Graves' Patient with Thymic Expression of Thyrotropin Receptors and Dynamic Changes in Thymic Hyperplasia Proportional to Graves' Disease Activity

Thymic hyperplasia is frequently observed in Graves'' disease. However, detectable massive enlargement of the thymus is rare, and the mechanism of its formation has remained elusive. This case showed dynamic changes in thymic hyperplasia on serial computed tomography images consistent with changes in serum thyrotropin receptor (TSH-R) antibodies and thyroid hormone levels. Furthermore, the patient''s thymic tissues underwent immunohistochemical staining for TSH-R, which demonstrated the presence of thymic TSH-R. The correlation between serum TSH-R antibody levels and thymic hyperplasia sizes and the presence of TSH-R in her thymus suggest that TSH-R antibodies could have a pathogenic role in thymic hyperplasia.     

THE PUTATIVE ANTI-THYROTROPIN RECEPTOR ANTIBODIES OF GRAVES' DISEASE:I. Gm ALLOTYPES *

The hyperthyroidism of Graves' disease is thought to be related to antithyrotropin receptor (TSH-R) antibodies. In order to study the degree of immunogenetic homogeneity of these antibodies, we carried out Gm typing of ‘receptor-purified’ IgG from patients with active Graves' disease and controls. The results were compared to those of serum, total IgG and IgG which failed to attach to TSH-R. We found that in five out of seven Gm heterozygote patients studied the receptor-purified antibodies were restricted to the products of one haplotype compared to three out of five similar controls. Such eluted antibodies were biologically active. Similar results in terms of immunogenetic restriction and activity were obtained when F(ab)₂ preparations were used. An unexpected finding was that sera and IgG from normal persons attached to thyroid membranes and that the attachment occurred via F(ab)₂. Normal whole serum and ‘receptor-purified’ IgG and F(₂ inhibited TSH binding in the receptor assay; however, this inhibition showed no specificity for TSH-R.     

Long-term Pathologic Changes of α1-Acid Glycoprotein (orosomucoid) Glycoforms in Autoimmune Thyroid Disease

Objective: We measured &#102 1-acid-glycoprotein (AGP) in patients with autoimmune thyroid disease to study a possible relationship between microheterogeneity of the naturally occurring glycoforms of AGP and autoimmune thyroid disease. Design, patients, measurements: In a group of 12 fasting thyrotoxic patients (11 females, mean age: 43 years) with newly diagnosed Graves' disease (subgroup 1), we measured serum concentrations of total AGP and its 3 glycoforms (&#119 mol/l, crossed affinity immunoelectrophoresis with con A in the first dimension gel) as well as total thyroxine, total triiodothyronine, thyrotropine, thyroid peroxidase antibodies (anti-TPO), antibodies against the TSH receptor (TRAb, TRAK ® ), at baseline and after 12 months of antithyroid drug therapy (ATD). For comparison, 4 subgroups of thyroid patients (patients with Graves' disease and thyroid associated ophthalmopathy (TAO) (subgroup 2, n = 10), radioiodine treated Graves' patients (subgroup 3, n = 7), Graves' patients without TAO (subgroup 4, n = 13), patients with Hashimoto's thyroiditis (subgroup 5, n = 8)) and 25 normal controls (17 females, mean age: 38 years) were studied. Results: In subgroups of TRAb positive Graves patients' serum levels of glycoform 1, 2 or 3 increased significantly (p < 0.005) after 12 months of ATD as compared to both baseline of that person or normal controls. No significant changes were found in the TRAb negative Hashimoto subgroup. Conclusion: Patients with autoimmune Graves' disease changed their relationship to AGP, and thus a role of AGP and its 3 glycoforms is suggested in the pathogenesis of Graves' disease.     

