Significance of thyroglobulin antibodies cross-reactive with thyroperoxidase (TGPO antibodies) in individual patients and immunized mice.

Thyroglobulin (TG) and thyroperoxidase (TPO), both involved in thyroid hormone synthesis, represent major autoantigens in thyroid autoimmune disease. Despite numerous studies, the emergence, pathophysiological significance and role of autoantibodies to TG and TPO remain elusive. The recent identification of a new category of thyroid-specific autoantibody interacting with both TG and TPO (TGPO autoantibodies) offers a new opportunity in the study of thyroid autoimmunity. To gain a better insight into the significance of these TGPO autoantibodies, measurement in individual samples appeared necessary. The unique property of TGPO autoantibodies, simultaneous binding to TG and TPO, was used to set up a sandwich method which combined coated TG and radio-iodinated TPO. This method was found to be strictly specific for TGPO autoantibodies and sensitive enough to assay TGPO autoantibodies in serum. In humans, TGPO autoantibodies were found in most of the sera with high TG and TPO autoantibody titres, but not in sera negative for TG autoantibodies, whatever the TPO autoantibody titre. Furthermore, high TGPO autoantibody titres were found in sera strongly cytotoxic for cultured porcine thyroid cells. However, significant correlation of TGPO autoantibody titre was observed neither with TG and TPO autoantibody titres (n = 48) nor with complement-dependent cytotoxicity (n = 50). TGPO antibody assay was also performed in individual plasma of CBA/J mice immunized with either human TG (n = 6) or human TPO (n = 6). Immunization with TG induced high levels of not only TG but also TGPO antibodies, which exhibited a strong reactivity for TPO and whose binding to TG and TPO was fully inhibited by TG. In contrast, immunization with TPO induced high levels of only specific TPO antibodies accompanied by low levels of specific TG antibodies. In this case TGPO antibodies were not detected. Of note, TG- and TPO-immunized mice mounted an immune response against their own TG, but did not exhibit histological signs of thyroiditis. Large panels of TG and TPO MoAbs were also investigated with this method: 18/25 TG MoAbs and only 1/13 TPO MoAbs were found cross-reactive. Taken together, these data provide evidence that TGPO antibodies are effectively present in individual patients and TG-immunized mice, are different from specific TG and TPO antibodies, and may derive from natural B cell repertoire by autoimmune processes involving TG and not TPO.     

Limited usefulness of antithyroperoxidase and antithyroglobulin assays in Jamaicans with Graves' disease

The clinical usefulness of commercially prepared haemagglutination kits for thyroperoxidase (TPO) antibody and thyroglobulin (TG) antibody was evaluated in 145 consecutive Jamaicans with Graves' disease. Sixty two (43%) of the patients were newly diagnosed, 12 (8%) were in remission and 71 (49%) had relapsed. Sera from 65 (45%) patients were positive for thyroid antibodies. The TPO antibodies were found in 43% (63/145), thyroglobulin antibodies in 12% (17/145) fifteen (10%) patients had both anti-TPO and TG antibodies. Similar prevalences of TPO antibody were found in newly diagnosed patients and those who had relapsed (44% v. 41%) but the prevalence in the patients in remission was significantly higher (75%; X2 = 4.8, P < 0.05). The prevalence of TPO antibody increased significantly with age at onset before age 35 years compared to later onset (56% v. 32%; X2 = 8.09, P < 0.005). The presence of both TPO antibody (64% v. 31%; X2 = 13.1, P < 0.001) and TG antibody (22% v. 6%; X2 = 8.8, P < 0.005) correlated positively with Graves' ophthalmopathy. Neither of the tests was adequately sensitive in detecting GD in Jamaicans but we recommend testing for TPO antibody without TG antibody as a cost-effective approach. The presence and titres of TPO antibody and TG antibody do not correlate with disease activity and are not reliable enough for monitoring antithyroid drug therapy in GD. There is a need for antibody tests which are efficacious in diagnosing and monitoring antithyroid drug therapy in GD, and suitable for use in developing countries.     

