The Formation of Thyrotropin Receptor (TSHR) Antibodies in a Graves ...

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 4 Printed in U.S.A.

The Formation of Thyrotropin Receptor (TSHR) Antibodies in a Graves’ Animal Model Requires the NTerminal Segment of the TSHR Extracellular Domain SHUICHI KIKUOKA, NAOKI SHIMOJO, KEN-ICHI YAMAGUCHI, YUKIHIKO WATANABE, AKIRA HOSHIOKA, AIZAN HIRAI, YASUSHI SAITO, KAZUO TAHARA, LEONARD D. KOHN, NAOKI MARUYAMA, YOICHI KOHNO, HIROO NIIMI

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Department of Pediatrics (S.K., N.S., K.Y., Ak.H., Y.K., H.N.) and Second Department of Internal Medicine (Ai.H., Y.S., K.T.), Chiba University School of Medicine, Chiba 260, Japan; SRL, Inc. (Y.W.), Hachioji 192, Japan; Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (L.D.K.), Bethesda, Maryland 20892; and Department of Molecular Biology, Tokyo Metropolitan Institute of Gerontology (N.M.), Tokyo 173, Japan ABSTRACT Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves’, including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves’ patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90 –165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves’

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HE TSH RECEPTOR (TSHR) plays a critical role in both the function and growth of the thyroid and is one of the major thyroid autoantigens. Anti-TSHR antibodies (antiTSHRAbs) of two types are the hallmark of Graves’ disease (GD): TSH-binding-inhibiting immunoglobulins (TBIIs) and stimulating TSHRAbs (reviewed in Refs. 1 and 2). Studies of monoclonal anti-TSHRAbs from a multiplicity of laboratories have concluded that the two types of antibodies are different (1– 8); nevertheless, separate evidence indicates that both types of TSHRAbs have their functional epitope on the N-terminus of the TSHR (1, 2, 8 –16). Thus, site-directed mutagenesis has defined important amino acid residues for stimulating TSHRAbs between residues 25– 61 (1, 2, 10, 15, 16). Studies with the Mc2 and Mc112 TSHR-lutropin/choriogonadotropin receptor (TSHR/LH-CGR) chimeras, with residues 90 –165 and 8 –165 of the TSHR, respectively, substituted by comparable LH-CGR residues, localized the functional epitope for stimulating TSHRAbs on residues 22–165 of the N-terminal segment of the TSHR extracellular domain and demonstrated the importance of residues 90 –165, indeReceived July 7, 1997. Address all correspondence and requests for reprints to: Dr. Naoki Shimojo, Department of Pediatrics and Clinical Research, National Shimoshizu Hospital, Yotsukaido, Chiba 284, Japan.

TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9 –165 (Mc112), 90 –165 (Mc2), or 261–370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc112 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves’ TSHRAbs, but it is important also in their formation in a mouse model of Graves’ disease. (Endocrinology 139: 1891–1898, 1998)

pendent of residues 25– 61, as a stimulating TSHRAb epitope (8, 11–14). Thus, 95% of patients lost all or most of their stimulating TSHRAb activity when large cohorts of patients were evaluated with the Mc2 chimera (12, 14); it is very likely that residues 30 – 61 and 90 –165 are conformationally linked and interdependent. Recent studies, using the TSHR/LHCGR chimeras, additionally identified two types of Graves’ TBIIs, each with different functional properties, whose epitopes are on the N-terminus of the TSHR extracellular domain; one type of Graves’ TBII has its epitope within residues 90 –165, and the other has its epitope on residues 25–90 (8, 13). Although we have progressed in our knowledge of the epitopes of the TSHRAbs in Graves’ patients, we know little of the regions on the TSHR important in their formation. Recently, we developed a model of GD by immunizing AKR/N mice with murine fibroblasts transfected with the TSHR and a murine major histocompatibility complex (MHC) class II molecule, having the same H-2k haplotype, but not either alone (17). Contrary to studies that immunized mice with the extracellular domain of the TSHR alone (18 – 27), we were able to induce immune thyroid disease with the humoral and histological features of human Graves’, including the presence of stimulating TSHRAbs and hyperthyroid-