Genetic immunization with mouse thyrotrophin hormone receptor plasmid breaks self‐tolerance for a murine model of autoimmune thyroid disease and Graves' orbitopathy

Experimental models of Graves' hyperthyroid disease accompanied by Graves' orbitopathy (GO) can be induced efficiently in susceptible inbred strains of mice by immunization by electroporation of heterologous human TSH receptor (TSHR) A‐subunit plasmid. In this study, we report on the development of a bona fide murine model of autoimmune Graves' disease induced with homologous mouse TSHR A‐subunit plasmid. Autoimmune thyroid disease in the self‐antigen model was accompanied by GO and characterized by histopathology of hyperplastic glands with large thyroid follicular cells. Examination of orbital tissues showed significant inflammation in extra‐ocular muscle with accumulation of T cells and macrophages together with substantial deposition of adipose tissue. Notably, increased levels of brown adipose tissue were present in the orbital tissue of animals undergoing experimental GO. Further analysis of inflammatory loci by ¹⁹F‐magnetic resonance imaging showed inflammation to be confined to orbital muscle and optic nerve, but orbital fat showed no difference in inflammatory signs in comparison to control β‐Gal‐immunized animals. Pathogenic antibodies induced to mouse TSHR were specific for the self‐antigen, with minimal cross‐reactivity to human TSHR. Moreover, compared to other self‐antigen models of murine Graves' disease induced in TSHR knock‐out mice, the repertoire of autoantibodies to mouse TSHR generated following the breakdown of thymic self‐tolerance is different to those that arise when tolerance is not breached immunologically, as in the knock‐out models. Overall, we show that mouse TSHR A‐subunit plasmid immunization by electroporation overcomes tolerance to self‐antigen to provide a faithful model of Graves' disease and GO.     

Peripheral Blood Lymphocyte Transformation to G2s in Patients with Autoimmune Thyroid Disease with and without Ophthalmopathy

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.     

Insulin-like growth factor-1 enhances the expression of functional TSH receptor in orbital fibroblasts from thyroid-associated ophthalmopathy

Thyroid-associated ophthalmopathy (TAO), an autoimmune disease, occurs in approximately 50 % of patients with Graves’ hyperthyroidism. Thyroid-stimulating hormone receptor (TSHR) that is expressed in orbital fibroblasts is the autoimmune target in the development of TAO. In addition to thyroid-stimulating immunoglobulin (TSI), insulin-like growth factor (IGF)-1 is also involved in the development of TAO. IGF-1 has been reported to potentiate the effects of thyroid-stimulating hormone (TSH) and TSI on TSHR signaling. In the current study, we investigated the effects of IGF-1 on the cell surface expression of the functional TSHR and its possible mechanism of action in human orbital fibroblasts. Our results show that orbital fibroblasts from the TAO patients expressed higher levels of IGF-1 receptor (IGF-R), compared to control subjects. Treatment with IGF-1 enhanced the expression of surface TSHR in orbital fibroblasts from TAO patients, but not from control subjects. In addition, treatment with IGF-1 increased the level of TSHR at both the protein and mRNA levels. Furthermore, pre-treatment with IGF-1 potentiated TSH-induced cAMP production, compared to cells that were treated with only TSH. Our results also show that pre-treatment with cycloheximide, an inhibitor of mRNA translation, partially, but not completely, inhibited the expression of TSHR on the cell surfaces of orbital fibroblasts from TAO patients. These collective results show that IGF-1enhances the cell surface expression of functional TSHR, not only by increasing TSHR expression, but also by inducing TSHR translocation to the plasma membrane in orbital fibroblasts from TAO.     

Unaltered thyroid function in mice responding to a highly immunogenic thyrotropin receptor: implications for the establishment of a mouse model for Graves' disease.

Grave's disease (GD) is a common disorder characterized by the presence of autoantibodies to the thyrotropin receptor. In the past, the exceedingly low expression of the thyrotropin receptor on thyrocytes has not allowed its purification in quantities sufficient to investigate the establishment of an animal model for this disease. In this study, we have purified the 398-amino acid, extracellular region of the human thyrotropin receptor (TSH-R.E) from insect cells using recombinant baculovirus, and explored its immunopathogenic properties in H-2b,d,q,k,s strains of mice. The receptor preparation was highly immunogenic since it elicited strong specific proliferative T cell responses as well as IgG responses in all strains tested. In addition, hyperimmunization with TSH-R.E induced (i) serum antibodies that blocked the binding of 125I-TSH to its receptor, a common feature of GD autoantibodies; and (ii) IgG that reacted with a synthetic peptide (residues 32-54) from the N-terminus of the receptor, a region implicated in the binding of thyroid stimulating antibodies. In SJL animals only, a weak antibody response to two other thyroid antigens, thyroglobulin and thyroid peroxidase, was also observed. The presence of these antibodies, however, was not accompanied by a detectable alteration in thyroid function as assessed by the measurement of serum TSH, T4 and iodine levels. Also mononuclear infiltration of the thyroid gland or morphological changes compatible with an activation state of thyrocytes were not apparent in TSH-R-challenged mice. In contrast, mice treated with the anti-oxidant aminotriazole showed a dramatic increase in serum TSH levels and an activated follicular epithelium. These data demonstrate that a highly immunogenic TSH-R.E in mice does not necessarily provide a proper stimulus for the induction of a hyper- or hypothyroid status as defined by hormonal or histological criteria. Main reasons for the inability to induce receptor-specific antibodies that affect thyroid function such as those generated in GD are likely to be the inappropriate folding of the recombinant extracellular domain of the receptor, or the xenogeneic nature of the autoantigen.     

