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Endocrinology Vol. 145, No. 3 1294-1301
Copyright © 2004 by The Endocrine Society

Thyrotropin Receptor Knockout Mice: Studies on Immunological Tolerance to a Major Thyroid Autoantigen

Pavel N. Pichurin, Oxana Pichurina, Russell C. Marians, Chun-Rong Chen, Terry. F. Davies, Basil Rapoport and Sandra M. McLachlan

Autoimmune Disease Unit (P.N.P., O.P., C.-R.C., B.R., S.M.M.), Cedars-Sinai Research Institute and UCLA School of Medicine, Los Angeles, California 90048; and Division of Endocrinology (R.C.M., T.F.D.), Diabetes and Bone Disease, Mount Sinai Medical School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: mclachlans{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Graves’ disease involves a breakdown in self-tolerance to the TSH receptor (TSHR). Central T cell tolerance is established by intrathymic deletion of immature T lymphocytes that bind with high affinity to peptides from autoantigens (like the TSHR) expressed ectopically in the thymus. In TSHR-knockout mice, tolerance cannot be induced to the TSHR, which should, therefore, be a foreign antigen for these animals. To test this hypothesis, TSHR-knockout mice and wild-type controls were vaccinated (three injections) with TSHR DNA or control DNA. TSHR antibodies, measured by ELISA, binding to TSHR-expressing eukaryotic cells, and TSH binding inhibition, developed in approximately 60% of TSHR-knockout mice, not significantly different from 80% in the wild-type mice. Antibody levels were also comparable in the two groups, and both strains recognized the same immunodominant linear antibody epitope at the amino terminus of the TSHR. Splenocyte responses to TSHR protein in culture, measured as interferon-{gamma} production, were similar in TSHR-knockout and wild-type mice. Moreover, T cells from both strains recognized the same two epitopes from a panel of 29 synthetic peptides encompassing the TSHR ectodomain and extracellular loops. This lack of difference in immune responses in TSHR-knockout and wild-type mice is unexpected and is contrary to observations in other induced animal models of autoimmunity. The importance of our finding is that the TSHR may not be similar to other model proteins used to define the concept of central immune tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTOIMMUNITY IMPLIES a breakdown in tolerance to self-proteins. In Graves’ disease, the development of thyroid stimulatory autoantibodies involves an immune response to a major thyroid autoantigen, the TSH receptor (TSHR). Development of tolerance by eliminating self-reactive lymphocytes is a complex process that occurs at different stages of development and involves both central and peripheral mechanisms (reviewed in Refs. 1, 2, 3). Central tolerance is established by intrathymic T cell education in which immature T lymphocytes are exposed to peptides processed from proteins expressed ectopically in the thymus. T cells with receptors that bind with high affinity to peptides from self-antigens undergo apoptosis and are deleted from the repertoire (4). Several studies have demonstrated that the TSHR is expressed in human and rodent thymic tissue, as detected by TSHR mRNA and protein (5, 6, 7). Against this background, it seems likely that T cells in humans and mice develop central tolerance for the TSHR by presentation of this antigen in the thymus during development.

A variety of novel protocols has been used to induce TSHR antibodies and Graves’ disease in mice and hamsters. One approach, vaccination with naked TSHR DNA in a plasmid, was found to induce detectable levels of TSHR antibodies in inbred mice (8) as well as antibodies and hyperthyroidism in some outbred mice (9). Subsequently, other researchers were unable to replicate these findings in the same strain of inbred mice (10, 11, 12, 13). These latter observations were surprising because, despite variability between animals, DNA vaccination has been used successfully to initiate substantial immune responses against a variety of antigens. However, it is notable that most studies involving naked DNA use foreign proteins, for example the human immunodeficiency virus protein gp120 (14).