Antithyroglobulin monoclonal and autoantibodies cross-react with an orbital connective tissue membrane antigen: a possible mechanism for the association of ophthalmopathy with autoimmune thyroid disorders.

The possibility that Graves' ophthalmopathy and autoimmune thyroid disorders may be associated because of autoimmune reactions against antigens shared between human orbital and thyroid tissues was investigated using anti-thyroglobulin (Tg) monoclonal and autoantibodies. Eleven of 16 mouse monoclonal antibodies (MCAB) tested reacted, in an enzyme-linked immunosorbent assay (ELISA), with an antigen in human orbital connective tissue membranes (OCTmem), but not with the OCT soluble fraction, or with membrane or soluble fractions of human eye muscle, lacrimal gland or skin connective tissue. The anti-OCTmem activity was absorbed by OCTmem and Tg, but not by liver membranes or bovine serum albumin (BSA). In preliminary studies four out of 113 human MCAB against thyroid or orbital tissue antigens showed reactivity restricted to Tg and OCTmem. Sera from approximately 50% of patients with autoimmune thyroid disorders, with or without ophthalmopathy, also reacted with OCTmem. The autoantibody activity correlated closely with serum titres of antithyroglobulin but not with the presence, duration, or severity of the eye disease. The OCTmem reactivity was absorbed by Tg, thyroid membranes, and OCTmem but not liver membranes, membranes prepared from other orbital tissues, or BSA. The OCTmem-Tg shared antigen site appeared not to be native thyroglobulin since, (i) MCAB and serum autoantibodies did not react with the cytosol fraction of OCT, and (ii) because the membrane antigen was not solubilizable. Because not all patients with ophthalmopathy have detectable anti-Tg antibodies and, conversely, because not all patients with detectable anti-Tg antibodies develop ophthalmopathy it is unlikely that autoimmunity against a OCTmem-Tg shared antigen is the primary mechanism of Graves' ophthalmopathy, although this possibility has not been excluded. On the other hand the reaction of anti-Tg autoantibodies with OCT membranes may be a model for other autoimmune reactions against other thyroid-orbital tissue-shared antigens. While the pathogenesis of Graves' ophthalmopathy is likely to be multifactorial, humoral and cellular reactions against primary orbital antigens, thyroid-orbitol tissue shared antigens, or both, are likely to play important roles.     

Clinical significance of a new autoantibody against a human eye muscle soluble antigen, detected by immunofluorescence.

The clinical significance of a circulating autoantibody against a recently identified soluble human eye muscle-derived antigen was studied in patients with Graves' ophthalmopathy and autoimmune thyroid disorders. Tests were positive in 73% of patients with Graves' ophthalmopathy, including six of seven with no associated thyroid disease (euthyroid Graves' disease). Tests were also positive in 27% of patients with hyperthyroidism but no clinically apparent eye disease, in 13% of patients with Hashimoto's thyroiditis without eye disease, in two of 12 patients with subacute thyroiditis, in one of 20 patients with nonimmunological thyroid disorders but in none of 39 normal subjects. There were significant positive correlations between serum levels of the antibody (expressed as a titre) and the severity of the eye muscle component quantified as an index as well as the duration of the eye disease. Antibodies were detected in three of five patients with only lid lag and state who subsequently developed active ophthalmopathy, in six of nine patients who developed eye disease after treatment of their hyperthyroidism and in one of eight first degree relatives of patients with Graves' ophthalmopathy. In addition three of the 12 patients with autoimmune thyroid disease without apparent eye involvement, but positive antibody tests, have developed ophthalmopathy since the time of testing. These findings suggest that tests for antibodies against a soluble human eye muscle antigen may be useful clinically as a diagnostic test and to predict the onset of eye disease in predisposed patients and subjects.     