Antibodies against 1D,a Recombinant 64-kDa Membrane Protein,Are Associated with Ophthalmopathy in Patients with Thyroid Autoimmunity

We have tested for serum antibodies reactive with ₁D, a recombinant 65-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluorescence and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. We also measured antibodies to a 64-kDa pig eye muscle membrane protein which is identified by SDS-PAGE and Western blotting, correlating the two reactivities. While antibodies to ₁D, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximately 40% of patients with ophthalmopathy, in both tests the greatest prevalence, by immunofluorescence, 73%, was demonstrated in patients with Graves' hyperthyroidism without clinically evident eye disease, although only 50% of these patients were positive in immunoblotting. When the two tests for anti-₁D antibodies were compared, immunofluorescence appeared to be the more specific and immunoblotting appeared to be the more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of less than 1 year duration; tests were positive in 49% of patients with more chronic ophthalmopathy and in 50% of patients with Graves' hyperthyroidism without evident eye disease. Antibodies reactive with ₁ D were detected in 17% of normals by immunofluorescence and 24% by immunoblots, while antibodies reactive with the 64-kDa pig eye muscle protein were detected in only 10% of the normal subjects tested. Lesser prevalences of antibodies to the two 64-kDa proteins in patients with established eye disease suggest that such antibodies may be an early abnormality in patients with Graves' hyperthyroidism who are predisposed to develop ophthalmopathy. Although the association was not close, reactivity against ₁D by immunoblotting, but not immunofluorescence, was significantly correlated with reactivity to a 64-kDa eye muscle membrane protein by immunoblotting. On the other hand, when sera containing antibodies reactive with both ₁D and the 64-kDa eye muscle protein were incubated with CHO (₁D) cell membrane, reactivity against ₁D was absorbed while that against the eye muscle protein was not. The precise relationship between the two 64-kDa proteins can only be clarified by cloning the 64-kDa protein from an eye muscle expression library and comparing the sequences with those of ₁D.     

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.     

Measurement of thyroid stimulating immunoglobulins using a novel thyroid stimulating hormone receptor–guanine nucleotide-binding protein, (GNAS) fusion bioassay

Hyperthyroidism, defined by overproduction of thyroid hormones, has a 2–3% prevalence in the population. The most common form of hyperthyroidism is Graves'' disease. A diagnostic biomarker for Graves'' disease is the presence of immunoglobulins which bind to, and stimulate, the thyroid stimulating hormone receptor (TSHR), a G-protein coupled receptor (GPCR). We hypothesized that the ectopically expressed TSHR gene in a thyroid stimulating immunoglobulin (TSI) assay could be engineered to increase the accumulation of the GPCR pathway second messenger, cyclic AMP (cAMP), the molecule measured in the assay as a marker for pathway activation. An ectopically expressing TSHR-mutant guanine nucleotide-binding protein, (GNAS) Chinese hamster ovary (CHO) cell clone was constructed using standard molecular biology techniques. After incubation of the new clone with sera containing various levels of TSI, GPCR pathway activation was then quantified by measuring cAMP accumulation in the clone. The clone, together with a NaCl-free cell assay buffer containing 5% polyethylene glycol (PEG)6000, was tested against 56 Graves'' patients, 27 toxic thyroid nodule patients and 119 normal patients. Using receiver operating characteristic analysis, when comparing normal with Graves'' sera, the assay yielded a sensitivity of 93%, a specificity of 99% and an efficiency of 98%. Total complex precision (within-run, across runs and across days), presented as a percentage coefficient of variation, was found to be 7·8, 8·7 and 7·6% for low, medium and high TSI responding serum, respectively. We conclude that the performance of the new TSI assay provides sensitive detection of TSI, allowing for accurate, early detection of Graves'' disease.     