Recently, a TSHR-knockout mouse has been developed (15). These mice survive normally if their diet is supplemented with desiccated thyroid extract. Assuming that central tolerance involves intrathymic expression of the receptor, such knockout animals would not be tolerant of the TSHR. Consequently, for the knockout mice, the TSHR would be a foreign antigen and, after immunization, would be expected to develop antibody responses of greater magnitude and/or T cell recognition of different epitopes than wild-type mice tolerant of a self-antigen. To test this hypothesis, we vaccinated TSHR-knockout mice and wild-type controls with TSHR DNA or control DNA and studied their antibody and T cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and DNA vaccination
TSHR-null mice were developed by injecting C57BL/6 mice with stem cells from a substrain of 129/Sv mice expressing a mutation designed to knock out TSHR expression (15). Knockout mice and wild-type controls, both on the same mixed genetic background (referred to as C57BL/6/129), were bred in a pathogen-free facility in the Cedars-Sinai Research Institute. From birth, the knockout animals were maintained on thyroid-supplemented mouse chow (modified Pico Rodent 20w/100 ppm thyroid powder, Newco Distributors, Rancho Cucamonga, CA). DNA vaccination was performed as previously described (10). Briefly, male and female mice (6–7 wk of age) were pretreated in the anterior tibialis muscle with cardiotoxin (100 µl per injection, 10 µM Naja nigricollis; Calbiochem, La Jolla, CA). Five to 7 d later, the mice were injected in the same muscle with TSHR DNA (100 µg) (10) or control DNA in the vector pcDNA3. Vaccination was repeated twice at 3-weekly intervals, and mice were euthanized 4 wk later to harvest blood and spleens. All animal studies were approved by the local Institutional Animal Care and Use Committee and were performed in accordance with the highest standards of care.

Purified TSHR antigen and synthetic TSHR peptides
TSHR-289 is an engineered recombinant variant of the receptor expressed in Chinese hamster ovary (CHO) cells that corresponds approximately to the extracellular A-subunit (16). Recombinant A-subunit protein was isolated from culture medium by affinity chromatography using mouse monoclonal antibody 3BD10 (16, 17) and analyzed by SDS-PAGE to determine its purity and concentration. Before use in ELISA or in lymphocyte cultures (below), TSHR A-subunit protein was dialyzed against 10 mM Tris (pH 7.4) and 50 mM NaCl.

Peptides encompassing amino acids in the extracellular domain and the three extracellular loops of the TSHR (a gift of Dr. John Morris, Mayo Clinic, Rochester, MN) were synthesized using an automated 431A peptide synthesizer and HPLC purified, and their structures were confirmed by mass spectrometry (18). Twenty-six peptides span the extracellular domain, each peptide being 20 amino acids in length and overlapping the subsequent peptide by five residues. Peptides were designated A, B, C, etc. corresponding to residues 22–41, 37–56, 42–71, etc. Residues 1–21 were omitted because they comprise the TSHR signal peptide that is not present in the mature protein (19). The three extracellular loop peptides, EC1, EC2, and EC3, corresponded to residues 474–494, 561–570, and 650–661, respectively. Peptides were resuspended in sterile distilled water and used at a final concentration of 10 µg/ml.

ELISA for TSHR antibodies
TSHR antibodies were measured by ELISA (sera diluted 1:100) using wells coated with recombinant TSHR A-subunit (see above; 1 µg/ml) as described (10). Antibody binding was detected with horseradish peroxidase-conjugated mouse anti-IgG (Sigma Chemical Co., St. Louis, MO). The signal was developed with o-phenylenediamine and H2O2, and optical density (OD) values were read at 490 nm.

Linear antibody epitopes were determined using ELISA wells coated with synthetic TSHR peptides (see above; 10 µg/ml peptide) as previously reported (20). Antibody binding (sera diluted 1:200) and detection was performed using peroxidase-conjugated antimouse IgG and substrate (described above). Peptide assays included TSHR A-subunit-coated wells as a positive control for potentially negative peptides and also serum from a control unimmunized mouse.