Assays of TSH-receptor antibodies in 576 patients with various thyroid disorders: their incidence, significance and clinical usefulness

The incidence and the significance of TSH-receptor antibodies in Graves' disease and in various thyroid disorders have been evaluated. TSH-binding inhibiting antibodies (TBIAb) and thyroid stimulating antibodies (TSAb) were detected in a large proportion of Graves' disease patients (TBIAb in 68.8% and TSAb in 77.8%), in a small number of patients with idiopathic myxoedema or Hashimoto's thyroiditis, and were not detected in patients with endemic euthyroid goitre, differentiated thyroid carcinoma and toxic adenoma. Furthermore, TSH-receptor antibodies were present in some patients with toxic multinodular goitre (TBIAb in 12.7% and TSAb in 15.9%). When TSH-receptor and other thyroid autoantibodies were compared, it was found that 13 of the 15 Graves' patients with negative tests for thyroglobulin and thyroid microsomal antibodies were positive for TSH-receptor antibodies. On the other hand, 9 of the 11 patients with toxic multinodular goitre who had positive TSH-receptor antibody tests, also had serum thyroglobulin and/or thyroid microsomal antibodies. No significant differences in the prevalence of TSH-receptor antibodies were found in Graves' patients irrespective of the presence of ophthalmopathy or pretibial myxoedema. Elevated TBIAb activity at the end of anti-thyroid drug treatment was found in 52.9% of Graves' patients who subsequently relapsed, while in Graves' patients in remission TBIAb was always negative. TSH-receptor antibody results were not predictive of the outcome of radioiodine treatment in Graves' disease. Finally no correlation could be found between TBIAb and TSAb in Graves' disease and Hashimoto's thyroiditis. In conclusion: the high incidence of TSH-receptor antibodies in Graves' disease confirms their pathogenetic role in the development of hyperthyroidism; TSH-receptor antibodies in Graves' disease are not significantly associated with the presence of ophthalmopathy or pretibial myxoedema; TSH-receptor antibody assays may be useful for the diagnosis of Graves' disease in the absence of other signs of autoimmunity. TBIAb seems to be a good predictor of relapse in Graves' patients treated with anti-thyroid drugs; a fraction of toxic multinodular goitre could be a nodular variant of Graves' disease.     

Prevalence of thyroid peroxidase, thyroglobulin and thyrotropin receptor antibodies in a long-term follow-up of juvenile Graves disease

OBJECTIVE: To relate the presence of thyroid peroxidase antibodies (TPO ab), thyroglobulin antibodies (Tg ab) and thyrotropin receptor antibodies (TSH-R ab) to the clinical course in a long-term follow-up of patients with juvenile Graves' disease (JGD). DESIGN: Patients with JGD were drawn from a Danish retrospective study and reexamined. RESULTS: A number of 105 patients were reexamined 4-21 years (median 10 years) after diagnosis. Three groups were formed: Gr.1: euthyroid patients with anti-thyroid drug (ATD) cessation more than 12 months before reexamination (n=41). Gr.2: patients still on ATD (n=24). Gr.3: subtotally thyroidectomized patients (n=40). Positive TPO ab titers were found in 75% of the patients. In 13% of the patients the titers were very high: >10,000 U/ml. Positive Tg ab were found in 51%. The prevalence of TPO ab and Tg ab was the lowest in group 3. Stimulating TSH-R ab titers were found in 13%. No patients had blocking TSH-R ab. The prevalence of TSH-R ab was 3% in the euthyroid patients, without surgery performed. 15% in the surgical patients, and 25% in the patients still on ATD. CONCLUSIONS: Many JGD patients were euthyroid at the long-term follow-up in spite of high TPO ab and Tg ab titers. Stimulating TSH-R ab were rare (13%). According to this presence of TPO ab, Tg ab or TSH-R ab does not predict the final outcome of JGD. Further studies are needed.     