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ism, in 17–25% of mice (17). We observed the formation of TBIIs in 90% of mice; the difference in stimulating TSHRAb and TBII formation supported conclusions that these were separate populations of antibodies. The Graves’ model opens the door to studies of how TSHRAbs are formed. In this report, we used the Graves’ model to question whether the functional epitopes for stimulating TSHRAbs and TBIIs on the N-terminus of the extracellular domain were critical also in TSHRAb formation. We immunized the AKR/N mice with murine fibroblasts transfected with each of three human (h) TSHR/rat LH-CGR chimeras with residues 9 –165 (Mc112), 90 –165 (Mc2), or 261–370 (Mc4) substituted by equivalent residues of the rat LH-CGR. The first two chimeras are missing the N-terminal portion of the extracellular domain of the TSHR wherein most Graves’ stimulating TSHRAbs and TBIIs have their functional epitope (8, 11–14). The Mc4 chimera is missing the C-terminal portion of the TSHR extracellular domain. We show that the N-terminal region of the TSHR, which is substituted in the Mc2 and Mc112 chimeras, is not only critical as an epitope for functional Graves’ TSHR autoantibodies, but also is critical for their formation. Materials and Methods Animals All experimental procedures were conducted in accordance with the policies of Chiba University’s Animal Care and Use Committee. All AKR/N (H-2k) mice were female and were used at 7 weeks of age in all experiments.

Fibroblasts The murine L cell fibroblast line (28), which expresses a hybrid gene containing Abk and Abd of murine MHC class II (RT4.15HP) and was kindly provided by Dr. R. N. Germain (NIAID, NIH), is the same as used in our previous study (17). The Abd determinant is membrane proximal and was shown not to be associated with antigen presentation (28); thus, this shuffled I-Ak molecule is not different from I-Ak in antigenpresenting activity, and there are no qualitative differences in either T cell or antibody-recognition of I-Ak molecules containing either hybrid or wild-type Abd (28). The control class II-untransfected murine L cell fibroblasts (DAP.3) also were provided by Drs. R. N. Germain and Prof. T. Saito (NIH and Chiba University School of Medicine).

TSHR gene transfection The cloning and characterization of the hTSHR, amplification of rat LH-CGR complementary DNA (cDNA) fragments by PCR, and creation of the TSHR/LH-CGR chimeras were reported previously (11). The chimeric receptors used in this report are designated to indicate the substituted segment of the hTSHR, numbered from the methionine start site (Fig. 1). In Mc112, residues 8 –165 of the hTSHR were replaced by residues 10 –166 of the LH-CGR; in Mc2, residues 90 –165 of the TSHR were replaced by residues 91–166; in Mc4, TSHR residues 261–370 were replaced by residues 261–329. Chimeric receptor cDNA, as well as the hTSHR and rat LH-CGR cDNAs, were subcloned into pSG5 expression vectors, and amplified DNA was purified by CsCl gradient centrifugation (11, 29). After subcloning into a pSG5 vector (Stratagene, La Jolla, CA), the hTSHR or the chimeras were transfected into RT4.15HP cells, together with pMAMneo (Clontech, Palo Alto, CA), using lipofectin (GIBCO BRL, Gaithersburg, MD), as described (17). Cells were selected for neomycin resistance using 500 mg/ml G418 (GIBCO BRL); stable transfectants were selected by their ability to increase cAMP levels in the presence of TSH (17). Positive cells were cloned by limiting dilution. Control RT4.15HP cells, transfected with the pSG5 vector alone, also were established.

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FIG. 1. Structure of the Mc112, Mc2, and Mc4 hTSHR/rat LH-CGR chimeras. The wild-type hTSHR is diagrammatically represented on top; the restriction sites used for chimera construction are noted. Open bars indicate the sequence of the extracellular domain of the TSHR; cross-hatched bars denote rat LH-CGR sequence. Numbers indicate the amino acid residue starting each segment, as numbered from the methionine start site. Chimera receptors are named according to the segment substituted by LH-CGR sequence.

Immunization of mice with transfectants Immunization of mice with the transfectants was as described (17). Briefly, mice were ip immunized every 2 weeks with 107 fibroblasts that had been pretreated with mitomycin C (Kyowa Hakko Kogyo, Tokyo). Two weeks after the sixth and final immunization, mice were killed and bled. These mice were chosen because they have the same class I and a homologous class II I-A molecule to that of the fibroblasts containing the transfected class II and TSHR cDNAs (17). The immunization protocol was chosen because mice first developed hyperthyroidism in significant numbers after the sixth immunization (17).