Flow cytometry for TSHR antibodies
Mouse sera (1:50 dilution) were studied for antibody binding to CHO cells expressing the TSHR ectodomain tethered to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor, TSHR-GPI cells (21). Binding was detected with fluorescein isothiocyanate-conjugated, affinity-purified, goat antimouse IgG (Caltag, Burlingame, CA). Assays included control untransfected CHO-K1 cells as well as cells incubated with second antibody alone. Flow cytometry was performed (10,000 events) using a FACScan with CELLQUEST Software (Becton Dickinson, San Jose, CA). Data are reported as geometric mean fluorescence for TSHR cells minus geometric mean fluorescence for CHO-K1 cells ({Delta} mean fluorescence).

Assays for TSH-binding inhibition (TBI) and thyroid-stimulating antibody (TSAb)
TBI was measured using a commercial kit (DYNOtest TRAK, ALPCO, Windham, NH). In brief, serum aliquots (25 µl, diluted 1:8 in assay buffer) were added to TSHR-coated tubes; after washing, [125I]TSH (~50,000 cpm) was added. After incubation and washing, radioactivity in the tubes was counted. TBI values were calculated as follows:


The normal range was defined as the mean ± 2 SD of TBI values from control-DNA-vaccinated mouse sera.

TSAb was measured and calculated as previously described (22). In brief, monolayers of CHO cells expressing the wild-type TSHR in 96-well plates were incubated with 3% test serum in 100 µl Hanks’ buffer without NaCl and supplemented with 20 mM HEPES (pH 7.4), 1 mM isobutylmethylxanthine, 220 mM sucrose, and 0.3% BSA. After 3 h at 37 C, total cAMP content (medium and cells) was measured by RIA. TSAb values are expressed as a percentage of basal cAMP generated in the presence of serum from normal, untreated mice.

Serum thyroxine levels
Total thyroxine (T4) in mouse sera was measured in undiluted serum (25 µl) by RIA using a kit (Diagnostic Products Corp., Los Angeles, CA).

Lymphocyte response to TSHR antigen and peptides
Splenocytes (duplicate 200-µl aliquots of ~5 x 105 cells) were incubated in round bottomed 96-well plates in the presence or absence of TSHR A-subunit protein (10 µg/ml), TSHR peptides (10 µg/ml), or concanavalin A (Sigma; 5 µg/ml). Culture medium was RPMI 1640, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamycin, 100 U/ml penicillin (all from Life Technologies, Inc., Carlsbad CA), and 50 µM ß-mercaptoethanol (EM Science, Gibbstown, NJ). After 5–6 d (37 C, 5% CO2), supernatants were centrifuged to remove cell debris and stored at -80 C until assayed. Interferon (IFN)-{gamma} was measured in culture supernatants by ELISA using capture and biotinylated detection antibodies from BD PharMingen (San Diego, CA) and reported as picograms per milliliter extrapolated from recombinant IFN-{gamma} standards (PharMingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TSHR antibodies in wild-type and TSHR-knockout mice
Vaccination with TSHR DNA in plasmid induced TSHR antibodies in both TSHR-knockout and wild-type mice as measured in four different assays. First, binding of IgG-class antibodies to ELISA wells coated with TSHR A-subunit protein was present in 58% (seven of 12) knockout mice and 80% (four of five) wild-type mice injected with TSHR DNA (Fig. 1AGo). No binding was detectable in sera from mice injected with control DNA. Second, on flow cytometry, sera from 67% (eight of 12) knockout mice and 80% (four of five) wild-type mice vaccinated with the TSHR had antibodies that bound to the TSHR expressed on CHO cells (Fig. 1BGo). In a third approach, TSHR antibodies were assayed for TBI. As for the ELISA, similar proportions of TSHR knockout and wild-type mice vaccinated with TSHR DNA had positive TBI values (greater than the mean + 2 SD for mice vaccinated with control DNA; Fig. 1CGo). Finally, TSAbs were present in one TSHR knockout mouse and two wild-type mice (Fig. 1DGo).



