Thyroid Antigens Research Articles

Sensitization of T lymphocytes to thyroid antigen in autoimmune thyroid disease as demonstrated by the monocyte procoagulant activity test.

Monocyte procoagulant activity (PCA) production has been reported to have a close relation to cell-mediated immunity (CMI), and the collaboration of T lymphocytes is necessary to induce PCA. Antigen-specific sensitization of lymphocytes in patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT) has been demonstrated by means of the production of cell-bound PCA by monocytes following antigen stimulation of whole peripheral blood mononuclear cells (PBM). These cells, obtained from both normal subjects and patients with autoimmune thyroid diseases, produced significant amounts of PCA with non-specific lectin, concanavalin A (Con A) stimulation; however, there was no significant difference between the two groups, suggesting that Con A stimulated T cells induced monocyte PCA nonspecifically. Peripheral mononuclear cells from patients with autoimmune thyroid diseases produced significantly greater amounts of PCA than PBM from normal subjects when stimulated with solubilized and IgG-free thyroid antigen. On the other hand, liver antigen did not induce significant amounts of PCA production in PBM from either normal subjects or patients. Significantly larger amounts of PCA were produced by PBM from patients following thyroid antigen stimulation than with liver antigen stimulation. Although monocytes were the major source of PCA, T cells were necessary to induce PCA in monocytes with thyroid antigen and Con A stimulation. Elimination of lymphocyte subsets in PBM from patients by negative selection (using monoclonal antibodies and complement) suggested that the collaboration of T lymphocytes, especially helper/inducer (T4+) T cells, was necessary to produce PCA with thyroid antigen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

How do T cells mediate autoimmune thyroiditis?

Susceptibility to experimental autoimmune thyroiditis (EAT) is linked to the mouse H-2 complex 1. Initiation of the condition by the thyroid antigen, thyroglobulin (Tg), requires T cells from susceptible mice 2. These T cells are autoreactive in that they recognize Tg and proliferate in vitro in response to it 3. The 1980s have brought forth confirmatory data from several laboratories on T-cell autoreactivity to mouse Tg both in vivo 4,5 and in vitro 6–13, and its correlation with EAT susceptibility. T cells from patients with Hashimoto's thyroiditis (HT), may proliferate in response to stimulation in vitro with human Tg 14–16. The capacity of murine T cells to expand and differentiate in vitro 17,18 and to serve as effector cells causing thyroiditis in vivo 7,8, 11,12 is strongly indicative of their pathogenic role. We are now in a position to discuss not only if T cells mediate autoimmune thyroiditis, but how they might contribute to the pathogenic process. At least two antigen-activated T-cell subsets seem to participate in initiating and perpetuating thyroid destruction. In this article, Yi-chi Kong and her colleagues present a synopsis of the supporting data from the mouse model and parallel findings from HT patients.

The production and characterization of thyroid-derived T-cell lines in Graves' disease and Hashimoto's thyroiditis

Although the thyroid gland itself is a major site of the autoimmune response, the study of T-cell function in autoimmune thyroid disease has usually relied on peripheral blood as a source of cells. In this study, we have established thyroid-derived T-cell lines from six patients with Graves' disease and one patient with Hashimoto's thyroiditis by culturing the thyroid lymphocytes on an autologous thyroid follicular cell monolayer in the presence of exogenous interleukin 2 (IL-2). These T-cell lines have allowed in vitro investigation of thyroid-derived T-cell function, an approach which was previously limited by the number of lymphocytes obtained from the gland. The lines were predominantly OKT3, OKT4, and HLA-DR positive but showed heterogenous proliferative responses. Some lines gave autologous or allogeneic mixed lymphocyte reactions but other did not. Only one of the seven lines responded well to the thyroid antigens thyroglobulin and microsomes presented by autologous monocytes. However, six of the lines proliferated in the presence of live but not dead autologous thyroid follicular cells, particularly when interferon-γ (IFN-γ) was added. This treatment has been shown to enhance HLA-DR and -DQ antigen expression by thyroid follicular cells in vitro. Furthermore, the proliferation induced by IFN-γ-treated thyroid follicular cells was increased when thyroglobulin was also added. Together these results support the hypothesis that the expression of Ia antigens such as HLA-DR by thyroid follicular cells in autoimmune thyroid disease may be important in enhancing the autoimmune response, conferring on these cells the ability to present thyroid autoantigens to T cells. The use of thyroid-derived T-cell lines should permit a more detailed evaluation of the disordered immunoregulation in Graves' disease and Hashimoto's thyroiditis than has been possible previously.