TSHRAb and thyroid function assays Commercial RIA kits (RSR Limited, Cardiff, UK) were used for the ability of antibodies in the serum to inhibit [125I]TSH binding (TBII activity) and to measure serum T4 levels (Dai-ichi Radioisotopes, Tokyo). Stimulating TSHRAb activity was measured using hTSHR-transfected Chinese hamster ovary (CHO) cells (13). In brief, 4,000 hTSHR-transfected CHO cells were plated in 96-well flat-bottom plates and cultured for 48 h in growth medium. Cells were washed with HBSS and incubated with 25 ml protein A-purified IgG (2 mg/ml) and 175 ml assay buffer (8 mm Na2PO4, 1.5 mm KH2PO4, 0.9 mm CaCl2, 2.7 mm KCl, 0.5 mm MgCl2, 222 mm sucrose, 0.5 mm 3-isobutyl-1-methylxanthine, 0.1% glucose, and 1% BSA). After a 3-h incubation at 37 C, supernatants were collected, and cAMP was measured with a commercial RIA kit (Yamasa Co. Ltd., Chiba, Japan). The ability of the IgG to inhibit TSH activity was measured using the same incubation conditions and hTSHR-transfected CHO cells, except that 10 mU/ml bovine TSH was present with each IgG. IgG was obtained from the sera of pairs of animals within each experimental group to overcome technical problems related to obtaining sufficient serum for all measurements in this report. In all cases, incubations were in duplicate, and all wells were assayed in duplicate. All experiments included normal mouse IgG as a negative control and both a known Graves’ stimulating TSHRAb and purified bTSH as positive controls.

Flow cytometry analysis of transfectants Fibroblasts (106 cells) were incubated with 1 mg monoclonal anti-I-Ak (MHC class II-specific) or anti-Dk (MHC class I-specific) antibodies obtained from the American Tissue Culture Collection, 10.2.16 or 15–5-S, respectively, or an isotype-specific control monoclonal antibody (Becton Dickinson, Mountain View, CA). After 30 min on ice, cells were washed with PBS at pH 7.4 and incubated for 30 min with fluorescein-isothiocyanate-conjugated goat antimouse IgG (Kirkegaard & Perry Labora-

FORMATION OF GRAVES’ TSHRAbs tories, Gaithersburg, MD), then analyzed by flow cytometry on a FACScan Cytometer using CellQuest software (Becton Dickinson).

Proliferation assay The spleens from the mice immunized with hTSHR-transfected RT4.15HP cells were excised, and a cell suspension was prepared. Thirty million cells were incubated with 106 mitomycin C-treated hTSHRtransfected RT4.15HP cells in a 25-cm2 culture flask, at 37 C, in 7% CO2, and in RPMI-1640 supplemented with 10% FBS, 2 mm l-glutamine, 50 mm 2-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin. After 10 days, cells were harvested and assayed for proliferative response to Mc2 or Mc112 chimera-transfected fibroblasts. In brief, 2 3 104 cells were incubated with 105 mitomycin C-treated fibroblasts in triplicate and in flat-bottom microplates. After a 72 h incubation, the cultures were pulsed with [3H]thymidine (0.5 mCi/well) for 16 h. Cells were collected with a harvester, and [3H]thymidine incorporation was measured by liquid scintillation spectrometry.

Other assays and statistics Protein concentration was determined by Bradford’s method (BioRad, Richmond, CA); recrystallized BSA was the standard. Statistical analysis was performed by one-way ANOVA to determine the P value between different groups and by Spearman’s rank correlation coefficient to validate the correlation between two series of data.

Materials Purified bovine TSH preparation was the kind gift of the NIH hormone distribution program (NIDDK-bTSH, 30 U/mg).

Results Characterization of the TSHR- and TSHR/LH-CGRtransfected fibroblasts

When the murine MHC class II-transfected fibroblast cell line, RT4.15HP, was transfected with the hTSHR or the TSHR/LH-CGR chimeras, all expressed the receptor in a functional array, exhibiting TSH-increased stimulation of the cAMP signal system (Fig. 2). The ability of the Mc2 or Mc112 chimeras to exhibit higher activity than the wild-type TSHR has been observed in CHO cells transfected with these chimeras (12). The transfection procedures did not result in different

FIG. 2. Bovine TSH-induced cAMP response of hTSHR-transfected fibroblasts. RT4.15HP cells transfected with the hTSHR (open squares), the Mc2 chimera (dark squares) Mc112 (dark diamonds), Mc4 (open circles), or vector alone (pSG5; closed circles) were stimulated with the indicated concentrations of bovine TSH for 3 h. Then the supernatants were collected, and cAMP in the supernatant was measured using a commercial RIA kit.

