3), DRB1*1101 (DR5), and DRB1*0701 (DR7). We scanned the TSHR extracellular domain using a T cell epitope-mapping algorithm, EpiMatrix. We compared these results with clinical studies of GD patients measuring in vitro T cell responses to the peptides. The study was conducted at a university laboratory. Patients included 200 serial adult clinic patients with GD. There were no interventions. Binding affinity of epitopes, predicted affinity, and reported T cell stimulation data were measured. Most peptides bound with intermediate or high affinity to one or more HLA-DR molecule. Peptides binding to HLA-DR3 and HLA-DR5, which predispose to GD, exhibited moderate binding affinities overall, whereas most peptides binding to GD-protective HLA-DR7 bound with high affinity. These differences may relate to T cell selection in the thymus. Binding affinity of peptides correlated strongly with EpiMatrix-predicted affinity for HLA-DRB1*0101, DRB1*1501, DR3, and DRB1*0701 but not HLA-DR5. Average IC(50) values correlated significantly with clinical T cell stimulation data. Three different methods for identifying immunogenic peptides did not provide a uniform picture of important TSHR epitopes. However, peptide 132-150 (GIFNTGLKMFPDLTKVYST) was identified by three methods as an important epitope in GD; the possible importance of peptides 145-163, 158-176, 207-222, 248-263, 272-291, and 343-362 was also identified.

Epitope mapping of tsh receptor-blocking antibodies in Graves' disease that appear during pregnancy.

Spontaneous remission of Graves' disease during pregnancy is thought to be due to a reduction of thyroid-stimulating antibody activity. We suspected, however, that a broader change in TSH receptor antibody characteristics might play an important role in modulating disease activity during pregnancy. We measured TSH binding inhibitory Ig, thyroid-stimulating antibody, and thyroid stimulating-blocking antibody activities in 13 pregnant Graves' disease patients at first, second, and third trimesters and 4 months postpartum. To measure and epitope-map thyroid-stimulating antibody and thyroid stimulating-blocking antibody activities, we used CHO cells transfected with wild-type human TSH receptor or with several TSH receptor-LH/hCG receptor chimeras: Mc1+2, Mc2, and Mc4. These chimeric cells have their respective TSH receptor residues 9-165, 90-165, and 261-370 substituted with equivalent residues of the LH/hCG receptor. Overall thyroid-stimulating antibody decreased, whereas thyroid stimulating-blocking antibody increased progressively during pregnancy. TSH binding inhibitory Ig fluctuated in individual patients, but overall the activities remained statistically unchanged. Thyroid stimulating-blocking antibody appeared in subjects who were either negative for thyroid-stimulating antibody or whose thyroid-stimulating antibody activity increased or decreased during pregnancy. Epitope mapping showed that the thyroid-stimulating antibodies were mainly directed against residues 9-165 of the N-terminus of the TSH receptor extracellular domain. All thyroid stimulating-blocking antibodies had blocking activities against residues 261-370 of the C-terminus of the ectodomain. However, the majority of the thyroid stimulating-blocking antibodies had a hybrid conformational epitope directed against N-terminal residues 9-89 or 90-165 as well. Despite a change in the activity level, we did not observe any change in the epitope of either the stimulatory or blocking Abs as pregnancy advanced. In conclusion, a change in the specificity of TSH receptor antibody from stimulatory to blocking activity was observed during pregnancy, and the appearance of thyroid stimulating-blocking antibody may contribute to the remission of Graves' disease during pregnancy.

Different bioactivities of human thyrotropin receptors with different signal peptides

For investigation of the mechanism and pathogenesis of Graves’ disease, availability of a large amount of functional human thyrotropin receptor (TSHR) capable of recognition by Graves’ autoantibodies is essential. Many attempts have been made to produce the extracellular domain of TSH receptor (TSHRE) in a baculovirus expression system. However, the receptor is expressed as an insoluble form and the refolded protein is often not recognized by the autoantibodies. In this study, we found that the TSHRE expressed with its own signal peptide (VL3-RE) in insect cells is retained inside of the cells and found in both soluble and insoluble fractions in equal proportion. The signal peptide is not removed. The receptor in the soluble fraction is not recognized by either TSH or Graves’ autoantibodies. The TSHRE with an insect-specific mellitin signal peptide (Mel-RE) is also retained inside of the cell and found in both the soluble and insoluble fractions in equal proportion. However, the signal peptide is removed and the receptor is recognized by the Graves’ autoantibodies but not by TSH. Also, the amount of Mel-RE expressed was 5–10-fold higher than VL3-RE. The two receptor preparations apparently have the same degree of glycosylation as evidenced by the same increased mass (∼15 kDa) due to glycosylation. However, the two receptors have different affinity for an anion-exchange resin and different p I. Deglycosylated receptors have the same p I. This suggests that the composition of sugars may be different. Taken together, the results suggest that the two receptors are modified and folded differently by different pathways due to the presence of different signal peptides. Use of an insect-specific signal peptide is recommended for expression of TSHR that is recognized by Graves’ autoantibodies in a baculovirus system.