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U mg and exhibited a peak in the Soret region. The ratio of A 411 to A 280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.

In vivo prevention of thyroid and pancreatic autoimmunity in the BB rat by antibody to class II major histocompatibility complex gene products.

Evidence is accumulating that the development of insulin-dependent diabetes mellitus involves autoimmune phenomena, both in the human and in the BB rat model. A strong association is observed in both cases with alleles of the class II major histocompatibility complex (MHC). Results of the present study show that autoimmune phenomena, as assessed by the presence of clinical diabetes or histological thyroiditis, are prevented by the injection of monoclonal antibodies to class II gene products in the BB rat. Immunosuppression was specifically obtained with a monoclonal antibody to the murine I-E equivalent, as opposed to the murine I-A equivalent, of the rat major histocompatibility complex. This represents indirect evidence for I-E subregion control of immune responses to islet cell and thyroid antigens in the BB rat model. The frequent occurrence of anaphylactic type deaths in young (1 month old) animals receiving more than six weekly injections of partially purified homologous (rat) monoclonal antibodies to rat class II gene products underscores the potential risks of this type of immunotherapy. The presumed immunologic mechanism (IgE antibody) and its specificity (anti-allotype, anti-idiotype, or anti-impurity) must be clarified to assess the risks and feasibility of this type of therapy.

2 The pathogenesis of graves' ophthalmopathy

The pathology of the orbital changes in Graves' ophthalmopathy (GO) has been discussed in detail. The target tissue is the eye muscle and the damage probably results from autoimmune processes. Cell-mediated immune responses have been demonstrated and an antibody to eye muscle is detectable in 70% of patients. There is no direct evidence for an effect of thyroid hormones or TSH on orbital tissues in GO. The relation between GO and autoimmune thyroid disease is discussed. Present evidence suggests that in GO autoimmune responses are directed to orbital tissue antigens and do not cross-react with thyroid antigens. Clinical studies suggest that all patients with hyperthyroidism have some abnormality of eye muscle, whereas not all patients with ophthalmopathy have evidence of thyroid disease. It is not possible at the present time to be certain whether GO is an integral part of Graves' disease or a separate entity.

Failure to demonstrate cell-mediated immune responses to thyroid antigens in Graves' disease using in vitro assays of lymphokine-mediated migration inhibition.

The use of in vitro assays of cell-mediated immunity has provided evidence of a defect in thyroid antigen-specific T suppressor cells in the peripheral blood of patients with autoimmune diseases. We employed a direct assay of T lymphocyte migration inhibition in an attempt to demonstrate cell-mediated immune responses to thyroid antigens in untreated patients with hyperthyroid Graves' disease and examined the influence of (1) a variety of antigen preparations, (2) different assay conditions, and (3) the HLA-DR3 status of normal subjects on this system. There was no significant difference in the migration of lymphocytes from 13 untreated patients with hyperthyroid Graves' disease to a crude 800 X g supernatant antigen preparation of normal human thyroid when incubated at either 20 or 37 C compared with the response of 13 normal subjects. The mean migration index was 57 +/- 22 (+/- SD) in the Graves' patients compared with 65.2 +/- 19.9 in the normal subjects at 20 C and 83 +/- 16.9 in the Graves' patients compared with 86.6 +/- 15.1 in the normal subjects at 37 C. Similarly, there was no significant difference in the migration indices obtained with T cells from Graves' patients and normal subjects using an 800 X g supernatant prepared from the thyroid glands of 2 patients with Graves' disease, incubated at 20 or 37 C (44.0 +/- 22.5 in the Graves' patients compared with 45.7 +/- 12.8 in the normal subjects at 20 C and 67.9 +/- 20.8 in the patients compared with 72 +/- 12.6 in the controls at 37 C). In contrast, the migration indices calculated for 20 C incubation were significantly lower than the corresponding value at 37 C using 800 X g normal thyroid antigen (P less than 0.05) and 800 X g Graves' thyroid antigen (P less than 0.01) in both patient and control groups. An identically prepared uterine antigen produced a similar reduction in the migration indices at 20 C compared with those at 37 C in 6 normal donors (P less than 0.05), but no temperature effect was found when 8 normal subjects were tested with purified protein derivative of tuberculin. The responses of 12 normal individuals to 800 X g supernatants of the normal and Graves' thyroid antigens were not influenced by the HLA-DR3 antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