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levels of class I expression, which were similar in all cells. Thus, flow cytometry analysis showed that hTSHR- and TSHR/LH-CGR chimera-transfected RT4.15HP cells expressed comparable levels of class I molecules on their cell surface, both with respect to each other and to the DAP.3 controls (Fig. 3, column 2). The hTSHR- and TSHR/LH-CGR chimera-transfected RT4.15HP cells all, however, exhibited increased surface expression of class II antigen, by comparison with the DAP.3 control cells, which exhibited no class II surface expression (Fig. 3, column 3). Mc112- or Mc4-transfected fibroblasts elicited a proliferative response when incubated with spleen cells from mice immunized with the TSHR, who do develop TBIIs and stimulating TSHRAbs (Fig. 4), indicating that Mc112- and Mc4transfected cells have T cell epitope(s). The hTSHR- and TSHR/LH-CGR chimera-transfected RT4.15HP and DAP.3 cells, or vector-transfected RT4.15HP cells, were used to immunize AKR/N mice. TSHRAbs produced by TSHR/LH-CGR chimeras

As previously reported (17), measurements of TBII activity showed that most mice immunized with hTSHR-transfected RT4.15HP cells developed serum TBII activity (Fig. 5). This was not true of the mice in the same experiment which were immunized with vector-transfected RT4.15HP cells (Fig. 5) or with DAP.3 cells expressing hTSHR alone (data not shown; see Ref. 17). Three independent experiments yielded the same results: most mice immunized with hTSHR-transfected RT4.15HP cells, but not with control cells, developed serum TBII activity. A reasonably constant proportion of mice, 17 to 25%, developed elevated T4 values and stimulating TSHRAbs (17). This was again not true of mice immunized with vector-transfected RT4.15HP cells or with DAP.3 cells expressing hTSHR alone (17). In neither case did immunization of mice with fibroblasts transfected with the Mc2 or Mc112 TSHR/LH-CGR chimeras result in the formation of TBII antibodies (Fig. 5). This was true in 3 separate experiments, with a total of 20 mice, and

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FIG. 3. Surface expression of MHC class I (column 2) and class II (column 3) molecules on the surface of murine fibroblasts used for immunization. Procedures were performed as described in Materials and Methods; experiments in column 1 were performed using a control antibody.

this was statistically significant (P , 0.01). No mouse immunized with fibroblasts containing the Mc2 or Mc112 chimera developed increased thyroid hormone levels (Fig. 6), nor did they develop IgGs with stimulating TSHRAb activity (Fig. 7A) or IgGs with the ability to inhibit TSH-increased cAMP levels in CHO cells transfected with the wild-type TSHR (Fig. 7B), as illustrated for the Mc112-immunized mice. As previously reported (17), the ability to inhibit TSHincreased cAMP levels is an activity associated with Graves’ TBII activity (8). The absence of hyperthyroidism and stimulating TSHRAb activity relative to mice immunized with wild-type TSHR was statistically significant (P , 0.05). Mice immunized with the Mc4 chimera developed TBIIs with the same frequency as mice immunized with the wild-type TSHR (Fig. 5). Discussion

GD is an autoimmune thyroid disease characterized by the presence of antibodies against the TSHR that stimulate the thyroid to cause hyperthyroidism and/or goiter. By immu-

nizing mice with fibroblasts transfected with the hTSHR and a MHC class II molecule, but not by either alone, we have induced immune hyperthyroidism that has the major humoral and histological features of GD in AKR/N mice: stimulating TSHRAbs, TBIIs which are different from the stimulating TSHRAbs, increased thyroid hormone levels, thyroid enlargement, thyrocyte hypercellularity, and thyrocyte intrusion into the follicular lumen (17). The development of a GD model in mice enabled us to examine questions concerning the mechanism by which TSHR autoantibodies might form. Evidence had accumulated that the N-terminus of the extracellular domain of the TSHR, residues 25–165, contained critical functional epitopes for both stimulating TSHRAbs and TBIIs (8 –16), both of which develop in the AKR/N mice immunized with murine fibroblasts transfected with the TSHR and murine MHC class II cDNAs (17). Though the importance of the N-terminal region as epitope for the stimulating TSHRAbs to exert their function is clear, its role in induction of TSHRAb in GD, so far, has not been studied. In this report, to see whether the