Euthyroid Graves' ophthalmopathy with negative autoantibodies

Graves'' disease is an autoimmune-based hyperthyroidism in which a number of different antibodies directed against thyroid tissue plays a role. Graves'' ophthalmopathy is thought to be a consequence of this autoimmune basis and occurs in some patients with Graves'' disease. On occasional cases, the disease may present only with ophthalmopathy without hyperthyroidism. A 32-year-old woman with euthyroid Graves'' ophthalmopathy and negative thyroid autoantibodies, including TSH receptor antibody, is presented here.     

Is the measurement of inferior thyroid artery blood flow velocity by color-flow Doppler ultrasonography useful for differential diagnosis between gestational transient thyrotoxicosis and Graves' disease? A prospective study

OBJECTIVE:

To determine the role of peak systolic velocity, end-diastolic velocity and resistance indices of both the right and left inferior thyroid arteries measured by color-flow Doppler ultrasonography for a differential diagnosis between gestational transient thyrotoxicosis and Graves'' disease during pregnancy.

METHODS:

The right and left inferior thyroid artery-peak systolic velocity, end-diastolic velocity and resistance indices of 96 patients with thyrotoxicosis (41 with gestational transient thyrotoxicosis, 31 age-matched pregnant patients with Graves'' disease and 24 age- and sex-matched non-pregnant patients with Graves'' disease) and 25 age- and sex-matched healthy euthyroid subjects were assessed with color-flow Doppler ultrasonography.

RESULTS:

The right and left inferior thyroid artery-peak systolic and end-diastolic velocities in patients with gestational transient thyrotoxicosis were found to be significantly lower than those of pregnant patients with Graves'' disease and higher than those of healthy euthyroid subjects. However, the right and left inferior thyroid artery peak systolic and end-diastolic velocities in pregnant patients with Graves'' disease were significantly lower than those of non-pregnant patients with Graves'' disease. The right and left inferior thyroid artery peak systolic and end-diastolic velocities were positively correlated with TSH-receptor antibody levels. We found an overlap between the inferior thyroid artery-blood flow velocities in a considerable number of patients with gestational transient thyrotoxicosis and pregnant patients with Graves'' disease.

CONCLUSIONS:

This study suggests that the measurement of inferior thyroid artery-blood flow velocities with color-flow Doppler ultrasonography does not have sufficient sensitivity and specificity to be recommended as an initial diagnostic test for a differential diagnosis between gestational transient thyrotoxicosis and Graves'' disease during pregnancy.     

Characterization of the murine immune response to the murine TSH receptor ectodomain: induction of hypothyroidism and TSH receptor antibodies

The thyrotropin receptor (TSHR) is the major autoantigen of human Graves' disease. In order to define the antigenicity of the TSHR in a defined model, we examined the immune response of BALB/c mice to immunization with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25–50 μg of insect cell expressed, purified and refolded, mTSHR-ecd in alum adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. Control mice received wild-type baculovirus-infected insect cell protein lysate, in a similar way. After 28 days, murine serum contained high titres of antibodies specific to mTSH-ecd and their titres continued to increase over 90 days. Antibody epitope mapping, using 26 peptides spanning the human TSHR-ecd, showed that a variety of regions of the ectodomain were antigenic. The earliest epitope included aa 22–41, but later two regions of reactivity were noted clustered towards the mid portion and carboxyl terminus of the ectodomain. The murine TSHR autoantibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of blocking the native TSHR. We showed that these TSHR antibodies acted, in vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with the hTSHR. Hence, the antibody response to mTSHR-ecd was potentially antagonistic in its influence on the TSHR. Assessment of thyroid function in the immunized mice showed a fall in serum total T3 by 90 days and markedly elevated murine TSH levels (from 64.0 to 239.6 ng/ml), confirming the onset of thyroid failure. However, thyroid histology remained grossly normal. These data demonstrate that mTSHR-ecd is a potent antigen with three major immunogenic regions. The induced mTSHR-Abs blocked TSH action in vivo and reduced murine thyroid function.     