Nonthyroid autoantibodies in Obese strain (OS) chickens

Organ-specific autoantibodies (AAb) to thyroid and non-thyroid antigens of various endocrine and exocrine glands (glandular stomach, pancreas, adrenal, parathyroid, and striated muscle) were determined by different serological procedures in sera from Obese strain (OS), Cornell C strain (CS), normal inbred strains (CC and CB), and outbred normal White Leghorn (NWL) chickens. Thyroglobulin autoantibodies (Tg-AAbs), evaluated by immunodiffusion, passive hemagglutination, enzyme-linked immunosorbent assay, and indirect immunofluorescence, as well as other organ-specific AAbs determined by indirect immunofluorescence, predominated in OS chickens. Tg-AAbs were found in the highest frequency, thyroid microsomal AAbs in intermediate frequency, and the other organ-specific AAbs in low frequency in OS chickens. Thyroid and nonthyroid organ-specific AAbs were found only occasionally in control chickens and then only in low titers. Thus, spontaneous autoimmune thyroiditis of OS chickens correlates closely with human Hashimoto thyroiditis not only in respect to AAbs to thyroid antigens but also to nonthyroid organ-specific antigens. Non-organ-specific AAbs, such as antinuclear antibodies, antibodies to chicken red blood cell nuclei, mitochondrial AAbs, smooth muscle antibodies, and reticulin AAbs occur in high frequency in all strains of chickens tested. Even a slight prevalence in NWL chickens was seen, indicating that the abnormal immune response in OS chickens is restricted to organ-specific antigens of the thyroid gland and in some cases also to other exocrine or endocrine glands.

A specific IgG in Graves' ophthalmopathy and its relation to retro-orbital and thyroid autoimmunity.

An IgG (ophthalmopathic immunoglobulin) that binds to retro-orbital antigen was identified in serum from patients with active Graves' ophthalmopathy, and its nature and specificity were investigated. Dose related binding of this immunoglobulin to retro-orbital antigens prepared from guinea pig harderian gland or porcine eye muscle was found, which could be abolished by prior incubation with antigen. The immunoglobulin did not bind to thyroid membranes, thyroid microsomes, or thyroglobulin or interact with liver, skeletal muscle, or fat membranes. Serum with high activity of thyrotrophin binding inhibiting immunoglobulin did not react with retro-orbital antigen, and this activity was not affected by preincubation of the serum with retro-orbital antigen. Thyroid stimulating hormone was also without effect on retro-orbital antigen. It is concluded that Graves' ophthalmopathy is associated with a specific ophthalmopathic immunoglobulin that reacts with retro-orbital antigen as distinct from thyroid antigens, and that the autoimmune response is directed towards retro-orbital antigens. This suggests that the ophthalmopathy is an entity distinct from autoimmune thyroid disease.

Immunological profiles in alopecia areata.

Cell-mediated immunity and auto-immune phenomena were investigated in sixty patients with active alopecia areata of various degrees of severity. Serum auto-antibodies to thyroid antigens were detected in twenty-three patients. Examination of T-lymphocyte populations, lymphocyte DNA synthesis, and lymphokine production in response to mitogen stimulation revealed no differences between the sixty patients and matched healthy control subjects. However, patients with thyroid auto-immunity and/or the presence of alopecia totalis or universalis showed significant reductions in interactive T lymphocytes (recognized by rosette formation with human B lymphoblastoid cells) and diminished production of leukocyte migration inhibition factor in response to stimulation with phytohaemagglutinin. This suggests that immune mechanisms may be involved in the pathogenesis of alopecia areata which is associated with thyroid auto-immunity or which progresses to total hair loss.

Evidence for cell-mediated immunity and specific suppressor T lymphocyte dysfunction in Graves' disease and diabetes mellitus.