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  		FIG. 1. TSHR antibodies are induced in TSHR-knockout (KO) and wild-type mice by TSHR DNA vaccination. A, Antibody binding to ELISA wells coated with TSHR A-subunit protein; data are reported as OD values at 490 nm. B, Antibody binding to TSHR-expressing CHO cells detected by flow cytometry. Values are the net mean fluorescence after subtraction of binding to untransfected CHO cells. C, TBI values reported as percent inhibition of radiolabeled TSH binding. D, TSAb reported as percent cAMP production relative to sera from control mice. In all panels, data are shown for individual mice after immunization with TSHR or control DNA. The shaded area represents the mean ± 2 SD for normal mice. Con, Control.

 
Overall, there were no significant differences between TSHR knockout and wild-type mice in their antibody responses to TSHR DNA vaccination in terms of mean levels or proportions of mice positive by ELISA, flow cytometry, TBI, or TSAb (summarized in Table 1Go). Some TSHR-knockout mice had higher levels of binding to TSHR-CHO cells than wild-type mice (Fig. 1BGo), and most wild-type mice had higher TBI values than some knockout animals (Fig. 1CGo). However, comparing the responses for the two groups of mice, these differences were not significantly different. TSAb activity was detectable in two wild-type mice, but neither animal was thyrotoxic: mean serum T4 levels (± SE) were similar in TSHR-DNA- and in control-DNA-vaccinated wild-type mice [4.0 ± 0.2 µg/dl (n = 5) and 3.0 ± 0.2 µg/dl (n = 7), respectively]. TSHR-knockout mice were not studied for T4 because they were maintained on thyroid hormone-supplemented mouse chow. Moreover, any TSAb that may be present in these mice obviously cannot induce hyperthyroidism in the absence of the TSHR.


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  		TABLE 1. Antibody and lymphocyte responses in TSHR-knockout and wild-type mice vaccinated with TSHR DNA

 
Linear TSHR antibody epitopes
The antibody epitopes measured by TBI, TSAb, and flow cytometry are typically conformational and are not easily amenable to analysis of their amino acids. In contrast, linear antibody epitopes can be determined using synthetic peptides. For this purpose, we used ELISA wells coated with 29 synthetic overlapping peptides corresponding to the TSHR ectodomain (peptides A–Z, encompassing amino acid residues 22–415; residues 1–21 correspond to the signal peptide not present in the mature protein) and the three extracellular loops (peptides EC1, EC2, and EC3) (18). The major peptide recognized by sera from TSHR-DNA-vaccinated knockout and wild-type mice, namely peptide A (residues 22–41) at the amino terminus of the TSHR, was not recognized by sera from control-DNA-vaccinated mice (Fig. 2Go). In contrast, sera from all mice regardless of strain or type of vaccination bound nonspecifically (although at a lower level) to peptide F (97–116). Recognition of other peptides in the panel was more variable, with moderate binding to a number of peptides in the C-terminal and extracellular loop regions. Peptide R (residues 277–296) was recognized by sera from some TSHR-vaccinated mice (Fig 2Go, upper and middle panels), with ELISA OD levels >0.8 attained in two of seven knockout and two of four wild-type mice (data not shown). In addition, serum from one of seven TSHR-vaccinated knockout mice, but none of the wild-type mice, bound to peptide W (residues 352–371).



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  		FIG. 2. Recognition of linear antibody epitopes by TSHR-knockout (KO) and wild-type mice vaccinated with TSHR DNA in a plasmid. IgG class antibody binding was measured using ELISA wells coated with 29 peptides (A–Z covering the TSHR ectodomain; EC1, EC2, and EC3 corresponding to the extracellular loops). Top, TSHR-KO (n = 7) mice vaccinated with TSHR DNA; middle, TSHR wild-type mice (n = 4) vaccinated with TSHR-DNA; bottom, TSHR-KO (n = 2) and TSHR wild-type mice (n = 2) vaccinated with control (Con) DNA. Data are reported as the mean + SEM (sera diluted 1:200). Peptides recognized by TSHR-vaccinated mice are indicated by letters. Peptide F, which gave an elevated nonspecific signal from all control and immunized mice, is also indicated. Horizontal dotted line, Mean OD for peptide F in control DNA immunized mice.