A study of human-human hybridomas from patients with autoimmune thyroid disease

Human-human B-cell hybridomas were established using peripheral blood lymphocytes from patients with autoimmune thyroiditis and Graves' disease. Peripheral mononuclear cells (PMC), with or without mitogen prestimulation, were fused with HGPRT-negative human myeloma cell lines (Gm4672 and Gm0462) using 44% polyethylene glycol. Developing hybridomas were screened by enzyme-linked immunosorbent assays (ELISAs) for human IgG and IgM and antibodies to human thyroglobulin (hTg) and microsomal antigen (M-Ag). A¹²⁵I-TSH binding inhibition assay was utilized for detecting antibodies to TSH receptor (TSH-R) protein. Hybridoma formation was observed only after prior mitogen stimulation of PMC. The amount of antibody secreted by the human-human hybridomas was highly variable (10 ng-100 µg/ml IgG/IgM). Nine and six-tenths percent of the hybrids secreted anti-hTg and 8.4% secreted anti-M-Ag. A 5% cloning efficiency was achieved, with detection of specific thyroid autoantibody secretion in one-third of the clones derived from positive hybridomas. Immunoglobulin secretion decreased with time and long-term stable clones were not achieved. Thyroid monoclonal autoantibodies to hTg, M-Ag, and TSH-R (IgG and IgM) detected during these studies were of a low affinity. In addition, antibodies were identified which exhibited marked specificity crossover between hTg, M-Ag, and nonthyroid antigens, suggesting the presence of recurrent epitopes. Such observations may help explain the multiplicity of thyroid autoantibodies in human thyroid disease and indicate a common defect in immunoregulation. We suggest that cross-reacting epitopes may be important in the derivation of thyroid-specific B-cell clones.     

Control of human thyroid autoantibody production in SCID mice.

In order to determine the phenotype of the cells required for thyroid autoantibody production, peripheral blood mononuclear cells (PBMC) from patients with autoimmune thyroid disease (AITD) were transferred to severe combined immunodeficient (SCID) mice. The production of human IgG, thyroglobulin (Tg) antibody and thyroid peroxidase (TPO) antibody in the SCID recipients was monitored for up to 4 months. PBMC from 10 of 13 AITD patients produced substantial IgG (> or = 100 micrograms/ml) and detectable Tg and TPO antibodies in recipient mice. PBMC pretreated to deplete or enrich T cells produced low or undetectable thyroid-specific antibody in SCID mice. Depletion of CD4+ T cells resulted in much lower or undetectable IgG, Tg and TPO antibodies compared with levels seen in recipients of control PBMC. By contrast, depletion of CD8+ T cells from the PBMC had no overall effect on autoantibody production, although with PBMC from some patients CD8+ depletion possibly enhanced both IgG and autoantibody production. In eight of 10 experiments, autoantibody levels reached maximal titres before total IgG levels peaked. It is considered that thyroid autoantibodies are produced from memory B cells activated in SCID mice and that this activation is T cell- and CD4+ T cell-dependent.     

Insight into antibody responses induced by plasmid or adenoviral vectors encoding thyroid peroxidase,a major thyroid autoantigen

Plasmid and adenoviral vectors have been used to generate antibodies in mice that resemble human autoantibodies to the thyrotrophin receptor. No such studies, however, have been performed for thyroid peroxidase (TPO), the major autoantigen in human thyroiditis. We constructed plasmid and adenovirus vectors for in vivo expression of TPO. BALB/c mice were immunized directly by intramuscular injection of TPO-plasmid or TPO-adenovirus, as well as by subcutaneous injection of dendritic cells (DC) infected previously with TPO-adenovirus. Intramuscular TPO-adenovirus induced the highest, and TPO-plasmid the lowest, TPO antibody titres. Mice injected with TPO-transfected DC developed intermediate levels. Antibodies generated by all three approaches had similar affinities (Kd approximately 10(-9)M) and recognized TPO expressed on the cell-surface. Their epitopes were analysed in competition assays using monoclonal human autoantibodies that define the TPO immunodominant region (IDR) recognized by patients with thyroid autoimmune disease. Surprisingly, high titre antibodies generated using adenovirus interacted with diverse TPO epitopes largely outside the IDR, whereas low titre antibodies induced by DNA-plasmid recognized restricted epitopes in the IDR. This inverse relationship between antibody titre and restriction to the IDR is likely to be due to epitope spreading following strong antigenic stimulation provided by the adenovirus vector. However, TPO antibody epitope spreading does not occur in Hashimoto's thyroiditis, despite high autoantibody levels. Consequently, these data support the concept that in human thyroid autoimmunity, factors besides titre must play a role in shaping an autoantibody epitopic profile.     