Migration inhibition of purified peripheral T lymphocytes in response to pancreatic islet cell antigen or thyroid antigen was used to study cell-mediated immune mechanisms in patients with diabetes mellitus (IDDM) and Graves' disease (GD). In response to islet cell antigen, T lymphocytes of subjects with IDDM for less than 3 yr exhibited migration inhibition, whereas those of normal subjects, noninsulin dependent diabetics, and subjects with IDDM for longer than 3 yr did not. Admixture of T lymphocytes from normal subjects with T lymphocytes from patients with IDDM for less than 3 yr substantially ameliorated the migration inhibition of the IDDM subjects to islet cell antigen. Migration of T lymphocytes from GD subjects was markedly inhibited by thyroid antigen and marginally inhibited by islet cell antigen. Admixture of GD T lymphocytes significantly ameliorated the migration inhibition of IDDM T lymphocytes to islet cell antigen, despite sensitization to thyroid antigen of the GD T lymphocytes. We conclude: 1) sensitization to islet cell antigen in IDDM of recent onset is confirmed; 2) the ability of normal and GD T lymphocytes to ameliorate the migration inhibition of IDDM T lymphocytes strongly suggests correction of deficient suppressor T lymphocyte function; 3) the ability of GD T lymphocytes to ameliorate migration inhibition of IDDM T lymphocytes to islet cell antigen is evidence for an antigen-specific rather than a generalized suppressor T lymphocyte defect in GD; and 4) similarly, the normalization of migration index of GD T lymphocytes in response to thyroid antigen by those IDDM T lymphocytes not sensitized to thyroid antigen is again evidence for an antigen-specific and not a generalized suppressor T lymphocyte defect in IDDM.

The histopathologic features of lithium-associated thyroiditis

The case of a 62-year-old woman with lithium-associated thyroiditis is presented. Lithium can produce goiters associated with hypothyroidism and, less commonly, hyperthyroidism and euthyroidism. The characteristic histopathologic features of the affected thyroid gland included fibrosis, lymphoid follicles with atrophy, and hyperplasia of thyroid follicles. The pathogenetic mechanism appears to be immunologic, with lithium acting as a haptene with a thyroid antigen to induce an "autoimmune" type of thyroiditis.

5 Diseases of the TSH receptor

We have reviewed the impact of cellular and molecular biology on our understanding of the immune system and thyroid autoimmune disease and presented the evidence that MHC, TCR and immunoglobulin genes are involved in susceptibility to such disease. At the level of the target thyroid epithelial cell, the identification of the genes for Tg and TPO (the microsomal antigen) using recombinant DNA techniques are evidence of dramatic progress. On the humoral side of the immune response, the investigation of restricted clonality is still hampered by the technical difficulties in obtaining immortal B cell lines producing thyroid antigen specific high affinity IgG antibodies, although the advent of tools to sequence the immunoglobulin V genes from small quantities of DNA will overcome this difficulty (e.g. by polymerase chain reactions). T cells have also begun to be characterized both phenotypically, thanks to the advent of ever better characterized monoclonal antibodies, and functionally at the clonal level, thanks to refinement of cell culture techniques. Future studies in this area will also need to focus on cell immortalization and maintenance of antigen-specific responses although, major strides in the direct sequencing of the TCR are taking place and investigation of T cell receptors in antigen-specific T cells should be feasible. MHC associations with thyroid autoimmune disease, as studied by analysis of RFLP patterns, have not significantly improved already established serological HLA associations and direct MHC gene sequencing will be required. Analysis of the organ-specific regulation of MHC class II gene expression has led to a better understanding of the functional role of MHC class II genes in thyroid autoimmune disease at the target cell level. Such studies have pointed out important local immune responses within the thyroid gland but have not yet provided the initiating factor or factors for human autoimmune thyroid disease in genetically susceptible individuals.

T lymphocyte sensitization and suppressor T lymphocyte defect in patients long after treatment for Graves' disease.