 
T cell responses in TSHR-knockout and wild-type mice
To study cellular immunity, we first examined the response of lymphocytes from knockout and wild-type mice challenged in vitro with TSHR A-subunit protein or with the mitogen concanavalin A. Splenocyte culture supernatants from TSHR-DNA-vaccinated knockout mice and wild-type mice generated significantly more IFN-{gamma} when cultured with TSHR A-subunit protein than when cultured in medium alone (P = 0.03; Fig. 3Go, right panel). Importantly, there was no difference in the magnitude of the IFN-{gamma} response to the receptor protein in knockout or wild-type mice. Control-DNA-vaccinated mice did not respond to TSHR A-subunit protein (Fig. 3Go, left panel). However, all mice (regardless of strain or DNA used in vaccination) generated substantial amounts of IFN-{gamma} after incubation with concanavalin A.



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  		FIG. 3. Similar responses of lymphocytes from TSHR-knockout (KO) and wild-type mice to in vitro challenge with TSHR A-subunit protein. Spleen cell suspensions from both mouse strains vaccinated with control or TSHR DNA were incubated (6 d) in medium, with TSHR protein or with concanavalin A. IFN-{gamma} production analyzed by ELISA is reported as picograms per milliliter. *, Values significantly different at P = 0.03 (t test); ns, not significantly different.

 
Next, we analyzed the epitopes recognized by T cells from TSHR-knockout and wild-type mice with the same panel of synthetic TSHR peptides used to determine linear antibody epitopes. After vaccination with TSHR DNA, splenocytes from the knockout mice produced IFN-{gamma} in response to peptides G and J (residues 112–131 and 157–176, respectively) (Fig. 4AGo). Splenocytes from similarly vaccinated wild-type mice responded to the same peptides, G and J (Fig. 4BGo). Unlike the TSHR-vaccinated mice, no particular peptides stimulated splenocyte cytokine production in control-DNA-injected mice; splenocytes from knockout mice responded variably to multiple peptides (Fig. 4CGo), whereas splenocytes from wild-type mice had minimal responses to all peptides (Fig. 4DGo).



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  		FIG. 4. Responses of spleen cells from TSHR-knockout (KO) and wild-type mice vaccinated with TSHR or control (Con) DNA. Splenocytes were incubated for 6 d with 26 peptides (20-mers) covering the TSHR ectodomain (peptides A–Z) and three peptides corresponding to the extracellular loops (EC1, EC2, and EC3). Culture supernatants were analyzed for IFN-{gamma} production (ELISA) and expressed as picograms per milliliter. A, TSHR DNA-vaccinated TSHR-KO mice (n = 4); B, TSHR DNA-vaccinated wild-type mice (n = 4); C, control DNA-vaccinated TSHR-KO mice (n = 3); D, control DNA-vaccinated wild-type mice (n = 3). The peptides inducing a response are indicated in capital letters. The dotted lines indicate the mean + SD for splenocytes incubated in medium alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Major differences have previously been documented between the immune response to a self-protein in wild-type animals vs. animals lacking the same antigen during embryogenesis. For example, different T cell epitopes are recognized after immunization with myelin basic protein (MBP) and adjuvant by mice lacking MBP vs. wild-type mice (23, 24). Even more striking, mice lacking the cell-adhesion protein desmoglein 3 (but not their wild-type counterparts), when immunized with this protein, develop immune responses that can be adoptively transferred to wild-type mice, leading to antibody-mediated pemphigus vulgaris (25).