Ultrastructural localization of endogenous peroxidase activity in Hashimoto's thyroiditis

Ultrastructural localization and intensity of endogenous thyroid peroxidase (TPO) in Hashimoto's thyroiditis were examined in relation to the serum thyroid hormone level, thyroid-stimulating hormone (TSH) concentration and anti-thyroid autoantibody titer. In Hashimoto's thyroiditis, TPO activity on the microvilli of follicular cells was more intense than that of normal thyroid tissue, but the intensity of the intracytoplasmic peroxidase reaction was generally weaker than that of Graves' or normal thyroid tissue. Microvillar TPO reaction products were positive in all thyroid follicular cells in patients with increased TSH levels, but no TPO activity was observed on the microvilli of patients with normal or low TSH levels, irrespective of their histological type or serum anti-microsomal antibody titer. It is suggested that TPO activity on the surface of microvilli of thyroid follicular cells in Hashimoto's thyroid gland is modulated by thyrotropin but is not affected by anti-thyroid autoantibodies.     

Antibodies targeting the calcium binding skeletal muscle protein calsequestrin are specific markers of ophthalmopathy and sensitive indicators of ocular myopathy in patients with Graves' disease

We have identified several eye muscle antigens and studied the significance of the corresponding serum autoantibodies in patients with Graves' disease. Of these antigens, only calsequestrin is expressed more in eye muscle than other skeletal muscles, which could explain at least partly the specific involvement of eye muscle in patients with Graves' disease. Earlier, we found a modest relationship between anti-calsequestrin antibodies and ophthalmopathy, but in that study we used calsequestrin prepared from rabbit heart muscle and measured antibodies by immunoblotting. We have reinvestigated the prevalences of anti-calsequestrin antibodies in larger groups of well-characterized patients with thyroid autoimmunity with and without ophthalmopathy and control patients and healthy subjects, using standard enzyme-linked immunosorbent assay incorporating highly purified rabbit skeletal muscle calsequestrin, which has a 97% homology with human calsequestrin, as antigen. Anti-calsequestrin antibodies were detected in 78% of patients with active congestive ophthalmopathy, in 92% of those with active inflammation and eye muscle involvement, but in only 22% of patients with chronic, 'burnt out' disease. Tests were also positive in 5% of patients with Graves' hyperthyroidism without evident ophthalmopathy (two patients) and one patient with 'watery eyes' but no other clear signs of congestive ophthalmopathy and IgA nephropathy and no known thyroid disease, but in no patient with Hashimoto's thyroiditis, toxic nodular goitre, non-toxic multi-nodular goitre or diabetes, or age- and sex-matched healthy subjects. In serial studies of all 11 patients with Graves' hyperthyroidism who had active ophthalmopathy at the time of the first clinic visit, or developed eye signs during the first 6 months, and positive anti-calsequestrin antibodies in at least one sample, anti-calsequestrin antibodies correlated with the onset of ocular myopathy in six patients. Antibodies targeting calsequestrin appear to be specific markers for ophthalmopathy and sensitive indicators of the ocular myopathy subtype of ophthalmopathy in patients with thyroid autoimmunity. However, these results must be considered preliminary until a large prospective study of patients with newly diagnosed Graves' hyperthyroidism, in which serum levels of calsequestrin antibodies are correlated with clinical changes and orbital eye muscle and connective tissue/fat volumes, has been carried out.     

