S. M. McLachlan

TSH RECEPTOR ANTIBODY SYNTHESIS BY THYROID LYMPHOCYTES

Several indirect observations have indicated that lymphocyte in the thyroid may be an important site of TSH receptor antibody synthesis in Graves’disease and we now describe an investigation of this possibility using improved lymphocyte isolation and TSH receptor antibody assay procedures. Our studies demonstrate that thyroid Iymphocytes spontaneously produce TSH receptor antibody in culture. Furthermore, experiments with mitogen tend to suggest that these cells, in contrast to lymphocytes from lymph nodes draining the thyroid, are part of an active immune response to the TSH receptor.

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 auloantibodies to thyroglobulin (Tg) and/or thyroid peroxidasc (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 × I06 cells mouse and the highest levels were recorded 2–3 weeks later. In contrast, Tg and/or TPO antibodies were undctcctablc 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 SCTD recipients of thyroid lymphocytes were of subclasses IgG1, IgG2 and IgG4 and the proportions closely resembled those of the donors scrum Tg antibodies. Blood lymphocytes transferred to SCiD recipients were also able to produce Tg antibodies of subclasses1, 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.

Evidence for a Potential Common T-Cell Epitope Between Human Thyroid Peroxidase and Human Thyroglobulin with Implications for the Pathogenesis of Autoimmune Thyroid Disease

In order to explore the possibility that, in autoimmune thyroid disease, anti-thyroglobulin (Tg) and anti-thyroid peroxidase (TPO) antibodies arise concurrently because they share a common T-cell epitope, we performed a detailed comparative analysis of the cDNA nucleotide sequences corresponding to these two genes. We discovered an 8 amino acid region (Leu-Ser-Glu-Asp-Leu-Leu-Ser- Ile in human TPO) in which there were 6 identical and 2 conserved amino acid residues when compared with human Tg. This remarkably similar region is near the amino-terminus of human TPO (residues 119-126) and the carboxyl-terminus of human Tg (residues 2763-2770). A second feature of interest was that this region of homology conforms to the Rothbard algorithm for a T-cell epitope. Third, probing of the Swiss-protein data bank (10,008 proteins containing 2,952,765 amino acids) with the human TPO region revealed greater homology for human Tg than for any other eukaryotic protein. Two bacterial proteins (E. coli aminopeptidase N and stringent starvation protein) had higher homology scores from human TPO than did human Tg. Our findings provide circumstantial evidence that human TPO and human Tg, and possibly certain bacterial proteins, do indeed share common T-cell epitopes that may play a role in the pathogenesis of autoimmune thyroid disease.

Thyroid autoantigens and human T cell responses.

We investigated the ability of T cells from patients with Hashimotos thyroiditis and with Graves disease as well as control donors to proliferate in response to thyroid peroxidase (TPO) and thyroglobulin using (i) lymphoid cells from different lymphoid organs; (ii) unfractionated or CD8‐depleted lymphoid suspensions or T cells+autologous low density cells (LDC); (iii) 200‐μI well cultures and 20‐μl hanging‐drop microcultures; and (iv) intact TPO and thyroglobulin, denatured thyroglobulin and 12 synthetic peptides predicted on the basis of the amino acid sequence of TPO to be T cell epitopes. In 200‐μl well cultures, proliferative responses (assessed in terms of 3H‐thymidine uptake) to intact TPO or thyroglobulin, digested thyroglobulin or synthetic TPO peptides were not significantly different in unfractionated or CD8‐depleted lymphoid suspensions from blood, thyroid or lymph nodes of TPO/thyroglobulin autoantibody‐positive patients, autoantibody‐negative patients or control donors. In contrast, blood T cells from some high titre patients with Hashimotos thyroiditis (but not from healthy individuals) proliferated in response to intact thyroglobulin or TPO presented by autologous LDC in hanging‐drop microcultures. Heat denatured thyroglobulin (with which thyroglobulin autoantibodies do not interact) did not stimulate proliferation and this observation, together with the ability of T cells from some patients to respond to intact thyroglobulin in the absence of LDC, indicated that thyroglobulin‐specific B cells may be involved in antigen presentation. As we were unable to demonstrate proliferation by blood T cells + LDC from all thyroglobulin antibody‐positive patients with Hashimotos thyroiditis, our studies suggest that the presence of sufficient precursor T cells, as well as the number and type of antigen‐presenting cells, are critical for T cell proliferative responses to human TPO and thyroglobulin.

Human Monoclonal Thyroglobulin Autoantibodies of High Affinity. I. Production, Characterisation and Interaction with Murine Monoclonal Thyroglobulin Antibodies

Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor‐α(TNF‐α), interferon‐gamma (IFN‐γ), interleukin‐1 (IL‐1 α and β) and interleukin‐2 (IL‐2) in the presence or absence of thyroid‐stimulating hormone (TSH). Neither TNF‐α nor IL‐2 affected thyroid cell survival, IFN‐γ was usually inhibitory and IL‐lα slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN‐γ, IL‐2 and TNF‐α inhibited the ability of thyroid lymphocytes from patients with Graves disease and Hashimotos thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well‐recognized ability to induce MHC class II antigens on thyroid cells, IFN‐γ may inhibit thyroid cell proliferation and TNF‐α, IFN‐γ and IL‐2 may down‐regulate thyroid autoantibody synthesis.