Based on findings of this type, we anticipated that the magnitude (or titer) of antibody responses and/or the spectrum of T cell epitopes would differ markedly in TSHR-knockout mice vs. wild-type mice after multiple injections of naked TSHR DNA. Indeed, our initial rationale for studying the TSHR-knockout mice was the possibility that naked TSHR DNA vaccination would induce high antibody levels in these animals, unlike the low or undetectable antibody levels we and others observed in BALB/c mice (10, 11, 12, 13). However, we found that TSHR-knockout mice and wild-type mice had similar antibody responses measured by ELISA, flow cytometry, TBI, and thyroid stimulation (Table 1Go). Because fewer wild-type than TSHR-knockout mice were characterized in these assays, minor differences between the two types of mice may have been obscured. Against this possibility, in TSHR-knockout and wild-type mice with comparable levels of antibody binding to the TSHR by ELISA, the same linear antibody epitopes were recognized. Moreover, the magnitude of splenocyte responses to TSHR protein was similar and the same dominant TSHR T cell epitopes were recognized by wild-type and knockout mice (Table 1Go). Unexpectedly, therefore, we found no major differences between TSHR-knockout mice, for whom the TSHR should be a foreign antigen, and wild-type controls in their response to vaccination with TSHR DNA.

The epitopes recognized by the TSHR-knockout and wild-type mice vaccinated with TSHR DNA deserve a comment. The dominant linear antibody epitope recognized by TSHR-knockout and wild-type mice is located at the extreme amino terminus and contains four cysteine residues (Fig. 5Go). The same peptide was immunodominant in several mouse strains immunized with TSHR protein and adjuvant as well as in BALB/c and C57BL/6 mice vaccinated with TSHR DNA and in BALB/c mice immunized with TSHR adenovirus (20). In addition to this dominant epitope, sera from approximately 50% of TSHR-DNA-vaccinated knockout mice and wild-type mice bound to peptide R, and serum from one knockout mouse recognized peptide W. Peptide W (but not peptide R) was previously found to be a linear antibody epitope in BALB/c mice immunized with TSHR adenovirus (20). In terms of cellular immune responses, splenocytes from TSHR-knockout and wild-type mice responded to peptides G and J (Fig. 5Go). We found that both peptides induced responses in TSHR-DNA-vaccinated mice of other strains. However, the G and J peptide combination differed from the patterns for BALB/c mice (peptides C, D, and J) and NOD.H-2h4 mice (peptides G and O) (26).



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  		FIG. 5. Amino acid sequences of synthetic human TSHR peptides recognized by T cells or antibodies. The sequences are aligned with the corresponding sequences of the following receptors: human TSHR (19 ), murine TSHR (38 ), murine FSHR (39 ), and murine LHR (40 ). Dots indicate identity; dashes indicate gaps. Note that amino acid residues 1–21 comprise the TSHR signal peptide, which is not present in the mature protein.

 
Our surprising finding, namely the similarity between responses to TSHR DNA vaccination in TSHR-knockout and wild-type mice, negates our initial hypothesis concerning the role of central tolerance. Like the studies for MBP and desmoglein (see above), our investigation was not designed to test the important issue of peripheral tolerance. Numerous studies have addressed the role of suppressor T cells in human thyroid autoimmunity (27 ; reviewed in Ref. 28), and T cells specific for the autoantigen thyroid peroxidase were shown to be resistant to tolerance induction (29). However, besides peripheral tolerance, there are a number of other potential explanations for our observations in TSHR-knockout and wild-type mice. First, the TSHR DNA used for vaccination expresses human rather than mouse receptor. Despite 87% amino acid homology (30), the difference may be sufficient for the human receptor to be foreign even in wild-type mice. Although an appealing hypothesis, it should be appreciated that injecting a related but foreign antigen may in itself be insufficient to induce an immune response. For example, wild-type C3H mice do not develop memory T cell responses after immunization with adjuvant and guinea pig MBP, the latter protein differing from mouse MBP by 15 residues distributed over the 168-amino-acid sequence (24). Moreover, injecting mice with transfected B cells expressing either the murine or the human TSHR induced similar immune responses (31). A second possibility concerns the properties of the adjuvant used in different protocols. Conventional immunization, which did reveal major differences between wild-type and MBP-knockout mice (23), involved Freund’s complete adjuvant (mycobacterium and oil). This powerful adjuvant is capable of breaking tolerance to MBP (or other self-antigens) in wild-type mice. In contrast, the adjuvant effect of plasmid DNA is very weak, being limited to intrinsic CpG motifs (32), and is, therefore, less likely to break tolerance to the TSHR in the wild-type mice. Nevertheless, we cannot exclude the possibility that the weak adjuvant effect of the plasmid in combination with the human TSHR is sufficient to break tolerance in wild-type mice.