The formation of thyrotropin receptor (TSHR) antibodies in a Graves' animal model requires the N-terminal segment of the TSHR extracellular domain.

Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves', including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves' patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90-165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves' TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9-165 (Mc1+2), 90-165 (Mc2), or 261-370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc1+2 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves' TSHRAbs, but it is important also in their formation in a mouse model of Graves' disease.

Thyrotropin-receptor and thyroid peroxidase-specific T cell clones and their cytokine profile in autoimmune thyroid disease.

We studied the cytokine profile and the immune responses to thyroid antigens of specific T cell clones (TCC) isolated from patients with Hashimoto's thyroiditis (HT) and Graves' disease (GD). Antigen-specific TCC were reactive to thyroid peroxidase (TPO), thyroglobulin (Tg) or human recombinant TSH-receptor extracellular domain (TSH-R), and/or their respective peptides. Of the 43 clones derived from HT patients, 65% were reactive to TPO, and 59% of the 32 clones derived from GD patients were reactive to TSH-R. TPO epitopes 100-119 and 625-644 were recognized by 75% of HT-derived clones, whereas TSH-R epitopes 158-176, 207-222, and 343-362/357-376 were recognized by 85% of GD-derived TCC. The TCC were classified according to their cytokine profile into T helper cell (Th)0 [secreting interleukin (IL)-4, IL-5, interferon (IFN)-gamma], Th1 (secreting IFN-gamma) and Th2 (secreting IL-4 and/or IL-5). Tumor necrosis factor-beta and IL-10 were produced by all subsets. The specific TCC were predominantly Th1-like cells in HT, and were Th0- and Th1-like cells in GD. Fifty three percent of Th0 clones were derived from GD patients and were reactive to TSH-R, whereas 50% of Th1 clones were derived from HT patients and were reactive to TPO or Tg. Most Th2 clones (82%) were reactive to TPO and were established from peripheral blood. All these clones produced IL-5, and 64% produced IL-4 and IL-10. Interestingly, IFN-gamma was highly produced by TPO- or Tg-specific clones established from HT thyroid tissue. These results confirm at the clonal level our previous studies regarding T cell epitopes on TPO and TSH-R molecules and support the concept that immunodominant T cell epitopes are located on amino acid residues 100-119 and 625-644 of TPO in HT and amino acid residues 158-176, 207-222 and 343-362/357-376 of TSH-R in GD. Our studies also demonstrate that thyroid-specific T cells can be classified into Th0, Th1, and Th2 subsets. TPO- or Tg-specific clones with Th1 phenotype appear to be involved in the pathogenesis of HT, mediating thyroid tissue destruction, whereas TSH-R clones with Th0 phenotype may induce thyroid-stimulating autoantibodies in GD.

Thyrotropin (TSH) receptor autoantibodies do not appear to bind to the TSH receptor produced in an in vitro transcription/translation system.

An in vitro transcription/translation (TnT) system was used to produce 35S-labeled full-length TSH receptor (TSHR) and TSHR extracellular domain (TSHRex). The interaction of the labeled proteins with TSHR autoantibodies in Graves' sera was then studied using an immunoprecipitation assay. In the assay, 35S-labeled TSHR or TSHRex were incubated with test sera, and any immune complexes formed were precipitated with protein A-Sepharose (in the case of mouse monoclonal antibodies, antimouse IgG-agarose was used). Rabbit antibodies to the TSHR and a mouse monoclonal antibody precipitated as much as 50% of the 35S-labeled TSHR preparations compared with about 2% for normal rabbit serum and 4% for a control monoclonal antibody. However, none of 34 Graves' sera (TSHR autoantibody levels ranging from 14-95% inhibition of [125I]TSH binding) were able specifically to immunoprecipitate 35S-labeled TSHR or TSHRex. These negative findings were confirmed by analysis of the immunoprecipitates on SDS-PAGE followed by autoradiography. Our results indicate that the TnT system is not useful for producing labeled TSHR preparations that can bind TSHR autoantibodies well. This is in contrast to TnT produced 35S-labeled glutamic acid decarboxylase, thyroid peroxidase, and 21-hydroxylase, which react well with their respective autoantibodies. One main difference between these 3 autoantigens and the TSHR is that the receptor is highly glycosylated, and this extensive glycosylation may be of critical importance for correct folding of the receptor. Consequently, the inability of the TnT system to glycosylate proteins could explain in part why TnT-produced 35S-labeled TSHR and TSHRex do not bind TSHR autoantibodies.