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FIG. 4. Proliferative responses of spleen cells from mice immunized with TSHR-transfected fibroblasts to chimera receptor-transfected fibroblasts. Spleen cells from mice immunized with RT4.15HP cells transfected with wild-type TSHR were stimulated with 106 mitomycin C-treated fibroblasts transfected with wild-type TSHR for 10 days, then tested for their responses to chimera receptor-transfected fibroblasts in a 3-day proliferation assay. Data are presented as mean 6 SD.

FIG. 5. TBII levels in the sera of mice immunized with fibroblasts transfected with hTSHR/rat LH-CGR chimeras. Each dot indicates the TBII titer of an individual mouse. Inhibition activity exceeding 20% (12 SD . mean of controls) was considered positive, based on experiments in which mice were immunized with vector-transfected RT4.15HP (21).

functional epitope was related to the formation of the TSHRAbs, we immunized mice with the Mc2 and Mc112 TSHR/LH-CGR chimeras that substitute TSHR residues 90 – 165 and 8 –165 with comparable LH-CGR residues, the same chimeras that were used to identify these critical functional epitopes (8, 11–14). We anticipated that N-terminal substitution by LH-CGR might lead to inability to generate stimulating TSHRAb and, in fact, we found no such activity in the

IgG fraction from mice immunized with Mc112- or Mc2transfected fibroblasts. These data are in accord with, and support, previous results that indicate that the N-terminal region is important for stimulating TSHRAb recognition (8 –16). Of special interest was the fact that mice immunized with the N-terminal region-substituted chimeric receptors did not, at all, form TSHRAb detected by the TBII assay. Mc112

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or Mc2 has no substitution at the C-terminal portion of the extracellular domain of the TSHR including immunodominant epitope, residues 357–372. This last point is of interest for the following reasons. Several groups reported that immunization of animals with the immunodominant peptide led to the induction of TSHRAb (30 –33). Furthermore, the immunodominant peptide could adsorb TSHRAb in Graves’

FIG. 6. Serum T4 levels in mice immunized with fibroblasts transfected with Mc112 and Mc2 hTSHR/rat LH-CGR chimeras. Each dot indicates serum T4 of an individual mouse. T4 levels higher than 4 mg/dl were considered significantly elevated, based on the T4 levels in the control mice immunized with vector-transfected RT4.15HP cells (mean 1 3 SD). Data are from a representative of 3 independently performed experiments. Whereas in all experiments, 24% of mice immunized with hTSHR-transfected RT4.15HP cells had elevated T4 values, no mice immunized with Mc112- or Mc2-transfected RT4.15HP cells (n 5 40 total) had elevated T4s. Serum T4 in mice immunized with Mc4-transfected RT4.15HP cells are not shown because of an insufficient amount of sera to be measured.

FIG. 7. Ability of IgG from mice immunized with fibroblasts transfected with hTSHR or TSHR/LH-CGR chimeras to increase cAMP levels (A) or to inhibit TSH-increased cAMP levels, an activity associated with TBII activity (B). Because only small amounts of serum could be obtained from individual mice, the sera from 2 mice were pooled, and the IgG was purified on a protein ASepharose column. The data presented were obtained from vector-transfected (pSG5) RT4.15HP-immunized mice whose TBII and T4 levels were within normal limits (Fig. 4), from mice immunized with hTSHR-transfected RT4.15HP cells who had high serum T4 levels with positive TBII activity, or from mice immunized with the Mc112 chimera. Data are shown as the mean of duplicate determinations. These data were duplicated by multiple different pools (.6) of IgG from comparable animals in each group.