Thyroid peroxidase and the induction of autoimmune thyroid disease

Animal models of autoimmune thyroid disease are associated with thyroglobulin (Tg) as autoantigen whereas in man the autoimmune response to microsomal antigen/thyroid peroxidase (TPO) appears to play a major role in thyroiditis. Consequently, we have compared the ability of TPO and Tg to induce thyroid autoantibodies and thyroid damage in mice known to be susceptible (CBA/J) or resistant (BALB/c) to thyroiditis induced using murine Tg. Groups of three to five mice were immunized twice using Freunds complete adjuvant with 80–100μg highly purified porcine (p) TPO, pTg, rat (r) Tg, human Tg, bovine serum albumin (BSA) or BSA + 0.2 μg pTg (the level of Tg contamination of TPO). Four weeks after immunization with TPO, plasma from CBA/J (but not BALB/c) mice contained IgG class antibodies which bound to TPO‐coated tubes in the presence or absence of excess Tg (and could therefore be clearly distinguished from Tg antibodies) but there was no evidence of thyroiditis in either strain of mice. In contrast, in CBA/J mice immunized with rTg and, to a lesser extent in mice that had received pTg, thyroid tissue was infiltrated with lymphoid cells and/or neutrophils and antibodies to pTg (but not pTPO) were present. Our observations demonstrate that induction of TPO antibody alone is insufficient to lead to thyroiditis in CBA/J mice. Further, these studies emphasize the complex interactions between MHC and different thyroid antigens in the processes leading to thyroid destruction.

A Human-Mouse Hybridoma which Secretes Monoclonal Thyroglobulin Autoantibody with Properties Similar to those of the Donor Patient's Serum Autoantibody

Human monoclonal antibodies produced by Epstein Barr (EB) virus transformation and/or cell fusion are frequently IgM antibodies which tend to cross react with a range of antigens and often bear little relationship to the highly specific IgG antibodies associated with human autoimmune disease. By fusing EB virus transformed B lymphocytes from a Hashimoto patient with a mouse myeloma line and selecting for synthesis of IgG class thyroglobulin (Tg) antibody, we have developed a hybridoma (VB/5) secreting Tg antibody of IgG2 subclass and lambda light chain type which has the characteristics of a monoclonal antibody on isoelectric focussing. The antibody has a high affinity for human Tg and recognises Tg from other primates but not non-primate Tg. However, it does not react with human thyroid peroxidase or a panel of other autoantigens. In terms of affinity constant, functional affinity and affinity heterogeneity, the antibody closely resembles the IgG2 lambda Tg antibody present in the serum of the Hashimoto patient whose B lymphocytes were used to develop the hybridoma. In addition to providing a useful reference standard for Tg antibody IgG subclass assays, VB/5 antibody and the hybridoma line provide a valuable starting point for detailed studies of Tg autoantibodies and the genes coding for the variable regions of their heavy and light chains.

T Cell Epitopes and Thyroid Peroxidase

Investigations of immune responses to exogenous antigens suggest that antibodies tend to interact with conformational, non-linear regions of polypeptides whereas T cells recognize short, lenear amino acid sequences of denatured antigen in association with MHC molecules. Further, detailed analyses of peptides stimulatory for T cells have indicated that such T cell “epitopes” have characteristic patterns of amino acids which may be predicted according to the algorithms of Rothbard [1] or Berzofsky [2].

A Search for Linkage Between Phenylthiocarbamide Tasting, the Kell Blood Group Locus and Autoantibodies to Thyroglobulin and Thyroid Peroxidase

Using new ultrasensitive assays to measure autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg) in the sera of families with autoimmune thyroid disease, it was recently demonstrated that the inheritance of TPO and Tg autoantibodies was consistent with Mendelian inheritance [1]. The tendency to produce TPO and Tg autoantibodies appeared to be inherited as an autosomal dominant trait fully penetrant in women but with reduced penetrance in men, and therefore a genetic linkage analysis with 28 polymorphic serological markers (located on different chromosomes) was carried out. The analysis revealed that several loci, notably the HLA antigens -A, -B, -DR, -DQ, as well as Bf on chromosome 6, could be excluded while markers for other loci (such as the immunoglobulin heavy chain marker Gm on chromosome 14) were uninformative [2]. However, there was a suggestion of weak linkage between the blood group Kell and TPO autoantibody. Kell has been reported to be linked to the locus governing the ability to taste phenylthiocarbamide (PTC) [3] which in turn may be associated with certain forms of thyroid disease [4]. In order to explore further the genetic basis of autoimmune thyroid disease, the study was extended, using a larger group of families, to determine whether autoimmune thyroid disease, Kell and PTC tasting are linked.