Effect of TSH and anti-TSH receptor antibodies on the plasma membrane potential of polymorphonuclear granulocytes

Effects of thyrotropin hormone (TSH) and anti-TSH receptor antibodies on the plasma membrane potential of polymorphonuclear granulocytes (PMN) were analyzed by means of flow cytometry. Both TSH and the autoantibody caused a rapid, dose-dependent hyperpolarization of the plasma membrane of PMNs. TSH was also able to mask (revert) the depolarizing effect of a chemotactic peptide, fMLP, on PMNs. No detectable rise in the cytosolic free calcium level accompanied the observed hyperpolarization. Quinine, a blocker of Ca 2+-activated and voltage-gated K + channels did not affect the hyperpolarization by TSH and antibodies. Decreasing the [K +] gradient across the plasma membrane by valinomycin, however, blocked the hyperpolarizing effect. Peptide 362–376 (derived from the extracellular domain of TSH receptor) also blocked the hyperpolarization induced by both TSH and anti-TSHR antibodies. These data suggest that the observed hyperpolarization is a specific, receptor-mediated early signal during interaction of PMNs with TSH or anti-TSHR antibodies.

Identification of the peptides that inhibit the stimulation of thyrotropin receptor by Graves' immunoglobulin G from peptide libraries.

Graves' disease is characterized by the overproduction of thyroid hormones due to the persistent stimulation of TSH receptor by autoantibodies. To determine the epitopes recognized by the autoantibodies, an enzyme-linked immunosorbent assay was developed that uses the human TSH receptor extracellular domain attached to plastic wells. The total IgG from some of the Graves' patients interacted with the bound TSH receptor (TSHR) at a significantly higher level than that in normal individuals. The IgG preparation that showed the highest binding activity was used for the identification of peptide sequences that prevent binding of Graves' IgG to TSHR from positional scanning synthetic peptide combinatorial libraries. A hexapeptide mixture, X1X2FDDA (X1 is a mixture of E, M, and Y; X2 is a mixture of E, H, and T), was found to be effective for inhibiting the binding of Graves' IgG to the TSHR. Further fractionation of X1X2FDDA showed that the following three sequences were highly effective: EEFDDA, ETFDDA, and EHFDDA. The second position of the three peptides did not appear to be important. The peptides also inhibited the cAMP synthesis induced by IgG of four of eight patients with Graves' disease tested. The synthesis of cAMP by TSH was also inhibited by the peptides to some extent. The peptide sequences most likely mimic a part of the conformational epitopes recognized by at least one class of Graves' IgG.

Graves' disease in severe combined immunodeficient mice.

Graves' disease (GD) is an autoimmune thyroid disorder involving an antibody (TSAb) directed against the TSH receptor (TSHR) producing thyroid stimulation. We have developed an animal model of GD by engrafting peripheral blood mononuclear cells or T cell lines plus autologous thyroid tissue into severe combined immunodeficient (SCID) mice. We xenografted Graves' thyroid tissue from six patients into six groups of SCID mice. Autologous PBMC and T cell lines reactive to recombinant human TSHR extracellular domain and non-TSHR lines were injected ip into the designated groups. In some of the studies, thyroid tissue was irradiated with 2000 rads before xenografting. Irradiation of xenografts induced thyroid tissue damage and release of thyroid antigens and hormones. Mice reconstituted with peripheral blood mononuclear cells or nonspecific T cell lines did not simulate GD. However, we achieved production of TSAb, elevation of serum T3, and TSAb-dependent survival and function of human Graves' thyroid tissue in SCID mice reconstituted with TSHR-specific T cell lines. We reconstituted SCID mice with PBMC and TSHR-specific T cell lines that recognized TSHR peptide 158-176. This may be in vivo evidence of the importance of peptide 158-176 as an immunodominant epitope on the TSHR extracellular domain.

Thyrotropin receptor-specific antibodies in BALB/cJ mice with experimental hyperthyroxinemia show a restricted binding specificity and belong to the immunoglobulin G1 subclass.