Autoantibodies to thyroid peroxidase in patients with chronic thyroiditis: effect of antibody binding on enzyme activities.

Using thyroid peroxidase (TPO), which was purified from the thyroid of patients with Graves' disease, we attempted to determine whether sera from patients with chronic thyroiditis contained antibodies to the enzyme. When the binding was tested by ELISA, sera from patients with chronic thyroiditis revealed high binding activities to TPO. When TPO was incubated with IgG from sera followed by treatment with protein A-Sepharose and centrifugation, the remaining TPO activities in the supernatant fraction were lower in most of the patients, as compared to normal controls. Moreover, IgG purified by DEAE-cellulose chromatography from sera in patients interfered with the TPO activities. Titres of anti-TPO antibodies correlated well with those of anti-microsome antibodies. These results indicate the presence of autoantibodies to TPO in sera of most patients with chronic thyroiditis and that TPO may be one component of microsome antigen complexes recognized by the autoantibodies. Studies on the inhibition of TPO by IgG isolated from sera of patients using guaiacol and iodide assays revealed that at least three epitopes of TPO molecule were recognized by autoantibodies and that the antigenic determinants on TPO molecule recognized by autoantibodies could be heterogeneous in patients.     

Comparative study on IgG and IgA antibodies against human thyroid and eye-muscle antigens in Graves' ophthalmopathy.

Circulating IgG and IgA anti-thyroid and anti-eye muscle antibodies were investigated in 87 patients with Graves' disease (60 cases with ophthalmopathy). The ELISA method was used. Both IgG and IgA antibodies were demonstrated against human thyroid and eye-muscle membrane or cytosol antigens. Anti-eye-muscle antibodies of the IgA type were observed more frequently than those of the IgG type (25 cases vs. 18 were demonstrated with membrane antigens and 37 cases vs. 23 with cytosol antigens). The respective distributions for thyroid antigens the cytosol fraction were 55 cases vs. 13 and 18 cases vs. 36. A significant difference was observed in the anti-thyroid IgG levels and the anti-eye-muscle membrane or cytosol levels between the patients with Graves' disease and those in control group (P less than 0.001). The difference in the IgA antibody to thyroid and eye-muscle antigens was significant between the patients with and without ophthalmopathy (P less than 0.002). The strong correlation between the levels of IgA antibodies to thyroid and those to the eye-muscle cytosol fractions might be connected with the theory of the common aetiology of the thyroid and eye diseases in Graves' ophthalmopathy (P less than 0.001). Circulating IgA anti-human thyroid and eye-muscle antibodies seemed to have a diagnostic relevance in the development of ophthalmopathy in Graves' ophthalmopathy.     

Isotype and immunoglobulin subclass distribution of eye muscle membrane reactive antibodies in the serum of patients with thyroid-associated ophthalmopathy as detected in Western blotting.

We have determined the immunoglobulin (Ig) class (isotype) and IgG subclass of autoantibodies in the serum of patients with thyroid-associated ophthalmopathy (TAO) or autoimmune thyroid disorders without evident ophthalmopathy reactive in Western blotting with antigens of 55, 64, 75 and 95 kDa in pig eye muscle membrane (PEMM). The 22 sera studied were shown, previously, to contain IgG antibodies reactive with one or more of the four antigens. The majority of sera antibodies reactive with PEMM antigens were of two or more IgG subclasses. Of the IgG subclass specificities IgG3 and IgG4 subclass antibodies were, overall, the most common. We were unable to demonstrate IgG subclass restriction for antibodies reactive with the 95 or 55 kDa antigens in PEMM, antibody activity being equally distributed in all four subclasses tested. While most of the sera which recognized a 64 kDa antigen did so with an IgG4 antibody, all other subclasses were also represented. On the other hand all 13 sera reactive with a 75 kDa antigen did so using Ig of the IgG3 subclass and 12 of these used the IgG4 subclass as well, IgG1 and IgG2 subclasses being represented in only 3 and 4 sera, respectively. There were no differences, in respect to Ig class or IgG subclass distribution of eye muscle reactive antibodies between patients with Graves' hyperthyroidism with ophthalmopathy and those with Hashimoto's thyroiditis, and eye disease. Control sera from five normal subjects and three patients with nonautoimmune thyroid disorders did not contain antibodies reactive with these PEMM antigens of any Ig class or IgG subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II