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patients (34 –36), suggesting that this region might be quite important for the formation of the TSHRAb. If this assumption is true, immunization with Mc112 or Mc2 should lead to induction of TSHRAb; however, this was not the case in our system. The discrepancy in results, i.e. TSHRAb production by peptide immunization vs. our present result, may be caused by the difference in systems employed; however, TSHR peptide immunization also gave different results, compared with immunization with the soluble TSHR extracellular domain. Thus, immunization with peptides at the N-terminal region of the TSHR extracellular domain induced stimulating TSHRAb without any TBII activity (37), whereas immunization of the entire extracellular domain of the TSHR largely resulted in production of TBII but not stimulating TSHRAb (18 –24). These data suggest that synthetic peptides are not necessarily antigens presented to the immune system in vivo and that production of TSHRAb by immunization with the TSHR peptides may not reflect the mechanism of TSHRAb formation in Graves’, nor may they represent functional antibodies, because they are not associated with significant hypo- or hyperthyroidism (37). Because our system to immunize mice with TSHR-transfected fibroblasts induces not only stimulating TSHRAb and TBII, both found in Graves’, but also hyperthyroidism and morphological changes in the thyroid consistent with Graves’ (17), in vivo mechanisms for generation of TSHRAb, may strictly require the N-terminal region of the TSHR. Immunization with the Mc4 chimera, which retains the N-terminal sequence of the extracellular domain but substitutes the C-terminus, resulted in the formation of TBIIs, in contrast to immunization with Mc2 and Mc112 chimeras. The importance of the N-terminus of the extracellular domain of the TSHR, and not the Cterminus, in the formation of TBIIs, therefore, was reinforced. There are several possible explanations for the inability of fibroblasts expressing N-terminal region-substituted chi-


meric receptor to raise TSHRAb. First, a different primary and/or tertiary structure in the N-terminal region-substituted chimeric receptors might not allow the generation of antibodies with substantial binding affinity for conformational epitope(s) in native TSHR, because some Graves’ IgGs span the N- and C-terminus and recognize determinants on both termini (2, 9, 34 –36). It would be possible that immunization with N-terminal region-substituted chimeric receptors raise antibodies to N-terminus of the LH-CGR and/or those to TSHR amino acids 166 – 415. We tried to detect antibodies to chimera-receptor by flow cytometry analysis; however, this procedure did not work, perhaps because of low expression levels of transfected receptor or because this procedure is not very sensitive, as recently reported (38, 39). Thus, less than 50% of Graves’ patients have antibodies detectable by this procedure, and there is no correlation with either TBII or stimulating TSHRAb. Detection of antibodies to the immunogenic peptide, aa 352–366, by Western blotting, also failed; but again, this could reflect poor assay sensitivity. A second possibility is that the N-terminal region contains B cell epitope(s) initially recognized by the immune system. In contrast to epitope spreading from C- to N-terminus of the extracellular domain suggested by some reports (14, 34, 35, 40), the Davies group has suggested, by studying the binding ability of TSHRAb to a panel of TSHR peptides, that TSHRAb epitopes in CBA/J mice spread from N- to C-terminus during serial immunization with the extracellular domain of TSHR (27). Because AKR/N mice have the same H-2 haplotype (H-2k) with CBA/J, the N-terminal region may be a primary B cell epitope for AKR/N mice, the strain used in these experiments. Deletion of a primary B cell epitope on the TSHR may be the cause for inability in epitope spreading, which subsequently leads to high affinity TSHRAb that inhibit TSH binding to the TSHR. This hypothesis and the first possibility are not mutually exclusive. Finally, a role of the N-terminal region as T cell epitopes may exist. Although we showed Mc112 contained epitopes recognized by T cells from mice immunized with native TSHR, suggesting that T cell epitope(s) exist on the portion other than segment 112, T cells that recognize the N-terminal region may be critical in generation of TSHRAb, in terms of help for B cells. There have been no data reported on the relationship between T cell epitope specificity and their functions in terms of help for TBII vs. stimulating TSHRAb. Because our murine model induces both types of antibodies, isolation and characterization of TSHR-specific T cells will give important information, to understand the mechanisms for generation of TSHRAbs in GD. In summary, our present report clearly shows that the same N-terminal region of the extracellular domain that is important as epitope for TBIIs and stimulating TSHRAbs, but not C-terminal region, is important in their formation in the mouse model of GD. Acknowledgment We thank Dr. R. N. Germain and Prof. T. Saito for providing RT4.15HP and DAP.3 cells; Ms. S. Uchiyama for help in measuring TBII and thyroid hormone levels; Dr. T. Ban for helpful discussions; and Ms. Y. Tsuchikawa and Ms. Y. Okuda for excellent technical assistance.

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