Immunization with the extracellular domain of TSH receptor (TSHR) led to the development of hyperthyroxinemia in BALB/cJ, but not C57BL/6J, SJL/J, and B10.BR, mice. Earlier, human studies had shown that thyroid-stimulating antibodies are predominantly of the immunoglobulin G1 (IgG1) subclass with a narrow specificity to TSHR, and antibodies that block thyroid function could be of any subclass with a broader specificity. Therefore, antibody responses in susceptible (BALB/cJ) and resistant (SJL/J) mice were characterized. There were no significant differences in the titers, relative affinities, or isotypes of antibodies against the TSHR. BALB/cJ and SJL/J sera reacted with 2 and 7 of 26 overlapping peptides from the extracellular domain of the TSHR. The ability of sera from BALB/cJ and SJL/J mice to block TSH binding to TSHR was reversed by 1 and 6 of the reactive peptides, respectively. BALB/cJ mice showed predominantly an IgG1 response against the TSHR and peptides, whereas SJL/J mice showed varying levels of all IgG subclasses. Although SJL/J sera reacted with peptides to which blocking antibodies bind, they did not show hypothyroidism, suggesting that their sera contained a mixture of blocking and stimulating antibodies that negated the effects of each other. In contrast, some TSHR-specific antibodies in BALB/cJ probably represented stimulating antibodies.

Thyrotropin receptor-specific antibodies in BALB/cJ mice with experimental hyperthyroxinemia show a restricted binding specificity and belong to the immunoglobulin G1 subclass

Immunization with the extracellular domain of TSH receptor (TSHR) led to the development of hyperthyroxinemia in BALB/cJ, but not C57BL/6J, SJL/J, and B10.BR, mice. Earlier, human studies had shown that thyroid-stimulating antibodies are predominantly of the immunoglobulin G1 (IgG1) subclass with a narrow specificity to TSHR, and antibodies that block thyroid function could be of any subclass with a broader specificity. Therefore, antibody responses in susceptible (BALB/cJ) and resistant (SJL/J) mice were characterized. There were no significant differences in the titers, relative affinities, or isotypes of antibodies against the TSHR. BALB/cJ and SJL/J sera reacted with 2 and 7 of 26 overlapping peptides from the extracellular domain of the TSHR. The ability of sera from BALB/cJ and SJL/J mice to block TSH binding to TSHR was reversed by 1 and 6 of the reactive peptides, respectively. BALB/cJ mice showed predominantly an IgG1 response against the TSHR and peptides, whereas SJL/J mice showed varying levels of all IgG subclasses. Although SJL/J sera reacted with peptides to which blocking antibodies bind, they did not show hypothyroidism, suggesting that their sera contained a mixture of blocking and stimulating antibodies that negated the effects of each other. In contrast, some TSHR-specific antibodies in BALB/cJ probably represented stimulating antibodies.

T-cells recognize multiple epitopes in the human thyrotropin receptor extracellular domain.

In Graves' disease (GD), the TSH receptor (TSHR) is believed to be the major target of an autoimmune response. T-Lymphocytes regulate the immune system. To assess the interaction of T-cells with TSHR in the pathogenesis of GD, we tested the T-cell response of peripheral blood mononuclear cells and T-cell lines to the recombinant human TSHR extracellular domain (rhTSHR-ECD) and 31 synthetic peptides corresponding to the entire TSHR-ECD in 20 patients with GD, 8 patients with Hashimoto's thyroiditis, 7 with colloid nodular goiter (CNG), and 20 normal controls. Comparing patients from different groups with normal subjects, there was a significant response to rhTSHR-ECD and thyroglobulin in GD patients (P < 0.001) and HT patients (P < 0.05), but not in CNG patients (P > 0.1). All 20 patients with GD responded to at least one peptide. The reactivity in GD patients was heterogeneous and spanned the entire TSHR-ECD. However, the reactivity was significantly different from that in controls for peptide regions 44-88, 119-176, 227-263, and 343-376, and the stimulation index (SI) values were significantly different for peptides 272-291 and 301-320. Significant differences were confined to peptides 158-176 and 343-362 and the region 227-263 for comparison of the number of positive responses in patients and controls to individual peptides. Forty-six percent of human leukocyte antigen-DQA1 0501 allele-positive Graves' patients responded to peptides 158-176 and 248-263 (SI = 3 or more) compared to 14% of allele-negative patients. In HT and CNG patients, the response was mainly to peptides in the carboxy-terminal half of the TSHR-ECD. Concordance of the reactivity in T-cell lines and peripheral blood mononuclear cells was observed in 36% of direct comparisons in GD. Eighty-five percent and 90% of GD patients were positive for microsomal antibody and TSHR antibody, respectively, and 59% of microsomal antibody-positive and 67% of TSHR antibody-positive patients responded to rhTSHR-ECD (SI = 2 or more). However, there was no significant correlation between antibody-positive patients and reactivity to specific peptides. Using multiple criteria to define immunodominance, peptides 158-176, 237-252, 248-263, and 343-362 seem to be important epitopes and may be critical for T-cell triggering in GD.