Injection of AKR/N mice with fibroblasts co-expressing MHC class II and TPO in the absence of adjuvant induces IgG-class TPO antibodies that resemble spontaneously arising human thyroid autoantibodies. We have used this model to examine the effect of iodide on TPO antibody induction as well as to analyse the interaction between T and B cells. Despite its importance as a major environmental factor in thyroid autoimmunity, variable iodide intake had no detectable effects on TPO antibody levels, lymphocytic infiltration of the thyroid or thyroid hormone levels. In terms of T cell responsiveness, splenocytes from TPO fibroblast-injected mice, but not from control mice, proliferated in response to TPO. Intriguingly, B cell-depleted splenocytes (mainly T cells without reduction of macrophages) proliferated in response to TPO only when co-cultured with irradiated autologous splenocytes from TPO fibroblast-injected mice but not from control mice. These data suggest that TPO-specific B cells are involved in antigen presentation to sensitized T cells and are supported by the ability of spleen cells from TPO cell-injected (but not control) mice to secrete TPO antibodies spontaneously in culture. In conclusion, we provide the first evidence for the presence of thyroid autoantigen-specific B cells and their ability to present their autoantigen to sensitized T cells in mice induced to develop TPO antibodies resembling autoantibodies in humans.     

Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.

Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.     

Severe combined immunodeficient (SCID) mice: a model for investigating human thyroid autoantibody synthesis

We have studied the ability of lymphocytes from the blood, thyroid and lymph nodes of patients with autoimmune thyroid disease (AITD) to produce autoantibodies to thyroglobulin (Tg) and/or thyroid peroxidase (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were detectable in plasma from SCID mice 7 days after transfer of 15-25 x 10(6) cells/mouse and the highest levels were recorded 2-3 weeks later. In contrast, Tg and/or TPO antibodies were undetectable in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the most antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounts of Tg and/or TPO antibody detected were in accordance with the ability of thyroid and lymph node lymphocytes to secrete these autoantibodies spontaneously in culture (indicating the presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tg and/or TPO antibodies in culture in response to pokeweed mitogen. Tg antibodies in plasma from SCID recipients of thyroid lymphocytes were of subclasses IgG1, IgG2 and IgG4 and the proportions closely resembled those of the donor's serum Tg antibodies. Blood lymphocytes transferred to SCID recipients were also able to produce Tg antibodies of subclasses 1, 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from patients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of human antibodies to Tg and TPO.     

Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease

Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.     

Relationship between thyroid autoantibody spectrotype and IgG subclass

The relationship between spectrotype and IgG subclass of autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO) has been investigated using sera from Hashimoto and Graves' patients. Isoelectric focussing (IEF) was carried out in gels over the range pI 3.5-9.5 followed by transblotting to nitrocellulose and probing the filters using 125I-labelled TPO and Tg. As has been shown previously for Tg antibodies, TPO antibody focussed over different pI values in different patients but the spectrotypes for individuals were constant over 2-5 years. Further, the pI values for TPO and Tg autoantibodies appeared to be related to IgG subclass, for example IgG1 Tg antibodies tended to focus nearer the cathode (pI 8.0-9.5) than IgG4 antibodies (pI 6.5-8.5) while antibodies of subclasses IgG1 + 2 + 4 produced spectrotypes covering a broad pI range (5.7-9.5). Consequently, it seems likely that the characteristic spectrotypes described by others for Tg autoantibodies, and those we now report for TPO antibodies, reflect the IgG subclass "fingerprints" which we suggest may be a measure of the ability of an individual to respond to different epitopes on these two thyroid antigens.