SUMMARYCirculating thyroid autoantibodies, T lymphocyte sensitization and suppressor T lymphocyte function were examined in thirty‐nine patients who had been treated for a mean of 10±2 years previously for hyperthyroid Graves’disease. Nineteen had been treated with radioactive iodine (131I), two with subtotal thyroidectomy, and eighteen were in long‐term remission after previous propylthiouracil (PTU) therapy. Sensitization of T lymphocytes to human thyroid antigen was studied by means of a modified migration inhibition factor test, using T lymphocyte enriched preparations, and antigen‐specific suppressor T lymphocyte function was assessed by the ability of T lymphocytes to suppress the production of migration inhibition factor by sensitized T lymphocytes of patients with active Graves’disease in response to human thyroid antigen. Despite effective correction of the hyperthyroid state in all patients, T lymphocyte sensitization to human thyroid antigen was still demonstrable in twelve of nineteen patients treated with 131I (63%), in nine of eighteen patients in remission after PTU treatment (50%) and in one of two patients after thyroidectomy. All those showing such sensitization also manifested a suppressor T lymphocyte defect. In three of the patients (all after 131I therapy) showing no such sensitization, there was nevertheless evidence of a suppressor T lymphocyte defect. On the other hand, thirteen patients (nine in the post‐PTU group, four in the post‐131I group) showed evidence of complete immunological remission. There was no correlation between the abnormal immunological findings and the number of years after treatment of the hyperthyroidism.The present study indicates that T lymphocyte sensitization and the underlying defect in suppressor T lymphocyte function are persistent in the majority of patients with Graves’disease years after 131I destructive therapy, and occur independently of the hyperthyroid state. These findings are consistent with the concept that an inherited defect in suppressor T lymphocyte function plays a primary role in the pathogenesis of Graves’disease. In addition, long‐term remissions following non‐ablative antithyroid drug therapy appear to be of two types, namely, those in which immunoregulation has been restored, and those with remission in spite of a continuingly active immunological process.

Isolation and characterization of mononuclear cells from various thyroid tissue specimens

Extensive studies of humoral and cell mediated autoimmune responses to thyroid antigens have been performed in order to understand the underlying mechanisms of autoimmune thyroid disorders. Very little is known, however, about the nature of the lymphocyte subpopulations in the thyroid gland and their possible involvement in the pathogenesis of thyroid diseases. We have developed a Percoll gradient technique to separate mononuclear cells from thyroid cells of resected thyroid glands. Thyroid tissue was minced, incubated with Dispase and passed through a tissue sieve. The filtrate was layered onto a four step discontinuous Percoll gradient (densities 1.140, 1.077, 1.061, 1.030 g/ml). Thyroid cells appeared in band II and mononuclear cells in band III. Mononuclear cells were characterized using the monoclonal antibodies OKT-3, OKT-8, OKI-a and OKM-1, and the levels of these populations in peripheral blood and thyroid tissue compared. Patients have been classified by conventional clinical, immunological and histological criteria. The studies involved thyroid tissues from 8 patients with euthyroid nodular goitre, 7 patients with Graves' disease and 1 with Hashimoto's thyroiditis. In the thyroid tissue of non-autoimmune thyroid diseases we find significantly less OKT-3+ cells compared to peripheral blood. In thyroid tissue of autoimmune thyroid diseases there are significantly less OKT-8+ cells compared to peripheral blood. These preliminary results might be linked to the hypothesis of decreased suppressor T-cell activity in autoimmune thyroid disease.

In vitro lymphocyte proliferation in response to polyclonal activators and microbial antigens, and production of immunoglobulins stimulating thyroid adenylate cyclase in Graves' disease.

Cultures of blood lymphocytes from 16 patients with Graves' disease (GD) and 14 matched controls were studied. Incorporation of [14C]thymidine was significantly increased in unstimulated cultures of GD lymphocytes, while the incorporation after stimulation with polyclonal activators (concanavalin A, pokeweed mitogen, phytohaemagglutinin), microbial antigens (E. coli, Candida albicans extract, purified protein derivative of tuberculin, Yersinia enterocolitica serotype 3) and subcellular fractions of human thyroid antigens did not differ from the controls. Due to the increased incorporation in unstimulated cultures, stimulation index is not suitable as an indicator of lymphocyte sensitization. After polyclonal activation or stimulation with thyroid antigens the lymphocytes were cultured for up to 21 days, and the supernatants were investigated for thyroid adenylate cyclase stimulating immunoglobulins (TACSI). No TACSI were demonstrated in supernatants of the lymphocyte cultures neither afer polyclonal activation nor after specific stimulation with several thyroid antigens.