A third possible explanation involves the presence in the TSHR-knockout mice of the homologous gonadotropin hormone receptors. These proteins, if expressed in the thymus, could cross-tolerize T cells to subsequent immunization with the TSHR. The wild-type mice expressing the TSHR would not require such cross-tolerance. Both TSHR-knockout and wild-type mice may already have comparable levels of tolerance to the TSHR. Therefore, the immune response to TSHR DNA vaccination would induce comparable responses in both strains of mice. Against the likelihood of cross-tolerance is the low level of homology between the linear antibody epitopes and T cell epitopes recognized by TSHR-knockout and wild-type mice (Fig. 5Go).

A fourth hypothesis to explain the lack of difference between wild-type and TSHR-knockout mice may relate to the magnitude of TSHR expression in the thymus. TSHR expression in the thymus of normal humans and rodents is considerably lower than in the thyroid (5, 6, 7). Low levels of thymic TSHR expression in the wild-type mice could preclude central tolerance to the TSHR. Such a phenomenon has been reported in transgenic mice that have a reduced level of transgene expression. For example, resistance to induced autoimmune uveitis correlated with a high level of antigen expression in the thymus (33). Moreover, a membrane-bound form of a model transgenic self-antigen was less effective than the soluble form of the same antigen in inducing T cell tolerance (34). Turning to human disease, a type I diabetes susceptibility locus maps to a variable number of tandem repeats upstream of the insulin gene. These polymorphisms are associated with high vs. low intrathymic insulin expression, thereby suggesting that tolerance to insulin may involve the protective effect of particular alleles (35, 36). Of particular interest in this regard is a recent paper, published after completion of our studies, that supports our observations with the TSHR. After immunization, mice lacking myelin oligoendrocyte glycoprotein (MOG) responded to the same MOG T and B cell epitopes as the wild-type mice (37). MOG transcript expression in the wild-type thymus was detected, albeit in trace amounts and was, of course, absent in the knockout mice. The authors concluded that low intrathymic MOG expression precludes the development of tolerance to this autoantigen in wild-type mice.

In conclusion, TSHR-knockout mice vaccinated with TSHR DNA generate cellular and humoral immune responses to the TSHR that resemble those in wild-type mice in terms of magnitude and epitopes. This lack of difference is an unexpected finding and is contrary to observations in some other induced animal models of autoimmunity. Our studies suggest that, as for MOG, central tolerance to the TSHR does not develop in wild-type mice, at least in animals on the C57BL6/129 background. These findings provide a novel and important message. Besides obvious differences among autoantigens in their protein structure, glycosylation, and cellular location and function, these targets of the autoimmune response also differ markedly with respect to induction of central tolerance.


   Acknowledgments
 
We thank Dr. Alan P. Johnstone (St. George’s Hospital Medical School, London, UK) for his TSHR-GPI cell line, Dr. John Morris (Mayo Clinic, Rochester MN) for providing the panel of TSHR peptides, and Dr. Boris Catz (Los Angeles, CA) for his generous support.


   Footnotes
 
This work was supported by NIH Grants DK 54684 (to S.M.M.) and DK 19289 (to B.R.).

Present address for R.M.: University of Colorado Health Sciences Center, Denver, Colorado 80262.

Abbreviations: CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; IFN, interferon; MBP, myelin basic protein; MOG, myelin oligoendrocyte glycoprotein; OD, optical density; TBI, TSH-binding inhibition; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.

Received October 16, 2003.

Accepted for publication November 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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