Antibodies to TSH-receptor in thyroid autoimmune disease interact with monoclonal antibodies whose epitopes are broadly distributed on the receptor

The hyperthyroidism of Graves' disease (GD) is caused by TSH-receptor (TSH-R) stimulating autoantibodies (TSAb), leading to overproduction of thyroid hormones. We present evidence for TSAb interaction with three distinct regions of the TSH-R, one in immediate vicinity of the carboxy terminal serpentine. Three murine monoclonal antibodies (MoAbs 28.1, A9 and 31.7) directed to amino acids 36-40, 147-228 and 382-415 were labelled and tested for their binding to human recombinant TSH-R on solid phase. All MoAbs bound to TSH-R with a K(d) of 8-12 nm and showed no competition among themselves. We tested 114 sera from euthyroid controls, 118 TBII positive sera from patients with GD (containing TSAb confirmed by bioassays), 16 TBII positive sera from patients with autoimmune thyroid disease (AIT), who were hypothyroid and had TSH blocking antibodies (TBAb), and 20 patients with AIT, who were hypothyroid but negative for all TRAb. Mid-regional MoAb A9 tracer achieved the highest sensitivity in the GD group (72.0%), whereas C-terminal MoAb 31.7 found most sera positive in the AIT group (87.5%). Surprisingly, the N-terminal MoAb 28.1 had the lowest sensitivity in the GD (10.4%) and AIT group (43.8%). Using a mixture of all three tracer MoAbs did not increase the sensitivity in the GD or AIT group, compared to the best single MoAb alone. Median inhibition of MoAb A9 was significantly (P < 0.001) higher than inhibition of MoAbs 28.1 or 31.7 in the group of GD patients but not in other groups. Almost all patient sera with positive reactivity in the MoAb tracer assays had TBII values in the higher range. However, there were many highly TBII positive sera, which did not show a displacement of the MoAb tracers. We conclude that, contrary to some reports, the binding of TSAb and TBAb to the TSH-R is not restricted to distinct and distant epitopes. The middle part of the TSH-R seems to be more relevant for TSAb binding than the N-terminal part, while a proportion of TSAb autoantibodies also binds to a C-terminal epitope of the TSH-R. The method described here is a TSH independent competitive assay for the detection of TSH-R autoantibodies.     

The Putative Role of Fibroblasts in the Pathogenesis of Graves' Disease: Evidence for the Involvement of the Insulin-like Growth Factor-1 Receptor in Fibroblast Activation

Graves' disease when fully expressed affects the thyroid gland and connective tissues of the orbit and pretibium. While the glandular disease is relatively well-characterized, the pathogenesis of the orbital and dermal components remains enigmatic. In the following article, we review some of the evidence suggesting that fibroblast activation in Graves' disease might play an integral role in the tissue remodeling associated with ophthalmopathy. The thyrotropin receptor (TSHR) is expressed at low levels in several connective tissue depots and by their derivative fibroblasts, including those from the orbit. Little direct evidence currently links extra-thyroidal TSHR expression with Graves' disease. Very recent observations now implicate the insulin-like growth factor-1 receptor (IGF-1R) as a fibroblast activating antigen. When immunoglobulins from patients with the disease, with or without clinical ophthalmopathy, bind IGF-1R on the surface of fibroblasts, the receptor becomes activated and upregulates the expression of two T lymphocyte chemoattractants, IL-16 and RANTES. Thus, IGF-1R may represent a second self-antigen with a pathogenic role in extra-thyroidal Graves' disease.     

Binding and functional effects of thyroid stimulating hormone on human immune cells

The expression and functional relevance of thyroid stimulating hormone (TSH) receptors on human immune cells were studied. Flow cytometric analysis was used to study the binding of biotinylated TSH to human peripheral blood mononuclear cells (PBMC) and various purified lymphoid populations. Our results indicate that the hormone binds well to monocytes and natural killer (NK) cells and marginally to purified tonsillar T and B lymphocytes. There was a significant increase in the binding of TSH to purified B cells that were activatedin vitro withStaphylococcus aureaus Cowan. In contrast, the binding of TSH to T cells was unaltered when they were stimulated with phytohemagglutinin (PHA). While TSH increases DNA synthesis and intracellular cAMP levels of FRTL-5 rat thyroid cells, it did not have such stimulatory effects on lymphocytes. However, there was a moderate increase in Ig production by activated B lymphocytes when they were cultured in the presence of the hormone. A possible function for TSH as a link between the immune system and the thyroid is discussed.