Autoimmunity and hypothyroidism.

Primary myxedema and hypothyroid Hashimoto's disease provide a well-documented example of organ-specific autoimmunity in man. Very slight modifications or increased release of thyroglobulin or thyroid antigens in the circulation may cause the rupture of autotolerance for the normal thyroid components, at least when individuals have a genetic predisposition to autoimmune thyroiditis (possibly associated with a predisposition to other autoimmune diseases). The demonstration of an association between HLA and thyroiditis, however, requires additional studies. The basic immunological abnormality responsible for autoimmunization against thyroid components is a defect in suppressor T cells, shown in experimental animals but not firmly established in man. The result of autoimmunization will be the appearance of cytotoxic mechanisms that lead to destruction of the thyroid follicle with progressive fibrosis, antibody-dependent cell-mediated cytotoxicity apparently being of major importance. A recent report shows, in addition, that thyroid atrophy in primary hypothyroidism is associated with the production of antibodies that block the thyroid-growth-promoting activity of TSH. The recent progress made in our understanding of autoimmune thyroiditis will certainly contribute to improving our knowledge of how and when autoimmunization might develop in man.

Triiodothyronine (T3) binding to immunoglobulin G subclasses in a slightly hypothyroid woman

A 44-year-old woman, with an earlier history of myxoedema, had stopped taking thyroxine and was found to have both a slightly elevated basal TSH level and an increased TSH response after thyroliberin stimulation. The T4 value was low borderline, and using a radioimmunoassay that separated free and antibody-bound hormone by polyethylene glycol precipitation, the T3 value was found to be less than zero. This last finding was explained by the existence of T3 antibodies in the patient's serum. The antibodies, being of the IgG class, did not bind T4, thyroglobulin or cytoplasmic thyroid antigen. The subclass IgG2, and to a lesser extent IgG1, IgG3 and IgG4, did bind T3. A Scatchard plot gave an antibody avidity constant of 4.28 x 10(8) l/mol and a binding site concentration of 3.8 x 10(-9) mol/l. Radioimmunoassay of total T3, after extraction with ethanol gave a high value. The concentration of free T3 was, however, just above the lower reference limit.

Triiodothyronine (T3) binding to immunoglobulin G subclasses in a slightly hypothyroid woman

A 44-year-old woman, with an earlier history of myxoedema, had stopped taking thyroxine and was found to have both a slightly elevated basal TSH level and an increased TSH response after thyroliberin stimulation. The T4 value was low borderline, and using a radioimmunoassay that separated free and antibody-bound hormone by polyethylene glycol precipitation, the T3 value was found to be less than zero. This last finding was explained by the existence of T3 antibodies in the patient's serum. The antibodies, being of the IgG class, did not bind T4, thyroglobulin or cytoplasmic thyroid antigen. The subclass IgG2, and to a lesser extent IgG1, IgG3 and IgG4, did bind T3. A Scatchard plot gave an antibody avidity constant of 4.28 × 108 1/mol and a binding site concentration of 3.8 × 10-9 mol/1. Radioimmunoassay of total T3, after extraction with ethanol gave a high value. The concentration of free T3 was, however, just above the lower reference limit.

IMMUNOLOGICAL DISTURBANCES IN TOXIC MULTINODULAR GOITRE AND ACTIVE GRAVES’DISEASE

Cell-mediated and humoral immunity were studied in seventeen patients with toxic multinodular goitre, ten with active Graves' disease and fourteen healthy controls. The study included determination of sheep erythrocyte and complement rosette-forming cells in the peripheral blood, immunoglobulin levels, titres of microsomal antibodies and migration inhibition test using thyroid extract and phytohaemagglutinin. When compared with controls the patients showed a positive response to thyroid antigen in the leucocyte migration inhibition test. Microsomal antibodies were detected in seven out of ten active Graves' disease patients against two out of seventeen of those with toxic multinodular goitre. Significantly increased IgG and IgA and deceased IgM levels were found only in the toxic multinodular group. These data provide further evidence for immunological disturbances in toxic multinodular goitre.